Inhibitory potency of 4-carbon alkanes and alkenes toward CYP2E1 activity

Abstract

CYP2E1 has been implicated in the bioactivation of many small molecules into reactive metabolites which form adducts with proteins and DNA, and thus a better understanding of the molecular determinants of its selectivity are critical for accurate toxicological predictions. In this study, we determined the potency of inhibition of human CYP2E1 for various 4-carbon alkanes, alkenes and alcohols. In addition, known CYP2E1 substrates and inhibitors including 4-methylpyrazole, aniline, and dimethylnitrosamine were included to determine their relative potencies. Of the 1,3-butadiene-derived metabolites studied, 3,4-epoxy-1-butene was the strongest inhibitor with an IC50 of 110 μM compared to 1700 μM and 6600 μM for 1,2-butenediol and 1,2:3,4-diepoxybutane, respectively. Compared to known inhibitors, inhibitory potency of 3,4-epoxy-1-butene is between 4-methylpyrazole (IC50 = 1.8 μM) and dimethylnitrosamine (IC50 = 230 μM). All three butadiene metabolites inhibit CYP2E1 activity through a simple competitive mechanism. Among the 4-carbon compounds studied, the presence and location of polar groups seems to influence inhibitory potency. To further examine this notion, the investigation was extended to include structurally and chemically similar analogues, including propylene oxide and various butane alcohols. Those results demonstrated preferential recognition of CYP2E1 toward the type and location of polar and hydrophobic structural elements. Taken together, CYP2E1 metabolism may be modified in vivo by exposure to 4-carbon compounds, such as drugs, and nutritional constituents, a finding that highlights the complexity of exposure to mixtures.

Keywords: Butadiene; CYP2E1; Cytochrome P450; Epoxide.

Figure 1

Metabolic pathways of 1,3-butadiene. Metabolites included in this study are shown in solid boxes. 1,2-epoxybutane-3,4-diol (shown in dashed box) was not commercially available and was not included in the study as a result.

Figure 2

Structural depiction of analogue compounds tested for importance of features in inhibitory potency with CYP2E1.

Figure 3

Panel A represents IC₅₀ plots for butadiene metabolites with recombinant CYP2E1 enzyme. Panel B represents IC₅₀ plots for butadiene metabolites with human liver microsomes. Nonlinear plots represent the IC₅₀ nonlinear regression for the normalized activity vs. log of inhibitor concentration. Individual tracings represent different metabolites, specifically, EB (circles), BD-diol (triangles), and DEB (squares). Reactions were performed at least four times at 37 °C and pH 7.4. Further reaction conditions are described in Experimental Procedures.

Figure 4

Dixon and Cornish-Bowden plots for epoxide metabolite inhibition of 4-nitrophenol metabolism by recombinant CYP2E1. Linear plots represent the Cornish-Bowden (left) and Dixon (right) transformation of the DynaFit non-linear regression for the reported model of inhibition for each butadiene metabolite. Individual tracings represent different concentrations of 4-nitrophenol, namely, 10 μM 4NP (circles), 25 μM 4NP (squares), 100 μM 4NP (triangles), and 500 μM 4NP (inverted triangles). Inhibitor concentrations were set at 1/3 IC₅₀, IC₅₀, and 3-fold above the IC₅₀. Reactions were performed at least three times at 37 °C and pH 7.4. Further reaction conditions are described in Experimental Procedures.

Figure 5

Dixon and Cornish-Bowden plots for epoxide metabolite inhibition of 4-nitrophenol metabolism by human liver microsomes. Linear plots represent the Cornish-Bowden (left) and Dixon (right) transformation of the DynaFit non-linear regression for the reported model of inhibition for each butadiene metabolite. Individual tracings represent different concentrations of 4-nitrophenol, namely, 10 μM 4NP (circles), 25 μM 4NP (squares), 100 μM 4NP (triangles), and 500 μM 4NP (inverted triangles). Inhibitor concentrations were set at 1/3 IC₅₀, IC₅₀, and 3-fold above the IC₅₀. Reactions were performed at least three times at 37 °C and pH 7.4. Further reaction conditions are described in Experimental Procedures.