Hepatic and pulmonary microsomal benzene metabolism in CYP2E1 knockout mice

Abstract

Benzene is an occupational and environmental toxicant. The major health concern for humans is acute myelogenous leukemia. To exert its toxic effects, benzene must be metabolized via cytochrome P450. CYP2E1 has been identified as the most important cytochrome, P450 isozyme in hepatic benzene metabolism in mice, rats, and humans. In pulmonary microsomes CYP2E1 and members of the CYP2F subfamily are both significantly involved. In the current study CYP2E1 knockout mice and wild-type controls were used to further examine the cytochrome P450 isozymes involved in metabolism of 24 μM benzene. The results show that CYP2E1 is the most important isozyme in the liver, accounting for 96% of the total hydroxylated metabolite formation. However, in the lung CYP2E1 was responsible for only 45% of the formation of total hydroxylated metabolite. Chemical inhibitors of CYP2E1 and CYP2F2 were used to further examine the contributions of these isozymes to benzene metabolism. The results confirmed the finding that while CYP2E1 is the most important isozyme in the liver, CYP2F2 and CYP2E1 are both significantly involved in the lung.

Introduction

The main concern for humans exposed to benzene is acute myelogenous leukemia (Infante et al., 1977, Aksoy, 1980, Aksoy, 1981, Yin et al., 1996). There is a mouse model that develops acute leukemia following benzene exposure (Rithidech et al., 1999), but no animal model fully mimics the leukemogenic process observed in humans. Studies have shown an increase in various types of tumors in mice, including lung tumors, (NTP, 1986, Snyder et al., 1988, Maltoni et al., 1989, Farris et al., 1993) and in rats (NTP, 1986, Maltoni et al., 1989) exposed to benzene. A few studies suggest that lung cancer may also occur in humans following benzene exposure (Aksoy, 1980, Aksoy, 1981, Yin et al., 1996).

For benzene to exert most of its toxic effects, it must be metabolized by the cytochrome P450 enzyme system. Benzene is first metabolized to benzene oxide (Jerina et al., 1968). Phenol is formed from the non-enzymatic rearrangement of benzene oxide (Jerina et al., 1968) and is metabolized to hydroquinone and catechol (Sawahata and Neal, 1983). Hydroquinone and catechol are further metabolized by myeloperoxidase in bone marrow to form p-benzoquinone and o-benzoquinone (Eastmond et al., 1987, Sadler et al., 1988).

Several cytochrome P450 isozymes may be involved in benzene metabolism. Using reconstituted rabbit cytochromes P450, Koop et al. (1989) characterized CYP2E1 as a low K_m, low V_max isozyme responsible for metabolizing both benzene and phenol. CYP2E1 formed greater amounts of all metabolites versus other cytochrome P450 isozymes with 0.3 mM benzene. However, with 2 mM benzene, CYP2B1 approached CYP2E1 in phenol formation but not hydroquinone formation. Other researchers have identified CYP2B1 as being capable of catalyzing the hydroxylation of both benzene and phenol in purified isozymes from rat liver (Snyder et al., 1993) and in rat liver microsomes (Gut et al., 1996).

The contribution of CYP2E1 to benzene metabolism has been further evaluated in numerous studies. Using diethyldithiocarbamic acid (DDTC, 300 μM), a known CYP2E1 inhibitor, Chaney and Carlson (1995) found metabolism of 17.5 μM benzene decreased by 96% in liver microsomes and 54% in lung microsomes from rat. Gut et al. (1996) found a significant decrease in benzene metabolism using DDTC (100–300 μM) The CYP2E1 inducers ethanol and acetone increased benzene metabolism in rat liver microsomes, and acetone did so in rabbit liver microsomes (Johansson and Ingelman-Sundberg, 1988). Ethanol also increased benzene metabolism in rats in vivo and in the 10 000×g fraction (Nakajima et al., 1985). Another CYP2E1 inducer, isopropanol, increased benzene metabolism in rat hepatocytes (Schrenk et al., 1991). Seaton et al. (1994) examined the metabolism of 3.4 μM benzene in human, mouse, and rat liver microsomes and found that human liver samples with higher CYP2E1 activities formed greater amounts of hydroquinone and catechol than did human liver samples with lower CYP2E1 activities. Nedelcheva et al. (1999) also found that human liver samples with higher CYP2E1 activities formed greater amounts of benzene metabolites.

