Depurinating naphthalene–DNA adducts in mouse skin related to cancer initiation

Abstract

Naphthalene has been shown to be a weak carcinogen in rats. To investigate its mechanism of metabolic activation and cancer initiation, mice were topically treated with naphthalene or one of its metabolites, 1-naphthol, 1,2-dihydrodiolnaphthalene (1,2-DDN), 1,2-dihydroxynaphthalene (1,2-DHN), and 1,2-naphthoquinone (1,2-NQ). After 4 h, the mice were sacrificed, the treated skin was excised, and the depurinating and stable DNA adducts were analyzed. The depurinating adducts were identified and quantified by ultraperformance liquid chromatography/tandem mass spectrometry, whereas the stable adducts were quantified by 32P-postlabeling. For comparison, the stable adducts formed when a mixture of the four deoxyribonucleoside monophosphates was treated with 1,2-NQ or enzyme-activated naphthalene were also analyzed. The depurinating adducts 1,2-DHN-1-N3Ade and 1,2-DHN-1-N7Gua arise from reaction of 1,2-NQ with DNA. Similarly, the major stable adducts appear to derive from the 1,2-NQ. The depurinating DNA adducts are, in general, the most abundant. Therefore, naphthalene undergoes metabolic activation to the electrophilic ortho-quinone, 1,2-NQ, which reacts with DNA to form depurinating adducts. This is the same mechanism as other weak carcinogens, such as the natural and synthetic estrogens, and benzene.

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

“…Under this view, a genotoxic MoA could be adopted for animals and potentially extrapolated as such to humans� (3) Postulated dual MoA for naphthalene Another MoA put forth was discussed by Bogen (2008)� Bogen proposed a "dual" MoA including both cytotoxicity and genotoxicity that happen concurrently, suggesting that a relationship exists whereby naphthalene-induced increased cytotoxicity and DNA damage can occur as independent events, but that the DNA damage occurs at the same time as the cytotoxicity and is not an initiating event� The proposed MoA is based on data from a study by Wilson et al� (1996) in which cytotoxicity and genotoxicity (sister-chromatid exchange [SCE]) were assayed in human mononuclear leukocytes (MNLs) exposed to naphthalene and various naphthalene metabolites in vitro� It is also based on the known potential for genotoxicity of the downstream naphthalene metabolites (i�e�, 1,2-naphthoquinone and 1,4-naphthoquinone) that has been shown in vitro, and recently in vivo (in mouse skin) (Saeed et al�, 2009), in combination with histopathology findings in the rat and mouse bioassays that clearly link region-specific cytotoxicity with observed sites of tumorigenesis� The proposal was put forth, in part, to illustrate that a purely genotoxic MoA will overestimate risk when there is a clear cytotoxic component to the MoA�…”

“…The following proposed key events-metabolism of naphthalene to the epoxide and other metabolites by CYP2F (and/or other CYPs), cytotoxicity (including GSH depletion in cells where cytotoxicity occurs), chronic inflammation, and regenerative hyperplasia-are plausibly necessary but not evidently sufficient for the animal MoA� If these are the only critical events in the MoA, we would expect to observe nasal tumors in mice, but this was not observed in the mouse bioassay� Further explanations or assumptions are therefore necessary to help account for the lack of tumors in the mouse nose� This requires that we look harder at the current data, perhaps to modify the proposed MoAs to reflect all available data� With regard to genotoxicity as a potential key event, no evidence suggests that genotoxicity is necessary (i�e�, critical early event) for naphthalene-induced tissue injury and carcinogenesis� If genotoxicity were a critical early event (i�e�, before cytotoxicity), one would expect tumors to be observed more systemically, i�e�, in other tissues where naphthalene is present and metabolized by CYP2F and where detoxification mechanisms are potentially depleted (i�e�, the mouse nose), but this was not observed in the animal bioassays� In vitro and in vivo evidence does suggest that 1,2-naphthoquinone (1,2-naphthoquinone) is genotoxic (ATSDR, 2005;Saeed et al�, 2009)� Is there a role for genotoxicity of 1,2-naphthoquinone in the animal MoA? If so, does it occur subsequent to or concurrent with cytotoxicity?…”

