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. 2026 Feb 23;39(3):339–346. doi: 10.1021/acs.chemrestox.5c00428

Naphthalene-DNA Adduct Formation in a Lung Airway Explant Model: The Role of Bioactivation and Naphthalene Metabolites

Morgan C Domanico †, Sarah A Carratt †, Nicole Collette ‡, Esther A Ubick ‡, Patricia C Edwards †, Weizhu Yang §, Bruce A Buchholz ∥, Xinxin Ding §,*, Laura S Van Winkle †,⊥,*
PMCID: PMC12997242  PMID: 41730518

Abstract

Humans are widely exposed to naphthalene. Once inhaled or ingested, naphthalene is metabolized by cytochrome P450 and other enzymes to form toxic metabolites known to harm lung epithelial cells. Naphthalene metabolites circulate in the blood. Chronic naphthalene inhalation promotes lesions in the epithelium of the mouse lung and rat nose. Oral naphthalene exposure leads to DNA adduct formation in mouse lung, but the contributions of different enzymatic pathways and the metabolites they generate are not fully understood. This study explores the influence of naphthalene metabolites on DNA adduct formation in the lungs of two species (mice and primates). To isolate the lung response, conducting airway explants containing Club cells, a target for pulmonary naphthalene toxicity, were microdissected from live lung tissue and incubated with 14C-naphthalene or its metabolites: 14C-1,2-naphthoquinone or 14C-naphthalene-1,2-dihydrodiol. Explants were incubated for 1 h, then processed immediately (T1), or were transferred to clean media for the remainder of the 24 h (T24), to monitor 14C in DNA over time. Accelerator mass spectrometry analysis revealed the formation of DNA adducts by all three radiolabeled compounds by T24. Our results support the notion that P450 enzymes of the Cyp2abfgs subfamily contribute to naphthalene-induced DNA adduct formation (approximately 4-fold reduction in male mice lacking the Cyp2abfgs genes, P < 0.01). The finding that naphthalene-1,2-dihydrodiol, a stable metabolite, formed DNA adducts (102–117 adducts/108 nucleotides) at 24 h following addition to the culture media validates the concern that circulating naphthalene metabolites can contribute to DNA adduct formation in the lung. DNA adducts persisted to 24 h after exposure in both mouse and primate airways and at comparable levels between species (77.8 vs 129 adducts/108 nucleotides, respectively). Together, these results support the importance of a potential genotoxic mechanism of naphthalene and its metabolites in vivo in both mice and nonhuman primates, and possibly also in humans.

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Introduction

Naphthalene is a ubiquitous environmental contaminant classified as an International Agency for Research on Cancer (IARC) 2B and U.S. Environmental Protection Agency (EPA) Class C possible human carcinogen. The U.S. National Toxicology Program has shown in chronic two-year bioassays that naphthalene promotes tumor-like lesions in female mouse lung , and rat nose. , Metabolism of naphthalene by cytochrome P450s (CYPs) is required for reactivity with macromolecules and subsequent toxicity; the field’s current understanding of naphthalene metabolism is summarized in Figure . While naphthalene has been shown to cause lung cytotoxicity, which may lead to tumor formation through repeated cycles of injury and repair, information on naphthalene metabolites’ ability to form DNA adducts in the target tissue (lung) has been limited. Evidence of a genotoxic mechanism of action by naphthalene or its metabolites has implications for human risk assessment.

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Metabolic pathways of naphthalene and metabolite formation. Active enzymes are indicated by abbreviations in diagram bubbles, including: P450 for cytochrome P450, mEH for microsomal epoxide hydrolase, and GST for glutathione-S-transferase. Reactive metabolites are highlighted by shading with gray boxes. Detoxification pathways are summarized within the boxes. Solid arrows denote major bioactivation pathways, with dashed arrows for minor pathways that may contribute small amounts of metabolites to the next step.

We previously demonstrated that naphthalene could form DNA adducts in B6C3F1 and NIH Swiss mice, as well as nonhuman primate airways immediately following a 1 h incubation period ex vivo. , This was tested in an airway explant model that contains the cells from the lung target microenvironment identified in the NTP assays. In this explant model, viable conducting airway cells, including abundant Club cells, can be maintained in vitro for up to 1 week. − In the current study, we expanded the earlier explant study by testing a set of new hypotheses.

