Introduction

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Cocaine abuse and dependence are among the most prevalent drug abuse problems in the United States (National Institute on Drug Abuse, 1999). Chronic cocaine abuse is associated with a number of medical problems, including cardiovascular, cerebral vascular, and infectious disorders, and disruption of reproductive function (Mello, 1998). However, it has been difficult to attribute the reproductive dysfunctions observed clinically to cocaine alone (Mello and Mendelson, 1997; Mello, 1998), because polydrug abuse is very common among cocaine users (Schütz et al., 1994; National Institute on Drug Abuse, 1999). Cocaine also can disrupt the menstrual cycle in rhesus monkeys (Mello et al., 1997a; Potter et al., 1999, 1998) and the estrous cycle in rats (King et al., 1993). For example, chronic cocaine self-administration over 1 year disrupted menstrual cycle duration with concomitant amenorrhea, anovulation, and luteal phase dysfunction in otherwise healthy female rhesus monkeys (Mello et al., 1997a). Daily cocaine administration during the follicular phase of the menstrual cycle also resulted in anovulatory cycles (Potter et al., 1998) as well as disruption of subsequent menstrual cycles (Potter et al., 1999).

The ways in which chronic cocaine exposure disrupts the menstrual cycle are poorly understood (for review, see Mello and Mendelson, 1997). One approach to this question is to systematically examine the effects of acute cocaine administration on basal levels of hormones that are essential for the normal menstrual cycle. Preclinical studies have consistently shown that cocaine stimulates significant increases in luteinizing hormone (LH) levels. In rhesus monkeys, an acute dose of cocaine (0.4 and 0.8 mg/kg i.v.) significantly increased LH in normally cycling early follicular and mid-luteal phase females and in males (Mello et al., 1990a, 1993). LH increased significantly within 10 to 20 min after i.v. cocaine administration and remained above baseline levels for 40 to 50 min. Moreover, cocaine enhanced luteinizing hormone-releasing-hormone (LHRH)-stimulated increases in LH in follicular phase rhesus females (Mello et al., 1990b). Deconvolutional analysis showed that the half-life of LH was not significantly altered after cocaine administration and the LH increase appears to reflect a burst of hypothalamic LHRH release (Mello and Mendelson, 1997). These findings were surprising because cocaine acts centrally as an indirect dopamine agonist by blocking dopamine reuptake by the dopamine transporter (Kuhar et al., 1991; Woolverton and Johnson, 1992). Moreover, exogenous dopamine agonist administration suppresses LH in both men and women (Yen, 1979). In contrast, dopamine administration, at a dose that significantly suppressed prolactin secretion, did not alter LH levels in follicular phase female rhesus monkeys (Mello et al., 1997b). Similarly, in ovariectomized rhesus monkeys, infusion of dopamine did not decrease basal or LHRH-stimulated LH (Spies et al., 1980; Pavasuthipaisit et al., 1981). These discrepant findings may represent a species difference in the effects of exogenous dopamine on LH.

Intravenous cocaine administration stimulated LH in cocaine- and opioid-dependent men (Mendelson et al., 1992) and intranasal cocaine stimulated LH in cocaine-naive men (Heesch et al., 1996). The effects of cocaine on LH in women are unknown, and the potential significance of LH stimulation for the menstrual cycle dysfunction associated with chronic cocaine exposure is unclear. A binge pattern of cocaine use, with doses taken as often as every 15 min, has been reported by cocaine abusers (Gawin and Kleber, 1985; Ward et al., 1997). If repeated episodes of cocaine abuse were associated with increased levels of LH in women during the follicular phase, this could contribute to the menstrual cycle abnormalities observed. Specifically, alterations in LH pulsatile release patterns and/or abnormal LH levels could disrupt gonadotropin and ovarian steroid feedback systems. This in turn could delay or impede normal follicle maturation and result in anovulatory menstrual cycles, luteal phase dysfunction, and amenorrhea (Hotchkiss and Knobil, 1994). Higher rates of pulsatile LH release were observed in rhesus females during chronic cocaine self-administration (Mello et al., 2000a), a finding consistent with cocaine's acute stimulation of LH release.

