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Effects of delta-9-tetrahydrocannabinol on in vitro energy substrate metabolism in mouse and rat testis
Physiology & Behavior, Vol. 46, pp. 65-68. ©Pergamon Press plc, 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Effects of Delta-9-Tetrahydrocannabinol on In Vitro Energy Substrate Metabolism in Mouse and Rat Testis I SYED HUSAIN
Department o f Pharmacology, University o f North Dakota, School o f Medicine, Grand Forks, N D 58201
HUSAIN, S. Effects of delta-9-tetrahydrocannabinol on in vitro energy substrate metabolism in mouse and rat testis. PHYSIOL BEHAV 46(1) 65-68, 1989.--In this in vitro radiorespirometric study, the effects of delta-9-tetrahydrocannabinol (THC) on glucose and fructose metabolism in the rat and mouse testes were compared. In the rat testis, THC caused a dose-dependent decrease of 15, 18, 20 and 25% in glucose metabolism with 0.1, 0.2 and 0.3 rnM concentrations. Similarly, a 39% inhibition in fructose metabolism was observed when rat testes were exposed to 0.3 mM THC. The comparative studies with mouse testis indicated that 0.1, 0.2 and 0.3 mM THC produced a 4, 12 and 30% decrease in glucose utilization where as a much greater decrease of 13, 30 and 41% in fructose utilization was observed with the same doses of THC. On the basis of these observations in these two animal models, it is concluded that THC has the ability to inhibit energy substrates utilization in the rat and mouse testis. This inhibition of cellular energetics may, in part, be responsible for most of the gonadal effects of THC. Delta-9-tetrahydrocannabinol
Glucose
Fructose
Energy substrate utilization
IN the last two decades, a good deal of research has been done to evaluate the effects of marijuana on different body systems (12,16). These studies have included its effects on the reproductive system both in the male and in the female. Studies on male reproduction has brought a wealth of information which indicates that both marijuana and delta-9-tetrahydrocannabinol (THC) decrease testosterone levels (1, 17, 20), reduce sperm counts (5,13), and sperm motility (18), change sperm morphology (21,22), decrease luteinizing hormone (LH) levels and follicle stimulating hormone (FSH) levels (3,19) and impair potency in different species, including humans (6,14). The effects on LH and FSH have partially helped to explain these actions of THC on gonadal functions. However, a more definitive biochemical basis to help explain most testicular effects of THC remains elusive. This is because THC has also been shown to cause direct effects on the germinal epithelium of the testis as well as on other gonadal functions (7,12). Previously, we have conducted several studies to evaluate the role of cellular energetics in the gonadal effects of THC.(8-11). This was done to substantiate the hypothesis that the effects of marijuana on gonadal functions are, in part, due to an inhibition of energy substrate utilization in the testis. Although these studies were done in rats, it was realized that other animal models such as mice have also been used in different laboratories in many phases of marijuana research. Therefore, this study was designed to compare the effects of THC on in vitro glucose and fructose
In vitro effects
metabolism in the testis of these two species. METHOD For this study, male albino mice and Sprague-Dawley rats were obtained from Bio Lab Corp., Minneapolis, MN. The mice were 5 to 6 weeks old and weighed between 25 to 30 g. The rats, on the other hand, weighed 250 ± 25 g and were approximately 60 days old. These animals were maintained on a rat chow and water ad lib under an alternate light/dark cycle of 12 hr each. All chemicals used in this study were of analytical grade. The glucose-6-14C and fructose-U-laC (specific activities 2.96 and 3.0 mCi/mmol each) were obtained from Amersham Corp., Arlington Heights, IL. The THC was kindly provided in ethanol by the National Institute on Drug Abuse, Washington, DC. On the day of the experiment, these animals were randomly sacrificed by either cervical dislocation (mice) or decapitation (rats) and their testes were rapidly removed. Each testis was stripped of its tunica and rat testes were sectioned into several small pieces. These testicular tissues were divided into control and test groups and placed in separate Warburg flask which contained 2 mi of Tris-buffered medium with 5.5 mM radiolabeled glucose or fructose. The center well and the side arm of the flasks contained 0.2 ml of 3.5 N KOH and 0.2 mi of 70% HC104 (w/v), respectively. Before receiving the testicular tissue, the medium was aerated for an hour with 100% oxygen and maintained at
'Supported by NIDA grant DA03595.
