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L R Pohl

Publications and source records attributed to L R Pohl.

At least 73 records · Page 4Linked to original sources
PubMed1985

Immunochemical evidence of trifluoroacetylated cytochrome P-450 in the liver of halothane-treated rats.

Four hours after the administration of halothane to phenobarbital-pretreated rats, subcellular fractions of liver were isolated and the proteins in the fractions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, and immunochemically stained with anti-trifluoroacetylated antibodies. The microsomal fraction contained the highest level of trifluoroacetylated adducts. Its major trifluoroacetylated component was immunochemically identified as a phenobarbital-inducible form of cytochrome P-450 (54 kDa), whereas the other observed trifluoroacetylated protein fraction (59 kDa) was not identified. The plasma membrane fraction also contained a 54-kDa trifluoroacetylated adduct, which was immunochemically related to the 54-kDa cytochrome P-450. Microsomes from untreated rats that were administered halothane contained only the 59-kDa trifluoroacetylated protein fraction. The specificity of the immunochemical staining for the bound oxidative metabolite of halothane was confirmed by the finding that rats treated with deuterated halothane had considerably less stained liver proteins than did those treated with halothane. These results suggest that the CF3COX oxidative metabolite of halothane is so reactive that it binds predominantly to the cytochrome P-450 that produced it.

Acetylation↗
PubMed1984

Nephrotoxicity of chloroform: metabolism to phosgene by the mouse kidney.

In this investigation, we have attempted to determine whether chloroform (CHCl3)-induced nephrotoxicity might be due to its metabolism to phosgene (COCl2) in the kidney. We have found that kidney homogenates from DBA/2J male mice in the presence of glutathione metabolize CHCl3 to 2-oxothiazolidine-4-carboxylic acid (OTZ). This product appears to be formed by the initial trapping of COCl2 by two molecules of GSH to form diglutathionyl dithiocarbonate (GSCOSG). Kidney gamma-glutamyl transpeptidase can rapidly metabolize GSCOSG to N-(2-oxothiazolidine-4-carbonyl)-glycine which is then hydrolyzed, possibly by cysteinyl glycinase to OTZ. The finding that deuterium-labeled chloroform (CDCl3) was less nephrotoxic and depleted less renal GSH than did CHCl3 suggests that the metabolism of CHCl3 to COCl2 may also occur in the kidney in vivo and lead to nephrotoxicity.

Animals↗
PubMed1984

Reductive-oxygenation mechanism of metabolism of carbon tetrachloride to phosgene by cytochrome P-450.

The mechanism of metabolism of carbon tetrachloride (CCl4) to phosgene (COCl2) in rat liver microsomes was investigated. When the oxygen dependency of the reaction was studied, it was found that the rate of the reaction increased as the oxygen concentration in the reaction atmosphere was decreased from 100% to 5%. Decreasing the oxygen concentration below 5% caused a decrease in the rate of the reaction. The reaction was not inhibited by superoxide dismutase or catalase nor was it supported by cumene hydroperoxide. A reconstituted form of cytochrome P-450 from phenobarbital-pretreated rats metabolized CCl4 to COCl2. These results are consistent with a mechanism we call reductive oxygenation. The first step of the reaction is the cytochrome P-450-dependent reductive dechlorination of CCl4 to trichloromethyl radical (CCl3.). This intermediate is trapped by oxygen to form trichloromethylperoxyl radical (CCl3OO.), which decomposes to COCl2 and possibly an electrophilic form of chlorine.

Animals↗
PubMed1983

Identification of dichloromethyl carbene as a metabolite of carbon tetrachloride.

Although indirect evidence has suggested that liver microsomal cytochrome P-450 can reductively dehalogenate several compounds to carbene metabolites, there has been no direct proof for the formation of these reactive species. We report in this paper that carbenes can be chemically trapped and identified as metabolites. For example, 1,1-dichloro-2,2,3,3-tetramethylcyclopropane was identified as a metabolite by gas chromatography mass spectrometry when carbon tetrachloride (CCl4) was incubated anaerobically with rat liver microsomes, NADPH and 2,3-dimethyl-2-butene. The reaction required NADPH and was inhibited by carbon monoxide. These findings show that cytochrome P-450 in rat liver microsomes can reductively metabolize CCl4 to dichloromethyl carbene (:CCl2) which can be trapped with 2,3-dimethyl-2-butene to form 1,1-dichloro-2,2,3,3-tetramethylcyclopropane. A similar approach may be used for the identification of carbene metabolites of other compounds.

Alkenes↗
PubMed1983

A continuous-flow detector for cytochrome P-450 and cytochrome P-420.

