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Gender differences in cocaine pharmacokinetics in CF-1 mice
Toxicology Letters 155 (2005) 35–40
Gender differences in cocaine pharmacokinetics in CF-1 mice Thomas Visallia , Rita Turkalla,b , Mohamed S. Abdel-Rahmana,∗ a
b
Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Room I-681, Newark, NJ 07103-2714, USA Department of Clinical Laboratory Sciences, School of Health Related Professions, University of Medicine and Dentistry of New Jersey, 65 Bergen Street, Newark, NJ 07107-3001, USA Received 6 July 2004; received in revised form 12 August 2004; accepted 12 August 2004 Available online 18 September 2004
Abstract Hepatocellular damage is thought to occur as a result of cytochrome P450-mediated oxidation of cocaine to norcocaine (NC), a precursor of the hepatotoxic nitrosonium ion. However, this damage occurs only in male mice, with females exhibiting minimal biochemical and histological signs of hepatocellular stress. The objective of this study was to determine the plasma time course and tissue disposition of cocaine and its metabolites to further investigate the role that metabolism may play in the gender difference observed. Male and female CF-1 mice were orally administered 20 mg/kg cocaine hydrochloride once daily for 7 days. Blood samples were withdrawn at various time points post-injection and analyzed for cocaine and its metabolites benzoylecgonine (BE), norcocaine, ecgonine methyl ester (EME), and ecgonine (E). In addition, tissue concentrations of cocaine and its metabolites were determined in liver, heart, brain, and kidney tissue. The results demonstrated that the plasma elimination half-life of cocaine is nearly three times longer in males versus females. Non-hepatotoxic hydrolysis metabolites BE, EME, and E were higher in female tissues while norcocaine was detected in tissues of male animals only. This study revealed that differences in cocaine pharmacokinetics and the resultant differences in the biodisposition of cocaine and its metabolites in tissues contribute to the mechanism of gender difference seen in cocaine hepatotoxicity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Ecgonine; Norcocaine; Hepatotoxicity
1. Introduction Oral exposure to cocaine causes hepatocellular damage in both humans and experimental animals (Boyer ∗ Corresponding author. Tel.: +1 973 972 6568; fax: +1 973 972 4554. E-mail address: [email protected] (M.S. Abdel-Rahman).
and Petersen, 1992; Labib et al., 2001; Perino et al., 1987; Wanless et al., 1990). Production of reactive oxygen species during the sequential oxidation of cocaine has been proposed as a requirement for cocaine hepatotoxicity (Bornheim, 1998; Labib et al., 2002a, 2003; Schuster et al., 1977). Excessive production of these reactive species is thought to contribute to oxidative stress that depletes the liver’s antioxidative capacity resulting
0378-4274/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2004.08.008
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T. Visalli et al. / Toxicology Letters 155 (2005) 35–40
in damage (Boelsterli and Goldlin, 1991). The oxidative metabolism of cocaine is facilitated by hepatic cytochrome P450s and yields norcocaine (NC), which may be oxidized to the highly reactive nitrosonium ion (Charkoudian and Shuster, 1985; Ndikum-moffer et al., 1997). However, cocaine metabolism also occurs by hydrolytic as well as oxidative pathways (Whittington et al., 1999). Plasma esterase-mediated hydrolysis, the major metabolic pathway, yields pharmacologically inactive and non-hepatotoxic metabolites (Ambre, 1985; Freeman and Harbison, 1981; Karch, 2001). It has been demonstrated in our laboratory that mice administered cocaine exhibit a gender difference in the occurrence of cocaine hepatotoxicity. Biochemical indices of liver damage as well as histological examination of liver sections show that female mice are less susceptible to cocaine hepatotoxicity after oral administration of the drug than are males (Visalli et al., 2004). Gender-dependent differences in metabolism may result in differences in pharmacokinetic profiles of cocaine and its metabolites, thus altering the degree of cocaine hepatotoxicity. In this study, the plasma time-course and tissue concentrations of cocaine and its metabolites were evaluated to investigate the role that metabolism may play in the gender difference observed for cocaine hepatotoxicity in mice.
