Investigation of the cumulative tissue doses of naphthoquinones in human serum using protein adducts as biomarker of exposure

Abstract

Both 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) are reactive metabolites of naphthalene that are thought to be responsible for the naphthalene-induced cytotoxicity and genotoxicity. The aim of this study was to investigate the cumulative tissue dose of 1,2-NPQ and 1,4-NPQ in human serum derived from blood donors in Taiwan via measurements of albumin adducts by a methodology, which employs trifluoroacetic acid anhydride and methanesulfonic acid to selectively cleave cysteinyl adducts on proteins. Both 1,2-NPQ and 1,4-NPQ adducts were detected in all male and female subjects ( n = 22). The median levels of 1,2-NPQ adduct in human subjects were estimated to be 268 (range 139–857) and 203 (range 128–1352) (pmol/g) in male ( n = 11) and female ( n = 11) subjects, respectively. In contrast, the median levels of 1,4-NPQ adduct were estimated to be 45.0 (range 22.0–117) and 38.9 (range 21.5–172) (pmol/g) in male and female subjects, respectively. We noticed that levels of 1,2-NPQ adduct were significantly correlated with those of 1,4-NPQ adduct (correlation coefficient r = 0.643, p < 0.01). Results from in vitro experiments confirmed that the production of naphthoquinones-derived adducts on serum albumin increased with increased concentration of naphthoquinones (0–100 μM). Linear relationships were observed over the range of concentration. Time-course experiments suggested that both 1,2-NPQ and 1,4-NPQ-derived adducts rapidly reached maximum values at 10 min mark and remained constant thereafter. The reaction rate constant analyses indicated that the second-order rate constants, representing in vitro reactions between naphthoquinones and cysteine residues of serum albumin, were estimated to be 0.0044/0.0002 L(g protein) −1 h −1, respectively. Overall, the cumulative tissue doses of 1,4-NPQ (217–316 nM h) in male and female subjects were ∼3-fold greater than those of 1,2-NPQ (76–98 nM h) in the study population. The initial concentrations of serum 1,2-NPQ and 1,4-NPQ in the study population were estimated to be between 145–188 and 807–1175 nM, respectively. We conclude that the relatively large amounts of naphthoquinones present in human serum may point to toxicological consequences.