One goal of the present study was to examine the acute effects of cocaine on LH in women who were occasional cocaine abusers and to determine whether these effects were cocaine-dose related. Women were studied during the follicular and the luteal phases of the menstrual cycle to evaluate the possible contribution of the hormonal milieu to cocaine's effects. Preclinical studies suggest that ovarian steroid hormones are essential for cocaine to stimulate anterior pituitary hormones (Mello et al., 1995; Sarnyai et al., 1995). Cocaine had no effect on LH or ACTH in ovariectomized monkeys, even though synthetic LHRH and corticotropin-releasing factor stimulated release of LH and ACTH, respectively (Mello et al., 1995; Sarnyai et al., 1995).

A second goal was to compare the effects of cocaine on LH in men and women studied under identical conditions. Gender differences in cocaine's cerebrovascular effects in humans (Levin et al., 1994; Kaufman et al., 2001) and cardiovascular and behavioral effects in rodents have been reported (for review, see Mello and Mendelson, 1997). However, we are unaware of any previous clinical reports of the possible contribution of gender to cocaine's effects on LH.

	Materials and Methods

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Subjects

Twenty-two adult females and 12 males provided informed consent for participation in this study. The study was approved by the Institutional Review Board of the McLean Hospital. Only men and women who fulfilled American Psychiatric Association Diagnostic and Statistical Manual (DSM-IV) criteria for a diagnosis of cocaine abuse (305.6) were selected. Volunteers with any lifetime DSM-IV Axis 1 disorder other than cocaine abuse and nicotine dependence were excluded. Women who were using oral contraceptive medication were excluded. Pregnancy tests (hCG C6 subunit blood tests) were completed on the morning before cocaine administration to ensure that no women had become pregnant since prestudy screening. All men and women selected for study were in good physical health and had normal medical and laboratory screening examinations. All subjects were drug-free on the study day as assessed by a qualitative urine drug screen described below (Triage Biosite Diagnostics, San Diego, CA).

Women were studied at two phases of the menstrual cycle. The follicular phase was defined as 5 to 9 days following the onset of menses. The luteal phase of the menstrual cycle was defined as 18 to 22 days after the onset of menses. Menstrual cycle phase was estimated from each woman's self-reports. Progesterone and estradiol levels were obtained on the day of the study to verify menstrual cycle phase. Two doses of intravenous cocaine (0.2 and 0.4 mg/kg) were studied at each phase of the menstrual cycle. The characteristics of men and women at each menstrual cycle phase in each of the two cocaine dose groups are summarized in Table 1. These subjects did not differ significantly with respect to age and body mass index. As indicated in Table 1, each dose of cocaine was studied in a group of six men. The 0.2-mg/kg dose of cocaine was studied in five women during the follicular phase of the menstrual cycle, and in five women during the luteal phase of the menstrual cycle. The 0.4-mg/kg dose of cocaine was studied in six women during the follicular phase of the menstrual cycle, and in six women during the luteal phase of the menstrual cycle.

Screening Procedures

Pregnancy Tests. Serum pregnancy tests were completed on the morning before cocaine or placebo administration to ensure that no women had become pregnant since the prescreening. The Stanbio QuPID Plus Test (Stanbio Laboratory, Inc., San Antonio, TX) is a qualitative immunoassay for the detection of human chorionic gonadotropin (hCG) in serum. The test uses a combination of monoclonal and polyclonal antibody reagents to selectively detect elevated levels of hCG. The Stanbio QuPID Plus Test for Pregnancy detects hCG concentrations of 20 mIU/ml and greater in serum.

Urine Drug Screens. It is important for subject safety, as well as to avoid confounding of the dependent variables, to ensure that subjects have not used any drugs before administration of intravenous cocaine. On the morning of each study day, urines were collected and analyzed with a Triage screen. The Triage Panel for Drugs of Abuse (Triage Biosite Diagnostics) is a rapid multiple immunoassay system for the qualitative detection of the major metabolites of these drugs of abuse in urine at the following cut-off concentrations (recommended screening cut-off concentrations by the Substance Abuse and Mental Health Services Administration): phencyclidine, 25 ng/ml; benzodiazepines, 300 ng/ml; cocaine, 300 ng/ml; amphetamines, 1000 ng/ml; tetrahydrocannabinol, 50 ng/ml; opiates, 300 ng/ml; and barbiturates, 300 ng/ml.

