Jacob Bell
New Member
Editors:
Monique C. Braude, Ph.D.
Jacqueline P. Ludford, M.S.
National Institute on Drug Abuse
NIDA Research Monograph 44
A RAUS Review Report
DEPARTMENT OF HEALTH AND HUMAN SERVICES
Public Health Service
Alcohol, Drug Abuse, and Mental Health Administration
National Institute on Drug Abuse
5600 Fishers Lane
Rockville, Maryland 20857
NIDA Research Monographs are prepared by the research divisions of the
National Institute on Drug Abuse and published by its Office of Science. The
primary objective of the scenes is to provide critical reviews of research
problem areas and techniques. the content of state of-the-art conferences,
Integrative research reviews and signifigant original research Its
dual publication emphasis IS rapid and targeted dissemination to the
scientific and professional community
Editorial Advisory Board
Avram Goldstein, M.D.
Addiction Research Foundation
Palo Alto. Colifornia
Jerome Jaffe, M.D.
University of Connecticut School of Medicine
Formington, Connecticut
Reese T. Jones, M.D.
Langley Porter Neuropsychiatric lnstitute
University of California
San Francisco, California
Jack Mendelson, M.D.
Alcohol once Drug Abuse Research Center
Harvard Medical School
McLeon Hospital
Belmont, Massachusetts
Helen Nowlis, Ph.D.
Rochester. New York
Lee Robins, Ph.D.
Washington University School of Medicine
St Louis, Missouri
NIDA Research Monograph Series
William Pollin, M.D.
DIRECTOR, NIDA
Jack Durell, M.D.
ASSOCIATE DIRECTOR, OFFICE OF SCIENCE, NIDA
EDITOR-IN-CHIEF
Eleanor W. Waldrop
MANAGING EDITOR
Parklawn Building, 5600 Fishers Lane Rockville Maryland 20857
ACKNOWLEDGMENT
This monograph is based upon papers and discussion from the RAUS
Review Conference on the Endocrine and Reproductive Effects of
Marijuana, held March 1 and 2, 1983, in Rockville, Maryland. The
conference was sponsored by the Office of Science and the Division
of Preclinical Research, National Institute on Drug Abuse.
COPYRIGHT STATUS
The National Institute on Drug Abuse has obtained permission from
the copyright holders to reproduce certain previously published
material as noted in the text. Further reproduction of this
material is prohibited without specific permission of the copyright
holders. All other material in this volume except quoted passages
from copyrighted sources is in the public domain and may be used or
reproduced without permission from the Institute or the authors.
Citation of the source is appreciated.
Opinions expressed in this volume are those of the authors and do
not necessarily reflect the opinions or official policy of the
National Institute on Drug Abuse or any other part of the U.S.
Department of Health and Human Services.
The U.S. Government does not endorse or favor any specific
commercial product or commodity. Trade or proprietary names
appearing in this publication are used only because they are
considered essential in the context of the studies reported herein.
Library of Congress catalog card number 83-,600600
DHHS publication number (HDM)84-1278
Printed 1984
NIDA Research Monographs are indexed in the Index Medicus. They
are selectively included in the coverage of American Statistics
Index, Biosciences Information Service, Chemical Abstracts, Current
Contents, Psychological Abstracts, and Psychopharmacology Abstracts.
Preface
The Research Analysis and Utilization System (RAUS) is designed to
serve four functions:
Collect and systematically classify the findings of all
intramural and extramural research supported by the
hational institute on Drug Abuse (NIDA);
Evaluate the findings in selected areas of particular
interest and formulate a state-of-the-art review by a
panel of scientific peers;
Disseminate findings to researchers in the field and to
administrators, planners, instructors, and other
interested persons;
Provide a feedback mechanism to NIDA staff and planners so
that the administration and monitoring of the NIDA
research program reflect the very latest knowledge gleaned
from research in the field.
Since there is a limit to the number of reseach findings that can
be intensively reviewed annually, four subject areas are chosen
each year to undergo a thorough examination. Distinguished
scientists in the selected field are provided with copies of
reports from NIDA-funded research and invited to add any
information derived from the literature and from their own research
in order to formulate a comprehensive vick of the field. Each
reviewer is charged with writing a state-of-the-art paper in his or
her particular subject area. These papers, together with a summary
of the discussions and recommendations which take place at the
review meeting, make up a RAUS Review Report in the NIDA Research
Monograph series.
The subject of the effects of marijuana on the endocrine and reproductive
systems was chosen for a RAUS review in Fiscal Year 1983
because marijuana use is so widespread among American youth and,
therefore, is of great programmatic importance to NIDA. Increased
prevalence of marijuana use over the past decade has been accompanied
by decreasing age of first use, and there is grave public health
concern about its effects on youth who are undergoing maturation of
their reproductive systems at about the same time as they are likely
to begin using marijuana. Since there is a growing body of research
on the subject, it became incumbent upon NIDA to gather the knowledge
that was available, evaluate it, and disseminate it. The results of
the RAUS review are presented in this monograph.
Dr. Monique C. Braude served as the scientific chairperson for the
meeting . Jacqueline P. Ludford is the RAUS coordinator
Executive Summary
Jacqueline P. Ludford, M.S., and Monique C. Braude, Ph.D.
Isolated reports of impaired sexual behavior, lowered hormone
levels, and abnormal offspring in animals after administration of
marijuana or its active principles, such as delta-9-tetrahydrocannabinol
(THC), prompted a review of current findings
relevant to the effects of marijuana on genetics and reproduction.
A RAUS review meeting was held on March l-2, 1983, and reviewers
were charged with evaluating the state of the art in the following
areas:
Effects of Cannabinoids
on Gene Expression
Effects of Cannabis and
Natural Cannabinoids on
Chromosomes and Ova
Effects of Marijuana in the
Male: Preclinical Studies
Endocrine Aspects of Cannabinoid
Action in Female Subprimates:
Search for Site of Action
Acute, Short Term, and Chronic
Effects on the Female
Primate Reproductive Function
Effects of Marijuana on Dr. Jack Mendelson
Neuroendocrine Function McLean Hospital/
in Human Males and Females Harvard University
Marijuana: Prenatal
Exposure in the Human
Dr. Gary Stein
University of
Florida
Dr. Akira Morishima
Columbia University
Dr. Jack Harclerode
Bucknell University
Dr. Lee Tyrey
Duke University
Dr. Carol Smith
Uniformed Services
University of the
Health Sciences
Dr. Katherine Tennes
University of
Colorado
Dr. Stein first discussed the importance of the preferential
expression of specific genes which can be associated with
modifications in the organization and/or representation of genetic
sequences. To address regulation of eukaryotic genetic sequences,
one must consider control at several cellular levels in the nucleus
and cytoplasm. Gene expression encompasses an extensive range of
cellular structures and biochemical processes starting in the
nucleus with DNA and terminating with the RNA molecule. Dr. Stein
then reviewed his studies on the effect of cannabinoids on the
genome, and on gene expression. To assess more definitively the
influences of cannabinoids on gene expression, Stein's group
examined the effect of Delta-9-THC on the representation of RNA
transcripts from two defined genetic sequences, histone genes and
ribosomal genes, in several human cell lines. They found that THC
causes a dose-dependent reduction in the cellular representation of
histone mRNA sequences at the higher concentrations used in their
assay. The extent to which cannabinoids affect the expression of
specific genetic sequences other than histone sequences is still an
open-ended question. Understanding the manner in which druginduced
alterations in gene expression are brought about should,
Stein believes, provide insight into the molecular basis of
cannabinoid-related modifications in cellular function.
Dr. Morishima reviewed the various reports on studies of the
effects of marijuana and natural cannabinoids on chromosomes. The
evidence from the available cytogenetic studies suggests that
cannabis and cannabinoids are extremely weak clastogens, i.e.,
produce little chromosome breakage and that their clastogenic
effects become apparent only in appropriately sensitive test
systems such as primary spermatocytes and bone marrow cells,
whereas the human lymphocyte system is relatively insensitive to
their clastogenic effects. He then reported the results of his
recent studies on the effects of THC on mice ova, showing that
chronic administration of THC to sexually developing mice produced
an increase in abnormal ova, although the percentage of increase
was small. It appears that this increase in degenerated ova was
caused by their inability to successfully undergo first cleavage
division, probably affecting the process of meiosis. Following his
review of the studies which reported errors of chromosome
separation (ECS) and of this recent data, Morishima now proposes a
new concept that cannabis and cannabinoids in vitro and in vivo act
by disrupting the meiotic as well as the mitotic processes.
Dr. Harclerode focused on the effect of chronic exposure of
laboratory animals to cannabinoids, with emphasis on the male
reproductive system. The reports in the literature of reduction in
reproductive organ weights are accompanied by reports that show
that the auality and ouantity of sperm produced by the testis are
affected by cannabinoids. Treatment of mice with THC for as little
as 5 days resulted in reduction of sperm production and appearance
of abnormal sperm. This was often accompanied by a decrease in
testicle and seminal vesicle weights. Two gonadotropins, LH and
FSH, secreted by the pituitary gland are of major importance to
reproduction in the male. A single hypothalamic factor, the
gonadotropin-releasing hormone (GnRH) is believed to be responsible
for the release of LH and FSH. THC induces a block of GnRH release
which results in lowered LH and FSH, thus reducing testosterone
production by the Leydig cells of the testis. Other hormones that
might have a synergistic or antagonistic effect upon reproduction
in the male are the adrenal cortical hormones, thyroid hormones,
growth hormones, and prolactin. THC appears to depress prolactin,
thyroid gland function, and growth hormone while elevating adrenal
cortical steroids.
Dr. Tyrey reviewed the effects of cannabinoids, primarily THC, on
the female reproductive function in subprimates and discussed
cannabinoid action on the target organs (uterus and ovary) as well
as on the CNS and the hypothalamic-pituitary axis. He concluded
that the search for a site of cannabinoid action in subprimates has
raised the possibility of cannabinoid effects at each level of the
female reproductive system. He feels that the early suggestion
that THC may have a direct "estrogen-like action on the uterus" has
not been substantiated by later studies which failed to show that
THC interacts with the estrogen cytoplasmic receptor. However,
there is now evidence that THC alters the secretion of reproductive
pituitary hormones (LH, prolactin) and of ACTH through effects in
the brain.
Dr. Smith reviewed the acute and chronic effects of THC on the
reproductive function of the female primate. She pointed out that
studies in these species show that cannabinoids inhibit secretion
of LH and FSH as well as prolactin. These changes in pituitary
hormones produce decreases in sex steroid hormones and cause
changes in ovulation. Dr. Smith also emphasized that the principal
site of action of cannabinoids is the hypothalamus. Her recent
findings show, however, that these cannabinoid effects are
reversible in sexually mature animals when drug treatment is
terminated and that there is development of tolerance to the
effects of THC after chronic administration. This may explain why
evidence of disrupting effects on female reproductive function has
been scarce. She observed that, in humans, it is not yet known how
much disruption of reproductive hormone levels is necessary for
changes in human fertility and sexual function to become apparent,
and she emphasized the need for clinical studies in female
marijuana smokers.
There are technical problems in obtaining data on the effect of
marijuana on human reproductive systems, but Dr. Mendelson reported
on the available data and on his own considerable work in this
area. In his study of the effects of marijuana on pituitarygonadal
hormones in human males, he found that the use of marijuana
alone did not suppress testosterone levels. Similar carefully
controlled studies of human female hormone levels are scarce,
although reports from animals suggest, as mentioned above, that THC
may produce a significant decrease in prolactin and LH. There have
been reports that marijuana users had shorter menstrual cycles and
lower prolactin levels than age-matched nonusers. A residential
ward study in human females is now underway, and some preliminary
observations were reported at the meeting.
Dr. Tennes reviewed the current knowledge about the effect of
marijuana on human pregnancy and fetal development. Although 10%
to 37% of pregnant women report use of marijuana, evidence
regarding its effect is confounded by the use of other substances,
nutrition, truthfulness of the woman's recall and report of the
amount of use, changes in use during pregnancy and the trimester
when these changes occurred, and a host of other technical
problems. There is suggestive evidence that marijuana may alter
the delivery process, reduce intrauterine weight gain by the fetus,
or affect visual and neurological excitatory responses. All of
these findings need to be confirmed in their relationship to
marijuana use, especially since marijuana is freauently used in
conjunction with tobacco and alcohol, which have their own
deleterious effects on the fetus.
