Date of Award

Winter 1983

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

Abstract

This study was undertaken to investigate whether the priority pollutant benzidine could be biotransformed by bacteria from natural environments and to determine what effect such biotransformation would have on the mutagenicity of the compound. Bacteria were collected from sites of toxic waste dumping and from estuarine, lake, and ocean locations. Sediment and water samples as well as bacterial isolates were incubated in media containing 100 (mu)g ml('-1 14)C(U)-benzidine (specific activity 6.76 mCi/mmol) supplemented with ammonia, phosphate and ferrous iron in aged seawater (salinity 26('o)/oo). Pure cultures were grown overnight in reduced-nutrient broth and washed prior to resuspending in the same volume of experimental media and incubating at ambient temperature (22-28(DEGREES)C) in darkness with shaking. Detection of metabolites by thin layer chromatography and subsequent autoradiography revealed that benzidine is biotransformed by unacclimated bacteria from polluted sites and from estuaries. Benzidine could not serve as a sole source of carbon and energy for growth. At a low concentration (1 (mu)g ml('-1)) of benzidine all the substrate was transformed to one major product; at 100 (mu)g ml('-1) two transformation products appeared but most of the benzidine remained unchanged. Benzidine was also transformed abiotically to mutagenic compounds but not the same ones seen after bacterial biotransformation. Upon extended incubation, bacteria transformed the metabolites back to benzidine indicating that the biotransformations were ring substitutions but not ring cleavage. Benzidine was mutagenic after microsomal activation in the Ames test, the fluctuation test using Ames tester strain TA98, and the inductest-(beta)-galactosidase assay. The transformation products were mutagenic without requiring prior microsomal activation. The inductest-(beta)-galactosidase assay (Elespuru and Yarmolinsky, 1979) was modified extensively to increase its sensitivity for detecting benzidine as a mutagen.

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