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FIG. 1. Rat testicular tissues were incubated in the presence or absence of 0. l, 0.2, 0.3, and 0.4 mM THC for 100 rnin at 37°C. The incubations were carried out in Tris-buffered medium containing 5.5 mM glucose with 6-14C glucose as the tracer. The open bars represent the CO= production from control tissues, whereas the hatched bars show CO2 production from rat testicular tissues exposed to THC dissolved in different volumes of ethanol.
37°C. Prior to incubations, THC was introduced into the test flask medium in 0.1, 0.2, 0.3 and 0.4 mM concentrations dissolved in 3, 6, 9 and 12 Ixl of ethanol. Control flasks received equal volume of ethanol. Incubations were carried out for 100 min at which time, the reaction was terminated by acidifying the medium with HC104 from the side arm of the flask. The 1 4 C O 2 produced due to the catabolism of the radiolabeled substrates was trapped in KOH of the center well for another hour. It was then counted in a scintillation counter and the rates of energy substrates utilization from control and THC-exposed tissues were expressed as ixmol 14CO2 produced/g dry tissue/100 min incubation. RESULTS AND DISCUSSION Using the above protocol, the effects of different concentrations of THC on glucose utilization by the rat testis were studied first. Four doses of THC were tested for this purpose. At 0.1 mM dose, which was dissolved in 3 ILl of ethanol, THC caused a significant inhibition of 15% in 14CO2 production as compared to controls. When rat testicular tissues were exposed to 0.2, 0.3 and 0.4 mM THC, dissolved in 6, 9 and 12 ~1 ethanol, all these concentrations of THC produced a significant reduction in glucose catabolism as compared to controls. This inhibition in the utilization of glucose was dose related and caused an 18, 20 and 25% decrease in ~4CO2 production (Fig. 1). In this study, a separate investigation with ethanol was conducted since ethanol served as a vehicle for THC. These experiments showed no significant effects of 3, 6, 9 or 12 txl of ethanol on glucose metabolism in the testis. Fructose is another important substrate for energy needs of the testis. Besides its normal supply to the testis, the synthesis of this substrate in the male accessory gland is testosterone dependent (15); however, testosterone production in itself is inhibited by THC (1, 17, 20). Fujimoto et al. (4) reported earlier that adult
E T H A N O L , p.I FIG. 2. Rat testicular tissues were incubated with or without 0.3 mM THC for 100 min at 37°C. The incubations were carried out in Tris-buffered medium containing 5.5 mM fructose with U-14C fructose as the tracer. The open bars represent CO 2 production from control tissue, whereas hatched bars show CO2 production from testicular tissues exposed to 0.3 mM THC dissolved in 9 Ixl of ethanol.
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FIG. 3. Mouse testicular tissues were incubated in the presence or absence of 0.1, 0.2 and 0.3 mM THC for 100 rain at 37°C. The incubations were carried out in Tris-buffered medium containing 5.5 mM glucose with 6-14C glucose as the tracer. The dotted bars represent the CO2 production from control tissues, whereas the hatched bars show CO 2 production from mouse testicular tissues exposed to THC dissolved in different concentrations of ethanol,
THC EFFECTS OF GLUCOSE AND FRUCTOSE METABOLISM
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rats treated chronically with crude marijuana extract contained less sperms in epididymis as well as had a decrease in fructose levels. In light of these observations, the following studies were undertaken to determine the effects of THC on fructose utilization by the rat testis. In these experiments, rat testicular tissues were exposed to 0.3 mM THC which caused a significant inhibition in ]4CO2
67
