Gas uptake studies of deuterium isotope effects on dichloromethane metabolism in female B6C3F1 mice in vivo.

In common with a diverse group of low-molecular-weight volatile substrates, dichloromethane (DCM; methylene chloride) is a high-affinity, low-capacity substrate for oxidation by several cytochrome P450 isoenzymes in vivo. DCM oxidation, catalyzed primarily by the 2E1 and 2B1 cytochrome P450 isoforms, yields carbon monoxide (CO) and carbon dioxide. We have studied the characteristics of DCM oxidation in vivo by examining the metabolism of DCM and of both deuterated forms ([2H2]-DCM and [2H]DCM) in female B6C3F1 mice with gas uptake methods. Gas uptake and CO production curves were analyzed by physiologically based pharmacokinetic (PBPK) modeling techniques, permitting differentiation of isotope effects on specific metabolic parameters from those associated with blood flow or diffusion limitations in vivo. A marked isotope effect was observed on the moles of CO produced per mole of DCM oxidized (0.76 +/- 0.06, 0.33 +/- 0.006, and 0.31 +/- 0.07, with DCM, [2H]DCM, and [2H2]DCM, respectively). Based on these ratios, the calculated kH/kD ratio for the rate constant of disproportionation of the putative formyl chloride intermediate was about 7, indicating a significant role of C-H bond breaking in this reaction. Deuterium substitution altered the apparent Km for metabolism; there was 14-fold increase in the apparent Km between DCM and [2H2]DCM (6.5 +/- 0.69 to 97 +/- 3.5 microM) with little effect on Km with [2H]DCM (14.4 +/- 0.015 microM). Vmax was not greatly affected by deuteration (151 +/- 1.2, 116 +/- 0.82, and 149 +/- 2.3 mumol/hr/kg with DCM, [2H]DCM, and [2H2]DCM, respectively). Two kinetic mechanisms are discussed, both of which are consistent with these observations. One, a conventional cytochrome P450 mechanism has a rate-limiting product-release step after the isotopically sensitive step; a second, more like a peroxidase mechanism, has a flux-limiting oxygen activation step followed by a second-order reaction between an activated oxygen-enzyme complex and DCM. Regardless of the correct mechanism, the in vivo kinetic constants for oxidation of DCM are complex and represent more than simple rate-limiting bond-breaking (Vmax) and enzyme-substrate binding (Km). Current PBPK models for metabolism of these volatiles may have to be restructured to account for this unusual kinetic mechanism.