Induction of rat hepatic cytochrome P-450 by ketamine and its toxicological implications

Ketamine is a common intravenous anesthetic and a frequent drug of abuse, alone or in combination with cocaine. However, the pharmacokinetic effects of ketamine have not been fully investigated. This study determined the effects of ketamine on cytochrome P-450 (P-450)-dependent catalytic activities, protein levels, and hepatotoxicity using male Wistar rats treated with 10, 20, 40, or 80 mg/kg ketamine intraperitoneally twice daily for 4 d. Treatment with ketamine produced a dose-dependent increase of pentoxyresorufin O-dealkylation activity of liver microsomes. Treatment with 80 mg/kg ketamine resulted in 14-, 3-, and 2-fold rise in O-dealkylation of pentoxyresorufin, ethoxyresorufin, and methoxyresorufin of rat liver microsomes, respectively. The treatment produced 31% and 86% increases in 7-ethoxycoumarin O-deethylation and erythromycin N-demethylation, respectively. In addition, aniline hydroxylation activity was elevated by 62%. Protein blot analysis of liver microsomal proteins revealed that 80 mg/kg ketamine induced P-450 1A, 2B, 2E1, and 3A proteins by 2-, 13-, 2-, and 2-fold, respectively. In reversibility study, ketamine-induced pentoxyresorufin O-dealkylation, 7-ethoxycoumarin O-deethylation, erythromycin N-demethylation, and methoxyresorufin O-demethylation activities of liver microsomes prepared from rats 4 d after ketamine treatment were 75%, 48%, 29%, and 38% lower than the respective activities of liver microsomes prepared from rats 1 d after treatment. Protein blot analysis showed that ketamine-induced P-450 2B1/2 proteins also decreased in a time-dependent manner in 4 d. In hepatotoxicity study, treatment of rats with 1 ml/kg CCl4 produced a 7-fold increase in serum alanine aminotransferase activity level and a 17-fold rise in rats pretreated with 80 mg/kg ketamine for 4 d. Treatment of ICR mice with 120 mg/kg cocaine produced a 17% mortality, whereas the same dose of cocaine produced a 50% mortality in mice pretreated with ketamine. Treatment of mice with 100 mg/kg cocaine produced a 76-fold increase in serum alanine aminotransferase activity level and a 260-fold rise in mice pretreated with 80 mg/kg ketamine for 4 d. The present study shows that ketamine induces the expression of multiple forms of P-450 in rat liver microsomes and increases CCl4-induced liver toxicity and cocaine-mediated acute toxicity. Other potential pharmacological or toxicological events related to ketamine use need to be further explored.     

Pharmacokinetic analysis of the metabolism of cocaine to norcocaine and N-hydroxynorcocaine in mice.

Cocaine is hepatotoxic in humans and is a very effective hepatotoxin in the mouse. Previous in vitro studies have shown that the mixed function oxidase system is very important in the metabolism of cocaine to the hepatotoxic species. Activation of cocaine to the cytotoxic species is thought to proceed through the N-demethylation and subsequent N-hydroxylation of the bridgehead amine. The in vivo metabolism of cocaine involves not only oxidative but also hydrolytic mechanisms. Therefore the principle aim of this study was to establish a system in which the in vivo metabolism of cocaine to norcocaine and N-hydroxynorcocaine could be determined. The in vivo metabolism of acutely administered cocaine to norcocaine and N-hydroxynorcocaine was assessed in two inbred mouse strains; one that is relatively sensitive to cocaine hepatotoxicity but shows little enhancement in toxicity when pretreated with phenobarbital (DBA/2lbg), and one that shows little hepatotoxicity unless pretreated with phenobarbital (C57BL/6lbg). Although no significant differences between the strains were seen in the pharmacokinetics of cocaine, the half-life of both norcocaine and N-hydroxynorcocaine was significantly longer in DBA mice. Accordingly, the area under the curve values for hepatic norcocaine and N-hydroxynorcocaine were approximately 5- and 3-fold greater, respectively, in DBA as compared with C57BL mice. The higher levels of both norcocaine and N-hydroxynorcocaine in the DBA mouse are consistent with previously established differences in cocaine hepatotoxicity in these strains of mice. Pretreatment of C57BL mice with phenobarbital prolonged the half-life of norcocaine and N-hydroxynorcocaine 5- to 8-fold over C57BL control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of coumarin and 7-ethoxycoumarin by rat,mouse, guinea pig,Cynomolgus monkey and human precision-cut liver slices

1. The metabolism of 50 μM 7-ethoxycoumarin and 50 μM [3-¹⁴C]coumarin has been studied in precision-cut liver slices from the male Sprague-Dawley rat, female DBA/2 mouse, male Dunkin-Hartley guinea pig, male Cynomolgus monkey and man.

