In Vitro Metabolism of Naphthalene by Human Liver Microsomal Cytochrome P450 Enzymes

Abstract

ABSTRACT:The polycyclic aromatic hydrocarbon naphthalene is an environmental pollutant, a component of jet fuel, and, since 2000, has been reclassified as a potential human carcinogen. Few studies of the in vitro human metabolism of naphthalene are available, and these focus primarily on lung metabolism. The current studies were performed to characterize naphthalene metabolism by human cytochromes P450. Naphthalene metabolites from pooled human liver microsomes (pHLMs) were trans-1,2-dihydro-1,2-naphthalenediol (dihydrodiol), 1-naphthol, and 2-naphthol. Metabolite production generated K m values of 23, 40, and 116 M and V max values of 2860, 268, and 22 pmol/mg protein/min, respectively. P450 isoform screening of naphthalene metabolism identified CYP1A2 as the most efficient isoform for producing dihydrodiol and 1-naphthol, and CYP3A4 as the most effective for 2-naphthol production. Metabolism of the primary metabolites of naphthalene was also studied to identify secondary metabolites. Whereas 2-naphthol was readily metabolized by pHLMs to produce 2,6-and 1,7-dihydroxynaphthalene, dihydrodiol and 1-naphthol were inefficient substrates for pHLMs. A series of human P450 isoforms was used to further explore the metabolism of dihydrodiol and 1-naphthol. 1,4-Naphthoquinone and four minor unknown metabolites from 1-naphthol were observed, and CYP1A2 and 2D6*1 were identified as the most active isoforms for the production of 1,4-naphthoquinone. Dihydrodiol was metabolized by P450 isoforms to three minor unidentified metabolites with CYP3A4 and CYP2A6 having the greatest activity toward this substrate. The metabolism of dihydrodiol by P450 isoforms was lower than that of 1-naphthol. These studies identify primary and secondary metabolites of naphthalene produced by pHLMs and P450 isoforms.

Naphthalene metabolism in relation to target tissue anatomy, physiology, cytotoxicity and tumorigenic mechanism of action

“…CYP isoform screening in this study identified CYP1A2 as the most efficient isoform for producing dihydrodiol and 1-naphthol, and CYP3A4 as the most effective for 2-naphthol production. Whereas further pHLM metabolism of 2-naphthol was found to produce 2,6-and 1,7-dihydroxynaphthalene, dihydrodiol and 1-naphthol were not efficiently metabolized by pHLMs (Cho et al, 2006). CYP1A2 and 2D6*1 were identified as the most active isoforms for producing 1,4-naphthoquinone, and CYP3A4 and CYP2A6 the most active at metabolizing dihydrodiol, though at rates less than those at which 1-naphthol was observed to be metabolized (Cho et al, 2006).…”

“…Whereas further pHLM metabolism of 2-naphthol was found to produce 2,6-and 1,7-dihydroxynaphthalene, dihydrodiol and 1-naphthol were not efficiently metabolized by pHLMs (Cho et al, 2006). CYP1A2 and 2D6*1 were identified as the most active isoforms for producing 1,4-naphthoquinone, and CYP3A4 and CYP2A6 the most active at metabolizing dihydrodiol, though at rates less than those at which 1-naphthol was observed to be metabolized (Cho et al, 2006). Necrosis of bronchial epithelial (Clara) cells in mice (O'Brien et al, 1985(O'Brien et al, , 1989Tong et al, 1981) and necrosis of olfactory epithelial cells in mice, rats and hamsters following intraperitoneal injection of naphthalene strongly indicate that metabolic activation in target tissues plays a dominant, and possibly exclusive, role in site-specific naphthalene cytotoxicity.…”

“…Pooled human liver microsomes (pHLMs) were found to metabolize naphthalene to trans-1,2-dihydro-1,2-naphthalenediol (dihydrodiol), 1-naphthol, and 2-naphthol, with corresponding kinetics characterized by K m values of 23, 40, and 116 µM and V max values of 2860, 268, and 22 pmol/mg protein/min, respectively (Cho et al, 2006). CYP isoform screening in this study identified CYP1A2 as the most efficient isoform for producing dihydrodiol and 1-naphthol, and CYP3A4 as the most effective for 2-naphthol production.…”

