Abstract
This study was conducted to investigate the effect of a 7-day treatment as well as the influence of gender on cocaine hepatotoxicity (CH). Lipopolysaccharide (LPS) potentiation of CH was also investigated. Male and female CF-1 mice were orally administered 20 mg/kg body weight cocaine hydrochloride once daily for 7 days. Four hours after the last cocaine administration, the mice were administered 12 × 106 EU LPS (or equal volume of sterile saline) intraperitoneally. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were evaluated as indices of liver injury. Blood and liver glutathione (GSH), glutathione reductase (GRx), and catalase (CAT) activities were also determined to investigate the oxidation stress induced by the treatment. Plasma ALT and AST concentrations were elevated in all males receiving cocaine alone or cocaine + LPS. Furthermore, blood GSH and CAT were decreased and GRx activity was elevated in the same males. Histological analysis revealed a high degree of focal necrosis in the male cocaine group, and severe necrosis in the male cocaine + LPS group. Unlike males, females showed no effect of either cocaine alone or cocaine + LPS treatments. These results indicate that gender plays a significant role in CH and its potentiation by LPS and lengthening the administration by two treatments increased the severity of cocaine + LPS hepatotoxicity dramatically in male mice.
Cocaine is one of the most powerfully addictive drugs. It is derived from the leaves of the Erythroxylum coca plant, and is a potent central nervous system stimulant (Evans 1981; Karch 2001). In the United States, cocaine abuse remains an extensive problem, with an estimated 15.4% of adults aged 18 to 25 using cocaine at least once in their lifetime. It is the most frequent cause of drug-related deaths. In 1999, an estimated 1.5 million Americans were current cocaine users, however, these figures may not account for all users (Das 1993; Karch 2001).
It is well known that cocaine is hepatotoxic in both humans and in experimental animals. It produces significant abnormalities in liver function tests, as well as perivenous, midzonal, or periportal necrosis (Afifi and Abdel-Rahman 1998; Kanel et al. 1990; Karch 2001; Labib, Turkall, and Abdel-Rahman 2001; Mehanny and Abdel-Rahman 1991; Perrino, Warren, and Levine 1987; Shuster et al. 1977; Silva et al. 1991; Wanless et al. 1990). The hepatotoxicity of cocaine is a result of its metabolism (Benowitz 1993; Boelsterli and Godlin 1991; Cone 1995). Cocaine metabolism occurs by a hydrolytic pathway and an oxidative pathway. Hydrolysis, the major pathway, yields pharmacologically inactive and nontoxic metabolites (Ambre 1985). Sequential oxidation of cocaine by cytochrome P450 to norcocaine, N-hydroxynorcocaine, norcocaine nitroxide, and possible oxidation to the nitrosonium ion has been proposed as a requirement for cocaine hepatotoxicity (CH) (Bornheim 1998; Labib, Turkall, and Abdel-Rahman 2002b, 2003a). Reactive oxygen species (ROS), including H2O2 and superoxide anion radicals, are also produced during cocaine oxidative metabolism (Bouis and Boelsterli 1990; Kloss, Rosen, and Raukman 1983). The amount of drug subject to metabolism by either pathway is dependent upon the route of administration. Pharmacokinetic studies have shown that cocaine concentration in the liver is 20 times higher after oral versus intravenous administration (Ma, Falk, and Lau 1999). Previous studies in our laboratory demonstrated that CH results following the once-daily oral administration of cocaine to male CF-1 mice for 5 days (Labib, Turkall, and Adbel-Rahman 2001).
Cocaine users are at great risk of exposure to bacterial infection for a number of reasons including needle sharing and bacterial translocation from the gastrointestinal (GI) tract to the systemic circulation (Ganey and Roth 2001; Van Leeuwen et al. 1994). Moreover, the vasoconstrictive effect of cocaine upon the bowel vasculature may cause an ischemic condition that facilitates the translocation of bacteria from the lumen of the GI tract to the circulation via the blood in the portal vein (Ganey and Roth 2001).