First, we hypothesized that the ability of naphthalene to form DNA adducts in the mouse airway explant culture is mainly mediated by P450 enzymes in the Cyp2a, 2b, 2f, 2g, and 2s gene subfamilies. This hypothesis was tested by comparing explants from wild-type mouse to those from Cyp2abfgs-null mouse, which is missing the Cyp2a, 2b, 2f, 2g, and 2s genes; both are on the C57BL/6 genetic background. The CYP2A and 2F enzymes play important roles in naphthalene metabolism and cytotoxicity in vivo, − but their specific contributions in the airways have not been directly examined in an ex vivo or in vivo model.

Second, we tested the hypothesis that circulating naphthalene-1,2-dihydrodiol can form DNA adducts in lung airways by adding radiolabeled naphthalene-1,2-dihydrodiol to the explant culture. Carratt et al. found that the toxic naphthalene metabolite, 1,2-naphthoquinone, was a highly potent DNA adductor in primate airway at 1 h but did not assess the ability of its more abundant and stable precursor, naphthalene-1,2-dihydrodiol, to form DNA adducts. There is potential for naphthalene-1,2-dihydrodiol generated in the liver to circulate to the lung, where it could undergo additional biotransformation into 1,2-naphthoquinone, which can then form DNA adducts. This would be an important consideration in characterizing human risk, as there is emerging evidence that hepatic bioactivation can contribute to naphthalene lung toxicity. −

Finally, we compared the abundance and persistence of DNA adducts formed from naphthalene, naphthalene-1,2-dihydrodiol, and 1,2-naphthoquinone in wild-type mouse lung airway explants to gauge their relative importance in the ultimate DNA lesion. This was tested by exposing explants to the compounds individually at equal concentrations for 1 h and measuring DNA adduct formation either immediately or 23 h after the 1 h exposure (Figure ). We have recently detected naphthalene-DNA adducts in vivo in the lungs and livers of wild-type mice following an oral naphthalene exposure and showed persistence of the adducts at 24 and 72 h after exposure. We also compared mouse and primate airway explants for DNA adduct levels at 23 h following a 1 h naphthalene exposure (T24) to test the hypothesis that the adducts are equally persistent in rodent and primate lung airways.

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Use of an ex vivo airway explant model to measure DNA adducts from rodents and nonhuman primates. We utilized a live ex vivo tissue method of conducting airway explants to analyze levels of naphthalene (NA)-, 1,2-naphthoquinone (1,2-NQ)-, or naphthalene-1,2-dihydrodiol (NA-1,2-diol)-derived 14C-label covalently bound to the DNA of conducting airway epithelium from mice or primates. Processing occurred directly after a 1 h incubation (T1) or a 1 h incubation followed by a recovery period in analyte-free media for the remainder of 24 h (T24). Tissue was washed with ethanol (15–20 washes) to remove any unbound analyte and then analyzed via accelerator mass spectrometry to quantify total DNA adducts in the tissue.

Overall, these studies provide crucial new data for determining the relevance of DNA adduct formation to human cancer risk from naphthalene exposure. Our results support the ability of circulating naphthalene-1,2-dihydrodiol, as well as 1,2-naphthoquinone, to form DNA adducts and show persistence of the DNA adducts in the target organ in both murine and primate models.

Experimental Procedures

Animals

All animal experiments were conducted in accordance with the National Institutes of Health guidelines and were performed following the protocols evaluated and approved by the University of California Davis (UCD) and Lawrence Livermore National Laboratory (LLNL) Institutional Animal Care and Use Committees (IACUC: UCD #18243, UCD #22779, and LLNL #314). All rodents were housed in filtered air barrier facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), with food and water ad libitum on a 12 h light/dark cycle. Wild-type adult (8–16 weeks) male and female C57BL/6J mice were purchased from Envigo (UCD) or Jackson Laboratories (LLNL). Cyp2abfgs-null mice (Cyp2a, Cyp2b, Cyp2f, Cyp2g, and Cyp2s knockout on B6 background) were produced at LLNL from breeding pairs obtained from the University of Arizona and genotyped by using PCR. All rhesus macaque tissues were from adult animals 3–16 years of age (average age 8.2 ± 4.1 years) that were euthanized for normal colony maintenance at the California National Primate Research Center (UC Davis).

