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J Doehmer

Publications and source records attributed to J Doehmer.

At least 37 records · Page 2Linked to original sources
PubMed1996

Sequence of the rat PB-inducible CYP2B1 promoter.

The 5' flanking region of the rat PB-inducible CYP2B1 gene was isolated and the sequence from +27 to -3878 was determined. This sequence contains several putative binding sites for the liver-enriched factors HNF3 as well as an AP1, two NF-kappa B and several possible STAT sites. The promoter sequence of the CYP2B1 gene was compared to that of the CYP2B2 sequence, published by Hoffman et al. ((1992) Gene Exp. 2, 353-362) and was found to be almost identical up to -2300 bp, beyond which it diverges significantly from the remaining published sequence of CYP2b2 gene. Transient transfection experiments in the differentiated hepatoma cell line, FGC4, showed that the 3.9 kb promoter was expressed, however, an increase in reporter activity was not observed in the presence of phenobarbital.

Animals↗
PubMed1996

The use of macroporous microcarriers for the large-scale growth of V79 cells genetically designed to express single human cytochrome P450 isoenzymes and for the characterization of the expressed cytochrome P450.

In this study, macroporous microcarriers were used for the large-scale growth of parental V79 cells and V79 cells genetically engineered to express a single human cytochrome P4501A1 isoenzyme (V79h1A1). Starting from 2 x 10(5) cells/ml, approximately 1 x 10(7) cells/ml could easily be harvested after 6 days in the case of the V79h1A1 cells, resulting in a total of 3.6 x 10(10) cells. For the first time, the presence of cytochrome P450 (CYP) in the expressed V79 cells could be demonstrated by CO difference spectra with a Soret maximum around 450 nm. CYP levels in microsomes derived from the V79h1A1 cells of 14 pmol/mg protein were achieved. Importantly, no CYP was detected in microsomal fractions of the parental V79 cells. Cytochrome b5 levels could also be measured by difference spectrophotometry. No significant differences were found between cytochrome b5 levels in microsomes derived from the large-scale growth of V79h1A1 cells and parental V79 cells, i.e., 16.7 +/- 7.9 vs 14.5 +/- 7.6 pmol/mg protein. The presence of human cytochrome P4501A1 (CYPh1A1) in microsomal fractions derived from the large-scale growth of V79h1A1 cells was further substantiated by measuring 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-dealkylase (ECOD), and testosterone-6 beta-hydroxylation activities. EROD, ECOD, and testosterone-6 beta-hydroxylation activities of the V79h1A1 microsomes were 40 pmol resorufin/min/pmol CYPh1A1, 13 pmol hydroxy-coumarin/min/pmol CYPh1A1, and 0.16 pmol 6 beta-hydroxytestosterone/min/pmol CYPh1A1, respectively, indicating the presence of a highly active human CYP1A1 enzyme system. Further confirmation that the CYP protein was correctly expressed was obtained by Western blotting. In conclusion, the use of macroporous microcarriers is suitable for large-scale growth of V79 cells expressing human CYP isoenzymes. The present method may provide an easy and rather inexpensive tool in obtaining large quantities of microsomes containing human CYP isoenzymes, which are involved in the bioactivation and bioinactivation of xenobiotics. High yields of microsomes containing human CYP isoenzymes may substantially facilitate the production of sufficient quantities of human metabolites to allow isolation and identification in an early stage of development of pharmacologically interesting drugs.

Animals↗
PubMed1996

Paracetamol-induced spindle disturbances in V79 cells with and without expression of human CYP1A2.

