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Cocaine impairs gonadotropin secretion in oophorectomized monkeys
Cocaine impairs gonadotropin secretion in oophorectomized monkeys Melin S. Canez, MD; Mary H. Samuels, MD,b Michael F. Luther, MS,d Thomas S. King, PhD,"' and Robert S. Schenken, MD' C
San Antonio, Texas OBJECTIVE: Our objective was to determine whether cocaine alters gonadotropin secretion in oophorectomized monkeys. STUDY DESIGN: Oophorectomized monkeys with elevated gonadotropin levels were chronically cannulated to allow blood sampling every 15 minutes. Monkeys received either saline solution or 2 or 4 mg/kg cocaine hydrochloride as an intravenous bolus. Other oophorectomized monkeys were pretreated with either saline solution or 4 mg/kg cocaine 2 hours before bolus gonadotropin-releasing hormone administration, and plasma luteinizing hormone and follicle-stimulating hormone levels were measured every 15 minutes for 3 hours. Monkeys were also given either saline solution or 4 mg/kg of cocaine with gonadotropin-releasing hormone simultaneously, and plasma gonadotropin levels were measured every 15 minutes for 3 hours. Serum luteinizing hormone and follicle-stimulating hormone levels were measured by radioimmunoassay. RESULTS: Both doses of cocaine resulted in a significant decrease in luteinizing hormone levels compared with controls. Follicle-stimulating hormone levels were significantly decreased only with the 4 mg/kg dose of cocaine. There was no difference in luteinizing hormone and follicle-stimulating hormone responses to gonadotropin-releasing hormone in the cocaine-treated monkeys compared with saline solution-treated monkeys by using repeated-measures analysis of variance. CONCLUSION: These findings demonstrate that acute cocaine administration to oophorectomized primates inhibits basal luteinizing hormone-follicle-stimulating hormone secretion but not gonadotropin-releasing hormone-stimulated luteinizing hormone and follicle-stimulating hormone release. In the absence of an effect on gonadotropin-releasing hormone-stimulated gonadotropin release, we conclude that the impaired luteinizing hormone-follicle-stimulating hormone secretion after cocaine administration is due in part to a direct effect of cocaine on gonadotropin-releasing hormone neurons or on hypothalamic neurotransmitter modulation of gonadotropin-releasing hormone release. (AM J OSSTET GVNECOL 1992;167:1785-93.)
Key words: Cocaine, reproductive dysfunction, primates Cocaine abuse has become an epidemic in recent years. 1 The use of cocaine in women of reproductive age has been associated with many complications of pregnancy, including sudden death, acute myocardial infarction, cerebrovascular accident, seizures, abruptio placentae, teratogenicity, intrauterine growth retardation, intrauterine fetal death, neonatal addiction, sudden infant death syndrome, and early childhood neurologic disorders.2. 3 Sexual dysfunction, irregular menstrual cycles, and amenorrhea have also been reported
From the Departments of Obstetrics and Gynecology, a Medicine, band Cellular and Structural Biology,' The University of Texas Health Science Center at San Antonio, and the Department of Research and Development, Audie L. Murphy Veterans Hospital. d Presented in part at the Thirty-eighth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23, 1991. Reprint requests: Robert S. Schenken, MD, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7836. 6/6/41910
nonpregnant women addicted to cocaine!·6 However, cocaine's effects on ovarian-menstrual cyclicity and reproductive functions in nongravid humans or subhuman primates are poorly understood. Preliminary data in rodents have shown that chronic cocaine treatment disrupts estrous cyclicity, causing prolonged periods of diestrus, repetitive days of estrus, decreased ovulation rates, and reduced luteinizing hormone (LH) levels. 7 This disruption of estrous cyclicity has recently been shown to be dose related. 6 Isolated and perfused hypothalami from rats chronically treated with cocaine demonstrate increased basal secretion of norepinephrine and serotonin and attenuated gonadotropin-releasing hormone (GnRH) response to pulsed norepinephrine. This alteration of hypothalamic aminergic activity necessary for pulsatile GnRH release suggests that cocaine may interrupt estrous cyclicity by altering GnRH pulsatility and gonadotropin release. This study was conducted to assess whether cocaine alters gonadotropin secretion in subhuman primates. III
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Initial studies were conducted to determine whether intravenous cocaine administration to monkeys resulted in serum cocaine levels equivalent to those levels reported in humans feeling "high." To assess whether cocaine altered basal or pulsatile gonadotropin secretion in primates, serum LH and follicle-stimulating hormone (FSH) levels were measured before and after saline solution or low- or high-dose cocaine administration. To determine whether cocaine altered GnRHstimulated gonadotropin secretion, saline solution or cocaine was given with GnRH and serum concentrations of gonadotropins were measured. Results suggested that cocaine impairs pituitary LH and FSH secretion via a direct hypothalamic effect. Material and methods
Animals. Long-term oophorectomized adult female cynomolgus (Macaca fascicularis) monkeys with elevated serum gonadotropin levels were studied. Weights ranged from 3.2 to 3.8 kg, and all monkeys had regular menstrual cycles before ovariectomy. Monkeys were free of chronic drug exposure. They were housed individually, provided food (Purina Monkey Chow; Ralston Purina, St. Louis) twice daily, and given free access to water. Rooms were climate controlled, with light and dark cycles of 14 hours light and 10 hours dark (lights on at 7 AM, lights off at 9 PM). Catheterization. Monkeys were anesthetized with intramuscular injections of 10 mg/kg ketamine (Ketaset; Aveco, Fort Dodge, la.) and 2 mg/kg xylazine (Rompun; Mobay Corp., Shawnee, Kan.). A 20-gauge cannula was inserted into the external jugular vein and advanced to the superior vena cava. The cannula was tunneled subcutaneously, exiting in the interscapular area of the animal. A primate jacket was placed on the monkey. The cannula was threaded out the back of the jacket and through a mobile tether consisting of a flexible metal tube connected from the jacket to a standardsized, single-channel fluid swivel. 9 Each monkey received an intramuscular injection of 300,000 IV of penicillin G benzathine (Bicillin; Wyeth, Philadelphia) postoperatively. Catheter patency was maintained by continuous infusion of lactated Ringer's solution containing 2000 IU/L heparin at a rate of 0.004 ml/min. Blood sampling. All blood samples were collected in heparinized syringes after the catheter was cleared of infusion solution. Plasma was harvested, snap frozen in acetone on dry ice, and stored at - 20° C. Red blood cells were reconstituted with saline solution and returned to the monkeys every 2 hours. Samples collected for cocaine analysis were aliquoted into tubes containing 200 f..LI of potassium phosphate buffer 0.05 mol/L (pH 5) plus sodium fluoride. All blood sampling and drug administrations were through the external jugular catheter.