A spectrophotometric detector for automatically monitoring levels of cytochrome P-450 and cytochrome P-420 in chromatographic column effluents is described. Levels of cytochrome P-450 and P-420 as low as 30 pmol/ml can be detected above an absorbance background noise equivalent to 10 pmol/ml of these heme proteins. Spectra and the concentrations of cytochrome P-450 and cytochrome P-420 are determined every 5 sec. The automatic flow reactor and detector offer the following advantages over existing manual methods: (i) greatly reduced analysis time, (ii) measurement of a larger number of independent samples than is practical manually, (iii) simultaneous measurement of cytochrome P-450 and its degradation product cytochrome P-420 immediately after elution from the column, thus avoiding further sample degradation, and (iv) greatly increased accuracy and threshold resolution due to highly reproducible reaction conditions and constant optics.

Animals↗
PubMed1983

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.

Aerobiosis↗
PubMed1983

Comparison of the effects of methyl-N-butyl ketone and phenobarbital on rat liver cytochromes P-450 and the metabolism of chloroform to phosgene.

It was previously shown that treatment of rats with methyl-n-butyl ketone (MBK) produced an increase in the total level of liver microsomal cytochromes P-450 and an increase in the rate of metabolism of chloroform (CHCl3) to phosgene (COCl2). In the present study it was found that MBK also produced qualitative changes in the composition of microsomal cytochromes P-450 in rat liver as determined by anion-exchange chromatography. The degree of the chromatographic changes paralleled the effect of MBK on the rate of metabolism of CHCl3 to COCl2 and CHCl3-induced hepatotoxicity, suggesting that MBK potentiated the hepatotoxicity of CHCl3, at least in part, by inducing the formation of cytochromes P-450 that metabolized CHCl3 to the hepatotoxin COCl2. In this regard, reconstitution studies with a form of cytochrome P-450 isolated from rat liver microsomes from rats treated with MBK or phenobarbital (Pb) showed unequivocally that cytochrome P-450 can metabolize CHCl3 to COCl2. Although analysis of rat liver microsomes by SDS-polyacrylamide electrophoresis and anion-exchange chromatography suggested that MBK and Pb had similar effects on the composition of cytochromes P-450, metabolism studies indicated that differences did exist.

Animals↗
PubMed1982

Oxidation of carbon tetrachloride, bromotrichloromethane, and carbon tetrabromide by rat liver microsomes to electrophilic halogens.

In order to determine whether CCl4, CBrCl3, CBr4 or CHCl3 undergo oxidative metabolism to electrophilic halogens by liver microsomes, they were incubated with liver microsomes from phenobarbital pretreated rats in the presence of NADPH and 2,6-dimethylphenol. The analysis of the reaction mixtures by capillary gas chromatography mass spectrometry revealed that 4-chloro-2,6-dimethylphenol was a metabolite of CCl4 and CBrCl3 whereas 4-bromo-2,6-dimethylphenol was a metabolite of CBr4. The formation of the metabolites was significantly decreased when the reactions were conducted with heat denatured microsomes, in the absence of NADPH or under an atmosphere of N2. These results indicate that the chlorines of CBrCl3 and CCl4 and the bromines of CBr4 are oxidatively metabolized by rat liver microsomes to electrophilic and potentially toxic metabolites.

Animals↗
PubMed1982

Stereoselective formation of bromobenzene glutathione conjugates.

Two bromobenzene-glutathione conjugates have been detected as both in vivo and in vitro metabolites of bromobenzene. Separation and purification by high pressure liquid chromatography (HPLC) and analysis by 13C and 1H-NMR spectroscopy indicated that the metabolites are trans-3-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol and trans-4-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol. The two conjugates are formed in unequal amounts; over a dose range of 25-500 mg/kg the ratio of the two conjugates excreted into bile in 6 h was 1.6 +/- 0.1 (mean +/- S.E.). Pretreatment of rats with either phenobarbital or 3-methyl-cholanthrene did not significantly alter the ratio of the two conjugates excreted into bile. When bromobenzene was incubated with rat liver microsomes and glutathione, the same two conjugates were formed in the presence but not in the absence of 100 000 x g supernatant. Furthermore, in the presence of 100 000 x g supernatant from control animals, microsomes from rats treated with phenobarbital formed both conjugates 6 times more rapidly than did microsomes from control rats, whereas microsomes from rats treated with 3-methylcholanthrene formed both conjugates less rapidly than did those from control rats. Thus, the data suggest that both conjugates are formed via bromobenzene 3,4-oxide and that their formation requires in liver cytosol.

Animals↗