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 substr ates, dichloromethane (DCM; methylene chloride) is a high-affinity, lo w-capacity substrate for oxidation by several cytochrome P450 isoenzym es in vivo. DCM oxidation, catalyzed primarily by the 2E1 and 2B1 cyto chrome P450 isoforms, yields carbon monoxide (CO) and carbon dioxide. We have studied the characteristics of DCM oxidation in vivo by examin ing the metabolism of DCM and of both deuterated forms ([H-2(2)]DCM an d [H-2]DCM) in female B6C3F1 mice with gas uptake methods. Gas uptake and CO production curves were analyzed by physiologically based pharma cokinetic (PBPK) modeling techniques, permitting differentiation of is otope effects on specific metabolic parameters from those associated w ith blood flow or diffusion limitations in vivo. A marked isotope effe ct 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, [H-2]DCM, and [H-2(2)]DCM, respectively). Based on these ratios, the calculated k(H)/k(D) ratio for the rate constant of disproportionation of the put ative formyl chloride intermediate was about 7, indicating a significa nt role of C-H bond breaking in this reaction. Deuterium substitution altered the apparent K-m for metabolism; there was 14-fold increase in the apparent K-m between DCM and [H-2(2)]DCM (6.5 +/- 0.69 to 97 +/- 3.5 mu M) with little effect on K-m with [H-2]DCM (14.4 +/- 0.015 mu M ). V-max was not greatly affected by deuteration (151 +/- 1.2, 116 +/- 0.82, and 149 +/- 2.3 mu mol/hr/kg with DCM, [H-2]DCM, and [H-2(2)]DC M, 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 iso topically 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. Regardles s of the correct mechanism, the in vivo kinetic constants for oxidatio n of DCM are complex and represent more than simple rate-limiting bond -breaking (V-max) and enzyme-substrate binding (K-m). Current PBPK mod els for metabolism of these volatiles may have to be restructured to a ccount for this unusual kinetic mechanism. (C) 1994 Academic Press, In c.