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Biomedical subjects

P G Forkert

Publications and source records attributed to P G Forkert.

At least 19 recordsLinked to original sources
PubMed2001

Mechanisms of 1,1-dichloroethylene-induced cytotoxicity in lung and liver.

Exposure to 1,1-dichloroethylene (DCE) causes lung and liver toxicities in mice. The lesions are characterized by damage preferentially to bronchiolar Clara cells in the lung and necrosis of centrilobular hepatocytes in the liver. The primary metabolites formed from DCE in lung and liver microsomal incubations are the epoxide, 2,2-dichloroacetaldehyde and 2-chloroacetyl chloride, which undergo hydrolysis and/or conjugation with glutathione (GSH). The major products formed are the epoxide-derived GSH conjugates 2-(S-glutathionyl) acetyl glutathione [B] and 2-S-glutathionyl acetate [C]. The hydrate of 2,2-dichloroacetaldehyde (acetal) is also detected. These metabolites are detected in vivo in murine lung and liver cytosol and in bile, and importantly, also in human lung and liver microsomal incubations. Formation of the epoxide is mediated mainly by CYP2E1. Immunohistochemical studies localized the epoxide-derived GSH conjugate [C] and cysteine-containing proteins in Clara cells and centrilobular hepatocytes. These findings are consistent with the premise that the lung and liver cytotoxicities induced by DCE are associated with in situ formation of the epoxide within the target cells.

Animals↗
PubMed2001

Strain-related differences in bioactivation of vinyl carbamate and formation of DNA adducts in lungs of A/J, CD-1, and C57BL/6 mice.

Inbred strains of mice exhibit differing susceptibilities to formation of lung tumors induced by procarcinogens including ethyl carbamate (EC) and vinyl carbamate (VC). Strain A/J mice are susceptible, whereas C57BL/6 mice are resistant to lung tumor development. In this study, we tested the hypothesis that differential susceptibilities of A/J, CD-1, and C57BL/6 mice to lung tumor development are associated, in part, with their capacities for VC bioactivation and with the extents of DNA adduct formation. Previous studies have shown that the P450 isozyme CYP2E1 and microsomal carboxylesterases are involved in activation and detoxication of VC, respectively. Bioactivation capacity, as estimated by ratios of p-nitrophenol hydroxylase, a CYP2E1 catalytic marker, to carboxylesterase activities, was greater in control A/J (1.32 +/- 0.18 x 10(-6)) and CD-1 (1.25 +/- 0.29 x 10(-6)) mice than in control C57BL/6 (0.69 +/- 0.12 x 10(-6)) mice. The ratios were reduced in all three strains of mice treated with VC. Covalent binding of [(14)C-carbonyl]-VC to lung proteins was time- and dose-dependent, and was significantly higher in A/J and CD-1 mice than in C57BL/6 mice. Experiments using (32)P-postlabeling/thin-layer chromatography showed formation of the DNA adducts 1,N:(6)-ethenodeoxyadenosine and 3,N:(4)-ethenodeoxycytidine in lungs of mice treated with VC. The DNA adducts were detected at 30 min after treatment, peaked at 60 min, and declined thereafter. Levels of 1,N:(6)-ethenodeoxyadenosine and 3,N:(4)-ethenodeoxycytidine were about 70% higher in A/J and CD-1 mice than in C57BL/6 mice. These results indicated that formation of VC metabolites in these murine strains is linked to their bioactivation capacities, and suggested that this attribute may confer differing susceptibilities to lung tumor development.

Animals↗
PubMed2001

Involvement of CYP2E1 and carboxylesterase enzymes in vinyl carbamate metabolism in human lung microsomes.