December 1992 Am J Obstet Gynecol
Treatments. An overview of the experimental design and phased statistical analysis is presented in Fig. 1. Experiment 1. Monkeys (N = 5) were given either 2 or 4 mg/kg cocaine hydrochloride on alternating days. Serum samples were drawn every 5 minutes for 15 minutes, then every 15 minutes for 2 hours. All samples were assayed for cocaine and benzoylecgonine. Experiment 2. The effects of cocaine on basal and pulsatile pituitary gonadotropin secretion were studied in five monkeys. The day after cannulation, blood samples were collected every 15 minutes from 7 AM to 3 PM. At 8 AM 1 ml of normal saline solution was given to each monkey. Three or four days later blood samples were collected every 15 minutes from 7 AM to 3 PM, and 2 mg/kg cocaine hydrochloride was dissolved in 1 ml of normal saline solution was given at 8 AM. After 3 or 4 days blood sampling resumed as before, and 4 mg/kg cocaine in 1 ml of saline solution was given at 8
AM.
Experiment 3. The effects of cocaine on gonadotropin release after exogenous GnRH treatment were studied in two monkeys. The day after cannulation blood samples were collected every 15 minutes from 8 AM to 2 PM. One milliliter of normal saline solution was given at 9 AM, and 15 Ilg/kg GnRH was given at 11 AM. Two days later blood samples were collected as before, 4 mg/kg cocaine hydrochloride was given at 9 AM, and 15 Ilg/kg GnRH was given at 11 AM. This was repeated (saline solution-GnRH 1 day, no treatment 1 day, cocaineGnRH 1 day) three more times with at least 3 days between treatment regimens. GnRH was given 2 hours after cocaine treatment to avoid confusion between acute cocaine stimulation and exogenous GnRH stimulation of gonadotropins. 10 There was no significant stimulation of gonadotropins after acute cocaine treatment; therefore GnRH and cocaine were given simultaneously in experiment 4. Experiment 4. The effects of cocaine and GnRH given simultaneously were studied in two monkeys. Blood samples were collected every 15 minutes from 8 AM to noon. The day after cannulation 1 ml of saline solution and 15 Ilgikg GnRH were given at 9 AM. One day later 4 mg/kg cocaine hydrochloride and 15 Ilg/kg GnRH were given at 9 AM. This treatment was repeated three more times with at least 3 days between treatment regimens. Assays. High-performance liquid chromatography with ultraviolet detection was used to measure plasma levels of cocaine and its primary metabolite, benzoylecgonine, in extracted serum collected in experiment 1. Details of the assay have been described. 7 All serum samples collected in experiments 2, 3, and 4 were assayed in duplicate for LH and FSH by radioimmunoassay as previously described. 10. II Monkey LH determinations were performed with an antiserum to
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Time (h) Fig. 1. Overview of experimental design in experiments 2, 3, and 4. Phase 1 (I = 0 to 1 hours) served as baseline. Saline solution or cocaine was administered at 1 = 1 hour in all experiments. Phase 2 (I = 1 to 3 hours) was analyzed to assess possible acute stimulatory effects of cocaine in experiments 2 and 3. GnRH was administered 2 hours after cocaine or saline solution (I = 3 hours) in experiment 3. In experiment 4 cocaine or saline solution was administered with GnRH at 1 = 1 hour, and the LH and FSH response was assessed from 1 = 1 to 4 hours.
ovine LH (anti-OLH No. IS, provided by Dr. C. Niswender) that cross reacts indistinguishably with rhesus LH. The standard was LER-M-907, and iodine 125ovine LH was used as label. Monkey FSH determinations were performed with a heterologous radioimmunoassay. Antihuman FSH and 125I-recombinant FSH are used with monkey FSH (LER-1909-2) as a standard. All samples for LH and FSH determinations in individual monkeys were performed in the same assay. The intraassay coefficients of variation ranged from 4% to 12% for LH and from 4% to II % for FSH. Statistics Experiment 1. The serum elimination phase half-life (t'/2) of cocaine and benzoylecgonine was calculated from the slopes of their first-order kinetic rates. 12 Experiment 2. Data were assessed in three phases on the basis of the elimination time of cocaine (Fig. 1). Phase I (t = a to I hour) was baseline. Phase 2 (t = I to 3 hours) was the immediate response while cocaine was still detectable in the serum. Phase 3 (t = 3 to 8 hours) was the delayed response. Phase I LH and FSH levels
were compared among monkeys by repeated-measure analysis of variance. During the immediate response (phase 2), peak LH and FSH levels after cocaine or saline solution administration were compared by repeated-measures analysis of covariance, controlling for baseline when necessary. During the delayed response (phase 3), LH and FSH levels after cocaine or saline solution administration were analyzed in two ways. First, gonadotropin levels were compared among treatment groups by repeated-measures analysis of covariance. Second, hormone pulses were identified by cluster analysis,'3 an objective, computer-based, pulse-analysis program. Variance was calculated by using of dosedependent coefficients of variation calculated from sample replicates in each monkey's hormone series. Cluster parameters were two points for test nadirs and one point for test peaks. The t statistics were 1.0 for upstrokes and 1.0 for downstrokes. These parameters have been previously defined to provide optimal sensitivity and positive accuracy for gonadotropin pulse detection while constraining false-positive and false-
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negative rates to 5%.'3. '4 Pulse frequency and amplitude were compared among groups by repeated-measures analysis of variance. Experiment 3. The data were similarly divided into three phases: phase 1 (t = 0 to 1 hour) was baseline, phase 2 (t = 1 to 3 hours) was the immediate response after cocaine or saline solution administration, and phase 3 (t = 3 to 6 hours) was the time after GnRH administration. Phase 1 LH and FSH levels were compared between treatments (cocaine or saline solution) by repeatedmeasures analysis of variance. Phase 2 peak levels ofLH and FSH were compared by repeated-measures analysis of covariance. Quantification of gonadotropin secretion after GnRH (phase 3) was estimated in two ways: first, by the area under the curves compared with the Student 1 test, and, second, serum gonadotropin levels
were plotted against time and analyzed with repeatedmeasures analysis of covariance. Experiment 4. The data were divided into two phases. Phase 1 (t = 0 to 1 hour) was baseline, and phase 2 (I = 1 to 4 hours) was the time period after simultaneous cocaine or saline solution and GnRH administration. Phase 1 LH and FSH levels were compared by repeated-measures analysis of variance. Phase 2 LH and FSH levels were compared between groups by area under the curve and repeated-measures analysis of covariance. Results
Experiment 1. Serum levels of cocaine and benzoylecgonine after 2 or 4 mg/kg doses are shown in Fig. 2. Peak serum levels of cocaine and benzoylecgonine were 151 ± 49 and 158 ± 15 ng/ml (mean ± SE),
Cocaine impairs gonadotropin secretion
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Time (h) Fig. 3. Experiment 2. Serum LH (solid line) and FSH (broken line) concentrations (lLg/ml) in a representative monkey after saline solution (upper panel), 2 mglkg cocaine (middle panel), or 4 mg/kg (lower panel) administered intravenously at t = I hour. Circles and triangles, respectively, indicate LH and FSH pulses as assessed by cluster analysis.
