Truth Seeker
New Member
1 Introduction
This Part II of the THC study deals with the methodical, and especially the biological basis for THC limits in food. The essential ideas and results of Part II are summarized in Chapter 3 of Part I and provide the basis for the proposed THC limits. Part II addresses specifically the scientifically interested, who would like to explore individual aspects of the matter-or the whole topic-in more detail, in order to obtain a better understanding of the fundamentals. Here, the reader also finds numerous references.
The majority of the extensive data on the pharmacological effects of THC were collected in animal and cellular experiments, mostly using very high THC doses, often not by oral but by other routes of administration. Chapter 2 tackles the transferability of these results to oral THC administration to humans. A further section of this chapter reviews the methodical basis commonly used in the establishment of limits for other substances and hazards. In the course of this, some aspects are examined that are of special importance in connection with THC.
Chapter 3 contains a survey of the pharmacological effects of THC and of its overall toxicity. Furthermore, matters of bioavailability, metabolism, mode of action, as well as development of tolerance are dealt with. A separate section deals with physical parameters, such as effects of temperature on the biological efficacy of THC.
Chapter 4 examines the matter of the psychotropic threshold, i.e. the placebo threshold for the oral administration of THC. For the purpose of fixing a limit, this serves for the determination of the maximum daily quantity of THC that will not lead to undesired acute or chronic psychological reactions.
Chapter 5 deals with possible physical effects below the psychotropic threshold. It serves for the determination of the maximal daily dose that does not lead to undesirable acute or chronic physical effects.
Some Excursuses discuss special questions that may arise in the context of the application of industrial hemp products to man: Among them is a short comparison of THC and alcohol in food, matters of assimilation of THC by the skin, for instance through cosmetics, as well as the detection of THC in body tissues after the ingestion of hemp-based food in comparison to the impact of marijuana ingestion.
Alcohol-containing products such as beer, wine or spirits that are used for intoxication typically contain 3-50% alcohol (ethanol). Chronic heavy use of alcohol may irreversibly damage brain cells and other organs (liver, heart). There is evidence of serious alcohol-induced malformations in the fetus (alcohol-embryopathy). Alcohol-containing food products such as fruit drinks, sweets, and meat dishes, generally contain less than 0.3% ethanol, this being an amount ten times smaller than what is contained in intoxicating products. This alcohol content, which is generally considered low and innocuous, needs not be declared in this context. According to the WHO (World Health Organization), a regular ingestion of 7 g of ethanol per day is harmless (Verbraucherzentrale 1998).
THC-containing drug products such as marijuana and hashish, applied for drug use, typically contain 1-20% by weight of THC, sometimes more. THC shows some potential for physical impairment, though this is much weaker than in alcohol. (Hall et al. 1994b, WHO 1997).
Cannabis-based food products, such as edible oil based on seeds from low-THC hemp and hemp-seed-based cereal bars, contain less than 0.005% THC (50 ppm) on average. Thus their THC content is less than 1/200 of the amount found in drug products. In Switzerland, precisely this value (i.e. 0.005%) was set as the limit for the concentration of THC in edible oil derived from hemp seeds. In other products of which greater amounts can be consumed, lower limits were set: for pastries it is 0.0005% and for alcoholic beverages it is 0.00002%. Thus the difference between these concentrations and the THC concentration in marijuana amounts to a factor of between 2,000-50,000.
2 Methodology
2.1 Extrapolation of different routes of administration to oral ingestion
A large portion of data on the toxicology of THC for humans and animals were not obtained following oral administration but after inhalative or parenteral (intravenous, subcutaneous, intraperitoneal) application. Different routes of administration result in a different bioavailability and in different pharmacokinetics (surveys: Wall et al. 1983, Maykut 1985, Agurell et al. 1986, Harvey 1991). This has to be taken into account when dosage and plasma concentrations are translated to the situation of an oral administration, i.e. the relevant exposure route for food considered in the following.
Intravenous administration: Intravenous administration results in a 100% bioavailability and an immediate rise of THC concentration in the blood. The onset of THC effects occurs within a few minutes. In order to achieve minimum psychotropic effects, 0.02 mg THC/kg BW (body weight) are necessary; as a function of body weight this typically corresponds to 1 mg THC. In the case of doses that lead to psychotropic effects, short-term peak plasma levels are reached that markedly decrease within minutes. The intravenous administration of 5 mg THC led to a plasma concentration of about 200 ng/ml THC, which decreased rapidly to 15 ng/ml after an hour and 3 ng/ml after 4 hours. The psychotropic effects had vanished after three hours. Smith and Asch (1984) intravenously administered monkeys with 2.5 mg THC/kg BW three times a week. This produced an average maximum plasma concentration of 300 ng/ml THC and an average long-term concentration of 15 ng/ml.
Inhalative administration: After inhalative administration, THC is quickly absorbed and the time course of plasma concentration is similar to the situation after intravenous administration. Bioavailability only reaches 10-30%, so that about five times the dose of intravenous administration is required to achieve the same effects. About 0.7 mg/kg, that is about 5 mg THC, are necessary to produce minimum psychotropic effects. An intoxication desired by cannabis consumers requires inhalation of at least 10-15 mg THC, which would lead to a maximum plasma concentration of 100 ng/ml after about 5 minutes. The concentration decreases rapidly, so that little THC will be detected after 2-3 hours. Chronic consumers need higher doses because of their development of tolerance to the active agent. This was shown in a study where 47 chronic Cannabis users tolerated inhaled THC doses of up to 180 mg without undesired side effects or nausea (Stefanis 1978).
Oral administration: The systemic bioavailability of THC reaches 10-20% after oral administration in a lipophilic vehicle. To achieve minimum psychotropic effects in man, 0.2-0.3 mg/kg are required, which equals 10-20 mg depending on the body weight. This is 10-15 times the dose of intravenous administration. The maximum plasma level after oral administration of this dose is of the order of 5 ng/ml and is reached after 2-4 hours. The psychotropic effect sets in after 30-60 minutes, peaks after 1-3 hours and lasts for 6-8 hours. The average maximum plasma concentration of THC in six cancer patients after oral ingestion of 15 mg THC was 3.9 ng/ml and was typically attained after two hours (Frytak et al. 1984). With the exception of one patient, the plasma level of THC in all cancer patients had dropped below 1 ng/ml or, respectively, no THC could be detected in the plasma any more after 6 hours. Three patients received three doses of 15 mg THC a day. The maximum plasma level ranged between 3.6 and 6.3 ng/ml; thus, it did not differ much from that following a single administration. Ohlsson et al. (1980) observed a maximum THC concentration of 5 to 6 ng/ml between 1 and 1.5 h after experienced marijuana smokers had ingested a chocolate cookie containing 20 mg THC. Similar results were found by Brenneisen et al. (1996) and Frytak et al. (1979), though they occasionally found higher plasma concentrations of more than 10 ng/ml.
The above comparison suggests that, in order extrapolate data from intravenous and inhalative administration to the ingestion route, different conversion factors for acute single application and chronic effects must be applied. The systematic bioavailability is relevant to chronic effects, whereas in relation to acute effects further aspects must be considered, such as the faster resorption and the considerably higher peak plasma concentrations of THC after smoking and intravenous intake relative to those after oral administration.
2.2 Extrapolation of animal data to man
One advantage of animal studies is that they allow a thorough control of the conditions of THC exposure, such as time and duration, as well as the control of possible confounding factors. Therefore they constitute an important element of toxicological research. However, for several reasons, caution is still required when using "data produced by those that continue to extrapolate animal data to humans without some attempt to discuss in detail the validity of their assumptions" (Campbell 1996).
The principle of phylogenetic continuity of species, including similarity of cell structure and energy metabolisms, is the common denominator for the cross-species extrapolation of toxicological data. Nevertheless there are some significant pharmacokinetic and other relevant differences between species that render extrapolation more difficult.
There are various methods for the extrapolation of animal data to man (Voisin et al. 1990, Winneke and Lilienthal 1992, Ings 1990). These shall only briefly be reviewed here:
2.2.1 Interspecies comparison based on body weight
The first approach to comparing toxicological data from various animal species with that of man is based on the body weight. Many biologic parameters such as water intake, creatinine clearance and synthesis of hemoglobin can be expressed as mathematical functions of body weight. These relations are fairly consistent over a wide range of species. The corresponding unit is milligram per kilogram (mg/kg). Results obtained from an application of 10 mg/kg THC to rats would be translated to man in the ratio of one to one if such a toxicological comparison based on body weight was employed. Hence 2 mg THC in a rat of 0.2 kg would correspond to 700 mg THC in a man weighing 70 kg.
However, many biological processes such as metabolic rate, cardiac function, and renal function are not directly proportional to body weight. Thus toxicological comparisons on the basis of body weight are likely to be inaccurate.
2.2.2 Interspecies comparison based on body surface
The body surface is another potential basis for the comparison of toxic effects between species. For instance, the heat loss from a warm-blooded animal is approximately proportional to its body surface. In studies on anti-cancer drugs it was demonstrated that the maximum tolerated dose in different animal species (mouse, rat, dog, monkey) correlated quite well with that in man, if adjusted to body surface. The corresponding unit is milligram per square meter (mg/m2) or milligram per square centimeter, respectively (mg/ cm2).
2.2.3 Interspecies comparison based on pharmacokinetics
If the specific toxicity of a compound is unknown, the best correlations can be achieved on the basis of pharmacokinetic data, such as absorption (body intake), distribution, metabolism and excretion. Classical pharmacokinetic models are based on plasma concentration and the AUC (area under the curve) as plasma concentration over time, produced by known doses. Such data allow the determination of extent and duration of systemic exposure to the analyzed substance.
Pharmacokinetics in man and in animals show some parallels and a number of conspicuous differences (Agurell 1986, Harvey and Brown 1991).
Unfortunately the available data on plasma concentrations after oral administration of THC in man and rat are available for different doses: the human subjects received lower doses whereas in animals the applied concentrations were higher (see Table 6). An oral intake of 0.2-0.3 mg/kg produced a peak plasma concentration of 3-10 ng/ml in man (Frytak et al. 1984, Brenneisen et al. 1996). In animal studies with rats, plasma concentrations peaked at about 100 ng/ml after oral administration of 15 mg/kg THC (Hutchings et al. 1991) and at about 150 ng/ml after 20 mg/kg THC (Scallet 1991). The concentrations maintained a high level for 6 hours. In another study where rats had received THC at doses of 15, 25 or 50 mg every day for two years, the THC concentration had finally reached a level of about 400, 1300 or 3000 ng/ml at the end of the test period (Chan et al. 1996). This is the result of the accumulation of THC, as known in man as well, that causes an augmented plasma level after chronic application.
Extrapolation of pharmacological data to different doses is problematic even if corresponding mathematical models exist (Inges 1990). It is desirable to have more animal data that would involve doses corresponding to human consumption patterns.
2.2.4 Interspecies comparison based on precise toxicological data
Unfortunately the results achieved by extrapolation on the grounds of the models presented above can be quite astonishing. Often toxicity in different species does not correspond to the toxicity determined by means of pharmacokinetic data. One example is the lethal dose of THC.
In a study with rats, the median lethal dose (LD50)was established to range between 800 and 1,900 mg/kg oral THC, depending on sex and strain (Thompson et al. 1973). When this dose is extrapolated on the basis of body surface, the oral LD50 in dogs would be a quarter of this dose (200-500 mg/kg) per kg BW and half of this dose (400-950 mg/kg) per kg BW in monkeys. However, the experimental studies revealed contrary results. No deaths were observed when orally administering the maximum THC doses either to dogs (up to 3,000 mg/kg THC) or to monkeys (up to 9,000 mg/kg THC) (Thompson et al. 1973). Instead of being 50% more sensitive, primates turned out to be at least five to ten times more resistant to THC. It has not yet become quite clear how, at low doses, the relevant target parameters (effects on hormonal system, immune system and fetus) would compare between species. Probably mice are especially predisposed to fetal malformations (Abel 1985). Also regarding the sensitivity of the hormonal system to THC, clear species-related distinctions were found that diminish the extrapolation of those results to man (Mendelson and Mello 1984).
Other examples of chemical substances demonstrate the wide range of possible conversion factors. Alcohol, methyl mercury and polychlorinated biphenyls cause embryonic malformations in man. The toxic doses per kg body weight in animal studies ranged from 0.2 to 8.0 times the toxic dose in man (Hemminki and Vineis 1985).
Conclusion: Animal data can provide an indication of the toxicity of THC to humans. However, the extrapolation of animal data to man is problematic, not only because of the use of high dosing regimens but also because of the lack of reliable conversion factors for the target parameter under examination. Smaller animals possibly experience a stronger THC toxicity compared to larger animals and primates. Thus, reliable data on the toxicity of THC in man should be based on studies with humans.
2.3 Methodical basis for the determination of thresholds
For those chemical substances whose undesirable effects are either known or suspected, certain concepts for the protection of health against impairments, such as the LOAEL ("lowest observed adverse effect level") and the NOAEL ("no observed adverse effect level"), have been established. In order to provide a safe margin of protection against potential harm, safety factors of 10 are typically applied. For each particular case, the precise value of a safety factor may vary with the reliability of the determined threshold, the relevance of the observed effects in animals and cells for man, the severity of effects above the threshold, and the understanding of the causal relationship between drug intake and observed effects. With regard to THC limits in food, the data situation is fortunate, as the daily THC intake of chronic Cannabis consumers (50-200 mg or 1-3 mg/kg BW, respectively) already clearly exceeds the daily doses of relevance to this study (0.1-0.2 mg/kg) by a factor of 10-15. Acute effects in man were mostly examined at THC doses that cause psychotropic effects. Thus, a NOAEL in this dose range constitutes a proper margin of protection for the ingestion of THC with food. Dosage and concentration in animal and cell studies mostly exceed this level by another one to three orders of magnitude.
Appropriate limits can be based only upon sufficient or at least probable evidence of effects and not on hypothetical effects. Early toxicological research with THC in the seventies often discovered health-impairing effects that could not be confirmed in the following years. Such inconsistent experimental findings cannot be utilized for the determination of limits for detrimental effects. Early studies were often short of well-designed methods concerning procedure, choice of controls and the consideration of possible confounding factors. For instance, insufficient imaging techniques (pneumoencephalography) suggested a cerebral atrophy as consequence of chronic Cannabis use, which later was refuted. Also, when "pair-feed controls" were employed in the eighties in animal studies, it was realized that even after high THC doses fetal impairments were not induced by THC toxicity, but rather resulted from the mother's reduced intake of food and water (Abel 1984). (Pair-feed controls are those that receive the same amount of food and water as the examined animals, who took in less food and water because of the deprivation caused by the administration of THC.)
At this point, two particularities of the toxicological evaluation of THC shall be emphasized:
1. Development of tolerance and accumulation: For most toxic agents, the toxicity increases with the duration of exposure and the NAOEL decreases correspondingly (Voisin et al. 1990). Such an increase of toxicity can be expected, especially for a substance which possesses a comparatively long half life (THC: 20-30 hours) and which accumulates with chronic administration. However, opposite effects were observed with THC, since tolerance develops for most effects, and this overcompensates for any accumulation that may occur. With an augmenting administration of THC, the psychological as well as most-if not all-physical effects mediated by specific receptors decrease (see Chapter 3.5). For instance, in THC studies of female rhesus monkeys, hormonal changes and a disruption of the menstrual cycle occurred (Smith et al. 1983). However, after six months of a persistent high-dosing schedule, the hormonal values and the menstrual cycle had returned to normal. Development of tolerance has also been established for most of the other effects (mood changes, cardiovascular effects, etc.).
2. Damage to children: Children generally respond more severely to chemical toxins and are rated as "sensitive persons," requiring larger margins of protection (Winnecke and Lilienthal 1992). A child's brain is, for instance, more susceptible to impairments than the brain of an adult. Contrary to most noxious substances, however, THC in relevant concentrations does not operate in an unspecified manner but acts on specific receptors on body cells (especially brain cells and immune cells). The number of cannabinoid receptors in adults is several times the number in children (see Chapter 5.2). This is also beneficial to the therapeutic uses of THC. Given the aforementioned reasons, children who were given THC (in this case delta-8-THC) in the course of a chemotherapy tolerated considerably higher doses (18 mg/m2 body surface) than adults would presumably have tolerated (Abrahamov 1995). Elderly people are more responsive to THC concerning psychotropic effects, even though the aging process leads to a slight reduction of THC receptors (Romero et al. 1998). However, caloric intake also usually decreases with age, causing a reduction in THC intake with food.
3 Pharmacology and pharmacokinetics
For a proper understanding of Cannabis effects, and for the sake of comparability of different routes of administration, a basic understanding of the pharmacokinetics of THC is needed (reviews: Agurell et al. 1986, Harvey 1991). It will be provided in the following sections.
3.1 Resorption and plasma level
Cannabis drug products (marijuana, hashish) are preferably inhaled (cigarettes, pipes) and only seldom orally ingested (tea, pastries, tincture). Besides these routes, the intravenous (injection into the blood vessel), the subcutaneous (injection under the skin), and the intraperitoneal (into the abdominal region) routes are widely applied in animal studies. This present study focuses on the toxicity from oral administration with food, as well as the transferability to the ingestion route of biological effects observed with other routes of administration.
Several studies determined the systemic bioavailability of THC after the smoking of a marijuana cigarette as ranging between 2 and 56% of the total amount of THC present in the cigarette. Generally, bioavailability appears to range between 10-30%, with inexperienced smokers achieving lower rates. Hence Lindgren et al. (1981) established a systemic bioavailability in heavy smokers of 23% (+/-16%) compared to 10% (+/-7%) in occasional users. The smoking of a marijuana cigarette containing 10-20 mg THC produces a maximum plasma level of about 100 ng/ml after three minutes. Subsequently, the plasma concentration drops rapidly. The subjective effect sets in after only a few puffs, and the maximum high is reached after 15-30 minutes, when the plasma level has already begun to decline. The psychotropic effects typically cease after three hours.
When orally ingested, the systemic bioavailability of the lipophilic THC molecule is typically 5-10%. However, it may be doubled when simultaneously applying a lipophilic carrier (fat, oil), thereby improving THC resorption. Thus the bioavailability is typically somewhat less than that via inhalation. The rate of intake also depends on additional factors, such as the fullness of the stomach, and varies between individuals. After oral administration of 15-20 mg THC, plasma levels peak at approximately 5 ng/ml THC, typically after 1-3 hours, with a large inter- and intra-individual variability. The subjective psychotropic effect has its onset after 30-90 minutes and lasts for about 6 hours. In order to achieve acute effects, considerably higher oral doses are required than with inhalation because of the slower intestinal resorption of THC and the somewhat lower bioavailability.
Substances that are applied to the skin can be systemically absorbed to an unknown extent. However, there have not yet been any quantitative studies of the dermal absorption of THC that would allow quantification. Nevertheless, this question is vital for the use of THC-containing products that are externally applied (cosmetics, dermatics for the treatment of neurodermitis). The physico-chemical characteristics of THC, however, allow a rough estimation of the amount of THC assimilated (Kalbitz et al. 1996, 1997).
Generally, the human skin is well protected against penetration by external substances. Many topically applied substances attain a systemic bioavailability of only a few percent (Hadgraft 1996). The main barrier to penetration is the cornea (stratum corneum), or more accurately, the cornified layer of the stratum corneum. In principle, substances can penetrate the space between the cells of the stratum corneum (intercellular), the cells themselves (intracellular), or the sebaceous and perspiratory glands and hair follicles. Only the first pathways of penetration generally play a relevant role (Hadgraft 1996, Kalbitz et al. 1996, Berti et al. 1995). For example, the route through the hair follicles and glands is of importance for polar molecules only. As a lipophilic molecule, THC does not belong to that group.
The permeation coefficient or, respectively, the permeability constant (Kp) constitute a quantitative expression for the ability of a substance to permeate the skin. The flux or absorption rate of a chemical results from a multiplication of the concentration (C) of a chemical on the skin surface with the permeability constant: flux = KpC (Mattie et al. 1994). The basic principles of absorption through the skin correspond to those of diffusion through semi-permeable membranes (Berti et al. 1995). Factors that influence the penetration through the skin are the thickness and condition of the skin, as well as the size of the penetrating substance and the carrier.
Molecules that enter and diffuse through the skin have to penetrate a number of lipid bilayers in the intercellular space, thereby repeatedly alternating from lipophilic to hydrophilic areas. Those molecules that are sufficiently lipophilic, such as glucocorticoids, easily cross those lipophilic phases. Thus, most publications still maintain as a rule that "highly lipophilic compounds with low molecular weights demonstrate the greatest flow rate through the stratum corneum" (Berti et al. 1995).
Occasionally, a direct relation between the coefficient of permeation and the octanol/water distribution coefficient is postulated (Guy 1995). The latter is a measure of a chemical's lipophilic and hydrophilic properties, respectively. A higher coefficient indicates stronger lipophilic characteristics. However, such a correlation could not be verified in experimental studies. Mattie et al. (1994) examined 13 substances with octanol/water coefficients ranging from zero to 1,400. The constant of permeability correlated only weakly with the octanol/water distribution coefficient (r2 = 0.04).
Instead, there is evidence that only a small fraction of strongly lipophilic substances, such as THC, overcomes the hydrophilic phases of the intercellular space. Gabriele Bast (1997) carried out a large number of experiments that involved different substances of differing lipophilic characteristics in different carriers, and stated: "When substances are applied in a lipophilic carrier the permeation coefficient Kp is notably decreased when the distribution coefficient (n-octanol/perfusion buffer (pH 7.4)) Poct exceeds 2000."
Conclusion: It may validly be assumed that, with an octanol/water distribution coefficient of 6,000 (Agurell et al. 1986), i.e. a strong lipophilic tendency, only a small amount of THC permeates the skin-and is systemically absorbed only on a small scale-when administered in an oily base, such as in cosmetics containing hemp oil. Based on experimental evidence obtained for other chemicals with known physico-chemical properties, the transdermal systemic bioavailability of THC thus is likely considerably less than the oral systemic bioavailability. Corresponding experimental studies should be conducted that quantify the exact rate of skin permeation.
3.2 Transport and metabolism
After absorption and infusion into the blood, more than 97% of THC and its metabolites are bound to plasma proteins. The octanol/water distribution coefficient is 6,000, the apparent volume of distribution in the body comes to 10 l/kg, typical of a lipophilic drug (Agurell 1986). THC crosses the blood-brain barrier comparatively easily and accumulates in fatty tissues from where it is re-released only slowly into other tissues, such as the blood. It is exponentially eliminated from the plasma, consistent with a multi-compartment model (2-4 compartments) with a terminal (ß-) plasma half-life of about 20-30 hours.
Ninety-five percent of THC is metabolized in the liver. Through microsomal hydroxylation, THC is converted to the equally pharmacologically effective 11-hydroxy-delta-9-THC, which in turn is converted into 11-nor-9-carboxy-delta-9-THC (THC-COOH) by the operation of alcohol dehydrogenase enzymes. Especially after oral administration, the 11-hydroxy-metabolite contributes considerably to the pharmacological effects, equaling those of THC. Besides the main metabolites, more than 20 other decomposition products are formed. The end products are 11-nor-acids and similar, more polar acids. About one third of those metabolites is excreted through the kidneys and about two thirds of them through the feces. The non-metabolized THC (5%) is defecated as well. The excretion through the urine is limited to acid metabolites only. THC-COOH has an elimination half life of 4-5 days. The complete elimination of a single THC dose may take up to 2-5 weeks. In chronic, heavy marijuana users, THC metabolites were still detected in the urine after 2-3 months after cessation of consumption.
