Interindividual variation in biotransformation and cytotoxicity of bromobenzene as determined in primary hepatocyte cultures derived from monkey and human liver.

1. Bromobenzene-evoked hepatotoxicity resulting from cytochrome P450-mediated epoxidation has been studied extensively in rodents in vivo and in rodent hepatocytes. In this paper we present data concerning the formation of bromphenols, glutathione (GSH) depletion and cytotoxicity observed in primate hepatocytes in primary culture after exposure to bromobenzene (BrB). 2. After pre-incubation for 2 or 24 h, hepatocytes were exposed to BrB in concentrations up to 2 mM for 4 or 24 h. 3. In both human and cynomolgus monkey hepatocytes BrB cytotoxicity and GSH depletion were found after exposure to 2 mM BrB. The degree of the observed effects was not influenced by the duration of pre-incubation and/or exposure periods. 4. Major inter-individual differences were observed, which could not be attributed to differences in cytochrome P450-mediated bioactivation rates. This suggests that the variation in individual susceptibility to BrB may be related to inter-individual differences in the activity of de-activating (metabolic) pathways. 5. The study of the background of these inter-individual differences may contribute to a more complete understanding of the factors ruling sensitivity to BrB or related chemicals.

Effect of alkylating and redox cycling quinones on insulin receptor autophosphorylation.

Autophosphorylation is thought to be an essential step in the insulin receptor signal transduction cascade. Previous studies have shown that thiol alkylation of the receptor can block this receptor autophosphorylation, whereas an oxidative environment can increase this process. Since the toxicity of quinones can be related to two mechanisms-redox cycling resulting in oxidative stress, and arylation of cellular nucleophilic groups-the effects of 1,4-naphthoquinone and menadione on insulin receptor autophosphorylation were investigated. The results show that these two quinones have a dual effect: lower concentrations leading to oxidative stress increase insulin receptor autophosphorylation, whereas higher concentrations cause a thiol depletion and inhibit the normal insulin receptor autophosphorylation.

Biotransformation of scoparone used to monitor changes in cytochrome P450 activities in primary hepatocyte cultures derived from rats, hamsters and monkeys.

The coumarin derivative scoparone is regioselectively demethylated yielding isoscopoletin and scopoletin. The ratio of the formation rates of these two metabolites (isoscopoletin/scopoletin; I/S ratio) is reported to mirror the contribution of several cytochrome P450 (P450) isoenzymes to the biotransformation of scoparine. The metabolism of scoparine has been studied in primary liver cell cultures of rats, hamsters, cynomolgus monkeys and in human liver cells. Rat hepatocytes appeared to metabolize scoparone 7 to 10 times slower than those of hamsters and monkeys. In hepatocyte monolayers of all three species the loss of P450 was paralleled by a decrease in total scoparone metabolism. In hamsters but not in rats, a decrease of the I/S ratio was found during primary culture of liver cells. A similar shift in the metabolic pattern of scoparine observed with the monkey hepatocytes was statistically not significant. Most likely, in hamster and possibly in monkey hepatocyte cultures the different P450s involved in scoparone metabolism decrease at unequal rates. In rat liver cells, however, the pattern of these P450 isoenzymes remains more or less unaltered. In contrast to liver cells from the other species, human hepatocytes did not secrete scopoletin in detectable amounts. Scoparone demethylation in humans may be qualitatively different from that in other mammals.

Comparative cytotoxicity of bromobenzene in primary cultures of rat and hamster hepatocytes and its relation to biotransformation.