Previous studies have shown that CYP2E1 and carboxylesterase enzymes contributed to vinyl carbamate (VC) metabolism in murine lung. Moreover, these studies have implicated CYP2E1 and the carboxylesterases in bioactivation and detoxication, respectively. Here we have tested the hypothesis that CYP2E1 and carboxylesterase enzymes are involved also in VC metabolism in human lung. Demethylation of N-nitrosodimethylamine (NDMA) is an enzyme activity associated with CYP2E1, and was used as a catalytic marker for this P450 in human lung microsomes. NDMA demethylase activity in lung microsomes from 10 patients ranged from 36.9 +/- 1.0 to 82.4 +/- 2.4 pmol/mg protein/min. Significant decreases (40-65%) in demethylase activity were detected in lung microsomes incubated with VC and NADPH, compared with the controls in which incubations were performed with only VC or only NADPH. Preincubation with the CYP2E1 inhibitor diallyl sulfone also significantly decreased demethylase activity, and abrogated the VC-induced effect. Similarly, preincubation of lung microsomes with a human CYP2E1 inhibitory monoclonal antibody ameliorated the VC-induced reduction in demethylase activity. Microsomal carboxylesterase activity in lung microsomes from 10 patients ranged from 19.02 +/- 2.28 to 48.18 +/- 4.34 nmol/mg protein/min, and was significantly decreased (25-45%) in microsomes incubated with phenylmethylsulfonyl fluoride, an inhibitor of the carboxylesterase enzyme. Preincubation of lung microsomes with phenylmethylsulfonyl fluoride and subsequent incubation with VC and NADPH exacerbated the reduction (60-80%) in demethylase activity evoked by reaction with VC and NADPH. These results are consistent with a role for the CYP2E1 enzyme and microsomal carboxylesterases in VC metabolism.

Allyl Compounds↗
PubMed2001

Pulmonary bioactivation of 1,1-dichloroethylene is associated with CYP2E1 levels in A/J, CD-1, and C57BL/6 mice.

1,1-Dichloroethylene (DCE) elicits lung cytotoxicity and selectively targets Clara cells of bronchioles. The toxic effects are ascribed to CYP2E1-mediated formation of reactive intermediates including the DCE epoxide. Here we tested the hypothesis that differential CYP2E1 levels in the lungs of A/J, CD-1, and C57BL/6 mice lead to differences in the extents of DCE bioactivation and lung damage. Our results showed that lung CYP2E1 levels differed significantly in the three murine strains, and followed the rank order A/J > CD-1 > C57BL/6. Covalent binding of [(14)C]DCE to lung proteins in A/J mice was significantly higher than in either CD-1 or C57BL/6 mice. HPLC analysis of lung cytosol from DCE-treated mice showed that 2-S-glutathionyl acetate, a glutathione (GSH) conjugate derived from the epoxide (conjugate [C]), was the major metabolite formed. Levels of [C] detected in cytosol from A/J and CD-1 mice were significantly higher than in C57BL/6 mice. Immunohistochemical staining for [C] was pronounced in the lungs of A/J mice, was lower in CD-1 mice, and was lowest in C57BL/6 mice. Levels of GSH were similar in the lungs of all untreated mice. However, significant reduction in GSH was found in DCE-treated mice, with decreases comparable in all three strains. Bronchiolar Clara cell damage was more severe in A/J and CD-1 mice than in C57BL/6 mice. These results showed differences in CYP2E1 levels in the lungs of A/J, CD-1, and C57BL/6 mice that correlated with the extent to which the DCE epoxide is formed as well as with the severity of lung cytotoxicity.

Animals↗
PubMed2001

Differential metabolism of 1,1-dichloroethylene in livers of A/J, CD-1, and C57BL/6 mice.

1,1-Dichloroethylene (DCE) causes hepatocellular necrosis that preferentially affects centrilobular hepatocytes. The cytotoxic lesion has been attributed to DCE oxidation mediated mainly by CYP2E1, resulting in formation of reactive intermediates including the DCE epoxide. Here, we have tested the hypothesis that differing levels of hepatic CYP2E1 in A/J, CD-1, and C57BL/6 (B6) mice lead to differences in magnitudes of DCE metabolism and severities of hepatotoxicity. Our results showed that amounts of the CYP2E1 protein were higher in A/J mice than in B6 and CD-1 mice. Covalent binding of DCE to liver proteins was variable in the three strains of mice and was higher in A/J than in B6 mice; intermediate levels were found in CD-1 mice. Levels of a DCE epoxide-derived glutathione conjugate detected in liver cytosol correlated with those present in bile extracts and were significantly higher in A/J than in CD-1 and B6 mice. Immunohistochemical studies showed that formation of DCE epoxide-cysteine protein adducts was enhanced in the livers of A/J mice, compared with those produced in the livers of CD-1 and B6 mice. Similarly, centrilobular necrosis was more severe in the livers of A/J mice than in those in either CD-1 or B6 mice. Levels of glutathione were similar in the three strains of untreated mice and were diminished at comparable levels in all mice. These results indicated that high expression of hepatic CYP2E1 in A/J mice coincided with increased DCE metabolism and enhanced severity of hepatotoxicity, relative to those in CD-1 and B6 mice.