Excursus III: Detection of THC after ingestion of hemp-containing food
The intake of a single oral dose of 16 or 33 mg THC by different experimentees caused a urine concentration of the THC metabolite THC-COOH of 170-240 ng/ml in an immunoassay. According to a GC/MS analysis (gas chromatography/mass spectrometry), these concentrations reached about 400 ng/ml (Lehman et al. 1997). With a chronic intake of such THC quantities, the metabolites accumulate. Consequently, a urine assay of heavy chronic Cannabis users might find THC-COOH concentrations of 500 to 1,000 ng/ml (Solowij et al. 1995). Because of the long elimination half-life of the THC metabolites and an accumulation in body tissues, they might remain detectable in the urine for weeks or even months after the last use (Bell et al. 1989, Ellis et al. 1985). In 86 chronic Cannabis users, THC metabolites were detected in an immunoassay up to 77 days after the last intake.
The ingestion of THC with food may also result in the detection of THC metabolites in the urine. A single oral intake of 40 ml of a hemp oil with a comparatively high THC content of 151 mg/ml, corresponding to about 6 mg THC, produced a maximum urinary THC-COOH concentration of about 100 ng/ml in an immunoassay (Alt and Reinhardt 1996). The intake of 135 ml of hemp oil containing an unknown THC concentration over a period of 4.5 days led to a THC-COOH concentration of 55 ng/ml (Struempler et al. 1997). It took two days after the last ingestion before the immunoassay tested negative. In another study, different patients tested positive for THC-COOH in the immunoassay after the intake of 15 ml hemp oil (Constantino et al. 1997). The daily intake of 10 ml hemp oil of an unknown concentration for 25 days caused a THC-COOH-concentration of 36 ng/ml, measured by GC/MS (Callaway et al. 1997).
Energy bars containing hemp seeds show only low THC concentrations. For example, a THC concentration of 4.4 mg/ml was found in the "Hempy-bar" energy bar of Green Machine Ltd. (Alt and Reinhardt 1997). After ingestion of one or two hemp seed bars, the urinary immunoassay tested positive (cutoff of 20 ng/ml). However, if measured quantitatively by CG/MS, only low concentrations, typically 1-2 ng/ml were detected (Fortner et al. 1997).
Conclusion: After the ingestion of hemp-based food, THC metabolites may be detectable in the urine. The observed THC-COOH concentration corresponds to the to the amount of THC ingested with food. Relevant THC-COOH concentrations are detected in the urine especially after intake of hemp oil, whereas other hemp products produce only low levels of metabolites as a result of their low THC content. Because of the accumulation of THC metabolites in body tissues, a long-term intake of small THC quantities may cause significant urine concentrations. In general, those results cannot be distinguished from those obtained after low-level drug consumption. However, the high concentrations that are found after chronic heavy marijuana use cannot be detected following ingestion of hemp seed foods.
3.3 Influence of physical factors on THC content
Ninety-five percent of the THC present in the Cannabis plant is found in a pharmacologically inactive form, i.e. one of two delta-9-tetrahydrocannabinolic acids (THCA) (Turner 1980), while the majority of biological effects are caused by the corresponding neutral phenolic forms of THC (Dewey 1986). Thus the question arises to what extent the total amount of THC species detected in food is pharmacologically active.
A conversion of THC acids into the pharmacologically active phenols-chemically a decarboxylation process (separation and release of carbon dioxide)-is accomplished most effectively by heating. A heating for five minutes to 200 to 210°C was found to be optimal for decarboxylation (Brenneisen 1984). Under these conditions, THCA was completely converted into neutral THC, while avoiding the subsequent oxidation to cannabinol (CBN). When marijuana is smoked and temperatures of 600° C are reached, obviously only a few seconds are sufficient for decarboxylation.
A much more gradual decarboxylation occurs at room temperature. Hence, after marijuana has been stored for a year, about 50% of its THCA had been converted into the active, neutral THC (Brenneisen 1984). However, storage also leads to a decrease in total THC content as THC oxidizes to neutral CBN, a non-psychotropic cannabinoid (Fairbairn 1976). The total THC content of marijuana dropped to 87% after 47 weeks of storage in the dark (20° C) and to 64% with exposure to light (Fairbairn 1976). Since THC in food is not protected by the plant's glands, as it is in marijuana, THC present in food is converted much faster into CBN. This is suggested by experiments with powdery Cannabis resin and alcoholic extracts (Fairbairn 1976).
Baker et al. (1981) analyzed 64 marijuana samples (Cannabis herb) and 26 hashish samples (Cannabis resin) for their relative amounts of THCA and THC, and found a wide range of ratios, especially in marijuana. In Cannabis resin, the ratio ranged between 0.5 to 1 and 6.1 to 1. Lower rates, corresponding to a low THCA fraction, were found in Cannabis samples from the Indian subcontinent, whereas samples originating from Mediterranean countries displayed higher rates. It may be assumed that much of the THC in cold-pressed hemp oil and other hemp based food is present in the form of the biologically inactive tetrahydrocannabinolic acid, as long as heating during the production process was insufficient for effective decarboxylation. Thus, for a realistic toxicological assessment of food, the concentration of phenolic THC must be determined. Only few studies have examined the percentage of pharmacologically inactive THC-acids in hemp based food. In a study of 10 commercially available hemp oils in Switzerland, THC-acids generally constituted less than 10% of the total THC-content (Lehmann et al. 1997). Thus, in this case the by far larger fraction was biologically active.
3.4 Mode of action
Most specific THC effects are mediated through cannabinoid receptors (reviews: Howlett 1995, Pertwee 1995, Matsuda 1997). At very high doses, non-specific effects on membrane fluidity and other non-specific effects may also become relevant (Martin 1986).
So far two types of THC receptors, CB1 and CB2, each with additional subtypes, have been identified and cloned. The CB1 receptor is found predominantly in brain cells, with a particularly high receptor density in motor, limbic, associative, cognitive, sensory and autonomic brain structures (basal ganglia, cerebellum, limbic system, hypothalamus, cerebral cortex). In addition, it was also found in the testes and other peripheral tissues.
The CB2 receptor has so far only been found outside the brain, particularly in cells of the immune system, such as in the spleen, tonsils, thymus, mast cells, and blood cells. Presumably it is involved modulating the operation of immune cells. Often CB1 and CB2 receptors are expressed from the same immune cells.
The endogenous ligands first discovered for the cannabinoid receptors were arachidonic acid derivatives (arachidonylethanolamides), which differ greatly from plant cannabinoids in their molecular structure (Devane 1992). They are called anandamides (reviews: Di Marzo and Fontana 1995, Mechoulam et al. 1996). Only recently another ligand, 2-arachidonylglycerol (2-AG) was identified (Stella et al. 1997).
3.5 Development of tolerance
Tolerance develops to most THC effects (Romero et al. 1997). This applies also to undesirable effects, such as effects on the psyche, the cardiovascular system and the hormonal system. An acute use of THC, for example, leads to a marked increase of the heart rate, whereas with chronic administration this effect no longer occurs, or at least not to the same extent. This tolerance phenomenon can be explained by two factors, i.e. enhancement of THC metabolism and down-regulation of brain cannabinoid receptors. The number of receptors decreases and their response to THC declines, such that only higher doses can produce the known THC effects. Thus, rats that had been administered THC over a period of five days exhibited a decreased specific binding in different receptor sites of the brain, ranging from 20 to 60% of that measured in controls. (Romero et al. 1997).
3.6 THC effects and total toxicity
THC has acute effects on almost every body system (reviews: Hall et al. 1994, Hollister 1986, Dewey 1986, Maykut 1985). The most conspicuous effects are those on the central nervous and cardiovascular systems. With regard to physical effects, THC produces an increased heart rate, reddened eyes and a dry mouth. As for psychotropic effects, a mild euphoria, an enhanced sensory perception, fatigue and eventually dysphoria together with anxiety have been observed.
As a function of dose, the following effects were observed in clinical studies in vivo (in living organisms) or in vitro (i.e. in laboratory dishes), respectively:
Psyche and perception: fatigue, euphoria, enhanced well-being, dysphoria, anxiety, disturbed orientation, increased sensory perception, heightened sexual experience, hallucinations, psychotic states.
Cognition and psychomotor performance: fragmented thinking, enhanced creativity, disturbed memory, unsteady walk, slurred speech.
Nervous system: attenuation of pain, muscle relaxation, appetite enhancement, decrease in body temperature, vomiting, anti-emetic effects, neuroprotective effects in brain schema.
Cardiovascular system: increased heart rate, enhanced heart activity and increase in oxygen demand, vasodilation, drop in blood pressure, collapse.
Eye: reddened conjunctivae, reduced tear flow, reduced intraocular pressure.
Respiratory system: bronchodilation, dry mouth.
Gastrointestinal tract: reduced bowel movements.
Hormonal system: effects on LH, FSH, testosterone, prolactin, somatotropin, TSH, reduced sperm count and sperm mobility and quality, suppressed ovulation and suppressed menstruation.
Immune system: impairment of cell-mediated and humoral immunity, anti-inflammatory and immune-stimulating effects.
Fetal development: fetal malformations, fetal growth retardation, impairment to fetal and postnatal cerebral development, improved postnatal development.
Despite these observed effects, the overall physical human toxicity of Cannabis is low. Human fatalities following acute Cannabis intoxication have not been reported. Chronic heavy marijuana use is also not related to mortality (Sidney et al. 1997). Long-term heavy marijuana users did not show any abnormal health features that distinguished them from the population as a whole (Gruber et al. 1997).
Strong side effects are rare, even with high THC doses. The median lethal dose (LD50) for rats is in the range of 800 to 1900 mg/kg (Thompson et al. 1973). No toxic deaths were observed in experiments of rhesus monkeys with an acute oral application of 9000 mg/kg (Thompson et al. 1973). For illustration purposes: 9000 mg/kg THC in a man weighing 70 kg corresponds to the consumption of 630 grams THC or 3 kg of high-percentage Cannabis resin (hashish), or 15 kg of marijuana of a medium quality.
Even a long-term high-dosing regimen of THC is tolerated relatively well. This was suggested by the example of rats that ingested 50 mg/kg THC every day for two years (Chan et al. 1996). At the end of the two-year period a mean of 45% of the controls and 70% of the dosed animals had survived. The higher survival in the THC group was primarily due to a decreased incidence of cancer.
4 THC thresholds for psychotropic effects
Some experimental and clinical studies report experiences with threshold values for psychotropic THC doses. Acute effects below the psychotropic threshold cannot be distinguished from placebo effects.
Lucas and Laszlo (1980) found pronounced psychotropic reactions (anxiety, marked visual distortions) in patients undergoing cancer chemotherapy who had received 15 mg THC/m2 (square meter of body surface), which corresponds to 25 mg THC in an average adult person (body surface: 1,7 m2). A reduction to 5 mg THC/m2, about 7.5-10 mg THC, produced only mild reactions.
No mood changes were observed in six cancer patients after administration of a single oral dose of 15 mg THC for antiemetic treatment (Frytak et al. 1984). Brenneisen et al. (1996) administered single oral doses of 10 or 15 mg THC to two patients. Physiologic parameters (heart rate) and psychological parameters (concentration, mood) were not modified by the administration. In a study by Chesher et al. (1990) of a healthy population dosed orally with 5 mg THC, no difference in the subjective level of intoxication was found compared to placebo controls. Doses of 10 and 15 mg THC caused slight differences compared to the placebo, and a dose of 20 mg, finally, caused marked differences in subjective perception.
In light of these findings, one may validly assume the psychotropic threshold to be in the range of 0.2-0.3 mg THC per kg body weight for a single oral dose taken in a lipophilic carrier, corresponding to an administration of 10-20 mg THC to an adult person. A single dose of 5 mg THC can be regarded as a placebo dose. In various clinical studies, psychotropic reactions were also observed following single doses of 5 mg THC. However, these cannot be distinguished from effects that occur after administration of placebos. As the duration of action of THC in therapeutic dosage ranges between 4 and 12 hours, a daily intake of 2 x 5 mg which equals 10 mg THC, administered orally in a lipophilic carrier, will not have any effects that could be distinguished from placebo effects.
5 Discussion of physical effects
Below the psychotropic threshold, an intake of THC cannot be distinguished from placebo substances with regard to physical parameters also. No perceptible acute effects as, for example, on the cardiovascular system, are observed. The question arises, however, whether undesired biological effects can still occur below the placebo threshold, especially with chronic intake.
Considering the effects observed in animal studies, four sectors must be examined in this context:
Effects on the genetic material (mutagenicity and carcinogenicity),
Effects on the immune system,
Effects on the hormonal system,
Effects on pregnancy.
Other aspects, such as the possible neurotoxicity of THC, will not be dealt with, since such effects were only found in animal experiments with a chronic administration of high doses that clearly exceeded the doses that are of relevance in the examined context (WHO 1997).
5.1 Genetic material and cell metabolism
5.1.1 Cell studies
Cannabis smoke can exert mutagenic activity as a result of carcinogens (benzpyrenes, nitrosamines). This was established in the Ames test. THC itself is not mutagenic (WHO 1997). THC may reduce the synthesis of DNA, RNA and proteins and modulate the normal cell cycle. To obtain those effects, however, very high doses were required in cell studies. Hence, in a study by Tahir et al. (1992) microtubules and microfilaments in PC12 cells, which are vital for cell division, were disrupted in a dose-dependent manner following treatment with 10-30 mM (micromol) THC.
5.1.2 Studies with Cannabis users
Studies with Cannabis users did not establish any increase in chromosomal breaks (Matsuyama et al. 1976, Matsuyama and Fu 1981, Cruickshank 1976, Cohen 1976). Thus, after 72 days of marijuana smoking, no increase in chromosomal breaks was found when compared to the breakage rate preceding administration.
Joergensen et al. (1991) evaluated the genotoxicity of Cannabis smoking by application of the sister-chromatid exchange (SCE) test, a sensitive tool for the discovery of genotoxic agents. They compared 22 tobacco smokers and 22 persons that smoked tobacco and marijuana. The smoking of tobacco in itself enhanced the SCE level significantly by 18.5% compared to non-smoking controls. The addition of marijuana did not further affect this level. Based on this observation the authors concluded that Cannabis smoke could not be considered genotoxic.
5.1.3 Conclusion
THC in doses used by marijuana smokers is neither mutagenic nor carcinogenic and it does not affect cell metabolism, either. The NOAEL ranges above concentrations relevant for the human consumption situation.
5.2 Pregnancy
In animals and man delta-9-THC crosses the placenta to the vascular system of the fetus. The course the THC concentration takes in fetal blood fairly coincides with that in the maternal blood, though fetal plasma concentrations were found to be lower compared to the maternal level in rats (Hutchings et al. 1987), in sheep (Abrams et al. 1985-1986), in dogs (Martin et al. 1977), and in monkeys (Bailey et al. 1987). In a study on dogs, the brain of the fetus showed a concentration that came to only one third of the mother's concentration half an hour after intravenous administration. This relationship was maintained with multiple administrations, indicating that the maternal plasma THC and not the fetal tissue is the actual source for the fetal plasma THC. Small quantities of THC also pass into the milk of the mothers. In a study on monkeys, 0.2% of the THC ingested by the mother appeared in the milk (Chao et al. 1976). Chronic administration leads to a THC accumulation in milk (Perez-Reyes and Wall 1982).
THC concentrations corresponding to normal marijuana use act on compound-specific binding sites (receptors). In early gestation, these have not yet been developed in the fetal brain so that, during this phase, cannabinoids lack a specific binding target (Hernandez et al. 1997). First cannabinoid receptors are detected at fetal age. However, their number progressively increases in postnatal life. In a study investigating the number of cannabinoid receptors during rat brain development, the receptors were found to multiply by five between the day of birth and the sixtieth day (grown-up rat) (Belue et al. 1995).
Non-receptor mechanisms such as the disruption of cell membranes require extremely high doses of THC. Thus, one can assume that no relevant THC effects will appear during early pregnancy though, they might occur in later gestation.
Four of the THC effects on pregnancy will be further analyzed:
1) Increase in birth complications
2) Increase in birth defects (i.e. heart defects, cleft palate)
3) Adverse pregnancy outcome (i.e. more premature births, low birth weight)
4) Adverse postnatal development or, respectively, impaired fetal brain development.
First epidemiological studies examining the prenatal effects of marijuana on man were published in the early eighties. Human studies are often subject of a number of methodical weaknesses that are not easily corrected and that may lead to inconsistent findings. Among these are:
The examined samples being too small. Especially if marijuana users and abstinent controls might differ only slightly, or if the relevant disorders are of a rare kind, such differences become evident only by examination of a large number of pregnant women and their children.
Uncertainties concerning the exact daily marijuana intake. As marijuana is an illicit drug, study participants may not be truthful in their answers to inquiries about their use. Therefore Shiono et al. (1995), in addition to the interviews, also took blood samples to be screened for cannabinoids. This often revealed a discrepancy between the results of the inquiry and those of the blood tests.
A lack of controlling and adjustment for confounding factors that may influence pregnancy. Thus, Cannabis consumption is often associated with a range of factors that, by themselves, may have effects on pregnancy, such as the consumption of other legal and illegal drugs. Education and socioeconomic status are of further relevance as they may influence the quality of nutrition during pregnancy and the prenatal care.
5.2.1 Birth complications
For many centuries, Cannabis has been used to alleviate the discomforts of childbirth. In the Western medicine of the 19th century, Cannabis preparations were employed to this end, also as they supported birth contractions. This effect, however, is not reliable according to historical reports (Mechoulam 1986).
A study by Greenland et al. (1982) established an elevated risk of abnormal progress of labor in 35% marijuana users when compared to 36 controls (Greenland et al. 1982). A frequent meconium staining (57% versus 25%) and an average longer duration of labor were found. In a second study of the same research group with a slightly enlarged population, these effects were much less significant (Greenland et al. 1983). The rate of a dysfunctional labor (43% versus 35%) and meconium staining (17% vs 13%) was only slightly elevated in marijuana users. Other researchers did not find any abnormalities in pregnancy. Thus, in a study of 291 women, Fried did not detect any significant differences in marijuana users under labor. Also Dreher et al. could not detect any significant features in the progress of labor when comparing thirty prenatally exposed mother/child pairs to thirty non-consuming pairs (Dreher et al. 1994). The collectives of Greenland et al. might have been selected and the higher rate of complications was possibly attributable to other circumstances.
Conclusion: There are isolated observations that suggest marijuana use during pregnancy might have adverse effects on the progress of labor. These were obtained by one research group in the early eighties and to date have not been confirmed. On the other hand, there are historical clinical reports of a beneficial effect that could be used therapeutically. These, however, were not fully reliable. There is no reason to believe that sub-psychotropic THC doses would have a negative effect on the progress of labor.
5.2.2 Birth defects
Animal studies: In some early animal studies, congenital malformations were found subsequent to the administration of high doses of THC (tabular review: Abel 1980). No pair-fed controls had been employed. A daily oral administration of up to 150 mg of THC in sesame oil, however, failed to have any effects on prenatal mortality, fetal weight, and the rate of internal and skeletal malformations in mice (Fleischmann et al. 1975). Subcutaneous injection of up to 100 mg/kg THC proved not to be fetotoxic (Keplinger 1973).
The marijuana-induced fetotoxicity in animals is enhanced by alcohol (Abel 1986). However, extremely high doses of marijuana, equaling 50-100 mg/kg THC, and relatively small quantities of alcohol (1 g/kg) were required to encounter this effect. Small doses of marijuana did not enhance fetotoxicity.
Hollister (1986) points out that "virtually every drug that has been studied for dysmorphogenic effects has been found to have them if the doses are high enough, if enough species were tested, or if treatment is prolonged" (p. 4).
Abel emphasizes the fact that findings of malformations were consistent only following exposure to relatively high doses and following the intraperitoneal route (direct delivery to the abdominal region) (Abel 1985). Not only due to the direct effects of THC, but also to the reduced maternal food and water consumption associated with high dosage, THC administration may account for many effects. According to Abel, the only reliably documented postnatal effect on offspring is a decrease in birth weight.
Human studies: In most epidemiological studies, evidence could not support any increase in congenital malformations following marijuana use during gestation. The only exception is a single early study (Hingson et al. 1982). Hingson and colleagues examined 1,690 mother/child pairs for effects of alcohol and marijuana use on embryonic development and fetal growth. Marijuana use was associated with the increase of a fetal syndrome known as alcoholembryopathy or fetal alcohol syndrome.
In all further epidemiological studies with many thousands of children, no such relation between marijuana use and fetal malformations was established (Astley 1992, Gibson et al. 1983, Knight et al. 1994, Linn et al. 1983, Witter and Niebyl 1990). Neither did a study investigating for various minor physical anomalies (MPAs) detect any significant Cannabis-related differences (O´Connel and Fried 1984).
Conclusion: Evidence today supports the fact that marijuana use during pregnancy does not produce any fetal malformations.
5.2.3 Pregnancy outcome
Most studies for the evaluation of marijuana-induced effects on pregnancy outcome examined the influence on duration of gestation and on birth weight or infant size, respectively. The results are inconsistent. Whereas some studies found a shorter duration of gestation or a decrease in birth weight, others did not discover any interference.
Duration of gestation: In a study of 7,301 births, the rate of premature births was found to be greatly enhanced by 25% in 36 mothers that self-reportedly smoked marijuana once a week (Gibson et al. 1983). Fried et al. (1984), after accounting for other potentially confounding factors, stated a dose-related decline in the length of gestation in 84 marijuana users when compared to abstinent women. In an extensive study by Linn et al. (1983) of 12,424 individuals, 10% of whom reported use of Cannabis, the only significant difference stated was a higher rate of precipitated labor in Cannabis users. Most studies, however, could not find any marijuana-induced modulation of the duration of gestation.
Birth weight and infant size: In a study by Abel (1984) pregnant rats were intubated with augmenting THC doses of 5 to 50 mg/kg per day until gestation day five and, subsequent to this, either 50 or 150 mg/kg from day six to parturition. In the high-dose group, no viable pups were born (the fetomortality was 100%). In the 50-mg-offspring, a decreased weight gain and a decreased birth weight was found. Hutchings et al. (1987) observed a decreased birth weight following a daily oral administration of 15 and 50 mg/kg THC. The decrement in birth weight and postnatal weight gain were dose-related. The THC-15 group reached the weight of the controls within a period of 11 days, whereas for the THC-50 group it took 32 days. Overall, the decrease in birth weight was not due to the THC-administration, but rather attributable to the reduced food and water intake of the exposed dams, as no significant difference was found compared to pair-fed controls.
In a study with 1,226 women, Zuckermann et al. (1989) found the neonatal weight significantly decreased by a mean of 79 grams and a decrease in size by a mean of half a centimeter in the newborns of marijuana users. Also, the study of Hingson et al. (1982) associated marijuana use during gestation with a lower birth weight. Another study found the elevated risk of a low birth weight only among white regular marijuana users whereas nonwhites (of Hispanic or African descent) were generally not at an increased risk (Hatch and Bracken 1986).
