Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene

Schmalix, Wolfgang A.; Mäser, Hildegard; Kiefer, Franz; Reen, Rashmeet K.; Wiebel, Friedrich J.; Gonzalez, Frank J.; Seidel, Albrecht; Glatt, Hansruedi; Greim, Helmut; Doehmer, Johannes

doi:10.1016/0926-6917(93)90052-r

Cited by 47 publications

(26 citation statements)

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“…V79MZ cells transfected with human CYP1A1 or human CYP1B1 were developed by Dr. Johannes Doehmer and colleagues at the Technical Institute for Toxicology, Munich, Germany [18,28]. The human GSTA1 cDNA (generously provided by Dr. C-P. Tu, Penn State University) was ligated into the modified CMV promoter-driven ΔpCEP4 expression vector, and generation of the V79MZh1A1 or V79MZh1B1 cell lines stably expressing human glutathione-S-transferase A1 was by the method of calcium phosphate-mediated transfection followed by hygromycin selection, verification of similar growth rates, and GST analysis as described previously [26].…”

Section: Cell Culture and Cell Linesmentioning

confidence: 99%

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (±)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

Kushman

Kabler

Ahmad

et al. 2007

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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We have used V79MZ hamster lung fibroblasts stably transfected with human cytochrome P450-1A1 (hCYP1A1; cell line designated V79MZh1A1) or P450-1B1 (hCYP1B1; cell line designated V79MZh1B1) alone, or in combination with human GST alpha-1 (hGSTA1), in order to examine GST protection against cytotoxicity and mutagenicity of dibenzo [a,l]pyrene (DBP) and the intermediate dihydrodiol metabolite (+/−)- . At comparable expression levels of hCYP1A1 and hCYP1B1, both DBP and DBPD were more cytotoxic in V79MZ1A1 (IC 50 = 2.7 nM and 0.7 nM, respectively) than in V79MZh1B1 (IC 50 = 6.0 nM and 4.8 nM, respectively). In contrast, both DBP and DBPD were 2-fold to 4-fold more mutagenic in V79MZh1B1 than in V79MZ1A1. Co-expression of hGSTA1 with hCYP1A1 decreased DBP cytotoxicity 2-fold compared to V79MZh1A1 with hCYP1A1 alone, and provided a small, yet still statistically significant, 1.3-fold protection against DBPD. Protection against mutagenicity of these compounds was comparable to that for cytotoxicity in cells expressing hCYP1A1. In V79MZh1B1 cells, co-expression of hGSTA1 conferred up to 5-fold protection against DBP cytotoxicity, and up to 9-fold protection against the (+/−)-DBP-dihydrodiol cytotoxicity relative to the cells expressing hCYP1B1 alone. Co-expression of hGSTA1 also reduced mutagenicity of DBP or its dihydrodiol to a lesser extent (1.3-fold to 1.8-fold) than the protection against cytotoxicity in cells expressing hCYP1B1. These findings demonstrate that the protective efficacy of hGSTA1 against DBP and DBPD toxicity is variable, depending on the compound or metabolite present, the specific cytochrome P450 isozyme expressed, and the specific cellular damage endpoint examined.Keywords glutathione transferase; cytochrome P-450; polycyclic aromatic hydrocarbon; cytotoxicity; mutagenicity; transfection *Corresponding Author: Alan J. Townsend, Ph.D., Biochemistry Department, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem N.C. 27157, Phone (336)-713-7215; FAX (336)-716-7671, E-mail: atown@wfubmc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. . These reactive products include diol-epoxides with potent mutagenic and carcinogenic activity, some of which are substrates for conjugation by isozymes of the glutathione-S-transferase (GST) superfamily of genes [5]. Conjugation with the nucleophilic thiol group of the tripeptide glutathione (GSH) at the electrophilic centers of activated PAH renders their reactive groups such as epoxides less toxic, more water-soluble and hence more readily excretable [5,6]. Conjugation with GSH also fla...

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Section: Cell Culture and Cell Linesmentioning

confidence: 99%

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (±)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

Kushman

Kabler

Ahmad

et al. 2007

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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“…This has been studied most intensively for benzo[a]pyrene (BaP), which is also a USEPA priority PAH. Usually, human and rodent cells have been used [9][10][11][12] and the cytochromes P450s (CYPs), including CYP1A, play important roles in the metabolic activation of BaP [13]. In this article, we have sought to optimize the alamar Blue and CFDA-AM assays for the detection of cytotoxicity arising from cellular metabolism by fish cells.…”

Section: Introductionmentioning

confidence: 99%

Transitory metabolic disruption and cytotoxicity elicited by benzo<[>a<]>pyrene in two cell lines from rainbow trout liver

