Abstract
The purpose of the current study is to determine whether sex differences in metabolism of cocaine (COC) exist that could contribute to the greater behavioral sensitivity of females to COC administration. To investigate this question, concentrations of COC and its two principle metabolites benzoylecgonine (BE) and ecgonine methyl ester (EME) were measured by gas chromatography/mass spectroscopy in brain and plasma collected from male and female rats that were sacrificed between 5 and 90 min after injection COC (15 mg/kg i.p.). COC concentrations did not differ in plasma or brain tissue of males and females, but sex-specific patterns of metabolite distribution were detected. BE was 2-fold higher in plasma and brain of males than females, whereas EME was much higher in brain and plasma of females. The influence of gonadal hormones on COC metabolite patterns were determined using gonadectomized and prepubertal rats. Castration of male or female rats did not alter brain or plasma COC, but did decrease BE concentrations. Seven-day-old pups injected with 15 mg/kg of COC had higher blood and brain COC than adults and relatively low levels of metabolites. No sex differences were found for COC, BE, or EME in brain or plasma of pups. These findings indicate that although gonadal steroids influence COC metabolism, these effects do not explain sex differences in COC-induced behaviors.
Current estimates indicate that cocaine (COC) is used by over 1.5 million people in the United States. Of these, about 30% are women, a majority of whom are of childbearing age. This translates to over 450,000 women who are at risk for exposing a fetus to COC (National Institute of Drug Abuse Household Study, 1997). Despite these large numbers, few studies have investigated whether women respond differently to COC than men.
Animal studies have found that male and female rats have different sensitivities to the psychomotor effects of peripherally injected stimulants like amphetamine (AMPH). Beatty and Holzer (1978), Robinson et al. (1980), and Brass and Glick (1981) have reported that female rats rotate more than males when given AMPH. Becker et al. (1982)confirmed these results and found that higher locomotion scores in females relative to males after AMPH treatment were partly due to higher AMPH levels in females due to sex differences in the metabolism of AMPH by the liver cytochrome P-450 (Meyer and Lytle, 1978). The sex difference persisted even though AMPH injections were adjusted to produce equivalent brain levels in males and females (Becker et al., 1982).
Sex differences in behaviors elicited by COC have also been found.Glick et al. (1983) and van Haaren and Meyer (1991) have shown that females show substantially greater locomotor responses to COC than males. Recent studies from our laboratory (Bowman and Kuhn, 1996) replicated this result and showed that these sex differences emerged after puberty. In addition, we have shown that females exhibit greater activation of the hypothalamo-pituitary-adrenal axis than males after COC administration (Kuhn and Francis, 1997). As was the case for AMPH, these sex effects may reflect pharmacokinetic differences. Major metabolites of COC in the rat include benzoylecgonine (BE) and ecgonine methyl ester (EME). BE is reportedly formed nonenzymatically as a hydrolytic product of COC (Stewart et al., 1979; Isenschmid et al., 1989), although there is evidence for carboxylesterase transformation of COC to BE in the liver (Brzezinski et al., 1994; Dean et al., 1995). EME is formed by plasma and liver esterases (Stewart et al., 1977;Stewart et al., 1979). Both EME and BE are bioactive metabolites of COC. BE is approximately equipotent to COC in stimulating locomotor activity in rats when injected directly into rats (Misra et al., 1975;Schuelke et al., 1996). BE can vasoconstrict cerebral arteries at concentrations that are achieved following COC administration (Madden et al., 1995). EME at high doses is reported to impair COC-stimulated behaviors (Schuelke et al., 1996). Therefore, potential sex differences in COC metabolism could mediate sex differences in physiologic and locomotor responses to COC through differences either in blood levels of COC, BE, or EME.
The purpose of this study was to investigate whether sex differences in COC metabolism exist in rats that could mediate reported sex differences in the locomotor response to COC. COC, BE, and EME were analyzed in brain and plasma of adult male and female rats over a 90-min time course. In addition, the control of COC metabolism by gonadal hormones was assessed by examining COC metabolism in developing male and female rats, and after gonadectomy in adults.
Materials and Methods
Animals.
