CYP2E1 metabolizes many substrates through cooperative mechanisms; however, the impact of these processes on the bioactivation of mixtures of compounds remains to be explored. We hypothesize that mixtures of acetaminophen and styrene undergo cooperative modulation of CYP2E1 metabolism rather than simple Michaelis-Menten competition. This outcome would then alter the overall bioactivation of styrene and acetaminophen to toxic metabolites and resulting potential toxic response from co-exposures. We previously reported that CYP2E1 metabolism of the common pollutant styrene yields sigmoidal kinetics (positive cooperativity) involving a two-binding site mechanism. Similarly, CYP2E1 metabolism of acetaminophen deviates from Michaelis-Menten kinetics and likely involves multiple binding events. Mixtures of styrene and acetaminophen altered their metabolism through these cooperative mechanisms as demonstrated through in vitro steady-state studies. Acetaminophen activated styrene oxidation at low concentrations up to 3-fold. This effect seems to reflect the ability of acetaminophen to mimic the positive cooperative interactions responsible for styrene activation of its own metabolism. At higher styrene concentrations, the two compounds competed for common binding sites leading to inhibition of styrene metabolism. Overall, the presence of acetaminophen caused a shift of the kinetic profile for styrene from sigmoidal (Hill coefficient 1.5) to hyperbolic (Hill coefficient ~1.0). Conversely, the presence of styrene did not have a significant effect on acetaminophen oxidation. Ongoing studies are further investigating the concentration dependence of this cooperative effect on the formation of metabolites from both substrates. These transformations of organic molecules are only part of the CYP2E1 reaction; this enzyme also generates reactive oxygen species (ROS) through a poorly coupled metabolic reaction. We are currently assessing the impact of cooperativity on the formation of ROS for styrene, acetaminophen, and mixtures of the two compounds. Taken together, these findings will provide crucial mechanistic information about the CYP2E1-dependent toxicity of two biologically important compounds present as a mixture.

CYP2E1 cooperativity impacts bioactivation of mixtures of styrene and acetaminophen

248th National Meeting (2014)

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