Cocaine Dose Selection. The two doses of cocaine (0.2 and 0.4 mg/kg i.v.) were selected on the basis of previous clinical studies. The low dose of cocaine (0.2 mg/kg i.v.) usually produces peak plasma cocaine levels of approximately 100 ng/ml. The higher dose (0.4 mg/kg i.v.) usually produces peak plasma cocaine levels over 200 ng/ml. These doses of cocaine have proved to be safe and induce significant changes in mood states and physiological responses in our previous clinical studies (Kaufman et al., 1998; Mendelson et al., 1998; Sholar et al., 1998).

Cocaine Administration Procedures. These studies were carried out on a clinical research ward. Cocaine was administered intravenously over an interval of 1 min. Subjects were studied in a semisupine position, and heart rate, blood pressure, and EKGs were continuously monitored with a Hewlett-Packard EKG monitor (model 78 352A) for 10 min before intravenous cocaine administration and for 2 h following intravenous injection. A physician certified in cardiopulmonary resuscitation was present during each study, and a cardiac defibrillator and appropriate emergency treatment medications were located in the study room.

Sample Collection Procedures. Baseline samples for analysis of LH, estradiol, and progesterone in women and testosterone and LH in men were collected 4 min before i.v. cocaine injection. Samples for LH and plasma cocaine analysis were collected at 2, 4, 8, 12, 16, 20, 30, 40, 60, 80, and 120 min following completion of the cocaine injection. This sampling frequency was based on previous observations that cocaine levels in plasma increase rapidly within 2 min after intravenous administration and reach peak levels within 4 to 5 min (Evans et al., 1996; Mendelson et al., 1999b; Sholar et al., 1998). All plasma samples for hormone and cocaine analysis were collected from an intravenous catheter in the arm opposite the arm in which cocaine was injected intravenously. Blood samples for hormone analysis were collected in heparinized tubes. Blood samples for cocaine analysis were transferred to heparinized Vacutainer tubes containing sodium fluoride and acetic acid (to prevent the hydrolysis of cocaine). All samples were iced immediately, centrifuged, and plasma was removed and frozen at 70°C for cocaine analysis.

Cocaine Hydrochloride Preparation. Cocaine hydrochloride was acquired from the National Institute on Drug Abuse in powder form and was dissolved in sterile water for intravenous injection by the McLean Hospital pharmacy. Sterility was ensured by passing the solution through a 0.22-µm Millipore filter and subjecting it to a Limulus Amebocyte Lysate test for detection of gram negative bacterial endotoxins. The test kit is manufactured by Whittaker Bioproducts (Walkersville, MD).

LH Assay. Serum LH was determined in duplicate by a direct, double antibody RIA method, using kits purchased from Incstar Corporation (Stillwater, MN). The assay sensitivity was 9.2 ng/ml and the intra- and interassay coefficients of variation were 2.9 and 5.3%, respectively.

Estradiol Assay. Serum estradiol was determined in duplicate by a direct, double antibody RIA method, using kits purchased from Diagnostic Product Corporation (Los Angeles, CA). The assay sensitivity was 1.1 pg/ml and the intra- and interassay coefficients of variation were 2.0 and 7.8%, respectively.

Progesterone Assay. Serum progesterone was determined in duplicate by Coat-A-Count RIA method, using kits purchased from Diagnostic Products Corporation. The assay sensitivity was 0.06 ng/ml and the intra- and interassay coefficients of variation were 4.0 and 5.1%, respectively.

Testosterone Assay. Serum total testosterone was determined in duplicate by Coat-A-Count RIA method, using kits purchased from Diagnostic Products Corporation. The assay sensitivity was 2.3 ng/dl and the intra- and interassay coefficients of variation were 3.0 and 5.9%, respectively.

Plasma Cocaine Analysis. Plasma cocaine levels were measured in duplicate using a solid-phase extraction method described by SPEC Instruction Manual by Ansys with a Hewlett-Packard 5890 Series II gas chromatograph equipped with a capillary column and a Hewlett-Packard 5971 Series Mass Selective detector (Abusada et al., 1993). The assay sensitivity was 10 ng/ml and the intra- and interassay coefficients of variation were 2.0 and 2.9%, respectively.