Explant Preparation

Mice were euthanized with an intraperitoneal overdose of pentobarbital sodium (Fatal-Plus C IIN; Vortech Pharmaceuticals). Methods for tissue collection were described previously. , In brief, lungs were removed en bloc and inflated with a 50:50 solution of prepared Waymouth’s MB 752/1 medium (Sigma, W1625) and 2% agarose (Sigma). Fresh tissue was kept on ice in Ham’s F12 Nutrient Mixture Media (Gibco, 11765047) and microdissected within 2 h of tissue collection. Lung tissue was microdissected on ice, as previously described, to yield conducting airway explants from rodents , and primates. Microdissected airways remain in their 3D configuration, are still metabolically active, and act as an enriched model for enzymes present exclusively in the airway epithelium.

Radiochemicals

Radiochemicals utilized in this study include: 14C-naphthalene, 14C-1,2-naphthoquinone, and 14C-1,2-dihydroxy-1,2-dihydronaphthalene (14C-naphthalene-1,2-dihydrodiol). A working solution of 14C-naphthalene was prepared by mixing primary stock 14C-naphthalene (1,4,5,8-14C) in methanol (MC 2147) (58 mCi/mmol; 100% purity) from Moravek Inc. (Brea, CA) with unlabeled naphthalene (99%, Sigma-Aldrich) in acetonitrile (99.9% HPLC-grade; ThermoFisher Scientific) to achieve a final concentration of 25 mM total naphthalene. A working solution of 14C-1,2-naphthoquinone was prepared by mixing primary stock 14C-1,2-naphthoquinone (1-14C) in acetonitrile (55 mCi/mmol; 97–99% purity) from American Radiolabeled Chemicals (St. Louis, MO) with unlabeled 1,2-naphthoquinone (Sigma-Aldrich) in acetonitrile to achieve a final concentration of 25 mM total 1,2-naphthoquinone. A working solution of 14C-naphthalene-1,2-dihydrodiol was prepared by mixing primary stock of 14C-1,2-dihydroxy-1,2-dihydronaphthalene (50 mCi/mmol; 98% purity) synthesized by American Radiolabeled Chemicals with racemic trans-1,2-dihydroxy-1,2-dihydronaphthalene (94% purity) from Toronto Research Chemicals suspended in acetonitrile, to achieve a final concentration of 25 mM total naphthalene-1,2-dihydrodiol.

Explant Incubations and DNA Isolation

Radioisotope explant exposure methods have been described in detail previously. , Briefly, the exposures were conducted by placing metabolically active, microdissected airway tissue in a 20 mL scintillation vial with 1 mL of dosed F12+6 medium containing 25 or 250 μM 14C-naphthalene, 250 μM 14C-1,2-naphthoquinone, or 250 μM 14C-naphthalene-1,2-dihydrodiol and incubated in a water bath for 1 h at 37 °C. Dosed F12+6 media was comprised of 10 μL 14C-dosing solution added into 990 μL of F12+6 media; 10 μL of acetonitrile was used as the vehicle control for each radiochemical exposure.

In this study, two exposure durations were tested (Figure ): T1, a 1 h exposure followed by immediate processing, or T24, a 1 h exposure followed by a recovery period until 24 h after the start of exposure in fresh, analyte-free media to assess adduct stability. T1 explants were immediately washed with 100% ethanol after exposure cessation, until radioactivity was indistinguishable from the background ethanol level using a liquid scintillation counter. T24 explants were washed with cold media five times, after the 1 h exposure, and then transferred to air–liquid interface culture (Corning, 1 μm Pore) for the recovery period. Explants were kept alive and metabolically active using supplemented F12+6 media, with 1 mL media in the basal well and 75 μL media in the apical well to facilitate liquid exchange across the Transwell membrane and incubated at 37 °C with ∼5% CO2. At 24 h postexposure, T24 tissue was washed repeatedly with 100% ethanol until radioactivity was comparable to background ethanol levels. During ethanol washes for both T1 and T24 explants, microfuge tubes were changed every five rounds to remove surface-bound 14C.

