The requirement of the gut flora in nitrobenzene-induced methemoglobinemia in rats.
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Animals↗
Biomedical subjects
L R Pohl
Publications and source records attributed to L R Pohl.
Warfarin: stereochemical aspects of its metabolism in vivo in the rat.
The biotransformation of the R and S isomers of warfarin was investigated in the rat. The formation of 7-hydroxywarfarin was stereoselective for the R enantiomer, while the formation of 4'-hydroxywarfarin was stereoselective for the S enantiomer. The 6-, 8-, and benzylic hydroxylation of both isomers was approximately the same. The reduction of the side chain ketone function of warfarin to the corresponding diastereomeric warfarin alcohols was stereoselective for the S isomer. The reduction also displayed a degree of stereospecificity with S reduction occurring predominantly. The results of the in vivo study agree in many cases with a previous in vitro investigation. However, differences between the in vitro and in vivo studies do exist and suggest that secondary stereoselective biotransformation routes occur in vivo and that the microsomal and soluble enzymes employed in the in vitro study may have been disrupted during isolation. Large amounts of polar labile conjugates of R and S warfarin, and metabolites were found in the urine. The 4-hydroxyl group of the coumarin ring appears to be the position of conjugation and this process appears to be regio and stereoselective.
Animals↗
The rapid identification of a new metabolite of warfarin via a chemical ionization mass spectrometry ion doublet technique.
An ion doublet chemical ionization mass spectrometry technique was utilized for the rapid detection and elucidation of the structure of a new hydroxylated metabolite of the oral anticoagulant warfarin. This technique involves the use of a 50:50 mixture of stable isotope labeled and unlabeled drug. With this procedure ions found in the mass spectrum which are associated with the administered drug can be identified unambiguously. The unknown metabolite from rat liver microsomal preparations was identified as benzylic hydroxywarfarin by reincubating the microsomes with warfarin specifically labeled in the benzylic position and observing the subsequent loss of label in the product. The ion doublet technique was also employed in a single human study, and benzylic hydroxywarfarin was detected and identified.
Animals↗
Synthesis and thin-layer chromatographic ultraviolet, and mass spectral properties of the anticoagulant phenprocoumon and its monohydroxylated derivatives.
Phenprocoumon and all of its aromatic monohydroxylated derivatives have been synthesized and analyzed by TLC, uv, and chemical ionization mass spectroscopy. By utilization of various combinations of these analytical techniques all of the titled compounds can be uniquely identified.
Chromatography, Thin Layer↗
Primary and beta-secondary deuterium isotope effects in N-deethylation reactions.
Lidocaine (1), labeled specifically with deuterium in the alpha-methylene (lidocaine-d4,2) and beta-methyl (lidocaine-d6,3) carbon atoms of the terminal amino group, was used to probe the mechanism of oxidative N-deethylation by rat liver microsomes. The reaction rates were determined by measuring the formation of acetaldehyde colorimetrically. This general assay for oxidative N-deethylation reactions has the advantages of being rapid, producing a relatively stable colored derivative and being linear over the range of 0.25-4 mug of acetaldehyde formed per milliliter of incubate. Deuterium substitution at the methylene carbon atoms, the presumed site of initial oxygen insertion, revealed a kH/kD = 1.49 +/- 0.11 and a KmD/KmH = 1.23. Deuterium substitution on the terminal methyl groups showed a kH/kD = 1.52 +/- 0.10 and a KmD/KmH = 0.92. The results are explained in terms of both primary and secondary isotope effects on a possible rate-determining step in the N-deethylation sequence.
Acetaldehyde↗
Biotransformation of phenprocoumon in the rat.
The metabolic fate of phenprocoumon [3-(alpha-ethylbenzyl)-4-hydroxycoumarin] in the rat is described. The major metabolites, 4',-6-,7-, and 8-hyproxyphenprocoumon, have been identified yb mass spectrometry, TLC, and uv and compared with authentic smaples. Metabolites are mainly excreted via the feces. The results are compared with those previously reported for warfarin.
