The in vitro biotransformation of hexachlorobenzene in relation to its toxicity

Hexachlorobenzene (HCB) has become a major environmental pollutant due to its formation as an unwanted byproduct in the industrial production of a number of chlorinated compounds, and because of its former use as fungicide. In laboratory animals, HCB induces tumor formation. In man and animals, HCB disturbes the hepatic heme biosynthesis, resulting in massive excretion of porphyrins. This porphyrinogenic action of HCB was shown to be due to a selective inhibition of the enzyme uroporphyrinogen decarboxylase. Evidence has been presented for the involvement of cytochrome P-450 in the porphyrinogenic action of HCB: in vivo induction of this enzyme by phenobarbital increased, and inhibition with piperonyl butoxide decreased the amount of excreted porphyrins. This led to the assumption that the inactivation of uroporhyrinogen decarboxylase could be attributed to a metabolite or a reactive intermediate formed during the oxidative biotransformation of HCB. The aim of the present study was to investigate the in vitro oxidative biotransformation of HCB, with special attention to the formation of reactive intermediates. In doing so, a more balanced evaluation could be made regarding the involvement of biotransformation in the toxic action of HCB.As tools in the present study of the biotransformation of HCB, use has been made of, on the one hand, rat liver microsomes (particles of the endoplasmatic reticulum, which contain the cytochrome P-450 complex) and purified cytochrome P-450 isoenzymes, and on the other hand, a primary culture of chick embryo hepatocytes. Throughout the investigations, radiolabeled HCB and metabolites have been applied. This proved to be of great advantage in the tracing and quantification of very small amounts of products.Evidence was obtained that rat liver microsomes were able to hydroxylate HCB. Pentachlorophenol (PCP) was detected in very small amounts (10 - 350 pmoles, depending on the type of microsomes used, in a 30 minute incubation of 50 nmoles of HCB). The hydroxylation of HCB appeared to be cytochrome P-450 dependent, as saturation of the microsomes with carbon monoxide, an inactivator of cytochrome P- 450 almost completely inhibited the reaction. The formation of PCP amounted to 80-90% of the total metabolite formation. As a minor metabolite, tetrachloro-1,4-hydroquinone (1,4-TCHQ) was identified. The apparent Km-values for the formation of PCP and 1,4-TCHQ were both determined to be 34 μM, with Vmax-values of 24 pmoles PCP/min/mg protein and 1.9 pmole 1,4-TCHQ/min/mg protein. Using 18O-labeled H 2 O, the origin of the oxygen atoms incorporated in PCP and 1,4-THCQ was determined to be molecular oxygen, indicating a sequential hydroxylation by cytochrome P-450. Furthermore, a small amount (5- 10% of all metabolites) was detected to be covalently bound to microsomal protein. Since this binding was metabolism-dependent, attention became focussed on the identity of the metabolite or reactive intermediate involved.Reductive dechlorination of HCB, resulting from a one electron reduction of HCB by cytochrome P-450, and giving rise to a pentachlorophenyl radical, was proven not to be involved in the process of covalent binding. Under anaerobic conditions, which in general stimulate reductive dehalogenation, the enzymatic hydroxylation was greatly reduced, while covalent binding also disappeared. Pentachlorobenzene, as a product of reductive dehalogenation, could not be detected.Covalent binding to microsomal protein was also detected as a result of microsomal PCP-hydroxylation. If PCP was incubated at concentrations comparable to those formed in HCB-incubations, the amount of covalent binding was similar to the amount detected after HCB-incubations. This indicated that the covalent binding resulting from the microsomal conversion of HCB was due to a reaction product of PCP, formed in these incubations. Because no covalent binding could be attributed to the hydroxylation of HCB to PCP, the formation of reactive species during this reaction is improbable.The covalent binding as a result of microsomal conversion of HCB could be prevented by addition of ascorbic acid to the incubation mixtures. The disappearance of covalent binding was accompanied by a proportional increase in the amount of TCHQ formed. Glutathione also inhibited covalent binding, but no increase in formation of 1,4-TCHQ was observed. These results led to the conclusion that the benzoquinone or semiquinone forms of the tetrachlorohydroquinones are the species involved in covalent binding to protein. Ascorbic acid prevents the oxidation of hydroquinones, while glutathione reacts with the benzoquinone, forming conjugates. The reaction of glutathione with tetrachloro-1,4-benzoquinone was studied chemically, and evidence was obtained for the formation of mono-, di- and tri-substituted conjugates, still In the oxidized form.Microsomal hydroxylation of PCP resulted, in addition to covalent binding, in the formation of tetrachloro-1,4-hydroquinone and tetrachloro-1,2-hydroquinone. The