2. Materials and methods 2.1. Animals Adult male and female CF-1 mice (Charles River Laboratories, Wilmington, MA), weighing 25–30 and 20–25 g, respectively, were used in these studies. Cocaine-induced liver necrosis has been well documented in this strain (Mehanny and Abdel-Rahman, 1991; Bornheim, 1998; Labib et al., 2002, 2003). The animals were quarantined for 1 week before the initiation of the experiment. They were housed in plastic cages in environmentally controlled rooms with a 12 h light/dark cycle at a constant temperature (23–25 ◦ C) and a relative humidity of 50%. The animals were maintained on Rodent Diet 5001 (Lab Diet, St. Louis, MO) and water ad libitum. The use of animals for this experiment was approved by the Institutional An-
imal Care and Use Committee (IACUC) of the University of Medicine and Dentistry of New Jersey, Newark, NJ. 2.2. Reagents and chemicals Cocaine hydrochloride was provided by the National Institute on Drug Abuse (NIDA), Bethesda, MD. Cocaine hydrochloride was dissolved in an appropriate volume of physiological saline prior to administration. Unless otherwise stated, all the chemicals were reagent grade and were purchased from Sigma. 2.3. Treatment protocol Mice were food fasted 12 h before all treatments. Cocaine hydrochloride was prepared fresh daily in saline. Mice were administered cocaine hydrochloride (20 mg/kg) by gavage once daily for seven consecutive days. Selection of cocaine dosage was based on previously published data from our laboratory (Labib et al., 2001, 2002). 2.4. Plasma time-course Hundred microliters of blood was withdrawn from the lateral tail vein of at least five animals per treatment via a heparinized syringe at 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240 min after the last cocaine administration. All samples were collected in ice-chilled test tubes. Plasma was obtained by centrifuging the blood at 1000 × g for 15 min at 4 ◦ C. Half-lives and area under the curve (AUC) were calculated utilizing SigmaPlot 7.0 (Richmond, CA) and Kinetica (Philadelphia, PA) software. 2.5. Tissue concentrations of cocaine and metabolites Liver, kidney, brain, and heart samples were obtained upon sacrifice, 30 min after the last cocaine dose. This time point was selected since it coincides with the peak plasma norcocaine concentration following oral administration (Ma et al., 1999). Tissue samples were removed and immediately washed in ice-cold saline and homogenized in sodium phosphate buffer (pH 7.4).
T. Visalli et al. / Toxicology Letters 155 (2005) 35–40
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2.6. HPLC Detection of cocaine and its metabolites in tissue and plasma samples was performed by reversed-phase high-pressure liquid chromatography (Waters, Milford, MA) utilizing a method developed and validated in this laboratory (Rofael and Abdel-Rahman, 2002). 2.7. Statistical analysis Data were represented as the mean ± S.E.M. Multiple comparisons were performed by analysis of variance (ANOVA) and followed by the Tukey–Kramer honestly significant difference (HSD) test. Statistical analysis between two groups was performed by Student’s independent t-test. In all analyses, the level of significance was set to p < 0.05. Statistical analyses were performed with the JMP 4.0.4 statistical software package (SAS Institute Inc., Cary, NC).
3. Results 3.1. Plasma time course Peak plasma cocaine concentrations were 2.5 times higher in males versus females and the area under the curve was nearly six-fold greater in males as well. Further, the plasma half-life of cocaine was approximately 50 min in males and 17 min in females despite similar absorption half-lives (Fig. 1). Norcocaine was not detected in females (Fig. 2) while benzoylecgonine (BE), ecgonine (E), and ecgonine methyl ester (EME) were all elevated 2.5–3-fold in females versus males (Figs. 3–5). No significant gender-related differences exist in absorption/formation half-lives or elimination half-lives of any of cocaine’s hydrolytic metabolites.
Fig. 1. Plasma time course of cocaine in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. Area under the curve (AUC) is 19.32 and 3.28 g min/ml for males and females, respectively. (a) Half-life of absorption in male animals = 15.9 min. (b) Half-life of elimination in male animals = 50.5 min. (a ) Half-life of absorption in female animals = 14.7 min. (b ) Half-life of elimination in female animals = 16.9 min.
in male animals versus females (Table 1). The liver consistently displayed the highest concentration of cocaine and its metabolites in the tissues of males and females (Tables 1–3).
3.2. Tissue concentrations of cocaine and its metabolites With the exception of norcocaine, which was not detected in female animals (Table 1), the tissue concentrations of other cocaine metabolites were consistently two to three-fold higher in females when compared with males (Tables 1–3). Conversely, tissue concentrations of cocaine were correspondingly higher
Fig. 2. Plasma time course of norcocaine in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. Norcocaine was not detected in female animals. (a) Half-life of formation in male animals = 4.6 min. (b) Half-life of elimination in male animals = 6.0 min.
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T. Visalli et al. / Toxicology Letters 155 (2005) 35–40
Fig. 3. Plasma time course of benzoylecgonine in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. (a) Half-life of formation in male animals = 6.8 min. (b) Half-life of elimination in male animals = 67.7 min. (a ) Half-life of formation in female animals = 5.8 min. (b ) Half-life of elimination in female animals = 63.0 min.
