Suicidal inactivation of haemoproteins by reductive metabolites of halomethanes: a structure-activity relationship...

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at microM concentrations with 1 mM carbon tetrachloride (CCl4), trichlorobromomethane (CCl3Br), chloroform (CHCl3) or methylene chloride (CH2Cl2) in presence of 1 mM sodium dithionite as the reducing agent. At the end of a 5-min incubation, haem was measured by various methods, i.e. binding spectrum with CO, pyridine-haemochromogen haem assay and porphyrin fluorescence, and compared for the four analogues. Statistically significant losses were observed, with all three haemo-protein systems, for CCi3Br, CCl4 and CHCl3, but not CH2Cl2. For Hb, the loss was greater with CCl3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl4 (haem assay, 31%) or CHCl3 (haem assay, 13%). On the other hand, with MHA, CCl4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl3. Finally, with microsomes, the inactivation was larger with CCl4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl3Br (CO binding, 33%; haem assay, 10%) or CHCl3 (haem assay, 9%; CO binding, 6%). In a separate set of similar experiments, an ion-pairing reverse phase HPLC method showed the formation of substrate-dependent hae-derived products during incubation of CCl3Br with Hb or microsomes, and of CCl4 with Hb. A correlation between potential for free radical formation (CCl3Br > CCl4 > CHCl3 > CH2Cl2) and extent of haem inactivation was observed with all methods for Hb, but not for microsomal P-450 or MHA. The results indicate that these halomethanes may be activated differently by different haemoproteins and suggest that their potential ability to undergo reductive metabolism may not be the only critical factor involved in P-450 haem inactivation by these chemicals.