DNA Extraction

Samples were stored dry at −20 °C until DNA isolation was performed as previously described. , Briefly, airway explants underwent bead homogenization immediately prior to DNA isolation using DNeasy Blood & Tissue Kits (Qiagen), with additional Proteinase K incubations to account for high formation of naphthalene-protein adducts. DNA concentration and purity were measured using a NanoDrop spectrophotometer.

Accelerator Mass Spectrometry

To prepare DNA samples for accelerator mass spectrometry (AMS) analysis, DNA was first dried in a quartz combustion vial with excess CuO, evacuated, sealed, combusted to CO2, and reduced to graphite via the Ognibene Method. All glass used in sample preparation was baked before use to remove residual carbon. Tributyrin was added to all samples as a carbon carrier to certify robust graphite formation, as previously described. , Graphite isotope ratios were measured using a National Electrostatics Corporation (Middleton, WI) 250 kV single-stage AMS spectrometer at Lawrence Livermore National Laboratory. AMS measurement times were typically 5–10 min/sample, with a counting precision of 0.5–3% (relative standard deviation, RSD) and a standard deviation among 3–10 measurements of 1–3%. Sample 14C/13C ratios were normalized to measurements of four identically prepared isotopic standard samples of known isotope concentration (IAEA C-6 or ANU sucrose). Isotope ratios were then converted to biologically relevant units using the carbon concentration, isotope ratio and mass of carbon carrier, specific activity of the labeled compound, and sample mass. Additional information is provided in the Supporting Information.

Statistics

Statistical analysis was performed with GraphPad Prism V10.5.0 (GraphPad Software Inc.). Data are provided as mean ± standard error of the mean (SEM) and analyzed by two-way analysis of variance (ANOVA) with Tukey’s post hoc test, except for the species comparison (Figure ), which utilized an aligned ranks transformation analysis of variance (ART-ANOVA) with Tukey’s post hoc test in R v4.5.1. Outliers were identified using Grubbs’ outlier test and removed at a maximum of one per group. Statistical significance was determined using a criterion of P < 0.05.

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DNA adduct levels in the naphthalene-exposed conducting airways of C57BL/6 mice and rhesus macaques were comparable and increased over 24 h. Mouse and primate airway explants were incubated with 250 μM 14C-naphthalene for 1 h and then immediately processed (T1) or 1 h followed by a 23 h recovery period in analyte-free media (T24). Data are reported as mean ± standard error (N = 8–10 treated, N = 5–7 controls). Mouse data are pooled males and females from Figure A. Statistical analyses used an aligned ranks transformation analysis of variance (ART-ANOVA) with Tukey’s post hoc test: ** P < 0.01.

Results

We first tested whether the ability of naphthalene to form DNA adducts in the mouse airway explant culture is dependent on P450 enzymes. As shown in Figure , airway explants from mice with whole-body knockout of Cyp2a, 2b, 2f, 2g, and 2s gene subfamilies had decreased DNA adduct formation compared to wild-type C57BL/6 mice, when exposed to 250 μM naphthalene for 1 h; the difference was significant in males (12.9 ± 5.14 vs 54.0 ± 18.4 DNA adducts/108 nucleotides; P < 0.01), though not in females (15.4 ± 7.72 vs 38.0 ± 20.4 DNA adducts/108 nucleotides; P = 0.17). This result indicates that under the test conditions knockout of the Cyp2abfgs gene cluster attenuates but does not fully ablate naphthalene-DNA adduct formation in airway explants.

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Naphthalene-DNA adduct formation is attenuated in the airways of male Cyp2abfgs-null mice compared to those of wild-type C57BL/6 mice. Airway explants were incubated with 250 μM 14C-naphthalene for 1 h and then immediately processed (T1). Data reported as mean ± standard error, with no outliers detected (N = 4–5). Mean background 14C level was derived from the sex-mixed vehicle control group of each strain and was subtracted to determine the elevation of the 14C signal present in the DNA of individual treated samples. Statistical analyses used a two-way analysis of variance (ANOVA) with Tukey’s post hoc test: ** P < 0.01.