production (39%, p < 0 . 0 0 1 ) from 5.5 m M radiolabeled fructose in the medium (Fig. 2). In the previous studies, with this concentration, THC had produced a 20% inhibition in glucose metabolism. Glucose and fructose are important energy substrates and fructose is a major sugar found in the testis. An inhibition in the utilization of these substrates could significantly affect many cellular functions of the testis. To test this hypothesis in another species, similar experiments were designed in mouse which has also been used most commonly as an animal model in different phases of marijuana research. At the lower dose of 0.1 mM THC, the testicular tissue of this species showed a 4% inhibition in glucose utilization as compared to its respective controls (Fig. 3). This inhibitory effect of THC became significant in subsequent doseresponse studies when mouse testicular tissues were exposed to higher concentrations of THC. At 0.3 m M THC, a 30% inhibition was observed which was higher than the 20% inhibition observed with rat testicular tissues in earlier experiments (Figs. 1 and 3). The dose-response effects of THC were therefore, similar in mice as observed in the rat. However, it did not demonstrate a significant inhibition in glucose utilization at each doses of THC tested. In other experiments, the effects of different concentrations of THC on fructose utilization by the mouse testis were evaluated. In these in vitro studies, with fructose as a substrate, THC produced much higher inhibition in 14CO2production. A dose of 0.1 m M THC caused 13% inhibition, whereas 0.2 m M and 0.3 m M THC produced a 30 to 41% inhibition, respectively (Fig. 4). This inhibition of 41% with 0.3 mM THC was similar to the 39% inhibition observed with rat testicular tissue (Figs. 2 and 4). Although, the lower concentration of THC (0.1 mM) produced a 13% decrease in fructose utilization, this inhibition did not achieve statistical significance. It is speculated that this may possibly be due to a comparative higher rate of substrate utilization by the mouse testis which overcomes the THC inhibition at lower doses or it may simply be a species dependent phenomena in terms of dose-response effects. It is, therefore, concluded that THC has the property to inhibit the utilization of energy rich substrates in rat and mouse testis. This inhibition of cellular energetics may, in part, be responsible for some of the gonadal effects of THC.
REFERENCES 1. Dalterio, S.; Bartke, A.; Robertson, C.; Watson, D.; Bertstein, S. Direct and pituitary mediated effects of delta-9-tetrahydrocannabinol on the testis. Pharmacol. Biochem. Behav. 8:673-678; 1978. 2. Dewey, W. L. Cannabinoid pharmacology. Pharmacol. Rev. 38: 151-177; 1986. 3. Dixit, V. P.; Sharma, V. N.; Lohiya, N. K. The effects of chronically administered cannabis extract on the testicular function of mice. J. Pharmacol. 26:111-114; 1974. 4. Fujimoto, G. I.; Kostellow, A. B.; Rosenbaum, R.; Morill, G. A.; Block, E. Effects of Cannabinoids on reproductive organs in the female Fischer rat. In: Nahas, G. G.; Paton, W. D. M., eds. Marijuana: Biological effects. Oxford: Pergamon Press; 1979:441447. 5. Hembree, W. C., III; Nahas, G. G.; Zeidenberg, P.; Huang, H. F. S. Changes in human spermatozoa associated with high dose marijuana smoking. In: Nahas, G. G.; Paton, W. D. M., eds. Marijuana: Biological effects. Oxford: Pergamon Press; 1979:429-439. 6. Hembree, W. C., HI; Nahas, G. G.; Zeidenherg, P.; Dyrenfurth, I. Evidence for a direct effect of marijuana smoking upon the characteristic of ejaculated human spermatozoa. Clin. Res. 24:272A; 1976. 7. Hembree, W. C., HI; Zeidenberg, P.; Nahas, G. G. Marijuana's effects on human gonadal function. In: Nahas, G. G., ed. Marijuana: Chemistry, biochemistry and cellular effects. New York: Springer Verlag; 1978:521-532. 8. Husaln, S.; Lame, M.; DeBoer, B. Alterations in rat testicular tissue
9. 10.
11.
12.
13. 14.
15.
