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Effects of Δ9-tetrahydrocannabinol administration on marker proteins of rat testicular cells
Life Sciences, Vol . 22, pp . 7-14 Printed in the U .S .A .
Pergamon Press
EFFECTS OF ~ 9 -TETRAHYDROCANNABINOL ADMINISTRATION ON MARKER PROTEINS OF RAT TESTICULAR CELLS Scott Schwarz, Jack Aarclerode and S . E . Nyquist Department of Biology, Bucknell University, Lewisburg, PA
17837
(Received in final form September 6, 1977) Summax~ The effects of chronic administration of 2 mg ~ 9-tetrahydrocannabinol per kg body weight upon rat testicular cell function was examined by use of selected testicular cell marker proteins . y-Glutamyl tranapeptidase was used as a marker of Sertoli cell plasma membranes ; sorbitol dehydrogenase was used as a marker of pachytene apermatocytea . The interstitial cells were marked by cytochrome P-450, a microsomal component, and ß-glucuronidase, a lysoeomal component . The results of this study show a rapid reduction in microsomal P-450 content following 2 days of tetrahydrocannabinol administration . In addition, y-glutamyl tranapeptidase was significantly reduced at 2 days and continued to decline to day 9 . ß-Glucuronidase and sorbitol dehydrogenase exhibited no aigaificant change over the course of the experiment . It is suggested that the reduction of testosterone synthesis in testes of tetrahydrocannabinol treated rata may be the result of a reduction in P-450 content . Recent studies have indicated that chronic or acute exposure to delta-9tetrahydrocannabinol (THC), a psychoactive ingredient of marijuana (Cannabis sativa ), may alter testicular function in both humans and laboratory animals . Using human subjects, Nahas et al . (1) observed decreased sperm counts in cannabis users, while Dixit _et al . (2) observed both a reduction in spermatogenesis and a general reductionin Leydig cell function in mice treated with a cannabis extract . Several investigators have also reported reduced levels of plasma testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH), as well as reduced testicular weights after chronic exposure to THC (3, 4,5,6,7,8,9) . List et al . (10) further demonstrated a reduction of testosterone formation _in vitro by testicular microsomes obtained from THC treated rate as compared to controls . Other investigators, however, have observed no significant reductions in testicular weight, levels of plasma testosterone, LH, or FSH in THC treated rata (11,12) . The experiments reported here were designed to clarify the mechanism of THC induced alterations in the testes . Marker proteins were utilized to monitor the effects of THC on the various cell types which comprise testicular tissues, i .e . germ cells, Sertoli cells, and Leydig cells, as well as to establish the sequence of these effects . Four marker proteins were chosen for the experiment based on previous studies which established the use of these proteins as markers of certain testicular cell types . Sorbitol dehydrogenase (SDH) (E .C . 1 .1 .1 .14), a cytoplaemic enzyme catalyzing the NADH2-dependent interconversion of fructose and sorbitol, has been established as a marker of pachytene spermatocytea by Mills and Means (13) . Gamma-glutamyl tranapeptidase (GTP) (E .C . 2 .3 .2 .1), a membrane bound enzyme involved in the amino acid exchange of glutamyl peptides, 7
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has been established as a marker of Sertoli cells by Lu _et _al . (14) and Hodgen and Sherina (15) . Menard _et al . (16) using fractionation techniques showed that cytochrome P-450 can be used as a marker for interstitial cells . Betaglucuronidase (E .C . 3 .7 .1 .31), a lysosomal enzyme, has been reported, based largely on hiatochemical studies, to be present in a variety of testicular cell types (17,18) . However, fractionation of testicular components (Rodgers, personal communications) into interstitial and tubular elements followed by a colorimetric assay for beta-glucuronidase using phenolphthalein glucuronic acid as the substrate, showed quite conclusively that the specific activity of the interstitial tissue is approximately five times higher than tubular activity ; thus although beta-glucuronidase is present in various types of testicular cells, and therefore not an ideal marker enzyme, it is useful as a crude marker of an interstitial cell activity . Materiale and Methods Experimental Design : The experiment was conducted twice under similar procedural specifications except ae otherwise noted . The initial experiment will hereafter be referred to as Experiment I, with the replicate experiment referred to ae Experiment II . Animals : Male Wietar rats were injected intraperitoneally with 2 mg/kg of THC or a vehicle containing lOX propylene glycol, 1X Tween 80, and 89X physiological saline . Animals were fed Purina Lab Chow and water _ad libitum and sub jected to a 14-10 hour light-dark schedule . All animals were weighed and sacrificed 24 hours after the last injection. In Experiment I animals weighing between 300 and 440 gm were injected daily between 11 :00 PM and 12 :00 AM, while is Experiment II, animals weighing between 247 gm and 315 gm were injected daily between 1 :00 PM and 2 :00 PM . Testicular weight was also recorded in Experiment II . Assay Procedure : Assay procedures were designed such that GTP, SDH, and ß-glucuronidase assays could all be performed on a single rat . An entire rat testes was required for a CO-hemoprotein (cytochrome P-450 and P-420) determi nation . Kinetic studies were performed for the