In most studies no relation between marijuana use and fetal growth was found (Day et al. 1991, Fried et al. 1984, Fried and O´Connell 1987, Gibson et al. 1983, Knight et al. 1994, Linn et al. 1983, Shiono et al. 1985, Tennes et al. 1985). Moreover, the question arises whether an average reduced birth weight of 79 grams, as observed by Zuckermann et al., is of any practical significance. Even though it was a perceptible statistical variation, it probably failed to be of any biological relevance (Pisacane 1989).
How does growth develop after birth? According to a study by Fried and O´Connell, (1987) the children of Cannabis users were on average heavier and taller than non-exposed children. In contrast to these results, another research group found that maternal use of marijuana was significantly and negatively related to a decreased infant size at eight months but not to weight and head circumference (Barr et al. 1984). Finally, a further study did not find any growth retardations at the age of one year (Tennes et al. 1985).
Conclusion: Animal studies found a dose-related decrement in birth weight induced by marijuana. These findings were obtained, however, at doses clearly beyond the range of a human consumption situation. Various epidemiological studies stated inconsistent effects of Cannabis use on length of gestation, birth weight and infant size. The majority of those studies, however, could not provide any evidence that the outcome of pregnancy was affected. Furthermore, there is no reference to an influence on postnatal growth development.
5.2.4 Brain development
The cannabinoid-anadamide receptor system might play an important part in cerebral development. The daily administration of 5 mg/kg THC to pregnant rats generated a doubling of activity of the enzyme tyrosine hydroxilase (TH) in specific brain cells of their fetuses (Hernandez et al. 1997). This enzyme is assumed to be a key factor in the development of TH-containing neurons and other neurons. Furthermore, animal studies established a disturbance of mesolimbic dopaminergic neurons among perinatally THC-exposed males, which persisted in adult animals (Garcia-Gil et al. 1997). Further mechanisms with effects on brain development remain under discussion (Navarro et al. 1995).
Animal studies: It is assumed that THC might have stronger toxic effects during the period of brain development than it does in adults. However, in animal studies behavioral alterations were only found in the offspring of those dams that had been exposed to extremely high THC doses during gestation. In a study of pregnant rats that had received 50 mg THC/kg per day, this gestational exposure did not affect the behavioral tests of their offspring (Abel 1984). By contrast, Hutchings et al. (1987) observed significantly longer latencies to attach to a nipple and impaired nipple attachment (repeatedly missing the nipple) in the offspring of rat dams that had been exposed to a daily oral administration of 50 mg/kg THC. No such impairments were observed among 15mg/kg-offspring when compared to controls. However, the authors presume that the alterations among the high-dose offspring were not a primary effect of THC toxicity but rather were secondary to the significant THC-induced reduction of food and water intake among the dams. This was borne out by the fact that no significant differences were found between THC-exposed animals and non-exposed controls when those were provided with an equally reduced food and water supply. Besides this, the activity level in the offspring was not impaired by high THC doses. Kwash et al. (1980) observed a decrease in learning abilities among the offspring following an injection of Cannabis resin in pregnant rats and they attributed this to the impeded postnatal weight gain. Navarro et al. (1995) observed that behavioral deficits and impairments of learning in the offspring were associated to comparatively low THC concentrations (1 and 5 mg/kg THC), whereas no such association existed with high concentrations (20 mg/kg). Other authors did not find any impaired learning (or memory) in animals (Charlebois and Fried 1980, Uyeno 1973, Abel 1984).
The concentrations of DNA, RNA and proteins in the brains of offspring whose mothers had been administered daily doses of 15 mg/kg THC did not differ from controls on day 7, 14 and 21 postnatal (Hutchings et al. 1991) A group, however, with a daily gestational exposure of 50 mg/kg THC showed a lower protein concentration on day 7 and 14 postnatal. Since the protein content correlates with the growth of neurons and the formation of neuronal links (synapses), a reduced protein synthesis may be considered as indices for the inhibition of neural processes. On day 21 postnatal the decrease was equalized.
Human studies: A study by Fried et al. (1987 b) found increased tremors and startles in those children whose mothers had regularly used marijuana during gestation in comparison to non-exposed controls on day 9 and 30 postnatal. In a sleep study, marijuana use was found to be associated with alterations in the sleep cycle of neonatals (Scher et al. 1988). Children aged three years from a different marijuana-using population showed a disturbance in their nocturnal sleep, waking up more often during the night (Dahl et al. 1995). Marijuana-exposed children aged 9 months achieved slightly lower mental test scores than non-exposed controls (Richardson et al. 1995). However, a difference was no longer found at the age of 19 months. In another study, children aged one year did not show any significant differences in their sleeping or eating habits, their mental functions, or psychomotor abilities (Tennes at al. 1985). The gestational exposure to marijuana was concluded not to produce a higher mortality rate by increasing the rate of SIDS (sudden infant death syndrome) (Ostrea et al. 1997). Dreher et al. on day three postnatal could not detect any differences between neonatals of marijuana users and those of non-consuming mothers in neuro-behavior assessments (Dreher et al. 1994, Dreher 1997). After one month, the differences became apparent in favor of the marijuana population: In the prenatally exposed children this was manifested in a greater liveliness, less irritability, less tremors; these children were more easily quieted and scored higher in their reaction to different stimuli (sound, light and touch).
Fried and colleagues pursued a longitudinal study of children until they reached school age (Fried 1995). However, between 6 months and 3 years of age no behavioral consequences of marijuana exposure were noted. At the age of 4 to 6 years, global intelligence still proved to be at a normal standard, though slight significant variations in their verbal abilities and their memory were stated. At the age of 9 to 12 their ability to speak and spell did not differentiate the exposed from the non-exposed children (Fried et al. 1997). At the end of the study the prenatally exposed children were not found to differ significantly in their neurobehavior from other children.
Conclusion: Animal studies even with high THC doses failed to establish any consistent supportive evidence for an impairment of brain development. The early neurologic symptoms found in neonatals by some researchers can be interpreted as withdrawal symptoms. Possible subtle inhibitions of cognitive functioning could appear in the sequel. However, these are inconsistent findings, not confirmed by other authors. Still, there is no reason to believe that sub-psychotropic THC doses could possibly affect the development of fetuses or neonates.
5.2.5 Summary
In their review on influences of Cannabis on pregnancy Levy and Koren (1990) pointed out the tendency of preferential publishing of those studies that find noxious effects, and leaving unpublished those that evaluate THC as a safe drug. However, even with this taken into account, there are only weak references to pregnancy being adversely affected by marijuana use. Animal studies found merely inconsistent evidence of health-impairing effects when administering doses of 10-20 mg/kg THC or more, that is 100 times the dose relevant to this study. There are references to a light impairment of brain development among children of chronic Cannabis users that, however, could not be validated by other authors. Also in consideration of the above mentioned animal findings, the NOAEL for different pregnancy-related parameters lies safely beyond or within the range of the human consumption situation of chronic marijuana users.
5.3 Hormonal system and reproduction
Marijuana acts on the hypothalamo-hypophyseal axis. This is a functional unit in the brain which plays an important part in the interaction of different hormones. The hypophysis (pituitary gland) secretes the sex hormones LH (luteinizing hormone), FSA (follicle stimulating hormone), and prolactin; the thyroid hormone TSH (thyrotropin); ACTH (adrenocorticotropin); and somatotropin (STH). These hormones respond to releasing hormones (RH) of the hypothalamus. LH regulates the testosterone production in the testes. Testosterone and FSH are vital for the sperm production (sperm count, sperm motility and sperm function).
5.3.1 Sex hormones
5.3.1.1 Men
In animal studies it was shown that THC may impair the function of male sex hormones and induce a decrease in the weights of sex organs (Dewey 1986, Mendelson and Mello 1984). However, contrary to this, various animal studies described a gain or a constancy in the weights of male sex organs (Abel 1981). THC decreased the anterior and mediobasal hypothalamic LH-RH concentration in rats when administered at 2 mg, 15 mg and 30 mg/kg body weight in a dose-related manner (Kumar and Chen 1983). Furthermore, simultaneous decreases in serum testosterone were observed. In another study it was found that THC lowered testosterone and LH levels in rats (Harclerode 1984). In the sequel, a tolerance to this effect developed, and with chronic THC exposure the hormone-concentration returned completely to normal values. In another study of rats that were chronically administered 1 mg, 5 mg or 25 mg/kg THC, the testosterone level was unaltered 24 hours after the last administration in the low-dose group, whereas it was found to have doubled in the 5 mg group (Morrill et al. 1983). In a study of mice, THC in relation to dose caused a statistically higher incidence of abnormal sperms (Zimmermann et al. 1979). Without THC the number of abnormal sperms amounted to 1.5%. After a treatment for five consecutive days with 5 mg/kg THC, this percentage had risen to 3.8%, and with 10 mg/kg THC it rose to 5.3%.
Testosterone: First suspicions that Cannabis might affect sexual hormones arose from case reports of gynecomastia in male young heavy Cannabis users (Harmon and Aliapoulis 1972). This suspicion was substantiated by Kolodny et al. (1974) who observed reduced serum testosterone levels coupled with a decrease in sperm count and sperm motility in chronic marijuana users. This frequently quoted study, however, holds a number of methodological faults that have been repeatedly criticized (Abel 1981). In fact, the results of this study could not be confirmed by a larger well-controlled study with chronic marijuana users (Mendelson et al. 1974). No difference in serum testosterone level was found either at the beginning of the study or after three weeks of heavy marijuana consumption.
Hollister (1986) assumed that a change, if any, in testosterone level and sperm production would only occur after long-lasting exposure. In two separate studies, one research group found a low sperm count with normal motility and morphology in chronic marijuana users who, under observation, had smoked 8-10 marijuana cigarettes over a period of four weeks (Hembree et al. 1978, 1976). In a study of 66 chronic Cannabis users, a comparison with 44 controls did not suggest any Cannabis-induced long-term effects on the plasma testosterone level (Friedrich et al. 1990). Correspondingly, other authors also found normal testosterone levels in chronic marijuana users (Schaefer et al. 1975, Coggins et al. 1976, Cushman 1975, Block et al. 1991).
Dax et al. (1989) investigated the effects of three 10 mg oral THC doses per day or three 18 mg doses in a marijuana cigarette for three days on male chronic marijuana users after at least two weeks of abstinence. They did not find any alterations in the plasma testosterone concentration. Cone et al. (1986) did not find any decrease in testosterone after the smoking of two marijuana cigarettes (2.8% THC). Mendelson et al. (1978) could not detect any influence on the testosterone level in 27 marijuana users who had consumed a mean of 54 marijuana cigarettes (moderate users) or 120 marijuana cigarettes (heavy users) over a period of 21 days.
FSH: Acute THC exposure (two marijuana cigarettes of 2.8% THC) does not result in an alteration of the FSH level (Cone et al. 1986). Also chronic administrations did not have any significant influence (Cushman 1975, Hembree et al. 1976, Block et al. 1991, Vescovi et al. 1992).
LH: In a study by Cone et al. (1986), a decrease in the LH level after acute THC exposure (approx. 50 mg inhalative) was noted. In a study of 10 chronic marijuana users, their basal and GnRH (gonadotropin-releasing hormone) levers were stimulated and levels of LH were found reduced (Vescovi et al. 1992). However, in other studies using a different experimental design, the LH concentration was not affected by THC exposure or Cannabis consumption (Cushman 1975, Hembree et al. 1976, Kolodny et al. 1974, Mendelson et al. 1978, Block 1991).
Prolactin: After three days of abstinence a slight elevation of prolactin concentration was stated in six chronic Cannabis users (Markianos and Stefanis 1982). Dax et al. (1989) investigated the effect of three 10 mg/kg oral doses per day or 18 mg/marijuana cigarette three times per day for days on male chronic marijuana users after two weeks of abstinence. Though no difference was found in plasma concentrations of LH and testosterone, they found the plasma prolactin level to be altered. The authors attributed this last finding to the heavy marijuana use. Mendelson et al. (1984), however, did not observe any acute effects on the prolactin level. Neither did Cone et al. (1986) find any decrease in prolactin after the smoking of two marijuana cigarettes (2.8% THC). Chronic Cannabis users do not show any significant alteration in their prolactin levels (Kolodny et al. 1974, Cohen 1976, Vescovi et al. 1992).
Puberty: Copeland et al. (1980) observed a pubertal arrest in a boy aged 16 years, who had consumed at least five marijuana cigarettes per day since he was 11 years old. Three months after cessation of consumption, a normal entry into puberty was observed. This is the only observation of this kind so far.
Conclusion: It is not conclusive to assume that there was a causal connection between the observed gynecomastia of strong marijuana smokers and their use of marijuana, all the more so because no associations between marijuana consumption and prolactin levels or other relevant parameters were found in later studies. Considering the widespread use of marijuana, literature would have to be expected to hold more published observations of this kind. Also with respect to possible influences on puberty, only one single case has been described to date. In animal studies, high doses produced a slightly higher incidence of abnormal sperm and following daily smoking of 8-10 marijuana cigarettes (100-300 mg THC) over a period of several weeks, a slight reduction in sperm count occurred though no increase in abnormal sperms or any impairment of function was observed. Neither acute nor strongly chronic Cannabis usage caused any consistent effects on the serum level of FSH, LH, prolactin or testosterone in male subjects.
5.3.1.2 Women
Animal studies reflected a Cannabis-induced blockade of the interaction of the hypothalamus, hypophysis and sexual organs. Thus THC delayed the onset of puberty and retarded menstruation in those studies (Tyrey and Murphy 1984). Multiple effects on the secretion of hormones were observed (Abel 1981, Smith and Asch 1984, Mendelson and Mello 1984). Evidence suggest that these incidences are not directly attributable to effects on production and secretion of ovarian sex steroid hormones, but result of a pituitary suppression of the GnRH (gonadotropin releasing hormones). In a study of rats that were administered 12.5, 25 or 50 mg/kg THC every day for two years, their prolactin concentration was not altered (Chang et al. 1996). In a 13-week study of mice and rats that received 5 to 500 mg/kg THC, the menstrual cycle was markedly prolonged relative to the controls. However, in a study of female monkeys, a tolerance of those disruptive effects on the menstrual cycle developed even at high doses (thrice weekly injections of 1.5 or 2.5 mg/kg THC) (Smith et al. 1983).
In comparison to men, far less data are available that describe the effects of THC on the female hormonal profile.
Menstrual cycle: Kolodny et al. (1979) reported an abnormal cycle length in marijuana smokers, averaging 26.8 days as compared to 28.8 days in controls. Moreover, the cycle was more often anovulatory (12.5% vs 38.3%). Dornbush et al. (1978) also found a reduced cycle length but did not state an increase in THC-induced anovulation. Other researchers did not find any significant influence on cycle length (Mendelson and Mello 1984).
Estrogen and progesterone: The hormonal profile of estrogen and progesterone did not differentiate chronic marijuana users from controls (Kolodny et al. 1979). Dornbush et al. (1978) did not find any significant influence on estrogen and estradiol. No correlation was found between acute marijuana smoking (18 mg THC) and the course of estrogen and progesterone concentrations during the menstrual cycle (Mendelson et al. 1986).
Testosterone: Twenty-six female chronic Cannabis users showed an increased testosterone level when compared to 16 controls (Dornbush et al. 1978). In the most extensive study to date, Block et al. (1991) did not discover any significantly elevated serum testosterone concentrations in comparison to controls and no significant association with their being grouped in occasional, intermittent or heavy users.
Prolactin: The smoking of one marijuana cigarette (1.83% delta-9-THC) did not produce any significant changes in plasma prolactin levels during the follicular phase (between menstruation and ovulation) of the menstrual cycle. However, when smoked during the luteal phase (between ovulation and menstruation), a transient small suppression of the plasma prolactin levels occurred 1 to 3 hours after consumption. (Mendelson et al. 1985b). Chronic users did not show any change in prolactin levels (Block et al. 1991).
LH: Mendelson et al. (1985) did not find any change in the LH level in 10 women after the smoking of one marijuana cigarette. However, a light significant decrement (p < 0.02) was observed when marijuana was consumed during the luteal phase. Chronic users present a normal LH level (Kolodny et al. 1979, Dornbush et al. 1978, Block et al. 1991).
FSH: Mendelson et al. (1986) did not state any change in FSH level after acute exposure to 18 mg THC. Also, chronic female users possessed equally normal FSH levels (Kolodny et al. 1979, Dornbush et al. 1978, Block et al. 1991).
Conclusion: Significantly less research data exist that deal with the influences of THC on female sex hormones in comparison to the scientific material on male sex hormones. The research results are inconsistent. There are no conclusive indices to any THC-associated influences on the menstrual cycle length, the number of cycles without ovulation, or on the plasma concentrations of estrogens, progesterone, testosterone, prolactin, LH or FSH in female marijuana users. The transient THC-induced suppression of prolactin and LH levels during the luteal phase of the menstrual cycle should be further investigated. However, this effect occurred only following the inhalative route which, in comparison to oral administration, is associated with a faster absorption of the drug and higher plasma THC levels. Chronic marijuana users did not show any significantly altered hormone levels.
5.3.2 Glucocorticoids
In animal studies, THC stimulates the secretion of ACTH (adrenocorticotropin). ACTH is secreted by the adenohypophysis and stimulates the synthesis of the glucocorticoids (cortisol) in the suprarenal cortex. THC produced a significant increase in serum cortisol in rats at doses of 2 mg, 5 mg and 30 mg/kg body weight (Kumar and Chen 1983). Also, other animal studies measured an increase in the cortisol level when administering doses in the range of 2 to 50 mg/kg THC (Birmingham and Bartova 1976, Pertwee 1974, Eldridge 1991). With chronic administration, a tolerance developed quickly and values progressively returned to normal (Pertwee 1974, Eldridge 1991).
A single oral administration did not elevate plasma cortisol in man (Hollister 1970). However, the smoking of two marijuana cigarettes caused a transient significant increase in plasma cortisol level (Cone et al. 1986). In the above-mentioned study by Dax et al. (1989), a thrice daily oral administration of THC did not find any THC-induced influence on the ACTH level. Chronic heavy marijuana users did not show any significant differences in their cortisol levels (Cruickshank 1976).
5.3.3 Thyroid hormones
The acute treatment of rats with 10 mg THC/kg reduced serum levels of thyrotropin (TSH) and of the thyroid hormones triiodothyronine (T3) and thyroxine (T4), but had no effect on the pituitary or thyroid response to exogenous (Hillard et al. 1984). Intraperitoneal doses of THC greater than 3 mg/kg reduced serum TSH levels by more than 90%. The ED50 for THC was approximately 0.3 mg/kg. Thus, doses were required that clearly exceeded those of relevance to this study. The serum levels of thyroid hormones in chronic marijuana users fail to show any significant incidences (Cruickshank 1976).
5.3.4 Glucose metabolism
Fifty years ago, in a study with 62 volunteers, it was already demonstrated that Cannabis does not have any significant influence on glucose metabolism (Allentuck 1944). In another study, marijuana did not produce any relevant effects on glucose metabolism after 1 to 3 days of fasting. The glucose tolerance was not affected by marijuana (Permutt et al. 1976). However, in one other study a high THC dose (6 mg intravenous) influenced the glucose tolerance test scores in some probands (Hollister and Raven 1976).
5.3.5 Summary
Animal studies have illustrated that, given a sufficiently high dosage, THC may act on the hypothalamo-pituitary-adrenal (HPA) axis and thus adversely affect the function of sex steroid hormones with effects also on hormone-producing sexual accessory organs. However, there are no consistent findings of adverse effects on male or female sexual organs within the range of the human consumption pattern. Strongest references exist concerning a hormonal dysfunction during puberty and a transient influence on prolactin and LH concentration during a certain phase of the menstrual cycle. However, these observations remained singular. A tolerance develops to THC-induced effects on the endocrine system (sex hormones). Also other hormones such as the glucocorticoids and thyroid hormones are influenced by high THC doses in animals. In man, no significant alterations were found at relevant doses. The NOAEL of THC for the influences on sex hormones and other hormones is safely above. or ranges within, the human consumption situation. There is no reason to believe that sub-psychotropic THC doses could affect the function or concentration of sexual hormones or other parameters relevant for reproduction, such as sperm quantity and quality.
5.4 Immune system
The immune system is a complex functional system for protection against noxious foreign material (for instance: bacteria) or for the elimination of anomalous structures (such as tumor cells). The organs of the lymphatic system (spleen lymphatic nodes), the production sites of lymphocytes and other immune cells (thymus, bone marrow), a multitude of cells (lymphocytes, macrophages) and different molecules (immunoglobulines, cytokines etc.) contribute to the immune system.
Immunity is either unspecific or specific (acquired). Unspecific immunity does not require a preceeding sensitization (from previous exposures). For instance, the ingestion of bacteria by macrophages, and the killing of tumor cells by natural killer cells belong to this category. As opposed to this, acquired immunity is based on selective responses of antibodies specifically sensitized to certain substances (antigens) or of specifically sensitized cells (T-lymphocytes, macrophages). The B-lymphocytes produce antibodies and regulate humoral (mediated by specific atibodies) acquired immunity whereas the activity of T-lymphocytes controls cell-mediated immunity.
THC alters some immune parameters of humoral and cell-mediated immunity in a dose-related manner, acting either in an immunosuppressive or immunostimulating manner depending on its effects. At high THC concentrations, unspecifc effects on the cell membrane seem to play an important role, whereas at a lower dosage, THC effects on the immune system appear at least partly mediated by a cannabinoid-receptor-dependent pathway (Sanchez et al. 1997, Burnette-Curley 1995, Kaminsky et al. 1994). The CB2 receptor was found to be of special relevance to these activities (Patrini et al. 1997).
The natural cannabinoid-receptor-antagonist anandamide exerts biphasic dose-related effects on a number of biological parameters in mice (Sulcova et al. 1997). One of these parameters is the leucocyte-phagocytosis, which is inhibited by high doses (10-100mg/kg) but stimulated by the lowest measured dose (0.01 mg/kg). Similar effects are to be expected with exogenous ligands such as THC.
5.4.1 Cell-mediated immunity
The effects of THC on cell-mediated immunity are determined by a variety of different tests. To this end, one assesses: the number of lymphocytes and the transformation of lymphocytes as a response to certain substances that promote cell division (mitogens); the influence on the synthesis of macromolecules (proteins, DNA) in T-lymphocytes; the T-cell capacity to form rosettes with sheep erythrocytes; the functions of leucocytes, natural killer cells and macrophages; the action on different cytokines (interferon, interleukines, tumor necrosis factor); and finally the susceptibility to bacterial and viral infections, as well as skin response to antigens.
5.4.1.1 Number of T-lymphocytes
There are inconsistent results concerning the effects of THC on lymphocyte count (Nahas et al. 1974). Some researchers found significant decreases in the T-lymphocyte count (Nahas et al. 1974) whereas others did not find the number of lymphocytes significantly altered (Rachelefsky et al. 1976, Cushman and Khurana 1977, Dax et al. 1989, Zimmer et al. 1976). Dax et al. (1989) investigated the effect of thrice-daily 10 mg oral or 18 mg THC in marijuana cigarettes for three days in chronic marijuana users after two previous weeks of abstinence. Any differences in the quantity of B- and T-lymphocytes as well as T-cell subtypes previous to, during, and subsequent to administration were not observed. Wallace et al. (1988) did not find the number of T-lymphocytes to be significantly altered but determined a change in the ratio of helper T-cells to suppressor cells (CD4/CD8-ratio) with an increase in the CD4 percentage. This was interpreted as an immunoenhancement, as helper T-cells stimulate the proliferation and activation of other immune cells.