respectively, after the 2 mg/kg dose. Cocaine and benzoylecgonine levels then gradually declined and were no longer detectable by 1 hour 45 minutes. Peak serum levels of cocaine and benzoylecgonine were 302 ± 54 and 211 ± 31 ng/ml (mean ± SE), respectively, after the 4 mglkg dose. Levels then gradually decreased and were no longer detectable by 2 hours. The tV2 values for cocaine after the 2 and 4 mg/kg doses were 22 ± 2 and 21 ± 3 minutes (mean ± SE), respectively. The tV2 for benzoylecgonine after the 2 and 4 mg/kg doses were 37 ± 7 and 29 ± 2 minutes (mean ± SE), respectively. Experiment 2. The effects of cocaine on gonadotropin secretion throughout the blood sampling interval are shown for one respective monkey in Fig. 3. There was significant variability in baseline gonadotropin levels among monkeys (p = 0.005 for LH, p = 0.02 for FSH). Therefore phases 2 and 3 of this experiment were adjusted for this baseline covariate. In phase 2 there was no significant stimulation of gonadotropins after acute cocaine treatment. LH secretion in phase 3 was significantly decreased after low-dose and high-
dose cocaine treatment, whereas FSH secretion was decreased only after high-dose cocaine administration, as assessed by repeated-measures analysis of covariance (Table I). Pulse analysis showed no effect of cocaine on LH or FSH pulse frequency. There was a clear trend toward decreased LH (P = 0.07), but not FSH, pulse amplitude after cocaine administration, as compared with saline solution. Experiment 3. Fig. 4 shows mean gonadotropin responses to GnRH in cocaine- and saline-treated monkeys. There was significant variability among baseline gonadotropin levels (P < 0.001 for LH and FSH) during phase 1. This was adjusted for in phases 2 and 3. In phase 2 there was no significant stimulation of gonadotropins after acute cocaine treatment. In phase 3 mean GnRH-stimulated gonadotropin secretion was not significantly different in monkeys treated with cocaine compared with saline solution-treated controls. Repeated-measures analysis of covariance showed no difference in mean LH levels over time. Monkeys treated with saline solution had a peak LH level
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Time (h) Fig. 4. Experiment 3. Serum LH (upper panel) and FSH (lower panel) responses (mean ± SE) to intravenous GnRH (15 II-glkg) administration at t = 3 hours after treatment with saline solution (broken line) or cocaine, 4 mg/kg (solid line), intravenously at t = 1 hour.
Table I. Gonadotropin secretion, pulse amplitude, and pulse frequency after saline solution or low-dose (2 mglkg) or high-dose (4 mg/kg) cocaine administration Mean decrease in gonadotropins
Repeated-measures analysis of variance
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(mean ± SE) of 6.1 ± 0.2 lJ.g/ml, compared with 5.3 ± 0.3 lJ.g/ml in cocaine-treated monkeys. LH secretion (area under the curve) was 4.7 lJ.g/ml-1/hr-1 in saline solution-treated and 3.4 IJ.g' ml- 1. hr- 1 in cocaine-treated monkeys. Repeated-measures analysis of covariance showed no difference in mean FSH levels in cocaine- and saline solution-treated monkeys. Monkeys treated with saline
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frequency (pulses/3 hr)
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15.2 ± 7.5 13.4 ± S.6 12.2 ± 9.2
2.4 ± 1.5 I.S ± 0.4 2.3 ± 1.0
solution had a peak FSH level (mean ± SE) of 30.6 ± 1.6 lJ.g/ml compared with 33.1 ± 3.0 IJ.g/ml in cocaine-treated monkeys. FSH secretion (area under the curve) was 12.7 IJ.g' ml- 1. hr- 1 in saline solutiontreated animals and 14.1IJ.g· ml- 1. hr- 1in the cocainetreated monkeys. Experiment 4. Fig. 5 shows mean gonadotropin responses to GnRH in cocaine- and saline-treated mon-
Cocaine impairs gonadotropin secretion
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keys. There were no significant differences in LH or FSH levels after GnRH in cocaine- or saline solutiontreated monkeys. LH secretion (area under the curve) was 2.4 IJ.g' ml- I • hr- I in saline solution-treated and 3.2 IJ.g' ml- I • hr- I in cocaine-treated monkeys. FSH secretion (area under the curve) was 10.5 IJ.g' ml- I • hr- I in saline solution-treated and 8.9 IJ.g' ml- I • hr- I in cocaine-treated monkeys. Comment An important preliminary step in establishing an
animal model to study the effects of human drug abuse is to demonstrate that the dose and plasma levels of the drug in the experimental paradigm are similar to those found in humans. Thus our initial aim was to establish the dose of cocaine necessary to mimic levels seen in humans. The literature reports large intersubject variations in metabolism of low-dose and high-dose intravenous cocaine in both humans and animals. 14.2o Peak serum cocaine levels in our oophorectomized monkeys were 151 ± 49 and 302 ± 54 ng/ml after doses of 2 and 4 mg/kg, respectively. These levels and the respective calculated tl/2 (22 ± 2 and 21 ± 3 minutes) are similar to those reported in other studies.
To assess the effects of cocaine on basal and pulsatile gonadotropin secretion, we analyzed changes in hormone levels during three phases (experiment 2). Phase 1, before cocaine exposure, served as baseline. Gonadotropin levels during the first 2 hours after cocaine exposure, phase 2, were assessed because previous studies demonstrated acute increases in LH and FSH secretion after cocaine administration to cycling monkeys.21. 22 Phase 3 represented the delayed effects of cocaine, once circulating drug levels were no longer detectable. There were no significant changes in serum LH or FSH levels during phase 2. In contrast, gradual, significant declines in LH and FSH secretion were observed in phase 3. This decrease in gonadotropins was observed with both the 2 and 4 mglkg doses of cocaine. A clear trend (p = 0.07) toward a decreased LH pulse amplitude was also apparent during phase 3. The absence of an acute stimulatory effect of cocaine on gonadotropin secretion in oophorectomized monkeys is in contrast to previous findings in cycling monkeys.21. 22 There are several possible explanations for these disparate findings. First, the doses of cocaine used in our study were fivefold greater than those used by Mello et al. 21 With these doses we achieved serum
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cocame levels similar to those observed in humans. Serum cocaine levels were not reported in the other two studies in cycling monkeys!" 22 and it remains possible that cocaine is stimulatory at low concentrations and inhibitory at higher concentrations. The use of oophorectomized versus intact monkeys may also be an important factor. The effects of biogenic amines and opioids on pituitary LH and FSH secretion are clearly modulated by ovarian steroids, and the effects of cocaine on gonadotropin secretion may also be modified by circulating estrogen and progesterone levels. 