ascorbic-acid and 2-3-5-6-tetrachlorohydroquinone

The worldwide distribution and high bioaccumulation potential of pentachlorophenol (PCP) in aquatic organisms imply a high toxicological impact in aquatic systems. Firstly, our investigations show that, similar to mammalian cell lines, PCP can be metabolized to tetrachlorohydroquinone (TCHQ) in the permanent cell line derived from rainbow trout liver cells (RTL-W1). Moreover, we demonstrate that PCP as well as its metabolite TCHQ is capable of influencing the viability of these cells. Three cell viability assays were performed to assess possible cellular targets of these substances. Thus, the cytotoxicity of the PCP-derivative TCHQ was shown for the first time in a fish cell line. Further investigations revealed the involvement of ROS in the cytotoxicity of PCP and its metabolite TCHQ. The observation of oxidative stress provides a plausible explanation for the increased cytotoxicity at higher concentrations especially for PCP and implies possible mechanisms underlying these observations. In addition, antioxidants such as ascorbic acid and quercetin modulate the detrimental effects of PCP and TCHQ whereby both compounds exacerbate the cytotoxic effects of high PCP and TCHQ concentrations.

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Line; Cell Survival; Fish Proteins; Hepatocytes; Hydroquinones; Inhibitory Concentration 50; Oncorhynchus mykiss; Oxidative Stress; Pentachlorophenol; Quercetin; Reactive Oxygen Species; Superoxide Dismutase; Water Pollutants, Chemical

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.

Topics: Animals; Ascorbic Acid; Chloranil; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Glutathione; Hydroquinones; Male; Microsomes, Liver; NAD; NADP; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Oxygen Consumption; Rabbits; Rats

The microsomal metabolism of hexachlorobenzene is studied, with special attention to the covalent binding to protein. The metabolites formed are pentachlorophenol and tetrachlorohydroquinone. In addition, a considerable amount of covalent binding to protein is detected (250 pmoles pentachlorophenol, 17 pmoles tetrachlorohydroquinone and 11 pmoles covalent binding in an incubation containing 50 mumoles of hexachlorobenzene). In order to establish the potential role of reductive dechlorination in the covalent binding, the anaerobic metabolism of hexachlorobenzene was investigated. At low oxygen concentrations no pentachlorobenzene was detected, and only very small amounts of pentachlorophenol as well as covalent binding, indicating a relationship between covalent binding and the microsomal oxidation of hexachlorobenzene. Incubations with 14C-pentachlorophenol at low concentrations showed that a conversion-dependent covalent binding occurs to the extent of 75 pmole binding per nmole pentachlorophenol. This is almost enough to account for the amount of label bound to protein observed in hexachlorobenzene incubations. This indicates that less than 10% of the covalent binding occurs during conversion of hexachlorobenzene to pentachlorophenol, and the remainder is produced during conversion of hexachlorobenzene to pentachlorophenol, and the remainder is produced during conversion of pentachlorophenol. The major product of microsomal oxidation of pentachlorophenol is tetrachlorohydroquinone, which is in redox-equilibrium with the corresponding semiquinone and quinone (chloranil). The covalent binding is inhibited by addition of ascorbic acid or glutathione to the hexachlorobenzene incubations. Ascorbic acid decreases the covalent binding with a simultaneous increase in formation of tetrachlorohydroquinone, probably due to a shift in the redox-equilibrium to the reduced side. Glutathione does not act as a reducing agent, since the inhibition of covalent binding is not accompanied by an increase in tetrachlorohydroquinone formation. Instead, glutathione reacts with chloranil, producing at least three stable products, probably in a Michael-type reaction. These results strongly indicate the involvement of chloranil or the semiquinone radical in the covalent binding during microsomal hexachlorobenzene metabolism.

Topics: Animals; Ascorbic Acid; Chloranil; Chlorobenzenes; Glutathione; Hexachlorobenzene; Hydroquinones; In Vitro Techniques; Male; Microsomes; Pentachlorophenol; Protein Binding; Rats; Rats, Inbred Strains