In particular, lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, is a potent inducer of hepatic inflammation in both humans and experimental animals (Cadenas and Cadenas 2001; Champion et al. 1976; Coalson et al. 1979; Moulin et al. 2001). Exposure to a small concentration of LPS increased the hepatotoxicity of certain chemicals and has been suggested to influence the pathogenic outcome of chemical exposure (Roth et al. 1997). Previous studies in our laboratory show that small, noninjurious doses of LPS potentiate CH in male CF-1 mice (Labib, Turkall, and Abdel-Rahman 2002a).
Gender differences often influence the toxicity of drugs and chemicals in mammalian species and have been shown to affect the bioavailability, metabolism, and lethality of many drugs (Calabrese 1986; Davis 1998; Spitzer 1999). Studies from our laboratory have shown that estrogen and testosterone have a major role in the bioavailability of drugs and chemicals (McCormick and Abdel-Rahman 1991; Dahab and Abdel-Rahman 1995; Abou-Hadeed et al. 1998).
This study was conducted to investigate the effect of 7-day oral cocaine treatment as well as the influence of gender on CH and LPS potentiation of CH.
MATERIALS AND METHODS
Animals
Adult male and female CF-1 mice (Charles River Laboratories, Wilmington, MA), weighing 25 to 30 g and 20 to 25 g, respectively, were used in these studies. Cocaine-induced liver necrosis has been well documented in this strain (Bornheim 1998; Labib, Turkall, and Abdel-Rahman 2001; Mehanny and Abdel-Rahman 1991). 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 to 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.
Reagents and Chemicals
Cocaine hydrochloride was provided by the National Institute on Drug Abuse (NIDA), Bethesda, MD. LPS derived from Salmonella enteriditis with an activity of 3 × 106 EU/mg was purchased from Sigma Chemical Co. (St. Louis, MO). Drugs were dissolved in an appropriate volume of 0.9% physiological saline prior to administration. The reagent kits used for measuring serum markers of liver injury (alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) were purchased from Sigma. Unless otherwise stated, all the chemicals were reagent grade and were purchased from Sigma.
Treatment Protocol
Mice were randomly assigned to one of four treatment groups (5–6/group) with a male group and female group assigned to each treatment:
Treatment 1: Saline (0.1 ml/animal, per os [p.o.])
Treatment 2: Saline (0.1 ml/animal, p.o.) followed by LPS (3 × 106 EU, intraperitoneal [i.p.])
Treatment 3: Cocaine only (20 mg/kg, p.o.)
Treatment 4: Cocaine (20 mg/kg, p.o.) followed by LPS (12 × 106 EU, i.p.) 4 hours after the last cocaine administration
Mice were food fasted 12 h before each treatment. Cocaine hydrochloride (20 mg/kg) and LPS (12 × 106 EU i.p.) were prepared fresh daily in saline. Mice in treatment group 1 were administered 0.9% saline daily for 7 consecutive days by oral gavage. The mice in treatment group 2 were administered LPS 4 h after the last saline treatment. Mice from treatments 3 and 4 were administered cocaine hydrochloride by gavage once daily for 7 consecutive days. This dosing regimen was selected based upon previous findings from our laboratory indicating an increase in hepatotoxicity with 5-day treatment versus 3-day treatment. Therefore, a 7-day dosing regimen was selected to see if a plateau in hepatotoxic damage is reached after 5 days due to possible up-regulation of antioxidant mechanisms. In treatment 4, 4 h after the last cocaine treatment, the mice were intraperitoneally administered 12 × 106 EU LPS. The animals were then anesthetized and sacrificed by decapitation 18 h after LPS/saline administration. Selection of cocaine and LPS dosages were based on previously published data from our laboratory (Labib, Turkall, and Abdel-Rahman 2001 Labib, Turkall, and Abdel-Rahman 2002a).
Determination of Hepatotoxicity
Liver cell integrity was monitored by measuring the release of ALT and AST into plasma using Sigma Diagnostics ALT and AST test kits (Sigma/Aldrich). Plasma samples for the transaminase assays were obtained upon sacrifice. Whole blood was collected and centrifuged for 10 min at 1000 × g. Results were expressed as international units per liter (IU/L).