Sixty-day-old intact and gonadectomized male and female rats (Sprague-Dawley) were obtained from Charles River Laboratories (Raleigh, NC). Lactating dams were purchased from Charles River Laboratories with litters consisting of six males and six females. Animals were housed in a 12-h light/dark environment and given ad libitum access to standard lab chow and water. Females were used without regard to estrous cycle state. This choice was made carefully, weighing the possibility that estrous cycle stage might influence COC pharmacokinetics. Our recent preliminary findings suggest that sex differences in locomotor response to COC are robust in rodents, whereas variations with estrous cycle stage are subtle and not statistically significant (Q.D.W., M. Bunin, R. M. Wightman, C.M.K., unpublished observations). Furthermore, because of the large number of experimental groups in the present study and the large number (a total of 67 females were used), the likelihood of randomly assigning a disproportionate number of females from one particular estrous cycle stage to any given group is exceedingly small. Therefore, the possibility of detecting an apparent sex difference that reflected only animals in a particular estrous cycle stage was small. Animals were used 7 to 10 days after surgery. Gonadectomy was confirmed in a subset of animals both surgically and through evaluation of estradiol, progesterone, or testosterone levels in blood. For experiments involving gonadectomized animals, the controls received sham surgeries at the same time. All experimental procedures were approved by the Institutional Animal Care and Use Committee and were in accordance with National Institutes of Health Guidelines for the Care and Use of Animals.
Drugs and Chemicals.
Deuterated and nondeuterated COC (D5-COC), BE-HCl (D5-BE), and EME (D3-EME) were supplied by Research Triangle Institute (Research Triangle Park, NC) with National Institute for Drug Abuse approval. Pentaflouroproprionic anhydride (PFPA) was obtained from Pierce (Rockford, IL), and 2,2,3,3,3-pentafluoro-1-propanol (PFP-OH) was purchased from Aldrich Chemicals (Milwaukee, WI). All solvents were Burdick and Jackson (Muskegon, MI) UV grade and were obtained from VWR Scientific (Durham, NC). All other chemicals used were obtained from Sigma Chemicals (St. Louis, MO). All glassware used in these experiments was silylated by gas-phase silanization or by using Aqua-Sil (Aldrich Chemicals). All vial caps were Teflon coated.
COC and Metabolite Time Course.
Male and female rats were injected i.p. with 15 mg/kg COC (in 1 ml/kg saline) and were decapitated 5, 15, 30, 45, 60, or 90 min later. These time points were chosen to parallel the reported onset (5 min), maximum (15–30 min), and offset (60–90 min) of COC-stimulated locomotion and adrencorticotropin secretion in our laboratory, and span the times COC and metabolite levels are maximal after a single dose (Misra et al., 1975; Petit et al., 1990; Bowman et al., 1996; Kuhn and Francis, 1997). Trunk blood was collected into ice-cold tubes containing 250 μl of saturated NaF and 100 μl of heparin and placed on ice. Tubes were centrifuged at 1600g for 10 min at 4°C, and the plasma was transferred into silylated vials containing 50 μl of saturated NaF. Brain tissue (minus olfactory bulbs) was dissected, weighed, and placed in cold tubes containing 2 ml of 1 N HCl and 1 ml 1% of NaF. Brain tissue was homogenized by Polytron. All samples were stored at −70°C until assayed.
Effects of Gonadectomy.
Castrated and sham-operated male and female rats were injected i.p. with 15 mg/kg COC and were sacrificed 30 min later. This time point was chosen because sex differences in metabolite levels were easily distinguished at this time. Because no difference in COC levels was observed, earlier time points when COC levels were maximal were not tested. Tissue collection proceeded as described for the time course experiment.
Developmental Studies.
Testing was performed on postnatal day 7 (PND7). Pups were injected i.p. with 15 mg/kg COC (in 0.1 ml of saline) and decapitated as described for adults. Animals were removed once from the dam during the injection period and again 15 min before decapitation. At this time they were placed in a humidified infant incubator at 32°C to maintain pup body temperature. Blood and brain samples from six same-sex littermates were pooled, yielding ann of 1 for each sex per litter.
Standard Formulation.
