Trifluoroacetylated proteins in liver and plasma of guinea pigs treated with HCFC-123 and halothane.

Trifluoroacetylated (TFA)-protein adducts were investigated by immunoblotting in liver and plasma of guinea pigs treated with the hepatotoxic anaesthetic halothane or the chlorofluorocarbon replacement 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123). Male outbred Hartley guinea pigs (320-400 g) were administered HCFC-123 (1, 5, 10 and 15 mmol/kg b.w.) or halothane (positive control, 10 mmol/kg b.w.) i.p. in corn oil. Blood and liver samples were collected 24 h after administration of HCFC-123 or halothane. Immunoreactive bands were demonstrated in liver microsomes at all HCFC-123 and halothane concentrations, and in plasma of animals treated with 10 and 15 mmol/kg b.w. HCFC-123 and 10 mmol/kg b.w. halothane, while no alteration of microsomal P450 content or monooxygenase activities markers of the P450 2A, 2E1 and 2B isoforms was observed. Instead, when HCFC-123 was administered at doses of 1 and 5 mmol/kg b.w., the 2E1-dependent p-nitrophenol hydroxylase activity was enhanced. The presence of TFA-proteins in plasma was always associated with hepatic damage. However, mild liver damage in some animals treated with 1 or 5 mmol/kg b.w. HCFC-123 was not associated with the presence of TFA-proteins in plasma. This indicates a lower threshold dose for the appearance of TFA-proteins or damage in the liver (1 mmol/kg b.w.) than for the presence of TFA-proteins in plasma (10 mmol/kg b.w.), thus suggesting that the presence of TFA-proteins in plasma may be the result of liver damage.

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.