glucose metabolism in the presence of delta-9-tetrahydrocannabinol. Proc. West. Pharmacol. Soc. 22:355-358; 1979. Husain, S.; Lame, M. Inhibitory effects of delta-9-tetrahydrocannabinol on glycolytic substrates in the rat testis. Pharmacology 23:102-112; 1981. Husain, S.; Lame, M. Possible mechanism for the cellular effects of marijuana on male reproductive function. In: Agurell, S.; Dewey, W. L.; Willette, R. E., eds. The cannabinoids: Chemical, pharmacologic and therapeutic aspects. New York: Academic Press; 1984:453-470. Husain, S. Involvement of cellular energetics in the gonadal effects of delta-9-tetrahydrocannabinol (THC). In: Harvey, D. J., ed. Marijuana '84: Proceedings of the Oxford Symposium on cannabis. Oxford: IRL Press; 1985:391-398. Jakubovic, A.; McGeer, P. L. In vitro inhibition of protein and nucleic acid synthesis in the rat testicular tissue by cannabinoids. In: Nahas, G. G., ed. Marijuana: Chemistry, biochemistry and cellular effects. New York: Springer Verlag; 1976:223-241. Kolodny, R. C.; Masters, W. H.; Kolodner, R. M.; Toro. G. Depression of plasma testosterone levels after chronic intensive marijuana use. N. Engl. J. Med. 290:872-874; 1974. Kolodny, R. C.; Lessin, P.; Toro, G.; Masters, W. H.; Cohen, S. Depression of plasma testosterone with acute marijuana administration. In: Brande, M. C.; Szara, S., eds. The pharmacology of marijuana. New York: Raven Press; 1976:217-225. Mann, T.; Parsons, U. Effects of testicular hormone on the formation
68
of seminal fructose. Nature 160:294; 1947. 16. Martin, B. R. Cellular effects of cannabinoids. Pharmacol. Rev. 38:45-74; 1986. 17. Maskarinec, M. P.; Shipley, G.; Novotny, M.; Brown, D. J.; Forney, R. B. Endocrine effects of cannabis in male rats. Toxicol. Appl. Pharmacol. 45:615-628; 1978. 18. Perez, L. E.; Smith, C. G.; Asch, R. H. Delta-9-tetrahydrocannabinol inhibits fructose utilization and motility in human, rhesus monkey and rabbit sperm in vitro. Fertil. Steril. 35:703-705; 1981. 19. Smith, C. G.; Besch, N. F.; Asch, R. H. Effects of marijuana on the reproductive system. In: Thomas, J. A.; Singhal, R. L., eds. Advances in sex hormones research. Baltimore: Urban and Schwarzenberg; 1980:273-294.
HUSAIN
20. Symons, A. M.; Teale, J. D.; Marks, V. Effects of delta-9-tetrahydrocannabinol on the hypothalamic pituitary gonadal system in maturing male rat. J. Endocrinol. 68:43P-44P; 1976. 21. Tilak, S. K.; Zimmarman, A. M. Effects of cannabinoids on spermatogenesis in mice: in vivo and in vitro studies. In: Agurell, S.; Dewey, W. L.; Willette, R. E., eds. The cannabinoids: Chemical, pharmacologic and therapeutic aspects. New York: Academic Press; 1984:427-439. 22. Zimmerman, A. M.; Zimmerman, S.; Raj, A. Y. Effect of cannabinoids on spermatogenesis in mice. In: Nahas, G. G.; Paton, W. D. M., eds. Marijuana: Biological effects. Oxford: Pergamon Press; 1979:407-418.
0031-9384/89 $3.00 + .00
Effects of Delta-9-Tetrahydrocannabinol on In Vitro Energy Substrate Metabolism in Mouse and Rat Testis I SYED HUSAIN
Department o f Pharmacology, University o f North Dakota, School o f Medicine, Grand Forks, N D 58201
HUSAIN, S. Effects of delta-9-tetrahydrocannabinol on in vitro energy substrate metabolism in mouse and rat testis. PHYSIOL BEHAV 46(1) 65-68, 1989.--In this in vitro radiorespirometric study, the effects of delta-9-tetrahydrocannabinol (THC) on glucose and fructose metabolism in the rat and mouse testes were compared. In the rat testis, THC caused a dose-dependent decrease of 15, 18, 20 and 25% in glucose metabolism with 0.1, 0.2 and 0.3 rnM concentrations. Similarly, a 39% inhibition in fructose metabolism was observed when rat testes were exposed to 0.3 mM THC. The comparative studies with mouse testis indicated that 0.1, 0.2 and 0.3 mM THC produced a 4, 12 and 30% decrease in glucose utilization where as a much greater decrease of 13, 30 and 41% in fructose utilization was observed with the same doses of THC. On the basis of these observations in these two animal models, it is concluded that THC has the ability to inhibit energy substrates utilization in the rat and mouse testis. This inhibition of cellular energetics may, in part, be responsible for most of the gonadal effects of THC. Delta-9-tetrahydrocannabinol
