Naphthalene genotoxicity: DNA adducts in primate and mouse airway explants

Abstract

Naphthalene (NA) is a ubiquitous environmental pollutant and possible human carcinogen that forms tumors in rodents with tissue/regional and species selectivity. This study seeks to determine whether NA is able to directly adduct DNA in an ex vivo culture system. Metabolically active lung tissue was isolated and incubated in explant culture with carbon-14 labeled NA (0, 25, 250 μM) or 1,2-naphthoquinone (NQ), followed by AMS analyses of metabolite binding to DNA. Despite relatively low metabolic bioactivation in the primate airway, dose-dependent NA-DNA adduct formation was detected. More airway adducts were detected in female mice (4.7-fold) and primates (2.1-fold) than in males of the same species. Few adducts were detected in rat airway or nasal epithelium. NQ, which is a metabolic product of NA, proved to be even more potent, with levels of adduct formation 70-80-fold higher than seen when tissues were incubated with the parent compound NA. This is the first study to demonstrate NA-DNA adduct formation at a site of carcinogenesis, the mouse lung. Adducts were also detected in non-human primate lung and with a NQ metabolite of NA. Taken together, this suggests that NA may contribute to in vivo carcinogenesis through a genotoxic mechanism.

Keywords: DNA adducts; Naphthalene; Naphthoquinone; Respiratory toxicity.

Figure 1.. Methods and study design.

(A) An example of metabolically active microdissected airway tissue from a B6C3F1 mouse. (B) Study design: metabolically active tissue from male and female adult animals of three species was isolated and incubated in explant culture with ¹⁴C labeled NA or ¹⁴C labeled1,2-NQ for 1 hour, followed by ¹⁴C-AMS analyses of metabolite binding to DNA or deoxynucleosides (dN) that include dA, dG, dC, and dT. Each ¹⁴C exposure was run in parallel with non-radiolabeled controls. Abbreviations: N, number of samples per endpoint; F, female; M, male.

Figure 2.. Sex and species differences in NA DNA adducts.

Mouse and primate airway are susceptible to DNA adduct formation after a 250μM NA ex vivo incubation. (A) Pooled B6C3F1 mouse airway (N=12), pooled rhesus macaque primate airway (N=9), and male nasal epithelium (respiratory (N=6) and olfactory (N=6)). The dashed black line reflects the level of radioactivity present in male rat lung, which is not a known site of carcinogenesis. Neither rat olfactory or respiratory nasal epithelium has levels of ¹⁴C exceeding levels in rat lung. (B) Adduct formation is elevated in lung tissue from female mice and primates. *=significantly different from rat respiratory, #=significantly different from rat olfactory, †=significantly different from rat lung, ‡ significantly different from male mouse airway.

Figure 3.. Dose dependent NA DNA adduct formation.

NA DNA adduct levels in (A) male and (B) female primate airway explants after a 1 hour incubation. Sample size for these comparisons was 3 (25μM) to 4 (250μM) for the male dose response, and 5 (25μM) to 6 (250μM) for female dose response.

Figure 4.. In primate airway explants, the ability of 1,2-NQ to form DNA adducts, and remain bound to dG, greatly exceeds that of NA.

(A) The 250μM incubation with 1,2-NQ resulted in 70-fold greater adduct formation in males and 80-fold greater adduct formation in females (p<0.0001; N_1,2-NQ=3, N_NA=6) than was observed with 250μM NA incubations. (B) NA and 1,2-NQ DNA adducts are still detected in enzymatically digested DNA, bound to dN.