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

Abstract

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at μM concentrations with 1 mM carbon tetrachloride (CCl 4), trichlorobromomethane (CCl 3Br), chloroform (CHCl 3) or methylene chloride (CH 2Cl 2) 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 haemoprotein systems, for CCl 3Br, CCl 4 and CHCl 3, but not CH 2Cl 2. For Hb, the loss was greater with CCl 3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl 4 (haem assay, 31%) or CHCl 3 (haem assay, 13%). On the other hand, with MHA, CCl 4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl 3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl 3. Finally, with microsomes, the inactivation was larger with CCl 4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl 3Br (CO binding, 33%; haem assay, 10%) or CHCl 3 (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 haemderived products during incubation of CCl 3Br with Hb or microsomes, and of CCl 3 with Hb. A correlation between potential for free radical formation ( CCl 3 Br > CCl 4 > CHCl 3 > CH 2 Cl 2) 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.