Spindle disturbing effects in terms of c-mitosis and cytotoxicity of paracetamol were investigated in two Chinese hamster V79 cell lines, one of which (V79MZh1A2) was transfected with human CYP1A2. This enzyme catalyses the oxidative formation of the reactive paracetamol metabolite, NAPQI, believed to initiate hepatoxicity by covalent binding to proteins after overdose. In the native V79 cell line paracetamol increased c-mitosis frequency in a concentration dependent manner from 8.7 + or - 3.5% (control) to 66 + or - 18% at 20 mM. A significant increase to 13.3 + or - 3.5% was first seen at 2.5 mM in the native cell line (P<0.05). In the V79MZh1A2 cells the concentration-effect curve was slightly shifted to the left (P<0.05) with c-mitosis frequency increased to 12.1 + or - 2.6% (P<0.05) at 1 mM paracetamol. At 5 mM paracetamol the c-mitosis frequency was 14.4 + or - 5.0% and 19.0 + or - 3.8% in the native and CYP1A2 expressing cell lines, respectively (P<0.05). At 20 mM paracetamol the c-mitosis frequency was 61 + or - 10% in the V79MZh1A2 cells. Cell survival was reduced to approximately 50% at 5-10 mM paracetamol in both cell lines. At 20 mM paracetamol survival was further decreased to 39 + or - 9% in V79MZh1A2 cells only (P<0.05). The present study demonstrated that paracetamol may disturb the spindle of dividing cells conveying a risk of aneuploidy. The spindle disturbing effect was only slightly enhanced by expression of CYP1A2, suggesting that metabolic activation plays only a minor role in this genotoxic effect. The reduction of survival mirrored the increase in c-mitosis frequency.

Acetaminophen↗
PubMed1996

Stable expression and coexpression of human cytochrome P450 oxidoreductase and cytochrome P450 1A2 in V79 Chinese hamster cells: sensitivity to quinones and biotransformation of 7-alkoxyresorufins and triazines.

V79 Chinese hamster cell lines were genetically engineered for the stable expression of human NADPH-cytochrome P450 oxidoreductase (CYPOR) alone or for the combined expression of CYPOR and human cytochrome P450 1A2 (CYP1A2). As determined by immunoblotting, the expression level of CYP1A2 in the latter cell line was found to be the same as in a previously constructed V79 cell line expressing CYP1A2 only. The heterologous expression of CYPOR in V79 cells resulted in increased sensitivity to quinone-type cytotoxins, e.g. duroquinone and menadione, that exert their toxicity primarily through the production of reactive oxygen species during redox cycling. The metabolic properties of the cell line expressing both CYPOR and CYP1A2 were characterized regarding dealkylation and deethylation of 7-alkoxyresorufins and sulfoxidation of the triazine derivatives ametryne and terbutryne, in comparison with the cell line expressing only CYP1A2. Increased CYPOR activity impaired the CYP1A2-dependent fluorometric resorufin assay, presumably by conversion of the 7-alkoxyresorufins and resorufin to their one-electron-reduced semiquinoneimine forms. The CYP1A2-dependent metabolism of the triazine derivatives ametryne and terbutryne was moderately enhanced by increased CYPOR activity. Interestingly, with CYPOR overexpression sulfoxidation was increased 2-3-fold, compared with N-deethylation, with a 1.3-1.9-fold increase. Thus, the level of CYPOR not only had an influence on CYP1A2 activity rates but also affected the relative proportions of metabolites in CYP1A2-specific metabolite profiles.

Animals↗
PubMed1995

The cytotoxicity of mitomycin C and adriamycin in genetically engineered V79 cell lines and freshly isolated rat hepatocytes.

