Deviation from additivity in mixture toxicity: relevance of nonlinear dose-response relationships and cell line differences in genotoxicity assays with combinations of chemical mutagens and gamma-radiation.

Abstract

Sublinear dose-response relationships are often seen in toxicity testing, particularly with bioassays for carcinogenicity. This is the result of a superimposition of various effects that modulate and contribute to the process of cancer formation. Examples are saturation of detoxification pathways or DNA repair with increasing dose, or regenerative hyperplasia and indirect DNA damage as a consequence of high-dose cytotoxicity and cell death. The response to a combination treatment can appear to be supra-additive, although it is in fact dose-additive along a sublinear dose-response curve for the single agents. Because environmental exposure of humans is usually in a low-dose range and deviation from linearity is less likely at the low-dose end, combination effects should be tested at the lowest observable effect levels (LOEL) of the components. This principle has been applied to combinations of genotoxic agents in various cellular models. For statistical analysis, all experiments were analyzed for deviation from additivity with an n-factor analysis of variance with an interaction term, n being the number of components tested in combination. Benzo[a]pyrene, benz[a]anthracene, and dibenz[a,c]anthracene were tested at the LOEL, separately and in combination, for the induction of revertants in the Ames test, using Salmonella typhimurium TA100 and rat liver S9 fraction. Combined treatment produced no deviation from additivity. The induction of micronuclei in vitro was investigated with ionizing radiation from a 137Cs source and ethyl methanesulfonate. Mouse lymphoma L5178Y cells revealed a significant 40% supra-additive combination effect in an experiment based on three independent replicates for controls and single and combination treatments. On the other hand, two human lymphoblastoid cell lines (TK6 and WTK1) as well as a pilot study with human primary fibroblasts from fetal lung did not show deviation from additivity. Data derived from one cell line should therefore not be generalized. Regarding the testing of mixtures for deviation from additive toxicity, the suggested experimental protocol is easily followed by toxicologists.

Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro

“…The only study showing agreement with CA is an Ames test with various polycyclic hydrocarbons (38). In that same article, larger effects than predicted by CA were observed for induction of MN by ionising radiation combined with ethyl methanesulphonate; however, this synergism was cell line dependent.…”

Effect of retinol on radiation- and estrogen-induced neoplastic transformation of human breast epithelial cells

“…Changes in expression of gene p53, and also its mutations, cause variations of cellular protein p53 concentration. The finding that gene mutations and changes in the expression form the basis of cancer processes, has prompted molecular epidemiologists to use biomarkers for detecting damaged genes or proteins synthesized under their control in easily available cellular material or systemic liquids (22). Mutations in the suppressor gene p53 are thought to be essential for cancer development.…”

Genotoxic mixtures and dissimilar action: concepts for prediction and assessment

“…The experiences with a mixture of benzo[a]pyrene, benz[a]anthracene and dibenz[a, c]anthracene in a bacterial system (Ames assay) communicated by Lutz et al (2002) show that CA provided good approximations of the observed joint effects, which was explained in terms of the similarity of the mode of mutagenic action of the tested chemicals, i.e. formation of a similar type of DNA adducts.…”