2. In liver slices from all five species 7-ethoxycoumarin was metabolized to 7-hydroxycoumarin (7-HC), which was extensively conjugated with D-glucuronic acid and sulphate. In rat and mouse, 7-HC was preferentially conjugated with sulphate, whereas rates of glucuronidation and sulphation were similar in the other three species.

3. [3-¹⁴C]coumarin was metabolized by liver slices from all five species to various polar products and to metabolite(s) that bound covalently to liver slice proteins. In Cynomolgus monkey and both human subjects studied, 7-HC was the major metabolite that was conjugated with D-glucuronic acid and sulphate, whereas in rat the major metabolites were products of the 3-hydroxylation pathway and unknown metabolites. Major metabolites in mouse liver slices were 7-HC, 3-hydroxylation pathway products and unknown metabolites, and in guinea pig liver slices, 7-HC and unknown metabolites.

4. The metabolism of 7-ethoxycoumarin to free and conjugated 7-HC and [3-¹⁴C] coumarin to total polar products was greater in liver slices from mouse and Cynomolgus monkey than the other three species.

5. With liver slices from all five species there appeared to be little difference in the extent of metabolism of 7-ethoxycoumarin and [3-¹⁴C]coumarin to various products in either a complex tissue culture medium (RPMI 1640 plus foetal calf serum) or a simple balanced salt solution (Earle's balanced salt solution).

6. These results demonstrate that precision-cut liver slices are avaluable in vitro model system for investigating species differences in xenobiotic metabolism. Generally, the observed species differences in coumarin metabolism in vitro agree well with available in vivo data.     

Species differences in the substrate specificity of hepatic cytochrome P-448 from polycyclic hydrocarbon-treated animals.

The in vitro effects of α-naphthoflavone on four hepatic mono-oxygenase activities associated with aromatic hydrocarbon responsiveness in the mouse (aryl hydrocarbon hydroxylase, 2-acetylamino-fluorene N-hydroxylase, biphenyl 2-hydroxylase, and biphenyl 4-hydroxylase) were investigated before and after methylcholanthrene treatment of C57BL/6N and DBA/2N mice, rats, hamsters, guinea pigs and rabbits. The electrophoretic pattern of cytochrome P-450 subunits and reduced CO-hemoprotein difference spectra of the microsomal fractions were also studied. Pretreatment of animals with methylcholanthrene caused: (1) a 1.5 to 2 mm hypsochromic shift in the Soret peak of the reduced hemoprotein-CO complexes in liver microsomes from a C57BL/6N mouse, rat, hamster and rabbit; a 0.5-nm hypsochromic shift in the guinea pig and no shift in the DBA/2N mouse; and (2) an increase in cytochrome P-450 apoproteins of the following molecular weights on sodium dodecyl sulfate-polyaerylamide gel electrophoresis: 54,000 and 55,000 in the C57BL/6N mouse; 48,000, 54,000 and 55,000 in the rat; 49,000 and 54,000 in the hamster; and 54,000 and 57,000 in the rabbit; a small increase in the 54,000 band was seen in the DBA/2N mouse and no increase in the guinea pig. In vitro addition of α-naphthoflavone selectively inhibited all four mono-oxygenase activities from the methylcholanthrene-treated C57BL/6N mouse, rat and hamster; 2-acetylaminofluorene N-hydroxylase and biphenyl 4-hydroxylase activities in the rabbit; and aryl hydrocarbon hydroxylase, 2-acetylaminofluorene N-hydroxylase and biphenyl 4-hydroxylase activities in the guinea pig. The addition of α-naphthoflavone enhanced the activities of aryl hydrocarbon hydroxylase and biphenyl 2-hydroxylase in liver microsomes from both control and methylcholanthrenetreated rabbits, but only biphenyl 2-hydroxylase activity was increased in the guinea pig: the activitity of 2-acetylaminofluorene N-hydroxylase was increased in both control and methylcholan-threne-treated DBA/2M mice, but only in the control C57BL/6N mouse. These data indicate that hepatic cytochrome P-448 is composed of multiple cytochromes, which differ among animal species, each catalyzing different mono-oxygenase activities.     