We then tested the ability of a stable naphthalene metabolite, naphthalene-1,2-dihydrodiol, formed after P450-catalyzed epoxygenation of naphthalene to form DNA adducts in lung airways of WT mice and compared it to naphthalene and 1,2-naphthoquinone for the abundance and persistence of the adducts formed. After incubation with naphthalene, DNA adducts were detected in explants at both 1 and 24 h after the initiation of a 1 h exposure period (Figures and A). Calculated levels of DNA adducts did not significantly differ between the two time points for either females (at T1, 38.0 ± 20.4 adducts/108 nucleotides; at T24, 80.3 ± 23.6 adducts/108 nucleotides) or males (at T1, 54.0 ± 18.4 adducts/108 nucleotides; at T24, 74.6 ± 25.6 adducts/108 nucleotides) (Figure A). At both time points, adduct levels were significantly elevated (P < 0.05 or lower) above the vehicle control group of their respective sex (Figure A). Naphthalene-1,2-dihydrodiol also formed DNA adducts in the explants; however, it did not immediately form a significant level of DNA adducts at T1 but did so by T24 in both females (117 ± 75.6 DNA adducts/108 nucleotides) and males (102 ± 62.3 DNA adducts/108 nucleotides) (Figure B).

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DNA adduct levels in C57BL/6 murine airway explants persisted over time, suggesting continued formation of reactive naphthalene metabolites and resulting DNA adducts during the recovery period. Airway explants were incubated with 250 μM 14C-labeled naphthalene (A), naphthalene-1,2-dihydrodiol (B), or 1,2-naphthoquinone (C) for 1 h and then immediately processed (T1) or were permitted a recovery period for the remainder of 24 h in analyte-free media (T24). Data are reported as mean ± standard error, with an outlier excluded from each T24 group (N = 4–6 for treated, N = 2–4 for controls). Statistical analyses used a two-way analysis of variance (ANOVA) with Tukey’s post hoc test: * P < 0.05, ** P < 0.01, *** P < 0.001.

With 1,2-naphthoquinone, which can be produced from naphthalene-1,2-dihydrodiol and is chemically reactive (Figure ), DNA adduct levels were formed at much higher levels (by 24–58 fold) than with naphthalene or naphthalene-1,2-dihydrodiol. The adducts levels were significantly elevated (P < 0.01 or lower) above vehicle controls regardless of sex or time point, with the female means at 2220 ± 721 (T1) and 2380 ± 357 (T24) DNA adducts/108 nucleotides, and the male means at 2520 ± 925 (T1) and 1800 ± 909 (T24) DNA adducts/108 nucleotides (Figure C). There was no significant difference between time points, indicating persistence of adducts formed.

Naphthalene also forms stable DNA adducts in primates and at levels comparable to mice. DNA adducts were detected in airway explants from sex-pooled rhesus macaques following exposure to 250 μM naphthalene for both the T1 duration (52.8 ± 27.0 adducts/108 nucleotides) and the T24 duration (129 ± 75.3 adducts/108 nucleotides), and the adduct levels were significantly higher than in vehicle controls (Figure ). There was no difference in naphthalene-induced DNA adduct formation between mice and primate airway at either T1 (mouse mean: 46.0 adducts/108 nucleotides; primate mean: 52.8 adducts/108 nucleotides) or T24 (mouse mean: 77.8 adducts/108 nucleotides; primate mean: 129 adducts/108 nucleotides). Primates also had significantly higher DNA adduct levels at T24 than at T1, as was observed in mice, indicating persistence of DNA adducts in both species (Figure ).

Discussion

We demonstrate for the first time that DNA adducts in naphthalene-exposed lung airway explants are formed in part by P450 enzymes of the Cyp2abfgs gene subfamilies and that exposure to a stable and abundant naphthalene metabolite, naphthalene-1,2-dihydrodiol, can also lead to DNA adduct formation in airway explants. We confirmed our recent finding that DNA adducts can form in vivo from naphthalene (which may include adducts from both naphthalene epoxide and naphthoquinones) and can persist until at least 24 h after exposure. We also found that DNA adducts from 1,2-naphthoquinone and its precursor, naphthalene-1,2-dihydrodiol, persist, that primate airway explants are as active as mouse airway explants in forming naphthalene-DNA adducts, and that the adducts persist to 24 h in both species.