5.4.1.2 Transformation of T-lymphocytes
Some studies examined the influence of THC on T-cell proliferation as a response to substances stimulating cell division, so-called mitogens (PHA = phytohemagglutin, Con A = concavalin, anti-CD3-antibodies, etc.), and cells (MLC = allogenic mixed lymphocyte culture).
Cellular studies: THC (10-100 mM) induced in vitro suppression of T-cell proliferation responses to PHA and MLC (Nahas et al. 1977). Furthermore, a reduced uptake of thymidine, leucine and uridine was found, which indicates a depressed protein/RNA synthesis. THC suppressed the lymphoproliferative response of T-lymphocytes that had been stimulated by human interleukin-2 in a dose-dependent manner (Kawakami et al. 1988). A minimum dosage of 2.5 mg/ml THC was required to obtain any measurable effects. A threshold concentration of 5 mg/ml THC was necessary to achieve an influence on the mitogen-induced T-lymphocyte proliferation (Klein et al. 1985).
Rachelefsky and Opelz (1977) did not find THC (at concentrations up to 1.1 millimol) to have any influence on the stimulating effect and the protein synthesis of PHA and MLC. Pross et al. (1992) found lymphocyte proliferation either to be suppressed or to be enhanced, dependent upon the measured parameters and the organ source of the lymphocytes. THC suppressed the proliferation from Con A- or PHA-stimulated lymphocytes derived from either spleen or lymph nodes. Spleen cells stimulated with anti-CD3 antibody and treated with low doses of THC displayed an enhanced proliferation, whereas the response in lymph nodes did not change. The research group determined that the observed immunosuppression or -stimulation related to the THC-induced modulation of interleukin-2 activity (Nakano et al. 1992). Luo et al. (1992) found THC to affect the mitogen-induced transformation of lymphocytes in a dose-related manner. The stimulation of lymphocytes with PHA relative to the uptake of thymidine reached its peak at 0.4 mg/ml THC. The effect decreased rapidly at higher or lower doses. When stimulated with Con A, the maximum stimulation was reached at 0.05 mg/ml THC. Human lymphocytes and mouse splenocytes responded in similar patterns.
Cells of marijuana users: Nahas et al. (1974) found a reduced thymidine uptake in lymphocytes of marijuana users after stimulation with PHA and MLC. Segelmann and Segelmann (1974) disputed these findings as they had doubts about the method applied and therefore requested these results to be revised. White et al. (1975) could not confirm these results. The incorporation of 14C-thymidine by peripheral blood lymphocytes did not differentiate 12 long-term marijuana smokers from controls. Lau et al. (1976) did not find any altered lymphocyte response to PHA as measured by thymidine uptake after oral ingestion of 210 mg THC for 18 days. Humoral and cell-mediated immunity were investigated in a study by Rachelefsky et al. (1976) of 12 healthy marijuana smokers (6 moderate and 6 heavy smokers) who smoked a mean of 5.4 marijuana cigarettes (approx 20 mg THC/cigarette) over a period of 64 days. Initially the number of T-lymphocytes decreased, but returned to normal in the course of the test period. In vitro responses to PHA and MLC were normal throughout the study. The number of marijuana cigarettes smoked did not affect the immunologic test scores. The authors concluded that chronic use of marijuana did not have any adverse effects on the T-lymphocyte proliferative response in young, healthy adults. Similar statements were published by Cohen (1976), Kaklamani et al. (1978) and Wallace et al. (1988). In the aforementioned study by Dax et al. (1989) with a thrice daily oral intake of a 10 mg or 18 mg THC marijuana cigarette for three days, one did not detect any significant alterations in mitogen response as measured before, during and after THC treatment.
5.4.1.3 Rosette formation
T-lymphocytes possess the capacity to form rosettes of sheep erythrocytes (red blood cells from sheep). Then a minimum of four T-cell-receptor-bound sheep erythrocytes surround the immune cell. THC affected an in vitro suppression of the T-cell rosetting capacity (Cushman et al. 1976). Mice that had been immunized with sheep erythrocytes exhibited a significantly reduced rosette formation after injections of 25 mg/kg THC or more (Lefkowitz et al. 1978).
Cushman et al. (1975 b) compared the lymphocytes in 23 normal controls and in 23 marijuana users. No difference in rosetting capacity was found in B-lymphocytes, but rosette formation differed in T-lymphocytes. The same research team examined further collectives and found a reduced early rosette formation by normal T-lymphocytes in 35 marijuana users when compared to controls, whereas late formation was not affected (Cushman and Khurana 1976). In a follow-up study of 10 marijuana users the same result was obtained, so that the authors assumed an impairment of a T-lymphocyte subpopulation by marijuana. Petersen et al. (1976) diagnosed a reduced rosette formation in two of three marijuana smokers. A further study found rosetting suppressed in five of six subjects after smoking 10 mg THC, but after 24 hours it had normalised in five of the six subjects that were examined. In the latest study on this topic, 12 chronic marijuana smokers did not differ from 15 control subjects in the rosetting capacity of their peripheral T-lymphocytes (Kaklamani 1978). In the 80s and 90s no further studies of this kind were conducted, and the clinical significance of rosette formation has not yet been ascertained.
5.4.1.4 Delayed-type hypersensitivity
Delayed-type hypersensitivity is cell mediated, contrary to the other types of hypersensitivity (immediate type, cytotoxic type, immune complex type). The release of lymphokines from specifically sensitized T-lymphocytes activates macrophages and mononuclear cells. The study of Smith et al. (1978) elucidated that THC suppressed the delayed-type hypersensitivity response to sheep erythrocytes in mice. High doses of 100 mg/kg THC subcutaneous for four days suppressed the immune response from 35 to 64%. Levy and Heppner (1978-1979) also found delta-8-THC to posses only weak (to moderate) activity in the suppression of the delayed-type hypersensitivity response to sheep erythrocytes.
5.4.1.5 Cytokines
Cytokines are substances that are produced by different cells and function as messengers between cells. Interleukines are mediators of such kind; secreted by leukozytes, they activate other immune cells (B- and T-lymphocytes, natural killer cells). Interferons are produced by different cells as part of an immune response to various stimuli and exert antiviral (interferon-alpha and -beta) and immunomodulating (interferon-gamma) activity. Tumor necrosis factor (alpha and beta) are cytokines produced by immune cells that regulate response to inflammation, immune functions and tumor defense.
Interferon: Chronic administration of 50 mg/kg THC to mice over a period of 56 days significantly reduced their interferon-alpha and interferon-beta concentration in their lymphocyte cultures after stimulation by various mitogens (Blanchard et al. 1986). A dose of 5 to 10 mM THC effected a dose-related in vitro suppression on IFN production, whereas 5 mM did not have any effect (Blanchard 1986). If mice received more than 15 mg/kg THC intraperitoneal, as in the study by Cabral et al. (1986b) their interferon-alpha and -beta production in response to herpes viruses would decline. Watzl et al. (1991) found the effect of THC on interferon-gamma-induction by mitogens to be dose-related, as IFN production was increased at THC concentrations of 0.1 and 1 mg/ml, whereas a decrease occurred at concentrations above 5 mg/ml.
Interleukins: THC elevated interleukin-1-bioactivity in human macrophage cultures stimulated with lipopolysaccharide (Shivers et al. 1994). Interleukin-6 levels were decreased. Another study reported an increased expression of interleukin-2 (IL-2) receptor alpha and beta proteins (Daaka et al. 1997). Specter and Lancz (1991) reported that the addition of THC dose-dependently inhibited IL-2 attachment to IL-2 receptors of cytotoxic T-lymphocytes. At 10 mg/ml THC they measured a suppression to 5% relative to the basis value, at 5 mg/ml to 25% and, finally, at 1 mg/ml to 90% of the initial value. In the study of Watzl (1991), concentrations of up to 1 mg/ml THC did not affect the interleukin-1-secretion of mitogen-activated human peripheral blood mononuclear cells, whereas a dose-related suppression was noted at higher drug levels. No effect on interleukin-2-secretion was displayed after addition of 0.1 mg/ml THC. Also, 1 mg/ml THC elevated secretion by 100%, and any doses above this concentrations did not produce any significant effect.
Tumor necrosis factor: Kusher et al. (1994) found a dose-related influence of THC on candida albicans-induced tumor necrosis factor (TNF) production by lymphocytes, a suppression of TN induction, only at concentrations ranging from 0.05 to 1 mg/ml, while no effects were found at 0.005 mg/ml (=5 ng/ml), which is the level of concentration relevant to the subject of our study. The study of Watzl (1991) reported that even high THC concentrations did not have any effect on TNF secretion after mitogen activation of peripheral blood leucocytes.
5.4.1.6 Host resistance and cell toxicity
Cellular defense is related to other immune parameters that will be discussed. For instance, cytokines affect the cytotoxic activity of natural killer cells.
Lymphocytes: THC in a concentration range of 2 mg/ml depressed induction and cytolytic activity of cytotoxic T-lymphocytes, though it failed to do so in a range between 25 and 2000 ng/ml THC (Lu 1990, Lu and Ou 1989). THC was found to exert a biphasic effect on lymphocyte metabolism, with a stimulation at low doses and a suppression at high THC concentrations. Thus a THC concentration of 0.01 mM induced a 1.3 fold and a concentration of 1 mM a 2 fold stimulation of glucose oxidation to carbon dioxide in mice lymphocytes. A further increase in concentration to 10 mM downregulated glucose oxidation to 40%, and 20 mM produced a decrease to 25% of the initial value. Corresponding effects were found for the phospholipid synthesis from glucose as 1 mM produced maximum values of 250% and 20 mM produced minimum values of 30%. The authors found evidence that the depression on glucose metabolism at high non-physiological doses is mediated by an unspecific receptor-independent pathway. Furthermore, concentrations of about 10 mM were found to have cytotoxic effects, reducing lymphocyte function to 1%, whereas a concentration of 1 mM did not exhibit any of those effects (Sanchez et al. 1997). At high doses a disruption of cell membrane function is to be expected.
Leucocytes: THC-induced effects on leucocyte migration are concentration dependent. For leucocytes from five marijuana smokers, a minimum concentration of 2.0 mg/ml was required to display any alterations (Schwartzfarb et al. 1974). Maximal inhibition of neutrophil granulocyte function occurred at a dose of 4 mg/ml THC; at 2 mg/ml THC activity was suppressed by 50% and at THC concentrations below 1 mg/ml no suppression was noted (Djeu et al. 1991). Neutrophile granulocytes, for instance, retarded candida albicans growth by 64%. By addition of 4 mg/ml THC, the antifungal activity was suppressed to only 5%, at 0.5 mg/ml it was 61%, at 0.25 mg/ml 63% and at 0.125 mg/ml 69%, thus having even slightly improved when compared to activity without THC.
Natural killer cell activity: In a study by Patel et al. (1985), natural killer cell activty in rats was not modulated after acute administration of 3 mg/kg subcutaneous, whereas subchronic administration of the same concentration impaired killer cell activity. A number of studies determined quite high threshold values of the order of 2 mg/ml for any inhibitory effects on killer cell activity to become detectable. In a study of mice, suppressive action was stated at a tissue concentration of 5-10 mg/ml (Klein et al. 1987). In a study of cultured murine splenocytes, a dose-related suppression was found in a concentration range of 10-30 mg/ml (Klein et al. 1987). A concentration of 5 mg/ml and above was found to be inhibitory for activity against a human tumor cell line (Specter et al. 1986). In a further study, natural killer cell activity was nearly completely inhibited after addition of 20 mg/ml THC; at 10 mg/ml it was suppressed to 30% of the baseline value and 1 mg/ml did not induce any inhibition (Specter and Lancz 1991 b). Also at a concentration of 5 mg/ml, THC inhibited the cytolytic NK-cell activity against another cell line (Kawakami et al. 1988). In a concentration range of 25 to 2,000 ng/ml, NK-cell activity was not disturbed (Lu and Ou 1989).
At concentrations of 0.05-2 mg/ml, Kusher et al. (1994) found a dose-related inhibition of cytolytic NK-cell activity in large granulated lymphocytes against tumor cells, whereas no such inhibitory action was observed in the low nanomolar-range. At 2 mg/ml THC approached maximal inhibitory effect, at 0.05 mg/ml a 50% inhibition occurred, and at 0.01 mg/ml no such effect was measured.
Macrophages: THC was shown to suppress the macrophage contact-dependent cytolytic activity by inhibiting tumor necrosis factor (TNF)- and NO-mediated killing (Burnette-Curley 1995, Fischer-Stenger et al. 1993). THC suppressed macrophagelytic action in concentrations of more than 2 mg/ml THC (Lu 1990) but failed to have such effect within the nanogram range (Lu and Ou 1989). In another study, a minimum of 10 mg/ml THC was required in cultured macrophages to impair their functions (Friedman et al. 1986). Macrophages normally spread on glass surfaces. THC was found to adversely affect this ability in relation to dose in another study (Lopez-Cepero et al. 1986). By adding 20 mg THC to a culture of normal mice peritoneal cells, the spreading was almost completely inhibited. The lowest dose measured that still induced minimum effects was 0.05 mg THC per culture. Higher concentrations were necessary to inhibit phagocytosis in yeast particles. Another study of mice, however, did not report any modulation of macrophage activity (Munson et al. 1976). Cabral and Vasquez (1991) examined various macrophage functions. THC induced a dose-related inhibition of anti-tumor-factor production in response to stimulation by Propionibacterium acnes in mice. An intraperitoneal administration of 15 to 100 mg/kg THC over a period of several days decreased production to 50% at the maximal dose and to 85% of the initial value at the lowest THC dose. Also macrophage contact-dependent cytotoxic activity, extrinsic antiviral activity, macophage capacity to take up viruses, and protein expression were found reduced in a similar fashion. Studies generally employed extremely high doses that induced a dose-related immunosuppression. The authors regard unspecific (receptor-independent) toxic effects on the cell membranes as cause for the observed impaired immune function.
5.4.1.7 Susceptibility to infections
Bradley et al. (1977) observed an application of various bacterial endotoxins when applied in combination with THC to reduce the lethal THC dose from approximately 350 to 150 mg/kg.
Mishkin and Cabral (1985) examined the effect of THC on host resistance against a vaginal infection with a herpes virus (HSV2, herpes simplex virus type 2) in mice. The animals were administered high THC doses from one day before until two days after intravaginal introduction of viruses. Those animals that had received 100 mg/kg THC exhibited heavy infections, higher virus titers and higher animal mortality. A similar experimental design with guinea pigs also showed a decrease in host resistance to intravaginally introduced herpes viruses after the animals had been intraperitoneally treated with the drug at doses ranging from 4 to 10 mg/kg on day 1-4, 8-11 and 15-18 after infection (Cabral et al. 1986). Cumulative death rate was dose-dependent.
At concentrations of 10 and 50 mg/kg, THC suppressed the lymphocyte proliferative response to a second exposition to herpes viruses (HSV2) in mice (Cabral et al. 1987). Furthermore, the drug affected expression of proteins as elicited by bacterial lipopolysaccharide (Cabral and Mishkin 1989). Morahan et al. (1979) found a higher susceptibility to infection not only with herpes viruses (HSV2) but also with bacteria (Listeria monocytogenes). The decrease in host resistance to tested bacteria was dose-dependent. THC doses of 38, 75 and 150 mg/kg induced a 10-, 17-, and 657-fold decrease in resistance, measured as change of lethal dose (LD50). Ashfaq et al. (1987) observed a weakened local immune response to bacterial infections after four days of marijuana smoking (estimated daily dose: 3.2 to 6.4 mg/kg) in mice. This significantly deteriorated the immune response to a skin necrosis induced by a subcutaneous injection of Staphylococcus aureus. As a daily injection of THC (10 mg/kg) by itself had no such effect, the authors inferred that other marijuana constituents must be responsible for this immune suppression.
Klein et al. (1993) found a decrease in survival rate among mice that had received an injection of 8 mg/kg THC 24 hours before and after a sublethal intravenous infection with the bacterium Legionella pneumophila. Death generally occurred 30 minutes after the second injection and resembled a cytokine-mediated septic shock. In fact, a marked increase in acute-phase pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) was measured. A follow-up confirmed the augmented lethality and the altered serum levels of acute-phase proteins (Smith et al. 1997). However death rate was not affected by lower doses of 1-5 mg/kg THC (Klein et al. 1993).
5.4.1.8 Skin reactions to antigens
Silverstein and Lessin (1974) investigated the skin reactions after sensitization to Dinitrochlorobenzene, a compound contained in photochemicals. A sensitivity was found in 100% of 34 examined marijuana smokers compared to 96% of 279 healthy non-smokers and 70% of 384 cancer patents. According to Hollister, these findings "raise questions about the clinical significance of experiments that have shown evidence of impairement of cell-mediated immunity from cannabinoids" (Hollister 1992).
5.4.2 Humoral immunity
The effect of THC on humoral immunity was tested by measuring the number of B-lymphocytes after stimulation by mitogens, or by the presence of sheep erythrocytes, by measuring the capacity of rosette formation as well as production of antibodies.
5.4.2.1 Number of B-lymphocytes and B-lymphocyte proliferation
The number of B-lymphocytes is not affected by smoking marijuana (Wallace et al. 1988, Rachelefsky et al. 1976). At least 5 mg/ml THC were required in cell cultures to effect suppression on lipopolysaccharide-induced proliferative response in B-lymphocytes (Klein et al. 1985). Munson et al. (1976) discovered a dose-related reduction of lymphocyteresponsitivity after 50, 100 and 200 mg/kg THC. In contrast Sanchez et al. (1997) observed a stimulation of glucose metabolism in B-lymphocytes at low (physiologic) THC concentrations. At low nanomolar concentrations that can be reached in the live organism, THC was found to effect an enhanced B-lymphocyte proliferation in response to activation through cross-linking of surface immunoglobulins (Derocq et al. 1995). Micromole concentrations effected a suppression instead. Thus the authors proved that the increase in immune activity is receptor mediated and they assume that the CB2 receptor is involved.
5.4.2.2 Rosette formation
Cushman et al. (1975 b) compared the lymphocytes of 23 normal controls to 23 marijuana users. The B-lymphocyte rosetting capacity was not found to be different in either group.
5.4.2.3 Antibody production
One way to assess humoral immunity is to measure the capacity of plaque formation in mice lymphocytes that are exposed in vitro to sheep erythrocytes against which they are immunized. Smith et al. (1978) established a median effective dose (ED50) of 70 mg/kg THC for the reduction of this effect in mice. In a study of Levy et al. (1981) a dose-dependent reduction in hemolytic plaque-forming cells was found. Peak reactivity was, however, only delayed by 24-48 hours and the THC concentration required produced high behavioral toxicity in the animals.
Baczynsky and Zimmerman (1983) investigated the primary-like humoral immune response, the secondary-like humoral immune response, and the memory aspects of the humoral immune response in mice after stimulation with sheep erythrocytes. Mice treated with 10 or 15 mg/kg THC during primary immunization displayed a depressed primary immune response. Administration of THC during secondary immunization did not produce any signifiant suppression on secondary response. The memory aspect was assessed by measuring the secondary immune response after THC had been administered during primary response. At 10 or 25 mg/kg THC, the secondary immune response was significantly suppressed. THC treatment (10 or 15 mg/kg) during primary immune response caused a decrease in thymus weight and reduced the number of thymus cells.
Zimmerman et al. (1977) observed a dose-dependent depression of the immune response at doses of 1 mg/kg, 5 mg/kg and 10 mg/kg in mice that had been stimulated with sheep erythrocytes. The splenic weight, the percentage of splenic white pulp of total spleen volume, the number of splenic plaque-forming cells, and the hemagglutination titer were reduced. In a study by Schatz et al. (1993), THC suppressed the primary humoral immune response in mice. Oral administration of 50-200 mg/kg THC produced a dose-related inhibition of antibody-forming-cell (AFC) response. Rosenkrantz et al. (1975) intraperitoneally immunized rats with sheep erythrocytes during, before, and after ingestion of 10 mg/kg THC. The primary immune response was reduced by values of 33 to 40%.
Luthra et al. (1980) did not find any development of tolerance to the suppression of antigenic stimulation with sheep red blood cells in rats. Twenty-six days of pretreatment with 6 or 12 mg/kg THC prior to antigenic stimulation did not modulate suppression of antibody-forming-cell proliferation or change hemagglutinin and hemolysin titers. In contrast, Loveless et al. (1981-1982) observed a development of hyporesponsiveness to delta-8-THC-induced immunosuppresion. If mice were pretreated daily for five days with 5 or 10 mg/kg THC before they were immunized with sheep erythrocytes, significantly higher doses were required to produce the equivalent inhibitory effect on hemolytic plaque-forming cells.
5.4.3 AIDS infection
A major immunosuppressive activity in THC would adversely affect the outbreak or the course of AIDS in HIV-positive patients who use Cannabis. Hall et al. (1994) maintain that so far no epidemologic relation between marijuana consumption and infectious diseases has become conspicuous. This did not, however, "exclude the possibility that chronic heavy use may produce minor impairments in immunity" (p 67).
THC is therapeuically employed as an appetite stimulant as well as an antiemetic and antivomiting agent in cancer and AIDS patients. Especially long-term treatment with THC as used for AIDS patients to enhance appetite would be precarious if an immune suppression would thus further damage the already impaired immune system. Kaslow et al. (1989) conducted a prospective epidemiological study of 4,954 homo- and bisexual men to examine the development of AIDS. The use of Cannabis or other psychoactive drugs did not increase the risk of progression to full-blown AIDS in HIV-positive men. Also among seropositive men with lower initial T-helper-lymphocyte count, those who used drugs and those who were non-users did not significantly differ in their respective risk of AIDS. In a six-year epidemiologic study, Di Franco et al. (1996) could not establish that the outbreak of AIDS in HIV-positive men was in any way associated with the use of Cannabis.
5.4.4 Summary
Cellular experiments and animal studies demonstrate that THC has suppressive effects on the humoral and cell-mediated immunity. However, the majority of those can be attributed to toxic unspecific effects. Many analysed parameters required extremely high doses to exhibit any significant effect and the effects were dose-dependent, with the threshold concentration being precisely determinable. When applying lower doses, one often observed differentially immunostimulating effects or no effects at all. For many immune parameters, the NOAEL is within a range irrelevant to a human consumption situation. In studies of man or of cells of marijuana users, the effects observed were often contradictory. If such effects were found at all, they were weak, even in case of heavy Cannabis use and of questionable relevance to health. The World Health Organisation summarized in its most recent Cannabis report: "Many of their effects appear to be relatively small, totally reversible after removal of the cannabinoids, and produced only at concentrations or doses higher than those required for psychoactivity (more than 10 mM in vitro, or more than 5 mg/kg in vivo)" (WHO 1997, p 27).