23 The techniques of blood collection and statistical analyses are also crucial to the interpretation of findings. The use of a primate chair and ketamine anesthesia, a known modulator of prolactin secretion, in the previous studies may have had effects not apparent in chronically cannulated monkeys with a vest and mobile tether assembly.9 Finally, both studies in cycling monkeys assessed changes in gonadotropin secretion within 2 hours of cocaine exposure. Our observation of decreased gonadotropin secretion 2 to 8 hours after cocaine needs to be confirmed in cycling monkeys. To assess the effects of cocaine on GnRH-stimulated gonadotropin secretion, we administered cocaine 2 hours before or concurrent with GnRH. Cocaine, administered 2 hours before GnRH, was initially selected to differentiate between acute and delayed effects of cocaine. The lack of an effect of either cocaine treatment regimen suggests that cocaine does not directly impair pituitary gonadotropin secretion. Rather, the decrease in gonadotropin levels seen in experiment 2 probably indicates an effect of cocaine on hypothalamic GnRH release. These results are again contradictory to the data obtained in cycling rhesus monkeys 24 by Mello et aI., who reported that 0.4 mg/kg of cocaine given 10 minutes before GnRH resulted in a significantly higher LH release as compared with either a placebo or a cocaine dose of 0.8 mglkg. This may represent a dose-related effect whereby 0.4 mg/kg of cocaine affected gonadotropin release differently than did other doses tested. Although the current study suggests that cocaine's major effects on gonadotropin secretion are at the hypothalamic level, our findings do not exclude other mechanisms whereby cocaine could impair reproductive functions. For example, cocaine could affect LH and FSH secretion by altering prolactin concentrations!' In addition, cocaine, a potent sodium (Na+) channel blocker, may also have a direct suppressive effect on pituitary gonadotropins. GnRH-stimulated release of LH is dependent on N a + channels, and N a + channel inhibitors have been shown to reduce GnRHstimulated LH release in vitro. 25 These local effects could indirectly alter the modulatory role of other neurotransmitters and GnRH on pituitary gonadotropin secretion. Our study in oophorectomized monkeys
December 1992 Am J Obstet Gynecol
does not address the possibility that cocaine may directly impair ovarian sex steroid production or metabolism and disrupt the integral role these hormones play in regulating hypothalamic neurotransmitters and pituitary-gonadotropin release. Although there are no data confirming a direct effect of cocaine on ovarian function, adverse effects of cocaine on reproductive function may be explained by changes in ovarian steroids!6 In conclusion, administration of 2 to 4 mglkg of cocaine to oophorectomized primates results in serum cocaine levels that parallel those observed in human addicts. These doses of cocaine significantly decreased circulating levels of LH and FSH in our animal model. The absence of an effect of cocaine on GnRH-stimulated gonadotropin release suggests that cocaine may directly affect GnRH neurons or hypothalamic neurotransmitter modulation of GnRH release, thereby impairing LH and FSH secretion. We thank Arturo Moreno,julie Foreman, and Donna johnson for their technical assistance and Irma Garcia and Gretta Small for their assistance in preparing this manuscript. REFERENCES 1. National Institute on Drug Abuse, Division of Epidemiology and Prevention Research. National household survey on drug abuse: main findings 1990. Washington: United States Department of Health and Human Services, Public Health Service, Alcohol, Drug Abuse, and Mental Health Administration, 199.47-51. 2. Chasnoff IJ, Burns WJ, Schnoll SH, et al. Cocaine use in pregnancy. N Engl] Med 1985;313:666-8. 3. Little BB, Snell LM, Klein VR, Gilstrap LC. Cocaine abuse during pregnancy: maternal and fetal implications. Obstet Gynecol 1989;73:157-60. 4. Siegal RK. Cocaine and sexual dysfunction: the curse of moma coca. J Psychoactive Drugs 1982;14:71-4. 5. Cocores JA, Dackis CA, Gold MS. Sexual dysfunction secondary to cocaine abuse in two patients. J Clin Psychiatry 1986;136:384-5. 6. Maier HW. Der Kokainismus-Geschichte/Pathologie Medizinische und behiirdliche Bekampfung. Leipzig: Georg Thieme Verlag, 1926. [English translation: Kalant 0]. Cocaine addiction. Toronto: Alcoholism and Drug Addiction Research Foundation, 1987.J 7. King TS, Schenken RS, Kang IS, Javors MA, Riehl RM. Cocaine disrupts estrous cyclicity and alters the reproductive neuroendocrine axis in the rat. Neuroendocrinology 1990;51: 15-22. 8. King TS, Canez MS, Gaskill S, Schenken RS. Chronic cocaine disruption of estrous cyclicity in the rat: dosedependent effects. In: Proceedings of the thirty-ninth annual meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18-21, 1992. 9. Williams RF, Barber DL, Cowan BD, Lynch A, Marut EL, Hodgen GD. Hyperprolactinemia in monkeys: induction by an estrogen-progesterone synergy. Steroids 1981;38: 321-31. 10. Pauerstein CJ, Eddy C, Croxatto HD, Hess R, Siler-Khodr TM, Croxatto HB. Temporal relationship of estrogen, progesterone and luteinizing hormone levels in ovulation in women and infra-human primates. AM J OBSTET GyNECOL 1978;130:876-86.
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11. Boorman GA, Niswender DG, Gay VL, Reichert LE, Midgley AR. Radioimmunoassay for follicle stimulating hormone in the rhesus monkey using anti-human FSH serum and rat FSH l3I I'. Endocrinology 1973;92:618-23. 12. Gibaldi M. Pharmacokinetics. In: Biopharmaceutics and clinical pharmacokinetics. 2nd ed. Philadelphia: Lea & Febiger, 1977:1. 13. Veldhuis JD, Johnson ML. Cluster analysis: a simple, versatile and robust algorithm for endocrine pulse detection. Am J Physiol 1986;250:E486-93. 14. Urban RJ, Johnson ML, Veldhuis JD. In vitro biological validation and biophysical modeling of the sensitivity and positive accuracy of endocrine peak detection. I. The LH pulse signal. Endocrinology 1989;124:2541-7. 15. Javid JI, Fischman MW, Schuster CR, Dekirmenjian H, DavidJM. Cocaine plasma concentration: relation ofphysiological and subjective effects in humans. Science 1978; 202:227-8. 16. Barnett G, Hawks R, Resnick R. Cocaine pharmacokinetics in humans. J Ethnopharmacol 1981;3:353-66. 17. Chow MJ, AmbreJJ, Ruo 11, Atkinson AJ Jr, Bowsher DJ, Fischman MW. Kinetics of cocaine distribution, elimination, and chronotropic effects. Clin Pharmacol Ther 1985; 38:318-24. 18. Jeffcoat AR, Perez-Reyes M, HiIIJM, Sadler BM, Cook E. Cocaine disposition in humans after intravenous injection, nasal insuffiation (snorting), or smoking. Drug Metab Dispos 1989;17: 153-9.
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19. Cone EJ, Kumor K, Thompson LK, Sherer M. Correlation of saliva cocaine levels with plasma levels and with pharmacologic effects after intravenous cocaine administration in human subjects. J Anal Toxicol 1988;12:200-6. 20. Misra AL, Giri W, Patel MN, Alluri VR, Mule SJ. Disposition and metabolism of [3H] cocaine in acutely and chronically treated monkeys. Drug Alcohol Depend 1977; 2:261-72. 21. Mello NK, Mendelson JH, Drieze J, Kelly M. Acute effects of cocaine on prolactin and gonadotropins in female rhesus monkey during the follicular phase of the menstrual cycle. J Pharmacol Exp Ther 1990;254:815-23. 22. Scher PM, Almirez RG, Steger RW, Smith CG. The effects of cocaine on reproductive hormones in the primate. Pharmacologist 1982;24:185. 23. Knobil E. The neuroendocrine control of the menstrual cycle. Recent Prog Horm Res 1980;36:53-88. 24. Mello NK, Mendelson JH, Drieze J, Kelley M. Cocaine effects on luteinizing hormone-releasing hormone-stimulated anterior pituitary hormones in female rhesus monkey. J Clin Endocrinol Metab 1990;71:1434-41. 25. McArdle CA, Cragoe EJ Jr, Poch A. Na+ dependence of gonadotropin-releasing hormone action: characterization of the Na + /H+ antiport in pituitary gonadotropes. Endocrinology 1991;128:771-8. 26. Kaufmann RA, Savoy-Moore RT, Sacco AG, Subramanian MG. The effect of cocaine on oocyte development and the follicular microenvironment in the rabbit. Fertil Steril 1990;54:921-6.
