Is a liver comparable to a liver? A comparison of different rat-derived S9-fractions with a biotechnological animal-free alternative in the Ames fluctuation assay.

Affiliation

Brendt J(1), Crawford SE(2), Velki M(3), Xiao H(4), Thalmann B(5), Hollert H(6), Schiwy A(7).
Author information:
(1)Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
(2)Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; Department of Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, Max-von-Laue-Str. 13, 60438 Frankfurt am Main, Germany.
(3)Department of Biology, Josip Juraj Strossmayer University of Osijek, Cara Hadrijana 8/A, 31000 Osijek, Croatia.
(4)Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; EWOMIS GmbH, Schießstrasse 26c, 63486 Bruchköbel, Germany.
(5)EWOMIS GmbH, Schießstrasse 26c, 63486 Bruchköbel, Germany.
(6)Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; EWOMIS GmbH, Schießstrasse 26c, 63486 Bruchköbel, Germany; Department of Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, Max-von-Laue-Str. 13, 60438 Frankfurt am Main, Germany. Electronic address: [Email]
(7)Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; EWOMIS GmbH, Schießstrasse 26c, 63486 Bruchköbel, Germany; Department of Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, Max-von-Laue-Str. 13, 60438 Frankfurt am Main, Germany.

Abstract

Metabolism has to be considered during the toxicological assessment of chemical and environmental samples because it is an important process in the mammalian liver. It can be assessed in vitro via liver homogenates called S9-fractions, an external metabolic activation system. However, the external metabolic activation systems can vary greatly in their composition due to biological variations among individual animals and animal strains that the S9-fraction are derived as well as the differences in the production treatment. To gain more insight into these variances, three different but commonly used rat-derived S9-fractions were compared in the present study for their variance and performance with a reference compound in the Ames fluctuation assay with Salmonella typhimurium strains TA 98 and TA 100 according to ISO 11350. Severe shortcomings of conventional rat-derived S9-fractions were observed in the present study, such that S9-fractions differed significantly within the same rat strain and for different types of induction procedures in regards to the metabolic capability. An intrinsic mutagenic potential of the three rat-derived S9-fractions were identified in the Ames fluctuation assay with varying S9-fraction concentrations. To address some of the shortcomings of the animal-derived S9-fraction, the present study investigated the use and performance of a biotechnological, animal-free alternative, ewoS9R, in comparison to one of the rat-derived S9-fraction as the others showed a mutagenic potential themselves. Specifically, 12 different chemicals were used as a reference to determine if ewoS9R could serve as an adequate and more consistent replacement of traditional rat-derived metabolic activation systems: 8 pro-mutagenic compounds (i.e., require metabolic activation to show a mutagenic potential), one pro-mutagenic compound but not in the tested strains, one mutagenic compound without metabolic activation and two compounds that are equivocal in the literature. EwoS9R was evaluated as a promising approach in the Ames fluctuation assay with 5 compounds observed to have similar results with both rat-derived S9-fraction and ewoS9R (41%), for 3 compounds ewoS9R was a better metabolization system than the rat-derived S9-fraction (16%). Further research is necessary to determine the full potential of ewoS9R in comparison to rat-derived S9-fractions.