Source, Graphs and Figures: THC Limits for Food, Part II
This Part II of the THC study deals with the methodical, and especially the biological basis for THC limits in food. The essential ideas and results of Part II are summarized in Chapter 3 of Part I and provide the basis for the proposed THC limits. Part II addresses specifically the scientifically interested, who would like to explore individual aspects of the matter-or the whole topic-in more detail, in order to obtain a better understanding of the fundamentals. Here, the reader also finds numerous references.
The majority of the extensive data on the pharmacological effects of THC were collected in animal and cellular experiments, mostly using very high THC doses, often not by oral but by other routes of administration. Chapter 2 tackles the transferability of these results to oral THC administration to humans. A further section of this chapter reviews the methodical basis commonly used in the establishment of limits for other substances and hazards. In the course of this, some aspects are examined that are of special importance in connection with THC.
Chapter 3 contains a survey of the pharmacological effects of THC and of its overall toxicity. Furthermore, matters of bioavailability, metabolism, mode of action, as well as development of tolerance are dealt with. A separate section deals with physical parameters, such as effects of temperature on the biological efficacy of THC.
Chapter 4 examines the matter of the psychotropic threshold, i.e. the placebo threshold for the oral administration of THC. For the purpose of fixing a limit, this serves for the determination of the maximum daily quantity of THC that will not lead to undesired acute or chronic psychological reactions.
Chapter 5 deals with possible physical effects below the psychotropic threshold. It serves for the determination of the maximal daily dose that does not lead to undesirable acute or chronic physical effects.
Some Excursuses discuss special questions that may arise in the context of the application of industrial hemp products to man: Among them is a short comparison of THC and alcohol in food, matters of assimilation of THC by the skin, for instance through cosmetics, as well as the detection of THC in body tissues after the ingestion of hemp-based food in comparison to the impact of marijuana ingestion.
Alcohol-containing products such as beer, wine or spirits that are used for intoxication typically contain 3-50% alcohol (ethanol). Chronic heavy use of alcohol may irreversibly damage brain cells and other organs (liver, heart). There is evidence of serious alcohol-induced malformations in the fetus (alcohol-embryopathy). Alcohol-containing food products such as fruit drinks, sweets, and meat dishes, generally contain less than 0.3% ethanol, this being an amount ten times smaller than what is contained in intoxicating products. This alcohol content, which is generally considered low and innocuous, needs not be declared in this context. According to the WHO (World Health Organization), a regular ingestion of 7 g of ethanol per day is harmless (Verbraucherzentrale 1998).
THC-containing drug products such as marijuana and hashish, applied for drug use, typically contain 1-20% by weight of THC, sometimes more. THC shows some potential for physical impairment, though this is much weaker than in alcohol. (Hall et al. 1994b, WHO 1997).
Cannabis-based food products, such as edible oil based on seeds from low-THC hemp and hemp-seed-based cereal bars, contain less than 0.005% THC (50 ppm) on average. Thus their THC content is less than 1/200 of the amount found in drug products. In Switzerland, precisely this value (i.e. 0.005%) was set as the limit for the concentration of THC in edible oil derived from hemp seeds. In other products of which greater amounts can be consumed, lower limits were set: for pastries it is 0.0005% and for alcoholic beverages it is 0.00002%. Thus the difference between these concentrations and the THC concentration in marijuana amounts to a factor of between 2,000-50,000.
2 Methodology
2.1 Extrapolation of different routes of administration to oral ingestion
A large portion of data on the toxicology of THC for humans and animals were not obtained following oral administration but after inhalative or parenteral (intravenous, subcutaneous, intraperitoneal) application. Different routes of administration result in a different bioavailability and in different pharmacokinetics (surveys: Wall et al. 1983, Maykut 1985, Agurell et al. 1986, Harvey 1991). This has to be taken into account when dosage and plasma concentrations are translated to the situation of an oral administration, i.e. the relevant exposure route for food considered in the following.
Intravenous administration: Intravenous administration results in a 100% bioavailability and an immediate rise of THC concentration in the blood. The onset of THC effects occurs within a few minutes. In order to achieve minimum psychotropic effects, 0.02 mg THC/kg BW (body weight) are necessary; as a function of body weight this typically corresponds to 1 mg THC. In the case of doses that lead to psychotropic effects, short-term peak plasma levels are reached that markedly decrease within minutes. The intravenous administration of 5 mg THC led to a plasma concentration of about 200 ng/ml THC, which decreased rapidly to 15 ng/ml after an hour and 3 ng/ml after 4 hours. The psychotropic effects had vanished after three hours. Smith and Asch (1984) intravenously administered monkeys with 2.5 mg THC/kg BW three times a week. This produced an average maximum plasma concentration of 300 ng/ml THC and an average long-term concentration of 15 ng/ml.
Inhalative administration: After inhalative administration, THC is quickly absorbed and the time course of plasma concentration is similar to the situation after intravenous administration. Bioavailability only reaches 10-30%, so that about five times the dose of intravenous administration is required to achieve the same effects. About 0.7 mg/kg, that is about 5 mg THC, are necessary to produce minimum psychotropic effects. An intoxication desired by cannabis consumers requires inhalation of at least 10-15 mg THC, which would lead to a maximum plasma concentration of 100 ng/ml after about 5 minutes. The concentration decreases rapidly, so that little THC will be detected after 2-3 hours. Chronic consumers need higher doses because of their development of tolerance to the active agent. This was shown in a study where 47 chronic Cannabis users tolerated inhaled THC doses of up to 180 mg without undesired side effects or nausea (Stefanis 1978).
Oral administration: The systemic bioavailability of THC reaches 10-20% after oral administration in a lipophilic vehicle. To achieve minimum psychotropic effects in man, 0.2-0.3 mg/kg are required, which equals 10-20 mg depending on the body weight. This is 10-15 times the dose of intravenous administration. The maximum plasma level after oral administration of this dose is of the order of 5 ng/ml and is reached after 2-4 hours. The psychotropic effect sets in after 30-60 minutes, peaks after 1-3 hours and lasts for 6-8 hours. The average maximum plasma concentration of THC in six cancer patients after oral ingestion of 15 mg THC was 3.9 ng/ml and was typically attained after two hours (Frytak et al. 1984). With the exception of one patient, the plasma level of THC in all cancer patients had dropped below 1 ng/ml or, respectively, no THC could be detected in the plasma any more after 6 hours. Three patients received three doses of 15 mg THC a day. The maximum plasma level ranged between 3.6 and 6.3 ng/ml; thus, it did not differ much from that following a single administration. Ohlsson et al. (1980) observed a maximum THC concentration of 5 to 6 ng/ml between 1 and 1.5 h after experienced marijuana smokers had ingested a chocolate cookie containing 20 mg THC. Similar results were found by Brenneisen et al. (1996) and Frytak et al. (1979), though they occasionally found higher plasma concentrations of more than 10 ng/ml.
The above comparison suggests that, in order extrapolate data from intravenous and inhalative administration to the ingestion route, different conversion factors for acute single application and chronic effects must be applied. The systematic bioavailability is relevant to chronic effects, whereas in relation to acute effects further aspects must be considered, such as the faster resorption and the considerably higher peak plasma concentrations of THC after smoking and intravenous intake relative to those after oral administration.
2.2 Extrapolation of animal data to man
One advantage of animal studies is that they allow a thorough control of the conditions of THC exposure, such as time and duration, as well as the control of possible confounding factors. Therefore they constitute an important element of toxicological research. However, for several reasons, caution is still required when using "data produced by those that continue to extrapolate animal data to humans without some attempt to discuss in detail the validity of their assumptions" (Campbell 1996).
The principle of phylogenetic continuity of species, including similarity of cell structure and energy metabolisms, is the common denominator for the cross-species extrapolation of toxicological data. Nevertheless there are some significant pharmacokinetic and other relevant differences between species that render extrapolation more difficult.
There are various methods for the extrapolation of animal data to man (Voisin et al. 1990, Winneke and Lilienthal 1992, Ings 1990). These shall only briefly be reviewed here:
2.2.1 Interspecies comparison based on body weight
The first approach to comparing toxicological data from various animal species with that of man is based on the body weight. Many biologic parameters such as water intake, creatinine clearance and synthesis of hemoglobin can be expressed as mathematical functions of body weight. These relations are fairly consistent over a wide range of species. The corresponding unit is milligram per kilogram (mg/kg). Results obtained from an application of 10 mg/kg THC to rats would be translated to man in the ratio of one to one if such a toxicological comparison based on body weight was employed. Hence 2 mg THC in a rat of 0.2 kg would correspond to 700 mg THC in a man weighing 70 kg.
However, many biological processes such as metabolic rate, cardiac function, and renal function are not directly proportional to body weight. Thus toxicological comparisons on the basis of body weight are likely to be inaccurate.
2.2.2 Interspecies comparison based on body surface
The body surface is another potential basis for the comparison of toxic effects between species. For instance, the heat loss from a warm-blooded animal is approximately proportional to its body surface. In studies on anti-cancer drugs it was demonstrated that the maximum tolerated dose in different animal species (mouse, rat, dog, monkey) correlated quite well with that in man, if adjusted to body surface. The corresponding unit is milligram per square meter (mg/m2) or milligram per square centimeter, respectively (mg/ cm2).
2.2.3 Interspecies comparison based on pharmacokinetics
If the specific toxicity of a compound is unknown, the best correlations can be achieved on the basis of pharmacokinetic data, such as absorption (body intake), distribution, metabolism and excretion. Classical pharmacokinetic models are based on plasma concentration and the AUC (area under the curve) as plasma concentration over time, produced by known doses. Such data allow the determination of extent and duration of systemic exposure to the analyzed substance.
Pharmacokinetics in man and in animals show some parallels and a number of conspicuous differences (Agurell 1986, Harvey and Brown 1991).
Unfortunately the available data on plasma concentrations after oral administration of THC in man and rat are available for different doses: the human subjects received lower doses whereas in animals the applied concentrations were higher (see Table 6). An oral intake of 0.2-0.3 mg/kg produced a peak plasma concentration of 3-10 ng/ml in man (Frytak et al. 1984, Brenneisen et al. 1996). In animal studies with rats, plasma concentrations peaked at about 100 ng/ml after oral administration of 15 mg/kg THC (Hutchings et al. 1991) and at about 150 ng/ml after 20 mg/kg THC (Scallet 1991). The concentrations maintained a high level for 6 hours. In another study where rats had received THC at doses of 15, 25 or 50 mg every day for two years, the THC concentration had finally reached a level of about 400, 1300 or 3000 ng/ml at the end of the test period (Chan et al. 1996). This is the result of the accumulation of THC, as known in man as well, that causes an augmented plasma level after chronic application.
Extrapolation of pharmacological data to different doses is problematic even if corresponding mathematical models exist (Inges 1990). It is desirable to have more animal data that would involve doses corresponding to human consumption patterns.
2.2.4 Interspecies comparison based on precise toxicological data
Unfortunately the results achieved by extrapolation on the grounds of the models presented above can be quite astonishing. Often toxicity in different species does not correspond to the toxicity determined by means of pharmacokinetic data. One example is the lethal dose of THC.
In a study with rats, the median lethal dose (LD50)was established to range between 800 and 1,900 mg/kg oral THC, depending on sex and strain (Thompson et al. 1973). When this dose is extrapolated on the basis of body surface, the oral LD50 in dogs would be a quarter of this dose (200-500 mg/kg) per kg BW and half of this dose (400-950 mg/kg) per kg BW in monkeys. However, the experimental studies revealed contrary results. No deaths were observed when orally administering the maximum THC doses either to dogs (up to 3,000 mg/kg THC) or to monkeys (up to 9,000 mg/kg THC) (Thompson et al. 1973). Instead of being 50% more sensitive, primates turned out to be at least five to ten times more resistant to THC. It has not yet become quite clear how, at low doses, the relevant target parameters (effects on hormonal system, immune system and fetus) would compare between species. Probably mice are especially predisposed to fetal malformations (Abel 1985). Also regarding the sensitivity of the hormonal system to THC, clear species-related distinctions were found that diminish the extrapolation of those results to man (Mendelson and Mello 1984).
Other examples of chemical substances demonstrate the wide range of possible conversion factors. Alcohol, methyl mercury and polychlorinated biphenyls cause embryonic malformations in man. The toxic doses per kg body weight in animal studies ranged from 0.2 to 8.0 times the toxic dose in man (Hemminki and Vineis 1985).
Conclusion: Animal data can provide an indication of the toxicity of THC to humans. However, the extrapolation of animal data to man is problematic, not only because of the use of high dosing regimens but also because of the lack of reliable conversion factors for the target parameter under examination. Smaller animals possibly experience a stronger THC toxicity compared to larger animals and primates. Thus, reliable data on the toxicity of THC in man should be based on studies with humans.
2.3 Methodical basis for the determination of thresholds
For those chemical substances whose undesirable effects are either known or suspected, certain concepts for the protection of health against impairments, such as the LOAEL ("lowest observed adverse effect level") and the NOAEL ("no observed adverse effect level"), have been established. In order to provide a safe margin of protection against potential harm, safety factors of 10 are typically applied. For each particular case, the precise value of a safety factor may vary with the reliability of the determined threshold, the relevance of the observed effects in animals and cells for man, the severity of effects above the threshold, and the understanding of the causal relationship between drug intake and observed effects. With regard to THC limits in food, the data situation is fortunate, as the daily THC intake of chronic Cannabis consumers (50-200 mg or 1-3 mg/kg BW, respectively) already clearly exceeds the daily doses of relevance to this study (0.1-0.2 mg/kg) by a factor of 10-15. Acute effects in man were mostly examined at THC doses that cause psychotropic effects. Thus, a NOAEL in this dose range constitutes a proper margin of protection for the ingestion of THC with food. Dosage and concentration in animal and cell studies mostly exceed this level by another one to three orders of magnitude.
Appropriate limits can be based only upon sufficient or at least probable evidence of effects and not on hypothetical effects. Early toxicological research with THC in the seventies often discovered health-impairing effects that could not be confirmed in the following years. Such inconsistent experimental findings cannot be utilized for the determination of limits for detrimental effects. Early studies were often short of well-designed methods concerning procedure, choice of controls and the consideration of possible confounding factors. For instance, insufficient imaging techniques (pneumoencephalography) suggested a cerebral atrophy as consequence of chronic Cannabis use, which later was refuted. Also, when "pair-feed controls" were employed in the eighties in animal studies, it was realized that even after high THC doses fetal impairments were not induced by THC toxicity, but rather resulted from the mother's reduced intake of food and water (Abel 1984). (Pair-feed controls are those that receive the same amount of food and water as the examined animals, who took in less food and water because of the deprivation caused by the administration of THC.)
At this point, two particularities of the toxicological evaluation of THC shall be emphasized:
1. Development of tolerance and accumulation: For most toxic agents, the toxicity increases with the duration of exposure and the NAOEL decreases correspondingly (Voisin et al. 1990). Such an increase of toxicity can be expected, especially for a substance which possesses a comparatively long half life (THC: 20-30 hours) and which accumulates with chronic administration. However, opposite effects were observed with THC, since tolerance develops for most effects, and this overcompensates for any accumulation that may occur. With an augmenting administration of THC, the psychological as well as most-if not all-physical effects mediated by specific receptors decrease (see Chapter 3.5). For instance, in THC studies of female rhesus monkeys, hormonal changes and a disruption of the menstrual cycle occurred (Smith et al. 1983). However, after six months of a persistent high-dosing schedule, the hormonal values and the menstrual cycle had returned to normal. Development of tolerance has also been established for most of the other effects (mood changes, cardiovascular effects, etc.).
2. Damage to children: Children generally respond more severely to chemical toxins and are rated as "sensitive persons," requiring larger margins of protection (Winnecke and Lilienthal 1992). A child's brain is, for instance, more susceptible to impairments than the brain of an adult. Contrary to most noxious substances, however, THC in relevant concentrations does not operate in an unspecified manner but acts on specific receptors on body cells (especially brain cells and immune cells). The number of cannabinoid receptors in adults is several times the number in children (see Chapter 5.2). This is also beneficial to the therapeutic uses of THC. Given the aforementioned reasons, children who were given THC (in this case delta-8-THC) in the course of a chemotherapy tolerated considerably higher doses (18 mg/m2 body surface) than adults would presumably have tolerated (Abrahamov 1995). Elderly people are more responsive to THC concerning psychotropic effects, even though the aging process leads to a slight reduction of THC receptors (Romero et al. 1998). However, caloric intake also usually decreases with age, causing a reduction in THC intake with food.
3 Pharmacology and pharmacokinetics
For a proper understanding of Cannabis effects, and for the sake of comparability of different routes of administration, a basic understanding of the pharmacokinetics of THC is needed (reviews: Agurell et al. 1986, Harvey 1991). It will be provided in the following sections.
3.1 Resorption and plasma level
Cannabis drug products (marijuana, hashish) are preferably inhaled (cigarettes, pipes) and only seldom orally ingested (tea, pastries, tincture). Besides these routes, the intravenous (injection into the blood vessel), the subcutaneous (injection under the skin), and the intraperitoneal (into the abdominal region) routes are widely applied in animal studies. This present study focuses on the toxicity from oral administration with food, as well as the transferability to the ingestion route of biological effects observed with other routes of administration.
Several studies determined the systemic bioavailability of THC after the smoking of a marijuana cigarette as ranging between 2 and 56% of the total amount of THC present in the cigarette. Generally, bioavailability appears to range between 10-30%, with inexperienced smokers achieving lower rates. Hence Lindgren et al. (1981) established a systemic bioavailability in heavy smokers of 23% (+/-16%) compared to 10% (+/-7%) in occasional users. The smoking of a marijuana cigarette containing 10-20 mg THC produces a maximum plasma level of about 100 ng/ml after three minutes. Subsequently, the plasma concentration drops rapidly. The subjective effect sets in after only a few puffs, and the maximum high is reached after 15-30 minutes, when the plasma level has already begun to decline. The psychotropic effects typically cease after three hours.
When orally ingested, the systemic bioavailability of the lipophilic THC molecule is typically 5-10%. However, it may be doubled when simultaneously applying a lipophilic carrier (fat, oil), thereby improving THC resorption. Thus the bioavailability is typically somewhat less than that via inhalation. The rate of intake also depends on additional factors, such as the fullness of the stomach, and varies between individuals. After oral administration of 15-20 mg THC, plasma levels peak at approximately 5 ng/ml THC, typically after 1-3 hours, with a large inter- and intra-individual variability. The subjective psychotropic effect has its onset after 30-90 minutes and lasts for about 6 hours. In order to achieve acute effects, considerably higher oral doses are required than with inhalation because of the slower intestinal resorption of THC and the somewhat lower bioavailability.
Substances that are applied to the skin can be systemically absorbed to an unknown extent. However, there have not yet been any quantitative studies of the dermal absorption of THC that would allow quantification. Nevertheless, this question is vital for the use of THC-containing products that are externally applied (cosmetics, dermatics for the treatment of neurodermitis). The physico-chemical characteristics of THC, however, allow a rough estimation of the amount of THC assimilated (Kalbitz et al. 1996, 1997).
Generally, the human skin is well protected against penetration by external substances. Many topically applied substances attain a systemic bioavailability of only a few percent (Hadgraft 1996). The main barrier to penetration is the cornea (stratum corneum), or more accurately, the cornified layer of the stratum corneum. In principle, substances can penetrate the space between the cells of the stratum corneum (intercellular), the cells themselves (intracellular), or the sebaceous and perspiratory glands and hair follicles. Only the first pathways of penetration generally play a relevant role (Hadgraft 1996, Kalbitz et al. 1996, Berti et al. 1995). For example, the route through the hair follicles and glands is of importance for polar molecules only. As a lipophilic molecule, THC does not belong to that group.
The permeation coefficient or, respectively, the permeability constant (Kp) constitute a quantitative expression for the ability of a substance to permeate the skin. The flux or absorption rate of a chemical results from a multiplication of the concentration (C) of a chemical on the skin surface with the permeability constant: flux = KpC (Mattie et al. 1994). The basic principles of absorption through the skin correspond to those of diffusion through semi-permeable membranes (Berti et al. 1995). Factors that influence the penetration through the skin are the thickness and condition of the skin, as well as the size of the penetrating substance and the carrier.
Molecules that enter and diffuse through the skin have to penetrate a number of lipid bilayers in the intercellular space, thereby repeatedly alternating from lipophilic to hydrophilic areas. Those molecules that are sufficiently lipophilic, such as glucocorticoids, easily cross those lipophilic phases. Thus, most publications still maintain as a rule that "highly lipophilic compounds with low molecular weights demonstrate the greatest flow rate through the stratum corneum" (Berti et al. 1995).
Occasionally, a direct relation between the coefficient of permeation and the octanol/water distribution coefficient is postulated (Guy 1995). The latter is a measure of a chemical's lipophilic and hydrophilic properties, respectively. A higher coefficient indicates stronger lipophilic characteristics. However, such a correlation could not be verified in experimental studies. Mattie et al. (1994) examined 13 substances with octanol/water coefficients ranging from zero to 1,400. The constant of permeability correlated only weakly with the octanol/water distribution coefficient (r2 = 0.04).
Instead, there is evidence that only a small fraction of strongly lipophilic substances, such as THC, overcomes the hydrophilic phases of the intercellular space. Gabriele Bast (1997) carried out a large number of experiments that involved different substances of differing lipophilic characteristics in different carriers, and stated: "When substances are applied in a lipophilic carrier the permeation coefficient Kp is notably decreased when the distribution coefficient (n-octanol/perfusion buffer (pH 7.4)) Poct exceeds 2000."
Conclusion: It may validly be assumed that, with an octanol/water distribution coefficient of 6,000 (Agurell et al. 1986), i.e. a strong lipophilic tendency, only a small amount of THC permeates the skin-and is systemically absorbed only on a small scale-when administered in an oily base, such as in cosmetics containing hemp oil. Based on experimental evidence obtained for other chemicals with known physico-chemical properties, the transdermal systemic bioavailability of THC thus is likely considerably less than the oral systemic bioavailability. Corresponding experimental studies should be conducted that quantify the exact rate of skin permeation.
3.2 Transport and metabolism
After absorption and infusion into the blood, more than 97% of THC and its metabolites are bound to plasma proteins. The octanol/water distribution coefficient is 6,000, the apparent volume of distribution in the body comes to 10 l/kg, typical of a lipophilic drug (Agurell 1986). THC crosses the blood-brain barrier comparatively easily and accumulates in fatty tissues from where it is re-released only slowly into other tissues, such as the blood. It is exponentially eliminated from the plasma, consistent with a multi-compartment model (2-4 compartments) with a terminal (ß-) plasma half-life of about 20-30 hours.
Ninety-five percent of THC is metabolized in the liver. Through microsomal hydroxylation, THC is converted to the equally pharmacologically effective 11-hydroxy-delta-9-THC, which in turn is converted into 11-nor-9-carboxy-delta-9-THC (THC-COOH) by the operation of alcohol dehydrogenase enzymes. Especially after oral administration, the 11-hydroxy-metabolite contributes considerably to the pharmacological effects, equaling those of THC. Besides the main metabolites, more than 20 other decomposition products are formed. The end products are 11-nor-acids and similar, more polar acids. About one third of those metabolites is excreted through the kidneys and about two thirds of them through the feces. The non-metabolized THC (5%) is defecated as well. The excretion through the urine is limited to acid metabolites only. THC-COOH has an elimination half life of 4-5 days. The complete elimination of a single THC dose may take up to 2-5 weeks. In chronic, heavy marijuana users, THC metabolites were still detected in the urine after 2-3 months after cessation of consumption.