Animals↗
PubMed2000

Epoxide formation from diallyl sulfone is associated with CYP2E1 inactivation in murine and human lungs.

We tested the hypothesis that an epoxide formed from diallyl sulfone (DASO(2)) is responsible for inactivation of CYP2E1 in murine and human lungs. An epoxide (1,2-epoxypropyl-3,3'-sulfonyl-1'-propene [DASO(3)]) was synthesized from DASO(2) and conjugated with glutathione (GSH) to produce the conjugates S-(1R, S-[[1-hydroxymethyl-2,3' -sulfonyl]-1' -propenyl]ethyl)glutathione (diastereomers) and S-(1-[[2R,S-hydroxypropyl]-3, 3'-sulfonyl]-1'-propenyl)glutathione (diastereomers). Analysis of these conjugates by high performance liquid chromatography revealed a major peak eluting at 20.5 min. This peak was detected in incubations of murine and human lung microsomes containing DASO(2) and nicotinamide adenine dinucleotide phosphate (NADPH), and was not detected in incubations performed in the absence of DASO(2) or NADPH. The amounts of epoxide-derived GSH conjugates formed in the incubations were concentration-dependent and achieved saturation at 0.75 mM DASO(2). Formation of the conjugates was also time-dependent and peaked at 2.0 h after DASO(2). The peak containing the GSH conjugates was also detected in incubations of CYP2E1-expressed lymphoblastoid microsomes, NADPH, and DASO(2). Maximal amounts of DASO(3), as estimated by formation of a 4-(p-nitrobenzyl)pyridine derivatized product, were detected in murine lung microsomes incubated for 35 min with 1 mM DASO(2). The derivatized DASO(3) was not detectable in incubations of human lung microsomes. p-Nitrophenol hydroxylation, a catalytic activity associated with CYP2E1, was reduced in murine and human lung microsomes incubated with DASO(2), with decreases that were concentration-dependent. Dose-dependent decreases in hydroxylase activity were also found in microsomes from mice treated in vivo with DASO(2) (25 to 200 mg/kg). These results supported the premise that an epoxide formed from DASO(2) mediates inactivation of lung CYP2E1. Furthermore, the findings suggested that the mouse model is relevant for studies of DASO(2) in human lung.

Allyl Compounds↗
PubMed2000

Inactivation of hepatic CYP2E1 by an epoxide of diallyl sulfone.

Diallyl sulfone (DASO(2)) inhibits hepatic CYP2E1. In this investigation, we have tested the hypothesis that an epoxide formed from DASO(2) is responsible for inactivation of hepatic CYP2E1 in mice. An epoxide of DASO(2) (1,2-epoxypropyl-3, 3'-sulfonyl-1'-propene; DASO(3)) was synthesized and conjugated to glutathione (GSH) to produce the conjugates S-(1R,S-[[ 1-hydroxymethyl-2,3'-sulfonyl]-1'-propenyl]ethyl)glutathione (diastereomers) and S-(1-[[2R,S-hydroxypropyl]-3, 3'-sulfonyl]-1'-propenyl)glutathione (diastereomers). Their identities were confirmed by (1)H NMR analysis, and these were used as analytical standards. HPLC analysis revealed a major peak for the GSH conjugates that eluted at 20.5 min. This peak was detected in liver microsomal incubations performed with DASO(2) in the presence of NADPH. A similar peak also was detected in incubations of CYP2E1-expressed lymphoblastoid microsomes, NADPH and DASO(2). The generation of the epoxide-derived GSH conjugates in the microsomal incubations was concentration-dependent, and reached saturation at 0. 75 to 1.0 mM DASO(2). Formation of the conjugates was also time-dependent and peaked at 2.0 h after DASO(2). Levels of DASO(3) formed from DASO(2), as estimated by production of a 4-(p-nitrobenzyl)pyridine derivative, were maximal at 1 mM DASO(2) at 30 min. CYP2E1-dependent p-nitrophenol hydroxylase activity was decreased in microsomes incubated with DASO(2), with alterations that were proportional to the concentration of DASO(2) (0.25-1.0 mM) used. Dose-dependent decreases in hydroxylase activity also were found in microsomes from mice treated in vivo with DASO(2) (25-200 mg/kg). These DASO(2)-induced decreases corresponded with reduced amounts of immunodetectable CYP2E1. Levels of spectrally detectable P450 and heme were both diminished by DASO(2). These results supported the contention that an epoxide formed from DASO(2) mediates the inactivation of hepatic CYP2E1.

