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Cannabis demasculinizes rats but is not estrogenic
Pergamon
Life Sciences vol . 17, pp . 1113-1118 Printed in the U.S .A .
Press
CANNABIS DENASCULINIZES RATS BUT IS NOT ESTROGENIC Allan B . Okey and Greg S . Truant Department of Biology University of Windsor Windsor, Ontario CANADA, H9B 3P4
(Received
in final form
Septsimber 2,
1975)
Summary Carmabis resin (54% THC) caused dose-related decreases in the development of testis, prostate and seminal vesicles when administered in the diet to immature male rats . Bioassays and estrogen-receptor studies showed no estrogenic activity in cannabis resin . Demasculinization and possible feminization by cannabis are not due to simple estrogen-like action on male tissues . Introduction Intensive marihuana use by young men has been reported to depress their plasma testosterone levels and spermatogenesis (1) and to lead to abnormal breast growth (2) . In rodents, various cannabis preparations reduce male co latory behavior (3), inhibit spermatogenesis (4), reduce seminal vesicle (4) and prostate weights (5) and inhibit body growth (5-7) . Perhaps the simplest explanation for all these cannabis. effects would be that cannabis components (or their metabolites) have estrogenic properties ; this attractive hypothesis has been proposed (2,7) and widely disseminated . However, using repeated bioassays and in vitro competition for estrogen-receptor sites we find no evidence that canna possesses significant estrogenic activity . -
Ws
Methods and Results Male Wistar rats (Figure 1) fed cannabis resin in their chow during sexual maturation show a linear, dose-related decrease in development of testes, prostate and seminal vesicles . If this were due to estrogenic activity in cannabis, the same dietary doses would be expected to stimulate uterine growth in immature female rats or in ovariectomized adult females . As shown in Figure 2, however, cannabis resin causes a dose-related decrease in uterine weight, even when the uterine weights are corrected for tReloss in body weight which occured at high cannabis doses . By comparison, diethylstilbestrol (DES) caused significant uterine growth in these animals at doses as low as 12 .5 parts per billion in the diet . DES also caused a significant advancement in vaginal opening in immature females ; by day-7 of DES treatment (50 ppb) all vaginas were open while no vaginas opened by that time in any cannabis treated groups .
1113
1114
Dwaaculinization by Caanabir
Vol . 17, No . 7
%lit arase selsbt Memos U dietary lntap of (newels rain (ceMalnieq aaR'Oxirtely .7110 . eaanMnol) was allaitait JAao Purina laboratory Chow rSn to 914 final commentions of 0-500 ports lier olllion (pou) A9-TNC . mal*~Ylstar-stain rate . par cop, era fad tMp diets fm ago 20-days sscrlfto et ago 57-days. Nlgb cannabis door doprasW body 9roru 11p11 .f1tent1yl orge %MOISIS ara am sail as et par 9 body right. correon, 14tion pHfletallta ara gMter than (-) 0.84 for mach lino and NCI Is s19h1fleutiy 1111aar (p < 0.01) . At 100 lion Tm fa the clot sack rat hat M lntate of abolit 0 mal TIC/kg body wlght/doy. 19% ceeabldlol,
sax s
lait
a
o
Legend for Figure 2.
eat .
Uterine wleht assay for estrom activity.
(13)
3 ")=
A. Cannabis real . diets (0-150 We A9-TXC). prepared as 1n Figure 1, were Ired either to ismature female YIStar-Strain rata ( " ) from a 20-0ays through age 27-days or for 15 days to mare WsISr ferles had been ova eetaltod six watt or to Start the diets. Each point ropresents the assn value for 10 rats . Cdrrelat10s coofficonts are greater than (-) 0.83 for weh line and each Is significantly linear (p!(1 .051 . Diethylstilbestrol (DES) used as control standard was fed at âeaea of 0-100 parts per billion t $) =01", ar ovariacto.ited ( " rats as In part A. Correlation Coefficient$ are greater than 0.95 for each line and each 11 significantly linear (pt 0.05) .
a.
