A metabolically competent human cell line expressing five cDNAs encoding procarcinogen-activating enzymes: application to mutagenicity testing

Crespi, Charles L.; Gonzalez, Frank J.; Steimel, Dorothy T.; Turner, Thomas R.; Gelboin, Harry V.; Penman, Bruce W.; Langenbach, Robert

doi:10.1021/tx00023a013

Cited by 136 publications

(53 citation statements)

References 18 publications

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“…Indeed, the dimethylnitrosamine demethylase activity measured in DB-7 cells was about the (Table 1 of Patten et al, 1992). On the other hand, Crespi et al (1991) have used Epstein-Barr virus-based vectors to introduce multiple copies of an episome into B-lymphoblastoid cells. This is a valuable procedure to measure the mutagenic effects of the bioactivation of P450 substrates since it can be rapidly manipulated to express a variety of different P450 isoforms individually or in combination.…”

Section: Level Of Expressionmentioning

confidence: 99%

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Mammalian PC‐12 cell genetically engineered for human cytochrome P450 2E1 expression

Mapoles

Berthou

Alexander

et al. 1993

European Journal of Biochemistry

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The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC-12. This cell line expressed cytochrome b, (58 ? 12 pmol/mg microsomal protein vs 528 k 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140k 20 nmol . min-' . mg microsomal protein-' vs 68 ? 48 nmol . min-' -mg-' in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co-transfected with the plasmid RSVneo into PC-12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6-hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB-7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC-12 cells. Chlorzoxazone 6-hydroxylation, n-butanol oxidation and dimethylnitrosamine N-demethylation were localized in microsomes (62, 60 and 63 pmol * min-' mg microsoma1 protein-', respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB-7 cells has catalytic competence similar to human liver CYP2E1. DB-7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase.Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.

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Section: Level Of Expressionmentioning

confidence: 99%

“…Mammalian cell lines stably transfected with P450 isoforms are also available. Doehmer et al (1991Doehmer et al ( ,1992 have inserted human CYPlAl/lA2 into V-79 cells and Crespi et al, (1991) have expressed CYP2B1/2B2 into the human lymphoblastoid AHHl cells.…”

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confidence: 99%

Mammalian PC‐12 cell genetically engineered for human cytochrome P450 2E1 expression

Mapoles

Berthou

Alexander

et al. 1993

European Journal of Biochemistry

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“…MCL-5 cells stably express cDNAs for human microsomal epoxide hydrolase and four cytochrome P450s (CYP1A2, CYP2A6, CYP2E1, and CYP3A4), in addition to low levels of constitutive, inducible CYP1A1 (Crespi et al, 1991). This cell line is sensitive to several potent animal carcinogens, including dibenzo[a/]pyrene, benzo[a]pyrene, and 1,6-and 1,8-dinitropyrene (Busby et al, 1994a(Busby et al, , 1995.…”

Section: Discussionmentioning

confidence: 99%

“…Because of its widespread environmental occurrence and biological activity, phenalenone could pose a human health hazard. Therefore, experiments were carried out to define further the biological activity of this compound by: (1) confirming its reported mutagenicity in the bacterium S. typhimurium TM677, (2) measuring its mutagenicity in metabolically competent human B-lymphoblastoid cell lines (MCL-5 and hlAlv2 cells) that stably expressed activities for different complements of human liver cytochrome P450s and microsomal epoxide hydrolase (Crespi et al 1991;Penman et al, 1994), and (3) determining its lung tumorigenicity in a newborn mouse assay.…”

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confidence: 99%

Bacterial and Human Cell Mutagenicity and Mouse Lung Tumorigenicity of the Oxygenated Polynuclear Aromatic Hydrocarbon Phenalenone