The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and beta-naphthoflavone (beta NF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytotoxicity was observed in V79, SD1 and XEM2 cells. Addition of metyrapone (MP, an inhibitor of P450IIB1) and alpha-naphthoflavone (alpha NF, an inhibitor of P450IA1) had no effect on the ADR-induced cytotoxicity in SD1 and XEM2 cells, respectively. Likewise, MMC was equitoxic in V79 and SD1 cells. Co-incubation of SD1 cells with MP did not alter MMC-induced cytotoxicity. MMC, however, showed a decreased cytotoxicity in XEM2 cells when compared to the parental V79 cells. Unexpectedly, the cytotoxicity of MMC in XEM2 cells was increased by alpha NF to the same level as observed in the parental V79 cells. In contrast to V79- and V79-derived cells, in freshly isolated hepatocytes from PB or beta NF-induced rats, MMC was cytotoxic (measured as lactate dehydrogenase leakage) within 3 h of incubation. ADR, however, was only cytotoxic to the hepatocytes when intracellular glutathione was first depleted by diethylmaleate. The MMC- and ADR-induced cytotoxicity was found to be more pronounced in PB-hepatocytes than in beta NF-hepatocytes. Contrary to the findings in the V79-derived cells, MP afforded complete protection against both MMC- and ADR-induced cytotoxicity in PB-hepatocytes, whereas alpha NF only partially inhibited the cytotoxicity of MMC in beta NF-hepatocytes. In conclusion, we have demonstrated that PB-inducible P450s play a role in the cytotoxicity of both MMC and ADR in freshly isolated PB-hepatocytes but that P450IIB1 does not in genetically reconstituted SD1 cells. P450IA1, however, decreased the cytotoxicity of MMC in the XEM2 cells. The ADR-induced cytotoxicity, which was observed in XEM2 cells, was not mediated by P450IA1. The present study underscores the complexity in the comparison of ADR- and MMC-induced cytotoxicities in normal and tumor cells.

Animals↗
PubMed1995

Inhibition of biotransformation by nitric oxide (NO) overproduction and toxic consequences.

Hepatic nitric oxide (NO) biosynthesis is induced by local or systemic inflammation. The highly reactive NO radical binds to prosthetic iron groups such as heme or iron-sulfur clusters leading to either activation or inhibition of enzymes such as guanylate cyclase, cyclooxygenase and aconitase. It has been known for years that NO also binds to the heme moiety of cytochrome P450s (CYP) with high affinity. However, it was demonstrated recently that binding of NO to CYPs also inhibits their enzymatic activity. This is true for exogenously applied as well as for endogenously synthesized NO. Suppression of CYP-dependent metabolism, which is a major problem of inflammatory liver diseases, can be significantly reversed by inhibition of NO synthesis in vivo under experimental conditions. We investigated whether these findings are applicable as a novel therapeutic principle in severe inflammatory liver dysfunction.

Animals↗
PubMed1995

Genetically engineered mammalian cells and applications.

In general, cells genetically engineered for stable and defined expression of xenobiotic-metabolizing enzymes are useful tools whenever a metabolism-related problem in toxicology and pharmacology is to be solved. It is the genetic and phenotypic nature of a given cell that determines its applicability. Mammalian cells have useful characteristics not given in bacterial, yeast or insect cells, which also may express xenobiotic-metabolizing enzymes. It is the problem to be solved and the question to be answered which determine the optimal choice for the best-suited expression system. There may even be subtle differences between mammalian cells of different species and organ origin, which might play a role in choosing a mammalian expression system. Thus, the level and specificity of the xenobiotic-metabolizing enzyme, the experimental testing conditions, and the biological endpoints present in a chosen cell are the most important criteria to be observed in the application of the mammalian expression systems.

Animals↗
PubMed1995

Cloning and stable expression of the human mitochondrial cytochrome P45011B1 cDNA in V79 Chinese hamster cells and their application for testing of potential inhibitors.

V79 Chinese hamster cells are being genetically engineered to express human mitochondrial cytochromes P450 as an analytical tool for studying adrenal steroid synthesis. Here, a V79 derived cell line is presented expressing the enzymatically active human cytochrome P45011B1 (CYP11B1) in a stable and constitutive manner. Full length CYP11B1 cDNA was obtained from surgically removed normal adrenal gland by polymerase chain reaction. The cDNA was recombined with a SV40 early promoter containing plasmid for stable integration and expression in V79 cells upon gene transfer. The presence of the human CYP11B1 cDNA in the genome of the transfected cells was confirmed by Southern analysis. CYP11B1 cDNA directed expression was detected by Northern analysis. CYP11B1 dependent hydroxylation of deoxycorticosterone and 11-deoxycortisol was measured by HPLC analysis. Interestingly, the nonsteroidogenic lung fibroblast derived V79 Chinese hamster cell line was able to support human CYP11B1 mediated steroid hydroxylation without simultaneous heterologous expression of the human electron transfer system, adrenodoxin, and adrenodoxin reductase. CYP11B1 inhibitory potency of metyrapone, spironolactone, and the azole derivatives ketoconazole, clotrimazole, miconazole, and fluconazole was measured using the newly established V79MZh11B1 cell line. Thus, beside steroid metabolism studies in general, this cell line may also serve as an in vitro tool for monitoring the interference of drugs with 11 beta-hydroxylase activity.