Primary cultures of rat and hamster hepatocytes were exposed to 0.5-2 mm-bromobenzene (BrB). Exposure to 2 mm-BrB within 2 hr after isolation caused cellular damage to hepatocytes from both species. Exposure to BrB also resulted in reduced glutathione (GSH) levels. In hamster hepatocytes, GSH was depleted by 60-85% with 0.5 mm-BrB, whereas in rat hepatocytes 2 mm caused a GSH depletion of about 50%. Liver cell cultures from both species converted BrB into three bromophenols. Hamster hepatocytes and microsomes appeared to metabolize BrB more rapidly than those from rat liver. However, in hamster hepatocytes at BrB concentrations of less than 2 mm no cytotoxicity could be detected, despite a rapid biotransformation of BrB and a vast depletion of glutathione; both factors are thought to be prerequisites for BrB-induced hepatotoxicity. The results of this comparative in vitro study emphasize the importance of detoxification mechanisms other than conjugation to GSH, such as a high epoxide hydrolase activity, in the determination of sensitivity to BrB.

The effect of beclobric acid and clofibric acid on peroxisomal beta-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes.

The peroxisome-proliferating effects of clofibric acid and beclobric acid were studied in primary cultures of hepatocytes derived from rat, monkey (Macaca fascicularis) and human liver. Determination of peroxisomal fatty acid beta-oxidation and morphometrical analysis of the peroxisomal compartment were performed after incubation of 1-day-old hepatocyte cultures for 3 days with either compound. In rat liver cell cultures both compounds gave a 10-fold increase in peroxisomal beta-oxidation, a 3-fold increase in the relative number of peroxisomes and a 1.5-fold increase in the mean size of peroxisomes. Beclobric acid gave its maximal effect at a concentration of 10 microM, which is at least one order of magnitude lower than the maximum-effect concentration of clofibric acid. At concentrations greater than 300 microM beclobric acid was cytotoxic. No stimulation of peroxisomal fatty acid beta-oxidation was found in either monkey or human hepatocyte cultures. Morphometrical analysis also showed no increase in the peroxisomal compartment in cultures derived from these species, as indicated by the lack of increase in both relative number and size of peroxisomes. In all three species tested beclobric acid was equally cytotoxic for hepatocytes in vitro. These results are of relevance for the interpretation of the peroxisome-proliferating effects of clofibrate and similar compounds in rats. Since peroxisome proliferation may be correlated to increased hepatic tumour incidences in the rat, the absence of peroxisome proliferation in primates suggests the absence of tumourogenic activity by hypolipidemic compounds in these species.

Effects of sulphydryl reagents on the formation of the aniline metabolite 4-aminophenol and its sulphate and glucuronide conjugates in primary cultures of rat hepatocytes.

1. The effects of various sulphydryl-blocking reagents on aniline biotransformation and cytochrome P-450 levels were studied in cultured rat hepatocytes. 2. Exposure of aniline-metabolizing hepatocytes to p-chloro-mercuribenzoate (PCMB) or p-chloromercuribenzenesulphonic acid (PCMBS) resulted in decreased levels of cytochrome P-450, decreased glucuronidation of 4-aminophenol and increased levels of free 4-aminophenol. 3. Incubation of aniline-metabolizing hepatocytes with disulfiram resulted in decreased formation of 4-aminophenol, but this was not associated with impaired glucuronidation or cytochrome P-450 levels. 4. Exposure of aniline-metabolizing hepatocytes to mersalyl, 2,2'-dithiodipyridine (DTP), 6,6'-carboxydipyridine disulphide (CPDS) or N-ethylmaleimide did not affect the biotransformation of aniline or cytochrome P-450 levels. 5. Metyrapone prevented degradation of cytochrome P-450. Exposure of cells to SKF-525 A inhibited aniline biotransformation without altering cytochrome P-450 levels. 6. PCMB and PCMBS inhibited aniline metabolism, probably by binding to a cysteinyl-SH residue in cytochrome P-450 apoenzyme and 'active sites' of UDP-glucuronyl transferases. Disulfiram inhibited aniline biotransformation, probably indirectly by diminishing NADPH.

The cytotoxicity of halothane in isolated hepatocytes: Evidence for two different mechanisms.