Excursus III: Detection of THC after ingestion of hemp-containing food
The intake of a single oral dose of 16 or 33 mg THC by different experimentees caused a urine concentration of the THC metabolite THC-COOH of 170-240 ng/ml in an immunoassay. According to a GC/MS analysis (gas chromatography/mass spectrometry), these concentrations reached about 400 ng/ml (Lehman et al. 1997). With a chronic intake of such THC quantities, the metabolites accumulate. Consequently, a urine assay of heavy chronic Cannabis users might find THC-COOH concentrations of 500 to 1,000 ng/ml (Solowij et al. 1995). Because of the long elimination half-life of the THC metabolites and an accumulation in body tissues, they might remain detectable in the urine for weeks or even months after the last use (Bell et al. 1989, Ellis et al. 1985). In 86 chronic Cannabis users, THC metabolites were detected in an immunoassay up to 77 days after the last intake.
The ingestion of THC with food may also result in the detection of THC metabolites in the urine. A single oral intake of 40 ml of a hemp oil with a comparatively high THC content of 151 mg/ml, corresponding to about 6 mg THC, produced a maximum urinary THC-COOH concentration of about 100 ng/ml in an immunoassay (Alt and Reinhardt 1996). The intake of 135 ml of hemp oil containing an unknown THC concentration over a period of 4.5 days led to a THC-COOH concentration of 55 ng/ml (Struempler et al. 1997). It took two days after the last ingestion before the immunoassay tested negative. In another study, different patients tested positive for THC-COOH in the immunoassay after the intake of 15 ml hemp oil (Constantino et al. 1997). The daily intake of 10 ml hemp oil of an unknown concentration for 25 days caused a THC-COOH-concentration of 36 ng/ml, measured by GC/MS (Callaway et al. 1997).
Energy bars containing hemp seeds show only low THC concentrations. For example, a THC concentration of 4.4 mg/ml was found in the "Hempy-bar" energy bar of Green Machine Ltd. (Alt and Reinhardt 1997). After ingestion of one or two hemp seed bars, the urinary immunoassay tested positive (cutoff of 20 ng/ml). However, if measured quantitatively by CG/MS, only low concentrations, typically 1-2 ng/ml were detected (Fortner et al. 1997).
Conclusion: After the ingestion of hemp-based food, THC metabolites may be detectable in the urine. The observed THC-COOH concentration corresponds to the to the amount of THC ingested with food. Relevant THC-COOH concentrations are detected in the urine especially after intake of hemp oil, whereas other hemp products produce only low levels of metabolites as a result of their low THC content. Because of the accumulation of THC metabolites in body tissues, a long-term intake of small THC quantities may cause significant urine concentrations. In general, those results cannot be distinguished from those obtained after low-level drug consumption. However, the high concentrations that are found after chronic heavy marijuana use cannot be detected following ingestion of hemp seed foods.
3.3 Influence of physical factors on THC content
Ninety-five percent of the THC present in the Cannabis plant is found in a pharmacologically inactive form, i.e. one of two delta-9-tetrahydrocannabinolic acids (THCA) (Turner 1980), while the majority of biological effects are caused by the corresponding neutral phenolic forms of THC (Dewey 1986). Thus the question arises to what extent the total amount of THC species detected in food is pharmacologically active.
A conversion of THC acids into the pharmacologically active phenols-chemically a decarboxylation process (separation and release of carbon dioxide)-is accomplished most effectively by heating. A heating for five minutes to 200 to 210°C was found to be optimal for decarboxylation (Brenneisen 1984). Under these conditions, THCA was completely converted into neutral THC, while avoiding the subsequent oxidation to cannabinol (CBN). When marijuana is smoked and temperatures of 600° C are reached, obviously only a few seconds are sufficient for decarboxylation.
A much more gradual decarboxylation occurs at room temperature. Hence, after marijuana has been stored for a year, about 50% of its THCA had been converted into the active, neutral THC (Brenneisen 1984). However, storage also leads to a decrease in total THC content as THC oxidizes to neutral CBN, a non-psychotropic cannabinoid (Fairbairn 1976). The total THC content of marijuana dropped to 87% after 47 weeks of storage in the dark (20° C) and to 64% with exposure to light (Fairbairn 1976). Since THC in food is not protected by the plant's glands, as it is in marijuana, THC present in food is converted much faster into CBN. This is suggested by experiments with powdery Cannabis resin and alcoholic extracts (Fairbairn 1976).
Baker et al. (1981) analyzed 64 marijuana samples (Cannabis herb) and 26 hashish samples (Cannabis resin) for their relative amounts of THCA and THC, and found a wide range of ratios, especially in marijuana. In Cannabis resin, the ratio ranged between 0.5 to 1 and 6.1 to 1. Lower rates, corresponding to a low THCA fraction, were found in Cannabis samples from the Indian subcontinent, whereas samples originating from Mediterranean countries displayed higher rates. It may be assumed that much of the THC in cold-pressed hemp oil and other hemp based food is present in the form of the biologically inactive tetrahydrocannabinolic acid, as long as heating during the production process was insufficient for effective decarboxylation. Thus, for a realistic toxicological assessment of food, the concentration of phenolic THC must be determined. Only few studies have examined the percentage of pharmacologically inactive THC-acids in hemp based food. In a study of 10 commercially available hemp oils in Switzerland, THC-acids generally constituted less than 10% of the total THC-content (Lehmann et al. 1997). Thus, in this case the by far larger fraction was biologically active.
3.4 Mode of action
Most specific THC effects are mediated through cannabinoid receptors (reviews: Howlett 1995, Pertwee 1995, Matsuda 1997). At very high doses, non-specific effects on membrane fluidity and other non-specific effects may also become relevant (Martin 1986).
So far two types of THC receptors, CB1 and CB2, each with additional subtypes, have been identified and cloned. The CB1 receptor is found predominantly in brain cells, with a particularly high receptor density in motor, limbic, associative, cognitive, sensory and autonomic brain structures (basal ganglia, cerebellum, limbic system, hypothalamus, cerebral cortex). In addition, it was also found in the testes and other peripheral tissues.
The CB2 receptor has so far only been found outside the brain, particularly in cells of the immune system, such as in the spleen, tonsils, thymus, mast cells, and blood cells. Presumably it is involved modulating the operation of immune cells. Often CB1 and CB2 receptors are expressed from the same immune cells.
The endogenous ligands first discovered for the cannabinoid receptors were arachidonic acid derivatives (arachidonylethanolamides), which differ greatly from plant cannabinoids in their molecular structure (Devane 1992). They are called anandamides (reviews: Di Marzo and Fontana 1995, Mechoulam et al. 1996). Only recently another ligand, 2-arachidonylglycerol (2-AG) was identified (Stella et al. 1997).
3.5 Development of tolerance
Tolerance develops to most THC effects (Romero et al. 1997). This applies also to undesirable effects, such as effects on the psyche, the cardiovascular system and the hormonal system. An acute use of THC, for example, leads to a marked increase of the heart rate, whereas with chronic administration this effect no longer occurs, or at least not to the same extent. This tolerance phenomenon can be explained by two factors, i.e. enhancement of THC metabolism and down-regulation of brain cannabinoid receptors. The number of receptors decreases and their response to THC declines, such that only higher doses can produce the known THC effects. Thus, rats that had been administered THC over a period of five days exhibited a decreased specific binding in different receptor sites of the brain, ranging from 20 to 60% of that measured in controls. (Romero et al. 1997).
3.6 THC effects and total toxicity
THC has acute effects on almost every body system (reviews: Hall et al. 1994, Hollister 1986, Dewey 1986, Maykut 1985). The most conspicuous effects are those on the central nervous and cardiovascular systems. With regard to physical effects, THC produces an increased heart rate, reddened eyes and a dry mouth. As for psychotropic effects, a mild euphoria, an enhanced sensory perception, fatigue and eventually dysphoria together with anxiety have been observed.
As a function of dose, the following effects were observed in clinical studies in vivo (in living organisms) or in vitro (i.e. in laboratory dishes), respectively:
Psyche and perception: fatigue, euphoria, enhanced well-being, dysphoria, anxiety, disturbed orientation, increased sensory perception, heightened sexual experience, hallucinations, psychotic states.
Cognition and psychomotor performance: fragmented thinking, enhanced creativity, disturbed memory, unsteady walk, slurred speech.
Nervous system: attenuation of pain, muscle relaxation, appetite enhancement, decrease in body temperature, vomiting, anti-emetic effects, neuroprotective effects in brain schema.
Cardiovascular system: increased heart rate, enhanced heart activity and increase in oxygen demand, vasodilation, drop in blood pressure, collapse.
Eye: reddened conjunctivae, reduced tear flow, reduced intraocular pressure.
Respiratory system: bronchodilation, dry mouth.
Gastrointestinal tract: reduced bowel movements.
Hormonal system: effects on LH, FSH, testosterone, prolactin, somatotropin, TSH, reduced sperm count and sperm mobility and quality, suppressed ovulation and suppressed menstruation.
Immune system: impairment of cell-mediated and humoral immunity, anti-inflammatory and immune-stimulating effects.
Fetal development: fetal malformations, fetal growth retardation, impairment to fetal and postnatal cerebral development, improved postnatal development.
Despite these observed effects, the overall physical human toxicity of Cannabis is low. Human fatalities following acute Cannabis intoxication have not been reported. Chronic heavy marijuana use is also not related to mortality (Sidney et al. 1997). Long-term heavy marijuana users did not show any abnormal health features that distinguished them from the population as a whole (Gruber et al. 1997).
Strong side effects are rare, even with high THC doses. The median lethal dose (LD50) for rats is in the range of 800 to 1900 mg/kg (Thompson et al. 1973). No toxic deaths were observed in experiments of rhesus monkeys with an acute oral application of 9000 mg/kg (Thompson et al. 1973). For illustration purposes: 9000 mg/kg THC in a man weighing 70 kg corresponds to the consumption of 630 grams THC or 3 kg of high-percentage Cannabis resin (hashish), or 15 kg of marijuana of a medium quality.
Even a long-term high-dosing regimen of THC is tolerated relatively well. This was suggested by the example of rats that ingested 50 mg/kg THC every day for two years (Chan et al. 1996). At the end of the two-year period a mean of 45% of the controls and 70% of the dosed animals had survived. The higher survival in the THC group was primarily due to a decreased incidence of cancer.
4 THC thresholds for psychotropic effects
Some experimental and clinical studies report experiences with threshold values for psychotropic THC doses. Acute effects below the psychotropic threshold cannot be distinguished from placebo effects.
Lucas and Laszlo (1980) found pronounced psychotropic reactions (anxiety, marked visual distortions) in patients undergoing cancer chemotherapy who had received 15 mg THC/m2 (square meter of body surface), which corresponds to 25 mg THC in an average adult person (body surface: 1,7 m2). A reduction to 5 mg THC/m2, about 7.5-10 mg THC, produced only mild reactions.
No mood changes were observed in six cancer patients after administration of a single oral dose of 15 mg THC for antiemetic treatment (Frytak et al. 1984). Brenneisen et al. (1996) administered single oral doses of 10 or 15 mg THC to two patients. Physiologic parameters (heart rate) and psychological parameters (concentration, mood) were not modified by the administration. In a study by Chesher et al. (1990) of a healthy population dosed orally with 5 mg THC, no difference in the subjective level of intoxication was found compared to placebo controls. Doses of 10 and 15 mg THC caused slight differences compared to the placebo, and a dose of 20 mg, finally, caused marked differences in subjective perception.
In light of these findings, one may validly assume the psychotropic threshold to be in the range of 0.2-0.3 mg THC per kg body weight for a single oral dose taken in a lipophilic carrier, corresponding to an administration of 10-20 mg THC to an adult person. A single dose of 5 mg THC can be regarded as a placebo dose. In various clinical studies, psychotropic reactions were also observed following single doses of 5 mg THC. However, these cannot be distinguished from effects that occur after administration of placebos. As the duration of action of THC in therapeutic dosage ranges between 4 and 12 hours, a daily intake of 2 x 5 mg which equals 10 mg THC, administered orally in a lipophilic carrier, will not have any effects that could be distinguished from placebo effects.
5 Discussion of physical effects
Below the psychotropic threshold, an intake of THC cannot be distinguished from placebo substances with regard to physical parameters also. No perceptible acute effects as, for example, on the cardiovascular system, are observed. The question arises, however, whether undesired biological effects can still occur below the placebo threshold, especially with chronic intake.
Considering the effects observed in animal studies, four sectors must be examined in this context:
Effects on the genetic material (mutagenicity and carcinogenicity),
Effects on the immune system,
Effects on the hormonal system,
Effects on pregnancy.
Other aspects, such as the possible neurotoxicity of THC, will not be dealt with, since such effects were only found in animal experiments with a chronic administration of high doses that clearly exceeded the doses that are of relevance in the examined context (WHO 1997).
5.1 Genetic material and cell metabolism
5.1.1 Cell studies
Cannabis smoke can exert mutagenic activity as a result of carcinogens (benzpyrenes, nitrosamines). This was established in the Ames test. THC itself is not mutagenic (WHO 1997). THC may reduce the synthesis of DNA, RNA and proteins and modulate the normal cell cycle. To obtain those effects, however, very high doses were required in cell studies. Hence, in a study by Tahir et al. (1992) microtubules and microfilaments in PC12 cells, which are vital for cell division, were disrupted in a dose-dependent manner following treatment with 10-30 mM (micromol) THC.
5.1.2 Studies with Cannabis users
Studies with Cannabis users did not establish any increase in chromosomal breaks (Matsuyama et al. 1976, Matsuyama and Fu 1981, Cruickshank 1976, Cohen 1976). Thus, after 72 days of marijuana smoking, no increase in chromosomal breaks was found when compared to the breakage rate preceding administration.
Joergensen et al. (1991) evaluated the genotoxicity of Cannabis smoking by application of the sister-chromatid exchange (SCE) test, a sensitive tool for the discovery of genotoxic agents. They compared 22 tobacco smokers and 22 persons that smoked tobacco and marijuana. The smoking of tobacco in itself enhanced the SCE level significantly by 18.5% compared to non-smoking controls. The addition of marijuana did not further affect this level. Based on this observation the authors concluded that Cannabis smoke could not be considered genotoxic.
5.1.3 Conclusion
THC in doses used by marijuana smokers is neither mutagenic nor carcinogenic and it does not affect cell metabolism, either. The NOAEL ranges above concentrations relevant for the human consumption situation.
5.2 Pregnancy
In animals and man delta-9-THC crosses the placenta to the vascular system of the fetus. The course the THC concentration takes in fetal blood fairly coincides with that in the maternal blood, though fetal plasma concentrations were found to be lower compared to the maternal level in rats (Hutchings et al. 1987), in sheep (Abrams et al. 1985-1986), in dogs (Martin et al. 1977), and in monkeys (Bailey et al. 1987). In a study on dogs, the brain of the fetus showed a concentration that came to only one third of the mother's concentration half an hour after intravenous administration. This relationship was maintained with multiple administrations, indicating that the maternal plasma THC and not the fetal tissue is the actual source for the fetal plasma THC. Small quantities of THC also pass into the milk of the mothers. In a study on monkeys, 0.2% of the THC ingested by the mother appeared in the milk (Chao et al. 1976). Chronic administration leads to a THC accumulation in milk (Perez-Reyes and Wall 1982).
THC concentrations corresponding to normal marijuana use act on compound-specific binding sites (receptors). In early gestation, these have not yet been developed in the fetal brain so that, during this phase, cannabinoids lack a specific binding target (Hernandez et al. 1997). First cannabinoid receptors are detected at fetal age. However, their number progressively increases in postnatal life. In a study investigating the number of cannabinoid receptors during rat brain development, the receptors were found to multiply by five between the day of birth and the sixtieth day (grown-up rat) (Belue et al. 1995).
Non-receptor mechanisms such as the disruption of cell membranes require extremely high doses of THC. Thus, one can assume that no relevant THC effects will appear during early pregnancy though, they might occur in later gestation.
Four of the THC effects on pregnancy will be further analyzed:
1) Increase in birth complications
2) Increase in birth defects (i.e. heart defects, cleft palate)
3) Adverse pregnancy outcome (i.e. more premature births, low birth weight)
4) Adverse postnatal development or, respectively, impaired fetal brain development.
First epidemiological studies examining the prenatal effects of marijuana on man were published in the early eighties. Human studies are often subject of a number of methodical weaknesses that are not easily corrected and that may lead to inconsistent findings. Among these are:
The examined samples being too small. Especially if marijuana users and abstinent controls might differ only slightly, or if the relevant disorders are of a rare kind, such differences become evident only by examination of a large number of pregnant women and their children.
Uncertainties concerning the exact daily marijuana intake. As marijuana is an illicit drug, study participants may not be truthful in their answers to inquiries about their use. Therefore Shiono et al. (1995), in addition to the interviews, also took blood samples to be screened for cannabinoids. This often revealed a discrepancy between the results of the inquiry and those of the blood tests.
A lack of controlling and adjustment for confounding factors that may influence pregnancy. Thus, Cannabis consumption is often associated with a range of factors that, by themselves, may have effects on pregnancy, such as the consumption of other legal and illegal drugs. Education and socioeconomic status are of further relevance as they may influence the quality of nutrition during pregnancy and the prenatal care.
5.2.1 Birth complications
For many centuries, Cannabis has been used to alleviate the discomforts of childbirth. In the Western medicine of the 19th century, Cannabis preparations were employed to this end, also as they supported birth contractions. This effect, however, is not reliable according to historical reports (Mechoulam 1986).
A study by Greenland et al. (1982) established an elevated risk of abnormal progress of labor in 35% marijuana users when compared to 36 controls (Greenland et al. 1982). A frequent meconium staining (57% versus 25%) and an average longer duration of labor were found. In a second study of the same research group with a slightly enlarged population, these effects were much less significant (Greenland et al. 1983). The rate of a dysfunctional labor (43% versus 35%) and meconium staining (17% vs 13%) was only slightly elevated in marijuana users. Other researchers did not find any abnormalities in pregnancy. Thus, in a study of 291 women, Fried did not detect any significant differences in marijuana users under labor. Also Dreher et al. could not detect any significant features in the progress of labor when comparing thirty prenatally exposed mother/child pairs to thirty non-consuming pairs (Dreher et al. 1994). The collectives of Greenland et al. might have been selected and the higher rate of complications was possibly attributable to other circumstances.
Conclusion: There are isolated observations that suggest marijuana use during pregnancy might have adverse effects on the progress of labor. These were obtained by one research group in the early eighties and to date have not been confirmed. On the other hand, there are historical clinical reports of a beneficial effect that could be used therapeutically. These, however, were not fully reliable. There is no reason to believe that sub-psychotropic THC doses would have a negative effect on the progress of labor.
5.2.2 Birth defects
Animal studies: In some early animal studies, congenital malformations were found subsequent to the administration of high doses of THC (tabular review: Abel 1980). No pair-fed controls had been employed. A daily oral administration of up to 150 mg of THC in sesame oil, however, failed to have any effects on prenatal mortality, fetal weight, and the rate of internal and skeletal malformations in mice (Fleischmann et al. 1975). Subcutaneous injection of up to 100 mg/kg THC proved not to be fetotoxic (Keplinger 1973).
The marijuana-induced fetotoxicity in animals is enhanced by alcohol (Abel 1986). However, extremely high doses of marijuana, equaling 50-100 mg/kg THC, and relatively small quantities of alcohol (1 g/kg) were required to encounter this effect. Small doses of marijuana did not enhance fetotoxicity.
Hollister (1986) points out that "virtually every drug that has been studied for dysmorphogenic effects has been found to have them if the doses are high enough, if enough species were tested, or if treatment is prolonged" (p. 4).
Abel emphasizes the fact that findings of malformations were consistent only following exposure to relatively high doses and following the intraperitoneal route (direct delivery to the abdominal region) (Abel 1985). Not only due to the direct effects of THC, but also to the reduced maternal food and water consumption associated with high dosage, THC administration may account for many effects. According to Abel, the only reliably documented postnatal effect on offspring is a decrease in birth weight.
Human studies: In most epidemiological studies, evidence could not support any increase in congenital malformations following marijuana use during gestation. The only exception is a single early study (Hingson et al. 1982). Hingson and colleagues examined 1,690 mother/child pairs for effects of alcohol and marijuana use on embryonic development and fetal growth. Marijuana use was associated with the increase of a fetal syndrome known as alcoholembryopathy or fetal alcohol syndrome.
In all further epidemiological studies with many thousands of children, no such relation between marijuana use and fetal malformations was established (Astley 1992, Gibson et al. 1983, Knight et al. 1994, Linn et al. 1983, Witter and Niebyl 1990). Neither did a study investigating for various minor physical anomalies (MPAs) detect any significant Cannabis-related differences (O´Connel and Fried 1984).
Conclusion: Evidence today supports the fact that marijuana use during pregnancy does not produce any fetal malformations.
5.2.3 Pregnancy outcome
Most studies for the evaluation of marijuana-induced effects on pregnancy outcome examined the influence on duration of gestation and on birth weight or infant size, respectively. The results are inconsistent. Whereas some studies found a shorter duration of gestation or a decrease in birth weight, others did not discover any interference.
Duration of gestation: In a study of 7,301 births, the rate of premature births was found to be greatly enhanced by 25% in 36 mothers that self-reportedly smoked marijuana once a week (Gibson et al. 1983). Fried et al. (1984), after accounting for other potentially confounding factors, stated a dose-related decline in the length of gestation in 84 marijuana users when compared to abstinent women. In an extensive study by Linn et al. (1983) of 12,424 individuals, 10% of whom reported use of Cannabis, the only significant difference stated was a higher rate of precipitated labor in Cannabis users. Most studies, however, could not find any marijuana-induced modulation of the duration of gestation.
Birth weight and infant size: In a study by Abel (1984) pregnant rats were intubated with augmenting THC doses of 5 to 50 mg/kg per day until gestation day five and, subsequent to this, either 50 or 150 mg/kg from day six to parturition. In the high-dose group, no viable pups were born (the fetomortality was 100%). In the 50-mg-offspring, a decreased weight gain and a decreased birth weight was found. Hutchings et al. (1987) observed a decreased birth weight following a daily oral administration of 15 and 50 mg/kg THC. The decrement in birth weight and postnatal weight gain were dose-related. The THC-15 group reached the weight of the controls within a period of 11 days, whereas for the THC-50 group it took 32 days. Overall, the decrease in birth weight was not due to the THC-administration, but rather attributable to the reduced food and water intake of the exposed dams, as no significant difference was found compared to pair-fed controls.
In a study with 1,226 women, Zuckermann et al. (1989) found the neonatal weight significantly decreased by a mean of 79 grams and a decrease in size by a mean of half a centimeter in the newborns of marijuana users. Also, the study of Hingson et al. (1982) associated marijuana use during gestation with a lower birth weight. Another study found the elevated risk of a low birth weight only among white regular marijuana users whereas nonwhites (of Hispanic or African descent) were generally not at an increased risk (Hatch and Bracken 1986).