These data will be further discussed in the Discussion and
Recommendations Section.
AUTHORS
Jacqueline P. Ludford, M.S.
Research Analysis Branch
Office of Science
National Institute on Drug Abuse
Rockville, Maryland 20857
Monique C. Braude, Ph.D.
Biomedical Branch
Division of Preclinical Research
National Institute on Drug Abuse
Rockville, Maryland 20857
Effects of Cannabinoids on Gene
Expression
Gary S. Stein, Ph.D., and Janet L. Stein, Ph.D.
INTRODUCTION
In this article we will consider approaches that have been taken
and can be taken to assess the influence of cannabinoids and other
abused substances on the genome and on gene expression. This is a
problem central to understanding drug-induced effects on a broad
spectrum of biological processes since numerous modifications in
cell structure and function, which have been reported to be
associated with abused substances, either a) affect expression of
genetic sequence or b) are a reflection of modifications in gene
expression. Within this context we should emphasize that
drug-induced perturbations in gene expression can result from
alterations in the genome itself or from modifications in the
transcription, processing, or translation of genetic information.
This article will be divided into three parts. First, by way of
introduction, we will summarize the experimental basis for our
current concepts of the eukaryotic genome and eukaryotic gene
control. Second, we will review approaches that nave been taken
to address the influence of cannabinoids on gene expression. We
will then consider approaches, which can be taken and should be
pursued, to further define in molecular terms cannabinoid-induced
effects on the structure, organization, and regulation of specific
genes.
It is our strong conviction that there are many long-standing and
to date unresolved questions related to cannabinoid-induced
effects on genes and gene control. Answers to these questions are
essential to understand the influence of abused substances from
the standpoints of immediate health hazards and, perhaps even more
important, of hereditary effects. It is encouraging tnat during
the past several years our understanding of genes and gene
regulation in cells has evolved dramatically, largely through a
number of highly innovative cellular and molecular approaches that
have been taken to address the organization and regulation of
eukaryotic genes. We are therefore now in a position,
conceptually and technologically, to apply these approaches to
assessing the effects of abused substances on the genome and on
gene expression--particularly in human cells.
I. Genes and Gene Regulation
Several of the experimental observations which historically have
served as the basis for our current concept of gene expression are
summarized in table 1. While in general terms, these classical
observations have a direct bearing on the manner in which
eukaryotic genes are controlled, a number of subtle qualifications
based on recent results provide explanations for long-standing
inconsistencies in our understanding of eukaryotic gene regulation.
TABLE 1
Gene Expression in Eukaryotic Cells
1. All diploid cells in an organism contain the same amount of
DNA.
2. All diploid cells contain identical genetic information.
3. Limited expression of genes in all cells.
4. Differences and similarities in expression of specific genes
in differentiated cells.
5. Ability to modify expression of specific genes.
The initial experimental Observations which led to models for
eukaryotic gene control were that all diploid cells of an organism
contain the same amount of DNA and that the DNA sequences present
in all diploid cells are identical. Equally important were the
observations that all cells express only a limited number of
genetic sequences and that those genes expressed reflect general
metabolic requirements shared by all living cells as well as
specialized requirements of differentiated cells. For example,
almost all cells express genes encoding enzymes involved in
intermediary metabolism while expression of globin genes is
restricted to erythropoietic cells. Superimposed upon this
preferential expression of specific genes, which permits cells to
execute their specialized biological/biochemical functions, is the
flexibility to permit variation in those genes expressed in
response to modifications of cellular activities or cellular
requirements. It was these observations that led to experimental
pursuit of the mechanisms by which defined genetic sequences are
selectively expressed while others are held in a nontranscribed
structure, conformation, and transcriptional state. What we must
now additionally take into consideration is that expression of
genes can be associated with modifications in the organization
and/or the representation of genetic sequences
Our views of eukaryotic genes and eukaryotic gene regulation are
constantly evolving. Structural and functional properties of
genes are largely inseparable, as reflected by a functional
relationship between the organization and expression of genetic
sequences. The eukaryotic genome is a protein-DNA complex, both
chromosomal proteins and DNA being essential for genome structure,
and alterations in the interactions of chromosomal proteins with
DNA in turn affect transcription or the transcriptional potential
of specific genes. It is becoming increasingly apparent that the
eukaryotic genome is not a static macromolecular complex, but
rather is subject to modifications in organization, structure, and
conformation which influence expression. There are different
types of genes, those which encode proteins and those for which
the products are ribosomal or transfer RNAs. Moreover, there are
substantial differences in the organization of various genetic
sequences, ranging in complexity from genes whose encoded proteins
are represented by contiguous nucleotide sequences to genes from
which the transcripts must undergo numerous splicing steps to
generate functional messenger RNAs. It has been well documented
that different genes are under different types of regulation.
Likewise, there may be some differences in the structure and
regulation of the same genes in different biological situations.
It therefore follows that to address regulation of eukaryotic
genetic sequences it is necessary to consider control at several
levels, which have been delineated in table 2. By definition gene
expression encompasses an extensive range of cellular structures
and biochemical processes, beginning in the nucleus at the DNA
double helix and terminating with a completely processed and
functional protein or RNA molecule. This presents a problem of an
extremely complex nature, and cannabinoid-induced lesions may
reside at any one or a combination of cellular levels.
Within the nucleus key steps in control of gene readout reside at
the level of the genome and in the nucleoplasm. Cannabinoids may
influence the structure and/or function of DNA nucleotide
sequences which constitute structural genes or their components,
in which case regions of the genome coding for defined proteins
would not be transcribed or the transcripts would not be appropriately
processed and translated into functional proteins. In
addition, cannabinoid-induced alterations in genetic sequences
coding for the synthesis of ribosomal RNAs, tRNAs, or purported
"regulatory RNAs" must be considered. Cannabinoio-induced
alterations may also become apparent in the nucleotides contained
within regulatory sequences or within those sequences involved in
punctuating the genetic code. In an overall evaluation of tne
mechanisms by which cannabinoids may modify genes, one must bear
in mind that there are four general categories of changes in the
nucleotide bases which are prevalent--base substitutions,
modifications of preexisting bases, base additions, and base
deletions. Recent evidence for additions, deletions, and
amplification of nucleotide sequences, as well as rearrangements
of genetic sequences in conjunction with expression, necessitates
serious consideration of quantitative and qualitative modifications
in DNA as potential regulatory events, and hence targets for
drug-induced perturbations in gene expression. Within this
context drug-mediated effects on DNA methylation, which has been
implicated in structural/transcriptional properties of genetic
sequences, should not be overlooked.
In evaluating the implications of cannabinoid-associated DNA
sequence modifications, one must critically determine the
influence of these drugs on the capability of the cell to repair
its DNA correctly. The repair process may itself introduce or
amplify errors.
Cannabinoid-induced modifications in gene expression may also
result from changes in macromolecules, principally chromosomal
proteins, which interact with DNA and are intimately involved with
the structural and transcriptional properties of the genome.
Variations of these proteins and their mode of association with
other genome components may be attributable to alterations in
amino acid sequences as well as to post-translational modifications
such as acetylation, methylation, phosphorylation, and
ADP-ribosylation. It should be kept in mind that cannabinoidinduced
changes in the metabolism of acetate, methyl, phosphate,
and ADP-ribose groups may be caused by variations in genetically
coded enzymes which are responsible for the addition and removal
of these moieties from genome-associated proteins. In addition,
some of these post-translational modifications of chromosomal
proteins may occur, at least in part, by nonenzymatic mechanisms
Another class of macromolecules which possess the ability to
influence readout as as function of cannabinoid treatment are the
RNA polymerases. Here, cannabinoid-induced changes may reside in
any one or several of the polymerases, in any one or several of
the subunits of the given polymerase, or in "factors" which
influence the specificity or efficiency of the enzyme.
A complex system which contains numerous focal points for
cannabinoid-induced lesions in the expression of genetic
information is that which is utilized in the processing of RNA
molecules. This is a multicomponent system consisting of:
a) endo- and exonucleases which cleave and degrade ribonucleotide
sequences during RNA precursor processing; b) enzymes modifying
ribonucleotide bases; c) nucleotidyl exotransferases which utilize
the 3' and 5' ends of RNA molecules as primers for addition of
nontemplated ribonucleotides; and a) proteins which complex with
RNAs or precursors thereof and are involved with enzymatic
modifications of transcripts, export of transcripts from the
nucleus to the cytoplasm, or assembly of functional translational
complexes. Such processing occurs in the three principal classes
of RNA molecules--ribosomal RNAs, messenger RNAs, and transfer
RNAs. While these reactions generally occur in the nucleoplasm,
they have also been reported to take place, to some extent, in the
cytoplasm.
Cannabinoid-induced aberrations in gene expression may also result
from perturbations in the equally complex cellular protein synthesizing
and processing machinery which resides primarily in the
cytoplasm. This may involve lesions in ribosomal and transfer
RNAs, in ribosomal proteins, in the extensive range of "translational
factors," and in enzymes involved in the assembly and/or
activation of proteins. Enzymes involved with protein turnover
constitute targets often overlooked when considering potentially
important sites for cannabinoid-induced lesions in gene expression.
From the preceding discussion it should be apparent that
cannabinoid-induced modifications in gene expression may result
from perturbations in a broad spectrum of macromolecular,
biosynthetic processes in the nucleus as well as in the
cytoplasm. Any step in the elaboration and processing of genetic
information is a potential target for a drug-induced lesion. Do
cannabinoids modify the structure or composition of the genome?
Do cannabinoids modify which genes are transcribed and which
remain silent? Do cannabinoids affect the efficiency or fidelity
of transcription? Are RNA processing steps modified by
cannabinoids? Do these drugs act at the translational level? The
key to addressing these questions is availability of high
resolution procedures for detecting cannabinoid-induced changes in
gene expression at various levels, and equally important, for
determining if drug-induced perturbations in gene expression are
functional or nonfunctional
II. Effect of Cannaoinoids on the Genome
A. Composition of the Genome
The eukaryotic genome exists in the form of a protein-DNA complex
(Stein et al. 1974, 1975); hence, an assessment of cannabinoidinduced
effects on the composition of the genome requires
evaluating the influence of cannabinoids on both DNA and
chromosomal proteins. It is also necessary to consider the
influence of cannabinoids on both chromatin and on chromosomes
since these represent interchangeable modes of genome packaging.
Several laboratories have investigated the effects of cannabinoids
on chromosome morphology and on the cellular representation of
specific chromosomes. Yet, to date this remains an area where
considerable controversy exists. The critical issues are whether
cannabinoids exhibit clastogenic activity, that is, induce
chromosome breaks, and/or whether cannabinoids act as mitotic
poisons. The latter effect would imply drug-induced action,
direct or indirect, on the mitotic apparatus or on the region of
the chromosome where attachment of spindle fibers occurs--
centromeric DNA or centromere-associated chromosomal proteins.
The mutagenic nature of cannabinoid-induced chromosomal lesions
also remains to be resolved. An indepth review of these
chromosome-related effects of cannabinoids is covered in the
chapter by Morishima in this volume.
An examination of the influence of cannabinoids on chromosomal
proteins indicates that the relative composition of both histones
and nonhistone chromosomal proteins is not significantly altered.
However, psychoactive and nonpsychoactive cannabinoids appear to
bring about a dose-dependent decrease in the synthesis of some
chromosomal polypeptides (Mon et al. 1981a,b). These results tend
to suggest that while cannabinoids do not affect the relative
cellular levels of specific histones, which are the molecules
primarily responsible for DNA packaging, these drugs may affect
the ability of cells to express genes which code for histone
proteins and/or affect histone protein turnover. Nonhistone
chromosomal proteins, which are involved in structural, enzymatic,
and regulatory action at the level of the genome, may be similarly
affected following cannabinoid treatment. Variations observed in
the extent to which chromosomal proteins are acetylated following
cannabinoid treatment can be related to changes in the nature of
chromosomal protein-DNA interaction, which may in turn reflect
drug-induced modification in chromatin structure and/or in
transcriptional properties of the genome. The studies carried out
to date do indeed suggest possible drug-induced changes in genome
composition, structure, and function but the data are of a
correlative nature. Although as discussed above, cannabinoidinduced
alterations in gene organization are not an unrealistic
expectation, experimental data to substantiate or eliminate such a
possibility are lacking.