Allyl Compounds↗
PubMed1999

Expression and inducibility of alpha, pi, and Mu glutathione S-transferase protein and mRNA in murine lung.

This investigation sought to establish the cellular expression and distribution of the alpha, pi, and mu classes of glutathione S-transferase (GST) enzymes in murine lung under control conditions and after treatment with tert-butyl-4-hydroxyanisole (BHA). Immunohistochemical and in situ hybridization studies were used to identify lung cells that were labeled for the GST subunits Yp, Ya, and Yb1. Immunoblotting of cytosolic proteins produced single bands of 28, 29, and 31 kD for Ya, Yp, and Yb1, respectively, in samples from untreated and BHA-treated mice. Treatment with BHA increased Ya and Yp reactivity, but this was not as marked for Yb1. Immunohistochemical staining for the Yp, Ya, and Yb1 subunits was localized in bronchioles and parenchyma of untreated and BHA-treated mice. Bronchiolar Clara and alveolar type II cells were stained to the greatest extent for all of the GST subunits. BHA treatment produced increased staining that was most pronounced in the bronchiolar epithelium. Ya and Yp were localized in the cytoplasm and nucleus, whereas Yb1 was found mainly in the cytoplasm. Immunoblots of extracted nuclear proteins revealed a band of 29 kD for Ya, with increased immunoreactivity in BHA-treated mice. In situ hybridization done with oligonucleotide probes showed abundant silver grains representing Ya, Yp, and Yb1 messenger RNA (mRNA) transcripts in the bronchioles. Grains were also localized in alveolar septa, and were most numerous in type II cells. Quantitative image analysis confirmed good agreement between relative levels of protein and mRNA transcripts. Quantities of mRNA transcripts for all subunits were increased in the parenchyma by BHA treatment, but the magnitudes of induction were most striking for Ya and Yp in the bronchioles. These results demonstrated that Ya, Yp, and Yb1 reside in specific lung areas and cells, and that in induced states, their increased expression is accompanied by increased mRNA.

Animals↗
PubMed1999

1,1-Dichloroethylene-induced Clara cell damage is associated with in situ formation of the reactive epoxide. Immunohistochemical detection of its glutathione conjugate.

