Lance R. Pohl

Phosgene: a metabolite of chloroform.

Abstract Cysteine inhibited the in vitro covalent binding of [14C] chloroform, (CHCl3), to microsomal protein and concomitantly trapped a reactive metabolite, presumably phosgene (COCl2), as 2-oxothiazolidine-4-carboxylic acid. When the incubation was conducted in an atmosphere of [18O] O2, the trapped COCl2 contained [18O]. These findings suggest that the CH bond of CHCl3 is oxidized by a cytochrome P-450 monooxygenase to produce trichloromethanol, which spontaneously dehydrochlorinates to yield the toxic agent phosgene.

Acetaminophen-induced hepatotoxicity

Abstract In large doses the commonly used analgesic acetaminophen produces a centrilobular hepatic necrosis in man and experimental animals. The toxicity is mediated by a reactive metabolite formed by a cytochrome P-450 mixed-function oxidase system in hepatic microsomes. Following therapeutic doses the reactive metabolite is efficiently detoxified by glutathione. Following large doses, however, the total hepatic glutathione concentration is decreased to approximately 20% of normal and the reactive metabolite covalently binds to protein. Changes in protein covalent binding caused by various treatments correlates with changes in the incidence and severity of the hepatic necrosis. The reactive metabolite is believed to be N-acetylimidoquinone and is apparently formed by a previously uncharacterized mechanism for cytochrome P-450.

Mechanism of metabolic activation of chloroform by rat liver microsomes

Abstract In this investigation, we attempted to determine if reactive metabolites other than phosgene (COCl 2 ) are involved in the metabolic activation of chloroform (CHCl 3 ) in rat liver microsomes. This problem was approached by determining whether the formation of COCl 2 can account for the metabolism of CHCl 3 to covalently bound product, carbon dioxide (CO 2 ), and chloride ion (Cl − ). It was found that the levels of covalent binding of [ 14 CHCl 3 and [ 14 C]CO 2 formation decreased proportionately when [ 14 C]COCl 2 was trapped as 2-oxothiazolidine-4-carboxylic acid by the addition of cysteine to the incubation mixture. The amount of this product corresponded closely to the sum of the decreases in covalent binding and CO 2 formation. [ 36 Cl]Chloride was formed from [ 36 Cl]CHCl 3 under the same conditions that produced COCl 2 from CHCl 3 . In addition, when 14 C-, 3 H-, or 36 Cl-labelled CHCl 3 was incubated with liver microsomes under a variety of conditions, only the 14 C-label was appreciably bound irreversibly to microsomal protein. These results support the view that COCl 2 is the major, if not the only, reactive metabolite formed from CHCl 3 in rat liver microsomes.

Epidemic of liver disease caused by hydrochlorofluorocarbons used as ozone-sparing substitutes of chlorofluorocarbons.

BACKGROUND Hydrochlorofluorocarbons (HCFCs) are used increasingly in industry as substitutes for ozone-depleting chlorofluorocarbons (CFCs). Limited studies in animals indicate potential hepatotoxicity of some of these compounds. We investigated an epidemic of liver disease in nine industrial workers who had had repeated accidental exposure to a mixture of 1,1-dichloro-2,2,2-trifluoroethane (HCFC 123) and 1-chloro-1,2,2,2-tetrafluoroethane (HCFC 124). All nine exposed workers were affected to various degrees. Both compounds are metabolised in the same way as 1-bromo-1-chloro-2,2,2-trifluoroethane (halothane) to form reactive trifluoroacetyl halide intermediates, which have been implicated in the hepatotoxicity of halothane. We aimed to test whether HCFCs 123 and 124 can result in serious liver disease. METHODS For one severely affected worker liver biopsy and immunohistochemical stainings for the presence of trifluoroacetyl protein adducts were done. The serum of six affected workers and five controls was tested for autoantibodies that react with human liver cytochrome-P450 2E1 (P450 2E1) and P58 protein disulphide isomerase isoform (P58). FINDINGS The liver biopsy sample showed hepatocellular necrosis which was prominent in perivenular zone three and extended focally from portal tracts to portal tracts and centrilobular areas (bridging necrosis). Trifluoroacetyl-adducted proteins were detected in surviving hepatocytes. Autoantibodies against P450 2E1 or P58, previously associated with halothane hepatitis, were detected in the serum of five affected workers. INTERPRETATION Repeated exposure of human beings to HCFCs 123 and 124 can result in serious liver injury in a large proportion of the exposed population. Although the exact mechanism of hepatotoxicity of these agents is not known, the results suggest that trifluoroacetyl-altered liver proteins are involved. In view of the potentially widespread use of these compounds, there is an urgent need to develop safer alternatives.