B. Gene Expression
Two approaches have been undertaken in several laboratories,
including ours, to study cannabinoid-induced effects on gene
expression (Blevins and Regan 1976; Carchman et al. 1976a,b;
Desoize et al. 1979; End et al. 1977; Green et al. 1983; Nahas et
al. 1974a,b, 1977; Lemberger 1973; McClean and Zimmerman 1976; Mon
et al. 1978, 1981a,b; Nahas and Desoize 1974; Nahas and Paton
1979; White et al. 1976; Zimmerman and McClean 1973; Zimerman and
Zimmerman 1976; Zimmerman et al. 1979). Early in vivo studies
suggested that cannaoinoid treatment brings about dose-dependent
inhibition of 3H-thymidine incorporation into DNA, 3H-uridine
incorporation into RNA, and 3H-leucine incorporation into
protein. However, these results, particularly the 3H-uridine
and 3H-leucine results, are complicated by the influence of
cannabinoids on the ribonucleotide and amino acid precursor pools--
perhaps in part a reflection of cannabinoid-induced effects on
cellular membranes. In vitro transcription stuaies carried out
using isolated nuclei, DNA, or chromatin suggest that such
preparations from untreated control and cannabinoid-treated cells
do not differ significantly with respect to their ability to
synthesize RNA. Interpretation of the latter studies is not
complicated by drug-related effects on precursor pools; however,
from these in vitro experiments it is possible to conclude only
that the overall transcriptional capacity of the genome is
refractory to cannabinoid treatment, and no indication of possible
cannabinoid-induced effects on the qualitative nature of gene
transcription can be gleaned. Furthermore, caution should be
exercised in interpreting results from in vitro studies because
the fidelity of the transcription process and the transcripts by
necessity must be carefully evaluated.
Recently, to assess more definitively the influence of
cannabinoids on gene expression, we examined the effect of
THC on the representation of RNA transcripts from two defined
genetic sequences, histone genes and ribosomal genes, in several
human cell lines. Levels of cellular histone mRNAs and ribosomal
RNAs were assayed by hybridization with cloned genomic human
histone and ribosomal genes under conditions where quantitation
was not influenced by nucleotide precursor pools. Our results
suggest that -THC causes a dose-dependent reduction in the
cellular representation of histone mRNA sequences. This
drug-induced reduction is at least to some extent selective
because cellular levels of ribosomal RNAs are not affected. We
have also observed that the cannabinoid-induced effect on histone
gene expression is less pronounced in human cells with active
drug-metabolizing systems.
Human histone and ribosomal genes represent two distinct types of
genetic sequences which differ with respect to their organization,
regulation, and functions. Human nistone genes are a family of
moderately reiterated genetic sequences--approximately 40 copies
per haploid genome. Each histone mRNA is transcribed from a set
of contiguous nucleotide sequences (unspliced), and histone gene
expression is related to cell proliferation. The gene products,
the histone proteins, are required for packaging several yaras of
DNA into "nucleosomes" where they are contained in a nucleus only
several microns in diameter. These histone proteins are necessary
for genome replication (to package newly replicated DNA) and
additionally play a role in the control of gene expression. The
human ribosomal genes are represented as a reiterated set of
sequences and the final gene products are the major structural RNA
species associated with large and small ribosomal subunits. In
contrast to the histone genes, where the primary transcripts
undergo a minimal amount of processing, the 5.8S, 18S, and 28S
ribosomal RNAs are derived from a 45S precursor via a series of
post-transcriptional cleavages.
Initially, the steady state levels of histone mRNAs were
determined in exponentially growing human cervical carcinoma
cells, HeLa S3 cells, following treatment with increasing
concentrations of -THC. Total cellular RNAs were fractionated
electrophoretically in 1.5% agarose gels (Rave et al. 1979),
transferred to nitrocellulose (Southern 1975) and hybridized with
32p-labeled [nick-translated (Maniatis et al. 1975)] cloned
genomic human histone sequences (Sierra et al. 1982). The levels
of histone mRNAs were then assayed autoradiographically.
Isolation of total celluar RNA permits greater than 90% recovery,
circumventing loss of RNA through nuclease activity and physical
manipulations which generally occur during subcellular fractionation.
Because the hybridization probe is radiolabeled
in vitro rather than the cellular RNAs in vivo, quantitation of
RNAs is not complicated by the intracellular ribonucleotide
precursor pools. RNA samples are quantitated spectrophotometrically
prior to electrophoretic fractionation and the extent
of transfer to nitrocellulose is monitored by ethidium bromide
staining and/or ultraviolet shadowing prior to and following
diffusion transfer. The efficiency of transfer to nitrocellulose
by the procedure used in these experiments has been monitored by
transfer of 32p-labeled DNA and shown to be greater than 95%.
The data in figures 1 and 2 clearly indicate that -THC brings
about a dose-dependent decrease in the representation of mRNAs for
the four core histone proteins, H2A, H2B, H3, and H4. Shown in
figure 1A is a hybridization signal obtained when 50 μg of
nitrocellulose-immobilized, total cellular HeLa cell RNAs from
control, and -THC treated, cells are hybridized with a cloned
human DNA sequence (pFF435) encoding H2A, H2B, and H3 histone
mRNAs. While the levels of H2A, H2B, an H3 histone mRNAs
isolated from cells treated with 10 μM -THC are not below
those from nondrug-treated or vehicle-treated controls, a marked
inhibition (greater than 80%--see table 3) is observed in cells
treated with 30 μM and 40 μM drug concentrations. Verification
that equivalent amounts of all' RNA samples were fractionated can
be gleaned from figure 1B which shows similar levels of ethidium
bromide staining of all RNAs and from figure 1C which shows
similar levels of all RNAs by ultraviolet shadowing. It should be
noted that because equivalent amounts of RNA from control and
drug-treated cells were analyzed, the data in figure 1A reflect a
dose-dependent, -THC-mediated inhibition in the relative
representation of three core histone mRNA species. A
dose-dependent inhibition of the absolute amounts of H2A, H2B, and
H3 histone mRNA/cell, with pronounced inhibition evident at 30 and
40 uM drug concentrations, was also observed when equivalent
aliquots (by volume) of RNA extracts from equivalent numbers of
control and -THC-treated cells were similarly analyzed (see
figure 1D). The data in figure 2 are results from experiments in
which total cellular RNAs from control and -THC-treated
exponentially growing HeLa S3 cells were analyzed by hybriaization
with cloned genomic H3 (pF0 422) (figure 2A) or H4 (pF0 108A)
(figure 2B) histone sequences. Consistent with the results shown
in figure 1, a greater than 80% inhibition in the representation
of H3 and H4 histone mRNAs was observed following treatment with
30 and 40 μM drug concentrations
The influence of -THC on the levels of histone mRNAs was then
studied in normal human diploid cells (WI38 human diploid fibroblasts)
and in SV40-transformed WI38 cells. A dose-dependent,
drug-induced decrease in the levels of all four core nistone mRNAs
was observed in both normal human diploid fibroblasts and in
SV40-transformed human diploid fibroblasts--a cannabinoid-induced
inhibition similar to that seen in HeLa S3 cells. As shown in
figures 3A and B, when total cellular RNAs from control and
Ag-THC-treated WI38 cells are hybriaized with 32p-labeled
pFF435, a plasmid containing cloned human genomic H2A, H2B, and H3
histone coding sequences, decreased levels of histone mRNAs are
observed in both normal WI38 and in SV40-transformed WI38 cells
treated with 30 and 40 μM drug concentrations. Confirmation of
the -THC-mediated inhibition of core histone mRNA levels in
normal and SV40-transformed WI38 human diploid fibroblasts can be
seen in figures 3C and 3D as well as in figures 3E and 3F where
similar drug-induced inhibitions in the representation of H3 and
H4 mRNAs, respectively, were observed.
The levels of H2A, H2B, H3, and H4 histone mRNAs were similarly
assayed in A549 human lung carcinoma cells after treatment with
Ag-THC. These cells have been reported to have active drug
metabolizing systems and to efficiently metabolize polycyclic
hydrocarbon-containing carcinogens. A pronounced decrease in the
inhibitory effect of 9-THC on the representation of core
histone mRNAs was observed in A549 cells compared With HeLa S3
cells and WI38 cells (normal and SV40-transformed). It is
unlikely that the reduced sensitivity of A549 cells to cannabinoid
treatment is attributable to changes in drug uptake. Tne
intracellular levels of -THC in SV40-transformed WI38 cells
and in A549 cells, when monitored by intracellular incorporation
of 3H THC (table 4) do not reflect the differences seen in
histone mRNA levels (figures 3 and 4).
Several lines of experimental evidence suggest that the -THCinduced
reductions in histone mRNA levels we have observed in
normal and transformed human cells are not merely a reflection of
a general, nonspecific cannabinoid-induced inhibition in RNA
synthesis. As reported previously, the cannabinoid-induced
inhibition of 3H-uridine incorporation into total cellular RNAs
largely reflects a drug-induced influence on the intracellular
nucleotide precursor pool rather than an effect on cellular RNA
metabolism (Mon et al. 1981a,b). The absence of a significant
quantitative effect of psychoactive and nonpsychoactive
cannabinoids on levels of nuclear (Mon et al. 1981a,b) or
chromatin (Mon et al. 1981a,b) transcription in vitro further
suggests that these drugs do not interfere with the general levels
or rates of cellular RNA synthesis
The inability of -THC, at concentrations between 10 and 40 μM,
to modify the levels of ribosomal RNAs provides more direct
evidence for some extent of specificity to the cannabinoidmediated
decrease in histone mRNA levels. In all experiments
reported in this paper, the representation of 18S and 28S
ribosomal RNAs was monitored in control, and in -THC-treated,
cells by staining gels with ethidium bromide and by ultraviolet
shadowing. A typical example of a gel showing the levels of the
major ribosomal RNAs in control and in drug-treated cells is shown
in figures 1B and 1C. Additionally, when electrophoretically
fractionated cellular RNAs from control and -THC-treated cells
were hybridized with 32P-labeled cloned human 18S (LS-2) and 28s
(LS-6) ribosomal RNA coding sequences, a dose-dependent decrease
in the representation of these RNAs was not observed. Figures 5A
and 5B show no change in the levels of 28S ribosomal RNAs from
-THC-treated HeLa and SV40-transformed WI38 cells in the same
RNA samples where greater than 80% reduction was observed for the
representation of core histone mRNAs in treated cells. Figure 5C
shows unchanged levels of 18S ribosomal RNA in these same cells
following hybridization with 32P-labeled human 18s ribosomal DNA.
A long-standing question has been whether cannabinoids influence
the expression of specific genetic sequences. While cannabinoidinduced
effects on cell structure and function, coupled with
cannabinoid-mediated modification in macromolecular biosynthesis,
are consistent with such a contention, direct experimental
evidence for an effect of cannabinoids on expression of specific
genes has to date not been reported. In this paper we present
data which indicate that treatment of exponentially growing normal
diploid and transformed human cells with Ag-THC results in a
dose-dependent decrease in the representation of histone mRNAs,
with a decreased sensitivity of cells with highly developed drug
metabolizing systems. This cannabinoid-mediated reduction of
cellular histone mRNA levels does not simply reflect a general
decrease in cellular mRNA levels or in cellular RNA metabolism.
We also present data indicating that the levels of ribosomal RNAs
are not altered by the concentrations of -THC used in our
studies, and we have reported previously that general levels of in
vitro and in vivo RNA synthesis are not quantitatively affected by
either psychoactive or nonpsychoactive cannabinoids.
