Hisayoshi Ikatsu

Purification and characterization of 2-ethoxyphenol-induced cytochrome P450 from Corynebacterium sp. strain EP1

A soluble cytochrome P450 (P450EP1A) induced by 2-ethoxyphenol was purified to apparent homogeneity from Corynebacterium sp. strain EP1. The P450EP1A showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 45 kDa. The CO-reduced difference spectra of P450EP1A had a Soret maximum at 447.6 nm. The substrate difference spectra with 2-ethoxyphenol showed an absorption maximum at 394.0 nm. The purified P450EP1A degraded 2-ethoxyphenol in an assay system composed of spinach ferredoxin-NADP+ oxidoreductase and NADPH. The reaction activity decreased to 1.4% of its original activity by addition of CO. The existence of catechol in the reaction mixture was confirmed after the metabolic reaction, indicating that P450EP1A catalyzes O-dealkylation of 2-ethoxyphenol. In addition to 2-ethoxyphenol, the P450EP1A metabolized 2-methoxyphenol, 1,1,1-trichloroethane, carbon tetrachloride, benzene, and toluene.

CYP2E1 Level in Rat Liver Injured by the Interaction between Carbon Tetrachloride and Chloroform

CYP2E1 Level in Rat Liver Injured by the Interaction between Carbon Tetrachloride and Chloroform: Hisayoshi Ikatsu, et al. Center for Environmental Science and Technology, Okayama University—The toxic interaction between carbon tetrachloride (CCI4) and chloroform (CHCI3) was investigated in rats chronically pretreated with ethanol, and in rat liver microsomes. A combined exposure to 10 ppm CCI4 and 50 ppm CHCl3 increased plasma alanine aminotransferase (ALT) activity to a level similar to that by a single exposure to 50 ppm CCI4. The single exposure decreased liver reduced glutathione (GSH) and cytochrome P450 (CYP) content, and N‐nitrosodimethylamine N‐demethylase (NDMAD) activity. The effects of combined exposure, however, were little or less than those of the single exposure. Immunoblot analysis with monoclonal antibody to CYP2E1 indicated that CYP2E1 was appreciably decreased after the single exposure but the enzyme remained after the combined exposure. The antibody inhibited CCI4‐induced lipid peroxidation in microsomes from ethanol‐treated rats. Ethanol pretreatment increased the metabolic rate of CCI4 and CCI4‐induced lipid peroxidation, but the coexistence of CHCI3 and other hydrocarbons such as trichloroethylene and 1,1,1‐trichloroethane did not increase the in vitro metabolic rate or the lipid peroxidation. The addition of GSH decreased CCI4‐induced lipid peroxidation, and the effect reached its maximum at 0.3 mM of GSH, so that the hepatotoxic interaction between CCI4 and CHCI3 at low dose exposure is more than additive when judged by the plasma ALT activity, but this is not true of the changes in drug‐metabolizing enzyme activity and GSH depletion.

Guide for Handling of Hazardous Organic Chemicals at Laboratory.

The environmental pollution by hazardous chemicals has been concerned. The discharge of hazardous chemicals into the environment must be prevented since it is difficult to specify the environmental influence by the chemicals. From the viewpoint of the health protection for employees, the handling of a lot of chemicals is regulated by various laws. However, the discharge of chemicals is hardly regulated. The chemicals on which the effluent standard has been already determined are only limited to a part of chemicals used in the laboratory. Even if the chemicals where the effluent standard is not set, the discharge of harmful chemicals must be avoided. In order to prevent the environmental pollution by chemicals, researchers themselves must learn how to manage the handling of used chemicals. Laboratories are, therefore, required to reduce the amounts of chemicals used and chemical wastes discharged. The chemicals should be used safely in the laboratory, in consideration of the environmental conservation and public health. Separate collection of chemical wastes at the generation source is the most important means for the safe treatment of the wastes. The basic principle of the treatment is to treat the wastes properly at the generation source. It is important for the original dischargers to recognize their responsibility as generators of the wastes.