The effect of halothane on the functional properties of isolated hepatocytes was investigated. Glycogen levels and the oxygen consumption of hepatocytes, isolated from both control and phenobarbital-induced rats, decreased dose-dependently after exposure to halothane, irrespective of the oxygen concentration of the incubation (5, 12 or 95% oxygen and 5% CO(2)). Halothane did not affect albumin synthesis in hepatocytes, but there was a loss of albumin from cells isolated from control rats, after exposure to the anaesthetic. Hepatocytes from phenobarbital-induced rats, incubated under hypoxic conditions, showed an increased production of ethane and thiobarbituric acid reactants, indicating lipid peroxidation. The cytochrome P-450 contents of the hepatocytes from phenobarbital-induced rats were decreased by halothane. All these effects of halothane were detected without observing any effects on the integrity of the cells, as determined by the leakage of lactate dehydrogenase.

Formation and disposition of N-hydroxylated metabolites of aniline and nitrobenzene by isolated rat hepatocytes.

The formation and secretion of phenylhydroxylamine plus nitrosobenzene was studied in cultures of isolated rat hepatocytes after addition of aniline or nitrobenzene. With aniline concn. up to 10 mM, N-oxygenated metabolites were secreted linearly with time over 2 h. Phenobarbitone pretreatment in vivo for c. 5 d increased aniline N-hydroxylation by a factor of 2.8. Nitrobenzene reduction by isolated rat hepatocytes, yielding phenylhydroxylamine plus nitrosobenzene in the medium, was stimulated 1.9-fold and 4.3-fold after phenobarbitone pretreatment in vivo for 5 and 10 d, respectively. After reduction of nitrobenzene by isolated hepatocytes, the secretion of N-oxygenated products into the medium was non-linear with time for substrate concn. higher than 2.5 mM, probably due to the formation of cytotoxic concn. of nitrosobenzene. Isolated rat hepatocytes reduced phenylhydroxylamine to aniline. Results indicate that isolated rat hepatocytes are a reliable and sensitive system to demonstrate N-oxygenated metabolites of aromatic amino- and reduction of nitrocompounds.

Effects of phenylhydroxylamine and aminophenols in Japanese quail in vivo.

1. The effects of phenylhydroxylamine, and o- and p-aminophenol were studied in the Japanese quail. 2. Symptoms normally observed in aniline-treated birds were seen in quail after phenylhydroxylamine dosage at > 10 mg/kg. Aminophenols (up to 50 mg/kg) did not give these symptoms. 3. Injection of phenylhydroxylamine (50 mg/kg) resulted in formation of 70% ferrihaemoglobin after 5 min, following which a rapid reduction of ferrihaemoglobin was observed. 4. Phenylhydroxylamine reached highest blood concn. of 0.2 mumol/ml after 5 min. Phenylhydroxylamine was reduced to aniline within 5 min. 5. The effects of aniline in vivo are most probably due to O2 shortage caused by ferrihaemoglobin formation.

Ferrihemoglobin formation by monohydroxy aniline derivatives in erythrocytes of some avian species in comparison with mammals.

Ferrihemoglobin formation nitrosobenzene and p-, m- and o-animophenol was studied in erythrocyte supspensions from a number of avian and mammalian species. Compared with the mammalian erythrocytes, bird red cells show little species differences in susceptibility. In birds, dose-effect relationship are different for nitrosobenzene and the aminophenols: using low concentration nitrosobenzene is more active than the aminophenols, while at higher concentrations o-aminophenol is at least equally effective. In birds, o-aminophenol is about eight times more potent than p-aminophenol. m-Anminophenol is not an inducer of ferrihemoglobin in erythrocytes of the cat, the goat and the Japanese quail.

Nitrosobenzene-induced ferrihemoglobin formation in Japanese quail erythrocytes. The significance of ferrihemoglobin reduction.

Quantitative aspects of nitrosobenzene-induced ferrihemoglobin formation were studied in quail erythrocytes in comparison to goat red cells. Many similarities exist with the mechanism in mammalian red cells such as a NADPH-dependent enzymatic recycling of phenylhydroxylamine from nitrosobenzene. Also the mechanism of ferrihemoglobin reduction is similar in both species, it is a NADH-dependent process. However, the reducing activity of quail red cells exceeds by far the activity of the majority of mammalian erythrocytes.