While our results clearly indicate that -THC preferentially
inhibits expression of histone genes, the levels at which
regulation is perturbed and the biological implications of this
cannabinoid-mediated effect remain to be resolved. The reduction
in cellular levels of histone mRNAs after cannabinoid treatment
may be attributable to alterations in mRNA stability, transcription,
or processing of histone transcripts. Additionally,
drug-induced structural modifications in the histone genes and in
their flanking regulatory sequences should also be considered
within this context. By analogy with other moderately reiterated
eukaryotic sequences which have been shown to unaergo structural
modifications in conjunction with phenotypic changes, cannabinoidinduced
effects on the structural features of human histone genes
specific genetic sequences other than histone sequences is
affected by cannabinoids is also an open-ended question--one which
is particularly important because the organization and regulation
of the moderately reiterated human histone genes differ considerably
from those of the more complex spliced single copy genes.
From a biological standpoint the selective effect of -THC on
expression of histone genes may be understandable. Expression of
histone genes has been shown to be temporally and functionally
coupled with DNA replication (Stein et al. 1979; Stein and Borun
1972; Wu and Bonner 1981), and cannabinoids have been shown to
bring about a dose-dependent inhibition in cell proliferation
(Blevins and Regan 1976; Carchman et al. 1976a,b; Desoize et al.
1979; End et al. 1977; Green et al. 1983; Nahas et al. 1974a,b,
1977; Lemberger 1973; McClean and Zimmerman 1976; Mon et al. 1978,
1981a,b; Nahas and Desoize 1974; Nahas and Paton 1979; White et
al. 1976; Zimmerman and McClean 1973; Zimmerman and Zimmerman
1976; Zimmerman et al. 1979). In fact, in the normal and
transformed cell lines we have examined, the extent to which
histone mRNA levels are affected by -THC is paralleled by the
extent to which proliferative activity is affected by
cannabinoids. It remains to be determined whether expression of
other genetic sequences, whose expression is prerequisite for DNA
replication or mitotic division, are preferentially inhibited by
cannabinoids. Equally important is whether the cannabinoidmediated
modifications in cellular histone mRNA levels are
attributable to a direct effect on the histone genes or the
transcripts, or alternatively, whether the effects of cannabinoids
on histone gene expression are indirect, e.g., acting initially on
other genetic sequences or cellular macromolecules.
III. Approaches to Defining Effects of Cannabinoids on Specific
Genes
Cannabinoid-induced modifications in cell structure and function
have been well-documented as have a series of physiological
effects resulting from such drug-induced cellular changes. Two
pivotal biological processes which have been shown to be
dramatically influenced by cannabinoids are endocrine function and
cell proliferation--both of which have been reviewed in this
monograph. Moreover, these are not unrelated processes since in
many cases proliferation is responsive to hormonal control.
Understanding the manner in which drug-induced alterations in gene
expression are brought about should provide insight into the
molecular basis of cannabinoid-related modifications in cellular
function.
Since cannabinoid-induced alterations in gene expression can
result from changes in the organization of genetic sequences
and/or in the manner in which genetic information is transcribed
and processed, a critical and systematic evaluation of the
influence of cannabinoids on the structure and expression of
specific genetic sequences, particularly in human cells, should be
a high priority. Understanding cannabinoid-induced effects on
human gene organization and expression is prerequisite to
evaluating possible short-term, long-term, and hereditable
disorders that may arise from the use of these drugs either
therapeutically or as abused substances. Of equal importance,
despite the history of fragmentary and often controversial reports
of cannabinoid-induced modification in genome-related phenomena
(e.g., chromosomal changes, alterations in RNA synthesis, etc.),
we are now in a position to address these issues directly and
definitively. Availability of a series of cloned human genes
permits evaluation of drug-related effects on specific genes, on
defined regions of genes, and on transcription and processing of
genetic information. Examples of ways in which cloned genetic
sequences can be utilized as high resolution probes for the
identification and quantitation of several specific human gene
transcripts were presented in the previous section of this chapter.
It will be instructive to focus efforts where possible on human
studies; for example, drug-induced effects on the organization of
specific genetic sequences or regions thereof can be performed
using DNA from only 20 ml of blood. Thereby the opportunity is
available to determine the effects of cannabinoias on the genomes
of subjects participating in endocrine function and behavior
studies. A number of normal and tumor-derived human cell lines
are available and should be utilized to complement such an
approach. By combining both intact organism and cell culture
approaches, it is possible to draw on the physiological reality of
the organism and the biochemical simplicity of isolated cells. It
would also be appropriate to concentrate efforts on evaluating
drug-induced effects on a limited series of genetic sequences,
those related to proliferation and endocrine function, where
phenotypic effects are well understood and the information
obtained can be integrated with other ongoing investigations.
In summary, we have attempted to discuss a series of high
resolution approaches and procedures which can provide important
information regarding the influence of cannabinoids on genome
structure and function. Examples of the applications of several
of these approaches and procedures have been presented in an
attempt to document the feasibility of their implementation. We
are highly optimistic that in the next few years our understanding
of the genetic effects of cannabinoids at the cellular and
molecular level will be significantly enhanced. These same
approaches can be implemented for assaying the influence of
unfractionated marijuana extracts, psychoactive and
nonpsychoactive components of marijuana, natural and synthetic
cannabinoids, and cannabinoid metabolites as well as other abused
substances, individually or in conjunction with cannabinoids.
REFERENCES
Blevins, R.D., and Regan, J.D. -tetrahydrocannabinol: Effect
of macromolecular synthesis in human and other mammalian cells.
In: Nahas, G.G., ed. Marihuana: Chemistry, Biochemistry and
Cellular Effects. New York: Springer, 1976 p. 213.
Carchman, R.A.; Harris, L.S.; and Munson, A.E. The inhibition of
DNA synthesis by cannabinoids. Cancer Research 36:95, 1976a.
Carchman, R.A.; Warner, W.; White, A.C.; and Harris, L.S.
Cannabinoids and neoplastic growth. In: Nahas, G.G., ea.
Marihuana: Chemistry, Biochemistry and Cellular Effects.
New York: Springer, 1976b, p. 329.
Desoize, B.; Leger, C.; and Nahas, G. Plasma membrane inhioition
of macromolecular precursor transport by THC. Biochem Pharmac
28:1113, 1979.
End, D.W.; Thoursen, K.; Dewey, W.L.; and Carchman, R.A. A
comparative study of the disposition of (-)- -tetrahydrocannabinol
in neuroblastoma and glioma cells in tissue culture:
Relation to cellular impairment. Molec Pharmacol 13:864, 1977.
Green, L.; Marashi, F.; Stein, J.L.; and Stein, G.S. A decreased
influence of cannabinoids on macromolecular biosynthesis and
cell proliferation in human cells which metabolize polycyclic
hydrocarbon carcinogens. Anticancer Research, in press, 1983.
Lemberger, L. Tetrahydrocannabinol metabolism in man. Drug
Metabolism and Disposition 1:461, 1973.
Maniatis, T.; Jeffrey, A.; and Kleid, D.G. Nucleotide sequence of
the rightward operator of phage. Proc Natl Acad Sci USA
72:1184-1188, 1975.
McClean, D.K., and Zimmerman, A.M. Action of -tetrahydrocannabinol
on cell division and macromolecular synthesis in
division-synchronized protozoa. Pharmacology 14:307-321, 1976.
Mon, M.J.; Haas, A.E.; Stein, J.L.; and Stein, G.S. Influence of
psychoactive and nonpsychoactive cannabinoids on cell
proliferation and macromolecular biosynthesis in human cells.
Biochem Pharmacol 30:31, 1981a.
Mon, M.J.; Haas, A.E.; Stein, J.L.; and Stein, G.S. Influence of
psychoactive and nonpsychoactive-cannabinoids on cnromatin
structure and function in human cells. Biochem Pharmacol 30-45
1981b.
Mon, M.J.; Jansing, R.L.; Doggett, S.; Stein, J.L.; and Stein, G.S.
Influence of -tetrahydrocannabinol on cell proliferation and
macromolecular biosynthesis in human cells. Biochem Pharmacol
27:1759-1765, 1978.
Nahas, G.G., and Desoize, B. Effect inhibiteur du 5 n-amylresorcinol
sur la transformation lymphoblastique. C R Acad Sci
Paris Series D 279:1607-1608, 1974.
Nahas, G.C., and Paton, W.D.M. Marihuana: Biological Effects.
Oxford: Pergamon Press, 1979.
Nahas, G.G.; Armand, J.P.; and Hsu, J. Inhibition in vitro de la
blastogenese des lymphocytes T par le -tetrahydrocannabinol.
C R Acad Sci Paris Series D 278:679, 1974a.
Nahas, G.G.; Desoize, B.; Armand, J.P.; Hsu. J.; and Morishima, A.
Inhibition in vitro de-la transformation iymphocytaire par
divers cannaibinoids naturels. C R Acad Sci Paris Series D
279:785-787, 1974b.
Nahas, G.G.; Morishima, A.; and Desoize, B. Effects of cannabinoids
of macromolecular synthesis and replication of cultured
lymphocytes. Fed Proc 36:1748-1752, 1977.
Rave, N.; Crkvenjakov, R.; and Boedtker, H. Identification of
procollagen mRNAs transferred to diazobenzyloxymethyl paper from
formaldehyde agarose gels. Nucleic Acids Res 6:3559-3567, 1979.
Sierra, F.; Lichtler, A.; Marashi, F.; Rickles, R.; Van Dyke, T.;
Clark, S.; Wells, J.; Stein, G.; and Stein, J. Organization of
human histone genes. Proc Natl Acad Sci USA 79:1795, 1982.
Southern, E.M. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. Jour Mol Biol
98:503, 1975.
Stein, G.S., and Borun, T.W. The synthesis of acidic chromosomal
proteins during the cell cycle of HeLa S3 cells. I. The
accelerated accumulation of acidic residual nuclear protein
before the initiation of DNA replication. Jour Cell Biol
52:292, 1972.
Stein, G.S.; Hochhauser, S.; and Stein, J.L. Histone genes: Their
structure and control. In: Busch, H.; ed. The Cell Nucleus.
Chromatin, Part D. New York:
Stein, G.S.; Spelsberg, T.C.;
Academic Press 1979, p. 259
and Kleinsmith, L.J. Nonhistone
chromosomal proteins and gene regulation. Science 183:817, 1974.
Stein, G.S.; Stein, J.L.; and Kleinsmith, L.J. Chromosomal
proteins and gene regulation. Scientific Amer 232.46, 1575.
White, A.C.; Munson, J.A.; Munson, A.E.; and Carchman, R.A.
Effects of -tetrahydrocannabinol in lewis lung adenocarcinoma
cells in tissue culture. Jour Natl Cancer Inst 56:6556, 1976.
Wu, R.S., and Bonner, W.M. Separation of salistone syntnesis
from S-phase histone synthesis in dividing cells. Cell
27:321-331, 1981.
Zimmerman, A.M., and McClean, D.K. Action of narcotic and
hallucinogenic agents on the cell cycle. In: Zimmerman. A.M.:
Padilla, G.M.; and Cameron, I.L., eds. Drugs and the Cell Cycle
New York: Academic press, 1973, pp. 67-94.
Zimmerman, A.M., and Zimmerman, S.B. The influence of marihuana on
eukaryote cell growth and development. In: Nahas, G.G.. ed.
Marihuana: Chemistry, Biochemistry and Cellular Effects.
New York: Springer, 1976. pp. 195-205.
Zimmerman, A.M.; Bruce, W.R.; and Zimmerman, S. Effects of
cannabinoids on sperm morphology. Pharmacology 18(3):143, 1979.
ACKNOWLEDGMENTS
These studies were supported by research grants DA-01188 and
DA-02033 from the National Institute on Drug Abuse. We are
indebted to Drs. Roy Schmickel and Golder Wilson for providing us
with cloned human ribosomal genes.
AUTHORS
Gary S. Stein, Ph.D.
Department of Biochemistry and Molecular Biology
University of Florida College of Medicine
Gainesville, Florida 32610
Janet L. Stein, Ph.D.
Department of Immunology and Medical Microbiology
University of Florida College of Medicine
Gainesville, Florida 32610
Source: Marijuana Effects On The Endocrine And Reproductive Systems
Monique C. Braude, Ph.D.