Histological Evaluation
The livers were immediately excised upon sacrifice, washed in ice-cold saline, and fixed in neutral, buffered 10% formalin for at least 24 h. The liver was then trimmed, processed, embedded in paraffin, sectioned at 4 to 6 μm, and stained with hematoxylin and eosin (H&E) for light microscopic evaluation.
Measurement of CAT, GSH, and GSH-Related Enzymes
Glutathione (GSH) was determined in whole blood and in liver homogenate according to the method of Beutler, Duran, and Kelly (1963). The activities of related antioxidant enzymes, glutathione reductase (GRx) and catalase (CAT) were measured in liver supernatant and blood hemolysate. CAT activity was measured according to the method of Aebi (1981), by monitoring the decomposition of H2O2 spectrophotometrically at 240 nm. Enzyme activity was expressed per milligram of hemoglobin and milligram of protein for blood and liver samples, respectively. GRx activity in liver homogenate was assayed according to the method of Couri and Abdel-Rahman (1980). This assay is based on the reduction of oxidized GSH, coupled with oxidation of NADPH. The unit activity of GRx was defined as an increase in log of GSH absorbance at 340 nm versus time. Protein was determined with serum albumin as a standard (Bradford 1976) and total hemoglobin was measured in whole blood using a method by Tietz (1987).
Statistical Analysis
Data were represented as the mean ± SEM. 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 < .05. Statistical analysis was performed with the JMP 4.0.4 statistical software package (SAS Institute, Inc., Cary, NC).
RESULTS
Hepatotoxicity Assessment
Neither gender showed elevation in serum transaminases after treatment with saline or LPS alone (Figure 1A, B ), and histologically, the livers from these mice had no evidence of injury (Figure 2). The architecture of the liver was normal for both saline and LPS treatments, with slight increase in phagocytic cell presence in animals treated with LPS (Figure 2C, D ). Male animals treated with cocaine consistently exhibited localized areas of centrilobular, zone 3 coagulative necrosis (Figure 3A ) with a 15-fold and 2.5-fold increase in ALT and AST, respectively (Figure 1A, B ). Males receiving cocaine + LPS displayed severe, hemorrhagic necrosis with complete loss of cell boundaries (Figure 3C ). Necrotic areas spanned central vein to central vein in many cases. ALT and AST activity increased threefold and nearly twofold, respectively, in these animals (Figure 1A, B ). Hepatocytes from females receiving cocaine or cocaine + LPS displayed minor variation in nuclear size, but no structural liver damage and no increase in ALT or AST was apparent for either treatment (Figure 3B, D ). A small amount of phagocytic cell infiltration was present in females treated with cocaine + LPS, consistent with LPS exposure (Figure 3D ).
GSH and Antioxidant Enzymes
Cocaine and cocaine + LPS treatments caused blood GSH levels to decrease approximately 30% in male animals versus saline treatment (Figure 4A ). Further, hepatic GSH content doubled for males receiving cocaine only as compared to saline treatment (Figure 4B ). A similar pattern was observed in GRx activity with hepatic GRx almost doubling in males receiving cocaine only as compared to saline (Figure 5). LPS treatment did not significantly alter hepatic GSH, blood GSH, or GRx in any animals, and females did not exhibit significant changes in any of the above indices after cocaine or cocaine + LPS treatment (Figures 4A, B and 5).
In addition, cocaine and cocaine + LPS treatment reduced blood and liver CAT concentrations by 30% to 35% and 55% to 60%, respectively, in male animals, whereas LPS caused a slight but significant decrease in blood and liver CAT in both genders (Figure 6A, B ). Cocaine + LPS treatment in females caused a small decrease in blood and hepatic CAT, consistent with LPS exposure (Figure 6A, B ). Blood and liver CAT activity of females receiving cocaine were not significantly different from saline treatment (Figure 6A, B ).