Glucose
Fructose
Energy substrate utilization
IN the last two decades, a good deal of research has been done to evaluate the effects of marijuana on different body systems (12,16). These studies have included its effects on the reproductive system both in the male and in the female. Studies on male reproduction has brought a wealth of information which indicates that both marijuana and delta-9-tetrahydrocannabinol (THC) decrease testosterone levels (1, 17, 20), reduce sperm counts (5,13), and sperm motility (18), change sperm morphology (21,22), decrease luteinizing hormone (LH) levels and follicle stimulating hormone (FSH) levels (3,19) and impair potency in different species, including humans (6,14). The effects on LH and FSH have partially helped to explain these actions of THC on gonadal functions. However, a more definitive biochemical basis to help explain most testicular effects of THC remains elusive. This is because THC has also been shown to cause direct effects on the germinal epithelium of the testis as well as on other gonadal functions (7,12). Previously, we have conducted several studies to evaluate the role of cellular energetics in the gonadal effects of THC.(8-11). This was done to substantiate the hypothesis that the effects of marijuana on gonadal functions are, in part, due to an inhibition of energy substrate utilization in the testis. Although these studies were done in rats, it was realized that other animal models such as mice have also been used in different laboratories in many phases of marijuana research. Therefore, this study was designed to compare the effects of THC on in vitro glucose and fructose
In vitro effects
metabolism in the testis of these two species. METHOD For this study, male albino mice and Sprague-Dawley rats were obtained from Bio Lab Corp., Minneapolis, MN. The mice were 5 to 6 weeks old and weighed between 25 to 30 g. The rats, on the other hand, weighed 250 ± 25 g and were approximately 60 days old. These animals were maintained on a rat chow and water ad lib under an alternate light/dark cycle of 12 hr each. All chemicals used in this study were of analytical grade. The glucose-6-14C and fructose-U-laC (specific activities 2.96 and 3.0 mCi/mmol each) were obtained from Amersham Corp., Arlington Heights, IL. The THC was kindly provided in ethanol by the National Institute on Drug Abuse, Washington, DC. On the day of the experiment, these animals were randomly sacrificed by either cervical dislocation (mice) or decapitation (rats) and their testes were rapidly removed. Each testis was stripped of its tunica and rat testes were sectioned into several small pieces. These testicular tissues were divided into control and test groups and placed in separate Warburg flask which contained 2 mi of Tris-buffered medium with 5.5 mM radiolabeled glucose or fructose. The center well and the side arm of the flasks contained 0.2 ml of 3.5 N KOH and 0.2 mi of 70% HC104 (w/v), respectively. Before receiving the testicular tissue, the medium was aerated for an hour with 100% oxygen and maintained at
'Supported by NIDA grant DA03595.
65
66
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FIG. 1. Rat testicular tissues were incubated in the presence or absence of 0. l, 0.2, 0.3, and 0.4 mM THC for 100 rnin at 37°C. The incubations were carried out in Tris-buffered medium containing 5.5 mM glucose with 6-14C glucose as the tracer. The open bars represent the CO= production from control tissues, whereas the hatched bars show CO2 production from rat testicular tissues exposed to THC dissolved in different volumes of ethanol.