Some Metabolites of S-Pentyl-L-cysteine in the Rabbit and Other Species

Abstract

1. The metabolism of S-pentyl-L-cysteine has been investigated in the guinea pig, hamster, mouse, rabbit and rat and in vitro by liver slices of these animals and of the pigeon and by kidney slices of mouse, rabbit and rat.

2. The amounts of pentylmercapturic acid, 3-(pentylthio)lactic acid and 3-(pentylthio)pyruvic acid excreted have been measured.

3. All the species examined excreted pentylmercapturic acid, the amount varying from 2% of the dose by the guinea pig to 73% by the hamster. 3-(Pentylthio)pyruvic and 3-(pentylthio)lactic acids were not detected in the urine of the hamster or rat but were excreted by the other species in amounts approximately the same as those of pentylmercapturic acid.

4. ‘Hydroxypentylmercapturic’ acids were excreted by mouse, rabbit and rat and were identified in the case of the rabbit and rat.

5. 4-Carboxybutylmercapturic acid was identified in the urine of dosed mice, rabbits and rats. A further metabolite in rat urine was tentatively identified as 3-(4-carboxybutylthio)lactic acid.

6. Pentylmercapturic acid sulphoxide was detected in the urine of dosed guinea pigs and mice.

7. All tissue preparations examined converted pentyl-L-cysteine to pentylmercapturic acid. 3-(Pentylthio)lactic acid and 3-(pentylthio)pyruvic acid were formed by all tissues examined although only in trace amounts by hamster liver and rat kidney slices. All tissues examined formed hydroxymercapturic acids' although only traces were detected in digests containing hamster liver slices. 4-Carboxybutylmercapturic acid was formed in rabbit liver digests. With rabbit kidney a metabolite thought to be 3-(4-carboxybutylthio)lactic acid was produced.     

ENDOTOXIN POTENTIATES THE HEPATOTOXICITY OF COCAINE IN MALE MICE

Cocaine produces hepatotoxicity by a mechanism that remains undefined but that has been linked to its oxidative metabolism. Endotoxin (lipopolysaccharide, LPS) is also a well-known cause of hepatic damage, where exposure to non-injurious doses of LPS increases the toxicity of certain hepatotoxins. This study was conducted to investigate the possible potentiation of cocaine-mediated hepatotoxicity (CMH) by LPS. Male CF-1 mice were administered oral cocaine hydrochloride for 5 consecutive days at a dose of 20 mg/kg with and without 12 2 10 6 EU LPS/kg given intraperitoneally 4 h after the last cocaine injection. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured as markers of liver injury. Blood and liver glutathione (GSH) levels were determined, as well as the activities of glutathione peroxidase (GPx) and catalase (CAT). In addition, the activity of liver glutathione reductase (GRx) was measured. The results demonstrate that endotoxin potentiated the hepatotoxicity of cocaine. Serum ALT and AST were significantly elevated with the combined cocaine and LPS treatment versus all other treatments. While cocaine alone resulted in centrilobular necrosis, the cocaine and LPS combination produced submassive necrosis. The increased hepatic GSH content and GRx activity observed with cocaine alone were not observed with the combination treatment, rendering the liver more susceptible to oxidative stress. Moreover, there was a significant decrease in the activities of hepatic GPx and CAT, particularly with the combination treatment. In conclusion, this study demonstrates that LPS potentiates the hepatotoxicity of cocaine as revealed by an array of biochemical and morphological markers.     

