scholarly journals 3Rs‐friendly approach to exogenous metabolic activation that supports high‐throughput genetic toxicology testing

2020 ◽  
Vol 61 (4) ◽  
pp. 408-432
Author(s):  
Shuchang Tian ◽  
Aiyana Cyr ◽  
Karen Zeise ◽  
Steven M. Bryce ◽  
Nikki Hall ◽  
...  
Keyword(s):  
High Throughput ◽  
Metabolic Activation ◽  
Genetic Toxicology ◽  
Exogenous Metabolic Activation

High Content Analysis of an In Vitro Model for Metabolic Toxicity

2014 ◽  
Vol 19 (10) ◽  
pp. 1402-1408 ◽  
Author(s):  
Stephanie D. Cole ◽  
Janna S. Madren-Whalley ◽  
Albert P. Li ◽  
Russell Dorsey ◽  
Harry Salem
Keyword(s):  
Content Analysis ◽  
High Throughput ◽  
Metabolic Activation ◽  
Hepatic Metabolism ◽  
Cell Types ◽  
Multiple Organ ◽  
Compound Libraries ◽  
High Content Analysis ◽  
Calcein Am

In vitro models that accurately and rapidly assess hepatotoxicity and the effects of hepatic metabolism on nonliver cell types are needed by the U.S. Department of Defense and the pharmaceutical industry to screen compound libraries. Here, we report the first use of high content analysis on the Integrated Discrete Multiple Organ Co-Culture (IdMOC) system, a high-throughput method for such studies. We cultured 3T3-L1 cells in the presence and absence of primary human hepatocytes, and exposed the cultures to 4-aminophenol and cyclophosphamide, model toxicants that are respectively detoxified and activated by the liver. Following staining with calcein-AM, ethidium homodimer-1, and Hoechst 33342, high content analysis of the cultures revealed four cytotoxic endpoints: fluorescence intensities of calcein-AM and ethidium homodimer-1, nuclear area, and cell density. Using these endpoints, we observed that the cytotoxicity of 4-aminophenol in 3T3-L1 cells in co-culture was less than that observed for 3T3-L1 monocultures, consistent with the known detoxification of 4-aminophenol by hepatocytes. Conversely, cyclophosphamide cytotoxicity for 3T3-L1 cells was enhanced by co-culturing with hepatocytes, consistent with the known metabolic activation of this toxicant. The use of IdMOC plates combined with high content analysis is therefore a multi-endpoint, high-throughput capability for measuring the effects of metabolism on toxicity.

OPTIMIZATION OF AN EXOGENOUS METABOLIC ACTIVATION SYSTEM FOR FETAX. II. PRELIMINARY EVALUATION

2001 ◽  
Vol 24 (2) ◽  
pp. 117-127 ◽  
Author(s):  
Douglas J. Fort ◽  
Robert L. Rogers ◽  
Robbin R. Paul ◽  
Enos L. Stover ◽  
Robert A. Finch
Keyword(s):  
Metabolic Activation ◽  
Preliminary Evaluation ◽  
Activation System ◽  
Exogenous Metabolic Activation

Evaluation and Application of a Bioluminescent Bacterial Genotoxicity Test

1993 ◽  
Vol 76 (4) ◽  
pp. 893-898 ◽  
Author(s):  
Thomas S C Sun ◽  
Henry M Stahr
Keyword(s):  
Aflatoxin B1 ◽  
Vibrio Fischeri ◽  
Fumonisin B1 ◽  
Metabolic Activation ◽  
Good Alternative ◽  
Complex Compounds ◽  
Rapid Screening ◽  
Genotoxicity Test ◽  
Exogenous Metabolic Activation ◽  
Positive Controls

Abstract The Mutatox® test (commercial name for the bioluminescent bacterial genotoxicity test) has been shown to be a good alternative to the Ames test. The test uses dark mutants of luminous bacteria (Vibrio fischeri) and determines the ability of various genotoxic agents to restore the luminescence by inducing mutation. It provides a rapid screening test which can be used to assay the genotoxicity of large numbers of pure and complex compounds. The test is completed in 1 day, and by serially diluting the compound, dose response data plus toxicity data can be generated for a number of samples simultaneously. For the direct assay (without exogenous metabolic activation), the positive controls selected were 3,6-diaminoacridine (proflavine) and N-methyl-N-nitro-nitrosoguanidine. For the S-9 assay, which incorporated the microsome fraction (S-9) from rat liver as an exogenous metabolic activation system, the positive controls selected were aflatoxin B1 and benzo(a)pyrene. This study also indicated that methyl-imidazo-quinoline and tryptophan pyrolysates were genotoxic in the presence of S-9 activation, aflatoxin B1 epoxide and fumonisin B1 showed direct genotoxic activity, and aflatoxin B2 and ochratoxin A were not genotoxic.

scholarly journals Comparative Toxigenicity and Associated Mutagenicity of Aspergillus fumigatus and Aspergillus flavus Group Isolates Collected from the Agricultural Environment

Toxins ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 458
Author(s):  
Caroline Lanier ◽  
David Garon ◽  
Natacha Heutte ◽  
Valérie Kientz ◽  
Véronique André
Keyword(s):  
Ames Test ◽  
Metabolic Activation ◽  
Agricultural Environment ◽  
Activation System ◽  
Mutagenic Response ◽  
Exogenous Metabolic Activation ◽  
Potential Impact ◽  
Potential Interactions ◽  
Aspergillus Flavus Group ◽  
Higher Doses

The mutagenic patterns of A. flavus, A. parasiticus and A. fumigatus extracts were evaluated. These strains of toxigenic Aspergillus were collected from the agricultural environment. The Ames test was performed on Salmonella typhimurium strains TA98, TA100 and TA102, without and with S9mix (exogenous metabolic activation system). These data were compared with the mutagenicity of the corresponding pure mycotoxins tested alone or in reconstituted mixtures with equivalent concentrations, in order to investigate the potential interactions between these molecules and/or other natural metabolites. At least 3 mechanisms are involved in the mutagenic response of these aflatoxins: firstly, the formation of AFB1-8,9-epoxide upon addition of S9mix, secondly the likely formation of oxidative damage as indicated by significant responses in TA102, and thirdly, a direct mutagenicity observed for higher doses of some extracts or associated mycotoxins, which does not therefore involve exogenously activated intermediates. Besides the identified mycotoxins (AFB1, AFB2 and AFM1), additional “natural” compounds contribute to the global mutagenicity of the extracts. On the other hand, AFB2 and AFM1 modulate negatively the mutagenicity of AFB1 when mixed in binary or tertiary mixtures. Thus, the evaluation of the mutagenicity of “natural” mixtures is an integrated parameter that better reflects the potential impact of exposure to toxigenic Aspergilli.

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