Adrenal Glands↗
PubMed1995

Cytochrome P450 mediated reactions studied in genetically engineered V79 Chinese hamster cells.

V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYP have been shown to serve as analytical tools for studying metabolism related problems in toxicology and pharmacology. Here, the application of rat and human CYP1A1 and CYP1A2 is demonstrated for comparative studies on the oxidation of polycyclic aromatic hydrocarbons, such as phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Live cells were cultivated for 2 days in the presence of these chemicals. Thereafter, the supernatant medium was checked for metabolites by gas chromatography and mass spectrometry. Marked cytochromes P450 and species dependent differences in the metabolite profiles were observed. Most important was the finding, that human cytochrome P450 1A1 almost exclusively oxidized benzo[a]pyrene in the 7,8,9,10-position, yielding the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene whereas the rat cytochrome P450 1A1 oxidized benzo[]pyrene in the 4,5-position and 7,8,9,10-position. The importance of this finding is underlined by results from cytotoxicity studies. Benzo[a]pyrene was twice as cytotoxic in the human cytochrome P450 1A1 than in the rat cytochrome P450 1A1 expressing V79 cells. Species and cytochrome P450 specific metabolite profiles were also observed for phenanthrene and benz[a]anthracene.

Animals↗
PubMed1995

Kinetics and inhibition by fluvoxamine of phenacetin O-deethylation in V79 cells expressing human CYP1A2.

The kinetics of phenacetin O-deethylation and its inhibition by fluvoxamine was investigated in a V79 cell line (V79MZh1A2) transfected with human CYP1A2. In four sets of experiments the apparent Km values for phenacetin O-deethylation ranged from 35 to 95 microM and the Ki for fluvoxamine-mediated inhibition of the reaction ranged from 2.7 to 14.5 nM, i.e. comparable to values obtained in human liver microsomes. The kinetic performance of the V79MZh1A2 cell line demonstrates its usefulness as an analytical tool in a variety of toxicological and drug metabolism studies involving CYP1A2.

Acetaminophen↗
PubMed1994

Inhibition of cytochromes P4501A by nitric oxide.

Inflammatory stimulation of the liver leads to the induction of nitric oxide (NO) biosynthesis. Because NO binds to the catalytic heme moiety of cytochromes P450 (CYPs), we investigated whether NO interferes with specific CYP-dependent metabolic pathways. In a first experimental approach V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYP1A1 and -1A2 were used. Incubation with the NO donors sodium nitroprusside and S-nitrosylacetylpenicillamine led to a concentration-dependent inhibition of all four CYP enzymes. CYP1A1 was more sensitive to the inhibitory effect of NO than CYP1A2. In the second part of the study, endogenous NO synthesis was induced in rat hepatocytes by incubation with a mixture of cytokines and endotoxin. Concurrently, as NO production in hepatocytes increased within 24 hr, a decrease in CYP1A1-dependent benzo[a]pyrene turnover was observed to almost undetectable levels. The competitive inhibitor of NO synthesis, NG-monomethyl-L-arginine, was able to significantly restore CYP1A1 activity in the presence of cytokines and endotoxin. Inhibition of hepatocellular CYP activity by NO was predominantly due to a direct effect on the enzymes. However, NO-dependent inhibition of CYP expression at a transcriptional level was also demonstrated. Our results indicate that inhibition of NO biosynthesis in patients suffering from systemic inflammatory response syndromes may help to restore biotransformation capacity of the liver.