Serial dilutions of a stock solution containing equal concentrations of deuterated COC (2[H]5-COC), BE (2[H]5-BE), and EME (2[H]3-EME) were made to give a final concentration of 5 ng/μl. Standard curves were generated using concentrations of the three analytes ranging from 25,000 to 25 ng/ml in MeOH, which were serially diluted from a single stock solution. Separate standard curves were generated for brain and plasma and were used to calculate predicted values. Three controls ranging from 25 to 250 ng were used for each extraction to provide an internal quality control.
Analyte Extraction and Derivatization.
COC and its metabolites were extracted from tissues as described by Crouch et al. (1995). On the day of the experiment, brain tissue was thawed and centrifuged at 34,000g for 30 min at 4°C. A 1-ml aliquot of prepared brain or plasma was added to a vial containing 10 or 20 μl of internal standard solution and 5 ml of 0.1 M potassium phosphate buffer (1:1 mono and dibasic salts, pH 6.0). Each control was prepared using internal standard, 4.9 ml of 0.1 M phosphate buffer, 1 ml of blank plasma, and 100 μl of the control solution. The samples were vortex mixed and centrifuged for 10 min at 1600g at 4°C.
Solid-phase extraction was performed in Bond-Elut C18 columns (Chrom Technology, Apple Valley, MN) prewet with consecutive washes of 2 ml of freshly prepared elution solvent (78:20:2 MeCl2:isopropanol:conc NH4OH), 2 ml of MeOH, and 2 ml of 0.1 M potassium phosphate buffer. The samples were washed with 6 ml of distilled water, 3 ml of 0.1 M HCl, and 9 ml of MeOH. Analytes were eluted with 2.5 ml of elution solvent. Diluted samples were evaporated using a centrifugal evaporation system to dryness, washed with 0.5 ml of MeCl2, and evaporated again to dryness to remove remaining water.
PFPA/PFP-OH derivatization was performed as described by Aderjan et al. (1993). To each sample, 50 μl of PFP-OH and 50 μl of PFPA were added to derivatize the analytes. Vials were capped, vortex mixed, and heated for 20 min at 60°C. Vials were then cooled to room temperature and evaporated to dryness. Samples were reconstituted in 30 μl of toluene and transferred to an autosampler vial and capped. Vials were stored in an airtight container at −70°C until run on the gas chromatography/mass spectroscopy (GC/MS), no longer than 1 month.
GC/MS Analysis.
A Hewlett Packard (HP) 5989-A mass spectrometer and a 5890 series II gas chromatograph controlled by HP 9000/series 400 UNIX workstation using chemstation software (HP-UX version B.08.0) were used for this analysis. Conditions for the analysis are shown in Table 1. Quantitation of analytes was done using selected ion-monitoring (SIM) methodology using ratios of nondeuterated and deuterated pairs. The data system monitored m/z 346 and 349 for EME/2[H]3-EME,m/z 422 and 427 for BE/2[H]5-BE, andm/z 304 and 309 for COC/2[H]5-COC, respectively. Area ratios of nondeuterated and deuterated controls were used to construct a standard curve. Linear regression analysis (using a weighting factor of 1/[analyte]) was used to give a formula for a line that was then used to predict unknown values.
Statistical Analysis.
Animal number for each group was determined by power analysis based on the variance in COC and metabolite data in preliminary experiments. The numbers run were adequate to detect at least a 30% difference in drug level. Time course data were analyzed using appropriate two-way ANOVAs, using time and sex as main factors. Postcastration pharmacokinetic data were analyzed using two-way ANOVA, using sex and surgical treatment as main factors. Post hoc Fisher’s protected least-squares tests were used when ANOVAs were significant. For all statistical tests, significance was set at p < .05.
Results
Time Course of COC Metabolism in Adults.
Adult plasma COC levels decreased rapidly throughout the testing session (Fig.1, top) (p < .001 for time). No sex differences in the time course were found, with peak levels reaching 851 ± 134 and 798 ± 89 ng/ml plasma for males and females, respectively. Likewise, no sex differences in COC levels were detected in brain (Fig. 1, bottom). Brain COC levels peaked between 5 and 15 min after injection at 5299 ± 574 ng/g wet weight brain for females and 4644 ± 533 ng/g wet weight brain for males. COC levels decreased rapidly during the remaining time sampled (p < .001 for time). Although the variance at 15 min was large and values of females appeared to be somewhat higher, values at earlier at later times were identical, and the overall ANOVA revealed no effects of sex.