1
Vol . 17, No . 7
1115
Damaiculinisation by Caanabis
w ar a 012
001
0.04
9
ep
y
L f
tee
60
go
lu
nid
9aane t, molette revint
Fi
1
plndfn0 of ( )-ntnd101 .11D (( ( )- )) to redan abasol in the of 10 e . Uterine tytosoi (196 .009 n 9 supernatant. 1.5 tee pro ~a n per e rv s r Isoeelrto (rtes Uttar rate) rs 1nttWLW .IM (3,)-EZ at 14 mcontratlor beewen 20 and 4000 piweola per liter . At tir and of incubation . destra~coatod charcoal was toad to moue wlbotmd steroid . 1M amount bound at each concentration was deta, land by liquid scintillation counting of allowance before and after charcoal treatment. 1M p.o1Ue tyteaol protein) and mbar of high-affinity binding sites " aanlet/liter) dissociation constants far the EZratutor interaction (9td rua dots nod by analysis of lines of but fit obtained throvil 'least sgrrn' linear Mon on as previously described (14) . ( " ) binding in tubes containing ('x)r only . binding in seem containing cannabis The specificity nain (equivalent to 10 611 0~-TIC added prior te ('11) show and lteited binding capacity are by abolition of Nr.affialp binding of 10,7M added as With " -lK. in the pneenca ten-radioactive E!
Coll a',
w
(" )
I& )
We also have administered cannabis resin by intraperitoneal injection over an eight-day period to immature female rats ; there was no stimulation of uterine growth at doses up to toxic levels (200 mg/kg/day) . It is well-documented that estrogens (either natural or synthetic) stimulate true uterine growth by first binding to specific receptor proteins in target tissues (8) . Thus, a sensitive in vitro test for potentially estrogenic substances can be done by determiningwFeTier the substance competes with (3H)-estradiol-17f (( ine cytosol .
(3H-E2 )
)) for specific, high-affinity sites in uter-
We have performed this assay with uterine cytosol from various
age-groups of rats .
As shown in Figure 3, cannabis resin at concentrations 09 equivalent to 10 -5M -THC does not compete with ( 3H)-E2 for estrogen 09 receptor sites . Tests with 10 -3M -THC show some reduction in the affinity of the E2-receptor interaction, probably through non-specific effects on the receptor protein . Preliminary experiments do not reveal any significant change in estrogen-receptor levels in uteri of rats given large cannabis resin doses in .vivo .
1116
Demasculinization by Cannabis .
Vol . 17 . No . 7
Discussion The negative results of the uterine weight bioassay and the lack of competition for estrogen receptor sites in vitro make it very unlikely that cannabis' (or its metabolites') demasculTniz -~effects can be attributed to estrogenic activity . Many hydroxylated/phenolic metabolites of various cannabinoids have been identified in the rat (9), but none of these seems to have free hydroxyl or phenolic groups located at both extremities of the molecule as occurs in known estrogens (8) . It is clear that high doses of cannabis can demasculinize rodents (3-5), this report) if given continuously ; intermittent high doses of THC were reported not to significantly reduce seminal vesicle weights (15) . In humans, however, Mendelson et al . (10) were unable to confirm previous reports (1) that marihuana smoking7epressed plasma testosterone levels . It also seems that gynecomastia (breast growth) occurs only in a small proportion of men who are heavy cannabis users . If the demasculinizing/faminizing effects of cannabis in men are confirmed to be real effects of using the drug, effective treatment or prevention will require knowledge of the underlying causal mechanism(s) . The eventual explanation could range from altered pituitary function (15) through liver disease (11) to effects at the level of DNA/RNA (12) or interaction of several of these with other factors such as genetic predisposition . It is very unlikely that the demasculi.nizing/forinizing effects of cannabis are due to any direct estrogen action . Acknowledgements Cannabis resin was supplied through the courtesy of Mr . R .A . Graham, Health Protection Branch, Health and Welfare Canada . Supported in part by grant A4670 from the National Research Council of Canada (ASO) and a summer scholarship for Research on Drug Abuse awarded to GST by the Non-Medical Use of Drugs Directorate, Health and Welfare Canada . We thank G.P . Bondy, K.C . Silinskas and J . Kennedy for dedicated technical assistance .
References 1.
R.C . KOLOONY, W.H . MASTERS, R .N . KOLODNER, G. TORO,- N. Egl . J . Ned . 290 : R72-874 (1974) .
2.
