+ Author Affiliations
Biochemical Toxicology Unit, Comparative Toxicology and Ecotoxicology Laboratory, Istituto Superiore di Sanità, Rome, Italy
- Dr. Emanuela Testai, Istituto Superiore di Sanità, Comparative Toxicology and Ecotoxicology Laboratory-Biochemical Toxicology Unit, Viale Regina Elena 299, I-00161 Rome, Italy. E-mail: email@example.com
The oxidative and reductive cytochrome P450 (P450)-mediated chloroform bioactivation has been investigated in human liver microsomes (HLM), and the role of human P450s have been defined by integrating results from several experimental approaches: cDNA-expressed P450s, selective chemical inhibitors and specific antibodies, correlation studies in a panel of phenotyped HLM. HLM bioactivated CHCl3 both oxidatively and reductively. Oxidative reaction was characterized by two components, suggesting multiple P450 involvement. The high affinity process was catalyzed by CYP2E1, as clearly indicated by kinetic studies, correlation with chlorzoxazone 6-hydroxylation (r = 0.837; p < 0.001), and inhibition by monoclonal antihuman CYP2E1 and 4-methylpyrazole. The low affinity phase of oxidative metabolism was essentially catalyzed by CYP2A6. This conclusion was supported by the correlation with coumarin 7-hydroxylase (r = 0.777; p < 0.01), inhibition by coumarin and by the specific antibody, in addition to results with heterologously expressed P450s. Chloroform oxidation was poorly dependent on pO2, whereas the reductive metabolism was highly inhibited by O2. The production of dichloromethyl radical was significant only at CHCl3concentration ≥1 mM, increasing linearly with substrate concentration. CYP2E1 was the primary enzyme involved in the reductive reaction, as univocally indicated by all the different approaches. The reductive pathway seems to be scarcely relevant in the human liver, since it is active only at high substrate concentrations, and in strictly anaerobic conditions. The role of human CYP2E1 in CHCl3 metabolism at low levels, typical of actual human exposure, provides insight into the molecular basis for eventual difference in susceptibility to chloroform-induced effects due to either genetic, pathophysiological, or environmental factors.
Chloroform is a ubiquitous atmospheric and water contaminant. Beside its extensive use as a solvent in industrial processes, it is formed as a by-product during the chlorination of water intended for human use and paper bleaching. Due to its volatility, chloroform can be easily released from waste or chlorinated waters into the atmosphere or in the ambient air. Therefore, a large part of the human population may be chronically exposed to chloroform from different sources, although drinking water has been considered the main one. Recently, routes of exposure other than oral consumption of chlorinated water have been evaluated as relevant. Indeed, some indoor activities, such as showering or bathing as well as cooking and housekeeping, may significantly contribute to total chloroform body burden through dermal and inhalation exposure (Wallace, 1997; Backer et al., 2000). The main concern for public health arose with the carcinogenic potential of chloroform in experimental animals (NCI, 1976; Jorgenson et al., 1985), which is strain-, species- and gender-specific (IPCS, 1994).
No studies of chronic toxicity or cancer incidence in humans exposed exclusively to chloroform are available. Nevertheless, there are a number of epidemiological studies on populations exposed to chlorinated drinking water, some of which evidenced a weak association between water consumption and cancer of the bladder and lower gastrointestinal tract (Hogan et al., 1979; Gottlieb et al., 1981; Cantor et al., 1998;Hidelsheim et al., 1998). Moreover, some adverse reproductive outcomes have been recently reported in humans exposed to chloroform via drinking water (Gallagher et al., 1998; Waller et al., 1998). However, the poor assessment of exposure and the concomitant presence of many water contaminants, including other trihalomethanes and disinfection by-products, makes it difficult to establish a causal link between chloroform itself and adverse effects in humans.
The required step for CHCl3-induced toxicity is the cytochrome P450 (P4501)-mediated bioactivation to reactive metabolites (IPCS, 1994; Constan et al., 1999) (Fig. 1). Extensive in vitro and in vivo studies on rodents have demonstrated that chloroform may be metabolized oxidatively to trichloromethanol, which spontaneously decomposes to the electrophilic phosgene (Mansuy et al., 1977; Pohl et al., 1977). COCl2 is highly reactive and binds covalently to cell components containing nucleophilic groups, including proteins, phospholipid (PL) polar heads, and reduced glutathione (Pohl et al., 1981; Testai et al., 1990; De Biasi et al., 1992; Gemma et al., 1996; Vittozzi et al., 2000). Alternatively, phosgene may be hydrolyzed by reacting with water, yielding carbon dioxide and hydrochloric acid (Fig. 1). In anoxic or hypoxic conditions, chloroform may be reduced to dichloromethyl radical (Tomasi et al., 1985; Testai et al., 1995), which is able to bind to PL-fatty acyl chains (De Biasi et al., 1992;Gemma et al., 1996; Vittozzi et al., 2000) or to abstract a hydrogen atom from the biological environment, leading to dichloromethane (Testai et al., 1995) (Fig. 1). The relative ratio between the two pathways depends on the oxygen partial pressure, on chloroform concentration and is specie-, organ- and gender-specific (Ade et al., 1994; Gemma et al., 1996; Vittozzi et al., 2000).
The two pathways of chloroform bioactivation.
In rodents, it has been evidenced that at low concentration chloroform is oxidized by CYP2E1 (Testai et al., 1996; Constan et al., 1999). At higher CHCl3 concentration, in the presence of oxygen, phosgene formation is catalyzed by CYP2B1/2, while in anoxic conditions dichloromethyl radical production seemed to be mediated by constitutive P450s (Testai et al., 1996). These direct observations supported a number of studies, showing the potentiation effect of a variety of CYP2E1 and 2B1/2 inducers on chloroform-induced toxicity (Sato et al., 1980; Stevens and Anders, 1981; Branchflower et al., 1983).
Only rare data on chloroform metabolism in human tissues have been published (Fry et al., 1972; Testai et al., 1991). No direct information on the human P450(s) involved in the reaction is available at present, although CYP2E1 is suspected as the major catalyst of chloroform metabolism. The knowledge of the P450(s) responsible for CHCl3 bioactivation can provide useful information to identify the population group characterized by higher susceptibility to chloroform toxicity. Moreover, it would be possible to evaluate the environmental influence on susceptibility to chloroform-induced effects, through the study of metabolic interaction due to the combined exposure with other xenobiotics, affecting the rate of chloroform metabolism. Therefore, we have undertaken this study to characterize oxidative and reductive chloroform metabolism in human liver microsomes (HLM). In addition, we have identified the human P450(s) responsible for COCl2 and⋅CHCl2 formation, by using different experimental approaches, including cDNA-expressed enzymes, correlation studies in a panel of phenotyped HLM, and inhibition by either chemical-selective inhibitors or anti-human P450 antibodies (Abs).