San Antonio, Texas OBJECTIVE: Our objective was to determine whether cocaine alters gonadotropin secretion in oophorectomized monkeys. STUDY DESIGN: Oophorectomized monkeys with elevated gonadotropin levels were chronically cannulated to allow blood sampling every 15 minutes. Monkeys received either saline solution or 2 or 4 mg/kg cocaine hydrochloride as an intravenous bolus. Other oophorectomized monkeys were pretreated with either saline solution or 4 mg/kg cocaine 2 hours before bolus gonadotropin-releasing hormone administration, and plasma luteinizing hormone and follicle-stimulating hormone levels were measured every 15 minutes for 3 hours. Monkeys were also given either saline solution or 4 mg/kg of cocaine with gonadotropin-releasing hormone simultaneously, and plasma gonadotropin levels were measured every 15 minutes for 3 hours. Serum luteinizing hormone and follicle-stimulating hormone levels were measured by radioimmunoassay. RESULTS: Both doses of cocaine resulted in a significant decrease in luteinizing hormone levels compared with controls. Follicle-stimulating hormone levels were significantly decreased only with the 4 mg/kg dose of cocaine. There was no difference in luteinizing hormone and follicle-stimulating hormone responses to gonadotropin-releasing hormone in the cocaine-treated monkeys compared with saline solution-treated monkeys by using repeated-measures analysis of variance. CONCLUSION: These findings demonstrate that acute cocaine administration to oophorectomized primates inhibits basal luteinizing hormone-follicle-stimulating hormone secretion but not gonadotropin-releasing hormone-stimulated luteinizing hormone and follicle-stimulating hormone release. In the absence of an effect on gonadotropin-releasing hormone-stimulated gonadotropin release, we conclude that the impaired luteinizing hormone-follicle-stimulating hormone secretion after cocaine administration is due in part to a direct effect of cocaine on gonadotropin-releasing hormone neurons or on hypothalamic neurotransmitter modulation of gonadotropin-releasing hormone release. (AM J OSSTET GVNECOL 1992;167:1785-93.)
Key words: Cocaine, reproductive dysfunction, primates Cocaine abuse has become an epidemic in recent years. 1 The use of cocaine in women of reproductive age has been associated with many complications of pregnancy, including sudden death, acute myocardial infarction, cerebrovascular accident, seizures, abruptio placentae, teratogenicity, intrauterine growth retardation, intrauterine fetal death, neonatal addiction, sudden infant death syndrome, and early childhood neurologic disorders.2. 3 Sexual dysfunction, irregular menstrual cycles, and amenorrhea have also been reported
From the Departments of Obstetrics and Gynecology, a Medicine, band Cellular and Structural Biology,' The University of Texas Health Science Center at San Antonio, and the Department of Research and Development, Audie L. Murphy Veterans Hospital. d Presented in part at the Thirty-eighth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23, 1991. Reprint requests: Robert S. Schenken, MD, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7836. 6/6/41910
nonpregnant women addicted to cocaine!·6 However, cocaine's effects on ovarian-menstrual cyclicity and reproductive functions in nongravid humans or subhuman primates are poorly understood. Preliminary data in rodents have shown that chronic cocaine treatment disrupts estrous cyclicity, causing prolonged periods of diestrus, repetitive days of estrus, decreased ovulation rates, and reduced luteinizing hormone (LH) levels. 7 This disruption of estrous cyclicity has recently been shown to be dose related. 6 Isolated and perfused hypothalami from rats chronically treated with cocaine demonstrate increased basal secretion of norepinephrine and serotonin and attenuated gonadotropin-releasing hormone (GnRH) response to pulsed norepinephrine. This alteration of hypothalamic aminergic activity necessary for pulsatile GnRH release suggests that cocaine may interrupt estrous cyclicity by altering GnRH pulsatility and gonadotropin release. This study was conducted to assess whether cocaine alters gonadotropin secretion in subhuman primates. III
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Initial studies were conducted to determine whether intravenous cocaine administration to monkeys resulted in serum cocaine levels equivalent to those levels reported in humans feeling "high." To assess whether cocaine altered basal or pulsatile gonadotropin secretion in primates, serum LH and follicle-stimulating hormone (FSH) levels were measured before and after saline solution or low- or high-dose cocaine administration. To determine whether cocaine altered GnRHstimulated gonadotropin secretion, saline solution or cocaine was given with GnRH and serum concentrations of gonadotropins were measured. Results suggested that cocaine impairs pituitary LH and FSH secretion via a direct hypothalamic effect. Material and methods
Animals. Long-term oophorectomized adult female cynomolgus (Macaca fascicularis) monkeys with elevated serum gonadotropin levels were studied. Weights ranged from 3.2 to 3.8 kg, and all monkeys had regular menstrual cycles before ovariectomy. Monkeys were free of chronic drug exposure. They were housed individually, provided food (Purina Monkey Chow; Ralston Purina, St. Louis) twice daily, and given free access to water. Rooms were climate controlled, with light and dark cycles of 14 hours light and 10 hours dark (lights on at 7 AM, lights off at 9 PM). Catheterization. Monkeys were anesthetized with intramuscular injections of 10 mg/kg ketamine (Ketaset; Aveco, Fort Dodge, la.) and 2 mg/kg xylazine (Rompun; Mobay Corp., Shawnee, Kan.). A 20-gauge cannula was inserted into the external jugular vein and advanced to the superior vena cava. The cannula was tunneled subcutaneously, exiting in the interscapular area of the animal. A primate jacket was placed on the monkey. The cannula was threaded out the back of the jacket and through a mobile tether consisting of a flexible metal tube connected from the jacket to a standardsized, single-channel fluid swivel. 9 Each monkey received an intramuscular injection of 300,000 IV of penicillin G benzathine (Bicillin; Wyeth, Philadelphia) postoperatively. Catheter patency was maintained by continuous infusion of lactated Ringer's solution containing 2000 IU/L heparin at a rate of 0.004 ml/min. Blood sampling. All blood samples were collected in heparinized syringes after the catheter was cleared of infusion solution. Plasma was harvested, snap frozen in acetone on dry ice, and stored at - 20° C. Red blood cells were reconstituted with saline solution and returned to the monkeys every 2 hours. Samples collected for cocaine analysis were aliquoted into tubes containing 200 f..LI of potassium phosphate buffer 0.05 mol/L (pH 5) plus sodium fluoride. All blood sampling and drug administrations were through the external jugular catheter.