“…As summarized in recent reviews on the metabolism of naphthalene ( Baldwin et al, 2004;Bogen et al, 2008;Buckpitt et al, 1992;Buckpitt et al, 1995;Buckpitt et al, 2002;Genter et al, 2006;Lee et al, 2005;Nagata et al, 1990;North et al, 2008;NTP, 1992NTP, , 2000Phimister et al, 2004;Plopper et al, 1992 a,b;Saeed et al, 2009;Thornton-Manning and Dahl, 1997;Van Winkle et al, 1995, 2004West et al, 2001;Wilson et al, 1996. data indicate the toxicity of naphthalene is attributable to its metabolic activation by one or more forms of CYP� Metabolism of naphthalene to the naphthalene 1,2-epoxide is believed to be the principal first step in the cytotoxicity of naphthalene and subsequent cellular proliferation and carcinogenicity� CYP2F2 enzyme isolated from mouse liver has been shown to catalyze epoxidation of naphthalene (Nagata et al�, 1990)� Immunolocalization studies and naphthalene metabolism assays in microdissected airways in mice indicate that CYP2F2 in distal airways (the most susceptible site) correlates with the highest rate of naphthalene metabolism (measured by presence of the dihydrodiol and GSH conjugates) � This study also showed that very low CYP2F levels in Clara cells of rat and hamster were correlated with low rates of naphthalene metabolism� Immunoblot analysis also showed that mice have 30-to 40-fold higher levels of CYP2F than rats in terminal bronchioles and trachea (Baldwin et al�, 2004)� A straight comparison of the rat and mouse lung data suggests potential involvement of CYP2F in the primary metabolism of naphthalene because the low levels of CYP2F in rat lung are consistent with the lack of lung tumors in rats� Other data have been put forth as supportive of the involvement of CYP2F in naphthalene carcinogenicity� For example, wild-type mice treated (IP injection) with naphthalene and a CYP2F inhibitor (5-phenyl-1-pentyne [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 (Genter et al�, 2006)� Furthermore, immunolocalization studies indicated that the rat nasal mucosa (primarily the olfactory epithelium) contains high levels of CYP2F4 (Baldwin et al�, 2004)� Naphthalene has been shown to be metaboli...…”

“…We propose that the balance of these activities differs between the mouse and rat nose, ultimately resulting in the observed difference in tumor formation� These questions are further addressed in the next section on genotoxicity� Key event 4-Genotoxicity Current data and hypothesized MoA An understanding of the potential genotoxicity of naphthalene and/or its metabolites is critical in developing an accurate MoA for naphthalene carcinogenesis� The Agency for Toxic Substances and Disease Registry (ATSDR, 2005) presented a review of over 40 naphthalene genotoxicity studies, 80% of which reported no evidence for genotoxicity for naphthalene� And the majority of positive studies are consistent with secondary effects produced by cytotoxicity� Several recent reviews (Schreiner, 2003;Brusick, 2008;Brusick et al�, 2008) have discussed the naphthalene genotoxicity data and that the evidence does not suggest that naphthalene or any of its metabolites drive naphthalene tumorigenicity� However, as outlined in proposed MoA (2) above, US EPA has proposed a genotoxic MoA for naphthalene (US EPA, 2004), based on data indicating genotoxicity of the naphthalene metabolite 1,2-naphthoquinone� The basis of US EPA's proposed MoA is derived, in part, from a 1,2-naphthoquinone mutagenesis study by Flowers-Geary et al� (1996), in which 1,2-naphthoquinone was found to cause a small increase in reversion mutations in Salmonella� In addition, 1,2-naphthoquinone and 1,4-naphthoquinone induced SCE in human MNLs (Wilson et al�, 1996)� Recent studies have shown that 1,2-naphthoquinone reacts with DNA in vitro (Saeed et al�, 2007) and in vivo in mouse skin (Saeed et al�, 2009) (2007) also observed stable DNA adducts in mouse skin that appeared to derive from the 1,2-naphthoquinone� Although depurinating adducts are known to cause mutations in DNA, at least in vitro, a recent review of the literature evaluating the biological significance for the involvement of N7-guanine adducts in mutagenesis found that there is insufficient evidence that N 7 -guanine adducts, or their apurinic sites, are the cause of mutations in cells and tissues, and that the stable DNA adducts may be more relevant (Boysen et al�, 2009)� Therefore, the relevance of naphthalene-induced stable DNA adducts needs further investigation� Studies by Kim et al� (1995) and Kim et al� (2000) suggest that naphthalene can induce stable N 6 adducts of deoxyadenosine from the tetrahydrodiol epoxide ("diol epoxide") of naphthalene� As shown in Figure 1, the 1,2-naphthoquinone and diol epoxide adducts are well downstream of the initial naphthalene epoxide and would not likely form until GSH has been depleted and cytotoxicity has already occurred� No in vivo studies have examined possible DNA adducts in naphthalene-exposed rat or mouse lung or nasal epithelial tissue� As we have mentioned, the balance of naphthalenemetabolizing, -detoxifying, and repair enzymes likely varies between species and tissues, and therefore the potential for formation of DNA adducts likely varies as well and should be examined in each relevant tissue (or cell type)� Th...…”