Pulmonary Clara cells are selectively damaged in mice given 1, 1-dichloroethylene (DCE), a chemical used in the plastics industry. The cytotoxicity is attributed to formation of a reactive metabolite believed to be the DCE-epoxide, which was detected in vitro. We have undertaken in vivo studies to test the hypothesis that in situ formation of the DCE-epoxide within Clara cells mediates the cell-specific injury manifested after DCE exposure. Formation of the epoxide was estimated by trapping of the metabolite with glutathione (GSH) and identifying the conjugated products as 2-(S-glutathionyl) acetyl glutathione ([B]) and 2-S-glutathionyl acetate ([C]). High-pressure liquid chromatographic analyses showed that conjugates [B] and [C] were both detected in lung cytosol isolated from mice treated in vivo with [14C]DCE. Epoxide levels in the cytosol, as estimated by the total amount of conjugates formed, were dose-dependent at DCE doses ranging from 25 to 225 mg/kg. Pretreatment of mice with buthionine sulfoximine (BSO) decreased sulfhydryl levels and significantly inhibited the formation of the GSH conjugates. Epoxide levels were also reduced by pretreatment with diallyl sulfone (DASO2), an inhibitor of the P450 isozyme CYP2E1. A polyclonal antibody was developed that is specific for conjugate [C] and that recognizes an antigen consisting of the conjugate epoxide-GSH-glutaraldehyde-bovine serum albumin. Immunohistochemical studies with this antibody revealed staining in Clara cells of mice treated with DCE. Staining was also present in Clara cells of mice treated with both BSO and DCE, but at slightly reduced levels. Reduction of this staining was more pronounced in Clara cells of mice treated with both DASO2 and DCE. These results show that the DCE-epoxide is formed in vivo, is localized in Clara cells, and correlates with the cytotoxicity manifested in this cell type.

Animals↗
PubMed1999

Cytochrome P-450-dependent bioactivation of 1,1-dichloroethylene to a reactive epoxide in human lung and liver microsomes.

We investigated the cytochrome P-450-dependent metabolism of 1, 1-dichloroethylene (DCE) by human lung and liver microsomes and compared the results from analogous experiments in mice. Metabolites were identified by HPLC analysis of their glutathione conjugates and/or hydrolyzed products and were detected by using [14C]DCE. The role of human CYP2E1 in the metabolic reactions was examined by comparing p-nitrophenol hydroxylase activities with levels of metabolites formed and by using the CYP2E1-selective inhibitor diallyl sulfone. The major products formed in microsomal incubations containing NADPH were the DCE-epoxide-derived glutathione conjugates 2-(S-glutathionyl)acetyl glutathione and 2-S-glutathionyl acetate. Lower levels of the acetal of 2,2-dichloroacetaldehyde were also detected. In lung samples from eight patients, the amounts of epoxide-derived conjugates formed ranged from 15.6 +/- 4.23 to 34.9 +/- 12.75 pmol/mg protein/min. The levels in murine lung were higher at 40.0 +/- 3.8 pmol/mg protein/min. In liver samples from five patients, conjugate levels ranged from 46.5 +/- 8.3 to 240.0 +/- 10. 5 pmol/mg protein/min, whereas levels in murine liver were 83.0 +/- 6.2 pmol/mg protein/min. Conjugate levels formed in human liver correlated with the relative levels of p-nitrophenol hydroxylase activity present, but this relationship was equivocal in human lung. Diallyl sulfone inhibited the formation of the glutathione conjugates (20-65%) in liver samples from all four patients, whereas only one of five human lung samples exhibited this inhibition (27%). These results demonstrated that the DCE-epoxide is a major metabolite formed by human microsomes and is mediated by CYP2E1 in liver and in some individuals in lung.

Adolescent↗
PubMed1999

In vivo formation and localization of 1,1-dichloroethylene epoxide in murine liver: identification of its glutathione conjugate 2-S-glutathionyl acetate.

The hepatotoxic effects induced by 1,1-dichloroethylene (DCE) are ascribed to cytochrome P-450 (CYP) 2E1-dependent formation of metabolites including 2,2-dichloroacetaldehyde and the DCE-epoxide. The DCE metabolites detected in incubations of liver microsomes are the acetal, the hydrate of 2,2-dichloroacetaldehyde, and the epoxide-derived GSH conjugates 2-S-glutathionyl acetyl glutathione ([B]) and 2-S-glutathionyl acetate ([C]). This study was undertaken to determine whether these DCE metabolites are also formed in vivo in murine liver. HPLC analysis of cytosol isolated from the livers of mice treated with [(14)C]DCE showed that [C] was the major conjugate formed, with lower levels of formation of [B]. The acetal was not detected in the cytosol. The formation of the epoxide-derived GSH conjugates was dose-dependent at 25 to 225 mg/kg DCE and occurred coincidentally with levels of covalent binding of DCE at the same doses. The acetal and conjugates [B] and [C] were also detected in bile collected from mice treated with DCE. Pretreatment of mice with buthionine sulfoximine decreased sulfhydryl levels and formation of conjugate [C], and increased DCE binding to liver proteins. In contrast, the levels of [C] and DCE binding were both reduced significantly in mice pretreated with the CYP2E1 inhibitor diallyl sulfone. Immunohistochemical studies indicated that protein adducts and conjugate [C] were localized in centrilobular hepatocytes and corresponded with the sites where CYP2E1 resided. Pretreatment with buthionine sulfoximine increased the amount of immunostaining. However, pretreatment with diallyl sulfone markedly decreased immunostaining for [C] in the hepatocytes. These results showed that 2,2-dichloroacetaldehyde and the epoxide are formed from DCE in vivo.