three marker enzymes to assu=e first order kinetics with respect to time and protein concentration . In Experiment II, protein concentrations were determined for each assay by the method of Lowry et al . (19), using bovine serum albumin as a standard . Data for Experiment Î were expressed on a wet weight basis . GTP assays were performed according to the procedure of Glenner _et al . (20), using N-(y-L-glutamyl)-ß-naphthylamide as substrate. Testicular tissue was homogenized in physiological saline (1 :19 w/v) with a Polytron homogenizer . The reaction was stopped after twenty minutes and assay tubes were centrifuged to remove particulate material . Naphthylamine concentration was measured by reading abaorbance at 550 nm on a Beckman spectrophotometer . SDH activities were determined by an assay which measured changes in the concentration of NADH2, a coenzyme in the SDH catalyzed reduction of fructose to sorbitol (21) . Testicular tissue was homogenized in 3 ml of 0 .035 M sodium phosphate buffer, pH 7.5 (1 :2 w/v) with a Polytron homogenizes and centrifuged at 105,000 g for 60 minutes . The change in NADH2 concentration was measured over the first three minutes of the reaction by reading changes in abaorbance at 340 nm on a Beckman DB-G spectrophotometer . Beta-glucuronidase assays were performed according to the method of Talalay et al . (17) . Testicular tissue was homogenized in physiological saline (1 :19 w/v) with a Polytron homogenizes and centrifuged at 105,000 g for 60
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minutes . The resultant supernatant contained the B-glucuronidase enzyme . The reaction was stopped after 40 minutes and assay tubes were centrifuged to remove particulate proteins . Phenolphthalein concentration was measured by reading absorbante at 550 nm on a Beckman spectrophotometer . The microsomal CO hemoprotein (cytochrome P-450 + P-420) was measured with the Cary-14 spectrophotometer using a reduced-carbon monoxide versus reduced difference spectrum ss described by Omura and Sato (22) . Testes were homo genized in 0 .25 M sucrose and centrifuged at 1000 g for 10 minutes, 12,000 g for 10 minutes, and 105,000 g for 60 minutes . The 105,000 g microsomal pellet was washed with 0.15 M KC1, suspended in 8 .5 ml of 0.2 M trio buffer, pH 7 .4, and used directly in the CO-hemoprotein determination . The amount of microsomal cytochrome P-450 was determined by using an extinction coefficient of 91 mM- 1 cm 1 for the difference in absorbante between 490 nm and 450 nm . The P-420 content was determined by using an extinction coefficient of 111 mM-lcm 1 for the difference in absorbante between 490 nm and 420 nm . Results The average growth rates for the rate used in these experiments were monitored and shown to be significantly reduced following nine days of chronic THC administration . In Experiment I the average growth rate calculated over the 9-day period was 4 .29 _+ 1.79 g/day for vehicle treated rate and 1 .49 _+ 1 .34 g/day for the THC treated rats . In Experiment II using younger rata the average growth rate over the 9-day period was 9 .90 _+ 1 .28 g/day for the vehicle treated rate and 7 .10 + 1.00 g/day for the THC treated rate, showing a 30X reduction (a < .O1) . No significant (a > .50) changes, however, in testes weights were noticed between the THC and vehicle treated rata following either two or nine days of THC administration . A substantial reduction of the microsomal cytochrome P-450 and its breakIn down product cytochrome P-420 was observed in THC treated rata (Table I) . Experiment I, after a period of two days, the average hemoprotein concentration expressed on a wet weight basis in rats receiving THC fell to approximately half that of vehicle treated rats (a < .10) . In Experiment II, the concentration of microsomal P-450 expressed relative to total microsomal protein declined in THC treated rate to a level of about 70% (a < .10) that of vehicle treated rats following two days of THC administration and 60X (a < .10) after nine days . The total microsomal P-450 content was also cdrreapondingly reduced (Table I) . The Y^glutamyl tranapeptidase activity (Table II) in THC treated rats was aigaificantly reduced after two days of THC administration and further reduced after 9 days to a level of approximately 60% (a < .05) in both Experiments I and II . Statistically insignificant reductions in SDH levels of THC treated rata were noted after 9 and 18 days of THC administration in Experiment I and after 2 and 9 days of THC administration in Experiment II (Table III) . Beta-glucuronidase activities showed no significant differences between THC and vehicle rate in both Experiments I and II (Table IV) . Diacuseion Our finding of decreased microsomal P-450 is THC treated rats is consistent with previous reporta of rapid reductions of plasma LH levels (8,9) after THC injections in rata . Purvia et al . (23) concluded that microsomal cytochrome P-450 in rat tentes exhibited a half life of 3.3 days . In addition, Menard and Purvis (24) demonstrated that microsomal cytochrome P-450 in chick testes was controlled by plasma LH levels . Given the rapid response of cytochrome P-450 to
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TABLE I The Effect of THC Treatment on Rat Testis Microsomal P-450 Content P-450 Content THC
Vehicle
Experiment I 0 2 9 18
Days Days Days Days
(4) (4) (4) (4)
(3) (4) (4)
(4) (4)
3 .00 + 1,13 2 .59 + 0 .47 2 .29 + 0 .62
2 .47 _+ 0 .76 -
0 .48 0 .77 0.62
(nmolea/mg protein) 0 .124 ± .016 0 .090 + .019
Experiment II 2 Days 9 Days
THC/Veh .
