Quantitative analysis of cytotoxicity and mutagenicity of benzo[a]pyrene in mammalian cells (CHO/HGPRT system)

Machanoff, Richard; O’Neill, J. Patrick; Hsie, Abraham W.

doi:10.1016/0009-2797(81)90084-3

Cited by 47 publications

(15 citation statements)

References 14 publications

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“…1B) of B(a)P were assayed in detail, with S9 mix in the presence and absence of UDPGA. In the absence of UDPGA, B(a)P is cytotoxic exhibiting a dose-dependent cellular lethality, as we reported earlier [23]. The detoxication of B(a)P by UDPGA is shown by a broad shoulder in the cytotoxicity curve and a reduction of lethality; at 8 and 20 pM of B(a)P in the absence of UDPGA the relative survival is approximately 50% and 8 % , respectively, and with UDPGA the corresponding survival is 90 % and 65 % , respectively.…”

Section: Resultssupporting

confidence: 84%

“…In all subsequent studies, 1.0 mg of UDPGA per flask (final concentration = 0.318 mM) was used to insure adequate UDPGA concentrations. The buffer system for the S9 mix as previously used with the CHO/HGPRT assay [23] was modified by the addition of Tris-HC1 buffer. We found that a combination of two buffers, sodium phosphate (25.0 mM; pH 8.0) and Tris-HC1 (62.5 rnM; pH 7.0) was the optimal buffer system for B(a)P-induced cytotoxicity and detoxication ( Table 11).…”

Section: Resultsmentioning

confidence: 99%

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Glucuronide conjugation reduces the cytotoxicity but not the mutagenicity of benzo(a)pyrene in the CHO/HGPRT assay

Recio

Hsie

1984

Teratog Carcinog Mutagen

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Benzo(a)pyrene (B[a]P) is biotransformed by the mixed-function oxidase (MFO) system to numerous metabolites some of which are cytotoxic and/or mutagenic to mammalian cells. However, conjugation of B(a)P metabolites with glucuronic acid in vivo is a major pathway of detoxication and elimination. The effects of glucuronide conjugation on B(a)P-induced cytotoxicity and mutagenicity were studied using the CHO/HGPRT assay with a rat liver homogenate preparation containing MFO system cofactors (S9 mix) and uridine diphosphate alpha-D-glucuronic acid (UDPGA). B(a)P metabolites proximate to the biologically active B(a)P quinones (B[a]P 6-OH) and to the B(a)P 7,8-diol-9,10 epoxide isomers (B[a]P 7,8-diol), were also assayed with S9 mix in the absence and presence of UDPGA. The addition of UDPGA to S9 mix reduced B(a)P-induced cytotoxicity but did not affect mutagenicity. B(a)P 6-OH-mediated cytotoxicity was also reduced in the presence of UDPGA. UDPGA had no effect on B(a)P 7,8-diol-induced cytotoxicity or mutagenicity. B(a)P phenols have been shown to be the preferred B(a)P-metabolite substrates for UDP-glucuronyltransferase enzymes. Thus, the reduction of B(a)P and B(a)P 6-OH-induced cytotoxicity by glucuronide conjugation is likely due to the elimination of cytotoxic phenols and quinones. Since B(a)P 7,8-diol is a poor substrate for UDP-glucuronyltransferase enzymes, no effects on B(a)P-induced mutagenicity or B(a)P 7,8-diol-induced cytotoxicity and mutagenicity were observed.

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Glucuronide conjugation reduces the cytotoxicity but not the mutagenicity of benzo(a)pyrene in the CHO/HGPRT assay

Recio

Hsie

1984

Teratog Carcinog Mutagen

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“…As with the MLA, the chemical test agents were dissolved in DMSO, and delivered to give a final concentration of 1% DMSO in the treatment medium. S9 mix was prepared as described by Machanoff et al (1981) and used at a final concentration of 0.4 mg S9 protein/ml in the treatment medium. After treatment, the cells were centrifuged and washed twice with calcium-magnesium-free phosphate-buffered saline (PBS), resuspended in 5 ml of growth medium, and incubated for 32 hr so that the vehicle controls would go through 1.5-2.0 cell divisions.…”

Section: Cell Treatment With Tempo-mnmentioning

confidence: 99%

Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells

Guo

Mittelstaedt

Guo

et al. 2013

Toxicology in Vitro

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Abstract2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) is a low molecular weight nitroxide and stable free radical. In this study, we investigated the cytotoxicity and genotoxicity of TEMPO in mammalian cells using the mouse lymphoma assay (MLA) and in vitro micronucleus assay. In the absence of metabolic activation (S9), 3 mM TEMPO produced significant cytotoxicity and marginal mutagenicity in the MLA; in the presence of S9, treatment of mouse lymphoma cells with 1-2 mM TEMPO resulted in dose-dependent decreases of the relative total growth and increases in mutant frequency. Treatment of TK6 human lymphoblastoid cells with 0.9-2.3 mM TEMPO increased the frequency of both micronuclei (a marker for clastogenicity) and hypodiploid nuclei (a marker of aneugenicity) in a dose-dependent manner; greater responses were produced in the presence of S9. Within the dose range tested, TEMPO induced reactive oxygen species and decreased glutathione levels in mouse lymphoma cells. In addition, the majority of TEMPO-induced mutants had loss of heterozygosity at the Tk locus, with allele loss of ≤34 Mbp.These results indicate that TEMPO is mutagenic in the MLA and induces micronuclei and hypodiploid nuclei in TK6 cells. Oxidative stress may account for part of the genotoxicity induced by TEMPO in both cell lines.

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“…The standard S9 is available commercially or can be produced by published methods [29] . The S9 is supplemented with a standard mixture of cofactors as described by Machanoff and co -workers [63] and used at a concentration of 1 -10% v/v in the fi nal test medium. The concentration of S9 is determined by preliminary tests of the cytotoxicity of the S9 and its ability to activate known metabolism -dependent mutagens (see below for activation -dependent controls).…”

Section: Criteria For Negative/vehicle and Positive Controlsmentioning

confidence: 99%