Animals↗
A new chiral reagent for the determination of nantiomeric purity and absolute configuration of certain substituted -arylethylamines.
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Absorption↗
Covalent bonding of the prosthetic heme to protein: a potential mechanism for the suicide inactivation or activation of hemoproteins.
In this perspective we have described a newly characterized pathway for the metabolism of the prosthetic heme of cytochrome P-450, which results in the formation of protein-bound adducts. This reaction occurs when the cytochrome P-450 metabolizes a variety of xenobiotics as well as endogenous compounds such as hydrogen peroxide and lipid hydroperoxides. It also takes place during the reactions catalyzed by other hemoproteins, such as myoglobin and hemoglobin. In the case of the reaction of ferrous myoglobin with BrCCl3, under single-turnover conditions, an intact heme moiety becomes covalently bound to an active-site amino acid. This covalently altered protein has significantly enhanced reductive activity compared to that of native myoglobin, as demonstrated by its rapid reduction of molecular oxygen and CCl4. It also is more rapidly proteolyzed than myoglobin. These findings may have relevance to the P-450 cytochromes in which suicide inactivation, destruction of the heme prosthetic group, and loss of the protein is observed. The activation of hemoproteins to heme-protein adducts may also have toxicological significance, perhaps in oxygen reperfusion injury in the myocardium as well as other tissues by enhancing the production of oxygen-derived radicals from molecular oxygen and lipid hydroperoxides. Clearly, further research in the characterization of heme-protein adducts is necessary before their importance in protein turnover and oxygen-induced injury can be determined.
Animals↗
The calcium-binding protein calreticulin is covalently modified in rat liver by a reactive metabolite of the inhalation anesthetic halothane.
A general procedure is presented for the isolation of several liver microsomal target proteins of the reactive trifluoroacetyl halide metabolite of halothane. It was found that most of these proteins could be selectively extracted from microsomes with 0.1% sodium deoxycholate and separated into partially purified fractions by DEAE-Sepharose anion-exchange chromatography. Using this method, we describe the isolation and identification of a 63-kDa target protein of halothane in rat liver. Amino acid sequences of the N-terminal and of several internal peptides of the protein, as well as the deduced amino acid sequence of a nearly full-length rat liver cDNA clone of the protein, showed 98% identity with a reported murine cDNA that encodes for calreticulin, a major calcium-binding protein of the lumen of endoplasmic reticulum. Although it remains to be determined what role calreticulin has in the development of halothane hepatitis, this study has shown that calreticulin can be a target of reactive metabolites of xenobiotics.
Amino Acid Sequence↗
Pentahaloethane-based chlorofluorocarbon substitutes and halothane: correlation of in vivo hepatic protein trifluoroacetylation and urinary trifluoroacetic acid excretion with calculated enthalpies of activation.
The hydrochlorofluorocarbons (HCFCs) 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) and 2-chloro-1,1,1,2-tetrafluoroethane (HCFC-124) and the hydrofluorocarbon (HFC) pentafluoroethane (HFC-125) are being developed as substitutes for chlorofluorocarbons that deplete stratospheric ozone. The structural similarity of these HCFCs and HFCs to halothane, which is hepatotoxic under certain circumstances, indicates that the metabolism and cellular interactions of HCFCs and HFCs must be explored. In a previous study [Harris et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1407], similar patterns of trifluoroacetylated proteins (TFA-proteins) were detected by immunoblotting with anti-TFA-protein antibodies in livers of rats exposed to halothane or HCFC-123. The present study extends these results and demonstrates that in vivo TFA-protein formation resulting from a 6-h exposure to a 1% atmosphere of these compounds follows the trend: halothane approximately HCFC-123 much greater than HFC-124, greater than HFC-125. The calculated enthalpies of activation of halothane, HCFC-123, HCFC-124, and HFC-125 paralleled the observed rate of trifluoroacetic acid excretion in HCFC- or HFC-exposed rats. Exposure of rats to a range of HCFC-123 concentrations indicated that TFA-protein formation was saturated at an exposure concentration between 0.01% and 0.1% HCFC-123. Deuteration of HCFC-123 decreased TFA-protein formation in vivo. Urinary trifluoroacetic acid excretion by treated rats correlated with the levels of TFA-proteins found after each of these treatments. No TFA-proteins were detected in hepatic fractions from rats given 1,1,1,2-tetrafluoroethane (HFC-134a), which is not metabolized to a trifluoroacetyl halide.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Immunochemical detection of liver protein adducts of the nonsteroidal antiinflammatory drug diclofenac.