December 1992 Am J Obstet Gynecol
Treatments. An overview of the experimental design and phased statistical analysis is presented in Fig. 1. Experiment 1. Monkeys (N = 5) were given either 2 or 4 mg/kg cocaine hydrochloride on alternating days. Serum samples were drawn every 5 minutes for 15 minutes, then every 15 minutes for 2 hours. All samples were assayed for cocaine and benzoylecgonine. Experiment 2. The effects of cocaine on basal and pulsatile pituitary gonadotropin secretion were studied in five monkeys. The day after cannulation, blood samples were collected every 15 minutes from 7 AM to 3 PM. At 8 AM 1 ml of normal saline solution was given to each monkey. Three or four days later blood samples were collected every 15 minutes from 7 AM to 3 PM, and 2 mg/kg cocaine hydrochloride was dissolved in 1 ml of normal saline solution was given at 8 AM. After 3 or 4 days blood sampling resumed as before, and 4 mg/kg cocaine in 1 ml of saline solution was given at 8
AM.
Experiment 3. The effects of cocaine on gonadotropin release after exogenous GnRH treatment were studied in two monkeys. The day after cannulation blood samples were collected every 15 minutes from 8 AM to 2 PM. One milliliter of normal saline solution was given at 9 AM, and 15 Ilg/kg GnRH was given at 11 AM. Two days later blood samples were collected as before, 4 mg/kg cocaine hydrochloride was given at 9 AM, and 15 Ilg/kg GnRH was given at 11 AM. This was repeated (saline solution-GnRH 1 day, no treatment 1 day, cocaineGnRH 1 day) three more times with at least 3 days between treatment regimens. GnRH was given 2 hours after cocaine treatment to avoid confusion between acute cocaine stimulation and exogenous GnRH stimulation of gonadotropins. 10 There was no significant stimulation of gonadotropins after acute cocaine treatment; therefore GnRH and cocaine were given simultaneously in experiment 4. Experiment 4. The effects of cocaine and GnRH given simultaneously were studied in two monkeys. Blood samples were collected every 15 minutes from 8 AM to noon. The day after cannulation 1 ml of saline solution and 15 Ilgikg GnRH were given at 9 AM. One day later 4 mg/kg cocaine hydrochloride and 15 Ilg/kg GnRH were given at 9 AM. This treatment was repeated three more times with at least 3 days between treatment regimens. Assays. High-performance liquid chromatography with ultraviolet detection was used to measure plasma levels of cocaine and its primary metabolite, benzoylecgonine, in extracted serum collected in experiment 1. Details of the assay have been described. 7 All serum samples collected in experiments 2, 3, and 4 were assayed in duplicate for LH and FSH by radioimmunoassay as previously described. 10. II Monkey LH determinations were performed with an antiserum to
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t GnRH Experiment
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Time (h) Fig. 1. Overview of experimental design in experiments 2, 3, and 4. Phase 1 (I = 0 to 1 hours) served as baseline. Saline solution or cocaine was administered at 1 = 1 hour in all experiments. Phase 2 (I = 1 to 3 hours) was analyzed to assess possible acute stimulatory effects of cocaine in experiments 2 and 3. GnRH was administered 2 hours after cocaine or saline solution (I = 3 hours) in experiment 3. In experiment 4 cocaine or saline solution was administered with GnRH at 1 = 1 hour, and the LH and FSH response was assessed from 1 = 1 to 4 hours.
ovine LH (anti-OLH No. IS, provided by Dr. C. Niswender) that cross reacts indistinguishably with rhesus LH. The standard was LER-M-907, and iodine 125ovine LH was used as label. Monkey FSH determinations were performed with a heterologous radioimmunoassay. Antihuman FSH and 125I-recombinant FSH are used with monkey FSH (LER-1909-2) as a standard. All samples for LH and FSH determinations in individual monkeys were performed in the same assay. The intraassay coefficients of variation ranged from 4% to 12% for LH and from 4% to II % for FSH. Statistics Experiment 1. The serum elimination phase half-life (t'/2) of cocaine and benzoylecgonine was calculated from the slopes of their first-order kinetic rates. 12 Experiment 2. Data were assessed in three phases on the basis of the elimination time of cocaine (Fig. 1). Phase I (t = a to I hour) was baseline. Phase 2 (t = I to 3 hours) was the immediate response while cocaine was still detectable in the serum. Phase 3 (t = 3 to 8 hours) was the delayed response. Phase I LH and FSH levels
were compared among monkeys by repeated-measure analysis of variance. During the immediate response (phase 2), peak LH and FSH levels after cocaine or saline solution administration were compared by repeated-measures analysis of covariance, controlling for baseline when necessary. During the delayed response (phase 3), LH and FSH levels after cocaine or saline solution administration were analyzed in two ways. First, gonadotropin levels were compared among treatment groups by repeated-measures analysis of covariance. Second, hormone pulses were identified by cluster analysis,'3 an objective, computer-based, pulse-analysis program. Variance was calculated by using of dosedependent coefficients of variation calculated from sample replicates in each monkey's hormone series. Cluster parameters were two points for test nadirs and one point for test peaks. The t statistics were 1.0 for upstrokes and 1.0 for downstrokes. These parameters have been previously defined to provide optimal sensitivity and positive accuracy for gonadotropin pulse detection while constraining false-positive and false-
1788 Canez et al.
December 1992 Am J Obstet Gynecol
Serum Cocaine
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negative rates to 5%.'3. '4 Pulse frequency and amplitude were compared among groups by repeated-measures analysis of variance. Experiment 3. The data were similarly divided into three phases: phase 1 (t = 0 to 1 hour) was baseline, phase 2 (t = 1 to 3 hours) was the immediate response after cocaine or saline solution administration, and phase 3 (t = 3 to 6 hours) was the time after GnRH administration. Phase 1 LH and FSH levels were compared between treatments (cocaine or saline solution) by repeatedmeasures analysis of variance. Phase 2 peak levels ofLH and FSH were compared by repeated-measures analysis of covariance. Quantification of gonadotropin secretion after GnRH (phase 3) was estimated in two ways: first, by the area under the curves compared with the Student 1 test, and, second, serum gonadotropin levels
were plotted against time and analyzed with repeatedmeasures analysis of covariance. Experiment 4. The data were divided into two phases. Phase 1 (t = 0 to 1 hour) was baseline, and phase 2 (I = 1 to 4 hours) was the time period after simultaneous cocaine or saline solution and GnRH administration. Phase 1 LH and FSH levels were compared by repeated-measures analysis of variance. Phase 2 LH and FSH levels were compared between groups by area under the curve and repeated-measures analysis of covariance. Results
Experiment 1. Serum levels of cocaine and benzoylecgonine after 2 or 4 mg/kg doses are shown in Fig. 2. Peak serum levels of cocaine and benzoylecgonine were 151 ± 49 and 158 ± 15 ng/ml (mean ± SE),
Cocaine impairs gonadotropin secretion
Volume 167 Number 6
-..E
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Time (h) Fig. 3. Experiment 2. Serum LH (solid line) and FSH (broken line) concentrations (lLg/ml) in a representative monkey after saline solution (upper panel), 2 mglkg cocaine (middle panel), or 4 mg/kg (lower panel) administered intravenously at t = I hour. Circles and triangles, respectively, indicate LH and FSH pulses as assessed by cluster analysis.