DISCUSSION
Previously, our laboratory reported that small, noninjurious doses of LPS potentiated the hepatotoxic effects of cocaine in male CF-1 mice following a 5-day treatment period (Labib et al. 2002a). The degree of hepatotoxicity in male CF-1 mice after cocaine + LPS treatment was further increased in this study based on development of severe, hemorrhagic liver necrosis with approximately 70% loss of cells. However, only a slight increase in plasma ALT and AST activity was exhibited. This is not inconsistent with the severe hepatic necrosis exhibited, because severely reduced amounts of viable tissues are no longer able to contribute increased amounts of ALT and AST to the blood (Speicher 1993; Wallach 1992).
In females, minor variation in nuclear size of hepatocytes was noted, indicating slight oxidative stress on the liver following 7-day treatment with cocaine or cocaine + LPS. However, there was an absence of histological or of biochemical changes seen in males following this same treatment. This may reflect gender-dependent differences in blood esterase and hepatic cytochrome P450 activity seen in both humans and experimental animals (Davis 1998; Illsley and Lamartiniere 1981; Smolen and Smolen 1990; Thompson et al. 1984). Testosterone has been shown to inhibit blood esterase activity (Illsley and Lamartiniere 1981), leaving a greater portion of the cocaine dose available for P450 oxidation in male mice. In addition, male mice have been shown to develop CH only after 30 days of age, suggesting that the enzyme(s) responsible for toxic metabolite production are absent or at very low levels in female and immature male mice (Smolen and Smolen 1990).
Further, significant gender-related differences in norcocaine tissue:plasma concentration ratios following cocaine administration have been observed in male and female rats. Hepatic tissue: plasma ratios of norcocaine were threefold greater in males than in females (Whittington et al. 1999). This gender-dependent difference in cocaine metabolite binding in hepatic tissue may influence the degree of harmful metabolite exposure to the liver.
Recent studies in our laboratory show that hepatocellular damage resulting from LPS and cocaine is mitigated by a depletion of antioxidant systems due to the presence of excessive ROS (Labib, Turkall, and Abdel-Rahman 2003a, 2003b). Therefore, GSH and related antioxidants were investigated to elucidate a mechanism for gender influence. Blood GSH concentration was decreased only in male mice treated with cocaine or cocaine + LPS. This decrease contrasts with increases in hepatic GSH concentration that likely reflects increased GSH synthesis to replenish GSH depleted due to oxidative stress. Increased GRx activity in males administered cocaine further supports this. Lower hepatic GSH concentration and GRx activity in males treated with cocaine + LPS versus cocaine alone is consistent with a smaller number of viable liver cells remaining following cocaine + LPS treatment. Blood or hepatic GSH concentrations in females were unaffected by either cocaine or cocaine + LPS treatments.
CAT activity was decreased in the blood of male mice treated with LPS, cocaine and cocaine + LPS. This is in agreement with studies performed in male experimental animals where down regulation of CAT genes occurs due to cocaine or LPS exposure (Diez-Fernandez et al. 1999; Labib, Turkall, and Abdel-Rahman 2001; Sakaguchi et al. 1981, 1996; Zaragoza et al. 2000). On the other hand, females did not showa decrease in CAT after cocaine treatment, but did show a significant decrease after LPS and cocaine + LPS treatments. However, decrease in CAT activity after cocaine + LPS treatment was of lower magnitude than that seen in male mice and is likely the effect of LPS because cocaine alone had no effect on CAT activity. Male and female mice exhibited a similar pattern of decreased CAT activity in blood as was exhibited in liver. Lack of cocaine-induced hepatotoxicity in females may be partly explained by the fact that cocaine does not cause a decrease in CAT activity similar to that seen in males. In conclusion, the results of this study demonstrate a dramatic increase in the degree of hepatotoxicity in male mice after cocaine + LPS treatment for cocaine administration only. However, unlike males, females showed no hepatotoxic effects from either cocaine or cocaine + LPS treatments. Thus, gender is shown to have a profound influence on CH and its potentiation by LPS.