Fig. 5. Plasma time course of ecgonine in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. (a) Half-life of formation in male animals = 10.8 min. (b) Half-life of elimination in male animals = 41.8 min. (a ) Half-life of formation in female animals = 9.4 min. (b ) Half-life of elimination in female animals = 38.3 min.
due to the influence of sex hormones on cocaine metabolism in CF-1 mice (Visalli et al., 2004). These studies show that a gender-dependent difference in cocaine pharmacokinetics also occurs in the same mouse strain. While the time to peak plasma cocaine concentration was similar in males and females, the peak height was higher in males versus females and the AUC was nearly six times greater in males. This is consistent with the elimination half-life of cocaine being nearly three times longer in males versus Table 1 Comparison between the tissue concentration of cocaine and norcocaine in male and female CF-1 mice after cocaine administration Fig. 4. Plasma time course of ecgonine methyl ester in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. (a) Half-life of formation in male animals = 11.5 min. (b) Half-life of elimination in male animals = 40.8 min. (a ) Half-life of formation in female animals = 10.0 min. (b ) Half-life of elimination in female animals = 37.1 min.
4. Discussion Previously, our laboratory reported genderdependent differences in cocaine hepatotoxicity
Tissue
Cocaine
Norcocaine
Male
Female
Male
Female
Plasma Liver Heart Brain Kidney
0.20 ± 0.1 0.89 ± 0.1 0.37 ± 0.1 0.47 ± 0.2 0.58 ± 0.1
0.08 ± 0.01a 0.31 ± 0.09a 0.14 ± 0.01a 0.17 ± 0.02a 0.22 ± 0.08a
0.09 ± 0.01 0.20 ± 0.01 0.09 ± 0.01 0.08 ± 0.03 0.10 ± 0.02
ND ND ND ND ND
Values are presented as mean ± S.E.M. (g/g tissue or g/ml plasma) from five mice/sex administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Mice were sacrificed 30 min after the last cocaine treatment. ND: not detected. a Significantly different from male treatment group (p < 0.05).
T. Visalli et al. / Toxicology Letters 155 (2005) 35–40 Table 2 Comparison between the tissue concentration of benzoylecgonine in male and female CF-1 mice after cocaine administration Tissue
Male
Female
Plasma Liver Heart Brain Kidney
1.00 ± 0.1 4.20 ± 0.7 1.91 ± 0.6 1.89 ± 0.1 2.00 ± 0.1
3.20 ± 0.3a 11.8 ± 1.1a 5.68 ± 0.5a 5.63 ± 0.5a 8.30 ± 0.6a
Values are presented as mean ± S.E.M. (g/g tissue or g/ml plasma) from five mice/sex administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Mice were sacrificed 30 min after the last cocaine treatment. a Significantly different from male treatment group (p < 0.05).
females, despite similar absorption half-lives. Even though absorbed cocaine is exposed to liver enzymes after oral administration, the majority of the dose does in fact reach the blood un-metabolized (Karch, 2001; Ma et al., 1999). Gender-related differences were also seen in the pharmacokinetics of cocaine metabolite formation. Females formed higher amounts of non-hepatotoxic, hydrolytic metabolites, i.e. BE, E and EME, than males despite similar elimination half-lives of these metabolites in both genders. This pattern suggests a higher plasma esterase activity in female versus male mice. Higher amounts of hydrolytic metabolites formed in females are also consistent with an absence of NC, since less cocaine would be available for oxidative metabolism. Studies by Roberts et al., 1993, support these findings by showing that co-administration of coTable 3 Comparison between the tissue concentration of ecgonine methyl ester and ecgonine in male and female CF-1 mice after cocaine administration Tissue
Plasma Liver Heart Brain Kidney
Ecgonine methyl ester
Ecgonine
Male
Female
Male
Female
0.48 ± 0.1 2.00 ± 0.2 0.95 ± 0.1 0.91 ± 0.1 1.51 ± 0.2
1.50 ± 0.1a 6.20 ± 0.5a 2.80 ± 0.7a 2.74 ± 0.6a 4.53 ± 0.6a
0.64 ± 0.1 2.40 ± 0.2 1.28 ± 0.2 1.01 ± 0.1 2.04 ± 0.1
2.18 ± 0.1a 7.94 ± 0.6a 3.90 ± 0.4a 3.40 ± 0.3a 7.01 ± 0.6a
Values are presented as mean ± S.E.M. (g/g tissue or g/ml plasma) from five mice/sex administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Mice were sacrificed 30 min after the last cocaine treatment. a Significantly different from male treatment group (p < 0.05).