Jacqueline P. Ludford, M.S.
National Institute on Drug Abuse
NIDA Research Monograph 44
A RAUS Review Report
DEPARTMENT OF HEALTH AND HUMAN SERVICES
Public Health Service
Alcohol, Drug Abuse, and Mental Health Administration
National Institute on Drug Abuse
5600 Fishers Lane
Rockville, Maryland 20857
NIDA Research Monographs are prepared by the research divisions of the
National Institute on Drug Abuse and published by its Office of Science. The
primary objective of the scenes is to provide critical reviews of research
problem areas and techniques. the content of state of-the-art conferences,
Integrative research reviews and signifigant original research Its
dual publication emphasis IS rapid and targeted dissemination to the
scientific and professional community
Editorial Advisory Board
Avram Goldstein, M.D.
Addiction Research Foundation
Palo Alto. Colifornia
Jerome Jaffe, M.D.
University of Connecticut School of Medicine
Formington, Connecticut
Reese T. Jones, M.D.
Langley Porter Neuropsychiatric lnstitute
University of California
San Francisco, California
Jack Mendelson, M.D.
Alcohol once Drug Abuse Research Center
Harvard Medical School
McLeon Hospital
Belmont, Massachusetts
Helen Nowlis, Ph.D.
Rochester. New York
Lee Robins, Ph.D.
Washington University School of Medicine
St Louis, Missouri
NIDA Research Monograph Series
William Pollin, M.D.
DIRECTOR, NIDA
Jack Durell, M.D.
ASSOCIATE DIRECTOR, OFFICE OF SCIENCE, NIDA
EDITOR-IN-CHIEF
Eleanor W. Waldrop
MANAGING EDITOR
Parklawn Building, 5600 Fishers Lane Rockville Maryland 20857
ACKNOWLEDGMENT
This monograph is based upon papers and discussion from the RAUS
Review Conference on the Endocrine and Reproductive Effects of
Marijuana, held March 1 and 2, 1983, in Rockville, Maryland. The
conference was sponsored by the Office of Science and the Division
of Preclinical Research, National Institute on Drug Abuse.
COPYRIGHT STATUS
The National Institute on Drug Abuse has obtained permission from
the copyright holders to reproduce certain previously published
material as noted in the text. Further reproduction of this
material is prohibited without specific permission of the copyright
holders. All other material in this volume except quoted passages
from copyrighted sources is in the public domain and may be used or
reproduced without permission from the Institute or the authors.
Citation of the source is appreciated.
Opinions expressed in this volume are those of the authors and do
not necessarily reflect the opinions or official policy of the
National Institute on Drug Abuse or any other part of the U.S.
Department of Health and Human Services.
The U.S. Government does not endorse or favor any specific
commercial product or commodity. Trade or proprietary names
appearing in this publication are used only because they are
considered essential in the context of the studies reported herein.
Library of Congress catalog card number 83-,600600
DHHS publication number (HDM)84-1278
Printed 1984
NIDA Research Monographs are indexed in the Index Medicus. They
are selectively included in the coverage of American Statistics
Index, Biosciences Information Service, Chemical Abstracts, Current
Contents, Psychological Abstracts, and Psychopharmacology Abstracts.
Preface
The Research Analysis and Utilization System (RAUS) is designed to
serve four functions:
Collect and systematically classify the findings of all
intramural and extramural research supported by the
hational institute on Drug Abuse (NIDA);
Evaluate the findings in selected areas of particular
interest and formulate a state-of-the-art review by a
panel of scientific peers;
Disseminate findings to researchers in the field and to
administrators, planners, instructors, and other
interested persons;
Provide a feedback mechanism to NIDA staff and planners so
that the administration and monitoring of the NIDA
research program reflect the very latest knowledge gleaned
from research in the field.
Since there is a limit to the number of reseach findings that can
be intensively reviewed annually, four subject areas are chosen
each year to undergo a thorough examination. Distinguished
scientists in the selected field are provided with copies of
reports from NIDA-funded research and invited to add any
information derived from the literature and from their own research
in order to formulate a comprehensive vick of the field. Each
reviewer is charged with writing a state-of-the-art paper in his or
her particular subject area. These papers, together with a summary
of the discussions and recommendations which take place at the
review meeting, make up a RAUS Review Report in the NIDA Research
Monograph series.
The subject of the effects of marijuana on the endocrine and reproductive
systems was chosen for a RAUS review in Fiscal Year 1983
because marijuana use is so widespread among American youth and,
therefore, is of great programmatic importance to NIDA. Increased
prevalence of marijuana use over the past decade has been accompanied
by decreasing age of first use, and there is grave public health
concern about its effects on youth who are undergoing maturation of
their reproductive systems at about the same time as they are likely
to begin using marijuana. Since there is a growing body of research
on the subject, it became incumbent upon NIDA to gather the knowledge
that was available, evaluate it, and disseminate it. The results of
the RAUS review are presented in this monograph.
Dr. Monique C. Braude served as the scientific chairperson for the
meeting . Jacqueline P. Ludford is the RAUS coordinator
Executive Summary
Jacqueline P. Ludford, M.S., and Monique C. Braude, Ph.D.
Isolated reports of impaired sexual behavior, lowered hormone
levels, and abnormal offspring in animals after administration of
marijuana or its active principles, such as delta-9-tetrahydrocannabinol
(THC), prompted a review of current findings
relevant to the effects of marijuana on genetics and reproduction.
A RAUS review meeting was held on March l-2, 1983, and reviewers
were charged with evaluating the state of the art in the following
areas:
Effects of Cannabinoids
on Gene Expression
Effects of Cannabis and
Natural Cannabinoids on
Chromosomes and Ova
Effects of Marijuana in the
Male: Preclinical Studies
Endocrine Aspects of Cannabinoid
Action in Female Subprimates:
Search for Site of Action
Acute, Short Term, and Chronic
Effects on the Female
Primate Reproductive Function
Effects of Marijuana on Dr. Jack Mendelson
Neuroendocrine Function McLean Hospital/
in Human Males and Females Harvard University
Marijuana: Prenatal
Exposure in the Human
Dr. Gary Stein
University of
Florida
Dr. Akira Morishima
Columbia University
Dr. Jack Harclerode
Bucknell University
Dr. Lee Tyrey
Duke University
Dr. Carol Smith
Uniformed Services
University of the
Health Sciences
Dr. Katherine Tennes
University of
Colorado
Dr. Stein first discussed the importance of the preferential
expression of specific genes which can be associated with
modifications in the organization and/or representation of genetic
sequences. To address regulation of eukaryotic genetic sequences,
one must consider control at several cellular levels in the nucleus
and cytoplasm. Gene expression encompasses an extensive range of
cellular structures and biochemical processes starting in the
nucleus with DNA and terminating with the RNA molecule. Dr. Stein
then reviewed his studies on the effect of cannabinoids on the
genome, and on gene expression. To assess more definitively the
influences of cannabinoids on gene expression, Stein's group
examined the effect of Delta-9-THC on the representation of RNA
transcripts from two defined genetic sequences, histone genes and
ribosomal genes, in several human cell lines. They found that THC
causes a dose-dependent reduction in the cellular representation of
histone mRNA sequences at the higher concentrations used in their
assay. The extent to which cannabinoids affect the expression of
specific genetic sequences other than histone sequences is still an
open-ended question. Understanding the manner in which druginduced
alterations in gene expression are brought about should,
Stein believes, provide insight into the molecular basis of
cannabinoid-related modifications in cellular function.
Dr. Morishima reviewed the various reports on studies of the
effects of marijuana and natural cannabinoids on chromosomes. The
evidence from the available cytogenetic studies suggests that
cannabis and cannabinoids are extremely weak clastogens, i.e.,
produce little chromosome breakage and that their clastogenic
effects become apparent only in appropriately sensitive test
systems such as primary spermatocytes and bone marrow cells,
whereas the human lymphocyte system is relatively insensitive to
their clastogenic effects. He then reported the results of his
recent studies on the effects of THC on mice ova, showing that
chronic administration of THC to sexually developing mice produced
an increase in abnormal ova, although the percentage of increase
was small. It appears that this increase in degenerated ova was
caused by their inability to successfully undergo first cleavage
division, probably affecting the process of meiosis. Following his
review of the studies which reported errors of chromosome
separation (ECS) and of this recent data, Morishima now proposes a
new concept that cannabis and cannabinoids in vitro and in vivo act
by disrupting the meiotic as well as the mitotic processes.
Dr. Harclerode focused on the effect of chronic exposure of
laboratory animals to cannabinoids, with emphasis on the male
reproductive system. The reports in the literature of reduction in
reproductive organ weights are accompanied by reports that show
that the auality and ouantity of sperm produced by the testis are
affected by cannabinoids. Treatment of mice with THC for as little
as 5 days resulted in reduction of sperm production and appearance
of abnormal sperm. This was often accompanied by a decrease in
testicle and seminal vesicle weights. Two gonadotropins, LH and
FSH, secreted by the pituitary gland are of major importance to
reproduction in the male. A single hypothalamic factor, the
gonadotropin-releasing hormone (GnRH) is believed to be responsible
for the release of LH and FSH. THC induces a block of GnRH release
which results in lowered LH and FSH, thus reducing testosterone
production by the Leydig cells of the testis. Other hormones that
might have a synergistic or antagonistic effect upon reproduction
in the male are the adrenal cortical hormones, thyroid hormones,
growth hormones, and prolactin. THC appears to depress prolactin,
thyroid gland function, and growth hormone while elevating adrenal
cortical steroids.
Dr. Tyrey reviewed the effects of cannabinoids, primarily THC, on
the female reproductive function in subprimates and discussed
cannabinoid action on the target organs (uterus and ovary) as well
as on the CNS and the hypothalamic-pituitary axis. He concluded
that the search for a site of cannabinoid action in subprimates has
raised the possibility of cannabinoid effects at each level of the
female reproductive system. He feels that the early suggestion
that THC may have a direct "estrogen-like action on the uterus" has
not been substantiated by later studies which failed to show that
THC interacts with the estrogen cytoplasmic receptor. However,
there is now evidence that THC alters the secretion of reproductive
pituitary hormones (LH, prolactin) and of ACTH through effects in
the brain.
Dr. Smith reviewed the acute and chronic effects of THC on the
reproductive function of the female primate. She pointed out that
studies in these species show that cannabinoids inhibit secretion
of LH and FSH as well as prolactin. These changes in pituitary
hormones produce decreases in sex steroid hormones and cause
changes in ovulation. Dr. Smith also emphasized that the principal
site of action of cannabinoids is the hypothalamus. Her recent
findings show, however, that these cannabinoid effects are
reversible in sexually mature animals when drug treatment is
terminated and that there is development of tolerance to the
effects of THC after chronic administration. This may explain why
evidence of disrupting effects on female reproductive function has
been scarce. She observed that, in humans, it is not yet known how
much disruption of reproductive hormone levels is necessary for
changes in human fertility and sexual function to become apparent,
and she emphasized the need for clinical studies in female
marijuana smokers.
There are technical problems in obtaining data on the effect of
marijuana on human reproductive systems, but Dr. Mendelson reported
on the available data and on his own considerable work in this
area. In his study of the effects of marijuana on pituitarygonadal
hormones in human males, he found that the use of marijuana
alone did not suppress testosterone levels. Similar carefully
controlled studies of human female hormone levels are scarce,
although reports from animals suggest, as mentioned above, that THC
may produce a significant decrease in prolactin and LH. There have
been reports that marijuana users had shorter menstrual cycles and
lower prolactin levels than age-matched nonusers. A residential
ward study in human females is now underway, and some preliminary
observations were reported at the meeting.
Dr. Tennes reviewed the current knowledge about the effect of
marijuana on human pregnancy and fetal development. Although 10%
to 37% of pregnant women report use of marijuana, evidence
regarding its effect is confounded by the use of other substances,
nutrition, truthfulness of the woman's recall and report of the
amount of use, changes in use during pregnancy and the trimester
when these changes occurred, and a host of other technical
problems. There is suggestive evidence that marijuana may alter
the delivery process, reduce intrauterine weight gain by the fetus,
or affect visual and neurological excitatory responses. All of
these findings need to be confirmed in their relationship to
marijuana use, especially since marijuana is freauently used in
conjunction with tobacco and alcohol, which have their own
deleterious effects on the fetus.