Serious idiosyncratic hepatic injury has been associated with the use of many nonsteroidal antiinflammatory drugs, including the widely prescribed agent diclofenac. In order to investigate the possibility that covalent protein adducts of reactive metabolites of diclofenac might be responsible for the hepatotoxicity produced by this drug, we have developed a polyclonal antibody that can recognize such adducts in tissues. Immunoblotting revealed that protein adducts of reactive metabolites of diclofenac of 50, 70, 110, and 140 kDa were formed in the livers of mice treated with diclofenac. In the future, it will be determined whether these adducts can cause hepatotoxicity by either a hypersensitivity or metabolic mechanism. Similar approaches may be used to study the protein adducts and mechanisms of hepatotoxicity of other nonsteroidal antiinflammatory drugs.
Animals↗
Serum antibodies from halothane hepatitis patients react with the rat endoplasmic reticulum protein ERp72.
Immunoblotting studies have previously shown that serum antibodies from halothane hepatitis patients react with several liver microsomal proteins that have been modified by the trifluoroacetyl halide metabolite of halothane. In this study, an 80-kDa protein recognized by the patients' antibodies has been purified from rat liver microsomes and characterized. When the purified trifluoroacetylated 80-kDa and native 80-kDa proteins were employed as test antigens in an enzyme-linked immunosorbent assay, serum antibodies from halothane hepatitis patients reacted with both of these proteins to a significantly greater extent than did serum antibodies from control patients. Amino acid sequence analyses of several hydrolytic peptide fragments of the 80-kDa protein showed that the protein was 99% identical to the deduced amino acid sequence of a murine cDNA of the luminal endoplasmic reticulum protein ERp72. These results indicate that trifluoroacetylated ERp72 in the liver of halothane hepatitis patients may induce immune responses against epitopes present on the covalently altered protein and those present on the native protein and may have a role in halothane hepatitis. In addition, immunoblot and immunohistochemical studies revealed that the 80-kDa protein was present in all tissues studied, but was in highest concentration in liver, adipose tissue, ovaries, and testes and was enriched in specific cells of some organs. In the future, these findings should help define the physiological function of ERp72.
Amino Acid Sequence↗
An immunochemical approach of identifying and characterizing protein targets of toxic reactive metabolites.
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Animals↗
Metabolic activation and immunochemical localization of liver protein adducts of the nonsteroidal anti-inflammatory drug diclofenac.
Diclofenac is a nonsteroidal anti-inflammatory agent that is reported to cause serious hepatic injury in some patients. To investigate the possibility that protein adducts derived from reactive intermediates of diclofenac might be responsible for the hepatotoxicity produced by this drug, we recently developed polyclonal antisera that recognized protein adducts of diclofenac. In the present study, we have characterized further the diclofenac adducts in rat liver. Immunoblotting studies showed that diclofenac-labeled hepatic proteins were formed in a dose- and time-dependent manner in rats given diclofenac. Subcellular fractionation of liver homogenates from diclofenac-treated rats showed that a 50-kDa microsomal protein and 110-, 140-, and 200-kDa plasma membrane proteins were labeled preferentially. Immunofluorescence studies of isolated hepatocytes and immunohistochemical analysis of liver slices from diclofenac-treated mice and rats confirmed that plasma membrane proteins were labeled by diclofenac metabolites and showed that the bile canalicular domain of the plasma membrane was a major site of diclofenac adduct formation. Additionally, we found that cytochrome P-450 and UDP-glucuronosyltransferase, but not acyl-CoA synthase, catalyzed the formation of reactive intermediates of diclofenac that were bound covalently to proteins in vitro. The metabolites catalyzed by cytochrome P-450 in vitro were bound exclusively to a 50-kDa microsomal protein, even in the presence of albumin. In contrast, the 110-, 140-, and 200-kDa plasma membrane proteins as well as others appeared to be labeled when diclofenac was activated by UDP-glucuronosyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Human cytochrome P450 2E1 is a major autoantigen associated with halothane hepatitis.
