Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

Abstract

Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

Figure 1. Inhibition of CYP1A1 and cytochrome P450 reductase by CEES

Panels A and B. Effects of CEES on CYP1A1 activity. In panel A, human recombinant CYP1A1 co-expressed with recombinant cytochrome P450 reductase was pre-treated with CEES (0.1–10 mM) or vehicle control for 1 h and then analyzed for CYP1A1 by EROD activity. In panel B, rat liver microsomes from β-naphthoflavone-treated rats, which contain CYP1A1, were treated with 10 mM CEES to inhibit EROD activity. After 1 hr, cytochrome P450 reductase containing microsomes (3.6 or 10.8 U) from saline treated rats, which contain low or no CYP1A1, were added to the CEES-treated microsomes to restore CYP1A1 activity. The inset compares EROD activity in control- and 10 mM CEES-treated microsomes from β-naphthoflavone-treated rats. Panel C. Effects of CEES on NADPH utilization by human recombinant cytochrome P450 reductase. NADPH utilization was monitored in enzyme assays as described in Panel A. Panel D. Rat liver microsomes from corn oil-treated rats were treated with CEES (0.1–10 mM) and then analyzed for cytochrome P450 reductase activity using the cytochrome c reduction assay. Each point is the mean ± SE, n = 3.

Figure 2. Inhibition of human recombinant cytochrome P450 reductase by CEES

Recombinant human cytochrome P450 reductase was pretreated with CEES (0.1–10 mM) or vehicle control. After 1 hr, enzyme activity was assessed by reduction of cytochrome c (Panel A), reduction of DCPIP (Panel B), utilization of NADPH during redox cycling (Panel C), or production of hydrogen peroxide during redox cycling (Panel D). In panels A and C, the quinone menadione (10 μM) was used to stimulate redox cycling. Each point is the mean ± SE, n = 3.

Figure 3. Inhibition of cytochrome P450 reductase by CEES in type II lung epithelial cells

MLE-15 cells were treated with increasing concentrations of CEES (0.1–10 mM) or vehicle control. After 1 hr, lysates were prepared and assayed for cytochrome P450 reductase-mediated reduction of cytochrome c (Panel A), or chemical redox cycling in the presence of 10 μM menadione (Panel B). Lysates from type II cells were also treated with CEES and then assayed for chemical redox cycling (Panel C) or their ability to reduce DCPIP (Panel D).

Figure 4. CEES stimulates the production of ROS by cytochrome P450 reductase

Panel A. Basal production of hydrogen peroxide. Recombinant human cytochrome P450 reductase (1 or 2.5 U/ml) was incubated with and without NADPH (100 μM). Hydrogen peroxide formation was quantified using Amplex Red. Panel B. CEES increases hydrogen peroxide formation. Recombinant NADPH cytochrome P450 reductase (1 U/ml) was treated with vehicle control or increasing concentrations of CEES (0.1–10 mM) and then analyzed for its ability to produce hydrogen peroxide. Values are expressed as percentage increase relative to control. Each point is the mean ± SE, n = 3. *Significantly different (p < 0.05) from control.