Chemico-Biological Interactions 181 (2009) 107–114 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Investigation of the cumulative tissue doses of naphthoquinones in human serum using protein adducts as biomarker of exposure Po-Hsiung Lin a,∗ , Dar-Ren Chen b , Tzu-Wen Wang a , Chia-Hua Lin a , Ming-Chieh Chuang a a Department of Environmental Engineering, National Chung Hsing University, Taichung 402, Taiwan b Comprehensive Breast Cancer Center, Changhua Christian Hospital, Changhua, Taiwan article info Article history: Received 4 March 2009 Received in revised form 20 May 2009 Accepted 21 May 2009 Available online 6 June 2009 Keywords: Protein adducts Tissue dose Naphthalene Quinones Biomarker abstract Both 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) are reactive metabolites of naph- thalene 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 con- trast, 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 M). Linear rela- tionships 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 10min 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 807–1175nM, respectively. We conclude that the relatively large amounts of naphthoquinones present in human serum may point to toxicological consequences. © 2009 Elsevier Ireland Ltd. All rights reserved. Abbreviations: 1,2-NPQ, 1,2-naphthoquinone; 1,2-NPQ-Alb and 1,4-NPQ-Alb, adducts resulting from reaction of 1,2- and 1,4-NPQ with cysteinyl residues in Alb; 1,2-NPQ-NAC and 1,4-NPQ-NAC, reaction products of 1,2- and 1,4-NPQ with N-acetyl- l-cysteine; 1,2-NPQ-S-TFA, trifluoroacetyl derivative of 1,2-NPQ adduct after the MT assay; 1,4-NPQ, 1,4-naphthoquinone; 1,4-NPQ-S-TFA, trifluoroacetyl derivatives of 1,4-NPQ adduct after the MT assay; Alb, albumin; EI, electron impact; GC–MS, gas chromatograph and mass spectrometer; GSH, glutathione; Hb, hemoglobin; HPLC, high performance liquid chromatography; MSA, methanesulfonic acid; MT assay, methanesulfonic acid and trifluoroacetic acid anhydride assay; NAC, N-acetyl-l- cysteine; NICI, negative ion chemical ionization; NPO1-Alb and NPO2-Alb, adducts resulting from reaction of NPO with cysteinyl residues in Alb; PAHs, polycyclic aromatic hydrocarbons; SD, standard deviation; TFA, trifluoroacetyl; TFAA, triflu- oroacetic acid anhydride. ∗ Corresponding author. Tel.: +886 4 22840441x515; fax: +886 4 22862587. E-mail address: pohsiunglin@yahoo.com (P.-H. Lin). 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous occu- pational and environmental pollutants. Many PAH compounds are known carcinogens, including naphthalene and benzo[a]pyrene. Cumulative evidence suggests that chronic exposure to PAHs is a risk factor for developing skin, lung, and breast cancer in human [1]. These diseases are associated with oncogenic mutations derived from the depurinating DNA adducts and bulky stable adducts of reactive metabolites of PAHs [2,3]. Metabolic activation of PAHs to form diol epoxides can give rise to the formation of promuta- genic DNA adducts [4,5]. Some of the PAH metabolites were used as urinary biomarkers for exposure to PAHs. The urinary excre- tion of a monohydroxylated PAH metabolite, 1-hydroxypyrene, is classically measured for the determination of internal dose of PAH exposure in epidemiological studies [6]. In addition, measurements of the urinary phenanthrenes metabolites, such as 1-, 2-, 3-, 4- , and 9-hydroxyphenanthrenes and phenanthrenes-dihydrodiols, 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.05.016