Screening of dioxin-like toxicity equivalents for various matrices with wildtype and recombinant rat hepatoma H4IIE cells.
Determination of dioxin-like compounds utilizing in vitro bioassays such as ethoxyresorufin-O-deethylase (EROD) or chemical-activated luciferase expression (DR-CALUX) is an important tool to evaluate their Ah receptor-mediated toxic effects. The aim of this study is to describe advantages and limitations of these bioassays using rat hepatoma (H4IIE) wildtype and recombinant H4IIE cells in a 96 well microtiter plate format. We are using these bioassays for the evaluation of relative responses (REP) from several congeners of dioxins (e.g., 2,3,4,7,8-PCDF) or dioxin-like compounds (PCB-126, 2,3,4,7,8-PBDF) to 2,3,7,8-TCDD. In addition, total toxic equivalency factors (TEFs) of mixtures of these dioxin-like compounds from several kinds of matrices such as feed, sediment, or thermal waste residues are measured by both bioassays and additional chemical analysis. These samples were measured in a cross-validation study between two laboratories using the DR-CALUX technology in comparison to the H4IIE-EROD assay and chemical analysis. Improvement of the quality criteria of the newly developed DR-CALUX bioassays in comparison to the EROD bioassay was demonstrated (higher coefficient of determination r(2); better repeatability of TCDD and samples), while induction factor, limit of detection, and limit of quantification have been similar. The tested samples showed positive responses in both bioassays using different kinetics (EROD: 72 h; DR-CALUX: 24 h). Ratio of measured toxicity equivalent (TEQ) values varied around mean values of 0.89 (comparing both DR-CALUX laboratories, ranging from 0.68-3.1), 2.0 (comparing EROD and DR-CALUX, ranging from 0.57-8.1), and 1.6-2.5 (comparing EROD-TEQ and I-TEQ, mean 1.6, ranging from 1.0-3.9; for DR-CALUX/I-TEQ, 2.5; 0.61-8.3), respectively. This demonstrates that these bioassays can be used as alternative screening technology for monitoring I-TEQ values in various standards and matrices.
Behnisch
Peter A
PA
Life Science Research Laboratories, Kaneka Corporation, Takasago, Hyogo 676-8688, Japan. peter_behnisch@sgs.com
Hosoe
Kazunori
K
Brouwer
Abraham
A
Sakai
Shin-Ichi
S
D000818:Animals; D001681:Biological Assay; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D004147:Dioxins / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000235:genetics; D008113:Liver Neoplasms / Q000201:enzymology / Q000473:pathology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000473:pathology*; D008156:Luciferases / Q000235:genetics; D011786:Quality Control; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D004147:Dioxins; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1