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caine and ethanol, a known esterase inhibitor, yielded lower concentrations of BE and higher concentrations of the parent compound and norcocaine in humans and mice. Also in agreement with this pharmacokinetic profile are reports that indicate that male experimental animals have higher concentrations of certain isoforms of cytochrome P450 when compared with females (Gandhi et al., 2004; Kato and Yamazoe, 1992; Kedderis and Mugford, 1998). Although hydrolysis facilitated by esterases is the major pathway of cocaine metabolism, greater amounts of hepatic cytochrome P450 in males would certainly support findings of higher concentrations of norcocaine in the plasma and liver of male mice. Since esterases are also present in tissues as well as in plasma (Silver, 1974; Small et al., 1996), altered tissue metabolism of cocaine may contribute to altered cocaine hepatotoxicity. With the exception of NC, which was not detected in females, the tissue concentrations of cocaine were lower and its hydrolytic metabolites were higher in females than males. Although tissue concentrations reflect plasma concentrations, higher tissue esterase activity, in part, may support the higher tissue hydrolytic metabolite concentrations seen in females compared to males. Further, in males and females, concentrations of cocaine and its metabolites were consistently highest in the liver. While the liver does contain esterases (Bowman et al., 1999; Foldes, 1978), high hepatic concentrations are likely to be a result of the route of administration. After absorption of the oral dose, a significant portion of cocaine that enters the portal circulation will be hydrolyzed. However, the first pass effect will initially expose the liver to the highest concentrations of these newly produced metabolites, yielding elevated metabolite concentrations in this tissue. A role for testosterone is suggested in the gender differences observed in the pharmacokinetic profiles of cocaine and its metabolites and in cocaine hepatotoxicity. Smolen and Smolen (1990) show that young, pre-weanling mice are resistant to cocaine hepatotoxicity. Conversely, adult male mice are highly susceptible to cocaine hepatotoxicity, while 30-day-old males display an intermediate degree of cocaine hepatotoxicity (Labib et al., 2001; Smolen and Smolen, 1990). Difference in cocaine hepatotoxicity produced with age is consistent with increasing concentrations of testosterone, a known suppressor of plasma esterase (Illsley
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and Lamartiniere, 1981). In female and sexually immature male mice with low testosterone concentrations, increased hydrolytic metabolites and decreased NC would be expected. On the other hand, a reversed pattern of these metabolites would be expected in adult males. Results of this study support a link between testosterone concentration and cocaine hepatotoxicity. In conclusion, these findings demonstrate that rapid hydrolysis of cocaine to BE, E, and EME attenuates hepatic NC production in female CF-1 mice and provides a mechanism to account for the gender-related difference in cocaine hepatotoxicity between females and males.
References Ambre, J., 1985. The urinary excretion of cocaine and metabolites in humans: a kinetic analysis of published data. J. Anal. Toxicol. 9, 241–245. Bornheim, L.M., 1998. Effect of cytochrome P450 inducers on cocaine-mediated hepatotoxicity. Toxicol. Appl. Pharmacol. 150, 158–165. Boelsterli, U.A., Goldlin, C., 1991. Biomechanisms of cocaineinduced hepatocyte injury mediated by the formation of reactive metabolites. Arch. Toxicol. 65, 351–360. Bowman, B.P., Vaughn, S.R., Waker, Q.D., Davis, S.L., Little, P.J., Scheffler, N.M., Thomas, B.F., Kuhn, C.M., 1999. Effects of sex and gonadectomy on cocaine metabolism in the rat. J. Pharmacol. Exp. Ther. 290, 1316–1323. Boyer, S.C., Petersen, D.R., 1992. Pharmacokinetic analysis of the metabolism of cocaine to norcociane and n-hydroxynorcocaine in mice. Drug Metab. Dispos. 20, 863–868. Charkoudian, J.C., Shuster, L., 1985. Electrochemistry of norcocaine nitroxide and related compounds: implications for cocaine hepatotoxicity. Biochem. Biophys. Res. Commun. 130, 1044–1051. Foldes, F.F., 1978. In: Foldes, F.F. (Ed.), Enzymes in Anesthesiology. Springer, New York, pp. 91 and 168. Freeman, R.Q., Harbison, R.D., 1981. Hepatic periportal necrosis induced by chronic administration of cocaine. Biochem. Pharmacol. 45, 739–754. Gandhi, M., Aweeka, F., Greenblatt, R.M., Blaschke, T.F., 2004. Sex differences in pharmacokinetics and pharmacodynamics. Annu. Rev. Pharmacol. Toxicol. 44, 499–523. Illsley, N.P., Lamartiniere, C.A., 1981. Endocrine regulation of rat serum cholinesterase activity. Endocrinology 108, 1737–1743. Karch, S.B., 2001. Karch’s Pathology of Drug Abuse. CRC Press, New York. Kato, R., Yamazoe, Y., 1992. Sex-specific cytochrome P450 as a cause of sex- and species-related differences in drug toxicity. Toxicol. Lett. 64/65, 661–667.
