C. den Besten

The liver, kidney, and thyroid toxicity of chlorinated benzenes.

The acute toxicity of a number of chlorinated benzenes, ranging from monosubstituted to pentasubstituted benzenes, was studied in rats. Toxic effects on the liver, the kidneys, and the thyroid were monitored after a single ip administration of 1, 2, or 4 mmol/kg monochlorobenzene (MCB), 1,2-dichlorobenzene (1,2-DICB), 1,4-dichlorobenzene (1,4-DICB), 1,2,4-trichlorobenzene (1,2,4-TRCB), and pentachlorobenzene (PECB). Due to its low solubility, 1,2,4,5-tetrachlorobenzene (1,2,4,5-TECB) was tested at a highest dose of 0.8 mmol/kg. 1,2-DICB and 1,2,4-TRCB produced the most severe hepatotoxic effects when compared with an equimolar dose of the other chlorinated benzenes, as determined by plasma ALT profile and histopathological changes after 72 hr. MCB was considerably less hepatotoxic. Severe degenerative damage to the kidney was only observed in a few rats treated with 1,2,4-TRCB. However, protein droplets in the tubular epithelial cells were observed at 72 hr after administration of 1,4-DICB, 1,2,4-TRCB, 1,2,4,5-TECB, and PECB. In the latter two groups, these protein droplets were still observed 9 days after administration. All chlorinated benzenes tested excluding MCB induced a reduction in plasma thyroxine levels. The extent of decrease in plasma thyroxine was more severe in rats treated with 1,2,4-TRCB or PECB and correlated well with the relative binding affinities of the phenolic metabolites to the plasma transport protein for thyroxine, i.e., transthyretin. The present study indicates that the establishment of a structure-activity relationship with regard to toxicity depends on the sensitivity of the respective target organs. In the series of (poly)chlorinated benzenes studied, ranging from mono- to pentachlorobenzene, the most severe effects on liver, kidney, and thyroid were observed for 1,2,4-substitution.

The involvement of primary and secondary metabolism in the covalent binding of 1,2- and 1,4-dichlorobenzenes

The microsomal oxidation of 1,2-[14C]- and 1,4-[14C]dichlorobenzene (DICB) was investigated with special attention for possible differences in biotransformation that might contribute to the isomer-specific hepatotoxicity. Major metabolites of both isomers were dichlorophenols (2,5-DICP for 1,4-DICB and 2,3- and 3,4-DICP for 1,2-DICB, respectively) and dichlorohydroquinones. The formation of polar dihydrodiols appeared to be a major route for 1,2-DICB but not 1,4-DICB. Both the hepatotoxic 1,2-DICB and the non-hepatotoxic 1,4-DICB were oxidized to metabolites that covalently interacted with protein and only to a small extent with DNA. Protein binding could be inhibited by the addition of the reducing agent ascorbic acid with a concomitant increase in the formation of hydroquinones and catechols, indicating the involvement of reactive benzoquinone metabolites in protein binding. However, in the presence of ascorbic acid, a substantial amount of protein-bound metabolites of 1,2-DICB was still observed, in contrast to 1,4-DICB where binding was nearly completely inhibited. This latter effect was ascribed to the direct formation of reactive benzoquinone metabolites in a single P450-mediated oxidation of para-substituted dichlorophenols (such as 3,4-DICP) in the case of 1,2-DICB. In contrast, the major phenol isomer derived from 1,4-DICB (i.e. 2,5-DICP) is oxidized to its hydroquinone derivative, which needs prior oxidation in order to generate the reactive benzoquinone species. Residual protein binding in the presence of ascorbic acid could also indicate the involvement of reactive arene oxides in the protein binding of 1,2-DICB, but not of 1,4-DICB. However, MO computer calculations did not provide indications for differences in chemical reactivity and/or stability of the various arene oxide/oxepin tautomers that can be formed from either 1,2-DICB or 1,4-DICB. In conclusion, reactive intermediates in the secondary metabolism of 1,2-DICB lead to more covalent binding than those derived from 1,4-DICB, which correlates very well with their reported hepatotoxic potency.

Biotransformation and toxicity of halogenated benzenes

1 Multiple potentially harmful metabolites can be distinguished in the metabolic activation of halogenated benzenes: epoxides, phenols, benzoquinones and benzoquinone-derived glutathione conjugates. 2 The role of these (re-) active metabolites in the toxic effects induced by halogenated benzenes such as hepatotoxicity, nephrotoxicity, porphyria and thyroid toxicity is discussed. 3 Evidence is presented suggesting that the formation of reactive benzoquinone metabolites rather than the traditional epoxides is linked to halogenated benzene-induced hepatotoxicity. 4 A crucial role for the benzoquinone-derived glutathione adducts in halogenated benzene-induced nephrotoxicity is clearly established. 5 Although metabolic activation appears to be involved in porphyria, the nature of the ultimate porphyrinogenic metabolite has not been elucidated yet. 6 Disturbances in thyroid hormone (and retinoid) homeostasis can be (at least partially) explained by the formation of halogenated phenol metabolites. 7 In conclusion, for a relevant prediction of the ultimate fate of a compound in a living organism, one should know the chemical characteristics and reactivity of the parent compound and its metabolites, together with insight into the formation mechanism of each of the suspected metabolites, and an understanding of the interaction between a specific chemical (reactive) structure and its target molecule.