Pathogenic Role of Natural Killer T and Natural Killer Cells in Acetaminophen-Induced Liver Injury in Mice Is Dependent on the Presence of Dimethyl Sulfoxide

Dimethyl sulfoxide (DMSO) is commonly used in biological studies to dissolve drugs and enzyme inhibitors with low solubility. Although DMSO is generally thought of as being relatively inert, it can induce biological effects that are often overlooked. An example that highlights this potential problem is found in a recent report demonstrating a pathogenic role for natural killer T (NKT) and natural killer (NK) cells in acetaminophen‐induced liver injury (AILI) in C57Bl/6 mice in which DMSO was used to facilitate acetaminophen (APAP) dissolution. We report that NKT and NK cells do not play a pathologic role in AILI in C57Bl/6 mice in the absence of DMSO. Although AILI was significantly attenuated in mice depleted of NKT and NK cells prior to APAP treatment in the presence of DMSO, no such effect was observed when APAP was dissolved in saline. Because of this unexpected finding, the effects of DMSO on hepatic NKT and NK cells were subsequently investigated. When given alone, DMSO activated hepatic NKT and NK cells in vivo as evidenced by increased NKT cell numbers and higher intracellular levels of the cytotoxic effector molecules interferon‐γ (IFN‐γ) and granzyme B in both cell types. Similarly, when used as a solvent for APAP, DMSO again increased NKT cell numbers and induced IFN‐γ and granzyme B expression in both cell types. Conclusion: These data demonstrate a previously unappreciated effect of DMSO on hepatic NKT and NK cells, suggesting that DMSO should be used cautiously in experiments involving these cells. (HEPATOLOGY 2008.)

N-hydroxyacetaminophen: A microsomal metabolite of N-hydroxyphenacetin but apparently not of acetaminophen

Abstract High pressure liquid chromatography and gas chromatography-mass spectrometry revealed that hamster liver microsomes form N-hydroxyacetaminophen from N-hydroxyphenacetin but apparently not from acetaminophen. Nevertheless more covalent binding of radiolabel occured with 3H-acetaminophen as a substrate than with 14C N-hydroxyphenacetin. It, therefore, seems likely that the principle chemically reactive metabolite formed from acetaminophen does not arise through the formation of N-hydroxyacetaminophen as has been previously postulated.

Deuterium isotope effect in bioactivation and hepatotoxicity of chloroform.

Abstract Cytochrome P-450 appears to catalyze the in vitro formation of phosgene (COCl2) from chloroform (CHCl3) in rat liver microsomes, since this reaction is NADPH dependent and inhibited by carbon monoxide and SKF 525-A. Moreover, the cleavage of the C-H bond appears to be the rate-determining step in this process since deuterium labeled chloroform (CDCl3) is biotransformed into COCl2 slower than is CHCl3. CDCl3 was also less hepatotoxic than CHCl3 suggesting that a similar pathway of metabolism is responsible for the hepatotoxic properties of chloroform.