Participation of CYP2A in Cocaine‐Induced Hepatotoxicity in Female Mice

Female ICR mice were treated with cocaine either alone or in combination with one of several cytochrome P450 (CYP) inducers, i.e. phenobarbital, β‐ionone, dexamethasone and β‐naphthoflavone. Cocaine‐induced hepatotoxicity was first observed by pretreatment with phenobarbital, β‐ionone or dexamethasone in accordance with significant elevation of cocaine N‐demethylation, the first step of cocaine bioactivation. The hepatic lesions occured in the periportal region (zone 1) by phenobarbital and β‐ionone and in the perivenular region (zone 3) by dexamethasone. The activities of the enzyme specific for CYP isozyme were determined to elucidate the effects of pretreatment with CYP inducers. β‐Naphthoflavone induced CYP1A and 2B but had no effects on hepatotoxicity by cocaine. On the other hand, β‐ionone enhanced hepatotoxicity without induction of CYP3A. Activities of cocaine N‐demethylase correlated well with CYP2A (r=0.83) and CYP2B (r=0.81). Cocaine N‐demethylation was inhibited particularly by addition of the CYP2A specific inhibitor, 8‐methoxypsoralen. Moreover, pretreatment with 8‐methoxypsoralen produced a marked inhibition of the hepatotoxicity induced by cocaine in phenobarbital‐treated mice. These results suggest that cocaine‐induced hepatotoxicity in female mice was mediated in part by CYP2A, participating in cocaine N‐demethylation.     

Shifting necrosis: butylated hydroxytoluene (BHT) and phenobarbital move cocaine-induced hepatic necrosis across the lobule

Cocaine (60 mg/kg i.p.) caused centrilobular necrosis in the livers of 55% of DBA/2Ha mice. Pretreatment with phenobarbital (PB, 3 x 80 mg/kg i.p.) increased the incidence of necrosis to 70% and shifted this damage to the midzonal region. Pretreatment with butylated hydroxytoluene (BHT, 0.1% in diet) increased the severity of the centrilobular damage and increased the incidence to 90%. Combined treatment with both PB and BHT shifted the site of necrosis to the periportal region of the liver, and induced necrosis in all animals. Microsomal malondialdehyde (MDA) did not reflect the extent of the damage and/or correlate with the site of damage. These results argue against a causal role for lipid peroxidation in the mechanism of cocaine hepatotoxicity, and demonstrate that prior exposure to enzyme-inducing agents can increase sensitivity and dramatically alter the site of cell damage.     

Endotoxin potentiates the hepatotoxicity of cocaine in male mice

Cocaine produces hepatotoxicity by a mechanism that remains undefined but that has been linked to its oxidative metabolism. Endotoxin (lipopolysaccharide, LPS) is also a well-known cause of hepatic damage, where exposure to non-injurious doses of LPS increases the toxicity of certain hepatotoxins. This study was conducted to investigate the possible potentiation of cocaine-mediated hepatotoxicity (CMH) by LPS. Male CF-1 mice were administered oral cocaine hydrochloride for 5 consecutive days at a dose of 20 mg/kg with and without 12 x 10(6) EU LPS/kg given intraperitoneally 4 h after the last cocaine injection. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured as markers of liver injury. Blood and liver glutathione (GSH) levels were determined, as well as the activities of glutathione peroxidase (GPx) and catalase (CAT). In addition, the activity of liver glutathione reductase (GRx) was measured. The results demonstrate that endotoxin potentiated the hepatotoxicity of cocaine. Serum ALT and AST were significantly elevated with the combined cocaine and LPS treatment versus all other treatments. While cocaine alone resulted in centrilobular necrosis, the cocaine and LPS combination produced submassive necrosis. The increased hepatic GSH content and GRx activity observed with cocaine alone were not observed with the combination treatment, rendering the liver more susceptible to oxidative stress. Moreover, there was a significant decrease in the activities of hepatic GPx and CAT, particularly with the combination treatment. In conclusion, this study demonstrates that LPS potentiates the hepatotoxicity of cocaine as revealed by an array of biochemical and morphological markers.     