In most studies no relation between marijuana use and fetal growth was found (Day et al. 1991, Fried et al. 1984, Fried and O´Connell 1987, Gibson et al. 1983, Knight et al. 1994, Linn et al. 1983, Shiono et al. 1985, Tennes et al. 1985). Moreover, the question arises whether an average reduced birth weight of 79 grams, as observed by Zuckermann et al., is of any practical significance. Even though it was a perceptible statistical variation, it probably failed to be of any biological relevance (Pisacane 1989).
How does growth develop after birth? According to a study by Fried and O´Connell, (1987) the children of Cannabis users were on average heavier and taller than non-exposed children. In contrast to these results, another research group found that maternal use of marijuana was significantly and negatively related to a decreased infant size at eight months but not to weight and head circumference (Barr et al. 1984). Finally, a further study did not find any growth retardations at the age of one year (Tennes et al. 1985).
Conclusion: Animal studies found a dose-related decrement in birth weight induced by marijuana. These findings were obtained, however, at doses clearly beyond the range of a human consumption situation. Various epidemiological studies stated inconsistent effects of Cannabis use on length of gestation, birth weight and infant size. The majority of those studies, however, could not provide any evidence that the outcome of pregnancy was affected. Furthermore, there is no reference to an influence on postnatal growth development.
5.2.4 Brain development
The cannabinoid-anadamide receptor system might play an important part in cerebral development. The daily administration of 5 mg/kg THC to pregnant rats generated a doubling of activity of the enzyme tyrosine hydroxilase (TH) in specific brain cells of their fetuses (Hernandez et al. 1997). This enzyme is assumed to be a key factor in the development of TH-containing neurons and other neurons. Furthermore, animal studies established a disturbance of mesolimbic dopaminergic neurons among perinatally THC-exposed males, which persisted in adult animals (Garcia-Gil et al. 1997). Further mechanisms with effects on brain development remain under discussion (Navarro et al. 1995).
Animal studies: It is assumed that THC might have stronger toxic effects during the period of brain development than it does in adults. However, in animal studies behavioral alterations were only found in the offspring of those dams that had been exposed to extremely high THC doses during gestation. In a study of pregnant rats that had received 50 mg THC/kg per day, this gestational exposure did not affect the behavioral tests of their offspring (Abel 1984). By contrast, Hutchings et al. (1987) observed significantly longer latencies to attach to a nipple and impaired nipple attachment (repeatedly missing the nipple) in the offspring of rat dams that had been exposed to a daily oral administration of 50 mg/kg THC. No such impairments were observed among 15mg/kg-offspring when compared to controls. However, the authors presume that the alterations among the high-dose offspring were not a primary effect of THC toxicity but rather were secondary to the significant THC-induced reduction of food and water intake among the dams. This was borne out by the fact that no significant differences were found between THC-exposed animals and non-exposed controls when those were provided with an equally reduced food and water supply. Besides this, the activity level in the offspring was not impaired by high THC doses. Kwash et al. (1980) observed a decrease in learning abilities among the offspring following an injection of Cannabis resin in pregnant rats and they attributed this to the impeded postnatal weight gain. Navarro et al. (1995) observed that behavioral deficits and impairments of learning in the offspring were associated to comparatively low THC concentrations (1 and 5 mg/kg THC), whereas no such association existed with high concentrations (20 mg/kg). Other authors did not find any impaired learning (or memory) in animals (Charlebois and Fried 1980, Uyeno 1973, Abel 1984).
The concentrations of DNA, RNA and proteins in the brains of offspring whose mothers had been administered daily doses of 15 mg/kg THC did not differ from controls on day 7, 14 and 21 postnatal (Hutchings et al. 1991) A group, however, with a daily gestational exposure of 50 mg/kg THC showed a lower protein concentration on day 7 and 14 postnatal. Since the protein content correlates with the growth of neurons and the formation of neuronal links (synapses), a reduced protein synthesis may be considered as indices for the inhibition of neural processes. On day 21 postnatal the decrease was equalized.
Human studies: A study by Fried et al. (1987 b) found increased tremors and startles in those children whose mothers had regularly used marijuana during gestation in comparison to non-exposed controls on day 9 and 30 postnatal. In a sleep study, marijuana use was found to be associated with alterations in the sleep cycle of neonatals (Scher et al. 1988). Children aged three years from a different marijuana-using population showed a disturbance in their nocturnal sleep, waking up more often during the night (Dahl et al. 1995). Marijuana-exposed children aged 9 months achieved slightly lower mental test scores than non-exposed controls (Richardson et al. 1995). However, a difference was no longer found at the age of 19 months. In another study, children aged one year did not show any significant differences in their sleeping or eating habits, their mental functions, or psychomotor abilities (Tennes at al. 1985). The gestational exposure to marijuana was concluded not to produce a higher mortality rate by increasing the rate of SIDS (sudden infant death syndrome) (Ostrea et al. 1997). Dreher et al. on day three postnatal could not detect any differences between neonatals of marijuana users and those of non-consuming mothers in neuro-behavior assessments (Dreher et al. 1994, Dreher 1997). After one month, the differences became apparent in favor of the marijuana population: In the prenatally exposed children this was manifested in a greater liveliness, less irritability, less tremors; these children were more easily quieted and scored higher in their reaction to different stimuli (sound, light and touch).
Fried and colleagues pursued a longitudinal study of children until they reached school age (Fried 1995). However, between 6 months and 3 years of age no behavioral consequences of marijuana exposure were noted. At the age of 4 to 6 years, global intelligence still proved to be at a normal standard, though slight significant variations in their verbal abilities and their memory were stated. At the age of 9 to 12 their ability to speak and spell did not differentiate the exposed from the non-exposed children (Fried et al. 1997). At the end of the study the prenatally exposed children were not found to differ significantly in their neurobehavior from other children.
Conclusion: Animal studies even with high THC doses failed to establish any consistent supportive evidence for an impairment of brain development. The early neurologic symptoms found in neonatals by some researchers can be interpreted as withdrawal symptoms. Possible subtle inhibitions of cognitive functioning could appear in the sequel. However, these are inconsistent findings, not confirmed by other authors. Still, there is no reason to believe that sub-psychotropic THC doses could possibly affect the development of fetuses or neonates.
5.2.5 Summary
In their review on influences of Cannabis on pregnancy Levy and Koren (1990) pointed out the tendency of preferential publishing of those studies that find noxious effects, and leaving unpublished those that evaluate THC as a safe drug. However, even with this taken into account, there are only weak references to pregnancy being adversely affected by marijuana use. Animal studies found merely inconsistent evidence of health-impairing effects when administering doses of 10-20 mg/kg THC or more, that is 100 times the dose relevant to this study. There are references to a light impairment of brain development among children of chronic Cannabis users that, however, could not be validated by other authors. Also in consideration of the above mentioned animal findings, the NOAEL for different pregnancy-related parameters lies safely beyond or within the range of the human consumption situation of chronic marijuana users.
5.3 Hormonal system and reproduction
Marijuana acts on the hypothalamo-hypophyseal axis. This is a functional unit in the brain which plays an important part in the interaction of different hormones. The hypophysis (pituitary gland) secretes the sex hormones LH (luteinizing hormone), FSA (follicle stimulating hormone), and prolactin; the thyroid hormone TSH (thyrotropin); ACTH (adrenocorticotropin); and somatotropin (STH). These hormones respond to releasing hormones (RH) of the hypothalamus. LH regulates the testosterone production in the testes. Testosterone and FSH are vital for the sperm production (sperm count, sperm motility and sperm function).
5.3.1 Sex hormones
5.3.1.1 Men
In animal studies it was shown that THC may impair the function of male sex hormones and induce a decrease in the weights of sex organs (Dewey 1986, Mendelson and Mello 1984). However, contrary to this, various animal studies described a gain or a constancy in the weights of male sex organs (Abel 1981). THC decreased the anterior and mediobasal hypothalamic LH-RH concentration in rats when administered at 2 mg, 15 mg and 30 mg/kg body weight in a dose-related manner (Kumar and Chen 1983). Furthermore, simultaneous decreases in serum testosterone were observed. In another study it was found that THC lowered testosterone and LH levels in rats (Harclerode 1984). In the sequel, a tolerance to this effect developed, and with chronic THC exposure the hormone-concentration returned completely to normal values. In another study of rats that were chronically administered 1 mg, 5 mg or 25 mg/kg THC, the testosterone level was unaltered 24 hours after the last administration in the low-dose group, whereas it was found to have doubled in the 5 mg group (Morrill et al. 1983). In a study of mice, THC in relation to dose caused a statistically higher incidence of abnormal sperms (Zimmermann et al. 1979). Without THC the number of abnormal sperms amounted to 1.5%. After a treatment for five consecutive days with 5 mg/kg THC, this percentage had risen to 3.8%, and with 10 mg/kg THC it rose to 5.3%.
Testosterone: First suspicions that Cannabis might affect sexual hormones arose from case reports of gynecomastia in male young heavy Cannabis users (Harmon and Aliapoulis 1972). This suspicion was substantiated by Kolodny et al. (1974) who observed reduced serum testosterone levels coupled with a decrease in sperm count and sperm motility in chronic marijuana users. This frequently quoted study, however, holds a number of methodological faults that have been repeatedly criticized (Abel 1981). In fact, the results of this study could not be confirmed by a larger well-controlled study with chronic marijuana users (Mendelson et al. 1974). No difference in serum testosterone level was found either at the beginning of the study or after three weeks of heavy marijuana consumption.
Hollister (1986) assumed that a change, if any, in testosterone level and sperm production would only occur after long-lasting exposure. In two separate studies, one research group found a low sperm count with normal motility and morphology in chronic marijuana users who, under observation, had smoked 8-10 marijuana cigarettes over a period of four weeks (Hembree et al. 1978, 1976). In a study of 66 chronic Cannabis users, a comparison with 44 controls did not suggest any Cannabis-induced long-term effects on the plasma testosterone level (Friedrich et al. 1990). Correspondingly, other authors also found normal testosterone levels in chronic marijuana users (Schaefer et al. 1975, Coggins et al. 1976, Cushman 1975, Block et al. 1991).
Dax et al. (1989) investigated the effects of three 10 mg oral THC doses per day or three 18 mg doses in a marijuana cigarette for three days on male chronic marijuana users after at least two weeks of abstinence. They did not find any alterations in the plasma testosterone concentration. Cone et al. (1986) did not find any decrease in testosterone after the smoking of two marijuana cigarettes (2.8% THC). Mendelson et al. (1978) could not detect any influence on the testosterone level in 27 marijuana users who had consumed a mean of 54 marijuana cigarettes (moderate users) or 120 marijuana cigarettes (heavy users) over a period of 21 days.
FSH: Acute THC exposure (two marijuana cigarettes of 2.8% THC) does not result in an alteration of the FSH level (Cone et al. 1986). Also chronic administrations did not have any significant influence (Cushman 1975, Hembree et al. 1976, Block et al. 1991, Vescovi et al. 1992).
LH: In a study by Cone et al. (1986), a decrease in the LH level after acute THC exposure (approx. 50 mg inhalative) was noted. In a study of 10 chronic marijuana users, their basal and GnRH (gonadotropin-releasing hormone) levers were stimulated and levels of LH were found reduced (Vescovi et al. 1992). However, in other studies using a different experimental design, the LH concentration was not affected by THC exposure or Cannabis consumption (Cushman 1975, Hembree et al. 1976, Kolodny et al. 1974, Mendelson et al. 1978, Block 1991).
Prolactin: After three days of abstinence a slight elevation of prolactin concentration was stated in six chronic Cannabis users (Markianos and Stefanis 1982). Dax et al. (1989) investigated the effect of three 10 mg/kg oral doses per day or 18 mg/marijuana cigarette three times per day for days on male chronic marijuana users after two weeks of abstinence. Though no difference was found in plasma concentrations of LH and testosterone, they found the plasma prolactin level to be altered. The authors attributed this last finding to the heavy marijuana use. Mendelson et al. (1984), however, did not observe any acute effects on the prolactin level. Neither did Cone et al. (1986) find any decrease in prolactin after the smoking of two marijuana cigarettes (2.8% THC). Chronic Cannabis users do not show any significant alteration in their prolactin levels (Kolodny et al. 1974, Cohen 1976, Vescovi et al. 1992).
Puberty: Copeland et al. (1980) observed a pubertal arrest in a boy aged 16 years, who had consumed at least five marijuana cigarettes per day since he was 11 years old. Three months after cessation of consumption, a normal entry into puberty was observed. This is the only observation of this kind so far.
Conclusion: It is not conclusive to assume that there was a causal connection between the observed gynecomastia of strong marijuana smokers and their use of marijuana, all the more so because no associations between marijuana consumption and prolactin levels or other relevant parameters were found in later studies. Considering the widespread use of marijuana, literature would have to be expected to hold more published observations of this kind. Also with respect to possible influences on puberty, only one single case has been described to date. In animal studies, high doses produced a slightly higher incidence of abnormal sperm and following daily smoking of 8-10 marijuana cigarettes (100-300 mg THC) over a period of several weeks, a slight reduction in sperm count occurred though no increase in abnormal sperms or any impairment of function was observed. Neither acute nor strongly chronic Cannabis usage caused any consistent effects on the serum level of FSH, LH, prolactin or testosterone in male subjects.
5.3.1.2 Women
Animal studies reflected a Cannabis-induced blockade of the interaction of the hypothalamus, hypophysis and sexual organs. Thus THC delayed the onset of puberty and retarded menstruation in those studies (Tyrey and Murphy 1984). Multiple effects on the secretion of hormones were observed (Abel 1981, Smith and Asch 1984, Mendelson and Mello 1984). Evidence suggest that these incidences are not directly attributable to effects on production and secretion of ovarian sex steroid hormones, but result of a pituitary suppression of the GnRH (gonadotropin releasing hormones). In a study of rats that were administered 12.5, 25 or 50 mg/kg THC every day for two years, their prolactin concentration was not altered (Chang et al. 1996). In a 13-week study of mice and rats that received 5 to 500 mg/kg THC, the menstrual cycle was markedly prolonged relative to the controls. However, in a study of female monkeys, a tolerance of those disruptive effects on the menstrual cycle developed even at high doses (thrice weekly injections of 1.5 or 2.5 mg/kg THC) (Smith et al. 1983).
In comparison to men, far less data are available that describe the effects of THC on the female hormonal profile.
Menstrual cycle: Kolodny et al. (1979) reported an abnormal cycle length in marijuana smokers, averaging 26.8 days as compared to 28.8 days in controls. Moreover, the cycle was more often anovulatory (12.5% vs 38.3%). Dornbush et al. (1978) also found a reduced cycle length but did not state an increase in THC-induced anovulation. Other researchers did not find any significant influence on cycle length (Mendelson and Mello 1984).
Estrogen and progesterone: The hormonal profile of estrogen and progesterone did not differentiate chronic marijuana users from controls (Kolodny et al. 1979). Dornbush et al. (1978) did not find any significant influence on estrogen and estradiol. No correlation was found between acute marijuana smoking (18 mg THC) and the course of estrogen and progesterone concentrations during the menstrual cycle (Mendelson et al. 1986).
Testosterone: Twenty-six female chronic Cannabis users showed an increased testosterone level when compared to 16 controls (Dornbush et al. 1978). In the most extensive study to date, Block et al. (1991) did not discover any significantly elevated serum testosterone concentrations in comparison to controls and no significant association with their being grouped in occasional, intermittent or heavy users.
Prolactin: The smoking of one marijuana cigarette (1.83% delta-9-THC) did not produce any significant changes in plasma prolactin levels during the follicular phase (between menstruation and ovulation) of the menstrual cycle. However, when smoked during the luteal phase (between ovulation and menstruation), a transient small suppression of the plasma prolactin levels occurred 1 to 3 hours after consumption. (Mendelson et al. 1985b). Chronic users did not show any change in prolactin levels (Block et al. 1991).
LH: Mendelson et al. (1985) did not find any change in the LH level in 10 women after the smoking of one marijuana cigarette. However, a light significant decrement (p < 0.02) was observed when marijuana was consumed during the luteal phase. Chronic users present a normal LH level (Kolodny et al. 1979, Dornbush et al. 1978, Block et al. 1991).
FSH: Mendelson et al. (1986) did not state any change in FSH level after acute exposure to 18 mg THC. Also, chronic female users possessed equally normal FSH levels (Kolodny et al. 1979, Dornbush et al. 1978, Block et al. 1991).
Conclusion: Significantly less research data exist that deal with the influences of THC on female sex hormones in comparison to the scientific material on male sex hormones. The research results are inconsistent. There are no conclusive indices to any THC-associated influences on the menstrual cycle length, the number of cycles without ovulation, or on the plasma concentrations of estrogens, progesterone, testosterone, prolactin, LH or FSH in female marijuana users. The transient THC-induced suppression of prolactin and LH levels during the luteal phase of the menstrual cycle should be further investigated. However, this effect occurred only following the inhalative route which, in comparison to oral administration, is associated with a faster absorption of the drug and higher plasma THC levels. Chronic marijuana users did not show any significantly altered hormone levels.
5.3.2 Glucocorticoids
In animal studies, THC stimulates the secretion of ACTH (adrenocorticotropin). ACTH is secreted by the adenohypophysis and stimulates the synthesis of the glucocorticoids (cortisol) in the suprarenal cortex. THC produced a significant increase in serum cortisol in rats at doses of 2 mg, 5 mg and 30 mg/kg body weight (Kumar and Chen 1983). Also, other animal studies measured an increase in the cortisol level when administering doses in the range of 2 to 50 mg/kg THC (Birmingham and Bartova 1976, Pertwee 1974, Eldridge 1991). With chronic administration, a tolerance developed quickly and values progressively returned to normal (Pertwee 1974, Eldridge 1991).
A single oral administration did not elevate plasma cortisol in man (Hollister 1970). However, the smoking of two marijuana cigarettes caused a transient significant increase in plasma cortisol level (Cone et al. 1986). In the above-mentioned study by Dax et al. (1989), a thrice daily oral administration of THC did not find any THC-induced influence on the ACTH level. Chronic heavy marijuana users did not show any significant differences in their cortisol levels (Cruickshank 1976).
5.3.3 Thyroid hormones
The acute treatment of rats with 10 mg THC/kg reduced serum levels of thyrotropin (TSH) and of the thyroid hormones triiodothyronine (T3) and thyroxine (T4), but had no effect on the pituitary or thyroid response to exogenous (Hillard et al. 1984). Intraperitoneal doses of THC greater than 3 mg/kg reduced serum TSH levels by more than 90%. The ED50 for THC was approximately 0.3 mg/kg. Thus, doses were required that clearly exceeded those of relevance to this study. The serum levels of thyroid hormones in chronic marijuana users fail to show any significant incidences (Cruickshank 1976).
5.3.4 Glucose metabolism
Fifty years ago, in a study with 62 volunteers, it was already demonstrated that Cannabis does not have any significant influence on glucose metabolism (Allentuck 1944). In another study, marijuana did not produce any relevant effects on glucose metabolism after 1 to 3 days of fasting. The glucose tolerance was not affected by marijuana (Permutt et al. 1976). However, in one other study a high THC dose (6 mg intravenous) influenced the glucose tolerance test scores in some probands (Hollister and Raven 1976).
5.3.5 Summary
Animal studies have illustrated that, given a sufficiently high dosage, THC may act on the hypothalamo-pituitary-adrenal (HPA) axis and thus adversely affect the function of sex steroid hormones with effects also on hormone-producing sexual accessory organs. However, there are no consistent findings of adverse effects on male or female sexual organs within the range of the human consumption pattern. Strongest references exist concerning a hormonal dysfunction during puberty and a transient influence on prolactin and LH concentration during a certain phase of the menstrual cycle. However, these observations remained singular. A tolerance develops to THC-induced effects on the endocrine system (sex hormones). Also other hormones such as the glucocorticoids and thyroid hormones are influenced by high THC doses in animals. In man, no significant alterations were found at relevant doses. The NOAEL of THC for the influences on sex hormones and other hormones is safely above. or ranges within, the human consumption situation. There is no reason to believe that sub-psychotropic THC doses could affect the function or concentration of sexual hormones or other parameters relevant for reproduction, such as sperm quantity and quality.
5.4 Immune system
The immune system is a complex functional system for protection against noxious foreign material (for instance: bacteria) or for the elimination of anomalous structures (such as tumor cells). The organs of the lymphatic system (spleen lymphatic nodes), the production sites of lymphocytes and other immune cells (thymus, bone marrow), a multitude of cells (lymphocytes, macrophages) and different molecules (immunoglobulines, cytokines etc.) contribute to the immune system.
Immunity is either unspecific or specific (acquired). Unspecific immunity does not require a preceeding sensitization (from previous exposures). For instance, the ingestion of bacteria by macrophages, and the killing of tumor cells by natural killer cells belong to this category. As opposed to this, acquired immunity is based on selective responses of antibodies specifically sensitized to certain substances (antigens) or of specifically sensitized cells (T-lymphocytes, macrophages). The B-lymphocytes produce antibodies and regulate humoral (mediated by specific atibodies) acquired immunity whereas the activity of T-lymphocytes controls cell-mediated immunity.
THC alters some immune parameters of humoral and cell-mediated immunity in a dose-related manner, acting either in an immunosuppressive or immunostimulating manner depending on its effects. At high THC concentrations, unspecifc effects on the cell membrane seem to play an important role, whereas at a lower dosage, THC effects on the immune system appear at least partly mediated by a cannabinoid-receptor-dependent pathway (Sanchez et al. 1997, Burnette-Curley 1995, Kaminsky et al. 1994). The CB2 receptor was found to be of special relevance to these activities (Patrini et al. 1997).
The natural cannabinoid-receptor-antagonist anandamide exerts biphasic dose-related effects on a number of biological parameters in mice (Sulcova et al. 1997). One of these parameters is the leucocyte-phagocytosis, which is inhibited by high doses (10-100mg/kg) but stimulated by the lowest measured dose (0.01 mg/kg). Similar effects are to be expected with exogenous ligands such as THC.
5.4.1 Cell-mediated immunity
The effects of THC on cell-mediated immunity are determined by a variety of different tests. To this end, one assesses: the number of lymphocytes and the transformation of lymphocytes as a response to certain substances that promote cell division (mitogens); the influence on the synthesis of macromolecules (proteins, DNA) in T-lymphocytes; the T-cell capacity to form rosettes with sheep erythrocytes; the functions of leucocytes, natural killer cells and macrophages; the action on different cytokines (interferon, interleukines, tumor necrosis factor); and finally the susceptibility to bacterial and viral infections, as well as skin response to antigens.
5.4.1.1 Number of T-lymphocytes
There are inconsistent results concerning the effects of THC on lymphocyte count (Nahas et al. 1974). Some researchers found significant decreases in the T-lymphocyte count (Nahas et al. 1974) whereas others did not find the number of lymphocytes significantly altered (Rachelefsky et al. 1976, Cushman and Khurana 1977, Dax et al. 1989, Zimmer et al. 1976). Dax et al. (1989) investigated the effect of thrice-daily 10 mg oral or 18 mg THC in marijuana cigarettes for three days in chronic marijuana users after two previous weeks of abstinence. Any differences in the quantity of B- and T-lymphocytes as well as T-cell subtypes previous to, during, and subsequent to administration were not observed. Wallace et al. (1988) did not find the number of T-lymphocytes to be significantly altered but determined a change in the ratio of helper T-cells to suppressor cells (CD4/CD8-ratio) with an increase in the CD4 percentage. This was interpreted as an immunoenhancement, as helper T-cells stimulate the proliferation and activation of other immune cells.