(nmolea/gm wet wt .) 1 .45 + 0 .66 2 .00 + 0 .16 1 .42 + 0 .34
Experiment II 0 Days 2 Days 9 Days
Control
0.172 + .045 0.150 + .055
0 .150 + .041 -
0 .72 0 .60
(umoles/total testis) 0 .848 + .091 0 .514 + .110
1 .228 + .449 0.859 + .447
-
0 .69 0 .60
P-450 content is expressed as the sum of P-450 and P-420. Chronic dosages of 2 mg THC/kg body weight were administered daily as described in Materials and Methods . The numbers in parentheses indicate the number of experimental animals per group . Values are expressed as means + standard deviation .
TABLE II The Effect of THC Treatment on Rat Testis Y-Glutamyl Tranepeptidase Y-Glutamyl Tranepeptidase Activity THC Experiment 0 2 9 18
I
Days Days Days Days
78 + 13 (4) 63 _+ 23 (4) 105 + 13 (4)
THC/Veh .
105 _+ 2 .42 (4) -
0 .76 0 .64 0 .96
(nmolea naphthylamine/min ./mg protein)
-
0.700 + .166 0 .447 + .040
103 + 18 (3) 98 _+ 5 (4) 110 + 13 (3)
0 .949 + .017 0 .825 + .116
0 .795 _+ .073 -
0 .74 0 .54
(nmolea naphthylamine/min ./total testis)
Ex~riment II 2 Days (4) 9 Days (4)
Control
(nmolea naphthylamine/min ./gm wet wt .)
ExQeriment II 0 Days (3) 2 Days (4) 9 Days (4)
Vehicle
264 + 39 206 + 27
303 + 30 369 + 46
-
0 .87 0 .56
Chronic dosages of 2 mg THC/kg body weight were administered daily as described in Materials and Methods . The numb era in parentheses indicate the number of experimental animals per group . Values are expressed as means _+ standard deviation .
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TABLE III The Effect of THC Treatment on Rat Testis Sorbitol Dehydrogenase Activity Sorbitol Dehydrogenase Activity THC
Vehicle
Experiment I 0 2 9 18
Days Days Days Days
75 .2 + 15 .2 (4) 97 .2 + 19 .1 (4) 62 .0 + 5 .50 (4)
73 .9 + 3 .8 (3) 115 .7 + 15 .3 (3) 67 .3 + 30 .0 (3)
83 .8 _+ 30 .2 (4) -
1 .02 0 .84 0 .92
(nmoles NADH2 /min ./mg protein) 2 .08 + .20 1 .98 + .13
2 .34 + .45 2 .34 + .37 r
Experiment II 2 Days (4) 9 Days (4)
THC/Veh .
(nmoles NADH 2 /min ./mg wet wt .)
Experiment II 0 Days (3) 2 Days (4) 9 Days (4)
Control
185 + 18 294 + 31
2 .41 _+ .40 -
0 .89 0 .85
(nmoles NADH2 /min ./total testis) 240 + 70 329 + 29
-
0 .77 0 .89
Chronic dosages of 2 mg THC/kg body weight were administered daily as described in Materials and Methods . The numbers in parentheses indicate the ntmlber of experimental animals per group . Values are expressed as means _+ standard deviation . TABLE IV The Effect of THC Treatment on Rat Testis ß-Glucuronidase Activity ß-Glucuronidase Activity THC E xperiment I 0 2 9 18
Days Days Days Days
THC/Veh .
122 + 21 (3) 108 + 14 (4) 221 + 166 (3)
119 .0 _+ 8 .6 (4) -
1 .16 1 .14 0 .87
(nmoles phenolphthalein/min ./mg protein) 2 .44 + .22 2 .40 + .50
Experiment II 2 Days (4) 9 Days (4)
Control
(nmoles phenolphthalein/min ./gm wet wt .) 141 + 8 (4) 123 + 21 (4) 192 + 102 (4)
Ex periment II 0 Days (3) 2 Days (4) 9 Days (4)
Vehicle
2 .18 + .16 2 .66 + 1 .72
1 .93 + .29 -
1 .12 0 .90
(nmolea phenolphthalein/mia ./total testis) 488 + 59 456 + 71
410 + 35 500 + 370
1 .19 1 .10
Chronic dosages of 2 mg THC/kg body weight were administered daily as described in Materials and Methods . The numbers in parentheses indicate the number of experimental animals per group . Values are expressed as means _+ standard deviation .
