Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays

Rueff, José; Chiapella, Carles; Chipman, J.K.; Darroudi, F.; Silva, ID; Duvergervanbogaert, M.; Fonti, E.; Glatt, Hansruedi; Isern, P; Laires, A.; Léonard, A.; Llagostera, Montserrat; Mossesso, P; Natarajan, A. T.; Palitti, F.; Rodrigues, António Sebastião; Schinoppi, Angelo; Turchi, G.; Werle-Schneider, Gisela

doi:10.1016/0027-5107(95)00246-4

Cited by 62 publications

(11 citation statements)

References 94 publications

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“…Erythrocytes have a variety of enzymatic activities which can detoxify double bond of patulin, changing the molecular structure, which could account for the decrease in genotoxicity. Thus, numerous toxic agents and the presence of erythrocytes in whole blood cultures of lymphocytes may help to explain the the addition of ascorbic acid to apple juice which contains patulin might be of value, particularly in that highly conlower cytotoxicity and genotoxicity of patulin (for a review see Rueff et al, 1996). On the other hand, one cannot rule taminated with this mycotoxin.…”

Section: Resultsmentioning

confidence: 99%

Induction of micronuclei and chromosomal aberrations by the mycotoxin patulin in mammalian cells: role of ascorbic acid as a modulator of patulin clastogenicity

Alves¹

2000

Mutagenesis

109

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Section: Resultsmentioning

confidence: 99%

Induction of micronuclei and chromosomal aberrations by the mycotoxin patulin in mammalian cells: role of ascorbic acid as a modulator of patulin clastogenicity

Alves¹

2000

Mutagenesis

109

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“…In fact, they contain high concentrations of reduced glutathione and glutathione transferases, which participate in the local detoxification of various drugs and chemicals which could damage Hb and membranes [20][21][22]. This property of RBC has been used as a source of metabolic activation in in vitro toxicology studies [23]. In the present study the results suggest that RBC, besides its involvement in the protection of its main components, are involved in the protection of lymphocytes against DEB toxicity, and possibly other cell systems not yet studied.…”

Section: Discussionmentioning

confidence: 99%

Role of haemoglobin in the protection of cultured lymphocytes against diepoxybutane (DEB), assessed by in vitro induced chromosome breakage

Porto

Chiecchio

Gaspar

et al. 2003

Mutation Research/Genetic Toxicology and Environmental Mutagene

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Diepoxybutane (DEB) is an alkylating agent that can be used to assess chromosome instability in repair-deficient subjects. Previous authors investigated the role of red blood cells (RBC) in determining individual susceptibility to DEB in normal healthy donors, and demonstrated that a polymorphic enzyme in RBC, Glutathione S-transferase T1 (GSTT1), is involved in DEB detoxification. In the present work we studied the influence of individual GSTM1 and GSTT1 genotypes and the presence of RBC on the frequency of DEB-induced chromosome breakage in lymphocyte cultures from normal individuals and, in particular, the influence of isolated components of RBC: RBC membranes, RBC lysate, and haemoglobin. Our results confirm that individual GSTT1 genotypes modulate the level of genetic lesions induced by DEB; however, this effect was not sufficient to explain the highly significant variation in chromosome breakage between whole blood and RBC-depleted cultures. We showed that RBC can protect cultured lymphocytes against chromosome breakage induced by DEB and we demonstrated the particular role of haemoglobin in the protective effect.

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“…Gating logic required these events to exhibit propidium iodide-associated fluorescence corresponding to 2n 2 4n DNA content. To limit the influence that mitotic and Thompson et al, 1987;Aeschbacher and Ruch, 1989;Sasaki et al, 1992;Ramsey et al, 1998;Rueff et al, 1996;Knasm€ uller et al, 1999;Kim and Guengerich, 2004 N-Nitrosodiethylamine (DEN) 55-18-5 Clastogen, requires metabolic activation to form alkylating agent (likely involves CYP2E1 which is not highly expressed in rat liver S9); often only positive at high concentrations (10 mM apoptotic cells might have on gH2AX measurements, p-H3 positive cells and highly fluorescent gH2AX-positive events were excluded from analysis [Rogakou et al, 2000;McManus and Hendzel, 2005;Huang et al, 2006]. The p-H3 measurements were the proportion of p-H3-positive events that exhibited propidium iodide-associated fluorescence of 4n and greater DNA content relative to the number of total events with 2n and greater DNA content.…”

Section: Flow Cytometric Analysismentioning

confidence: 99%

γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9

Bernacki

Bryce

Bemis

et al. 2016

Environ and Mol Mutagen

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Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry-based assay (MultiFlow™ DNA Damage Kit—p53, γH2AX, Phospho-Histone H3) can distinguish direct-acting clastogens and aneugens from non-genotoxicants [Environ Mol Mutagen 57(2016)171-189]. The current work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments TK6 cells were exposed to 11 promutagens and 37 presumed non-genotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hrs after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hrs, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and non-genotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 non-genotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories in order to further evaluate the merits and limitations of this approach.

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Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays

Cited by 62 publications

References 94 publications

Induction of micronuclei and chromosomal aberrations by the mycotoxin patulin in mammalian cells: role of ascorbic acid as a modulator of patulin clastogenicity

Induction of micronuclei and chromosomal aberrations by the mycotoxin patulin in mammalian cells: role of ascorbic acid as a modulator of patulin clastogenicity

Role of haemoglobin in the protection of cultured lymphocytes against diepoxybutane (DEB), assessed by in vitro induced chromosome breakage

γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9

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