CYP2E1 knockout mice have been used to examine the significance of CYP2E1 in the metabolism or toxicity of several xenobiotics (Ghanayem et al., 2000), including benzene (Valentine et al., 1996). Following the inhalation of 200 ppm benzene for 6 h, knockout mice that did not express a functional CYP2E1 protein produced only 13% of the total metabolites found in the urine of wild-type mice. Furthermore, genotoxicity or cytotoxicity were not detected in bone marrow, spleen, thymus, or blood of CYP2E1 knockout mice but were detected in the wild-type controls, following inhalation exposure to 200 ppm benzene for 6 h per day for 5 days (Valentine et al., 1996).

All these studies suggest that CYP2E1 is the cytochrome P450 isozyme primarily responsible for metabolizing benzene although CYP2B1 may be involved at high substrate concentrations. Accordingly, physiologically based pharmacokinetic (PBPK) models assume that benzene metabolism is due only to CYP2E1 (Seaton et al., 1994, Lovern et al., 1999, Cole et al., 2001). However, another cytochrome P450 isozyme merits attention, especially in the lung. CYP2F2, found in the mouse, is capable of biotransforming xenobiotics, such as, styrene (Carlson, 1997), naphthalene (Chang et al., 1996, Shultz et al., 1999), ethoxyresorufin, pentoxyresorufin, 1-nitronaphthalene, p-nitrophenol (Shultz et al., 1999), 2-methylnaphthalene, anthracene, and benzo[a]pyrene (Shultz et al., 2001). Interestingly, CYP2F2 has approximately the same p-nitrophenol metabolizing activity as CYP2E1 (Shultz et al., 1999). This is significant because p-nitrophenol is considered a model substrate for examining CYP2E1 activity (Reinke and Moyer, 1985, Koop, 1986). Therefore, p-nitrophenol hydroxylation may not be specific to CYP2E1 as was previously thought.

We have earlier demonstrated that CYP2E1 is the cytochrome P450 isozyme most responsible for hepatic microsomal benzene metabolism in the mouse, rat, and human, with 24 and 1000 μM substrate (Powley and Carlson, 2000). Results of this study have also shown that members of the CYP2F subfamily, as well as CYP2E1 are involved in pulmonary metabolism. Isozymes of the CYP2B subfamily were not notably involved at the low benzene concentrations studied. This is not surprising since other researchers found CYP2B1 to be capable of metabolizing benzene to an extent comparable with CYP2E1 only at concentrations higher than were used in our study (Koop et al., 1989, Snyder et al., 1993).

In the current study we used CYP2E1 knockout mice and wild-type controls to identify the cytochrome P450 isozymes involved in the metabolism of 24 μM benzene. This is the lowest concentration of benzene used in our earlier studies (Powley and Carlson, 1999, Powley and Carlson, 2000), and clearly represents a value that is more environmentally relevant than millimolar concentrations. Microsomes from liver and lung were used to determine if different cytochrome P450 isozymes are involved in the metabolism of benzene in these two tissues. Specific chemical inhibitors of CYP2E1 and CYP2F2 were used to further examine the cytochrome P450 isozymes involved in benzene metabolism. Since α-methylbenzylaminobenzotriazole (MBA, 1 μM final concentration), a CYP2B1 inhibitor (Mathews and Bend, 1986) did not have a significant effect on benzene metabolism in an earlier study (Powley and Carlson, 2000), this inhibitor was not used in the current study. Diethyldithiocarbamic acid (DDTC, 300 μM final concentration) was used as an inhibitor of CYP2E1 (Ono et al., 1996), and 5-phenyl-1-pentyne (5P1P, 5 μM final concentration) was used to inhibit CYP2F2 (Chang et al., 1996) The degree of specificity of these inhibitors is not fully known. Roberts et al. (1998) showed that 5P1P in addition to inhibiting CYP2F2, may also inhibit both CYP2E1 and CYP2B1. Although they used a higher concentration of 5P1P than employed in the present study, 50 μM versus 5 μM, there may be some inhibitory effect on CYP2E1 and CYP2B1 at 5 μM.

Identification of the cytochrome P450 isozymes responsible for benzene metabolism has important implications in understanding the effects of benzene exposure on human health. Polymorphism of enzymes involved in xenobiotic metabolism, including cytochromes P450, can alter an individual's disposition to toxicity. Polymorphisms of CYP2E1 are known to exist (Hu et al., 1997). Finding polymorphisms of other cytochrome P450 isozymes involved in benzene metabolism could help identify at risk individuals. Another important use for identifying the cytochrome P450 isozymes involved in benzene metabolism is for use in physiologically based pharmacokinetic modeling.