These data will be further discussed in the Discussion and
Recommendations Section.
AUTHORS
Jacqueline P. Ludford, M.S.
Research Analysis Branch
Office of Science
National Institute on Drug Abuse
Rockville, Maryland 20857
Monique C. Braude, Ph.D.
Biomedical Branch
Division of Preclinical Research
National Institute on Drug Abuse
Rockville, Maryland 20857
Effects of Cannabinoids on Gene
Expression
Gary S. Stein, Ph.D., and Janet L. Stein, Ph.D.
INTRODUCTION
In this article we will consider approaches that have been taken
and can be taken to assess the influence of cannabinoids and other
abused substances on the genome and on gene expression. This is a
problem central to understanding drug-induced effects on a broad
spectrum of biological processes since numerous modifications in
cell structure and function, which have been reported to be
associated with abused substances, either a) affect expression of
genetic sequence or b) are a reflection of modifications in gene
expression. Within this context we should emphasize that
drug-induced perturbations in gene expression can result from
alterations in the genome itself or from modifications in the
transcription, processing, or translation of genetic information.
This article will be divided into three parts. First, by way of
introduction, we will summarize the experimental basis for our
current concepts of the eukaryotic genome and eukaryotic gene
control. Second, we will review approaches that nave been taken
to address the influence of cannabinoids on gene expression. We
will then consider approaches, which can be taken and should be
pursued, to further define in molecular terms cannabinoid-induced
effects on the structure, organization, and regulation of specific
genes.
It is our strong conviction that there are many long-standing and
to date unresolved questions related to cannabinoid-induced
effects on genes and gene control. Answers to these questions are
essential to understand the influence of abused substances from
the standpoints of immediate health hazards and, perhaps even more
important, of hereditary effects. It is encouraging tnat during
the past several years our understanding of genes and gene
regulation in cells has evolved dramatically, largely through a
number of highly innovative cellular and molecular approaches that
have been taken to address the organization and regulation of
eukaryotic genes. We are therefore now in a position,
conceptually and technologically, to apply these approaches to
assessing the effects of abused substances on the genome and on
gene expression--particularly in human cells.
I. Genes and Gene Regulation
Several of the experimental observations which historically have
served as the basis for our current concept of gene expression are
summarized in table 1. While in general terms, these classical
observations have a direct bearing on the manner in which
eukaryotic genes are controlled, a number of subtle qualifications
based on recent results provide explanations for long-standing
inconsistencies in our understanding of eukaryotic gene regulation.
TABLE 1
Gene Expression in Eukaryotic Cells
1. All diploid cells in an organism contain the same amount of
DNA.
2. All diploid cells contain identical genetic information.
3. Limited expression of genes in all cells.
4. Differences and similarities in expression of specific genes
in differentiated cells.
5. Ability to modify expression of specific genes.
The initial experimental Observations which led to models for
eukaryotic gene control were that all diploid cells of an organism
contain the same amount of DNA and that the DNA sequences present
in all diploid cells are identical. Equally important were the
observations that all cells express only a limited number of
genetic sequences and that those genes expressed reflect general
metabolic requirements shared by all living cells as well as
specialized requirements of differentiated cells. For example,
almost all cells express genes encoding enzymes involved in
intermediary metabolism while expression of globin genes is
restricted to erythropoietic cells. Superimposed upon this
preferential expression of specific genes, which permits cells to
execute their specialized biological/biochemical functions, is the
flexibility to permit variation in those genes expressed in
response to modifications of cellular activities or cellular
requirements. It was these observations that led to experimental
pursuit of the mechanisms by which defined genetic sequences are
selectively expressed while others are held in a nontranscribed
structure, conformation, and transcriptional state. What we must
now additionally take into consideration is that expression of
genes can be associated with modifications in the organization
and/or the representation of genetic sequences
Our views of eukaryotic genes and eukaryotic gene regulation are
constantly evolving. Structural and functional properties of
genes are largely inseparable, as reflected by a functional
relationship between the organization and expression of genetic
sequences. The eukaryotic genome is a protein-DNA complex, both
chromosomal proteins and DNA being essential for genome structure,
and alterations in the interactions of chromosomal proteins with
DNA in turn affect transcription or the transcriptional potential
of specific genes. It is becoming increasingly apparent that the
eukaryotic genome is not a static macromolecular complex, but
rather is subject to modifications in organization, structure, and
conformation which influence expression. There are different
types of genes, those which encode proteins and those for which
the products are ribosomal or transfer RNAs. Moreover, there are
substantial differences in the organization of various genetic
sequences, ranging in complexity from genes whose encoded proteins
are represented by contiguous nucleotide sequences to genes from
which the transcripts must undergo numerous splicing steps to
generate functional messenger RNAs. It has been well documented
that different genes are under different types of regulation.
Likewise, there may be some differences in the structure and
regulation of the same genes in different biological situations.
It therefore follows that to address regulation of eukaryotic
genetic sequences it is necessary to consider control at several
levels, which have been delineated in table 2. By definition gene
expression encompasses an extensive range of cellular structures
and biochemical processes, beginning in the nucleus at the DNA
double helix and terminating with a completely processed and
functional protein or RNA molecule. This presents a problem of an
extremely complex nature, and cannabinoid-induced lesions may
reside at any one or a combination of cellular levels.
Within the nucleus key steps in control of gene readout reside at
the level of the genome and in the nucleoplasm. Cannabinoids may
influence the structure and/or function of DNA nucleotide
sequences which constitute structural genes or their components,
in which case regions of the genome coding for defined proteins
would not be transcribed or the transcripts would not be appropriately
processed and translated into functional proteins. In
addition, cannabinoid-induced alterations in genetic sequences
coding for the synthesis of ribosomal RNAs, tRNAs, or purported
"regulatory RNAs" must be considered. Cannabinoio-induced
alterations may also become apparent in the nucleotides contained
within regulatory sequences or within those sequences involved in
punctuating the genetic code. In an overall evaluation of tne
mechanisms by which cannabinoids may modify genes, one must bear
in mind that there are four general categories of changes in the
nucleotide bases which are prevalent--base substitutions,
modifications of preexisting bases, base additions, and base
deletions. Recent evidence for additions, deletions, and
amplification of nucleotide sequences, as well as rearrangements
of genetic sequences in conjunction with expression, necessitates
serious consideration of quantitative and qualitative modifications
in DNA as potential regulatory events, and hence targets for
drug-induced perturbations in gene expression. Within this
context drug-mediated effects on DNA methylation, which has been
implicated in structural/transcriptional properties of genetic
sequences, should not be overlooked.
In evaluating the implications of cannabinoid-associated DNA
sequence modifications, one must critically determine the
influence of these drugs on the capability of the cell to repair
its DNA correctly. The repair process may itself introduce or
amplify errors.
Cannabinoid-induced modifications in gene expression may also
result from changes in macromolecules, principally chromosomal
proteins, which interact with DNA and are intimately involved with
the structural and transcriptional properties of the genome.
Variations of these proteins and their mode of association with
other genome components may be attributable to alterations in
amino acid sequences as well as to post-translational modifications
such as acetylation, methylation, phosphorylation, and
ADP-ribosylation. It should be kept in mind that cannabinoidinduced
changes in the metabolism of acetate, methyl, phosphate,
and ADP-ribose groups may be caused by variations in genetically
coded enzymes which are responsible for the addition and removal
of these moieties from genome-associated proteins. In addition,
some of these post-translational modifications of chromosomal
proteins may occur, at least in part, by nonenzymatic mechanisms
Another class of macromolecules which possess the ability to
influence readout as as function of cannabinoid treatment are the
RNA polymerases. Here, cannabinoid-induced changes may reside in
any one or several of the polymerases, in any one or several of
the subunits of the given polymerase, or in "factors" which
influence the specificity or efficiency of the enzyme.
A complex system which contains numerous focal points for
cannabinoid-induced lesions in the expression of genetic
information is that which is utilized in the processing of RNA
molecules. This is a multicomponent system consisting of:
a) endo- and exonucleases which cleave and degrade ribonucleotide
sequences during RNA precursor processing; b) enzymes modifying
ribonucleotide bases; c) nucleotidyl exotransferases which utilize
the 3' and 5' ends of RNA molecules as primers for addition of
nontemplated ribonucleotides; and a) proteins which complex with
RNAs or precursors thereof and are involved with enzymatic
modifications of transcripts, export of transcripts from the
nucleus to the cytoplasm, or assembly of functional translational
complexes. Such processing occurs in the three principal classes
of RNA molecules--ribosomal RNAs, messenger RNAs, and transfer
RNAs. While these reactions generally occur in the nucleoplasm,
they have also been reported to take place, to some extent, in the
cytoplasm.
Cannabinoid-induced aberrations in gene expression may also result
from perturbations in the equally complex cellular protein synthesizing
and processing machinery which resides primarily in the
cytoplasm. This may involve lesions in ribosomal and transfer
RNAs, in ribosomal proteins, in the extensive range of "translational
factors," and in enzymes involved in the assembly and/or
activation of proteins. Enzymes involved with protein turnover
constitute targets often overlooked when considering potentially
important sites for cannabinoid-induced lesions in gene expression.
From the preceding discussion it should be apparent that
cannabinoid-induced modifications in gene expression may result
from perturbations in a broad spectrum of macromolecular,
biosynthetic processes in the nucleus as well as in the
cytoplasm. Any step in the elaboration and processing of genetic
information is a potential target for a drug-induced lesion. Do
cannabinoids modify the structure or composition of the genome?
Do cannabinoids modify which genes are transcribed and which
remain silent? Do cannabinoids affect the efficiency or fidelity
of transcription? Are RNA processing steps modified by
cannabinoids? Do these drugs act at the translational level? The
key to addressing these questions is availability of high
resolution procedures for detecting cannabinoid-induced changes in
gene expression at various levels, and equally important, for
determining if drug-induced perturbations in gene expression are
functional or nonfunctional
II. Effect of Cannaoinoids on the Genome
A. Composition of the Genome
The eukaryotic genome exists in the form of a protein-DNA complex
(Stein et al. 1974, 1975); hence, an assessment of cannabinoidinduced
effects on the composition of the genome requires
evaluating the influence of cannabinoids on both DNA and
chromosomal proteins. It is also necessary to consider the
influence of cannabinoids on both chromatin and on chromosomes
since these represent interchangeable modes of genome packaging.
Several laboratories have investigated the effects of cannabinoids
on chromosome morphology and on the cellular representation of
specific chromosomes. Yet, to date this remains an area where
considerable controversy exists. The critical issues are whether
cannabinoids exhibit clastogenic activity, that is, induce
chromosome breaks, and/or whether cannabinoids act as mitotic
poisons. The latter effect would imply drug-induced action,
direct or indirect, on the mitotic apparatus or on the region of
the chromosome where attachment of spindle fibers occurs--
centromeric DNA or centromere-associated chromosomal proteins.
The mutagenic nature of cannabinoid-induced chromosomal lesions
also remains to be resolved. An indepth review of these
chromosome-related effects of cannabinoids is covered in the
chapter by Morishima in this volume.
An examination of the influence of cannabinoids on chromosomal
proteins indicates that the relative composition of both histones
and nonhistone chromosomal proteins is not significantly altered.
However, psychoactive and nonpsychoactive cannabinoids appear to
bring about a dose-dependent decrease in the synthesis of some
chromosomal polypeptides (Mon et al. 1981a,b). These results tend
to suggest that while cannabinoids do not affect the relative
cellular levels of specific histones, which are the molecules
primarily responsible for DNA packaging, these drugs may affect
the ability of cells to express genes which code for histone
proteins and/or affect histone protein turnover. Nonhistone
chromosomal proteins, which are involved in structural, enzymatic,
and regulatory action at the level of the genome, may be similarly
affected following cannabinoid treatment. Variations observed in
the extent to which chromosomal proteins are acetylated following
cannabinoid treatment can be related to changes in the nature of
chromosomal protein-DNA interaction, which may in turn reflect
drug-induced modification in chromatin structure and/or in
transcriptional properties of the genome. The studies carried out
to date do indeed suggest possible drug-induced changes in genome
composition, structure, and function but the data are of a
correlative nature. Although as discussed above, cannabinoidinduced
alterations in gene organization are not an unrealistic
expectation, experimental data to substantiate or eliminate such a
possibility are lacking.