Kedderis, G.L., Mugford, C.A., 1998. Sex-dependent metabolism of xenobiotics. Drug Metab. Rev. 30 (3), 441–498. Labib, R., Turkall, R., Abdel-Rahman, M.S., 2001. Oral cocaine produces dose-related hepatotoxicity in male mice. Toxicol. Lett. 125, 29–37. Labib, R., Turkall, R., Abdel-Rahman, M.S., 2002. Endotoxin potentiates the hepatotoxicity of cocaine in male mice. J. Toxicol. Environ. Health A 65, 977–993. Labib, R., Turkall, R., Abdel-Rahman, M.S., 2002a. Endotoxin potentiates cocaine-mediated hepatotoxicity by nitric oxide and reactive oxygen species. Int. J. Toxicol. 22, 1–12. Labib, R., Turkall, R., Abdel-Rahman, M.S., 2003. NAcetylcycsteine pretreatment decreases cocaine and endotoxin induced hepatotoxicity. J. Toxicol. Environ. Health A 66 (3), 223–240. Ma, F., Falk, J.L., Lau, C.E., 1999. Cocaine pharmacodynamics after intravenous and oral administration in rats: relation to pharmacokinetics. Psychopharmacology 44, 323–332. Mehanny, S.Z., Abdel-Rahman, M.S., 1991. Cocaine hepatotoxicity in mice: histologic and enzymatic studies. Toxicol. Pathol. 19 (1), 24–29. Ndikum-moffer, F.M., Schoeb, T.R., Roberts, S.M., 1997. Liver toxicity from norcocaine nitroxide an N-oxidative metabolite of cocaine. J. Pharmacol. Exp. Ther. 284, 413–419. Perino, L.E., Warren, G.H., Levine, J.S., 1987. Cocaine-induced hepatotoxicity in humans. Gastroenterology 93, 176–180. Roberts, S.M., Harbison, R.A., James, R.C., 1993. Inhibition by ethanol of the metabolism of cocaine to benzoylecgonine and ecgonine methyl ester in mouse and human liver. Drug. Metab. Dispos. 21 (3), 537–541. Rofael, H.Z., Abdel-Rahman, M.S., 2002. Development and validation of a high performance liquid chromatography method for determination of cocaine and its metabolites and lidocaine. J. Appl. Toxicol. 22, 123–128. Schuster, L., Quimby, F., Bates, A., Thompson, M.L., 1977. Liver damage from cocaine in mice. Life Sci. 20, 1035–1041. Silver, A., 1974. The Biology of Cholinesterases. North-Holland Publishing Company, New York. Small, D.H., Michaelson, S., Sberna, G., 1996. Invited review: nonclassical actions of cholinesterases: role in cellular differentiation, tumorgenesis, and Alzheimer’s disease. Neurochem. Int. 28, 453–483. Smolen, T.N., Smolen, A., 1990. Developmental expression of cocaine hepatotoxicity in the mouse. Pharmacol. Biochem. Behav. 36, 333–338. Visalli, T., Turkall, R., Abdel-Rahman, M.S., 2004. Cocaine hepatotoxicity and its potentiation by lipopolysaccharide. Int. J. Toxicol. 23 (3), 163–170. Wanless, I.R., Dore, S., Gopinath, N., Tan, J., Cameron, R., Heathcote, E.J., Blendis, L.M., Levy, G., 1990. Histopathology of cocaine hepatotoxicity. Gastroenterology 98, 497–501. Whittington, R.A., Iso, A., Khan, K., Cooper, T.B., Morishima, H.O., 1999. Role of gender in the toxicity of norcocaine. J. Lab. Clin. Med. 133, 590–596.
Gender differences in cocaine pharmacokinetics in CF-1 mice Thomas Visallia , Rita Turkalla,b , Mohamed S. Abdel-Rahmana,∗ a
b
Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Room I-681, Newark, NJ 07103-2714, USA Department of Clinical Laboratory Sciences, School of Health Related Professions, University of Medicine and Dentistry of New Jersey, 65 Bergen Street, Newark, NJ 07107-3001, USA Received 6 July 2004; received in revised form 12 August 2004; accepted 12 August 2004 Available online 18 September 2004
Abstract Hepatocellular damage is thought to occur as a result of cytochrome P450-mediated oxidation of cocaine to norcocaine (NC), a precursor of the hepatotoxic nitrosonium ion. However, this damage occurs only in male mice, with females exhibiting minimal biochemical and histological signs of hepatocellular stress. The objective of this study was to determine the plasma time course and tissue disposition of cocaine and its metabolites to further investigate the role that metabolism may play in the gender difference observed. Male and female CF-1 mice were orally administered 20 mg/kg cocaine hydrochloride once daily for 7 days. Blood samples were withdrawn at various time points post-injection and analyzed for cocaine and its metabolites benzoylecgonine (BE), norcocaine, ecgonine methyl ester (EME), and ecgonine (E). In addition, tissue concentrations of cocaine and its metabolites were determined in liver, heart, brain, and kidney tissue. The results demonstrated that the plasma elimination half-life of cocaine is nearly three times longer in males versus females. Non-hepatotoxic hydrolysis metabolites BE, EME, and E were higher in female tissues while norcocaine was detected in tissues of male animals only. This study revealed that differences in cocaine pharmacokinetics and the resultant differences in the biodisposition of cocaine and its metabolites in tissues contribute to the mechanism of gender difference seen in cocaine hepatotoxicity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Ecgonine; Norcocaine; Hepatotoxicity
1. Introduction Oral exposure to cocaine causes hepatocellular damage in both humans and experimental animals (Boyer ∗ Corresponding author. Tel.: +1 973 972 6568; fax: +1 973 972 4554. E-mail address: [email protected] (M.S. Abdel-Rahman).