respectively, after the 2 mg/kg dose. Cocaine and benzoylecgonine levels then gradually declined and were no longer detectable by 1 hour 45 minutes. Peak serum levels of cocaine and benzoylecgonine were 302 ± 54 and 211 ± 31 ng/ml (mean ± SE), respectively, after the 4 mglkg dose. Levels then gradually decreased and were no longer detectable by 2 hours. The tV2 values for cocaine after the 2 and 4 mg/kg doses were 22 ± 2 and 21 ± 3 minutes (mean ± SE), respectively. The tV2 for benzoylecgonine after the 2 and 4 mg/kg doses were 37 ± 7 and 29 ± 2 minutes (mean ± SE), respectively. Experiment 2. The effects of cocaine on gonadotropin secretion throughout the blood sampling interval are shown for one respective monkey in Fig. 3. There was significant variability in baseline gonadotropin levels among monkeys (p = 0.005 for LH, p = 0.02 for FSH). Therefore phases 2 and 3 of this experiment were adjusted for this baseline covariate. In phase 2 there was no significant stimulation of gonadotropins after acute cocaine treatment. LH secretion in phase 3 was significantly decreased after low-dose and high-
dose cocaine treatment, whereas FSH secretion was decreased only after high-dose cocaine administration, as assessed by repeated-measures analysis of covariance (Table I). Pulse analysis showed no effect of cocaine on LH or FSH pulse frequency. There was a clear trend toward decreased LH (P = 0.07), but not FSH, pulse amplitude after cocaine administration, as compared with saline solution. Experiment 3. Fig. 4 shows mean gonadotropin responses to GnRH in cocaine- and saline-treated monkeys. There was significant variability among baseline gonadotropin levels (P < 0.001 for LH and FSH) during phase 1. This was adjusted for in phases 2 and 3. In phase 2 there was no significant stimulation of gonadotropins after acute cocaine treatment. In phase 3 mean GnRH-stimulated gonadotropin secretion was not significantly different in monkeys treated with cocaine compared with saline solution-treated controls. Repeated-measures analysis of covariance showed no difference in mean LH levels over time. Monkeys treated with saline solution had a peak LH level
1790 Canez et al.
December 1992
Am J Obstet Gynecol
7
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Time (h) Fig. 4. Experiment 3. Serum LH (upper panel) and FSH (lower panel) responses (mean ± SE) to intravenous GnRH (15 II-glkg) administration at t = 3 hours after treatment with saline solution (broken line) or cocaine, 4 mg/kg (solid line), intravenously at t = 1 hour.
Table I. Gonadotropin secretion, pulse amplitude, and pulse frequency after saline solution or low-dose (2 mglkg) or high-dose (4 mg/kg) cocaine administration Mean decrease in gonadotropins
Repeated-measures analysis of variance
O.S
p < 0.0001 p < 0.0001
(JJg/ml)
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1.1
2.7 2.0
(mean ± SE) of 6.1 ± 0.2 lJ.g/ml, compared with 5.3 ± 0.3 lJ.g/ml in cocaine-treated monkeys. LH secretion (area under the curve) was 4.7 lJ.g/ml-1/hr-1 in saline solution-treated and 3.4 IJ.g' ml- 1. hr- 1 in cocaine-treated monkeys. Repeated-measures analysis of covariance showed no difference in mean FSH levels in cocaine- and saline solution-treated monkeys. Monkeys treated with saline
p p
= O.OS = 0.04
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Pulse
(JJg/ml)
frequency (pulses/3 hr)
4.7 ± 2.1 3.3 ±l.l 2.2 ± 1.0
3.0 ± 0.7 2.2 ± 1.1 2.5 ± 0.6
15.2 ± 7.5 13.4 ± S.6 12.2 ± 9.2
2.4 ± 1.5 I.S ± 0.4 2.3 ± 1.0
solution had a peak FSH level (mean ± SE) of 30.6 ± 1.6 lJ.g/ml compared with 33.1 ± 3.0 IJ.g/ml in cocaine-treated monkeys. FSH secretion (area under the curve) was 12.7 IJ.g' ml- 1. hr- 1 in saline solutiontreated animals and 14.1IJ.g· ml- 1. hr- 1in the cocainetreated monkeys. Experiment 4. Fig. 5 shows mean gonadotropin responses to GnRH in cocaine- and saline-treated mon-
Cocaine impairs gonadotropin secretion
Volume 167 Number 6
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7
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keys. There were no significant differences in LH or FSH levels after GnRH in cocaine- or saline solutiontreated monkeys. LH secretion (area under the curve) was 2.4 IJ.g' ml- I • hr- I in saline solution-treated and 3.2 IJ.g' ml- I • hr- I in cocaine-treated monkeys. FSH secretion (area under the curve) was 10.5 IJ.g' ml- I • hr- I in saline solution-treated and 8.9 IJ.g' ml- I • hr- I in cocaine-treated monkeys. Comment An important preliminary step in establishing an
animal model to study the effects of human drug abuse is to demonstrate that the dose and plasma levels of the drug in the experimental paradigm are similar to those found in humans. Thus our initial aim was to establish the dose of cocaine necessary to mimic levels seen in humans. The literature reports large intersubject variations in metabolism of low-dose and high-dose intravenous cocaine in both humans and animals. 14.2o Peak serum cocaine levels in our oophorectomized monkeys were 151 ± 49 and 302 ± 54 ng/ml after doses of 2 and 4 mg/kg, respectively. These levels and the respective calculated tl/2 (22 ± 2 and 21 ± 3 minutes) are similar to those reported in other studies.