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1978
changes in plasma LH levels, it ie feasible that our observed reductions in cytochrome P-450 after 2 days of THC injections in rats could be a result of reduced plasma LH levels . Our results also suggest that previously reported reductions of plasma testosterone after THC administration (10,11) could be mediated through such an LA-induced change in cytochrome P-450 activity in the testes . Various portions of the testosterone biosynthetic pathway occur in the mitochondria and the microsomal fractions of the Leydig cells . Our data showing reduced microsomal P-450 could account for some reduction in either testosterone synthesis or secretion in THC treated rats . Two important enzymes of testosterone synthesis, 17 a-hydroxylase and C17-C20 lyase, are localized on the microsomes and contain cytochroms P-450 as their active site (25) . These enzymes have been shown to be responsive to changes in plasma pituitary gonadotrophin levels, and their activities decay with half lives similar to those of microsomal cytochrome P-450 (23) . In a similar fashion Purvis et al . (25) demonstrated that mitochondrial P-450 levels, like microsomal P-450 levels, are controlled'by gonadotrophins . Mitochondrial P-450 is not normally detectable in rat testes but by exogenous administration of human chorionic gonadotrophin the P-450 levels were elevated to detectable concentrations . Purvia et _al . (25) also demonstrated that the half life of mitochondrial P-450 was even shorter than that of the microsomal P-450. This data, together with reports (26) that the cholesterol aide-chain cleavage enzyme complex, of which P-450 is an integral component, may be the rate limiting step in testosterone synthesis, raise some interesting questions about the possible effect of THC on mitochondrial P-450 content as well . The reduction of cytochrome P-450 levels in THC treated rats may reflect a selective effect of THC on cytochrome P-450 and does not necessarily indicate an overall reduction in size, number, or activity of Leydig cells, ae evidenced by maintenance of normal beta-glucuronidase levels and normal testicular weight in our experimental animals . Although the reduction in GTP activity after just two days of THC treatment was not as dramatic as the reduction of cytochrome P-450 content, they do suggest an early initiation of the events which lead to a substantial reduction of GTP activity by the ninth day of injections . The reduction in GfP activity may be the result of a THC induced alteration of FSH secretion. However, previous reports (6,8) indicate that changes in plasma FSH after THC administration are not apparent until well after changes in plasma LH and testosterone have occurred . These observations suggest that the reductions we found in GTP activity may be caused by some factor other than an alteration in plasma FSH . levels . The mounting evidence of partial regulation of Sertoli cells by testosterone (27) raises the possibility that reduction in GTP activity in THC treated rate was caused by an alteration in the level of testosterone synthe sis and/or secretion . This theory is a particularly attractive method of explaining the rapidness of the observed reduction in GTP activity . Our data indicate that while no major alterations in germ cell development were observed during the 18-day period of chronic THC administration, there was a substantial reduction in activity of both the Sertoli cell marker enzyme, GTP, and the Leydig cell marker protein, cytochrome P-450. The reduction in cytochrome P-450 suggests that this cytochrome may serve as the mediating mechanism between previously observed reductions in plasma LH and plasma testosterone following THC administration in rats . Although reductions of GTP activity in THC treated rats may be caused by reduced plasma FSH, the
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Tetrahydrocannabinol and Testis Enzymes
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rapidness of the reduction suggests that the initial effect of THC on Sertoli cells is probably caused by reductions in testosterone synthesis and/or secretion . Other experiments involving simultaneous administration of FSH and THC and LH and THC to rate would be helpful for further clarification of the effect of THC on the testis and on Sertoli cells in particular . Açknowle~ents The authors wish to thank Dr . Cheryl Lu for assistance with the GTP assay . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 .
