Reductive activation of HCFC-123 by methaemalbumin.
Hydrochlorofluorocarbon 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), a close structural analogue of the hepatotoxic anaesthetic halotane and a replacement for some ozone-depleting chlorofluorocarbons, is metabolized by liver cytochrome P450 (P450), both in vitro and in vivo. P450 activates HCFC-123, both oxidatively and reductively, to reactive species which attack P450 itself and also damage other targets leading to hepatotoxicity. Previous work in our laboratory has shown that some haloalkanes, including halomethanes CCl4, CCl3Br, CHCl4 and CH2Cl2 as well as halothane, are activated by different haemoproteins to reactive metabolites resulting in the protein's suicidal inactivation. Among these is methaemalbumin (MHA), a synthetic complex of haem with human albumin often used as a model for various natural haemoproteins, such as P450. The aim of this study was to use MHA as a model to investigate the mechanism of P450 inactivation by HCFC-123. We found that MHA can reductively activate HCFC-123 to reactive species resulting in the loss of its haem group. During anaerobic incubation of MHA with 10 mM HCFC-123, a typical reduced difference spectrum was observed with a 470-nm peak that increased with time, indicating an interaction between HCFC-123 or HCFC-123 metabolites and haem. In similar anaerobic incubations, a significant loss of haem was measured using both the pyridine-haemochromogen technique and an ion-pairing reverse-phase HPLC method (37 and 30%, respectively). The loss of haem was time-, but not dose-dependent. No statistically significant loss of protoporphyrin IX, as measured by a fluorescence technique, or of the absolute haem spectrum produced in presence of CO (CO-haem complex) was observed up to 10 mM HCFC-123. Finally, a small but statistically significant inorganic fluoride production was measured in the presence of 20 mM HCFC-123 using an F(-)-specific electrode. Taken together, these results indicate that incubation of the non-enzymatic P450 model MHA with HCFC-123 under anaerobic conditions leads to reductive activation of the substrate, resulting in the modification of haem, as was previously shown to occur for halothane. The haem modification is due to interaction of the prosthetic haem group of MHA with HCFC-123 metabolites. These data confirm the results of previous work with rat liver microsomal P450 and confirm suicidal destruction of haem to be the mechanism responsible for the HCFC-123-dependent loss of the enzyme's content and catalytic function.
Zanovello
Alberta
A
Department of Environmental Medicine and Public Health, University of Padua, Via Giustiniani 2, 35128 Padua, Italy.
Ferrara
Roberta
R
Manno
Maurizio
M
D000693:Anaerobiosis; D018685:Anesthetics, Inhalation / Q000378:metabolism; D000818:Animals; D001711:Biotransformation; D017402:Chlorofluorocarbons / Q000378:metabolism*; D064227:Chlorofluorocarbons, Ethane; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005459:Fluorides / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D006221:Halothane / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006801:Humans; D008705:Methemalbumin / Q000378:metabolism*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats
D018685:Anesthetics, Inhalation; D017402:Chlorofluorocarbons; D064227:Chlorofluorocarbons, Ethane; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005609:Free Radicals; D008705:Methemalbumin; C067411:2,2-dichloro-1,1,1-trifluoroethane; D006418:Heme; D005459:Fluorides; D006221:Halothane