and Petersen, 1992; Labib et al., 2001; Perino et al., 1987; Wanless et al., 1990). Production of reactive oxygen species during the sequential oxidation of cocaine has been proposed as a requirement for cocaine hepatotoxicity (Bornheim, 1998; Labib et al., 2002a, 2003; Schuster et al., 1977). Excessive production of these reactive species is thought to contribute to oxidative stress that depletes the liver’s antioxidative capacity resulting
0378-4274/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2004.08.008
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in damage (Boelsterli and Goldlin, 1991). The oxidative metabolism of cocaine is facilitated by hepatic cytochrome P450s and yields norcocaine (NC), which may be oxidized to the highly reactive nitrosonium ion (Charkoudian and Shuster, 1985; Ndikum-moffer et al., 1997). However, cocaine metabolism also occurs by hydrolytic as well as oxidative pathways (Whittington et al., 1999). Plasma esterase-mediated hydrolysis, the major metabolic pathway, yields pharmacologically inactive and non-hepatotoxic metabolites (Ambre, 1985; Freeman and Harbison, 1981; Karch, 2001). It has been demonstrated in our laboratory that mice administered cocaine exhibit a gender difference in the occurrence of cocaine hepatotoxicity. Biochemical indices of liver damage as well as histological examination of liver sections show that female mice are less susceptible to cocaine hepatotoxicity after oral administration of the drug than are males (Visalli et al., 2004). Gender-dependent differences in metabolism may result in differences in pharmacokinetic profiles of cocaine and its metabolites, thus altering the degree of cocaine hepatotoxicity. In this study, the plasma time-course and tissue concentrations of cocaine and its metabolites were evaluated to investigate the role that metabolism may play in the gender difference observed for cocaine hepatotoxicity in mice.
2. Materials and methods 2.1. Animals Adult male and female CF-1 mice (Charles River Laboratories, Wilmington, MA), weighing 25–30 and 20–25 g, respectively, were used in these studies. Cocaine-induced liver necrosis has been well documented in this strain (Mehanny and Abdel-Rahman, 1991; Bornheim, 1998; Labib et al., 2002, 2003). The animals were quarantined for 1 week before the initiation of the experiment. They were housed in plastic cages in environmentally controlled rooms with a 12 h light/dark cycle at a constant temperature (23–25 ◦ C) and a relative humidity of 50%. The animals were maintained on Rodent Diet 5001 (Lab Diet, St. Louis, MO) and water ad libitum. The use of animals for this experiment was approved by the Institutional An-
imal Care and Use Committee (IACUC) of the University of Medicine and Dentistry of New Jersey, Newark, NJ. 2.2. Reagents and chemicals Cocaine hydrochloride was provided by the National Institute on Drug Abuse (NIDA), Bethesda, MD. Cocaine hydrochloride was dissolved in an appropriate volume of physiological saline prior to administration. Unless otherwise stated, all the chemicals were reagent grade and were purchased from Sigma. 2.3. Treatment protocol Mice were food fasted 12 h before all treatments. Cocaine hydrochloride was prepared fresh daily in saline. Mice were administered cocaine hydrochloride (20 mg/kg) by gavage once daily for seven consecutive days. Selection of cocaine dosage was based on previously published data from our laboratory (Labib et al., 2001, 2002). 2.4. Plasma time-course Hundred microliters of blood was withdrawn from the lateral tail vein of at least five animals per treatment via a heparinized syringe at 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240 min after the last cocaine administration. All samples were collected in ice-chilled test tubes. Plasma was obtained by centrifuging the blood at 1000 × g for 15 min at 4 ◦ C. Half-lives and area under the curve (AUC) were calculated utilizing SigmaPlot 7.0 (Richmond, CA) and Kinetica (Philadelphia, PA) software. 2.5. Tissue concentrations of cocaine and metabolites Liver, kidney, brain, and heart samples were obtained upon sacrifice, 30 min after the last cocaine dose. This time point was selected since it coincides with the peak plasma norcocaine concentration following oral administration (Ma et al., 1999). Tissue samples were removed and immediately washed in ice-cold saline and homogenized in sodium phosphate buffer (pH 7.4).
T. Visalli et al. / Toxicology Letters 155 (2005) 35–40
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2.6. HPLC Detection of cocaine and its metabolites in tissue and plasma samples was performed by reversed-phase high-pressure liquid chromatography (Waters, Milford, MA) utilizing a method developed and validated in this laboratory (Rofael and Abdel-Rahman, 2002). 2.7. Statistical analysis Data were represented as the mean ± S.E.M. Multiple comparisons were performed by analysis of variance (ANOVA) and followed by the Tukey–Kramer honestly significant difference (HSD) test. Statistical analysis between two groups was performed by Student’s independent t-test. In all analyses, the level of significance was set to p < 0.05. Statistical analyses were performed with the JMP 4.0.4 statistical software package (SAS Institute Inc., Cary, NC).