G . G . NAHAS, N . SUEIU-FOCA, J . P . ARMANS, and A . MORISHIMA, Science 183, 419-420 (1974) . V . P . DIXIT, V . N . SHARMA, and N . R . LOHIYA, Eur . _J . Pharmacol . _26, 111114 (1974) . G . R . THOMPSON, M . M . MASON, H . ROSENRRANTZ, and M . C . BRAUDE, Toxicol . Appl . Pharmacol . 25, 373-390 (1973) . H . ROSENRRANPZ, R . A . SPRAGUE, R . W . FLEISCHMAN, and M . C .BRAUDE, Toxicol . Appl . Pharmacol . _32, 399-417 (1975) . M . C . Braude and S . Szara (eda .), The Pharmacoloxy _of Marihuana , Raven Press, New York (1976) . R . COLLU, J . LETARTE, G . LEBOEUF, and J . R . DUCHARME, Life Sci . 16, 533542 (1975) . R . C . KOLODNY, W . H . MASTERS, R . M . KOLODNER, and G . TORO, _N . Engl . _J . _Med . 290, 872-924 (1974) . R . C . RÛLODNY, G . TORO, and W . H . MASTERS, N . Eng1 .J .Med .292,868 (1975) . B . MARKS, ProA . Brain _Kea . _39, 331-338 (1973) . A . LIST, B . NAZAR, S . NYQUIST, and J . HARCLERODE, ~ Metab . Die os . 5, 268-272 (1977) . J . H . MENDELSON, J . KUEH[TLg, J . ELLINGBAE, and T . F . BABOR, N . ~ . J . Med . _29, 1051-1055 (1974) . liEMBREE, W. P . ZEIDENBERG, and G . NAHAS, in Marihuana : Chemistry , Biochemistry and Cellular Effects , G . Nahas (ed .), Springer-Verlag, New York, pp . 521-532 (1976) . N . MILLS and A . MEANS, Endocrin . _91, 147-156 (1972) . C . C . LU and A . STEINBERGER, 9th Ann . Meetings Soc . Study Reprod ., Philadelphia, Abs . 107 (1976) . G . HODGEN and R . SHERINS, Endocrin . 93, 985-989 (1973) . R . MENARD, S . LATIF and J . PURVIS, Endocrin . 97, 1587-1592 (1975) . P . TALALAY, W . H . FISHMAN and C . HUGGINS, _J . Biol . Chem . 166, 757-772 (1946) . W . FISHMAN and J . BARER, _J . Histochem . Cytochem . _4, 570-587 (1956) . 0 . LOWRY, N . ROSEBROUGH, A . FARR, and R . RANDALL, _J . Biol . Chem . 193, 265-275 (1951) . G . GLENNER, J . FOLD, and P . McMILLAN, _J . Histochem . tochem . _10, 481489 (1962) . D . BISHOP, J . Gen . Physiol . 50, 2504 (1967) . T . OMURA and R SATO, . _J . Biol. Chem . _239, 2370-2378 (1964) . J . PURVIS, J . CANICK, S . LATIF, J . ROSENBAUM, J . HOLOGGITAS and R . MENARD, Arch . Biochem . Biophys . 159, 39-49 (1973) . R . MENARD and J . PURVIS, Endocrin . 91 :1506-1512 (1972) . J . PURVIS, J . CANICR, J . ROSENBAUM, J . HOLOGGITAS, and S . LATIF, Arch . Biochem . Biophys . _159, 32-38 (1973) . P . F . HALL, in _The Androgens _of _the Testis , R . B . Eik-Nee (ed .), Marcel Dekker, New York . pp . 73-116 (1970) . A . MEANS, J . FAKUNDING, C . HUCKINS, D . TINDALL, and R . VITALE, in Recent Progress _in Hormoae Research , Vol . 32, R . Greep (ed .), Academic Press, New York . pp . 508-519 (1976) .
Pergamon Press
EFFECTS OF ~ 9 -TETRAHYDROCANNABINOL ADMINISTRATION ON MARKER PROTEINS OF RAT TESTICULAR CELLS Scott Schwarz, Jack Aarclerode and S . E . Nyquist Department of Biology, Bucknell University, Lewisburg, PA
17837
(Received in final form September 6, 1977) Summax~ The effects of chronic administration of 2 mg ~ 9-tetrahydrocannabinol per kg body weight upon rat testicular cell function was examined by use of selected testicular cell marker proteins . y-Glutamyl tranapeptidase was used as a marker of Sertoli cell plasma membranes ; sorbitol dehydrogenase was used as a marker of pachytene apermatocytea . The interstitial cells were marked by cytochrome P-450, a microsomal component, and ß-glucuronidase, a lysoeomal component . The results of this study show a rapid reduction in microsomal P-450 content following 2 days of tetrahydrocannabinol administration . In addition, y-glutamyl tranapeptidase was significantly reduced at 2 days and continued to decline to day 9 . ß-Glucuronidase and sorbitol dehydrogenase exhibited no aigaificant change over the course of the experiment . It is suggested that the reduction of testosterone synthesis in testes of tetrahydrocannabinol treated rata may be the result of a reduction in P-450 content . Recent studies have indicated that chronic or acute exposure to delta-9tetrahydrocannabinol (THC), a psychoactive ingredient of marijuana (Cannabis sativa ), may alter testicular function in both humans and laboratory animals . Using human subjects, Nahas et al . (1) observed decreased sperm counts in cannabis users, while Dixit _et al . (2) observed both a reduction in spermatogenesis and a general reductionin Leydig cell function in mice treated with a cannabis extract . Several investigators have also reported reduced levels of plasma testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH), as well as reduced testicular weights after chronic exposure to THC (3, 4,5,6,7,8,9) . List et al . (10) further demonstrated a reduction of testosterone formation _in vitro by testicular microsomes obtained from THC treated rate as compared to controls . Other investigators, however, have observed no significant reductions in testicular weight, levels of plasma testosterone, LH, or FSH in THC treated rata (11,12) . The experiments reported here were designed to clarify the mechanism of THC induced alterations in the testes . Marker proteins were utilized to monitor the effects of THC on the various cell types which comprise testicular tissues, i .e . germ cells, Sertoli cells, and Leydig cells, as well as to establish the sequence of these effects . Four marker proteins were chosen for the experiment based on previous studies which established the use of these proteins as markers of certain testicular cell types . Sorbitol dehydrogenase (SDH) (E .C . 1 .1 .1 .14), a cytoplaemic enzyme catalyzing the NADH2-dependent interconversion of fructose and sorbitol, has been established as a marker of pachytene spermatocytea by Mills and Means (13) . Gamma-glutamyl tranapeptidase (GTP) (E .C . 2 .3 .2 .1), a membrane bound enzyme involved in the amino acid exchange of glutamyl peptides, 7