B. Gene Expression
Two approaches have been undertaken in several laboratories,
including ours, to study cannabinoid-induced effects on gene
expression (Blevins and Regan 1976; Carchman et al. 1976a,b;
Desoize et al. 1979; End et al. 1977; Green et al. 1983; Nahas et
al. 1974a,b, 1977; Lemberger 1973; McClean and Zimmerman 1976; Mon
et al. 1978, 1981a,b; Nahas and Desoize 1974; Nahas and Paton
1979; White et al. 1976; Zimmerman and McClean 1973; Zimerman and
Zimmerman 1976; Zimmerman et al. 1979). Early in vivo studies
suggested that cannaoinoid treatment brings about dose-dependent
inhibition of 3H-thymidine incorporation into DNA, 3H-uridine
incorporation into RNA, and 3H-leucine incorporation into
protein. However, these results, particularly the 3H-uridine
and 3H-leucine results, are complicated by the influence of
cannabinoids on the ribonucleotide and amino acid precursor pools--
perhaps in part a reflection of cannabinoid-induced effects on
cellular membranes. In vitro transcription stuaies carried out
using isolated nuclei, DNA, or chromatin suggest that such
preparations from untreated control and cannabinoid-treated cells
do not differ significantly with respect to their ability to
synthesize RNA. Interpretation of the latter studies is not
complicated by drug-related effects on precursor pools; however,
from these in vitro experiments it is possible to conclude only
that the overall transcriptional capacity of the genome is
refractory to cannabinoid treatment, and no indication of possible
cannabinoid-induced effects on the qualitative nature of gene
transcription can be gleaned. Furthermore, caution should be
exercised in interpreting results from in vitro studies because
the fidelity of the transcription process and the transcripts by
necessity must be carefully evaluated.
Recently, to assess more definitively the influence of
cannabinoids on gene expression, we examined the effect of
THC on the representation of RNA transcripts from two defined
genetic sequences, histone genes and ribosomal genes, in several
human cell lines. Levels of cellular histone mRNAs and ribosomal
RNAs were assayed by hybridization with cloned genomic human
histone and ribosomal genes under conditions where quantitation
was not influenced by nucleotide precursor pools. Our results
suggest that -THC causes a dose-dependent reduction in the
cellular representation of histone mRNA sequences. This
drug-induced reduction is at least to some extent selective
because cellular levels of ribosomal RNAs are not affected. We
have also observed that the cannabinoid-induced effect on histone
gene expression is less pronounced in human cells with active
drug-metabolizing systems.
Human histone and ribosomal genes represent two distinct types of
genetic sequences which differ with respect to their organization,
regulation, and functions. Human nistone genes are a family of
moderately reiterated genetic sequences--approximately 40 copies
per haploid genome. Each histone mRNA is transcribed from a set
of contiguous nucleotide sequences (unspliced), and histone gene
expression is related to cell proliferation. The gene products,
the histone proteins, are required for packaging several yaras of
DNA into "nucleosomes" where they are contained in a nucleus only
several microns in diameter. These histone proteins are necessary
for genome replication (to package newly replicated DNA) and
additionally play a role in the control of gene expression. The
human ribosomal genes are represented as a reiterated set of
sequences and the final gene products are the major structural RNA
species associated with large and small ribosomal subunits. In
contrast to the histone genes, where the primary transcripts
undergo a minimal amount of processing, the 5.8S, 18S, and 28S
ribosomal RNAs are derived from a 45S precursor via a series of
post-transcriptional cleavages.
Initially, the steady state levels of histone mRNAs were
determined in exponentially growing human cervical carcinoma
cells, HeLa S3 cells, following treatment with increasing
concentrations of -THC. Total cellular RNAs were fractionated
electrophoretically in 1.5% agarose gels (Rave et al. 1979),
transferred to nitrocellulose (Southern 1975) and hybridized with
32p-labeled [nick-translated (Maniatis et al. 1975)] cloned
genomic human histone sequences (Sierra et al. 1982). The levels
of histone mRNAs were then assayed autoradiographically.
Isolation of total celluar RNA permits greater than 90% recovery,
circumventing loss of RNA through nuclease activity and physical
manipulations which generally occur during subcellular fractionation.
Because the hybridization probe is radiolabeled
in vitro rather than the cellular RNAs in vivo, quantitation of
RNAs is not complicated by the intracellular ribonucleotide
precursor pools. RNA samples are quantitated spectrophotometrically
prior to electrophoretic fractionation and the extent
of transfer to nitrocellulose is monitored by ethidium bromide
staining and/or ultraviolet shadowing prior to and following
diffusion transfer. The efficiency of transfer to nitrocellulose
by the procedure used in these experiments has been monitored by
transfer of 32p-labeled DNA and shown to be greater than 95%.
The data in figures 1 and 2 clearly indicate that -THC brings
about a dose-dependent decrease in the representation of mRNAs for
the four core histone proteins, H2A, H2B, H3, and H4. Shown in
figure 1A is a hybridization signal obtained when 50 μg of
nitrocellulose-immobilized, total cellular HeLa cell RNAs from
control, and -THC treated, cells are hybridized with a cloned
human DNA sequence (pFF435) encoding H2A, H2B, and H3 histone
mRNAs. While the levels of H2A, H2B, an H3 histone mRNAs
isolated from cells treated with 10 μM -THC are not below
those from nondrug-treated or vehicle-treated controls, a marked
inhibition (greater than 80%--see table 3) is observed in cells
treated with 30 μM and 40 μM drug concentrations. Verification
that equivalent amounts of all' RNA samples were fractionated can
be gleaned from figure 1B which shows similar levels of ethidium
bromide staining of all RNAs and from figure 1C which shows
similar levels of all RNAs by ultraviolet shadowing. It should be
noted that because equivalent amounts of RNA from control and
drug-treated cells were analyzed, the data in figure 1A reflect a
dose-dependent, -THC-mediated inhibition in the relative
representation of three core histone mRNA species. A
dose-dependent inhibition of the absolute amounts of H2A, H2B, and
H3 histone mRNA/cell, with pronounced inhibition evident at 30 and
40 uM drug concentrations, was also observed when equivalent
aliquots (by volume) of RNA extracts from equivalent numbers of
control and -THC-treated cells were similarly analyzed (see
figure 1D). The data in figure 2 are results from experiments in
which total cellular RNAs from control and -THC-treated
exponentially growing HeLa S3 cells were analyzed by hybriaization
with cloned genomic H3 (pF0 422) (figure 2A) or H4 (pF0 108A)
(figure 2B) histone sequences. Consistent with the results shown
in figure 1, a greater than 80% inhibition in the representation
of H3 and H4 histone mRNAs was observed following treatment with
30 and 40 μM drug concentrations
The influence of -THC on the levels of histone mRNAs was then
studied in normal human diploid cells (WI38 human diploid fibroblasts)
and in SV40-transformed WI38 cells. A dose-dependent,
drug-induced decrease in the levels of all four core nistone mRNAs
was observed in both normal human diploid fibroblasts and in
SV40-transformed human diploid fibroblasts--a cannabinoid-induced
inhibition similar to that seen in HeLa S3 cells. As shown in
figures 3A and B, when total cellular RNAs from control and
Ag-THC-treated WI38 cells are hybriaized with 32p-labeled
pFF435, a plasmid containing cloned human genomic H2A, H2B, and H3
histone coding sequences, decreased levels of histone mRNAs are
observed in both normal WI38 and in SV40-transformed WI38 cells
treated with 30 and 40 μM drug concentrations. Confirmation of
the -THC-mediated inhibition of core histone mRNA levels in
normal and SV40-transformed WI38 human diploid fibroblasts can be
seen in figures 3C and 3D as well as in figures 3E and 3F where
similar drug-induced inhibitions in the representation of H3 and
H4 mRNAs, respectively, were observed.
The levels of H2A, H2B, H3, and H4 histone mRNAs were similarly
assayed in A549 human lung carcinoma cells after treatment with
Ag-THC. These cells have been reported to have active drug
metabolizing systems and to efficiently metabolize polycyclic
hydrocarbon-containing carcinogens. A pronounced decrease in the
inhibitory effect of 9-THC on the representation of core
histone mRNAs was observed in A549 cells compared With HeLa S3
cells and WI38 cells (normal and SV40-transformed). It is
unlikely that the reduced sensitivity of A549 cells to cannabinoid
treatment is attributable to changes in drug uptake. Tne
intracellular levels of -THC in SV40-transformed WI38 cells
and in A549 cells, when monitored by intracellular incorporation
of 3H THC (table 4) do not reflect the differences seen in
histone mRNA levels (figures 3 and 4).
Several lines of experimental evidence suggest that the -THCinduced
reductions in histone mRNA levels we have observed in
normal and transformed human cells are not merely a reflection of
a general, nonspecific cannabinoid-induced inhibition in RNA
synthesis. As reported previously, the cannabinoid-induced
inhibition of 3H-uridine incorporation into total cellular RNAs
largely reflects a drug-induced influence on the intracellular
nucleotide precursor pool rather than an effect on cellular RNA
metabolism (Mon et al. 1981a,b). The absence of a significant
quantitative effect of psychoactive and nonpsychoactive
cannabinoids on levels of nuclear (Mon et al. 1981a,b) or
chromatin (Mon et al. 1981a,b) transcription in vitro further
suggests that these drugs do not interfere with the general levels
or rates of cellular RNA synthesis
The inability of -THC, at concentrations between 10 and 40 μM,
to modify the levels of ribosomal RNAs provides more direct
evidence for some extent of specificity to the cannabinoidmediated
decrease in histone mRNA levels. In all experiments
reported in this paper, the representation of 18S and 28S
ribosomal RNAs was monitored in control, and in -THC-treated,
cells by staining gels with ethidium bromide and by ultraviolet
shadowing. A typical example of a gel showing the levels of the
major ribosomal RNAs in control and in drug-treated cells is shown
in figures 1B and 1C. Additionally, when electrophoretically
fractionated cellular RNAs from control and -THC-treated cells
were hybridized with 32P-labeled cloned human 18S (LS-2) and 28s
(LS-6) ribosomal RNA coding sequences, a dose-dependent decrease
in the representation of these RNAs was not observed. Figures 5A
and 5B show no change in the levels of 28S ribosomal RNAs from
-THC-treated HeLa and SV40-transformed WI38 cells in the same
RNA samples where greater than 80% reduction was observed for the
representation of core histone mRNAs in treated cells. Figure 5C
shows unchanged levels of 18S ribosomal RNA in these same cells
following hybridization with 32P-labeled human 18s ribosomal DNA.
A long-standing question has been whether cannabinoids influence
the expression of specific genetic sequences. While cannabinoidinduced
effects on cell structure and function, coupled with
cannabinoid-mediated modification in macromolecular biosynthesis,
are consistent with such a contention, direct experimental
evidence for an effect of cannabinoids on expression of specific
genes has to date not been reported. In this paper we present
data which indicate that treatment of exponentially growing normal
diploid and transformed human cells with Ag-THC results in a
dose-dependent decrease in the representation of histone mRNAs,
with a decreased sensitivity of cells with highly developed drug
metabolizing systems. This cannabinoid-mediated reduction of
cellular histone mRNA levels does not simply reflect a general
decrease in cellular mRNA levels or in cellular RNA metabolism.
We also present data indicating that the levels of ribosomal RNAs
are not altered by the concentrations of -THC used in our
studies, and we have reported previously that general levels of in
vitro and in vivo RNA synthesis are not quantitatively affected by
either psychoactive or nonpsychoactive cannabinoids.