J . HAMM and M .A . ALIAPOULIOS, N. Enol . J . Mod. 287 :
3.
N .E . CORCORAN, A.AMIT, C.W . MALSBURY, S . DAYKIN, 7 : 779-783 (1974) . Pathol . Pharmacol .
4.
V .P . DIXIT, V .N . SHANA, N .K . LOHIYA, Eur. J . Pharmacol . 26 : 111-114 (1974) . _ G.R . THOMPSON, M.M . MASON, H . ROSENKRANTZ, N.C . BRAUDE, Toxicol . Appl . Pharmacol . 25 : 373-390 (1973) .
5.
936 (1972) .
els . Com. Chem .
6.
F.J . MANNIN9, J .H . MCDONOUGH, JR ., T .F . ELSMORE, C . SALLER, F.J . SODETZ, Science 174 : 424-426 (1971) .
7.
J . SOLOMOVAND D .X . SHATTUCK, N . Engl . J . Ned . 291 :
8.
E .V . JENSEN and E .R . DeSOMBRE, Annu . Rev . Biochem . 41 :
9.
M.E . WALL, Ann. N.Y . Acad . Sci .
191 :
309 (1974) . 203-230 (1972) .
23 (1971) .
10 .
J .H . MENDELSON, J . KUEHNLE, J . ELLZIÏGBOE, T .F . BABOR, N . En9l . J . Med . 291 : 1051-1055 (1974) .
11 .
DT. VAN THEIL and R . LESTER, N . Engl . J. Med . 291 :
251-253 (1974) .
Vol . 17, No . 7 12 . 13 . 14 . 15 .
Damasculinization by Cannabis
1117
G .G . NAHAS, N . SUCIU-FOCA, J .P . ARMAND, A . MORI3ATMA, Science 183 : 419-420 (1974) . J .G . CLEMENT and A .B . OKEY, Can . J . Physiol . Pharmacol . 50 : 971-975 (1972) . _ D .D . KEIGHTLEY and A .B . OKEY, Cancer Res . 33 : 2637-2642 (1973) . R . COLLU, J . LETARTE, G . LEBOEUF, J .R . DUCIIGRME, Life Sci . 16 : 542 (1975) .
533-542
Life Sciences vol . 17, pp . 1113-1118 Printed in the U.S .A .
Press
CANNABIS DENASCULINIZES RATS BUT IS NOT ESTROGENIC Allan B . Okey and Greg S . Truant Department of Biology University of Windsor Windsor, Ontario CANADA, H9B 3P4
(Received
in final form
Septsimber 2,
1975)
Summary Carmabis resin (54% THC) caused dose-related decreases in the development of testis, prostate and seminal vesicles when administered in the diet to immature male rats . Bioassays and estrogen-receptor studies showed no estrogenic activity in cannabis resin . Demasculinization and possible feminization by cannabis are not due to simple estrogen-like action on male tissues . Introduction Intensive marihuana use by young men has been reported to depress their plasma testosterone levels and spermatogenesis (1) and to lead to abnormal breast growth (2) . In rodents, various cannabis preparations reduce male co latory behavior (3), inhibit spermatogenesis (4), reduce seminal vesicle (4) and prostate weights (5) and inhibit body growth (5-7) . Perhaps the simplest explanation for all these cannabis. effects would be that cannabis components (or their metabolites) have estrogenic properties ; this attractive hypothesis has been proposed (2,7) and widely disseminated . However, using repeated bioassays and in vitro competition for estrogen-receptor sites we find no evidence that canna possesses significant estrogenic activity . -
Ws
Methods and Results Male Wistar rats (Figure 1) fed cannabis resin in their chow during sexual maturation show a linear, dose-related decrease in development of testes, prostate and seminal vesicles . If this were due to estrogenic activity in cannabis, the same dietary doses would be expected to stimulate uterine growth in immature female rats or in ovariectomized adult females . As shown in Figure 2, however, cannabis resin causes a dose-related decrease in uterine weight, even when the uterine weights are corrected for tReloss in body weight which occured at high cannabis doses . By comparison, diethylstilbestrol (DES) caused significant uterine growth in these animals at doses as low as 12 .5 parts per billion in the diet . DES also caused a significant advancement in vaginal opening in immature females ; by day-7 of DES treatment (50 ppb) all vaginas were open while no vaginas opened by that time in any cannabis treated groups .