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1978
has been established as a marker of Sertoli cells by Lu _et _al . (14) and Hodgen and Sherina (15) . Menard _et al . (16) using fractionation techniques showed that cytochrome P-450 can be used as a marker for interstitial cells . Betaglucuronidase (E .C . 3 .7 .1 .31), a lysosomal enzyme, has been reported, based largely on hiatochemical studies, to be present in a variety of testicular cell types (17,18) . However, fractionation of testicular components (Rodgers, personal communications) into interstitial and tubular elements followed by a colorimetric assay for beta-glucuronidase using phenolphthalein glucuronic acid as the substrate, showed quite conclusively that the specific activity of the interstitial tissue is approximately five times higher than tubular activity ; thus although beta-glucuronidase is present in various types of testicular cells, and therefore not an ideal marker enzyme, it is useful as a crude marker of an interstitial cell activity . Materiale and Methods Experimental Design : The experiment was conducted twice under similar procedural specifications except ae otherwise noted . The initial experiment will hereafter be referred to as Experiment I, with the replicate experiment referred to ae Experiment II . Animals : Male Wietar rats were injected intraperitoneally with 2 mg/kg of THC or a vehicle containing lOX propylene glycol, 1X Tween 80, and 89X physiological saline . Animals were fed Purina Lab Chow and water _ad libitum and sub jected to a 14-10 hour light-dark schedule . All animals were weighed and sacrificed 24 hours after the last injection. In Experiment I animals weighing between 300 and 440 gm were injected daily between 11 :00 PM and 12 :00 AM, while is Experiment II, animals weighing between 247 gm and 315 gm were injected daily between 1 :00 PM and 2 :00 PM . Testicular weight was also recorded in Experiment II . Assay Procedure : Assay procedures were designed such that GTP, SDH, and ß-glucuronidase assays could all be performed on a single rat . An entire rat testes was required for a CO-hemoprotein (cytochrome P-450 and P-420) determi nation . Kinetic studies were performed for the three marker enzymes to assu=e first order kinetics with respect to time and protein concentration . In Experiment II, protein concentrations were determined for each assay by the method of Lowry et al . (19), using bovine serum albumin as a standard . Data for Experiment Î were expressed on a wet weight basis . GTP assays were performed according to the procedure of Glenner _et al . (20), using N-(y-L-glutamyl)-ß-naphthylamide as substrate. Testicular tissue was homogenized in physiological saline (1 :19 w/v) with a Polytron homogenizer . The reaction was stopped after twenty minutes and assay tubes were centrifuged to remove particulate material . Naphthylamine concentration was measured by reading abaorbance at 550 nm on a Beckman spectrophotometer . SDH activities were determined by an assay which measured changes in the concentration of NADH2, a coenzyme in the SDH catalyzed reduction of fructose to sorbitol (21) . Testicular tissue was homogenized in 3 ml of 0 .035 M sodium phosphate buffer, pH 7.5 (1 :2 w/v) with a Polytron homogenizes and centrifuged at 105,000 g for 60 minutes . The change in NADH2 concentration was measured over the first three minutes of the reaction by reading changes in abaorbance at 340 nm on a Beckman DB-G spectrophotometer . Beta-glucuronidase assays were performed according to the method of Talalay et al . (17) . Testicular tissue was homogenized in physiological saline (1 :19 w/v) with a Polytron homogenizes and centrifuged at 105,000 g for 60
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Tetrahydrocannabinol and Testis Enzymes
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minutes . The resultant supernatant contained the B-glucuronidase enzyme . The reaction was stopped after 40 minutes and assay tubes were centrifuged to remove particulate proteins . Phenolphthalein concentration was measured by reading absorbante at 550 nm on a Beckman spectrophotometer . The microsomal CO hemoprotein (cytochrome P-450 + P-420) was measured with the Cary-14 spectrophotometer using a reduced-carbon monoxide versus reduced difference spectrum ss described by Omura and Sato (22) . Testes were homo genized in 0 .25 M sucrose and centrifuged at 1000 g for 10 minutes, 12,000 g for 10 minutes, and 105,000 g for 60 minutes . The 105,000 g microsomal pellet was washed with 0.15 M KC1, suspended in 8 .5 ml of 0.2 M trio buffer, pH 7 .4, and used directly in the CO-hemoprotein determination . The amount of microsomal cytochrome P-450 was determined by using an extinction coefficient of 91 mM- 1 cm 1 for the difference in absorbante between 490 nm and 450 nm . The P-420 content was determined by using an extinction coefficient