apparent Km-values for the formation of these hydroquinones was 13 μM, a value also measured for the formation of covalently bound products in PCP-incubations. Ascorbic acid influenced the covalent binding in the same way as observed in HCB-incubations. A conversion-dependent covalent binding to DNA was observed in incubations with DNA, which was 0.2 times the amount of binding to protein.Using liver microsomes from rats treated with different inducers of cytochrome P-450, not only different rates of conversion of HCR and PCP were measured, but indications were also obtained for a preferential formation of either of the two isomers of the hydroquinone by different isoenzymes of cytochrome P-450. This matter has been studied in more detail, using purified cytochrome P-450 isoenzymes and monoclonal antibodies against cytochrome P-450b+e and cytochrome P-450c. it appeared that at least three different isoenzymes are Involved In the hydroxylation of HCB. Purified cytochromes P-450b and P-450e exhibited a (very low) catalytic activity towards HCB, while selective inhibition of these isoenzymes in microsomes also resulted in a moderate decrease in formation of PCP. Moreover, microsomes from rats treated with dexamethasone, which induces cytochrome P-450p, displayed a 4.4 times higher rate of hydroxylation than microsomes treated with phenobarbital, which induces cytochromes P-450b and P-450e. It seems likely that P-450p is the major cytochrome P-450 isoenzyme involved in the hydroxylation of HCB. In a reconstituted system, PCP was converted to the hydroquinones by purified cytochromes P-450c and P-450d, and to a much lesser extent by cytochromes P-450b and P-450e. The first two isoenzymes mentioned preferentialy produced the 1,4-diol, while the latter two form more 1,2-TCHQ than 1,4-THCQ. Selective inhibition of the catalytic activity of P-450c in microsomes from rats treated with 3-methylcholanthrene (a potent inducer of cytochrome P-450c), and inhibition of P-450b+e in microsomes from phenobarbital treated rats did not result in a decrease in formation of diols, indicating that these isoenzymes were not involved in the microsomal hydroxylation. Microsomes from dexamethasone induced rats again were the most potent in converting PCP, indicating the importance of cytochrome P-450p. These microsomes almost exclusively produce the 1,4-diol.A high reactivity of tetrachloro-1,4-benzoqui none towards protein was detected. The oxidation of tetrachloro-1,4-hydroquinone to its benzoquinone was studied. Purified cytochrome P-450b appeared to be able to catalyze this reaction. However, in microsomes, cytochrome P-450 is not the only enzyme Involved: carbon monoxide only partially inhibited the oxidation, as measured by covalent binding to microsomal protein, while even under anearobic conditions binding was still 39% of the amount Found under aerobic conditions. Superoxide dismutase inhibited covalent binding in microsomes to almost the same extent. while oxidation by purified cytochrome P- 450 was completely inhibited. This indicated that all oxygen- mediated oxidation of 1,4-TCHQ can be ascribed to the superoxide anion radical. However, covalent binding as a result of microsomal hydroxylation of PCP was not influenced by superoxide dismutase. This might indicate that TCHQ, as formed from PCP, is oxidized in the active site of cytochrome P-450, by superoxide anion radicals generated by this enzyme. The finding that 1,4-TCHQ stimulated the oxidase activity of cytochrome P-450 supports this hypothesis.In order to obtain more insight In the biotransformation of HCB and the cellular protective mechanisms against alkylation damage, the metabolic route of HCB leading to the formation of the metabolites reacting with protein has been studied in a primary culture of chick embryo hepatocytes, using radiolabeled HCB, PCP and 1,4-TCHQ. Although covalent binding as a result of biotransformation of these compounds was detected, the relative amount was lower than found during microsomal incubations. It is clear that a number of mechanisms are available to protect against this binding. The hydroxylation of HCB and PCP in these cultures resembled the microsomal hydroxylation the same inducers were effective, and PCP was the only extractable product detected. However, biotransformation of PCP did not result in accumulation of diols. Instead, a number of conjugation reactions prevented covalent binding by either a reaction with PCP or with the benzoquinones. Incubations with 1,4-TCHQ supported these findings: although a high degree of biotransformation was measured, only a relatively small amount of covalent binding was detected.In view of these results, it is unlikely that covalent binding to protein, as caused by the oxidative biotransformation of HCB, is of major importance in vivo. A direct alkylation of uroporphyrinogen decarboxylase by TCBQ is most probably not involved in the process of HCB-induced porphyria. However, a relation between covalent binding of the tetrachlorobenzoquinones and the carcinogenicity of HCB and the mutagenicity of PCP may exist