Bilirubin inhibits bile acid induced apoptosis in rat hepatocytes.

BACKGROUND AND AIMS: Hydrophobic bile acids contribute to hepatocellular injury in cholestasis and rapidly induce apoptosis in vitro; however, unlike Fas agonists, cholestasis does not cause extensive hepatocyte apoptosis. As antioxidants provide protection against bile acid induced liver injury, our premise was that bilirubin, a free radical scavenger with increased plasma levels in the presence of liver disease, could protect hepatocytes against bile acid induced apoptosis. METHODS: Freshly isolated rat hepatocytes were incubated for four hours with 100 micromol/l glycochenodeoxycholate (GCDC) alone or with increasing concentrations of unconjugated (UCB) or conjugated (CB) bilirubin. RESULTS: Both UCB and CB inhibited GCDC induced apoptosis in a dose dependent fashion and suppressed the generation of reactive oxygen species by hepatocytes. CONCLUSIONS: The antiapoptotic effect of bilirubin associated with its antioxidant properties indicates that hyperbilirubinaemia may have a protective role in liver disease.

Inactivation of rat liver cytochrome P450 (P450) by N,N-dimethylformamide and N,N-dimethylacetamide.

N,N-dimethylformamide (DMF), an organic solvent widely used in industry, is bioactivated by cytochrome P450 (P450) to reactive metabolites which are believed to be responsible for the hepatotoxicity observed in animals and humans. A decrease of the activating enzyme has been reported in rats treated with DMF, although the specific P450 isoform(s) involved and the nature of the reactive species responsible for this and the other toxic effects are still being investigated. In the present work, the effect of DMF and of the structurally related N,N-dimethylacetamide (DMAc) on the activating enzyme and the nature of the reactive species involved in the mechanism of P450 inactivation by the two chemicals were investigated in vitro. Incubation of liver microsomes from pyridine-induced rats with either substrate resulted in a dose-dependent (0-20 mM) loss of P450 (up to 28 and 24% for DMF and DMAc, respectively), microsomal haem (up to 24 and 20% for DMF and DMAc, respectively), but not protoporphyrin IX content. Moreover, bubbling of CO through the incubation mixture gave almost complete protection against substrate-dependent P450 inactivation, and the spin trapping agent N-tert-butyl-alpha-phenylnitrone, but neither glutathione nor vitamin C, provided a significant protection against DMF- or DMAc-dependent haem loss. Finally, electron spin resonance analysis of microsomal incubations in presence of DMF or DMAc showed spectral evidence for a carbon centered radical intermediate. The results indicate, overall, that both compounds are metabolized in vitro by P450, probably CYP2E1, to free radical metabolites which attack the haem prosthetic group, leading to suicidal enzyme inactivation.

Bioactivation and toxicity in vitro of HCFC-123 and HCFC-141b: role of cytochrome P450.

The bioactivation and cytotoxicity in vitro of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) and 1,1-dichloro-1-fluoroethane (HCFC-141b), two replacements for some ozone-depleting chlorofluorocarbons (CFC), were investigated in rat liver microsomes and isolated rat hepatocytes. Both compounds were activated by cytochrome P450 to reactive metabolites, as indicated by: (i) the depletion of exogenous and cellular glutathione, (ii) the increased LDH release from hepatocytes, (iii) the loss of microsomal P450 content and activities, and (iv) the formation of free radical species observed in the presence of the two compounds. Moreover, the formation of two stable metabolites and an increased production of conjugated dienes, a marker of lipid peroxidation, were observed for both HCFC-123 and HCFC-141b. The biotransformation of both compounds by pyridine- and phenobarbital-induced rat liver microsomes and the inhibition of LDH release by 4-methylpyrazole and troleandomycin indicate that P450 2E1, 2B and, possibly, also 3A are the isoforms involved in the bioactivation and toxicity of HCFC-123 and HCFC-141b in the rat.

Bioactivation to free radicals and cytotoxicity of 1,1-dichloro-1-fluoroethane (HCFC-141b).