The partial dependence of DNA adduct formation on P450 enzymes of the Cyp2abfgs subfamilies (Figure ) is consistent with their selective expression in the respiratory tract and the known function of these enzymes, particularly CYP2A5 and CYP2F2, in naphthalene bioactivation (naphthalene oxide formation) and cytotoxicity. ,, The finding also validates the DNA adducts as biomarkers derived from P450-dependent naphthalene metabolic activation. The residual DNA adducts detected in the null mice may be explained by the presence of other CYPs in the lung, such as CYP2E1 and CYP3A, as their human orthologs are known to be active toward naphthalene bioactivation. ,,

Naphthalene-1,2-dihydrodiol, a stable intermediate metabolite of naphthalene, can form DNA adducts in the mouse airway (Figure B). The latency in formation suggests naphthalene-1,2-dihydrodiol can enter airway cells and undergo additional bioactivation to metabolites that form DNA adducts in the hours following exposure. This is of particular concern given the possibility of naphthalene to undergo trans-organ bioactivation: parent naphthalene, which even when inhaled does reach the circulating blood, may be bioactivated in one organ (e.g., the liver) and then could circulate through the blood to other tissues and undergo further bioactivation at these secondary sites (e.g., the lung). The most abundant naphthalene metabolite formed from pooled human liver microsomes is naphthalene-1,2-dihydrodiol. Circulation of naphthalene metabolites in vivo, and impact on lung cells, appears likely as naphthalene metabolites in the circulating medium of isolated perfused mouse lung led to lung Club cell cytotoxicity. Importantly, even reactive naphthalene epoxide can transit in the circulation, as the half-life of circulating naphthalene epoxide was found to be 11 min when albumin was present. The contribution of hepatic bioactivation of naphthalene to lung toxicity has previously been observed in liver-specific Cpr (cytochrome P450 reductase gene) knockout mice. The role of circulating metabolites is of particular importance due to the dominance of liver metabolism in humans, which produces ample naphthalene metabolites that can then circulate to impact other tissues.

The downstream metabolite of naphthalene-1,2-dihydrodiol, 1,2-naphthoquinone, was a potent DNA adductor in the C57BL/6 mouse airway (Figure C), consistent with our previous findings in primate airways. These 1,2-naphthoquinone-DNA adducts also persisted to 24 h postexposure without a significant decrease despite the recovery period. Naphthoquinone has a highly reactive chemical structure, able to bond readily to macromolecules, which explains the much greater amounts of adduct formed (Figure C) compared to naphthalene (Figure A) and naphthalene-1,2-dihydrodiol (Figure B). The higher potency of 1,2-naphthoquinone supports a greater importance of circulating 1,2-naphthoquinone than naphthalene-1,2-dihydrodiol as a contributor to DNA adduct formation in the lung. While circulating naphthalene-1,2-dihydrodiol will require further metabolism in the target tissue before it can form DNA adducts, 1,2-naphthoquinone can directly form DNA adducts upon arrival at a target tissue. Additionally, 1,2-naphthoquinone is also present as an atmospheric pollutant; , thus, these results support the likelihood of lung DNA damage resulting from acute or chronic exposures to 1,2-naphthoquinone in the inhaled air. It will be important to determine 1,2-naphthoquinone exposure levels in human populations in future studies for association with possible genotoxicity.