3. Results 3.1. Plasma time course Peak plasma cocaine concentrations were 2.5 times higher in males versus females and the area under the curve was nearly six-fold greater in males as well. Further, the plasma half-life of cocaine was approximately 50 min in males and 17 min in females despite similar absorption half-lives (Fig. 1). Norcocaine was not detected in females (Fig. 2) while benzoylecgonine (BE), ecgonine (E), and ecgonine methyl ester (EME) were all elevated 2.5–3-fold in females versus males (Figs. 3–5). No significant gender-related differences exist in absorption/formation half-lives or elimination half-lives of any of cocaine’s hydrolytic metabolites.
Fig. 1. Plasma time course of cocaine in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. Area under the curve (AUC) is 19.32 and 3.28 g min/ml for males and females, respectively. (a) Half-life of absorption in male animals = 15.9 min. (b) Half-life of elimination in male animals = 50.5 min. (a ) Half-life of absorption in female animals = 14.7 min. (b ) Half-life of elimination in female animals = 16.9 min.
in male animals versus females (Table 1). The liver consistently displayed the highest concentration of cocaine and its metabolites in the tissues of males and females (Tables 1–3).
3.2. Tissue concentrations of cocaine and its metabolites With the exception of norcocaine, which was not detected in female animals (Table 1), the tissue concentrations of other cocaine metabolites were consistently two to three-fold higher in females when compared with males (Tables 1–3). Conversely, tissue concentrations of cocaine were correspondingly higher
Fig. 2. Plasma time course of norcocaine in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. Norcocaine was not detected in female animals. (a) Half-life of formation in male animals = 4.6 min. (b) Half-life of elimination in male animals = 6.0 min.
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Fig. 3. Plasma time course of benzoylecgonine in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. (a) Half-life of formation in male animals = 6.8 min. (b) Half-life of elimination in male animals = 67.7 min. (a ) Half-life of formation in female animals = 5.8 min. (b ) Half-life of elimination in female animals = 63.0 min.
Fig. 5. Plasma time course of ecgonine in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. (a) Half-life of formation in male animals = 10.8 min. (b) Half-life of elimination in male animals = 41.8 min. (a ) Half-life of formation in female animals = 9.4 min. (b ) Half-life of elimination in female animals = 38.3 min.
due to the influence of sex hormones on cocaine metabolism in CF-1 mice (Visalli et al., 2004). These studies show that a gender-dependent difference in cocaine pharmacokinetics also occurs in the same mouse strain. While the time to peak plasma cocaine concentration was similar in males and females, the peak height was higher in males versus females and the AUC was nearly six times greater in males. This is consistent with the elimination half-life of cocaine being nearly three times longer in males versus Table 1 Comparison between the tissue concentration of cocaine and norcocaine in male and female CF-1 mice after cocaine administration Fig. 4. Plasma time course of ecgonine methyl ester in CF-1 mice. Each time point represents mean ± S.E.M. from five mice/sex. Mice were administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Plasma samples were collected after the last cocaine administration. (a) Half-life of formation in male animals = 11.5 min. (b) Half-life of elimination in male animals = 40.8 min. (a ) Half-life of formation in female animals = 10.0 min. (b ) Half-life of elimination in female animals = 37.1 min.
4. Discussion Previously, our laboratory reported genderdependent differences in cocaine hepatotoxicity
Tissue
Cocaine
Norcocaine
Male
Female
Male
Female
Plasma Liver Heart Brain Kidney
0.20 ± 0.1 0.89 ± 0.1 0.37 ± 0.1 0.47 ± 0.2 0.58 ± 0.1
0.08 ± 0.01a 0.31 ± 0.09a 0.14 ± 0.01a 0.17 ± 0.02a 0.22 ± 0.08a
0.09 ± 0.01 0.20 ± 0.01 0.09 ± 0.01 0.08 ± 0.03 0.10 ± 0.02
ND ND ND ND ND
Values are presented as mean ± S.E.M. (g/g tissue or g/ml plasma) from five mice/sex administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Mice were sacrificed 30 min after the last cocaine treatment. ND: not detected. a Significantly different from male treatment group (p < 0.05).