Hypothesis-based weight of evidence: A tool for evaluating and communicating uncertainties and inconsistencies in the large body of evidence in proposing a carcinogenic mode of action—naphthalene as an example

“…Wild-type mice treated (IP injection) with naphthalene and a CYP2F inhibitor (5-PIP) showed no signs of lung or olfactory toxicity, as compared to the mice treated with naphthalene and no inhibitor where toxicity was observed in lung (hemorrhagic lungs) and olfactory tissue (100% sloughing of olfactory mucosa) (Genter et al�, 2006)� This study has been frequently cited in the literature as support that CYP2F is the CYP involved in metabolic activation of naphthalene to its toxic metabolites in mouse lung� Although these studies are supportive, especially in combination with the immunolocalization studies correlating CYP2F with the naphthalene epoxide at the site of injury in mouse lung, it is important to consider that the CYP2F inhibitor (5-PIP) also inhibits CYP2E1, which is also present in mouse lung and rat nose� Therefore, one needs to consider the possibility that CYP2E1 could be involved in naphthalene metabolism� However, the relative activities of these enzymes toward naphthalene needs consideration� Recombinant CYP2F from mice and rats efficiently generates the naphthalene oxide: CYP2F4 from rats (K M = 3 µM, V max = 107 min −1 ) (Baldwin et al�, 2005); and CYP2F2 from mice (K M = 3 µM, V max = 104 min −1 ) (Shultz et al�, 1999)� We did not find any studies that evaluated the activity of rodent CYP2E1 toward naphthalene� However, recombinant CYP2E1 from humans metabolized naphthalene to its 1-naphthol metabolite at a much lower efficiency (K M = 10�1 µM, V max = 8�4 min −1 ) (Cho et al�, 2006)� These data suggest that CYP2F metabolizes naphthalene more efficiently than CYP2E1, but studies comparing both CYPs in humans and rodents would provide stronger evidence as to the relevance of these two CYPs in various species� Studies in CYP2E1 and CYP2F knockout mice, or with inhibitors specific to CYP2E1, would provide useful information in this regard� Accordingly, as shown in Table 3, the mouse lung data provide "some evidence" that CYP2F is the primary CYP involved in metabolism of naphthalene to the epoxide, but more studies are necessary before we can rule out the involvement of CYP2E1 in metabolic activation of naphthalene to its toxic metabolites in mouse lung� CYP2E1 is present in mouse nasal and lung tissues (Simmonds et al�, 2004;Green et al�, 2001)� One possibility might be that CYP2E1 and CY2F2 are both involved in metabolic activation of naphthalene and cytotoxicity� There is some precedent for considering this possibility; a recent study of 1,1-DCE bioactivation and cytotoxicity in murine Clara cells (Simmonds et al�, 2004) suggested that both CYP2F2 and CYP2E1 are involved in 1,1-dichloroethylene (1,1-DCE) bioactivation and lung toxicity� Immunolocalization studies indicated that the rat nasal mucosa (primarily the olfactory epithelium) contains high levels of CYP2F4 (Baldwin et al�, 2004)� In addition, a recent study by Morris and Buckpitt (2009) found that naphthalene metabolism in the rat nose was inhibited after pretreatment with 5-PIP, suggesting that CYP2F is likely responsible, at least in part, for naphthalene metabolism in the rat nose� Therefore, as shown in Table 3, the rat nasal data provide "some evidence" that CYP2F4 is responsible for naphthalene metabo...…”

Mode-of-Action Uncertainty for Dual-Mode Carcinogens:Lower Bounds for Naphthalene-Induced Nasal Tumors in Rats Implied byPBPK and 2-Stage Stochastic Cancer Risk Models

“…can metabolize naphthalene to trans-1,2-dihydro-1,2-naphthalenediol (dihydrodiol), 1-naphthol and 2-naphthol; further pHLM metabolism of 2-naphthol produces 2,6-and 1,7-dihydroxynaphthalene, whereas dihydrodiol and 1-naphthol are not efficiently metabolized by pHLMs (Cho et al, 2006).…”