Plasma BE levels were significantly different between males and females (p < .001 for sex, p < .05 for time), with males peaking higher than females and maintaining levels nearly 2-fold higher than those of females throughout the time course (Fig.2, top). Brain BE levels increased throughout the time period recorded (Fig. 2, bottom) (p< .001 for time) and also showed a sex difference (p< .05 for sex) due to a greater accumulation of BE in the brain of males than females at later time points.
Plasma EME levels were higher in females than in males (p < .001 for sex), with females having higher peak EME levels than males and higher levels throughout the time course (Fig. 3, top). In addition, brain EME was higher in females throughout the time course (p < .001 for sex and time) and increased along a different time course than males (p < .01 sex × time) (Fig. 3, bottom). EME was higher in female brain than in male brain (p < .01) and accumulated more rapidly there than in males (p < .001 sex × time).
Effect of Gonadectomy on COC Metabolism.
Plasma levels of COC and EME were not altered by gonadectomy (Fig.4). BE levels in the plasma were significantly decreased after gonadectomy (p < .05 for surgery) regardless of sex case-sensitive (p = 0.9 for sex case-sensitive × surgery). Brain COC, BE, and EME levels were similarly unaffected by gonadectomy (Fig. 4).
COC Metabolism during Development.
Brain and plasma levels of COC, BE, and EME were examined in immature males and females, as our behavioral data would predict that no sex differences would be observed. No sex differences existed in COC, BE, or EME levels in the plasma of 7-day-old pups (Table 2). COC levels in pup plasma were consistently 2- to 3-fold higher/relative to adults (p < .001 for age). BE levels in the plasma were conversely lower in pups (p < .01 for age), although they reached the level of adult females at the later time points due to gradual accumulation. As with adults, BE was the major metabolite found in pup plasma (p < .001 versus EME). EME levels were similar between male adults and male and female pups, but were significantly lower in male and female pups than adult females (ANOVA, p < .001 for age; ANOVA, p < .001 for sex × age).
As in plasma, no sex differences were found in COC, BE, or EME levels in the pup brain (Table 3). Pup brain COC levels peaked nearly 100% higher in pups than in adults (p < .001 for age), and brain COC concentration persisted longer in pups (p < .001 for age × time), with levels remaining near 5.0 μg/g wet weight brain throughout the time course. Brain BE levels were higher in adults than in pups (p < .01 for age). As in plasma, brain EME levels in the pups mirrored adult male levels. Both male and female pup EME brain levels were significantly below adult female levels (p < .001 for age; p < .001 for age × sex). As with adults, EME was the major metabolite found in pup brain (p < .001 versus BE).
Discussion
These studies show that female and male rats have comparable COC levels in both plasma and brain. This finding suggests that reported sex differences in the locomotor response to COC are not due to substantial differences in brain drug levels. Gonadectomy of male or female rats had no impact on plasma or brain COC levels. This study does show that the metabolic route of COC is influenced by both age and sex. Gonadectomy significantly reduced the amount of BE produced indicating that the production of BE is affected by gonadal steroids. In addition, the overall metabolism of COC is greatly reduced in neonatal rats, which most likely reflects delayed ontogeny of the enzymes responsible for its breakdown. This deficiency leads to elevated concentrations of COC in the plasma and the brain, which may partially explain the robust locomotor response to COC that developing animals exhibit (Bowman and Kuhn, 1996).
Role of COC Metabolism in the Locomotor Response to COC.
Enhanced responses by females to an acute challenge of COC have been described (Glick et al., 1983; van Haaren and Meyer, 1991; Bowman and Kuhn, 1996). Only a single study by Heyser et al. (1994) found slight sex differences in male and female 120-day-old rats given 10 mg/kg COC i.p, with females having higher brain COC levels than males at early time points. However, their data were collapsed across animals receiving three different prenatal treatments, so control animals could not be directly compared. In contrast, a separate report (van Luijtelaar et al., 1996) found that males had slightly higher plasma levels of COC than females after 20 mg/kg but not 10 mg/kg COC i.p. Effects were found only when data were collapsed across time, and samples were collected under urethane anesthesia, both of which could have complicated interpretation of the data. In addition, all animals in their studies received chronic COC treatment, which has been shown to increase brain COC levels (Pettit et al., 1990). In the present study, no significant sex differences in brain or plasma COC levels were observed following acute injection of COC into undisturbed, COC-naı̈ve animals following a dose of COC that has been shown to elicit different locomotor responses in males and females. This absence of sex differences in COC levels supports the importance of central mechanisms in the observed sex differences in the response to psychomotor stimulants.