T. Visalli et al. / Toxicology Letters 155 (2005) 35–40 Table 2 Comparison between the tissue concentration of benzoylecgonine in male and female CF-1 mice after cocaine administration Tissue
Male
Female
Plasma Liver Heart Brain Kidney
1.00 ± 0.1 4.20 ± 0.7 1.91 ± 0.6 1.89 ± 0.1 2.00 ± 0.1
3.20 ± 0.3a 11.8 ± 1.1a 5.68 ± 0.5a 5.63 ± 0.5a 8.30 ± 0.6a
Values are presented as mean ± S.E.M. (g/g tissue or g/ml plasma) from five mice/sex administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Mice were sacrificed 30 min after the last cocaine treatment. a Significantly different from male treatment group (p < 0.05).
females, despite similar absorption half-lives. Even though absorbed cocaine is exposed to liver enzymes after oral administration, the majority of the dose does in fact reach the blood un-metabolized (Karch, 2001; Ma et al., 1999). Gender-related differences were also seen in the pharmacokinetics of cocaine metabolite formation. Females formed higher amounts of non-hepatotoxic, hydrolytic metabolites, i.e. BE, E and EME, than males despite similar elimination half-lives of these metabolites in both genders. This pattern suggests a higher plasma esterase activity in female versus male mice. Higher amounts of hydrolytic metabolites formed in females are also consistent with an absence of NC, since less cocaine would be available for oxidative metabolism. Studies by Roberts et al., 1993, support these findings by showing that co-administration of coTable 3 Comparison between the tissue concentration of ecgonine methyl ester and ecgonine in male and female CF-1 mice after cocaine administration Tissue
Plasma Liver Heart Brain Kidney
Ecgonine methyl ester
Ecgonine
Male
Female
Male
Female
0.48 ± 0.1 2.00 ± 0.2 0.95 ± 0.1 0.91 ± 0.1 1.51 ± 0.2
1.50 ± 0.1a 6.20 ± 0.5a 2.80 ± 0.7a 2.74 ± 0.6a 4.53 ± 0.6a
0.64 ± 0.1 2.40 ± 0.2 1.28 ± 0.2 1.01 ± 0.1 2.04 ± 0.1
2.18 ± 0.1a 7.94 ± 0.6a 3.90 ± 0.4a 3.40 ± 0.3a 7.01 ± 0.6a
Values are presented as mean ± S.E.M. (g/g tissue or g/ml plasma) from five mice/sex administered cocaine (20 mg/kg, p.o.) for seven consecutive days. Mice were sacrificed 30 min after the last cocaine treatment. a Significantly different from male treatment group (p < 0.05).
39
caine and ethanol, a known esterase inhibitor, yielded lower concentrations of BE and higher concentrations of the parent compound and norcocaine in humans and mice. Also in agreement with this pharmacokinetic profile are reports that indicate that male experimental animals have higher concentrations of certain isoforms of cytochrome P450 when compared with females (Gandhi et al., 2004; Kato and Yamazoe, 1992; Kedderis and Mugford, 1998). Although hydrolysis facilitated by esterases is the major pathway of cocaine metabolism, greater amounts of hepatic cytochrome P450 in males would certainly support findings of higher concentrations of norcocaine in the plasma and liver of male mice. Since esterases are also present in tissues as well as in plasma (Silver, 1974; Small et al., 1996), altered tissue metabolism of cocaine may contribute to altered cocaine hepatotoxicity. With the exception of NC, which was not detected in females, the tissue concentrations of cocaine were lower and its hydrolytic metabolites were higher in females than males. Although tissue concentrations reflect plasma concentrations, higher tissue esterase activity, in part, may support the higher tissue hydrolytic metabolite concentrations seen in females compared to males. Further, in males and females, concentrations of cocaine and its metabolites were consistently highest in the liver. While the liver does contain esterases (Bowman et al., 1999; Foldes, 1978), high hepatic concentrations are likely to be a result of the route of administration. After absorption of the oral dose, a significant portion of cocaine that enters the portal circulation will be hydrolyzed. However, the first pass effect will initially expose the liver to the highest concentrations of these newly produced metabolites, yielding elevated metabolite concentrations in this tissue. A role for testosterone is suggested in the gender differences observed in the pharmacokinetic profiles of cocaine and its metabolites and in cocaine hepatotoxicity. Smolen and Smolen (1990) show that young, pre-weanling mice are resistant to cocaine hepatotoxicity. Conversely, adult male mice are highly susceptible to cocaine hepatotoxicity, while 30-day-old males display an intermediate degree of cocaine hepatotoxicity (Labib et al., 2001; Smolen and Smolen, 1990). Difference in cocaine hepatotoxicity produced with age is consistent with increasing concentrations of testosterone, a known suppressor of plasma esterase (Illsley
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and Lamartiniere, 1981). In female and sexually immature male mice with low testosterone concentrations, increased hydrolytic metabolites and decreased NC would be expected. On the other hand, a reversed pattern of these metabolites would be expected in adult males. Results of this study support a link between testosterone concentration and cocaine hepatotoxicity. In conclusion, these findings demonstrate that rapid hydrolysis of cocaine to BE, E, and EME attenuates hepatic NC production in female CF-1 mice and provides a mechanism to account for the gender-related difference in cocaine hepatotoxicity between females and males.
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