A metabolite of halothane covalently binds to an endoplasmic reticulum protein that is highly homologous to phosphatidylinositol-specific phospholipase C-α but has no activity

When the inhalation anesthetic halothane was administered to rats, a 58 kDa protein in the liver became covalently labeled by the trifluoroacetyl chloride metabolite of halothane. The amino acid sequences of the N-terminal and of several internal peptide fragments of the protein were 99% homologous to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha. The purified trifluoroacetylated 58 kDa protein or native 58 kDa protein, however, did not have phosphatidylinositol-specific phospholipase C activity. We conclude that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha may encode for a microsomal protein of unknown function.

Reductive oxygenation of carbon tetrachloride: trichloromethylperoxyl radical as a possible intermediate in the conversion of carbon tetrachloride to electrophilic chlorine.

Under aerobic conditions, rat liver microsomes convert carbon tetrachloride to an electrophilic form of chlorine that is trapped with 2,6-dimethylphenol to form 4-chloro-2,6-dimethylphenol. The mechanism of cytochrome P-450-catalyzed electrophilic chlorine formation from carbon tetrachloride was examined with structure-activity studies of electrophilic halogen formation and chemical and in vitro microsomal studies. 4-Chloro-2,6-dimethylphenol is not formed as a consequence of a reaction of 2,6-dimethylphenoxyl radical with carbon tetrachloride or carbon tetrachloride-induced lipid peroxyl radical formation. Only tetrahalomethanes were found to yield electrophilic halogens. The chemical oxidants hydrogen peroxide, cumene hydropheroxide, sodium periodate, and iodobenzene diacetate did not support electrophilic halogen formation from carbon tetrachloride, carbon tetrabromide, or hexachloroethane in microsomal studies. The addition of superoxide dismutase, catalase, sodium azide, or glutathione to microsomal incubations did not affect the rate of electrophilic chlorine formation, whereas Paraquat completely inhibited the reaction. The radical spin trap phenyl t-butyl nitrone (14 mM) completely inhibited electrophilic chlorine formation. The rate of electrophilic chlorine formation was highest at 2-5% atmospheric oxygen, whereas anaerobiosis completely inhibited electrophilic chlorine formation, and high oxygen tension impaired electrophilic chlorine formation. These results preclude direct oxidation of carbon tetrachloride or a reaction of superoxide anion radical with carbon tetrachloride as the initial step in electrophilic chlorine formation and suggest that the likely initial step is reductive dehalogenation of carbon tetrachloride to trichloromethyl radical which then traps oxygen to form trichloromethylperoxyl radical. Subsequent reaction of trichloromethyl peroxyl radical leads to electrophilic chlorine. These findings may have important implications concerning carbon tetrachloride-induced lipid peroxidation and carbon tetrachloride hepatotoxicity.

Macrophage migration inhibitory factor in drug-induced liver injury: a role in susceptibility and stress responsiveness

Idiosyncratic drug-induced hepatitis may depend upon many factors including a balance between pro- and anti-inflammatory mediator production levels. Using a guinea pig model of liver injury induced by bioactivation of the anesthetic drug, halothane, we found that toxicity was commensurate with an increase in serum macrophage migration inhibitory factor (MIF), a pro-inflammatory signal and counter-regulator of glucocorticoids, but only in susceptible animals. The pathogenic role of MIF was further investigated using a murine model in which liver injury was induced by the reactive metabolite of another drug, acetaminophen (APAP). MIF leakage from the liver into the sera preceded peak increases in toxicity following APAP administration. MIF null (-/-) mice were significantly less susceptible to this toxicity at 8 h. At 48 h following a 300 mg/kg dose, complete lethality was observed in wild-type mice, while 46% survival was noted in MIF-/- mice. The decreased hepatic injury in MIF-/- mice correlated with a reduction in mRNA levels of interferon-gamma and a significant increase in heat shock protein expression, but was unrelated to the APAP-protein adduct formation in the liver. These findings support MIF as a critical pro-toxicant signal in drug-induced liver injury with potentially important and novel effects on heat shock protein responsiveness.