5.4.1.2 Transformation of T-lymphocytes
Some studies examined the influence of THC on T-cell proliferation as a response to substances stimulating cell division, so-called mitogens (PHA = phytohemagglutin, Con A = concavalin, anti-CD3-antibodies, etc.), and cells (MLC = allogenic mixed lymphocyte culture).
Cellular studies: THC (10-100 mM) induced in vitro suppression of T-cell proliferation responses to PHA and MLC (Nahas et al. 1977). Furthermore, a reduced uptake of thymidine, leucine and uridine was found, which indicates a depressed protein/RNA synthesis. THC suppressed the lymphoproliferative response of T-lymphocytes that had been stimulated by human interleukin-2 in a dose-dependent manner (Kawakami et al. 1988). A minimum dosage of 2.5 mg/ml THC was required to obtain any measurable effects. A threshold concentration of 5 mg/ml THC was necessary to achieve an influence on the mitogen-induced T-lymphocyte proliferation (Klein et al. 1985).
Rachelefsky and Opelz (1977) did not find THC (at concentrations up to 1.1 millimol) to have any influence on the stimulating effect and the protein synthesis of PHA and MLC. Pross et al. (1992) found lymphocyte proliferation either to be suppressed or to be enhanced, dependent upon the measured parameters and the organ source of the lymphocytes. THC suppressed the proliferation from Con A- or PHA-stimulated lymphocytes derived from either spleen or lymph nodes. Spleen cells stimulated with anti-CD3 antibody and treated with low doses of THC displayed an enhanced proliferation, whereas the response in lymph nodes did not change. The research group determined that the observed immunosuppression or -stimulation related to the THC-induced modulation of interleukin-2 activity (Nakano et al. 1992). Luo et al. (1992) found THC to affect the mitogen-induced transformation of lymphocytes in a dose-related manner. The stimulation of lymphocytes with PHA relative to the uptake of thymidine reached its peak at 0.4 mg/ml THC. The effect decreased rapidly at higher or lower doses. When stimulated with Con A, the maximum stimulation was reached at 0.05 mg/ml THC. Human lymphocytes and mouse splenocytes responded in similar patterns.
Cells of marijuana users: Nahas et al. (1974) found a reduced thymidine uptake in lymphocytes of marijuana users after stimulation with PHA and MLC. Segelmann and Segelmann (1974) disputed these findings as they had doubts about the method applied and therefore requested these results to be revised. White et al. (1975) could not confirm these results. The incorporation of 14C-thymidine by peripheral blood lymphocytes did not differentiate 12 long-term marijuana smokers from controls. Lau et al. (1976) did not find any altered lymphocyte response to PHA as measured by thymidine uptake after oral ingestion of 210 mg THC for 18 days. Humoral and cell-mediated immunity were investigated in a study by Rachelefsky et al. (1976) of 12 healthy marijuana smokers (6 moderate and 6 heavy smokers) who smoked a mean of 5.4 marijuana cigarettes (approx 20 mg THC/cigarette) over a period of 64 days. Initially the number of T-lymphocytes decreased, but returned to normal in the course of the test period. In vitro responses to PHA and MLC were normal throughout the study. The number of marijuana cigarettes smoked did not affect the immunologic test scores. The authors concluded that chronic use of marijuana did not have any adverse effects on the T-lymphocyte proliferative response in young, healthy adults. Similar statements were published by Cohen (1976), Kaklamani et al. (1978) and Wallace et al. (1988). In the aforementioned study by Dax et al. (1989) with a thrice daily oral intake of a 10 mg or 18 mg THC marijuana cigarette for three days, one did not detect any significant alterations in mitogen response as measured before, during and after THC treatment.
5.4.1.3 Rosette formation
T-lymphocytes possess the capacity to form rosettes of sheep erythrocytes (red blood cells from sheep). Then a minimum of four T-cell-receptor-bound sheep erythrocytes surround the immune cell. THC affected an in vitro suppression of the T-cell rosetting capacity (Cushman et al. 1976). Mice that had been immunized with sheep erythrocytes exhibited a significantly reduced rosette formation after injections of 25 mg/kg THC or more (Lefkowitz et al. 1978).
Cushman et al. (1975 b) compared the lymphocytes in 23 normal controls and in 23 marijuana users. No difference in rosetting capacity was found in B-lymphocytes, but rosette formation differed in T-lymphocytes. The same research team examined further collectives and found a reduced early rosette formation by normal T-lymphocytes in 35 marijuana users when compared to controls, whereas late formation was not affected (Cushman and Khurana 1976). In a follow-up study of 10 marijuana users the same result was obtained, so that the authors assumed an impairment of a T-lymphocyte subpopulation by marijuana. Petersen et al. (1976) diagnosed a reduced rosette formation in two of three marijuana smokers. A further study found rosetting suppressed in five of six subjects after smoking 10 mg THC, but after 24 hours it had normalised in five of the six subjects that were examined. In the latest study on this topic, 12 chronic marijuana smokers did not differ from 15 control subjects in the rosetting capacity of their peripheral T-lymphocytes (Kaklamani 1978). In the 80s and 90s no further studies of this kind were conducted, and the clinical significance of rosette formation has not yet been ascertained.
5.4.1.4 Delayed-type hypersensitivity
Delayed-type hypersensitivity is cell mediated, contrary to the other types of hypersensitivity (immediate type, cytotoxic type, immune complex type). The release of lymphokines from specifically sensitized T-lymphocytes activates macrophages and mononuclear cells. The study of Smith et al. (1978) elucidated that THC suppressed the delayed-type hypersensitivity response to sheep erythrocytes in mice. High doses of 100 mg/kg THC subcutaneous for four days suppressed the immune response from 35 to 64%. Levy and Heppner (1978-1979) also found delta-8-THC to posses only weak (to moderate) activity in the suppression of the delayed-type hypersensitivity response to sheep erythrocytes.
5.4.1.5 Cytokines
Cytokines are substances that are produced by different cells and function as messengers between cells. Interleukines are mediators of such kind; secreted by leukozytes, they activate other immune cells (B- and T-lymphocytes, natural killer cells). Interferons are produced by different cells as part of an immune response to various stimuli and exert antiviral (interferon-alpha and -beta) and immunomodulating (interferon-gamma) activity. Tumor necrosis factor (alpha and beta) are cytokines produced by immune cells that regulate response to inflammation, immune functions and tumor defense.
Interferon: Chronic administration of 50 mg/kg THC to mice over a period of 56 days significantly reduced their interferon-alpha and interferon-beta concentration in their lymphocyte cultures after stimulation by various mitogens (Blanchard et al. 1986). A dose of 5 to 10 mM THC effected a dose-related in vitro suppression on IFN production, whereas 5 mM did not have any effect (Blanchard 1986). If mice received more than 15 mg/kg THC intraperitoneal, as in the study by Cabral et al. (1986b) their interferon-alpha and -beta production in response to herpes viruses would decline. Watzl et al. (1991) found the effect of THC on interferon-gamma-induction by mitogens to be dose-related, as IFN production was increased at THC concentrations of 0.1 and 1 mg/ml, whereas a decrease occurred at concentrations above 5 mg/ml.
Interleukins: THC elevated interleukin-1-bioactivity in human macrophage cultures stimulated with lipopolysaccharide (Shivers et al. 1994). Interleukin-6 levels were decreased. Another study reported an increased expression of interleukin-2 (IL-2) receptor alpha and beta proteins (Daaka et al. 1997). Specter and Lancz (1991) reported that the addition of THC dose-dependently inhibited IL-2 attachment to IL-2 receptors of cytotoxic T-lymphocytes. At 10 mg/ml THC they measured a suppression to 5% relative to the basis value, at 5 mg/ml to 25% and, finally, at 1 mg/ml to 90% of the initial value. In the study of Watzl (1991), concentrations of up to 1 mg/ml THC did not affect the interleukin-1-secretion of mitogen-activated human peripheral blood mononuclear cells, whereas a dose-related suppression was noted at higher drug levels. No effect on interleukin-2-secretion was displayed after addition of 0.1 mg/ml THC. Also, 1 mg/ml THC elevated secretion by 100%, and any doses above this concentrations did not produce any significant effect.
Tumor necrosis factor: Kusher et al. (1994) found a dose-related influence of THC on candida albicans-induced tumor necrosis factor (TNF) production by lymphocytes, a suppression of TN induction, only at concentrations ranging from 0.05 to 1 mg/ml, while no effects were found at 0.005 mg/ml (=5 ng/ml), which is the level of concentration relevant to the subject of our study. The study of Watzl (1991) reported that even high THC concentrations did not have any effect on TNF secretion after mitogen activation of peripheral blood leucocytes.
5.4.1.6 Host resistance and cell toxicity
Cellular defense is related to other immune parameters that will be discussed. For instance, cytokines affect the cytotoxic activity of natural killer cells.
Lymphocytes: THC in a concentration range of 2 mg/ml depressed induction and cytolytic activity of cytotoxic T-lymphocytes, though it failed to do so in a range between 25 and 2000 ng/ml THC (Lu 1990, Lu and Ou 1989). THC was found to exert a biphasic effect on lymphocyte metabolism, with a stimulation at low doses and a suppression at high THC concentrations. Thus a THC concentration of 0.01 mM induced a 1.3 fold and a concentration of 1 mM a 2 fold stimulation of glucose oxidation to carbon dioxide in mice lymphocytes. A further increase in concentration to 10 mM downregulated glucose oxidation to 40%, and 20 mM produced a decrease to 25% of the initial value. Corresponding effects were found for the phospholipid synthesis from glucose as 1 mM produced maximum values of 250% and 20 mM produced minimum values of 30%. The authors found evidence that the depression on glucose metabolism at high non-physiological doses is mediated by an unspecific receptor-independent pathway. Furthermore, concentrations of about 10 mM were found to have cytotoxic effects, reducing lymphocyte function to 1%, whereas a concentration of 1 mM did not exhibit any of those effects (Sanchez et al. 1997). At high doses a disruption of cell membrane function is to be expected.
Leucocytes: THC-induced effects on leucocyte migration are concentration dependent. For leucocytes from five marijuana smokers, a minimum concentration of 2.0 mg/ml was required to display any alterations (Schwartzfarb et al. 1974). Maximal inhibition of neutrophil granulocyte function occurred at a dose of 4 mg/ml THC; at 2 mg/ml THC activity was suppressed by 50% and at THC concentrations below 1 mg/ml no suppression was noted (Djeu et al. 1991). Neutrophile granulocytes, for instance, retarded candida albicans growth by 64%. By addition of 4 mg/ml THC, the antifungal activity was suppressed to only 5%, at 0.5 mg/ml it was 61%, at 0.25 mg/ml 63% and at 0.125 mg/ml 69%, thus having even slightly improved when compared to activity without THC.
Natural killer cell activity: In a study by Patel et al. (1985), natural killer cell activty in rats was not modulated after acute administration of 3 mg/kg subcutaneous, whereas subchronic administration of the same concentration impaired killer cell activity. A number of studies determined quite high threshold values of the order of 2 mg/ml for any inhibitory effects on killer cell activity to become detectable. In a study of mice, suppressive action was stated at a tissue concentration of 5-10 mg/ml (Klein et al. 1987). In a study of cultured murine splenocytes, a dose-related suppression was found in a concentration range of 10-30 mg/ml (Klein et al. 1987). A concentration of 5 mg/ml and above was found to be inhibitory for activity against a human tumor cell line (Specter et al. 1986). In a further study, natural killer cell activity was nearly completely inhibited after addition of 20 mg/ml THC; at 10 mg/ml it was suppressed to 30% of the baseline value and 1 mg/ml did not induce any inhibition (Specter and Lancz 1991 b). Also at a concentration of 5 mg/ml, THC inhibited the cytolytic NK-cell activity against another cell line (Kawakami et al. 1988). In a concentration range of 25 to 2,000 ng/ml, NK-cell activity was not disturbed (Lu and Ou 1989).
At concentrations of 0.05-2 mg/ml, Kusher et al. (1994) found a dose-related inhibition of cytolytic NK-cell activity in large granulated lymphocytes against tumor cells, whereas no such inhibitory action was observed in the low nanomolar-range. At 2 mg/ml THC approached maximal inhibitory effect, at 0.05 mg/ml a 50% inhibition occurred, and at 0.01 mg/ml no such effect was measured.
Macrophages: THC was shown to suppress the macrophage contact-dependent cytolytic activity by inhibiting tumor necrosis factor (TNF)- and NO-mediated killing (Burnette-Curley 1995, Fischer-Stenger et al. 1993). THC suppressed macrophagelytic action in concentrations of more than 2 mg/ml THC (Lu 1990) but failed to have such effect within the nanogram range (Lu and Ou 1989). In another study, a minimum of 10 mg/ml THC was required in cultured macrophages to impair their functions (Friedman et al. 1986). Macrophages normally spread on glass surfaces. THC was found to adversely affect this ability in relation to dose in another study (Lopez-Cepero et al. 1986). By adding 20 mg THC to a culture of normal mice peritoneal cells, the spreading was almost completely inhibited. The lowest dose measured that still induced minimum effects was 0.05 mg THC per culture. Higher concentrations were necessary to inhibit phagocytosis in yeast particles. Another study of mice, however, did not report any modulation of macrophage activity (Munson et al. 1976). Cabral and Vasquez (1991) examined various macrophage functions. THC induced a dose-related inhibition of anti-tumor-factor production in response to stimulation by Propionibacterium acnes in mice. An intraperitoneal administration of 15 to 100 mg/kg THC over a period of several days decreased production to 50% at the maximal dose and to 85% of the initial value at the lowest THC dose. Also macrophage contact-dependent cytotoxic activity, extrinsic antiviral activity, macophage capacity to take up viruses, and protein expression were found reduced in a similar fashion. Studies generally employed extremely high doses that induced a dose-related immunosuppression. The authors regard unspecific (receptor-independent) toxic effects on the cell membranes as cause for the observed impaired immune function.
5.4.1.7 Susceptibility to infections
Bradley et al. (1977) observed an application of various bacterial endotoxins when applied in combination with THC to reduce the lethal THC dose from approximately 350 to 150 mg/kg.
Mishkin and Cabral (1985) examined the effect of THC on host resistance against a vaginal infection with a herpes virus (HSV2, herpes simplex virus type 2) in mice. The animals were administered high THC doses from one day before until two days after intravaginal introduction of viruses. Those animals that had received 100 mg/kg THC exhibited heavy infections, higher virus titers and higher animal mortality. A similar experimental design with guinea pigs also showed a decrease in host resistance to intravaginally introduced herpes viruses after the animals had been intraperitoneally treated with the drug at doses ranging from 4 to 10 mg/kg on day 1-4, 8-11 and 15-18 after infection (Cabral et al. 1986). Cumulative death rate was dose-dependent.
At concentrations of 10 and 50 mg/kg, THC suppressed the lymphocyte proliferative response to a second exposition to herpes viruses (HSV2) in mice (Cabral et al. 1987). Furthermore, the drug affected expression of proteins as elicited by bacterial lipopolysaccharide (Cabral and Mishkin 1989). Morahan et al. (1979) found a higher susceptibility to infection not only with herpes viruses (HSV2) but also with bacteria (Listeria monocytogenes). The decrease in host resistance to tested bacteria was dose-dependent. THC doses of 38, 75 and 150 mg/kg induced a 10-, 17-, and 657-fold decrease in resistance, measured as change of lethal dose (LD50). Ashfaq et al. (1987) observed a weakened local immune response to bacterial infections after four days of marijuana smoking (estimated daily dose: 3.2 to 6.4 mg/kg) in mice. This significantly deteriorated the immune response to a skin necrosis induced by a subcutaneous injection of Staphylococcus aureus. As a daily injection of THC (10 mg/kg) by itself had no such effect, the authors inferred that other marijuana constituents must be responsible for this immune suppression.
Klein et al. (1993) found a decrease in survival rate among mice that had received an injection of 8 mg/kg THC 24 hours before and after a sublethal intravenous infection with the bacterium Legionella pneumophila. Death generally occurred 30 minutes after the second injection and resembled a cytokine-mediated septic shock. In fact, a marked increase in acute-phase pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) was measured. A follow-up confirmed the augmented lethality and the altered serum levels of acute-phase proteins (Smith et al. 1997). However death rate was not affected by lower doses of 1-5 mg/kg THC (Klein et al. 1993).
5.4.1.8 Skin reactions to antigens
Silverstein and Lessin (1974) investigated the skin reactions after sensitization to Dinitrochlorobenzene, a compound contained in photochemicals. A sensitivity was found in 100% of 34 examined marijuana smokers compared to 96% of 279 healthy non-smokers and 70% of 384 cancer patents. According to Hollister, these findings "raise questions about the clinical significance of experiments that have shown evidence of impairement of cell-mediated immunity from cannabinoids" (Hollister 1992).
5.4.2 Humoral immunity
The effect of THC on humoral immunity was tested by measuring the number of B-lymphocytes after stimulation by mitogens, or by the presence of sheep erythrocytes, by measuring the capacity of rosette formation as well as production of antibodies.
5.4.2.1 Number of B-lymphocytes and B-lymphocyte proliferation
The number of B-lymphocytes is not affected by smoking marijuana (Wallace et al. 1988, Rachelefsky et al. 1976). At least 5 mg/ml THC were required in cell cultures to effect suppression on lipopolysaccharide-induced proliferative response in B-lymphocytes (Klein et al. 1985). Munson et al. (1976) discovered a dose-related reduction of lymphocyteresponsitivity after 50, 100 and 200 mg/kg THC. In contrast Sanchez et al. (1997) observed a stimulation of glucose metabolism in B-lymphocytes at low (physiologic) THC concentrations. At low nanomolar concentrations that can be reached in the live organism, THC was found to effect an enhanced B-lymphocyte proliferation in response to activation through cross-linking of surface immunoglobulins (Derocq et al. 1995). Micromole concentrations effected a suppression instead. Thus the authors proved that the increase in immune activity is receptor mediated and they assume that the CB2 receptor is involved.
5.4.2.2 Rosette formation
Cushman et al. (1975 b) compared the lymphocytes of 23 normal controls to 23 marijuana users. The B-lymphocyte rosetting capacity was not found to be different in either group.
5.4.2.3 Antibody production
One way to assess humoral immunity is to measure the capacity of plaque formation in mice lymphocytes that are exposed in vitro to sheep erythrocytes against which they are immunized. Smith et al. (1978) established a median effective dose (ED50) of 70 mg/kg THC for the reduction of this effect in mice. In a study of Levy et al. (1981) a dose-dependent reduction in hemolytic plaque-forming cells was found. Peak reactivity was, however, only delayed by 24-48 hours and the THC concentration required produced high behavioral toxicity in the animals.
Baczynsky and Zimmerman (1983) investigated the primary-like humoral immune response, the secondary-like humoral immune response, and the memory aspects of the humoral immune response in mice after stimulation with sheep erythrocytes. Mice treated with 10 or 15 mg/kg THC during primary immunization displayed a depressed primary immune response. Administration of THC during secondary immunization did not produce any signifiant suppression on secondary response. The memory aspect was assessed by measuring the secondary immune response after THC had been administered during primary response. At 10 or 25 mg/kg THC, the secondary immune response was significantly suppressed. THC treatment (10 or 15 mg/kg) during primary immune response caused a decrease in thymus weight and reduced the number of thymus cells.
Zimmerman et al. (1977) observed a dose-dependent depression of the immune response at doses of 1 mg/kg, 5 mg/kg and 10 mg/kg in mice that had been stimulated with sheep erythrocytes. The splenic weight, the percentage of splenic white pulp of total spleen volume, the number of splenic plaque-forming cells, and the hemagglutination titer were reduced. In a study by Schatz et al. (1993), THC suppressed the primary humoral immune response in mice. Oral administration of 50-200 mg/kg THC produced a dose-related inhibition of antibody-forming-cell (AFC) response. Rosenkrantz et al. (1975) intraperitoneally immunized rats with sheep erythrocytes during, before, and after ingestion of 10 mg/kg THC. The primary immune response was reduced by values of 33 to 40%.
Luthra et al. (1980) did not find any development of tolerance to the suppression of antigenic stimulation with sheep red blood cells in rats. Twenty-six days of pretreatment with 6 or 12 mg/kg THC prior to antigenic stimulation did not modulate suppression of antibody-forming-cell proliferation or change hemagglutinin and hemolysin titers. In contrast, Loveless et al. (1981-1982) observed a development of hyporesponsiveness to delta-8-THC-induced immunosuppresion. If mice were pretreated daily for five days with 5 or 10 mg/kg THC before they were immunized with sheep erythrocytes, significantly higher doses were required to produce the equivalent inhibitory effect on hemolytic plaque-forming cells.
5.4.3 AIDS infection
A major immunosuppressive activity in THC would adversely affect the outbreak or the course of AIDS in HIV-positive patients who use Cannabis. Hall et al. (1994) maintain that so far no epidemologic relation between marijuana consumption and infectious diseases has become conspicuous. This did not, however, "exclude the possibility that chronic heavy use may produce minor impairments in immunity" (p 67).
THC is therapeuically employed as an appetite stimulant as well as an antiemetic and antivomiting agent in cancer and AIDS patients. Especially long-term treatment with THC as used for AIDS patients to enhance appetite would be precarious if an immune suppression would thus further damage the already impaired immune system. Kaslow et al. (1989) conducted a prospective epidemiological study of 4,954 homo- and bisexual men to examine the development of AIDS. The use of Cannabis or other psychoactive drugs did not increase the risk of progression to full-blown AIDS in HIV-positive men. Also among seropositive men with lower initial T-helper-lymphocyte count, those who used drugs and those who were non-users did not significantly differ in their respective risk of AIDS. In a six-year epidemiologic study, Di Franco et al. (1996) could not establish that the outbreak of AIDS in HIV-positive men was in any way associated with the use of Cannabis.
5.4.4 Summary
Cellular experiments and animal studies demonstrate that THC has suppressive effects on the humoral and cell-mediated immunity. However, the majority of those can be attributed to toxic unspecific effects. Many analysed parameters required extremely high doses to exhibit any significant effect and the effects were dose-dependent, with the threshold concentration being precisely determinable. When applying lower doses, one often observed differentially immunostimulating effects or no effects at all. For many immune parameters, the NOAEL is within a range irrelevant to a human consumption situation. In studies of man or of cells of marijuana users, the effects observed were often contradictory. If such effects were found at all, they were weak, even in case of heavy Cannabis use and of questionable relevance to health. The World Health Organisation summarized in its most recent Cannabis report: "Many of their effects appear to be relatively small, totally reversible after removal of the cannabinoids, and produced only at concentrations or doses higher than those required for psychoactivity (more than 10 mM in vitro, or more than 5 mg/kg in vivo)" (WHO 1997, p 27).
Source, Graphs and Figures: THC Limits for Food, Part II