The metabolism of pentachlorobenzene by rat liver microsomes: The nature of the reactive intermediates formed

Metabolism of [14C]-pentachlorobenzene by liver microsomes from dexamethasone-induced rats results in the formation of pentachlorophenol and 2,3,4,6-tetrachlorophenol as major primary metabolites in a ratio of 4:1, with 2,3,4,5- and 2,3,5,6-tetrachlorophenols as minor metabolites. The unsubstituted carbon atom is thus the favourite site of oxidative attack, but the chlorine substituted positions still play a sizable role. As secondary metabolites both para- and ortho-tetrachlorohydroquinone are formed (1.4 and 0.9% of total metabolites respectively). During this cytochrome P450-dependent conversion of pentachlorobenzene, 5-15% of the total amount of metabolites becomes covalently bound to microsomal protein. Ascorbic acid inhibits this binding to a considerable extent, indicating that quinone metabolites play an important role in the binding. However, complete inhibition was never reached by ascorbic acid, nor by glutathione, suggesting that other reactive intermediates, presumably epoxides, are also responsible for covalent binding.

Comparison of the urinary metabolite profiles of hexachlorobenzene and pentachlorobenzene in the rat

The urinary metabolite profile of hexachlorobenzene (HCB) and pentachlorobenzene (PCBz) in the rat is compared after dietary exposure for 13 weeks. Both HCB and PCBz are oxidized to pentachlorophenol (PCP) and tetrachlorohydroquinone (TCHQ), which were the only two mutual metabolites formed. Additional urinary metabolites of HCB are N-acetyl-S(pentachlorophenyl)cysteine (PCTP-NAC), which appeared to be quantitatively the most important product, and mercaptotetrachlorothioanisole (MTCTA), which was excreted as a glucuronide. PCBz is more extensively metabolized to the major metabolites 2,3,4,5-tetrachlorophenol (TCP), mercaptotetrachlorophenol (MTCP) and the glucuronide of pentachlorothiophenol (PCTP), and the minor metabolites methylthiotetrachlorophenol (MeTTCP), hydroxytetrachlorophenyl sulphoxide (HTCPS), and bis(methylthio)-trichlorophenol (bis-MeTTriCP). The biotransformation of HCB and PCBz was modulated by selective inhibition of cytochrome P450IIIA in rats which received combined treatment of HCB or PCBz with triacetyloleandomycin (TAO). Rats receiving this diet had a strongly diminished excretion of both PCP and TCHQ, as compared to rats fed HCB or PCBz alone, indicating the involvement of P450IIIA in the oxidation of both compounds. However, the excretion of 2,3,4,5-TCP was not diminished by co-treatment of rats with PCBz and TAO, indicating that: (i) the oxidation of PCBz to PCP and 2,3,4,5-TCP does not proceed via a common intermediate; and (ii) oxidation of PCBz to 2,3,4,5-TCP is not mediated by P450IIIA. Co-treatment of rats with PCBz and TAO had a differential effect on the excretion of sulphur-containing metabolites, resulting in a decrease in the excretion of PCTP glucuronide, whereas no change was observed in the excretion of MTCP, as compared to rats receiving PCBz alone. The observed differences in HCB and PCBz metabolites clearly deserve further in vitro studies to elucidate their origin.

Comparative biotransformation of hexachlorobenzene and hexafluorobenzene in relation to the induction of porphyria

The porphyrinogenic action of hexafluorobenzene was investigated and compared to that of hexachlorobenzene. Metabolite patterns in the urine of exposed rats were determined to quantify the extent of metabolism through cytochrome P450 catalysed oxidation and glutathione conjugation. Results obtained demonstrate an almost similar extent of formation of phenolic metabolites. However, in the urine of hexachlorobenzene exposed rats significantly higher levels of the N-acetyl-S-(pentahalophenyl)cysteine were observed than in the urine of hexafluorobenzene exposed rats. Hexafluorobenzene exposure did not result in induction of porphyria, whereas exposure to hexachlorobenzene did result in significantly elevated levels of urinary as well as liver porphyrins. Together these results indicate that if the reactive intermediate is indeed formed in the cytochrome P450 catalysed initial oxidative dehalogenation, the extent of its formation as well as its subsequent reactivity and reaction pathways vary with the type of the halogen substituents. Furthermore, the results seem to indicate that the extent of metabolism of hexahalogenated benzenes into urinary metabolites resulting from glutathione conjugation is a better indication of their porphyrinogenic action than their extent of metabolism to phenolic metabolites. Two explanations for this observation are presented.