The findings regarding sex differences in plasma COC levels and the physiologic effects of COC in other species are mixed. Lukas et al. (1996) have compared COC actions after metered nasal insufflation in men and in women at two stages of the menstrual cycle. COC levels were higher in men than women in either mid-follicular or luteal phases of their menstrual cycle. Males showed more intense subjective effects of COC than females, but heart rate did not differ according to sex. In contrast, recent preliminary reports after i.v. COC administration suggest that blood levels of COC are comparable in men and women, and do not vary significantly across the menstrual cycle (Mendelson et al., 1999a). Similarly, in rhesus monkeys, COC pharmacokinetics are similar in males and females after i.v. administration (Mendelson et al., 1999b). These findings are consistent with the present results in rodents. However, comparing our data to human studies should be done cautiously because of species-specific expression and control of cholinesterases. For example, men have higher plasma cholinesterase activity (opposite of rats) and lower erythrocyte esterase activity than women (Sidell and Kaminskis, 1975).
Impact of Estrous Cycle Stage on COC Metabolism.
Although the present study did not test COC pharmacokinetics at every stage of the estrous cycle, it is unlikely that the substantial sex reported difference in COC-stimulated behavior but not pharmacokinetics reflects the accidental selection of females in a particular estrous cycle stage. Findings from our laboratory suggest that the sex difference in locomotor response to COC is substantial but that estrous cycle stage differences using the same assessment strategy are not significant (Walker et al., 1998, 1999). These findings are consistent with previous studies of COC self-administration in which sex differences were substantial and detected with either an FR1 or progressive ratio schedule, whereas significant estrous cycle stage differences were detected only with a progressive ratio schedule (Roberts et al., 1989). Similarly, the single study which compared the locomotor response to AMPH in females in different estrous cycle stages to that observed in males, females responded more than males at every stage of the cycle, although at one stage (diestrous 1) this difference was not significant (Becker et al., 1982). Other studies report estrous cycle differences in COC, AMPH, or apomorphine responses (Steiner et al., 1980; Hruska et al., 1982; Joyce and Van Hartesveldt, 1984) but generally report females respond more than males, and that cycle differences are frequently reflected in differences in the specific topography of behaviors (postural deviation versus rotation) but not necessarily in horizontal locomotion. Furthermore, frequently such changes occur during very restricted time frames, especially during the dark cycle of proestrus, that would not extend to stimulant-induced locomotion conducted during the morning hours (Becker et al., 1982; Robinson et al., 1982). Therefore, although a substantial literature supports a role for estrous cycle stage in spontaneous and perhaps stimulant-induced locomotion (Beatty 1979), the present findings and previous studies suggest that sex differences are large and easily detected across estrous cycle stage.
Mechanisms of COC Metabolism.
COC is metabolized predominantly by two parallel pathways in humans and rats (Stewart et al., 1977) (Fig. 5). Both BE and EME are further metabolized to ecgonine by the same mechanisms responsible for the production of EME and BE from COC, respectively. EME is the product of de-esterification by plasma and liver cholinesterases (Stewart et al., 1977, 1979). Plasma cholinesterase activity is higher in female rats than in male rats, a sex difference that is lost after castration (Illsley and Lamartiniere, 1981; Morishima et al., 1993) and reversed with hormone replacement (Illsley and Lamartiniere, 1981). Although the production of more EME in intact females than intact males in the current study is consistent with this reported sex difference in cholinesterase activity, this same difference should have produced a fall in EME in ovariectomized females, but it did not. Perhaps our use of a 1-week recovery period was insufficient to permit a total loss of the effects of gonadal steroids on esterase activity. Alternatively, erythrocyte cholinesterase and liver esterases could be contributing to metabolism of COC to EME in a sex-neutral way. Regulation of these enzymes, and the relative proportion of metabolism of each, is less well characterized, and activity of these other enzymes could compensate for the potential effects of castration on plasma cholinesterase.