“…As shown in Figure 1, the naphthalene epoxide is metabolized by EH to the 1,2-dihydroxy-1,2-dihydronaphthalene ("dihydrodiol"), and then further by DD to the 1,2-naphthoquinone� Much of the focus on the MoA for naphthalene carcinogenesis has been on formation of the primary naphthalene metabolite, the naphthalene epoxide, by CYP2F� However, since there are studies indicating the involvement of the 1,2-napthoquinone in naphthalene cytotoxicity, and the potential for its genotoxic effects, understanding the tissue and species variability of the enzymes involved in formation of the 1,2-naphthoquinone, or other downstream naphthalene metabolites (such as the diol epoxide), is critical toward evaluating an MoA for naphthalene carcinogenicity� Saeed et al� (2009) suggests that metabolism of 1-naphthol to the 1,2-naphthoquinone may be metabolically similar to metabolism of estradiol to catechol estrogen quinones, which have been shown to react with DNA to form N 7 -guanine and N 3 -adenine adducts� As discussed by Brusick et al� (2008), CYP1B1 has been shown to metabolize estradiol to catechol estrogen quinones, resulting in N 7 -guanine and N 3 -adenine adduct formation in vitro (Belous et al�, 2007)� Therefore, examining the involvement of CYP1B1 in formation of the 1,2-naphthoquinone seems warranted� Again, it is very likely that species and tissue variability in the balance between the levels and activities of CYP2F (and/or CYP2E1, CYP1B1, or other CYPs) and other enzymes (GSH S-transferase, EH, DD) involved in formation of the 1,2-naphthoquinone or other downstream metabolites, or enzymes involved in repair of DNA damage induced by these metabolites, will help explain differences in species and tissue responses to naphthalene� For example, as shown in a study by Green et al� (2001), the activity of EH toward metabolism of the styrene metabolite, styrene epoxide, is about 10-fold higher in the rat than in the mouse nose� The authors suggest that this could be why the rat nose is less susceptible to styrene toxicity than the mouse nose, because the rat is able to more rapidly detoxify the epoxide� A higher activity of EH toward naphthalene would likely have a different outcome� If the activity of EH toward naphthalene is also higher in the rat nose, this might suggest formation of the dihydrodiol and 1,2-naphthoquinone at a higher rate in the rat versus the mouse nose� This might explain the difference in response to naphthalene injury between the mouse and rat nose� There are similar levels of CYP2F in rat and mouse nasal tissue, leading to similar levels of cytotoxicity and hyperplasia� However, upon high levels of naphthalene exposure, GSH depletion, and cytotoxicity, higher levels of EH activity in the rat nose could lead to higher levels of 1,2-naphthoquinone that could lead to genotoxic effects (either from direct reaction with DNA or redox cycling to generate DNA damage via ROS) on already hyperplastic tissue, potentially leading to atypical hyperplasia and tumors� So, unlike styrene for which EH is detoxifying, for naphthalene, high EH in rat nasal tissue may lead to increased levels of the toxic 1,2-naphthoquinone� Figure 3 illustrates potential combinations (yet to be tested) of CYP2F levels, GSH depletion, and EH and DD activities that could lead to the observed naphthalene-induced effects in rodents� If EH is more active toward naphthalene in the rat than the mouse nose, this could explain why there are no tumors in the mouse nose: As shown in row 3 of Figure 3, GSH depletion may lead to form...…”