Data Analysis. Progesterone and estradiol levels during the follicular and luteal phases of the menstrual cycle were analyzed with a two-way ANOVA for menstrual cycle phase and cocaine dose. Plasma cocaine and LH values for subjects were analyzed using a 3 (group) × 12 (time) repeated measures ANOVA. If significant main effects were detected, one-way ANOVAs were performed to identify the times at which groups differed significantly. The statistical significance of temporal covariance between LH and plasma cocaine levels was determined by regression analyses.

	Results

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Ovarian Steroid Hormone Levels during the Follicular and Luteal Phases of the Menstrual Cycle. Analysis of ovarian steroid hormone levels confirmed that women were in the mid-follicular phase or the mid-luteal phase of the menstrual cycle on the study day. Average baseline estradiol and progesterone levels during each phase of the menstrual cycle for each cocaine dose group are summarized in Table 2. Average estradiol and progesterone levels during the luteal phase were significantly higher than during the follicular phase (P < 0.0001) in both the 0.2 and the 0.4 mg/kg cocaine dose groups. Estradiol and progesterone levels were equivalent in the two cocaine dose groups during the follicular phase and during the luteal phase of the menstrual cycle.

Baseline Testosterone Levels in Men. Baseline testosterone levels did not differ significantly before cocaine administration. In the 0.2 mg/kg cocaine dose group, baseline testosterone levels averaged 537 ± 89 ng/dl. In the 0.4 mg/kg cocaine dose group, baseline testosterone levels averaged 424 ± 35 ng/dl.

Baseline LH Levels in Men and Women. In women studied during the follicular phase, precocaine baseline LH levels averaged 57 ± 9 ng/ml in the 0.2 mg/kg cocaine dose group and 69 ± 3 ng/ml in the 0.4 mg/kg cocaine dose group. In women studied during the luteal phase, precocaine baseline LH levels averaged 50 ± 11 ng/ml in the 0.2 mg/kg cocaine dose group and 67 ± 14 ng/ml in the 0.4 mg/kg cocaine dose group. In men, precocaine baseline LH levels averaged 55 ± 6 and 49 ± 5 ng/ml in the low- and high-cocaine dose groups. There were no statistically significant differences in baseline LH levels in follicular phase women or in luteal phase women before cocaine administration. Male LH levels were also equivalent to LH levels in women before low- and high-dose cocaine administration.

Cocaine Plasma Levels in Women. Plasma cocaine levels in women after intravenous administration of 0.2 and 0.4 mg/kg cocaine are shown in Fig. 1. Peak plasma cocaine levels were cocaine dose-dependent in women studied during the follicular and during the luteal phase of the menstrual cycle. Peak plasma cocaine levels after 0.4 mg/kg i.v. cocaine were significantly higher than after 0.2 mg/kg i.v. in women at both phases of the menstrual cycle (P < 0.003). Cocaine reached peak plasma levels within 4 min after i.v. administration of 0.2 mg/kg i.v. and averaged 124 ± 18 ng/ml during the follicular phase and 87 ± 21 during the luteal phase. Although peak plasma cocaine levels were lower during the luteal phase, these differences did not achieve statistical significance (P = 0.18). Peak plasma cocaine levels after 0.4 mg/kg cocaine averaged 287 ± 21 and 264 ± 37 ng/ml during the follicular and the luteal phase of the menstrual cycle, respectively, and these levels were not significantly different.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Plasma cocaine levels in women after intravenous cocaine administration. Cocaine levels are shown during the follicular phase (top) and luteal phase (bottom). Plasma cocaine levels (ng/ml) are shown on the left ordinates. The vertical line indicates when cocaine was administered. Time (min) after i.v. cocaine administration is shown on the abscissa. Five women were given 0.2 mg/kg i.v. cocaine during each menstrual cycle phase, and data (mean ± S.E.M.) are shown as open circles. Six women were given 0.4 mg/kg i.v. during each menstrual cycle phase, and data (mean ± S.E.M.) are shown as solid circles.