of 111 mM-lcm 1 for the difference in absorbante between 490 nm and 420 nm . Results The average growth rates for the rate used in these experiments were monitored and shown to be significantly reduced following nine days of chronic THC administration . In Experiment I the average growth rate calculated over the 9-day period was 4 .29 _+ 1.79 g/day for vehicle treated rate and 1 .49 _+ 1 .34 g/day for the THC treated rats . In Experiment II using younger rata the average growth rate over the 9-day period was 9 .90 _+ 1 .28 g/day for the vehicle treated rate and 7 .10 + 1.00 g/day for the THC treated rate, showing a 30X reduction (a < .O1) . No significant (a > .50) changes, however, in testes weights were noticed between the THC and vehicle treated rata following either two or nine days of THC administration . A substantial reduction of the microsomal cytochrome P-450 and its breakIn down product cytochrome P-420 was observed in THC treated rata (Table I) . Experiment I, after a period of two days, the average hemoprotein concentration expressed on a wet weight basis in rats receiving THC fell to approximately half that of vehicle treated rats (a < .10) . In Experiment II, the concentration of microsomal P-450 expressed relative to total microsomal protein declined in THC treated rate to a level of about 70% (a < .10) that of vehicle treated rats following two days of THC administration and 60X (a < .10) after nine days . The total microsomal P-450 content was also cdrreapondingly reduced (Table I) . The Y^glutamyl tranapeptidase activity (Table II) in THC treated rats was aigaificantly reduced after two days of THC administration and further reduced after 9 days to a level of approximately 60% (a < .05) in both Experiments I and II . Statistically insignificant reductions in SDH levels of THC treated rata were noted after 9 and 18 days of THC administration in Experiment I and after 2 and 9 days of THC administration in Experiment II (Table III) . Beta-glucuronidase activities showed no significant differences between THC and vehicle rate in both Experiments I and II (Table IV) . Diacuseion Our finding of decreased microsomal P-450 is THC treated rats is consistent with previous reporta of rapid reductions of plasma LH levels (8,9) after THC injections in rata . Purvia et al . (23) concluded that microsomal cytochrome P-450 in rat tentes exhibited a half life of 3.3 days . In addition, Menard and Purvis (24) demonstrated that microsomal cytochrome P-450 in chick testes was controlled by plasma LH levels . Given the rapid response of cytochrome P-450 to
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Tetrahydrocannabinol and Teatia Enzyme
Vol. 22~ No . 1, 1978
TABLE I The Effect of THC Treatment on Rat Testis Microsomal P-450 Content P-450 Content THC
Vehicle
Experiment I 0 2 9 18
Days Days Days Days
(4) (4) (4) (4)
(3) (4) (4)
(4) (4)
3 .00 + 1,13 2 .59 + 0 .47 2 .29 + 0 .62
2 .47 _+ 0 .76 -
0 .48 0 .77 0.62
(nmolea/mg protein) 0 .124 ± .016 0 .090 + .019
Experiment II 2 Days 9 Days
THC/Veh .
(nmolea/gm wet wt .) 1 .45 + 0 .66 2 .00 + 0 .16 1 .42 + 0 .34
Experiment II 0 Days 2 Days 9 Days
Control
0.172 + .045 0.150 + .055
0 .150 + .041 -
0 .72 0 .60
(umoles/total testis) 0 .848 + .091 0 .514 + .110
1 .228 + .449 0.859 + .447
-
0 .69 0 .60
P-450 content is expressed as the sum of P-450 and P-420. Chronic dosages of 2 mg THC/kg body weight were administered daily as described in Materials and Methods . The numbers in parentheses indicate the number of experimental animals per group . Values are expressed as means + standard deviation .
TABLE II The Effect of THC Treatment on Rat Testis Y-Glutamyl Tranepeptidase Y-Glutamyl Tranepeptidase Activity THC Experiment 0 2 9 18
I
Days Days Days Days
78 + 13 (4) 63 _+ 23 (4) 105 + 13 (4)
THC/Veh .
105 _+ 2 .42 (4) -
0 .76 0 .64 0 .96
(nmolea naphthylamine/min ./mg protein)
-
0.700 + .166 0 .447 + .040
103 + 18 (3) 98 _+ 5 (4) 110 + 13 (3)
0 .949 + .017 0 .825 + .116
0 .795 _+ .073 -
0 .74 0 .54
(nmolea naphthylamine/min ./total testis)
Ex~riment II 2 Days (4) 9 Days (4)
Control
(nmolea naphthylamine/min ./gm wet wt .)
ExQeriment II 0 Days (3) 2 Days (4) 9 Days (4)
Vehicle
264 + 39 206 + 27
303 + 30 369 + 46
-
0 .87 0 .56
Chronic dosages of 2 mg THC/kg body weight were administered daily as described in Materials and Methods . The numb era in parentheses indicate the number of experimental animals per group . Values are expressed as means _+ standard deviation .
Vol . 22, No . 1, 1978
Tetrahydrocannabinol and Testis Enzymes
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TABLE III The Effect of THC Treatment on Rat Testis Sorbitol Dehydrogenase Activity Sorbitol Dehydrogenase Activity THC
Vehicle
Experiment I 0 2 9 18
Days Days Days Days
75 .2 + 15 .2 (4) 97 .2 + 19 .1 (4) 62 .0 + 5 .50 (4)
73 .9 + 3 .8 (3) 115 .7 + 15 .3 (3) 67 .3 + 30 .0 (3)
83 .8 _+ 30 .2 (4) -
1 .02 0 .84 0 .92
(nmoles NADH2 /min ./mg protein) 2 .08 + .20 1 .98 + .13
2 .34 + .45 2 .34 + .37 r
Experiment II 2 Days (4) 9 Days (4)
THC/Veh .
(nmoles NADH 2 /min ./mg wet wt .)