While our results clearly indicate that -THC preferentially
inhibits expression of histone genes, the levels at which
regulation is perturbed and the biological implications of this
cannabinoid-mediated effect remain to be resolved. The reduction
in cellular levels of histone mRNAs after cannabinoid treatment
may be attributable to alterations in mRNA stability, transcription,
or processing of histone transcripts. Additionally,
drug-induced structural modifications in the histone genes and in
their flanking regulatory sequences should also be considered
within this context. By analogy with other moderately reiterated
eukaryotic sequences which have been shown to unaergo structural
modifications in conjunction with phenotypic changes, cannabinoidinduced
effects on the structural features of human histone genes
specific genetic sequences other than histone sequences is
affected by cannabinoids is also an open-ended question--one which
is particularly important because the organization and regulation
of the moderately reiterated human histone genes differ considerably
from those of the more complex spliced single copy genes.
From a biological standpoint the selective effect of -THC on
expression of histone genes may be understandable. Expression of
histone genes has been shown to be temporally and functionally
coupled with DNA replication (Stein et al. 1979; Stein and Borun
1972; Wu and Bonner 1981), and cannabinoids have been shown to
bring about a dose-dependent inhibition in cell proliferation
(Blevins and Regan 1976; Carchman et al. 1976a,b; Desoize et al.
1979; End et al. 1977; Green et al. 1983; Nahas et al. 1974a,b,
1977; Lemberger 1973; McClean and Zimmerman 1976; Mon et al. 1978,
1981a,b; Nahas and Desoize 1974; Nahas and Paton 1979; White et
al. 1976; Zimmerman and McClean 1973; Zimmerman and Zimmerman
1976; Zimmerman et al. 1979). In fact, in the normal and
transformed cell lines we have examined, the extent to which
histone mRNA levels are affected by -THC is paralleled by the
extent to which proliferative activity is affected by
cannabinoids. It remains to be determined whether expression of
other genetic sequences, whose expression is prerequisite for DNA
replication or mitotic division, are preferentially inhibited by
cannabinoids. Equally important is whether the cannabinoidmediated
modifications in cellular histone mRNA levels are
attributable to a direct effect on the histone genes or the
transcripts, or alternatively, whether the effects of cannabinoids
on histone gene expression are indirect, e.g., acting initially on
other genetic sequences or cellular macromolecules.
III. Approaches to Defining Effects of Cannabinoids on Specific
Genes
Cannabinoid-induced modifications in cell structure and function
have been well-documented as have a series of physiological
effects resulting from such drug-induced cellular changes. Two
pivotal biological processes which have been shown to be
dramatically influenced by cannabinoids are endocrine function and
cell proliferation--both of which have been reviewed in this
monograph. Moreover, these are not unrelated processes since in
many cases proliferation is responsive to hormonal control.
Understanding the manner in which drug-induced alterations in gene
expression are brought about should provide insight into the
molecular basis of cannabinoid-related modifications in cellular
function.
Since cannabinoid-induced alterations in gene expression can
result from changes in the organization of genetic sequences
and/or in the manner in which genetic information is transcribed
and processed, a critical and systematic evaluation of the
influence of cannabinoids on the structure and expression of
specific genetic sequences, particularly in human cells, should be
a high priority. Understanding cannabinoid-induced effects on
human gene organization and expression is prerequisite to
evaluating possible short-term, long-term, and hereditable
disorders that may arise from the use of these drugs either
therapeutically or as abused substances. Of equal importance,
despite the history of fragmentary and often controversial reports
of cannabinoid-induced modification in genome-related phenomena
(e.g., chromosomal changes, alterations in RNA synthesis, etc.),
we are now in a position to address these issues directly and
definitively. Availability of a series of cloned human genes
permits evaluation of drug-related effects on specific genes, on
defined regions of genes, and on transcription and processing of
genetic information. Examples of ways in which cloned genetic
sequences can be utilized as high resolution probes for the
identification and quantitation of several specific human gene
transcripts were presented in the previous section of this chapter.
It will be instructive to focus efforts where possible on human
studies; for example, drug-induced effects on the organization of
specific genetic sequences or regions thereof can be performed
using DNA from only 20 ml of blood. Thereby the opportunity is
available to determine the effects of cannabinoias on the genomes
of subjects participating in endocrine function and behavior
studies. A number of normal and tumor-derived human cell lines
are available and should be utilized to complement such an
approach. By combining both intact organism and cell culture
approaches, it is possible to draw on the physiological reality of
the organism and the biochemical simplicity of isolated cells. It
would also be appropriate to concentrate efforts on evaluating
drug-induced effects on a limited series of genetic sequences,
those related to proliferation and endocrine function, where
phenotypic effects are well understood and the information
obtained can be integrated with other ongoing investigations.
In summary, we have attempted to discuss a series of high
resolution approaches and procedures which can provide important
information regarding the influence of cannabinoids on genome
structure and function. Examples of the applications of several
of these approaches and procedures have been presented in an
attempt to document the feasibility of their implementation. We
are highly optimistic that in the next few years our understanding
of the genetic effects of cannabinoids at the cellular and
molecular level will be significantly enhanced. These same
approaches can be implemented for assaying the influence of
unfractionated marijuana extracts, psychoactive and
nonpsychoactive components of marijuana, natural and synthetic
cannabinoids, and cannabinoid metabolites as well as other abused
substances, individually or in conjunction with cannabinoids.
REFERENCES
Blevins, R.D., and Regan, J.D. -tetrahydrocannabinol: Effect
of macromolecular synthesis in human and other mammalian cells.
In: Nahas, G.G., ed. Marihuana: Chemistry, Biochemistry and
Cellular Effects. New York: Springer, 1976 p. 213.
Carchman, R.A.; Harris, L.S.; and Munson, A.E. The inhibition of
DNA synthesis by cannabinoids. Cancer Research 36:95, 1976a.
Carchman, R.A.; Warner, W.; White, A.C.; and Harris, L.S.
Cannabinoids and neoplastic growth. In: Nahas, G.G., ea.
Marihuana: Chemistry, Biochemistry and Cellular Effects.
New York: Springer, 1976b, p. 329.
Desoize, B.; Leger, C.; and Nahas, G. Plasma membrane inhioition
of macromolecular precursor transport by THC. Biochem Pharmac
28:1113, 1979.
End, D.W.; Thoursen, K.; Dewey, W.L.; and Carchman, R.A. A
comparative study of the disposition of (-)- -tetrahydrocannabinol
in neuroblastoma and glioma cells in tissue culture:
Relation to cellular impairment. Molec Pharmacol 13:864, 1977.
Green, L.; Marashi, F.; Stein, J.L.; and Stein, G.S. A decreased
influence of cannabinoids on macromolecular biosynthesis and
cell proliferation in human cells which metabolize polycyclic
hydrocarbon carcinogens. Anticancer Research, in press, 1983.
Lemberger, L. Tetrahydrocannabinol metabolism in man. Drug
Metabolism and Disposition 1:461, 1973.
Maniatis, T.; Jeffrey, A.; and Kleid, D.G. Nucleotide sequence of
the rightward operator of phage. Proc Natl Acad Sci USA
72:1184-1188, 1975.
McClean, D.K., and Zimmerman, A.M. Action of -tetrahydrocannabinol
on cell division and macromolecular synthesis in
division-synchronized protozoa. Pharmacology 14:307-321, 1976.
Mon, M.J.; Haas, A.E.; Stein, J.L.; and Stein, G.S. Influence of
psychoactive and nonpsychoactive cannabinoids on cell
proliferation and macromolecular biosynthesis in human cells.
Biochem Pharmacol 30:31, 1981a.
Mon, M.J.; Haas, A.E.; Stein, J.L.; and Stein, G.S. Influence of
psychoactive and nonpsychoactive-cannabinoids on cnromatin
structure and function in human cells. Biochem Pharmacol 30-45
1981b.
Mon, M.J.; Jansing, R.L.; Doggett, S.; Stein, J.L.; and Stein, G.S.
Influence of -tetrahydrocannabinol on cell proliferation and
macromolecular biosynthesis in human cells. Biochem Pharmacol
27:1759-1765, 1978.
Nahas, G.G., and Desoize, B. Effect inhibiteur du 5 n-amylresorcinol
sur la transformation lymphoblastique. C R Acad Sci
Paris Series D 279:1607-1608, 1974.
Nahas, G.C., and Paton, W.D.M. Marihuana: Biological Effects.
Oxford: Pergamon Press, 1979.
Nahas, G.G.; Armand, J.P.; and Hsu, J. Inhibition in vitro de la
blastogenese des lymphocytes T par le -tetrahydrocannabinol.
C R Acad Sci Paris Series D 278:679, 1974a.
Nahas, G.G.; Desoize, B.; Armand, J.P.; Hsu. J.; and Morishima, A.
Inhibition in vitro de-la transformation iymphocytaire par
divers cannaibinoids naturels. C R Acad Sci Paris Series D
279:785-787, 1974b.
Nahas, G.G.; Morishima, A.; and Desoize, B. Effects of cannabinoids
of macromolecular synthesis and replication of cultured
lymphocytes. Fed Proc 36:1748-1752, 1977.
Rave, N.; Crkvenjakov, R.; and Boedtker, H. Identification of
procollagen mRNAs transferred to diazobenzyloxymethyl paper from
formaldehyde agarose gels. Nucleic Acids Res 6:3559-3567, 1979.
Sierra, F.; Lichtler, A.; Marashi, F.; Rickles, R.; Van Dyke, T.;
Clark, S.; Wells, J.; Stein, G.; and Stein, J. Organization of
human histone genes. Proc Natl Acad Sci USA 79:1795, 1982.
Southern, E.M. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. Jour Mol Biol
98:503, 1975.
Stein, G.S., and Borun, T.W. The synthesis of acidic chromosomal
proteins during the cell cycle of HeLa S3 cells. I. The
accelerated accumulation of acidic residual nuclear protein
before the initiation of DNA replication. Jour Cell Biol
52:292, 1972.
Stein, G.S.; Hochhauser, S.; and Stein, J.L. Histone genes: Their
structure and control. In: Busch, H.; ed. The Cell Nucleus.
Chromatin, Part D. New York:
Stein, G.S.; Spelsberg, T.C.;
Academic Press 1979, p. 259
and Kleinsmith, L.J. Nonhistone
chromosomal proteins and gene regulation. Science 183:817, 1974.
Stein, G.S.; Stein, J.L.; and Kleinsmith, L.J. Chromosomal
proteins and gene regulation. Scientific Amer 232.46, 1575.
White, A.C.; Munson, J.A.; Munson, A.E.; and Carchman, R.A.
Effects of -tetrahydrocannabinol in lewis lung adenocarcinoma
cells in tissue culture. Jour Natl Cancer Inst 56:6556, 1976.
Wu, R.S., and Bonner, W.M. Separation of salistone syntnesis
from S-phase histone synthesis in dividing cells. Cell
27:321-331, 1981.
Zimmerman, A.M., and McClean, D.K. Action of narcotic and
hallucinogenic agents on the cell cycle. In: Zimmerman. A.M.:
Padilla, G.M.; and Cameron, I.L., eds. Drugs and the Cell Cycle
New York: Academic press, 1973, pp. 67-94.
Zimmerman, A.M., and Zimmerman, S.B. The influence of marihuana on
eukaryote cell growth and development. In: Nahas, G.G.. ed.
Marihuana: Chemistry, Biochemistry and Cellular Effects.
New York: Springer, 1976. pp. 195-205.
Zimmerman, A.M.; Bruce, W.R.; and Zimmerman, S. Effects of
cannabinoids on sperm morphology. Pharmacology 18(3):143, 1979.
ACKNOWLEDGMENTS
These studies were supported by research grants DA-01188 and
DA-02033 from the National Institute on Drug Abuse. We are
indebted to Drs. Roy Schmickel and Golder Wilson for providing us
with cloned human ribosomal genes.
AUTHORS
Gary S. Stein, Ph.D.
Department of Biochemistry and Molecular Biology
University of Florida College of Medicine
Gainesville, Florida 32610
Janet L. Stein, Ph.D.
Department of Immunology and Medical Microbiology
University of Florida College of Medicine
Gainesville, Florida 32610
Source: Marijuana Effects On The Endocrine And Reproductive Systems