1. The in vitro bioactivation by rat liver microsomes and the cytotoxicity in rat hepatocytes of 1,1-dichloro-1-fluoroethane (HCFC-141b), a replacement for some ozone depleting chlorofluorocarbons (CFC), have been investigated. 2. Anaerobic incubations of liver microsomes from pyridine-induced rats with HCFC-141b in the presence of the spin-trapping agent N-t-butyl-alpha-phenylnitrone (PBN) resulted in the formation of a typical ESR radical signal. 3. In the presence of HCFC-141b, a dose-dependent formation of conjugated dienes was observed that was partially inhibited by PBN, glutathione (GSH) and vitamin C. Moreover, HCFC-141b increased the release of lactate dehydrogenase (LDH) and the depletion of cellular glutathione in isolated rat hepatocytes under both normoxic and hypoxic conditions. 4. HCFC-141b-dependent cytotoxicity was completely prevented by PBN under both conditions and it was partially prevented under normoxic conditions by the broad-spectrum P450 inhibitor metyrapone, the P4502E1 specific inhibitor 4-methylpyrazole and the P4503A-specific inhibitor troleandomycin. Interestingly, HCFC-141b-dependent glutathione depletion was not prevented by PBN, metyrapone, 4-methylpyrazole or troleandomycin, whereas two glutathione depletors, 2,6-dimethyl-2,5-heptadien-4-one (phorone) and diethylmaleate, partially prevented LDH release. 5. The present results indicate that HCFC-141b is reductively metabolized in vitro to free radical intermediates by P450, in particular by the CYP2E1 and, to a lower extent, CYP3A isoforms, leading to peroxidative membrane damage and glutathione-independent cytotoxicity.

Reactive oxygen species and proinflammatory cytokine signaling in endothelial cells: effect of selenium supplementation.

The release of superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), induced by tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta), has been studied in the endothelial cell line ECV 304 in the presence and absence of selenium (Se) supplementation. Both cytokines elicit the production of both species. Selenium supplementation, which increases Se-enzyme activity, decreases the amount of H(2)O(2) but not O(2)(*-) detectable in the extracellular medium. Inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by diphenyliodonium (DPI) or phenylarsine oxide (PAO), largely prevents O(2)(*-) production, whereas H(2)O(2) remains above the amount accounted for by disproportion of residual O(2)(*-). Thus, a fraction of H(2)O(2) found in the medium, derives from an intracellular pool, which is under control of selenium-dependent peroxidases. This is further supported by the observation that in Se-supplemented cells, the rate of intracellular glutathione (GSH) depletion induced by cytokine treatment is faster and more extensive. Because Se supplementation decreases cytokine-induced NF-kappaB activity, whereas added H(2)O(2) is inactive and catalase does not affect the activation induced by TNF-alpha, it is concluded that only intracellularly generated H(2)O(2) has a role in transcription factor activation by both TNF-alpha and IL-1beta.

Cytochrome P450 inactivation during reductive metabolism of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) by phenobarbital- and pyridine-induced rat liver microsomes.

The reductive metabolic activation of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), one of the potential substitutes for the ozone-depleting chlorofluorocarbons and a close structural analogue of the hepatotoxic anesthetic halothane, was investigated in vitro. During incubation of liver microsomes from phenobarbital-(PB) or pyridine-induced (PYR) rats with 0-20 mM HCFC-123 under anaerobic conditions, a dose- and time-dependent depletion of added exogenous glutathione was observed, indicating the formation of reactive metabolites. Under similar incubation conditions, except for the absence of glutathione, 1-chloro-2,2,2-trifluoroethane and 1-chloro-2,2-difluoroethene were detected as products of reductive metabolism of HCFC-123, as previously reported for halothane. As shown previously in our laboratory for halothane, under these conditions HCFC- 123 also caused a statistically significant loss of microsomal cytochrome P450 (P450) as indicated by a decrease of the classical absorption spectrum in the presence of CO. Both glutathione depletion and P450 loss were almost completely prevented by previous saturation of the incubation mixture with CO and were partially prevented by the presence of the free-radical scavenger N-t-butyl-alpha-phenylnitrone or the carbene trapping agent 2,3-dimethyl-2-butene, suggesting that both types of intermediates may be involved. The loss of P450 was associated with a quantitatively similar loss of microsomal heme, as measured by the pyridine hemochromogen reaction, with PB but not with PYR microsomes. Finally, both the P4502E1-specific p-nitrophenol hydroxylase activity in PYR microsomes and the P4502B1/2-specific pentoxyresorufin O-depentylase activity in PB microsomes were significantly inhibited (58 and 53%, respectively) by prior incubation with HCFC-123, suggesting that both isoforms are able to catalyze the activation of this halogenated compound. These results indicate that indeed HCFC-123, like its analogue halothane, is activated reductively to reactive metabolites by at least two P450 isoforms, namely P4502E1 and P4502B1/2. These metabolites, probably free radicals and/or carbene species, may attack the enzyme resulting in modification of the heme group and subsequent loss of catalytic activity.

