Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique

When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl₃-derived free radical has been confirmed by using [¹³C]CHCl₃, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl₃) and bromodichloro-methane (CHBrCl₂) suggests that the free radical derived from CHCl₃ may be CHCl₂. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr₃), and iodoform (CHI₃). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl₃ < CHBrCl₂ < CHBr₃ < CHI₃. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihatomethanes might be of relevance to their established toxicity in the whole animal.