Schirmer

Chan

Bols

2000

J. Biochem. Mol. Toxicol.

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Two cell lines, RTL‐W1 and R1, from rainbow trout liver were used to investigate the effects of benzo[A]pyrene (BaP). BaP induced a catalytic measure of CYP1A, 7‐ethoxyresorufin‐O‐deethylase (EROD) activity, in the rainbow trout liver cell line RTL‐W1 but not in R1. Geldanamycin inhibited EROD induction by BaP. Potential BaP metabolites, BaP‐7,8‐dihydrodiol (BDP) and 6,12‐BaP quinone (BQ) also induced EROD activity in RTL‐W1. Very low BaP concentrations slightly stimulated cell proliferation in both cell lines. Higher BaP concentrations caused cytotoxicity in RTL‐W1 but not in R1. Cytotoxicity was detected in a cell viability assay with 5‐carboxyfluorescein diacetate acetoxymethyl ester, and as a decline in cell number. In both cell lines, BaP exposure impaired the reduction of the redox dye, alamar Blue (AB). After BaP removal, AB reduction recovered. Similar results were observed with BQ. As AB monitors metabolic activity, this novel phenomenon was termed transitory metabolic disruption. This decline in AB readings that was caused by BaP was ameliorated in RTL‐W1 by α‐naphthoflavone and geldanamycin, which suggests a role for CYP1A, and in R1 by indomethacin, which suggests involvement of prostaglandin‐H‐synthase. The significance of the response to BaP that is detected with AB and whether other PAHs cause it will be interesting future questions. © 2000 John Wiley & Sons, Inc. J Biochem Toxicol 14:262–276, 2000

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“…In particular, Cyp1a2 knockout mice do not have lower ABP-DNA adducts (Tsuneoka et al, 2003) or attenuated ABP-induced methemoglobinemia (Shertzer et al, 2002), nor do these animals display an increased protection against either ABP (Kimura et al, 1999)-or PhIP-induced carcinogenesis (Kimura et al, 2003). Furthermore, Cyp1a1 knockout mice exhibited a slower rate of clearance of benzo[a]pyrene upon oral exposure but a much greater toxicity (Uno et al, 2004), despite in vitro studies that indicate a facilitatory role for CYP1A1 in benzo[a]pyrene toxicity (Schmalix et al, 1993). These studies undoubtedly emphasize the importance of performing in vivo studies in conjunction with in vitro studies to be able to fully understand the potential interplay between multiple enzyme systems, their tissue-specific expression patterns, and the effect of clearance on activation.…”

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confidence: 99%

In Vivo and in Vitro Metabolism of Arylamine Procarcinogens in Acetyltransferase-Deficient Mice

Sugamori

Brenneman²,

Grant³

2006

Drug Metab Dispos

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ABSTRACT:Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a number of aromatic and heterocyclic amines, many of which are procarcinogenic agents. Interestingly, these enzymes are binary in nature, participating in both detoxification and activation reactions, and thus it is unclear what role NATs actually play in either preventing or enhancing toxic responses. The ultimate direction may be substrate-specific and dependent on its tissuespecific metabolism by competing, but genetically variable, drugmetabolizing enzymes. To investigate the effect of N-acetylation on the metabolism of some classical procarcinogenic arylamines, we have used our double knockout Nat1/2(؊/؊) mouse model to test both in vitro activity and the in vivo clearance of some of these agents. As expected, N-acetylation activity was undetectable in tissue cytosol preparations from Nat1/2(؊/؊) mice for 4-aminobiphenyl (ABP) and 2-aminofluorene (AF), whereas significant levels were measured in all wild-type tissue cytosols tested, indicating the widespread metabolism of these agents. Nat1/2(؊/؊) mice displayed a variable response with respect to in vivo pharmacokinetics. AF appeared to be most severely compromised, with a 3-to 4-fold increased area under the curve (AUC), whereas the clearance of ABP was found to be less dependent on N-acetylation, with no difference in ABP-AUC between wild-type and knockout animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine was neither N-acetylated nor was its clearance affected by NAT genotype, signifying a dependence on other drug-metabolizing enzymes. The elucidation of the role that N-acetylation plays in the clearance of procarcinogenic agents is the first step in attempting to correlate metabolism by NATs to toxic outcome prevention or augmentation.

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Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene

Cited by 47 publications

References 54 publications

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (±)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (±)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

Transitory metabolic disruption and cytotoxicity elicited by benzo<[>a<]>pyrene in two cell lines from rainbow trout liver

In Vivo and in Vitro Metabolism of Arylamine Procarcinogens in Acetyltransferase-Deficient Mice

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Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene

Cited by 47 publications

References 54 publications

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (±)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

Cytotoxicity and mutagenicity of dibenzo[a,l]pyrene and (±)-dibenzo[a,l]pyrene-11,12-dihydrodiol in V79MZ cells co-expressing either hCYP1A1 or hCYP1B1 together with human glutathione-S-transferase A1

Transitory metabolic disruption and cytotoxicity elicited by benzo<&lsqb;>a<&rsqb;>pyrene in two cell lines from rainbow trout liver

In Vivo and in Vitro Metabolism of Arylamine Procarcinogens in Acetyltransferase-Deficient Mice

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Transitory metabolic disruption and cytotoxicity elicited by benzo<[>a<]>pyrene in two cell lines from rainbow trout liver