Reductive activation of 1,1-dichloro-1-fluoroethane (HCFC-141b) by phenobarbital- and pyridine-induced rat liver microsomal cytochrome P450.

1. During anaerobic reductive incubation of liver microsomes, from either the pyridine- or phenobarbital-treated rat, with 1,1-dichloro-1-fluoroethane (HCFC-141b) in the presence of a NADPH-regenerating system, a time- and dose-dependent formation of reactive metabolites was detected as indicated by a depletion of added exogenous glutathione. 2. A statistically significant, dose-dependent loss of both cytochrome P450 and microsomal haem was also observed under these experimental conditions. Furthermore, a statistically significant decrease of p-nitrophenol hydroxylase and pentoxyresorufin O-depentylase activity was measured in microsomes from the pyridine- and phenobarbital-induced rat, respectively indicating that both P4502E1 and P4502B undergo substrate-dependent inactivation. 3. Both reactive metabolite formation and P450 inactivation were almost completely inhibited by previous bubbling of the incubation mixture with carbon monoxide, indicating that interaction of the substrate with a free and reduced P450 haem iron is required for substrate bioactivation and enzyme loss. 4. The presence in the incubation mixture of the spin-trap N-t-butyl-alpha-phenylnitrone (PBN) and the carbene trap 2,3-dimethyl-2-butene (DMB) largely prevented both glutathione depletion and P450 loss. This suggests that free radical and carbene intermediates formed by the metabolic activation of the substrate are involved in the inactivation of P450 and the loss of its prosthetic haem group.

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

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at microM concentrations with 1 mM carbon tetrachloride (CCl4), trichlorobromomethane (CCl3Br), chloroform (CHCl3) or methylene chloride (CH2Cl2) 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 haemo-protein systems, for CCi3Br, CCl4 and CHCl3, but not CH2Cl2. For Hb, the loss was greater with CCl3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl4 (haem assay, 31%) or CHCl3 (haem assay, 13%). On the other hand, with MHA, CCl4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl3. Finally, with microsomes, the inactivation was larger with CCl4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl3Br (CO binding, 33%; haem assay, 10%) or CHCl3 (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 hae-derived products during incubation of CCl3Br with Hb or microsomes, and of CCl4 with Hb. A correlation between potential for free radical formation (CCl3Br > CCl4 > CHCl3 > CH2Cl2) 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.

Reductive activation of halothane by human haemoglobin results in the modification of the prosthetic haem.

The metabolic activation of halothane by human haemoglobin (Hb) under reducing conditions in vitro is reported. Absolute spectra of sodium dithionite-reduced Hb, recorded during its anaerobic incubation in the presence of the substrate, showed decreasing concentrations of reduced Hb (Hb2+) with time. The loss of Hb2+ was accompanied, although only to some extent, by a concurrent oxidation to methaemoglobin (Hb3+), suggesting that electron transfer from Hb to the substrate had occurred. Reductive halothane metabolism was observed under these conditions as indicated by a dose-dependent inorganic fluoride (F-) production, which was, however, lower than that observed with heated Hb or a water soluble haem preparation (methaemalbumin). A rapid, partial loss of Hb was found upon addition of the substrate to the incubation mixture, as indicated by a decrease of the typical peak at 418 nm in the absolute spectra recorded in the presence of carbon monoxide (CO). This effect was associated with a loss of the Hb prosthetic group, haem, as shown by a decrease of the pyridine-haemochromogen reaction. Both effects were time and dose dependent. The inhibition of the Hb inactivation reaction by adding exogenous CO or the spin trapping agent N-t-butyl-alpha-phenylnitrone (PBN) to the incubation mixture beforehand indicated that (a) a reduced and free haem iron is required by Hb to activate halothane, and (b) the formation of free radical reactive metabolites of halothane is likely to be responsible for Hb inactivation. The mechanism of the reaction may involve the attack of these metabolites on the haem group of Hb, as indicated by the detection, with a reverse-phase ion-pairing HPLC system, of two Hb-derived products showing a typical haem-like absorption spectrum. The present results resemble those obtained recently with carbon tetrachloride (Ferrara et al., Alternatives to Laboratory Animals 21: 57-64, 1993) and suggest a common mechanism of activation of the two polyhalogenated alkanes by Hb.