Allyl Compounds↗
PubMed1999

Diminished CYP2E1 expression and formation of 2-S-glutathionyl acetate, a glutathione conjugate derived from 1,1-dichloroethylene epoxide, in murine lung tumors.

We hypothesized that resistance of lung tumors to the cytotoxic effects of xenobiotics is associated with loss of cytochrome P-450 expression, leading to defective formation of reactive intermediates. To test this hypothesis, we investigated 1,1-dichloroethylene (DCE), a chemical that causes Clara cell damage, in a urethane-induced model of lung tumorigenesis. Lung metabolism of DCE yields 2-S-glutathionyl acetate (conjugate [C]), a glutathione conjugate derived from DCE-epoxide, believed to be the ultimate toxic species. We used immunohistochemistry to investigate CYP2E1 expression in nontumor- and tumor-bearing lung to identify cells capable of generating [C]. CYP2E1 and [C] were colocalized in adjacent tissue sections to determine coincidence between CYP2E1 and [C] in lung cells. CYP2E1 was highly localized to the bronchiolar epithelium of nontumor-bearing lung and in uninvolved tissue of tumor-bearing lung and was concentrated in the Clara cells. In contrast, tumor foci including hyperplasias, adenomas, and carcinomas were deficient in CYP2E1 in both untreated and DCE-treated mice. Immunoreactivity for [C] was also detected in the bronchiolar epithelium in nontumor-bearing lung and uninvolved tissue of tumor-bearing lung of DCE-treated mice and was reduced in hyperplasias, adenomas, or carcinomas. Thus, there was a coincidence between the sites of CYP2E1 expression and [C] formation. Conjugate [C] accumulated only in lung cells in which CYP2E1 was expressed. Histochemical staining for glutathione confirmed its presence in tumor foci. Thus, bioactivation and conjugation of DCE occur in structurally normal tissue from both nontumor- and tumor-bearing lung but was lost in tumor tissue, irrespective of the stage of tumor development.

Adenoma↗
PubMed1999

Inactivation of cytochrome P-450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes by vinyl carbamate in murine pulmonary microsomes.

We tested the hypothesis that vinyl carbamate (VC) is metabolized in vitro by cytochrome P-450 and carboxylesterase enzymes in murine lung. Incubations with VC and an NADPH-generating system produced a 50% decrease in N-nitrosodimethylamine (NDMA) demethylation and a corresponding loss in the amounts of immunodetectable CYP2E1. Preincubation of microsomes with a CYP2E1 inhibitory antibody or the CYP2E1-selective inhibitor diallyl sulfone (DASO2) inhibited demethylase activity; no alterations were detected upon subsequent exposure to VC. Carboxylesterase-mediated hydrolysis of p-nitrophenyl acetate was reduced by 22% in microsomes incubated with VC. Decreased carboxylesterase activity also was detected in microsomes incubated with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of hydrolase A, a carboxylesterase isozyme. No change in enzyme activity was detected when microsomes were subsequently incubated with VC. The loss in carboxylesterase activity correlated with decreased immunodetectable hydrolase A in microsomes incubated with VC, PMSF, or PMSF and VC. The reduction in VC-induced NDMA demethylase activity was increased to 85% of the control in microsomes previously incubated with PMSF, and this corresponded with a marked decrease in CYP2E1 immunoreactivity in the immunoblots. Covalent binding of VC to proteins was detected in microsomes incubated with VC and an NADPH-generating system. Binding was inhibited in microsomes preincubated with either an inhibitory CYP2E1 antibody or DASO2. In contrast, binding levels were augmented in microsomes preincubated with PMSF. These data supported VC metabolism by CYP2E1 and hydrolase A in murine lung microsomes and is consistent with involvement of CYP2E1 and hydrolase A in the activation and detoxication of VC, respectively.