Participation of CYP2A in cocaine-induced hepatotoxicity in female mice

Female ICR mice were treated with cocaine either alone or in combination with one of several cytochrome P450 (CYP) inducers, i.e. phenobarbital, beta-ionone, dexamethasone and beta-naphthoflavone. Cocaine-induced hepatotoxicity was first observed by pretreatment with phenobarbital, beta-ionone or dexamethasone in accordance with significant elevation of cocaine N-demethylation, the first step of cocaine bioactivation. The hepatic lesions occured in the periportal region (zone 1) by phenobarbital and beta-ionone and in the perivenular region (zone 3) by dexamethasone. The activities of the enzyme specific for CYP isozyme were determined to elucidate the effects of pretreatment with CYP inducers. Beta-naphthoflavone induced CYP1A and 2B but had no effects on hepatotoxicity by cocaine. On the other hand, beta-ionone enhanced hepatotoxicity without induction of CYP3A. Activities of cocaine N-demethylase correlated well with CYP2A (r=0.83) and CYP2B (r=0.81). Cocaine N-demethylation was inhibited particularly by addition of the CYP2A specific inhibitor, 8-methoxypsoralen. Moreover, pretreatment with 8-methoxypsoralen produced a marked inhibition of the hepatotoxicity induced by cocaine in phenobarbital-treated mice. These results suggest that cocaine-induced hepatotoxicity in female mice was mediated in part by CYP2A, participating in cocaine N-demethylation.     

Biomechanisms of cocaine-induced hepatocyte injury mediated by the formation of reactive metabolites

Cocaine is an intrinsic hepatotoxin in laboratory animals, and there is growing evidence that high doses of cocaine can precipitate hepatic necrosis in humans. The rodent model of cocaine hepatotoxicity is commensurate with the concept that a multistep, mainly cytochrome P-450 dependentN-oxidative pathway is responsible for the expression of hepatocellular injury. Among the possible biomechanisms by which cocaine exerts its cytotoxic effects, direct oxidative damage by reactive oxygen species generated by redox cycling during the metabolic cascade seems most important. The role of the ensuing lipid peroxidation and protein thiol oxidatioin is less clear. Similarly, the functional role of irreversible (covalent) binding of a not yet defined electrophilic cocaine intermediate to hepatocellular proteins remains enigmatic so long as the critical molecular targets have not been identified. Finally, glutathione plays a pivotal protective role against cocaine-induced hepatic injury. Interactions with ethanol or inducers of the expression of the cytochrome P-450IIB subfamily can potentiate cocaine hepatotoxicity. Thus, the net amount of the ultimate reactive species seems to determine the severity of the hepatic lesions and to be responsible for the marked interspecies, interstrain, and sex differences. Recent advances in culture techniques of hepatocytes and precision-cut liver slices from various species including man have made it possible to correlate cocaine biotransformation with cytotoxicity and to selectively study the putative cellular mechanisms. Clearly, more studies are necessary to further illuminate our understanding of the role of the biochemical and molecular events precipitating hepatic necrosis during cocaine-mediated hepatotoxicity.Dedicated to Professor Dr. Gerhard Zbinden on the occasion of his retirement     

Administration of the calcineurin inhibitor cyclosporine modulates cocaine-induced locomotor activity in rats

RATIONALE: Cocaine administration in rats increases locomotor activity as a result of underlying changes in neurotransmitter dynamics and intracellular signaling. The serine/ threonine phosphatase, calcineurin, is known to modulate several signaling proteins that can influence behavioral responses to cocaine. OBJECTIVE: This study aimed to determine whether calcineurin plays a role in locomotor responses associated with acute and repeated cocaine exposure. Second, we examined cocaine-mediated changes in intracellular signaling to identify potential mechanism underlying the ability of calcineurin to influence cocaine-mediated behavior. METHODS: Locomotor activity was assessed over 17 days in male Sprague-Dawley rats (n = 48) that received daily administration of cocaine (15 mg/kg, s.c.) or saline in the presence or absence of the calcineurin inhibitor, cyclosporine (15 mg/kg, i.p.). Non-cocaine-treated animals from this initial experiment (n = 24) also received an acute cocaine challenge on day 18 of testing. RESULTS: Daily cyclosporine administration potentiated the locomotor response to repeated cocaine 5 min after cocaine injection and attenuated the sustained locomotor response 15 to 40 min after cocaine. Furthermore, cyclosporine pretreatment for 17 days augmented the acute locomotor response to acute cocaine 5 to 30 min after cocaine injection. Finally, repeated exposure to either cocaine or cyclosporine for 22 days increased synapsin I phosphorylation at the calcineurin-sensitive Ser 62/67 site, demonstrating a common downstream target for both calcineurin and cocaine. CONCLUSION: Our results suggest that calcineurin inhibition augments locomotor responses to cocaine and mimics cocaine-mediated phosphorylation of synapsin I.     