To assess the effects of cocaine on basal and pulsatile gonadotropin secretion, we analyzed changes in hormone levels during three phases (experiment 2). Phase 1, before cocaine exposure, served as baseline. Gonadotropin levels during the first 2 hours after cocaine exposure, phase 2, were assessed because previous studies demonstrated acute increases in LH and FSH secretion after cocaine administration to cycling monkeys.21. 22 Phase 3 represented the delayed effects of cocaine, once circulating drug levels were no longer detectable. There were no significant changes in serum LH or FSH levels during phase 2. In contrast, gradual, significant declines in LH and FSH secretion were observed in phase 3. This decrease in gonadotropins was observed with both the 2 and 4 mglkg doses of cocaine. A clear trend (p = 0.07) toward a decreased LH pulse amplitude was also apparent during phase 3. The absence of an acute stimulatory effect of cocaine on gonadotropin secretion in oophorectomized monkeys is in contrast to previous findings in cycling monkeys.21. 22 There are several possible explanations for these disparate findings. First, the doses of cocaine used in our study were fivefold greater than those used by Mello et al. 21 With these doses we achieved serum
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cocame levels similar to those observed in humans. Serum cocaine levels were not reported in the other two studies in cycling monkeys!" 22 and it remains possible that cocaine is stimulatory at low concentrations and inhibitory at higher concentrations. The use of oophorectomized versus intact monkeys may also be an important factor. The effects of biogenic amines and opioids on pituitary LH and FSH secretion are clearly modulated by ovarian steroids, and the effects of cocaine on gonadotropin secretion may also be modified by circulating estrogen and progesterone levels. 23 The techniques of blood collection and statistical analyses are also crucial to the interpretation of findings. The use of a primate chair and ketamine anesthesia, a known modulator of prolactin secretion, in the previous studies may have had effects not apparent in chronically cannulated monkeys with a vest and mobile tether assembly.9 Finally, both studies in cycling monkeys assessed changes in gonadotropin secretion within 2 hours of cocaine exposure. Our observation of decreased gonadotropin secretion 2 to 8 hours after cocaine needs to be confirmed in cycling monkeys. To assess the effects of cocaine on GnRH-stimulated gonadotropin secretion, we administered cocaine 2 hours before or concurrent with GnRH. Cocaine, administered 2 hours before GnRH, was initially selected to differentiate between acute and delayed effects of cocaine. The lack of an effect of either cocaine treatment regimen suggests that cocaine does not directly impair pituitary gonadotropin secretion. Rather, the decrease in gonadotropin levels seen in experiment 2 probably indicates an effect of cocaine on hypothalamic GnRH release. These results are again contradictory to the data obtained in cycling rhesus monkeys 24 by Mello et aI., who reported that 0.4 mg/kg of cocaine given 10 minutes before GnRH resulted in a significantly higher LH release as compared with either a placebo or a cocaine dose of 0.8 mglkg. This may represent a dose-related effect whereby 0.4 mg/kg of cocaine affected gonadotropin release differently than did other doses tested. Although the current study suggests that cocaine's major effects on gonadotropin secretion are at the hypothalamic level, our findings do not exclude other mechanisms whereby cocaine could impair reproductive functions. For example, cocaine could affect LH and FSH secretion by altering prolactin concentrations!' In addition, cocaine, a potent sodium (Na+) channel blocker, may also have a direct suppressive effect on pituitary gonadotropins. GnRH-stimulated release of LH is dependent on N a + channels, and N a + channel inhibitors have been shown to reduce GnRHstimulated LH release in vitro. 25 These local effects could indirectly alter the modulatory role of other neurotransmitters and GnRH on pituitary gonadotropin secretion. Our study in oophorectomized monkeys
December 1992 Am J Obstet Gynecol
does not address the possibility that cocaine may directly impair ovarian sex steroid production or metabolism and disrupt the integral role these hormones play in regulating hypothalamic neurotransmitters and pituitary-gonadotropin release. Although there are no data confirming a direct effect of cocaine on ovarian function, adverse effects of cocaine on reproductive function may be explained by changes in ovarian steroids!6 In conclusion, administration of 2 to 4 mglkg of cocaine to oophorectomized primates results in serum cocaine levels that parallel those observed in human addicts. These doses of cocaine significantly decreased circulating levels of LH and FSH in our animal model. The absence of an effect of cocaine on GnRH-stimulated gonadotropin release suggests that cocaine may directly affect GnRH neurons or hypothalamic neurotransmitter modulation of GnRH release, thereby impairing LH and FSH secretion. We thank Arturo Moreno,julie Foreman, and Donna johnson for their technical assistance and Irma Garcia and Gretta Small for their assistance in preparing this manuscript. REFERENCES 1. National Institute on Drug Abuse, Division of Epidemiology and Prevention Research. National household survey on drug abuse: main findings 1990. Washington: United States Department of Health and Human Services, Public Health Service, Alcohol, Drug Abuse, and Mental Health Administration, 199.47-51. 2. Chasnoff IJ, Burns WJ, Schnoll SH, et al. Cocaine use in pregnancy. N Engl] Med 1985;313:666-8. 3. Little BB, Snell LM, Klein VR, Gilstrap LC. Cocaine abuse during pregnancy: maternal and fetal implications. Obstet Gynecol 1989;73:157-60. 4. Siegal RK. Cocaine and sexual dysfunction: the curse of moma coca. J Psychoactive Drugs 1982;14:71-4. 5. Cocores JA, Dackis CA, Gold MS. Sexual dysfunction secondary to cocaine abuse in two patients. J Clin Psychiatry 1986;136:384-5. 6. Maier HW. Der Kokainismus-Geschichte/Pathologie Medizinische und behiirdliche Bekampfung. Leipzig: Georg Thieme Verlag, 1926. [English translation: Kalant 0]. Cocaine addiction. Toronto: Alcoholism and Drug Addiction Research Foundation, 1987.J 7. King TS, Schenken RS, Kang IS, Javors MA, Riehl RM. Cocaine disrupts estrous cyclicity and alters the reproductive neuroendocrine axis in the rat. Neuroendocrinology 1990;51: 15-22. 8. King TS, Canez MS, Gaskill S, Schenken RS. Chronic cocaine disruption of estrous cyclicity in the rat: dosedependent effects. In: Proceedings of the thirty-ninth annual meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18-21, 1992. 9. Williams RF, Barber DL, Cowan BD, Lynch A, Marut EL, Hodgen GD. Hyperprolactinemia in monkeys: induction by an estrogen-progesterone synergy. Steroids 1981;38: 321-31. 10. Pauerstein CJ, Eddy C, Croxatto HD, Hess R, Siler-Khodr TM, Croxatto HB. Temporal relationship of estrogen, progesterone and luteinizing hormone levels in ovulation in women and infra-human primates. AM J OBSTET GyNECOL 1978;130:876-86.
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11. Boorman GA, Niswender DG, Gay VL, Reichert LE, Midgley AR. Radioimmunoassay for follicle stimulating hormone in the rhesus monkey using anti-human FSH serum and rat FSH l3I I'. Endocrinology 1973;92:618-23. 12. Gibaldi M. Pharmacokinetics. In: Biopharmaceutics and clinical pharmacokinetics. 2nd ed. Philadelphia: Lea & Febiger, 1977:1. 13. Veldhuis JD, Johnson ML. Cluster analysis: a simple, versatile and robust algorithm for endocrine pulse detection. Am J Physiol 1986;250:E486-93. 14. Urban RJ, Johnson ML, Veldhuis JD. In vitro biological validation and biophysical modeling of the sensitivity and positive accuracy of endocrine peak detection. I. The LH pulse signal. Endocrinology 1989;124:2541-7. 15. Javid JI, Fischman MW, Schuster CR, Dekirmenjian H, DavidJM. Cocaine plasma concentration: relation ofphysiological and subjective effects in humans. Science 1978; 202:227-8. 16. Barnett G, Hawks R, Resnick R. Cocaine pharmacokinetics in humans. J Ethnopharmacol 1981;3:353-66. 17. Chow MJ, AmbreJJ, Ruo 11, Atkinson AJ Jr, Bowsher DJ, Fischman MW. Kinetics of cocaine distribution, elimination, and chronotropic effects. Clin Pharmacol Ther 1985; 38:318-24. 18. Jeffcoat AR, Perez-Reyes M, HiIIJM, Sadler BM, Cook E. Cocaine disposition in humans after intravenous injection, nasal insuffiation (snorting), or smoking. Drug Metab Dispos 1989;17: 153-9.
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