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Biotransformation of pollycyclic aromatic hydrocarbons by trout liver S9 fractions: Evaluation of competitive inhibition using a substrate depletion approach
Citation:
Nichols, J., M. Ladd, A. Hoffman, AND P. Fitzsimmons. Biotransformation of pollycyclic aromatic hydrocarbons by trout liver S9 fractions: Evaluation of competitive inhibition using a substrate depletion approach. Fish Biotransformation in Bioaccumulation: Technical Workshop, sponsored by HESI, Washington, D.C, October 01 - 03, 2019.
Impact/Purpose:
In this study we used an in vitro substrate depletion approach to evaluate competitive inhibition of biotransformation that occurs when polycyclic aromatic hydrocarbons (PAHs) are tested as binary mixtures. The results indicate that such inhibition occurs and that these interactions may be described using a classical competitive inhibition model. The methods outlined in this study may be used to evaluate the potential for competitive inhibition of PAH metabolism that occurs in a natural setting or controlled laboratory exposures. Because PAHs almost always occur in the environment as part of a complex chemical mixture, the potential for such inhibition, and by extension the potential for such effects to impact the persistence and bioaccumulation of these compounds is highly relevant.
Description:
Environmental contaminants frequently occur as part of a chemical mixture, potentially resulting in competitive inhibition among multiple substrates metabolized by the same enzyme. Trout liver S9 fractions were used to evaluate the biotransformation of three polycyclic aromatic hydrocarbons (PAHs): phenanthrene (PHEN), pyrene (PYR) and benzo[a]pyrene (BAP), tested as binary mixtures. Initial rates of biotransformation were determined using a substrate depletion approach. The resulting data were then fitted by simultaneous non-linear regression to a competitive inhibition model. In each case, the PAH possessing the lower Michaelis-Menten affinity constant (KM) competitively inhibited biotransformation of the other compound. Inhibition constants (Ki) determined for the lower-KM compound were generally close to previously determined KM values, consistent with the suggestion that phase I biotransformation of PAHs is largely catalyzed by one, or a small number of cytochrome P450 enzymes. The use of a substrate depletion approach to perform enzyme inhibition studies imposes practical limitations on experimental design and complicates the interpretation of derived kinetic constants. Nevertheless, the resulting information may have utility for chemical hazard assessments as well as the design and interpretation of controlled laboratory studies. Depletion experiments informed by measured chemical concentrations in tissues may also provide a means of determining whether enzyme inhibition occurs under relevant environmental conditions.