WANG

Busby

1996

Toxicol Sci

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Phenalenone (perinaphthenone) is a major oxygenated polynuclear aromatic hydrocarbon (oxy-PAH) atmospheric pollutant formed from the combustion of fossil fuels. Mutagenicity of phenalenone was measured in quantitative forward mutation assays with Salmonella typhimurium TM677 and metabolically competent human B-lymphoblastoid cell lines (MCL-S and hlAlv2 cells), and its tumorigenicity was also assessed in a newborn mouse assay. Phenalenone was mutagenic in Salmonella in the presence of rat liver postmitochondrial supernatant (PMS) at a minimum detectable mutagen concentration (MDMC) of 12 /jg/ml, but was not mutagenic in the absence of PMS at concentrations up to 100 /ig/ ml. Phenalenone was not significantly mutagenic in either human cell line after 28 hr treatment, although mutant fractions were increased by nearly fivefold in hlAlv2 cells (at the tk locus) exposed at 30 ^g/ml. However, after 72 hr treatment, phenalenone was mutagenic at the hprt locus in hlAlv2 cells with an MDMC of 3 /jg/mL Phenalenone was also tumorigenic in male BLU:Ha mice with a lung tumor incidence of 33% 6 months after injection with 4.2 mg phenalenone, the highest dose tested. Lung tumor multiplicity in this treatment group was 0.5 tumor/mouse. No increase in lung tumors in female mice was observed. Indices of lung tumor incidence (ED W ) and multiplicity (TMi.o) for male mice were 29.3 and 34.9 /xmol, respectively. These data suggest that phenalenone does not contribute significantly to the mutagenicity or carcinogenicity of combustion emission extracts, c 19% Society of ToxicologyPhenalenone, known also as phenaJen-1 -one and perinaphthenone, is a tricyclic aromatic ketone (Fig. 1) widely distributed in the environment as a pollutant from the combustion of fuels. It has been detected in extracts of urban air particles from Norway (Ramdahl, 1983), Spain (Aceves and Grimalt, 1992), Sweden (Strandell et al, 1994), and the United States, where the average ambient concentration at a single site over a 3-day period was 7.7 jxg/1000 m 3 (Stanley etal., 1969).Phenalenone was the major identified oxy-PAH 3 in the particle extract of emissions from light-duty diesel engines (Lies et al, 1986;Strandell et al, 1994) and from noncatalyst gasoline engines operated under a standard test-driving procedure (Alsberg et al, 1985;Strandell et al, 1994). Phenalenone emissions for both types of engines were approximately 15 jig/km. Phenalenone also was reported as a major polar component of particle extracts from a coal-fired fluidized bed combustor (Chiu et al, 1983), from a domestic natural gas-fired water heater and a space heater (Rogge et al, 1993), and from a residential oil burner (Leary et al, 1983(Leary et al, , 1987. Although it was not detected in the No. 2 fuel oil consumed in the oil burner study, phenalenone may form as an oxidation product in thermally or catalytically cracked middle distillate fuels (such as diesel fuel or number 2 fuel oil) during storage under ambient conditions (Pedley et al., 1988;Marshman, 1990). Because concentratio...

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“…These cells were derived from an L3 variant (30) (32). L3 cells have a 2-fold higher CYPlAI activity and a lower background mutation frequency at the tk locus than AHH-1 cells.…”

Section: Mutagenicity Assaysmentioning

confidence: 99%

Bacterial and human cell mutagenicity study of some C18H10 cyclopenta-fused polycyclic aromatic hydrocarbons associated with fossil fuels combustion.

Lafleur

Longwell²,

Marr³

et al. 1993

Environ Health Perspect

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A number of isomeric C18H10 polycyclic aromatic hydrocarbons (PAHs), thought to be primarily cyclopenta-fused PAHs, are produced during the combustion and pyrolysis of fossil fuels. To determine the importance of their contributions to the total mutagenic activity of combustion and pyrolysis samples in which they are found, we characterized reference quantities of four C18H10 CP-PAHs: benzo[ghi]fluoranthene (BF), cyclopenta[cd]pyrene (CPP), cyclopent[hi]acephenanthrylene (CPAP), and cyclopent[hi]aceanthrylene (CPAA). Synthesis of CPAA and CPAP is described. The availability of reference samples of these isomers also proved to be an essential aid in the identification of the C18H10 species often found in combustion and pyrolysis samples. Chemical analysis of selected combustion and pyrolysis samples showed that CPP was generally the most abundant C18H10 isomer, followed by CPAP and BF. CPAA was detected only in pyrolysis products from pure PAHs. We tested the four C18H10 PAHs for mutagenicity in a forward mutation assay using S. typhimurium. CPP, BF, and CPAA were roughly twice as mutagenic as benzo[a]pyrene (BaP), whereas CPAP was only slightly active. These PAHs were also tested for mutagenic activity in human cells. In this assay, CPP and CPAA were strongly mutagenic but less active than BaP, whereas CPAP and BF were inactive at the dose levels tested. Also, the bacterial and human cell mutagenicity of CPAA and CPAP were compared with the mutagenicity of their monocyclopenta-fused analogs, aceanthrylene and acephenanthyrlene. Although the mutagenicities of CPAP and acephenanthrylene are similar, the mutagenic activity of CPAA is an order of magnitude greater than that of aceanthyrlene.

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A metabolically competent human cell line expressing five cDNAs encoding procarcinogen-activating enzymes: application to mutagenicity testing

Cited by 136 publications

References 18 publications

Mammalian PC‐12 cell genetically engineered for human cytochrome P450 2E1 expression

Mammalian PC‐12 cell genetically engineered for human cytochrome P450 2E1 expression

Bacterial and Human Cell Mutagenicity and Mouse Lung Tumorigenicity of the Oxygenated Polynuclear Aromatic Hydrocarbon Phenalenone

Bacterial and human cell mutagenicity study of some C18H10 cyclopenta-fused polycyclic aromatic hydrocarbons associated with fossil fuels combustion.

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