Comparative studies of the in vitro metabolism and covalent binding of 14C-benzene by liver slices and microsomal fraction of mouse, rat, and human

Metabolism of benzene by the liver has been suggested to play an important role in the hepatotoxicity of benzene. The role of the different benzene metabolites and the causes of species differences in benzene hepatotoxicity are, however, not known. The metabolism and covalent binding of 14C-benzene by liver microsomal fractions and liver slices from rat, mouse, and human subjects have been studied. Rat microsomal fraction formed phenol at a rate of 0.32 nmol/min/mg of protein; mouse microsomal fraction formed phenol at 0.64 nmol/min/mg and hydroquinone at 0.03 nmol/min/mg; and human microsomal fraction formed phenol at 0.46 nmol/min/mg and hydroquinone at 0.07 nmol/min/mg. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver microsomal protein was 29, 113, and 169 pmol/min/mg of protein, respectively. The rates of metabolite formation from benzene by liver slices in nmol/min/g of tissue were: rat, phenol 0.15, hydroquinone 0.26, and phenylsulfate 1.22; mouse: phenol 0.13, hydroquinone 0.29, phenylsulfate 1.37, and phenylglucuronide 1.34; and human: phenol 0.16, hydroquinone 0.27, phenylsulfate 0.83, and phenylglucuronide 0.52. trans,trans-Muconic acid formation was not detected with liver slices of any species. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver slices was 8.2, 79.7, and 27.3 pmol/min/g liver, respectively. There was no correlation between ascorbic acid levels in the human liver slices and covalent binding of 14C-benzene metabolites. The results show that phenol and hydroquinone found in extrahepatic tissues, including bone marrow, of animals exposed to benzene could originate from the liver. There was no evidence for the release of highly reactive benzene metabolites such as trans,trans-muconaldehyde or p-benzoquinone from liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic determinants of susceptibility to the rewarding and other behavioral actions of cocaine

The role of genotype as a determinant of biologically based inter-individual differences in vulnerability to substance abuse has received little systematic investigation except in the case of alcohol. This report describes the use of an animal model, the inbred mouse, to identify and to characterize variants with inherently altered susceptibilities to the rewarding and other behavioral actions of cocaine. Among a battery of nine inbred strains chosen solely for their genetic diversity, genetic polymorphisms commonly occurred which altered the potency and/or efficacy of cocaine to induce conditioned place preference, oral self-administration, motor activity activation, seizures and lethality. These changes in cocaine sensitivity generally were of a behavior-specific and pharmacodynamic nature. One strain, DBA/2J, found to be markedly hyporesponsive to the rewarding action of cocaine, also was hyporesponsive to the rewarding effects of amphetamine, etonitazene, phencyclidine, caffeine and procaine. We speculate that this strain has an inherent generalized appetitive defect. The frequent occurrence and large magnitude of inherent phenotypic changes in cocaine responsiveness which we have identified among inbred mouse strains now permits an analytical genetic study of processes underlying cocaine-mediated reinforcement.     

Evidence of enhanced in vivo lipid peroxidation after acute cocaine administration

An acute intraperitoneal dose (60 mg/kg) of cocaine to DBA/2Ha male mice results in enhanced lipid peroxidation in vivo, as measured by an increase in conjugated diene absorption in hepatic microsomal lipids. The initiation of this lipid peroxidation is an early consequence of cocaine administration; as early as 1 h after cocaine, peroxidized lipids are significantly greater in treated animals than in controls. This cocaine-induced lipid peroxidation remains at a maximal level from 2 to 4 h and returns approximately to control levels by 8 h. The metabolites of cocaine also produce lipid peroxidation in vitro. Liver microsomes from phenobarbital-treated DBA/2Ha male mice, incubated aerobically in the presence of NADPH, cocaine or the cocaine oxidative metabolites, norcocaine and norcocaine nitroxide, induced lipid peroxidation as measured by an increase in the production of thiobarbituric acid (TBA)-reactive products. The extent of lipid peroxidation is greater for the oxidative metabolites of cocaine than for cocaine itself.     

Valproic acid hepatotoxicity in human liver slices.

