In vitro quantitative determination of phospholipid adducts of chloroform intermediates in hepatic and renal microsomes from different rodent strains

Abstract

We have comparatively studied in vitro the oxidative and reductive pathways of chloroform metabolism in hepatic and renal microsomes of rodent strains used for carcinogenicity testing (B6C3F1 mice, Osborne Mendel and Sprague Dawley rats). To this aim we exploited the regioselective binding of phosgene to phospholipid (PL) polar heads and of dichloromethyl radical to PL fatty acyl chains, using a method based on the chemical transmethylation of PL adducts, followed by phase partitioning of the resulting products (De Biasi et al., 1992). The analysis of results let us to conclude at first that a 14C label partitioning by 89.2 (±6.5)% or 13.7 (±5.0)% in the aqueous phase is typical of the PL adduct with phosgene (PL-PHOS) or with dichloromethyl radical (PL-RAD), respectively. Metabolism of 0.1 mM CHCl 3 was mainly oxidative in all the samples, being hepatic microsomes more active than renal ones by about one order of magnitude and levels of CHCl 3-derived PL adducts in B6C3F1 mouse liver microsomes higher than in rat samples. At 5 mM CHCl 3, total levels of PL adducts in renal microsomes reached levels almost similar to those found in liver microsomes. However, while B6C3F1 mouse kidney microsomes produced both reactive metabolites, similarly as the hepatic samples, Osborne Mendel rat kidney microsomes bioactivated CHCl 3 only reductiveiy, producing the radical. The relevance of this finding depends on the fact that phosgene is known to be the major cause of CHCl 3 toxicity, based on data with the rat liver and mouse liver and kidney, while nephrotoxicity in rats occurs with minimal production of COCl 2. Chloroform reductive bioactivation may therefore provide a reasonable explanation for the toxicity of chloroform to the rat kidney. The same finding may be of interest in elucidating the metabolic reasons of the chloroform-induced kidney tumors in Osborne Mendel rats.