Allyl Compounds↗
PubMed1998

Isozyme-selective metabolism of ethyl carbamate by cytochrome P450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes in murine liver microsomes.

Cytochrome P450 and carboxylesterase enzymes have been implicated in the metabolism of the carcinogen ethyl carbamate (EC). In this study, we have used a murine liver microsomal system to investigate the relative contributions of P450 and carboxylesterase isozymes to hepatic metabolism of EC. N-Nitrosodimethylamine (NDMA) demethylation and p-nitrophenyl acetate (PNA) hydrolysis were used as catalytic markers of CYP2E1 and carboxylesterase enzymes, respectively. Incubation of liver microsomes with EC (1 mM) produced slight but significant decreases in NDMA demethylation and PNA hydrolysis activities. Incubation of microsomes with paraoxon (PAX), a general carboxylesterase inhibitor, or phenylmethylsulfonyl fluoride (PMSF), a specific inhibitor of hydrolase A, produced decreases of 85 and 45%, respectively, in carboxylesterase activities; neither of the inhibitors elicited alterations in levels of NDMA demethylation. Reaction of microsomes with either PAX or PMSF and then with EC exacerbated the reduction (285%) of NDMA demethylation, and this loss corresponded to decreases in immunodetectable CYP2E1 content. The reduction in PNA hydrolysis activity induced by PAX, PMSF, or EC correlated with decreased immunodetectable hydrolase A in liver microsomes; however, reaction with PAX and not PMSF or EC resulted in loss of immunoreactivity for hydrolase B. These data correlated with levels of covalent binding of [ethyl-14C]EC to liver microsomes, which were significantly elevated in incubations conducted with PAX or PMSF. Antibody inhibition of the CYP2E1 enzyme significantly reduced levels of binding to microsomal proteins, compared with control levels. These results are consistent with the premise that EC is metabolized by CYP2E1 and hydrolase A in liver microsomes of mice.

Animals↗
PubMed1997

Conjugation of glutathione with the reactive metabolites of 1, 1-dichloroethylene in murine lung and liver.

Exposure to 1,1-dichloroethylene (DCE) elicits lung and liver cytotoxicities that are manifested in bronchiolar Clara cell injury and centrilobular necrosis, respectively. The tissue damage is associated with cytochrome P450-dependent bioactivation of DCE to reactive intermediates, and is consistent with the finding that the target cells coincided with the sites of high concentrations of cytochrome P450 enzymes. The metabolites formed from DCE bind covalently to cellular macromolecules, and the extent of binding and cell damage are inversely related to the content of intracellular glutathione (GSH). Histochemical studies showed that staining for GSH in the lung is localized in the bronchiolar epithelial and alveolar septal cells, with relatively strong staining in the Clara cells. In the liver, staining is observed rather uniformly in hepatocytes distributed across the hepatic lobule. Depletion of GSH correlates with the Clara cell damage and centrilobular necrosis observed in the lung and liver, respectively. The primary metabolites of DCE formed in lung and liver microsomal incubations have been identified as DCE-epoxide, 2,2-dichloroacetaldehyde and 2-chloroacetyl chloride. All are electrophilic metabolites that form secondary reactions including conjugation with GSH. Results of our studies indicated that the DCE-epoxide is the major metabolite forming conjugates with GSH, and this reaction is likely responsible for the depletion of GSH observed in vivo. Our findings support the premise that, following depletion of intracellular GSH, metabolites of DCE including the DCE-epoxide bind to cellular proteins, a process which leads to cell damage and suggests that conjugation with the thiol nucleophile represents a-detoxification mechanism.

Animals↗
PubMed1997

Coexpression of Ah receptor and CYP1A1 in hepatocytes of C57BL/6J and DBA/2J mice.