The present results indicate that hydrolysis of COC to BE is at least in part enzymatic, is under the control of gonadal steroids and occurs less during early development. BE production was significantly greater in males than in females, but dropped in both males and females after castration. Furthermore, BE production in neonates was dramatically below levels observed for both adult males and females. The explanation for these findings is complicated by the absence of definitive information about the mechanisms responsible for the production of BE. Several studies have shown that BE can be formed nonenzymatically simply by incubating COC in saline or buffer at physiologic pH and temperature (Stewart et al., 1979). Although this may be true in blood or buffer, carboxylesterases in the liver are capable of enzymatically converting COC to BE (Brzezinski et al., 1994; Dean et al., 1995).Zhang et al. (1996) have shown that males exhibit more carboxylesterase activity than females, which could explain the sex disparity in plasma BE levels seen in our study. Similar changes in enzymatic processes are consistent with the present findings. The drug-metabolizing enzymes that have been implicated in BE production are immature during this stage of development (Sterri et al., 1985). If BE production was simply a product of nonenzymatic hydrolysis, castrated and developing animals should not make less BE than adults. Therefore, the present results are most consistent with the possibility that endocrine regulation of enzymatic mechanisms mediates the observed changes in BE production with sex and age.
Sex Differences in BE and COC-Induced Locomotion.
The greater levels of BE formed in male rats probably do not contribute to the sex differences in behavior seen in adult rats. When delivered centrally, BE elicits behaviors similar to COC, such as increased locomotor behavior and seizures (Misra et al., 1975; Shuelke et al., 1996). However, the doses of BE needed to achieve 50% incidence of behaviors are similar to COC, in the 30- to 50-μg range after intracisternal administration (Misra et al., 1975; Shuelke et al., 1996). Our studies indicate that after peripheral injection of COC, concentrations of BE are so low in the brain that they are not likely to contribute to behavior. Similarly low levels were reported in mouse brain after i.p. administration of COC (Benuck et al., 1987). This conclusion is consistent with the failure of primates to self-administer BE or EME even at doses 200- to 300-fold greater than the EC50 for COC (Spealman et al., 1989). In addition, no significant sex differences occur in brain BE levels during the time period that sex differences in behaviors are seen, although BE levels increase in males over females late in the time course. Others have shown that BE does accumulate in the brains of male rats after repeated administration of COC, although it is not clear that BE accumulates to levels capable of influencing behavior (Estevez et al., 1978; Weiss and Gawin, 1988). Centrally injected EME does not stimulate behaviors on its own (Misra et al., 1975; Shuelke et al., 1996). Rather, it either sedates, or attenuates behaviors stimulated by COC and BE, with a dose required for 50% incidence around 500 μg after intracisternal administration (Shuelke et al., 1996). EME should attenuate COC-stimulated behavior, and therefore females would be expected to show lower COC-stimulated behaviors than males. Considering the relative metabolite profile in males and females, sex differences in COC metabolism, if anything, would attenuate the observed sex difference in stimulation of locomotion.
COC Metabolism and Sex-Specific Cardiovascular Effects of COC.
COC metabolites in the periphery may contribute to the toxic effects of COC use. When delivered in situ, BE causes vasoconstriction of cat cerebral arteries more than COC itself through nonnoradrenergic mechanisms (Madden et al., 1995). In addition, BE and EME are known to decrease the sinus node rate in the heart, although less than COC itself (Wang and Carpentier, 1994), via mechanisms other than blockade of fast sodium channels (Crumb and Clarkson, 1992). In the current study, concentrations of BE in plasma are elevated relative to COC during much of the time course. If BE is causing vasoconstriction of cerebral or cardiac arteries to a larger extent in males than females, the cardiovascular toxicity of COC could be more severe. Interestingly, a single clinical study does show that COC-dependent women were less likely to show cerebral perfusion effects than COC-dependent men (Levin et al., 1994), and another preliminary study showed less COC-induced vasoconstriction in luteal phase women than men (Kaufman et al., 1998). These are important issues in studying potential medical complications of COC use and abuse, and deserve further exploration.