1113
1114
Dwaaculinization by Caanabir
Vol . 17, No . 7
%lit arase selsbt Memos U dietary lntap of (newels rain (ceMalnieq aaR'Oxirtely .7110 . eaanMnol) was allaitait JAao Purina laboratory Chow rSn to 914 final commentions of 0-500 ports lier olllion (pou) A9-TNC . mal*~Ylstar-stain rate . par cop, era fad tMp diets fm ago 20-days sscrlfto et ago 57-days. Nlgb cannabis door doprasW body 9roru 11p11 .f1tent1yl orge %MOISIS ara am sail as et par 9 body right. correon, 14tion pHfletallta ara gMter than (-) 0.84 for mach lino and NCI Is s19h1fleutiy 1111aar (p < 0.01) . At 100 lion Tm fa the clot sack rat hat M lntate of abolit 0 mal TIC/kg body wlght/doy. 19% ceeabldlol,
sax s
lait
a
o
Legend for Figure 2.
eat .
Uterine wleht assay for estrom activity.
(13)
3 ")=
A. Cannabis real . diets (0-150 We A9-TXC). prepared as 1n Figure 1, were Ired either to ismature female YIStar-Strain rata ( " ) from a 20-0ays through age 27-days or for 15 days to mare WsISr ferles had been ova eetaltod six watt or to Start the diets. Each point ropresents the assn value for 10 rats . Cdrrelat10s coofficonts are greater than (-) 0.83 for weh line and each Is significantly linear (p!(1 .051 . Diethylstilbestrol (DES) used as control standard was fed at âeaea of 0-100 parts per billion t $) =01", ar ovariacto.ited ( " rats as In part A. Correlation Coefficient$ are greater than 0.95 for each line and each 11 significantly linear (pt 0.05) .
a.
1
Vol . 17, No . 7
1115
Damaiculinisation by Caanabis
w ar a 012
001
0.04
9
ep
y
L f
tee
60
go
lu
nid
9aane t, molette revint
Fi
1
plndfn0 of ( )-ntnd101 .11D (( ( )- )) to redan abasol in the of 10 e . Uterine tytosoi (196 .009 n 9 supernatant. 1.5 tee pro ~a n per e rv s r Isoeelrto (rtes Uttar rate) rs 1nttWLW .IM (3,)-EZ at 14 mcontratlor beewen 20 and 4000 piweola per liter . At tir and of incubation . destra~coatod charcoal was toad to moue wlbotmd steroid . 1M amount bound at each concentration was deta, land by liquid scintillation counting of allowance before and after charcoal treatment. 1M p.o1Ue tyteaol protein) and mbar of high-affinity binding sites " aanlet/liter) dissociation constants far the EZratutor interaction (9td rua dots nod by analysis of lines of but fit obtained throvil 'least sgrrn' linear Mon on as previously described (14) . ( " ) binding in tubes containing ('x)r only . binding in seem containing cannabis The specificity nain (equivalent to 10 611 0~-TIC added prior te ('11) show and lteited binding capacity are by abolition of Nr.affialp binding of 10,7M added as With " -lK. in the pneenca ten-radioactive E!
Coll a',
w
(" )
I& )
We also have administered cannabis resin by intraperitoneal injection over an eight-day period to immature female rats ; there was no stimulation of uterine growth at doses up to toxic levels (200 mg/kg/day) . It is well-documented that estrogens (either natural or synthetic) stimulate true uterine growth by first binding to specific receptor proteins in target tissues (8) . Thus, a sensitive in vitro test for potentially estrogenic substances can be done by determiningwFeTier the substance competes with (3H)-estradiol-17f (( ine cytosol .
(3H-E2 )
)) for specific, high-affinity sites in uter-
We have performed this assay with uterine cytosol from various
age-groups of rats .
As shown in Figure 3, cannabis resin at concentrations 09 equivalent to 10 -5M -THC does not compete with ( 3H)-E2 for estrogen 09 receptor sites . Tests with 10 -3M -THC show some reduction in the affinity of the E2-receptor interaction, probably through non-specific effects on the receptor protein . Preliminary experiments do not reveal any significant change in estrogen-receptor levels in uteri of rats given large cannabis resin doses in .vivo .
