Metabolism of chloroform in the human liver and identification of the competent P450s.
The oxidative and reductive cytochrome P450 (P450)-mediated chloroform bioactivation has been investigated in human liver microsomes (HLM), and the role of human P450s have been defined by integrating results from several experimental approaches: cDNA-expressed P450s, selective chemical inhibitors and specific antibodies, correlation studies in a panel of phenotyped HLM. HLM bioactivated CHCl(3) both oxidatively and reductively. Oxidative reaction was characterized by two components, suggesting multiple P450 involvement. The high affinity process was catalyzed by CYP2E1, as clearly indicated by kinetic studies, correlation with chlorzoxazone 6-hydroxylation (r = 0.837; p < 0.001), and inhibition by monoclonal antihuman CYP2E1 and 4-methylpyrazole. The low affinity phase of oxidative metabolism was essentially catalyzed by CYP2A6. This conclusion was supported by the correlation with coumarin 7-hydroxylase (r = 0.777; p < 0.01), inhibition by coumarin and by the specific antibody, in addition to results with heterologously expressed P450s. Chloroform oxidation was poorly dependent on pO(2), whereas the reductive metabolism was highly inhibited by O(2). The production of dichloromethyl radical was significant only at CHCl(3) concentration > or =1 mM, increasing linearly with substrate concentration. CYP2E1 was the primary enzyme involved in the reductive reaction, as univocally indicated by all the different approaches. The reductive pathway seems to be scarcely relevant in the human liver, since it is active only at high substrate concentrations, and in strictly anaerobic conditions. The role of human CYP2E1 in CHCl(3) metabolism at low levels, typical of actual human exposure, provides insight into the molecular basis for eventual difference in susceptibility to chloroform-induced effects due to either genetic, pathophysiological, or environmental factors.
Gemma
Simonetta
S
Biochemical Toxicology Unit, Comparative Toxicology and Ecotoxicology Laboratory, Istituto Superiore di Sanità, Rome, Italy.
Vittozzi
Luciano
L
Testai
Emanuela
E
D001711:Biotransformation; D002461:Cell Line, Transformed; D002725:Chloroform / Q000493:pharmacokinetics*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction
D007527:Isoenzymes; D002725:Chloroform; D003577:Cytochrome P-450 Enzyme System