It remains to be determined whether the higher adduct abundance with naphthoquinone exposure also reflects greater resistance of these adducts to DNA repair (and thus greater risk for genotoxicity) compared with adducts formed from other reactive naphthalene metabolites. Adducts formed following naphthalene-1,2-dihydrodiol exposure are likely the same as those formed from naphthoquinone exposure, as naphthalene-1,2-dihydrodiol is the precursor of 1,2-naphthoquinone; however, adducts formed after naphthalene exposure may include, in addition to 1,2-naphthoquinone adducts, other reactive naphthalene metabolites, including 1,4-naphthoquinone, naphthalene-1,2-epoxide, and 1,2,3,4-tetrahydro-1,2-hydroxy-3,4-epoxynaphthalene (Figure ). Ultimately, the structures of the DNA adducts detected here are unknown. However, stable and depurinating DNA adducts have been synthesized in vitro by reacting individual nucleosides or calf-thymus DNA with 1,2-naphthoquinone, creating multiple guanine and adenine adducts, including 1,2-dihydroxynaphthalene (DHN)-4-N3-adenine (A), 1,2-DHN-4-N6-A, 1,2-DHN-4-N1-A, 1,2-DHN-4-N6-deoxyadenosine (dA), 1,2-DHN-4-N7-guanine (G), 1,2-DHN-4-N7-deoxyguanosine (dG), 1,2-NQ-4-N7-dG (1,2), , and 1,2-NQ-4-N7-G, as well as 1,2-NQ-3-OH-4-N1-dG, 1,2-NQ-3-OH-4-N7-dG, and 1,2-NQ-4-OH-3-N2-dG. It remains to be determined whether these adducts are formed in the airway explants in our study or in the lung upon in vivo exposure, and the persistence of specific naphthalene-induced DNA adducts is unknown.

There appear to be mouse strain differences in DNA adduct formation in naphthalene-exposed airway explants, with C57BL/6 mice having fewer adducts. Previous work with B6C3F1 murine airways found a sex-pooled average of 1706 ± 409 adducts/pg DNA at T1, whereas sex-pooled C57BL/6 mice from this study only formed 442 ± 188 DNA adducts/pg DNA, increasing to 739 ± 214 DNA adducts/pg DNA by T24. Strain differences in DNA adduct formation may be attributable to differences in phase I enzymes capable of bioactivating naphthalene and/or in phase II detoxification pathways, with glutathione levels known to vary by mouse strain. It could also be due to sex-related variations in DNA repair, which have not yet been studied in relation to naphthalene-derived DNA adducts.

Interestingly, mean DNA adducts in naphthalene-exposed C57BL/6 mouse airway explants at T1 (females: 38 ± 20, males: 54 ± 18 adducts/108 nucleotides) were comparable to levels observed in whole lung 2 h after a 50 mg/kg oral exposure (females: 21 ± 15, males: 57 ± 15 adducts/108 nucleotides). By T24, however, there are far more DNA adducts remaining in the airway explants (females: 80 ± 24, males: 75 ± 26 adducts/108 nucleotides) than are present in mouse lung 24 h after a 50 mg/kg oral exposure (females: 5 ± 1, males: 12 ± 6 adducts/108 nucleotides). This difference likely reflects the rapid clearance of naphthalene in vivo by hepatic P450 enzymes, with a clearance half-life for serum naphthalene at ∼2 h following intraperitoneal naphthalene injection, whereas the lack of systemic blood circulation in the explant system would be expected to substantially extend the half-life of naphthalene and its metabolites in the airways. Indeed, in mice with liver-specific deletion of the Cpr gene and consequent abolishment of hepatic microsomal metabolism of naphthalene, the serum half-life of naphthalene increased to ∼5 h. Nonetheless, since naphthalene was unavailable from the culture media after the initial 1 h incubation, the amounts of tissue-bound naphthalene or its metabolites available for continued reaction with DNA would decrease over time throughout the recovery period and would unlikely sustain the DNA adduct levels seen at T1 if the adducts were rapidly removed through DNA repair. Overall, the ex vivo data support our in vivo data for DNA adduct persistence, though further studies are needed to identify conditions (when new adducts are no longer formed) to more accurately account for rates of adduct removal.