Experiment II 0 Days (3) 2 Days (4) 9 Days (4)
Control
185 + 18 294 + 31
2 .41 _+ .40 -
0 .89 0 .85
(nmoles NADH2 /min ./total testis) 240 + 70 329 + 29
-
0 .77 0 .89
Chronic dosages of 2 mg THC/kg body weight were administered daily as described in Materials and Methods . The numbers in parentheses indicate the ntmlber of experimental animals per group . Values are expressed as means _+ standard deviation . TABLE IV The Effect of THC Treatment on Rat Testis ß-Glucuronidase Activity ß-Glucuronidase Activity THC E xperiment I 0 2 9 18
Days Days Days Days
THC/Veh .
122 + 21 (3) 108 + 14 (4) 221 + 166 (3)
119 .0 _+ 8 .6 (4) -
1 .16 1 .14 0 .87
(nmoles phenolphthalein/min ./mg protein) 2 .44 + .22 2 .40 + .50
Experiment II 2 Days (4) 9 Days (4)
Control
(nmoles phenolphthalein/min ./gm wet wt .) 141 + 8 (4) 123 + 21 (4) 192 + 102 (4)
Ex periment II 0 Days (3) 2 Days (4) 9 Days (4)
Vehicle
2 .18 + .16 2 .66 + 1 .72
1 .93 + .29 -
1 .12 0 .90
(nmolea phenolphthalein/mia ./total testis) 488 + 59 456 + 71
410 + 35 500 + 370
1 .19 1 .10
Chronic dosages of 2 mg THC/kg body weight were administered daily as described in Materials and Methods . The numbers in parentheses indicate the number of experimental animals per group . Values are expressed as means _+ standard deviation .
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Tetrahydrocannabinol and Testis Enzymes
Vol . 22, No . 1,
1978
changes in plasma LH levels, it ie feasible that our observed reductions in cytochrome P-450 after 2 days of THC injections in rats could be a result of reduced plasma LH levels . Our results also suggest that previously reported reductions of plasma testosterone after THC administration (10,11) could be mediated through such an LA-induced change in cytochrome P-450 activity in the testes . Various portions of the testosterone biosynthetic pathway occur in the mitochondria and the microsomal fractions of the Leydig cells . Our data showing reduced microsomal P-450 could account for some reduction in either testosterone synthesis or secretion in THC treated rats . Two important enzymes of testosterone synthesis, 17 a-hydroxylase and C17-C20 lyase, are localized on the microsomes and contain cytochroms P-450 as their active site (25) . These enzymes have been shown to be responsive to changes in plasma pituitary gonadotrophin levels, and their activities decay with half lives similar to those of microsomal cytochrome P-450 (23) . In a similar fashion Purvis et al . (25) demonstrated that mitochondrial P-450 levels, like microsomal P-450 levels, are controlled'by gonadotrophins . Mitochondrial P-450 is not normally detectable in rat testes but by exogenous administration of human chorionic gonadotrophin the P-450 levels were elevated to detectable concentrations . Purvia et _al . (25) also demonstrated that the half life of mitochondrial P-450 was even shorter than that of the microsomal P-450. This data, together with reports (26) that the cholesterol aide-chain cleavage enzyme complex, of which P-450 is an integral component, may be the rate limiting step in testosterone synthesis, raise some interesting questions about the possible effect of THC on mitochondrial P-450 content as well . The reduction of cytochrome P-450 levels in THC treated rats may reflect a selective effect of THC on cytochrome P-450 and does not necessarily indicate an overall reduction in size, number, or activity of Leydig cells, ae evidenced by maintenance of normal beta-glucuronidase levels and normal testicular weight in our experimental animals . Although the reduction in GTP activity after just two days of THC treatment was not as dramatic as the reduction of cytochrome P-450 content, they do suggest an early initiation of the events which lead to a substantial reduction of GTP activity by the ninth day of injections . The reduction in GfP activity may be the result of a THC induced alteration of FSH secretion. However, previous reports (6,8) indicate that changes in plasma FSH after THC administration are not apparent until well after changes in plasma LH and testosterone have occurred . These observations suggest that the reductions we found in GTP activity may be caused by some factor other than an alteration in plasma FSH . levels . The mounting evidence of partial regulation of Sertoli cells by testosterone (27) raises the possibility that reduction in GTP activity in THC treated rate was caused by an alteration in the level of testosterone synthe sis and/or secretion . This theory is a particularly attractive method of explaining the rapidness of the observed reduction in GTP activity . Our data indicate that while no major alterations in germ cell development were observed during the 18-day period of chronic THC administration, there was a substantial reduction in activity of both the Sertoli cell marker enzyme, GTP, and the Leydig cell marker protein, cytochrome P-450. The reduction in cytochrome P-450 suggests that this cytochrome may serve as the mediating mechanism between previously observed reductions in plasma LH and plasma testosterone following THC administration in rats . Although reductions of GTP activity in THC treated rats may be caused by reduced plasma FSH, the
Vol . 22, No . 1, 1978
Tetrahydrocannabinol and Testis Enzymes
13
rapidness of the reduction suggests that the initial effect of THC on Sertoli cells is probably caused by reductions in testosterone synthesis and/or secretion . Other experiments involving simultaneous administration of FSH and THC and LH and THC to rate would be helpful for further clarification of the effect of THC on the testis and on Sertoli cells in particular . Açknowle~ents The authors wish to thank Dr . Cheryl Lu for assistance with the GTP assay . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 .
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