Effects of Cocaine on Luteinizing Hormone Levels in Women. Figure 2 shows that cocaine's effects on LH were dose-dependent in women during both phases of the menstrual cycle. LH levels did not change significantly from baseline after administration of 0.2 mg/kg cocaine in women studied at the follicular phase or at the luteal phase. After administration of 0.4 mg/kg cocaine, LH began to increase within 4 min and reached peak levels of 85 ± 10 and 84 ± 15 ng/ml within 16 min during the follicular and luteal phases, respectively. LH remained significantly above baseline for 4 min (16-20 min postcocaine) in follicular phase women and for 8 min (12-20 min postcocaine) in luteal phase women. LH increased significantly above baseline within 16 min in follicular phase women when plasma cocaine levels averaged 221 ± 19 ng/ml. In women studied during the luteal phase, LH increased significantly within 12 min after 0.4 mg/kg cocaine administration when plasma cocaine levels averaged 239 ± 35 ng/ml.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Cocaine's effects on luteinizing hormone levels in women. Luteinizing hormone levels (ng/ml) are shown on the left ordinates and time (min) after i.v. cocaine administration is shown on the abscissa. LH data collected during the follicular phase (top) and luteal phase (bottom) of the menstrual cycle are shown. The vertical line indicates when cocaine was administered. LH levels after 0.2 mg/kg i.v. cocaine are shown as open circles, and each data point is the average (±S.E.M.) of five subjects. LH levels after 0.4 mg/kg i.v. cocaine are shown as closed circles and each data point is the average (±S.E.M.) of six subjects.

Cocaine Plasma Levels and Effects on Luteinizing Hormone Levels in Men. Plasma cocaine levels in men after intravenous administration of 0.2 and 0.4 mg/kg cocaine are shown in Fig. 3, top. Cocaine plasma levels were maximal within 8 min after cocaine administration and remained elevated for 60 to 80 min. In men, the average peak cocaine levels reached after administration of 0.4 mg/kg cocaine (227 ± 22 ng/ml) were significantly higher than after administration of 0.2 mg/kg cocaine (95 ± 15 ng/ml) (P < 0.002). Both doses of cocaine significantly increased LH levels in men (P < 0.01) (Fig. 3, bottom). After administration of 0.2 mg/kg cocaine, LH levels increased significantly within 8 min and reached peak levels of 77 ± 9 ng/ml within 20 min when plasma cocaine levels averaged 86 ± 16 ng/ml. LH remained significantly higher than baseline for 32 min (between 8 and 40 min). After administration of 0.4 mg/kg cocaine, LH increased significantly within 8 min and reached peak levels of 72 ± 3 ng/ml within 20 min when plasma cocaine levels averaged 169 ± 17 ng/ml. LH remained significantly higher than baseline for 32 min (between 8 and 40 min). Thus, higher cocaine levels did not result in significantly greater increases in LH in men.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Plasma cocaine and luteinizing hormone levels in men after intravenous cocaine administration. Plasma cocaine levels (ng/ml) and luteinizing hormone levels (ng/ml) after administration of 0.2 and 0.4 mg/kg i.v. cocaine are shown on the left ordinate. Time (min) after i.v. cocaine administration is shown on the abscissa. The vertical line indicates when cocaine was administered. Plasma cocaine and LH levels after 0.2 mg/kg i.v. cocaine are shown as open circles. Plasma cocaine and LH levels after 0.4 mg/kg i.v. cocaine are shown as closed circles. Each data point is the average (±S.E.M.) of samples collected from six men. The asterisks indicate a significant change in LH from the precocaine baseline (**P < 0.01; ***P < 0.001).

Gender Comparisons of the Effects of Cocaine on LH. Figure 4 shows peak plasma cocaine levels and peak LH levels for men and follicular and luteal phase women after administration of 0.2 and 0.4 mg/kg cocaine. Although cocaine stimulated significant increases in LH in both men and women, cocaine was more potent in altering LH in men than in women. A low dose of cocaine (0.2 mg/kg i.v.) did not increase LH in women at either phase of the menstrual cycle but significantly increased LH in men. Peak cocaine levels after administration of 0.2 mg/kg cocaine were higher in women than in men, but these differences were not statistically significant.