The distribution of the hepatic aryl hydrocarbon receptor (AhR) and CYP1A1 was investigated in control and 3-methylcholanthrene (3-MC)-treated C57BL/6J (B6) and DBA/2J (D2) mice. The AhR was identified as protein bands of 95 and 104 kDa in cytosol of B6 and D2 mice, respectively, while a protein of 93 kDa was detected in nuclear extracts from both murine strains. CYP1A1 was recognized as a protein of 56 kDa in microsomes from B6 and D2 mice. Lower amounts of immunodetectable AhR and CYP1A1 were observed in D2 mice, compared to B6 mice. Immunohistochemical studies were used to colocalize the AhR and CYP1A1 in adjacent liver sections. Staining for the AhR was localized most prominently in the cytoplasm of centrilobular hepatocytes and was minimal in the nuclei. Periportal hepatocytes were also reactive for the AhR, but the staining was usually localized in the nuclei. Other hepatocytes contained moderate amounts of cytoplasmic staining, whereas nuclear staining was present at low levels. Immunodetectable CYP1A1 also predominated in centrilobular hepatocytes and was negligible in periportal hepatocytes. The staining was exclusively cytoplasmic and was not seen in the nuclei of hepatocytes. Observations of adjacent liver sections revealed that the AhR and CYP1A1 were coexpressed in individual hepatocytes. A similar regional distribution for the AhR and CYP1A1 was manifested in B6 and D2 mice, with the exception that staining was more pronounced in the former under both control and induced conditions. These results showed heterogeneity in the distribution of the AhR and CYP1A1, with comparable levels of both proteins residing in the same hepatocytes. They further demonstrated that the AhR is localized in both the cytoplasm and nuclei of hepatocytes.

Animals↗
PubMed1997

Metabolism of ethyl carbamate by pulmonary cytochrome P450 and carboxylesterase isozymes: involvement of CYP2E1 and hydrolase A.

The lung is highly susceptible to ethyl carbamate (EC)-induced tumorigenesis. Our goal in this study was to investigate the in vitro isozyme-selective metabolism of EC in lung microsomes by cytochrome P450 and carboxylesterase enzymes. Our results showed that incubations with EC produced significant reduction in p-nitrophenol (PNP) hydroxylation and N-nitrosodimethylamine (NDMA) demethylation; there were no alterations in 7-pentoxyresorufin- and 7-ethoxyresorufin O-dealkylase activities. Reaction of microsomes with an inhibitory CYP2E1 antibody and subsequent reaction with EC abolished the EC-induced diminution in NDMA demethylase activity. Carboxylesterase activity, as assessed by hydrolysis of p-nitrophenyl acetate, was significantly decreased in microsomes incubated with EC. Reactions with EC in conjunction with the carboxylesterase inhibitors, paraoxon (PAX) or phenylmethylsulfonyl fluoride (PMSF), abolished the EC-induced decrease in carboxylesterase activity; PAX is a broad-spectrum carboxylesterase inhibitor, whereas PMSF is a specific inhibitor of hydrolase A, a carboxylesterase isozyme. Incubations of EC in combination with either PAX or PMSF exacerbated the EC-induced reduction in PNP hydroxylase and NDMA demethylase activities. Alterations in immunodetectable CYP2E1 protein levels were not apparent in microsomes incubated with EC alone, but the amounts were decreased in reactions with EC in conjunction with either PAX or PMSF. Immunoblotting with antibodies for the carboxylesterase isozymes, hydrolase A and B, revealed loss of immunodetectable hydrolase A in microsomes incubated with EC, PAX, or PMSF. However, immunodetectable hydrolase B was only decreased in microsomes reacted with PAX but not with PMSF or EC. These findings correlated with our covalent binding data, which showed that levels of binding of [14C-ethyl]EC to lung microsomes were significantly higher in incubations conducted in conjunction with PAX or PMSF, compared with control levels. Antibody inhibition of the CYP2E1 enzyme significantly reduced the extent of binding. Our results demonstrated that EC metabolism in lung microsomes, as estimated from magnitudes of covalent binding, is mediated by the P450 isozyme CYP2E1 and the carboxylesterase isozyme hydrolase A.

Animals↗