If sex differences in brain or plasma levels of COC, BE, or EME are not contributing to the sex differences seen in the locomotor response to COC, other mechanisms must be responsible. One possible mechanism for these effects could be sex differences in either dopamine (DA) uptake or receptor function. Numerous reports have shown estrogen regulation of DA receptor expression (Hruska, 1986; Levesque and Di Paolo, 1991) as well as transporter binding (Morissette and Di Paolo, 1993a,b). In addition, Peris et al. (1991) reported that chronic estrogen treatment enhanced both COC-induced locomotor sensitization in ovariectomized female rats, as well as AMPH-induced [3H]DA release in the striatum. This breadth of effects of estrogen on striatal dopaminergic systems could help explain sex disparities in COC-stimulated behaviors. Additional studies elucidating what central effects are responsible for these sex differences in behavior may help provide insight into particular vulnerabilities in male and female populations at risk for COC abuse.
Ontogeny of COC Metabolism.
The second main finding of these studies concerns the development of COC metabolism in rats. Our data show that COC levels are significantly higher in the brain and in the plasma of developing postnatal rats than in adults. This finding could explain why developing animals exhibit an unexpectedly robust locomotor response to COC (Spear and Brick, 1979; Bowman and Kuhn, 1996) despite the fact that DA receptors, DA content, and DA transporter function are around 20% of adult levels during the first week of life (Broaddus and Bennett, 1990; Rao et al., 1991). These results also show the importance of determining effective doses of drugs when testing developing animals. Controlling dose across age simply by delivering equal milligram per kilogram doses may not be adequate in comparing the response in pups to that of adults. This is particularly important in light of concerns about the effects of developmental exposure to COC, both pre- and postnatally.
The current findings of reduced production of COC metabolites in neonatal rats confirm and extend a report by Morishima and Whittington (1995) who showed that 7-day-old females had higher plasma, brain, heart, and liver COC levels after a single 10-mg/kg s.c. injection of COC than adult females. No male data or metabolite data were reported in that study. Other reports of COC metabolism during development have concentrated on gestational exposure and are somewhat conflicting.Spear et al. (1989) showed that s.c. administration of COC to pregnant dams (gestation day 20, E20) led to plasma and brain COC levels 2- to 3-fold higher in the dam than in the fetus. This is contrasted by work of Robinson et al. (1994), who injected E18 dams with i.v. COC and found that COC levels did not differ significantly between fetal and dam brain or plasma. Our findings suggest that COC is metabolized in neonatal animals to a lesser degree than adults.
Conclusions.
These results indicate that levels of major metabolites but not COC differ between adult male and female rats. Sex differences in COC and metabolites were not present in developing animals. This finding, in combination with castration studies, provides physiological support from intact animals that BE is chiefly a product of enzymatic hydrolysis, and that this enzyme is under the control of gonadal steroids. The lack of sex differences in COC levels suggests that neurochemical mechanisms likely explain sex differences in the locomotor response to COC. In addition, the slower metabolism exhibited by developing animals in this study should be taken into account when assessing the effects of COC administration and exposure in the neonatal period. Finally, on the basis of the sex differences in plasma BE levels, males would be predicted to have more risk of cardiovascular complications than females, due to the peripheral cardiovascular effects of BE.
Acknowledgments
We thank Mary Myers for her technical assistance.
Footnotes
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Send reprint requests to: Dr. Cynthia M. Kuhn, Department of Pharmacology, Box 3813, Duke University Medical Center, Durham, NC 27710. E-mail: ckuhn{at}acpub.duke.edu
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↵1 This work was supported by Grant 09079 from the National Institute for Drug Abuse.
- Abbreviations:
- COC
- cocaine
- EME
- ecgonine methyl ester
- BE
- benzoylecgonine
- DA
- dopamine
- AMPH
- amphetamine
- GC/MS
- gas chromatography/mass spectroscopy
- PFPA
- pentaflouroproprionic anhydride
- PFP-OH
- 2,2,3,3,3-pentafluoro-1-propanol
- Received November 5, 1998.
- Accepted May 11, 1999.
- The American Society for Pharmacology and Experimental Therapeutics