Animals↗
PubMed1994

Biotransformation of the naturally occurring lignan (-)-arctigenin in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms.

V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450 1A1, P450 1A2, and P450 2B1, and human P450 1A2 were used to analyse the metabolism of (-)-arctigenin. Concentrations of metabolites formed in the medium were determined by TLC, HPLC, GC, and GC-MS. Only in the cell line expressing rat P450 2B1 was (-)-arctigenin metabolized to a significant extent. The lignan was converted by O-demethylation to (-)-3'-O-demethylarctigenin.

Animals↗
PubMed1994

Arylamines suppress their own activation and that of nitroarenes in V79 Chinese hamster cells by competing for acetyltransferases.

The effect of 2-aminofluorene (2-AF) on the toxicity of 2-aminoanthracene (2-AA) and 1,6-dinitropyrene (1,6-DNP) was studied in N-acetyltransferase-proficient V79-NHr1A2 cells genetically engineered for the expression of cytochrome P4501A2, and in wild-type V79-NH cells. 2-AA inhibited the growth of V79-NHr1A2 cells and induced the formation of micronuclei at concentrations of 0.1 to 1.0 microM, but was virtually without toxic effects at a concentration of 10 microM. Addition of 2-AF protected against the cytotoxic and genotoxic effects elicited by low concentrations of 2-AA. Half-maximum protection was observed at 0.2 to 0.5 microM 2-AF. The arylamine also prevented the cytotoxicity caused by 1,6-DNP in V79-NH cells and completely suppressed the formation of 1-acetylamino-6-nitropyrene from 1,6-DNP in these cells. The results indicate that arylamines and related N-hydroxyarylamines are substrates for the same acetyltransferase in V79-NH cells. In consequence, arylamines are capable of suppressing the activation of their proximate cytotoxic and genotoxic products in these cells and, presumably, in vivo.

Acetyltransferases↗
PubMed1994

Genetically engineered in vitro systems for biotransformation studies.

In order to understand cytochrome P450-mediated metabolism of xenobiotics such as drugs and pollutants, several cell systems are genetically engineered for metabolic competence by cloning cDNAs encoding cytochrome P450 and other enzymes and by heterologous expression in bacterial, yeast, and mammalian cells. Genetically engineered cell systems are defined for the cDNA enzyme function. In conjunction with cell intrinsic properties, these genetically engineered cell systems can be used for the assessment of metabolism-dependent pharmacological and/or toxicological effects.

Animals↗
PubMed1993

V79 Chinese hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies.

V79 Chinese hamster cells are being genetically engineered for stable expression of cytochromes P450 using a SV40 early promoter containing plasmid as the eukaryotic expression vector. V79 cells lack endogenous cytochrome P450 activity. Therefore, genetically engineered V79 cell lines are defined for the cytochrome P450 isoform acquired upon cDNA mediated gene transfer. V79 cells have a longstanding tradition as indicator cells for various biological endpoints, e.g. mutation, chromosomal aberration, cytotoxicity. The genetically engineered V79 cells combine these useful biological endpoints with metabolic competence. In this sense, V79 cell lines genetically engineered for stable expression of cytochromes P450 present newly developed tools for studying and understanding metabolism related problems in toxicology and pharmacology.

Animals↗
PubMed1993

Biotransformation of methylxanthines in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms. Allocation of metabolic pathways to isoforms and inhibitory effects of quinolones.

V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2, P450IIB1, human P450IA2, and rat liver epithelial cells expressing murine P450IA2 were used to allocate metabolic pathways of methylxanthines to specific isoforms and to test the suitability of such cell lines for investigations on drug interactions occurring at the cytochrome expressed. The cell lines were exposed to caffeine and/or theophylline and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. The results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity. Addition of the quinolone antibiotic agents pipemidic acid or pefloxacin, both known to inhibit caffeine metabolism in vivo and in human liver microsomes, reduced formation rates of all metabolites of caffeine in cells expressing rat and human P450IA2. Theophylline was mainly metabolized via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1.

Animals↗