View larger version (61K): [in this window] [in a new window]   Fig. 4.   Gender comparisons of the effects of cocaine on LH. Peak plasma cocaine levels are shown on the left ordinate (top). Peak LH levels (ng/ml) are shown on the left ordinate (bottom). Data obtained after administration of 0.2 mg/kg cocaine are shown as gray bars and data obtained after administration of 0.4 mg/kg cocaine are shown as black bars. The average precocaine baseline LH levels are indicated as a white horizontal bar in each group. The asterisks indicate a significant change in LH from the precocaine baseline (*P < 0.05; **P < 0.01).

	Discussion

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Stimulation of LH by Cocaine. LH increased significantly after administration of 0.4 mg/kg i.v. cocaine in both men and women. This is the first study of the effects of cocaine on LH in women and in male cocaine abusers who were not dependent on cocaine or other drugs. These findings extend the generality of previous reports that cocaine stimulates LH release in male and female rhesus monkeys (Mello et al., 1990a,b, 1993). Cocaine (30 mg i.v. or 4.28 mg/70 kg) also increased LH levels in cocaine- and opioid-dependent men (Mendelson et al., 1992) and intranasal cocaine (2 mg/kg) increased LH levels in cocaine-naive men (Heesch et al., 1996). In our previous studies of cocaine- and opioid-dependent men, significant increases in LH were detected within 5 min and reached peak levels within 15 min (Mendelson et al., 1992). A similar time course of LH increases was observed in male cocaine abusers in the present study. Significant increases in LH were measured within 8 min after both doses of i.v. cocaine, and peak levels of LH occurred after 20 min.

Gender-Related Differences in the Effects of Cocaine. Two gender differences were observed in the effects of cocaine on LH. Cocaine was more potent in stimulating LH in men than in women, and LH remained significantly elevated longer after cocaine administration in men than in women. It was surprising to find that a low dose of cocaine stimulated LH in men, but did not stimulate LH in women at either phase of the menstrual cycle. This gender difference did not appear to be accounted for by differences in baseline LH levels or peak plasma cocaine levels. Baseline LH levels were equivalent in all subjects, and there were no significant gender differences in plasma cocaine levels after 0.2 mg/kg cocaine. Moreover, peak plasma cocaine levels were slightly higher in follicular phase females than in males. Men and women were matched for body mass index, age, and reported history of cocaine use. We have previously reported that there were no significant gender differences in the pharmacokinetic profiles of intravenous cocaine users (Mendelson et al., 1999b) so it is unlikely that dispositional factors accounted for the greater potency of low doses of cocaine in men. Similarities in reported cocaine use patterns between men and women argue against the possibility that women were more tolerant to cocaine's effects. Cocaine stimulated significantly higher levels of another anterior pituitary hormone, ACTH, in occasional cocaine users than in cocaine-dependent men, and this appeared to reflect cocaine tolerance (Mendelson et al., 1998). However, the women in the present study were not cocaine-dependent.

Implications of Cocaine's Stimulation of LH. The adverse effects of repeated stimulation of LH by cocaine on reproductive function can be inferred from the disruptions of the menstrual cycle associated with chronic cocaine self-administration (Mello et al., 1997a; Mello, 1998). However, the mechanisms that account for cocaine's stimulation of LH are unknown. Cocaine's stimulation of LH in rhesus monkeys appears to reflect a burst of hypothalamic LHRH and not a change in LH disposition according to a deconvolution analysis (Mello and Mendelson, 1997). As noted earlier, cocaine's actions as an indirect dopamine agonist may not be directly relevant to its effects on LH. Exogenous dopamine administration did not increase or decrease LH in rhesus monkeys (Spies et al., 1980; Pavasuthipaisit et al., 1981; Mello et al., 1997a) but decreased LH in humans (Yen, 1979), and the role of endogenous dopamine in LH regulation remains controversial (Yen, 1999). LH release is controlled by many neuromodulatory systems in brain (Hotchkiss and Knobil, 1994; Yen, 1999) and the relative contribution of cocaine's effects on estradiol, dopamine, norepinephrine, and endogenous opioid systems remains to be determined (Mello and Mendelson, 1997).