1116
Demasculinization by Cannabis .
Vol . 17 . No . 7
Discussion The negative results of the uterine weight bioassay and the lack of competition for estrogen receptor sites in vitro make it very unlikely that cannabis' (or its metabolites') demasculTniz -~effects can be attributed to estrogenic activity . Many hydroxylated/phenolic metabolites of various cannabinoids have been identified in the rat (9), but none of these seems to have free hydroxyl or phenolic groups located at both extremities of the molecule as occurs in known estrogens (8) . It is clear that high doses of cannabis can demasculinize rodents (3-5), this report) if given continuously ; intermittent high doses of THC were reported not to significantly reduce seminal vesicle weights (15) . In humans, however, Mendelson et al . (10) were unable to confirm previous reports (1) that marihuana smoking7epressed plasma testosterone levels . It also seems that gynecomastia (breast growth) occurs only in a small proportion of men who are heavy cannabis users . If the demasculinizing/faminizing effects of cannabis in men are confirmed to be real effects of using the drug, effective treatment or prevention will require knowledge of the underlying causal mechanism(s) . The eventual explanation could range from altered pituitary function (15) through liver disease (11) to effects at the level of DNA/RNA (12) or interaction of several of these with other factors such as genetic predisposition . It is very unlikely that the demasculi.nizing/forinizing effects of cannabis are due to any direct estrogen action . Acknowledgements Cannabis resin was supplied through the courtesy of Mr . R .A . Graham, Health Protection Branch, Health and Welfare Canada . Supported in part by grant A4670 from the National Research Council of Canada (ASO) and a summer scholarship for Research on Drug Abuse awarded to GST by the Non-Medical Use of Drugs Directorate, Health and Welfare Canada . We thank G.P . Bondy, K.C . Silinskas and J . Kennedy for dedicated technical assistance .
References 1.
R.C . KOLOONY, W.H . MASTERS, R .N . KOLODNER, G. TORO,- N. Egl . J . Ned . 290 : R72-874 (1974) .
2.
J . HAMM and M .A . ALIAPOULIOS, N. Enol . J . Mod. 287 :
3.
N .E . CORCORAN, A.AMIT, C.W . MALSBURY, S . DAYKIN, 7 : 779-783 (1974) . Pathol . Pharmacol .
4.
V .P . DIXIT, V .N . SHANA, N .K . LOHIYA, Eur. J . Pharmacol . 26 : 111-114 (1974) . _ G.R . THOMPSON, M.M . MASON, H . ROSENKRANTZ, N.C . BRAUDE, Toxicol . Appl . Pharmacol . 25 : 373-390 (1973) .
5.
936 (1972) .
els . Com. Chem .
6.
F.J . MANNIN9, J .H . MCDONOUGH, JR ., T .F . ELSMORE, C . SALLER, F.J . SODETZ, Science 174 : 424-426 (1971) .
7.
J . SOLOMOVAND D .X . SHATTUCK, N . Engl . J . Ned . 291 :
8.
E .V . JENSEN and E .R . DeSOMBRE, Annu . Rev . Biochem . 41 :
9.
M.E . WALL, Ann. N.Y . Acad . Sci .
191 :
309 (1974) . 203-230 (1972) .
23 (1971) .
10 .
J .H . MENDELSON, J . KUEHNLE, J . ELLZIÏGBOE, T .F . BABOR, N . En9l . J . Med . 291 : 1051-1055 (1974) .
11 .
DT. VAN THEIL and R . LESTER, N . Engl . J. Med . 291 :
251-253 (1974) .
Vol . 17, No . 7 12 . 13 . 14 . 15 .
Damasculinization by Cannabis
1117
G .G . NAHAS, N . SUCIU-FOCA, J .P . ARMAND, A . MORI3ATMA, Science 183 : 419-420 (1974) . J .G . CLEMENT and A .B . OKEY, Can . J . Physiol . Pharmacol . 50 : 971-975 (1972) . _ D .D . KEIGHTLEY and A .B . OKEY, Cancer Res . 33 : 2637-2642 (1973) . R . COLLU, J . LETARTE, G . LEBOEUF, J .R . DUCIIGRME, Life Sci . 16 : 542 (1975) .
533-542