Autoantibodies against specific human cytochrome P450s have been found in the sera of patients suffering from a variety of diseases, including those caused by drugs. In the cases of tienilic acid- and dihydralazine-induced hepatitis, patients have serum autoantibodies directed against cytochromes P450 2C9 and P450 1A2, respectively. In the present study, we have found that 25 of 56 (45%) patients diagnosed with halothane hepatitis have autoantibodies that react with human cytochrome P450 2E1 that was purified from a baculovirus expression system. The autoantibodies inhibited the activity of cytochrome P450 2E1 and appeared to be directed against mainly conformational epitopes. In addition, because cytochrome P450 2E1 became trifluoroacetylated when it oxidatively metabolized halothane, it is possible that the covalently altered form of cytochrome P450 2E1 may be able to bypass the immunologic tolerance that normally exists against cytochrome P450 2E1. A similar mechanism may explain the formation of autoantibodies that have been found against other cellular targets of the reactive trifluoroacetyl chloride metabolite of halothane.
Acetylation↗
The use of stable isotopes to identify reactive metabolites and target macromolecules associated with toxicities of halogenated hydrocarbon compounds.
1. Halogenated compounds, such as the inhalation anaesthetics, halothane and enflurane, and the chemicals chloroform, carbon tetrachloride, and bromotrichloromethane can cause hepatotoxicity, nephrotoxicity, and inactivation of cytochromes P-450. Each of these toxicities is mediated by reactive metabolites. 2. Stable isotopes of hydrogen, carbon, chlorine and oxygen have been used in conjunction with mass spectrometry and n.m.r. spectrometry to identify the structures of these metabolites, to elucidate the mechanisms of their formation, and to characterize the structures of their macromolecular adducts. 3. In a number of cases, oxidative pathways of metabolism to toxic metabolites have been defined by kinetic deuterium isotope effects. 4. Recently, we have found that the trichloromethyl radical metabolite of bromotrichloromethane can activate myoglobin by causing the covalent cross-linking of haem to protein. The structure of a haem-myoglobin adduct has been defined by the use of stable isotope studies.
Animals↗
Determination of toxic pathways of metabolism by deuterium substitution.
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Animals↗
Factors affecting the expression of trifluoroacetylated liver microsomal protein neoantigens in rats treated with halothane.
Previous studies have shown that antibodies in the sera of halothane hepatitis patients recognize trifluoroacetylated liver microsomal proteins (neoantigens) of 100 kDa, 76 kDa, 59 kDa, 57 kDa, and 54 kDa. In the present investigation, factors that might affect the level of expression of the neoantigens were investigated. A study of the time course of neoantigen expression in halothane-treated rats revealed that the 100 kDa, 76 kDa, 59 kDa, and 57 kDa neoantigens were longer-lived than the 54 kDa neoantigen and could be detected in the liver up to a week after the administration of halothane. Pretreatment of rats with isoniazid, which is known to induce cytochrome P-450 IIE1, appeared to increase the expression of each of the neoantigens, whereas inducers of several other forms of cytochrome P-450 had either very little effect or decreased the expression of several of the neoantigens. Female rats appeared to express some of the neoantigens at a higher level than that found in males. Examination of the organ distribution of the trifluoroacetylated neoantigens showed that, of the tissues examined, only the liver contained appreciable levels of the neoantigens. These results indicate that the level of expression and possibly the immunogenicity of the trifluoroacetylated liver neoantigens may be influenced by their half-lives and the repertoire of cytochrome P-450 present in the liver.
Animals↗