Precision cut human liver slices were incubated in organ culture with valproic acid (VPA) to identify patterns of sensitivity to VPA-induced hepatotoxicity. The slices were incubated in Krebs-HEPES buffer supplemented with 25mM glucose and 84 micrograms/ml gentamycin. At 2, 4, 6, 12, 18 and 24 hr slices were taken and analyzed for K+ retention, synthesis of protein and LDH leakage. All three of these viability indicators showed that certain human livers were more susceptible to VPA-induced hepatotoxicity than others. In the limited group of human livers investigated (n = 9) we found one to be particularly sensitive and two relatively insensitive to VPA toxicity. The remaining tissues were of intermediate sensitivity towards VPA. At this time there is no correlation between the human livers that were susceptible to VPA induced hepatotoxicity and age or sex. This study was designed to show that VPA does induce hepatotoxicity in vitro at therapeutically relevant concentrations. Future studies will show whether VPA hepatotoxicity correlates with VPA metabolism, nutritional status or concomitant therapy.     

Modulation of cocaine metabolism in primary rat hepatocyte cultures: effects on irreversible binding and protein biosynthesis

To study mechanisms of cocaine-induced hepatotoxicity, short-term-cultured rat hepatocytes were exposed to cocaine or norcocaine at 10(-6) to 10(-4) M. Induction in vivo (with Aroclor 1254) or inhibition in vitro (with SKF-525A) of cytochrome P450 modulated the rate of oxidative biotransformation of cocaine to norcocaine and to other metabolites in vitro. The quantitative changes in the metabolic conversion of cocaine were paralleled by the amount of radiolabeled cocaine equivalents irreversibly bound to hepatocellular protein. Furthermore, induction of cytochrome P450-mediated cocaine or norcocaine metabolism was associated with inhibition of protein biosynthesis in cultured hepatocytes, whereas this effect was restored to normal when the oxidative metabolism was blocked by SKF-525A. Glutathione depletion with buthionine sulfoximine both increased the covalent binding of cocaine to hepatic macromolecules and augmented the inhibitory effect on protein biosynthesis. The integrity of the hepatocellular plasma membrane was not affected (no effect on lactate dehydrogenase leakage). The results indicate that in rat hepatocytes (a) a high proportion of intracellular cocaine is converted to a reactive metabolite which irreversibly binds to protein, and (b) irreversible binding of cocaine to hepatic protein is associated with impairment of hepatocellular function and could play a role in cocaine-mediated hepatotoxicity.     

Genetic Determinants of Suceptability to the Rewarding and Other Behavioral Actions of Cocaine

The role of genotype as a determinant of biologically based inter-individual differences in vulnerability to substance abuse has received little systematic investigation except in the case of alcohol. This report describes the use of an animal model, the inbred mouse, to identify and to characterize variants with inherently altered susceptibilities to the rewarding and other behavioral actions of their genetic diversity, genetic polymorphisc commonly occurred which altered the potency and/or efficacy of cocaine to induce conditioned place preference, oral self-administration, motor activity activation, seizures and lethality. These changes in cocaine sensitivity generally were of a behavior-specific and pharmacodynamic nature. One strain, DBA/2J, found to be markedly hyporesponsive to the rewarding action of cocaine, also was hyporesonsive to the rewarding effects of amphetamine, etonitazene, phencyclidine, caffeine and procaine. We speculate that this strain has an inherent generalized appetitive defect. The frequent occurence and large magnitude of inherent phenotypic changes in cocaine responsiveness which we have identified phenotypic changes in cocaine responsiveness which we have identified among inbred mouse strains now permits an analytical genetic study of prcoesses underlying cocaine-mediated reinforcement.     

Cocaine toxicity in cultured rat hepatocytes

Cocaine hydrochloride was added to primary cultures of hepatocytes isolated from naive and phenobarbital-induced (80 mg/kg i.p. for 3 d) Sprague-Dawley rats. Cocaine was cytotoxic, as measured by lactate dehydrogenase release, to cells from naive rats in concentrations of 1 mM or greater. Phenobarbital induction greatly increased the cytotoxic potency of cocaine in vitro, with nearly complete loss of cell viability at cocaine concentrations in culture as low as 0.01 mM. The addition of 10 μM SK&F-525-A to the cultures blocked cocaine cytotoxicity in cells from both naive and phenobarbital-induced rats. These results suggest that the metabolic pathways leading to cocaine hepatotoxicity identified in the mouse also exist in the rat hepatocyte.