37°C. Prior to incubations, THC was introduced into the test flask medium in 0.1, 0.2, 0.3 and 0.4 mM concentrations dissolved in 3, 6, 9 and 12 Ixl of ethanol. Control flasks received equal volume of ethanol. Incubations were carried out for 100 min at which time, the reaction was terminated by acidifying the medium with HC104 from the side arm of the flask. The 1 4 C O 2 produced due to the catabolism of the radiolabeled substrates was trapped in KOH of the center well for another hour. It was then counted in a scintillation counter and the rates of energy substrates utilization from control and THC-exposed tissues were expressed as ixmol 14CO2 produced/g dry tissue/100 min incubation. RESULTS AND DISCUSSION Using the above protocol, the effects of different concentrations of THC on glucose utilization by the rat testis were studied first. Four doses of THC were tested for this purpose. At 0.1 mM dose, which was dissolved in 3 ILl of ethanol, THC caused a significant inhibition of 15% in 14CO2 production as compared to controls. When rat testicular tissues were exposed to 0.2, 0.3 and 0.4 mM THC, dissolved in 6, 9 and 12 ~1 ethanol, all these concentrations of THC produced a significant reduction in glucose catabolism as compared to controls. This inhibition in the utilization of glucose was dose related and caused an 18, 20 and 25% decrease in ~4CO2 production (Fig. 1). In this study, a separate investigation with ethanol was conducted since ethanol served as a vehicle for THC. These experiments showed no significant effects of 3, 6, 9 or 12 txl of ethanol on glucose metabolism in the testis. Fructose is another important substrate for energy needs of the testis. Besides its normal supply to the testis, the synthesis of this substrate in the male accessory gland is testosterone dependent (15); however, testosterone production in itself is inhibited by THC (1, 17, 20). Fujimoto et al. (4) reported earlier that adult
E T H A N O L , p.I FIG. 2. Rat testicular tissues were incubated with or without 0.3 mM THC for 100 min at 37°C. The incubations were carried out in Tris-buffered medium containing 5.5 mM fructose with U-14C fructose as the tracer. The open bars represent CO 2 production from control tissue, whereas hatched bars show CO2 production from testicular tissues exposed to 0.3 mM THC dissolved in 9 Ixl of ethanol.
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FIG. 3. Mouse testicular tissues were incubated in the presence or absence of 0.1, 0.2 and 0.3 mM THC for 100 rain at 37°C. The incubations were carried out in Tris-buffered medium containing 5.5 mM glucose with 6-14C glucose as the tracer. The dotted bars represent the CO2 production from control tissues, whereas the hatched bars show CO 2 production from mouse testicular tissues exposed to THC dissolved in different concentrations of ethanol,
THC EFFECTS OF GLUCOSE AND FRUCTOSE METABOLISM
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rats treated chronically with crude marijuana extract contained less sperms in epididymis as well as had a decrease in fructose levels. In light of these observations, the following studies were undertaken to determine the effects of THC on fructose utilization by the rat testis. In these experiments, rat testicular tissues were exposed to 0.3 mM THC which caused a significant inhibition in ]4CO2
67
production (39%, p < 0 . 0 0 1 ) from 5.5 m M radiolabeled fructose in the medium (Fig. 2). In the previous studies, with this concentration, THC had produced a 20% inhibition in glucose metabolism. Glucose and fructose are important energy substrates and fructose is a major sugar found in the testis. An inhibition in the utilization of these substrates could significantly affect many cellular functions of the testis. To test this hypothesis in another species, similar experiments were designed in mouse which has also been used most commonly as an animal model in different phases of marijuana research. At the lower dose of 0.1 mM THC, the testicular tissue of this species showed a 4% inhibition in glucose utilization as compared to its respective controls (Fig. 3). This inhibitory effect of THC became significant in subsequent doseresponse studies when mouse testicular tissues were exposed to higher concentrations of THC. At 0.3 m M THC, a 30% inhibition was observed which was higher than the 20% inhibition observed with rat testicular tissues in earlier experiments (Figs. 1 and 3). The dose-response effects of THC were therefore, similar in mice as observed in the rat. However, it did not demonstrate a significant inhibition in glucose utilization at each doses of THC tested. In other experiments, the effects of different concentrations of THC on fructose utilization by the mouse testis were evaluated. In these in vitro studies, with fructose as a substrate, THC produced much higher inhibition in 14CO2production. A dose of 0.1 m M THC caused 13% inhibition, whereas 0.2 m M and 0.3 m M THC produced a 30 to 41% inhibition, respectively (Fig. 4). This inhibition of 41% with 0.3 mM THC was similar to the 39% inhibition observed with rat testicular tissue (Figs. 2 and 4). Although, the lower concentration of THC (0.1 mM) produced a 13% decrease in fructose utilization, this inhibition did not achieve statistical significance. It is speculated that this may possibly be due to a comparative higher rate of substrate utilization by the mouse testis which overcomes the THC inhibition at lower doses or it may simply be a species dependent phenomena in terms of dose-response effects. It is, therefore, concluded that THC has the property to inhibit the utilization of energy rich substrates in rat and mouse testis. This inhibition of cellular energetics may, in part, be responsible for some of the gonadal effects of THC.
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