The DNA adduct data for primates (Figure ) demonstrate a similarity in abundance of DNA adducts detected and apparent persistence of these adducts in both mice and primates. This reinforces the value of mouse genotoxicity data for assessing human risks. Carratt et al. also observed comparable DNA adduct formation between the airway explants of female primates and B6C3F1 mice 1 h after exposure. However, this similarity is in contrast to previous findings of large species differences between rodents and primates or humans in lung microsomal activity toward naphthalene metabolism. − For example, airway microsomes from primates metabolized 250 μM of naphthalene at less than 1% of the mouse airway microsomal rate. Notably, the abundance of DNA adduct formation in the explant model differs from metabolic rates measured using isolated microsomes and reaction products that reflect individual metabolic steps, as the former represents the cumulative activities in multiple enzymatic and nonenzymatic reactions leading from initial metabolism of naphthalene by P450s, to hydrolysis of naphthalene oxide, the formation of naphthoquinones, and eventually the formation of DNA adducts. Individual steps in this cascade of reactions may be impacted by limited availability of cofactors and cellular compartmentalization of reactions, and the abundance of the final product (DNA adduct) reflects the balance between bioactivation and detoxification and between DNA adduct formation and removal/repair in individual cells. Thus, the abundance data from the ex vivo study using primate airways are likely a more accurate depiction of the capacity of human airways to form DNA adducts following naphthalene exposure than in vitro microsomal activity data. Accordingly, risk assessment that focuses solely on enzymatic differences between humans and rodent models may improperly assess total genotoxic risk, as DNA adduct formation cannot be attributed to a sole bioactivation source.

In summary, this study extends our previous work by providing novel evidence of the role of P450 enzymes of the Cyp2abfgs gene subfamily in naphthalene-induced DNA adduction in airway explants. We demonstrate the ability of naphthalene-1,2-dihydrodiol to lead to the formation of DNA adducts, which supports the role of this abundant circulating metabolite contributing to DNA adduct formation in remote tissues. Additionally, we confirmed the high capability of primate airways to form naphthalene-DNA adducts and report the novel finding of persistence of the DNA adducts in primates, which suggests that the adducts are not completely repaired, indicating an increased likelihood to cause mutation. Our findings support the possibility of circulating naphthalene metabolites to contribute to DNA adduct formation in the lung and raise concerns for naphthalene’s potential for genotoxicity in humans. Further exploration of the DNA adducts formed by individual naphthalene metabolites in vivo is required to complete our understanding of how enzymatic differences between species, and individuals with genetic polymorphisms, may influence risk for a possible naphthalene genotoxic mode of action.

Supplementary Material

tx5c00428_si_001.pdf (250.5KB, pdf)

Acknowledgments

Reviewed and released as LLNL-JRNL-2007651. This work was partially performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Diagrams were created with BioRender.com. The authors would like to thank Ed Kuhn for his technical assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.chemrestox.5c00428.

  • Accelerator mass spectrometry formulas to convert isotope ratios to DNA adduct levels (PDF)

This manuscript was written through contributions from all authors. All authors have given approval to the final version of the manuscript. CRediT: Morgan C Domanico formal analysis, investigation, visualization, writing - original draft, writing - review & editing; Sarah A Carratt formal analysis, investigation, writing - original draft, writing - review & editing; Nicole Collette investigation, writing - review & editing; Esther A. Ubick investigation, writing - review & editing; Patricia C Edwards investigation, writing - review & editing; Weizhu Yang investigation, writing - review & editing; Bruce A. Buchholz conceptualization, formal analysis, investigation, methodology, project administration, resources, supervision, writing - review & editing; Xinxin Ding conceptualization, funding acquisition, methodology, project administration, supervision, writing - review & editing; Laura S. Van Winkle conceptualization, funding acquisition, methodology, project administration, supervision, writing - review & editing.

This manuscript is the result of funding in whole or in part by the National Institutes of Health (NIH). It is subject to the NIH Public Access Policy. Through acceptance of this federal funding, NIH has been given a right to make this manuscript publicly available in PubMed Central upon the Official Date of Publication, as defined by NIH. As part of the NIH’s Data Management and Sharing Policy, raw data will be made accessible via Dryad (https://datadryad.org/) upon official manuscript publication. Funding provided by NIEHS R01ES020867 and ES020867S1, NIH R24GM137748, P41GM103483, and DOD LC130820. During the period this research was conducted, M.C.D. was supported by NIEHS T32 ES007013, and S.A.C. was supported by the Robert Emrie Smith Memorial Research Fellowship and NIEHS T32 Fellowship ES007058. Additional support provided by the UC Davis Environmental Health Sciences Center Biostatistics Core P30ES023513 and the UC Davis Comprehensive Cancer Center P30CA093373.

The authors declare no competing financial interest.

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