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S. Alex Villalobos,¹ Michael J. Anderson,¹ Michael S. Denison,² David E. Hinton,¹ Katherine Tullis,² Ian M. Kennedy,³ A. Daniel Jones,⁶ Daniel P. Y. Chang,⁵ GoSu Yang,³ and Peter Kelly⁴

¹Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, ²Department of Environmental Toxicology, ³Department of Mechanical and Aeronautical Engineering, ⁴Department of Chemistry, ⁵Department of Civil and Environmental Engineering, and ⁶Facility for Advanced Instrumentation, University of California, Davis, CA 95616 USA.

Abstract

Conventional chemical analyses of incineration by-products identify compounds of known toxicity but often fail to indicate the presence of other chemicals that may pose health risks. In a previous report, extracts from soot aerosols formed during incomplete combustion of trichloroethylene (TCE) and pyrolysis of plastics exhibited a dioxinlike response when subjected to a keratinocyte assay. To verify this dioxinlike effect, the complete extract, its polar and nonpolar fractions, some containing primarily halogenated aromatic hydrocarbons, were evaluated for toxicity using an embryo assay, for antiestrogenicity using primary liver cell cultures, and for the ability to transform the aryl hydrocarbon receptor into its DNA binding form using liver cytosol in a gel retardation assay. Each of these assays detect dioxinlike effects. Medaka (Oryzias latipes) embryos and primary liver cell cultures of rainbow trout (Oncorhynchus mykiss) were exposed to concentrations of extract ranging from 0.05 to 45 g/l. Cardiotoxicity with pericardial, yolk sac, and adjacent peritoneal edema occurred after exposure of embryos to concentrations of 7 g/l or greater. These same exposure levels were associated with abnormal embryo development and, at the higher concentrations, death. Some of the fractions were toxic but none was as toxic as the whole extract. In liver cells, total cellular protein and cellular lactate dehydrogenase activity were not altered by in vitro exposure to whole extract (0.05-25 g/l) . However, induction of cytochrome P4501A1 protein and ethoxyresorufin O-deethylase activity occurred. In the presence of whole extract, estradiol-dependent vitellogenin synthesis was reduced. Of the fractions, only fraction 1 (nonpolar) showed a similar trend, although vitellogenin synthesis inhibition was not significant. The soot extract and fractions bound to the Ah receptor and showed a significantly positive result in the gel retardation/DNA binding test. Chemical analyses using GC-MS with detection limits for 2,3,7,8-tetrachlorodibenzo-p-dioxin and dibenzofuran in the picomole range did not show presence of these compounds. Our results indicate that other chemicals associated with TCE combustion and not originally targeted for analysis may also pose health risks through dioxinlike mechanisms. Key words: Ah receptor, antiestrogen, complex mixture, dioxinlike toxicity, dioxin-response element binding, embryo/cardiovascular toxicity, incomplete combustion by-products, liver, trichloroethylene, vitellogenin. Environ Health Perspect 104:734-743 (1996)

Address correspondence to I.M. Kennedy, Department of Mechanical and Aeronautical Engineering, University of California, Davis, Davis, CA 95616 USA.

Trout-specific antibodies and standards were gifts from Michael Miller, West Virginia University, and Ray Simon, formerly of the U.S. Fish Health Center, Kearneysville, West Virginia. Anti-scup CYP1A1 (Mab 1-12-3) was a gift of John Stegeman, Woods Hole Oceanographic Institution. 2,3,7,8-Tetrachlorodibenzo-p-dioxin was obtained from S. Safe, Texas A&M University. A.V. thanks Miguel González-Doncel and Swee Teh for their assistance in the histological preparations and evaluations. This research was supported by the NIEHS Superfund Basic Research Program (P42ESO4699) , by the Ecotoxicology Program of the University of California Toxic Substances Research and Teaching Program, and by the US EPA-UC Davis Center for Ecological Health Research (R819658) .

Received 29 March 1996 ; accepted 10 April 1996.