The aim of this study was to investigate the electrophilic tissue burden (ETB) formation, assessed as covalent binding of the ultimate carcinogen benzo( a)pyrene diolepoxide (BaPDE) with cellular proteins, in liver, lung and heart, as well as with haemoglobin (Hb) following repeated exposure to binary mixtures of benzo( a)pyrene (BaP) and pyrene (P). Male Sprague-Dawley rats were injected intraperitoneally, once daily for 10 consecutive days, with binary mixtures of BaP and P in three different exposure scenarios corresponding to BaP/P ratios of 0.2, 1 and 5 and with three dose levels of BaP (2, 6 and 20 mg/kg) for each scenario. ETB levels were measured as the ultimate analyte benzo( a)pyrene tetrol (BaPTeT) obtained after mild acid hydrolysis of BaPDE-adducts with proteins. A high-performance liquid chromatography fluorescence technique was used to quantify the analyte. Similar ETB levels (within a factor of 4) were observed in all tissues studied at any given binary dose. However, the ETB generally tended to be somewhat higher in metabolically active tissues (i.e. liver and lung) than in metabolically non-active tissues (i.e. heart and Hb). Lack of influence of pyrene on ETB levels in all tissues was confirmed over the binary dose range examined. Linear BaP-dose-dependent ETB formation in all tissues (at P

This study was aimed at investigating the correlation between biomarkers of exposure to polycyclic aromatic hydrocarbons and, more specifically, at examining the role of urinary 1-hydroxypyrene (1-OHP) as a reliable measure of internal dose linked to the electrophilic tissue burden (ETB), assessed as covalent binding of the ultimate carcinogen benzo( a)pyrene diolepoxide (BaPDE) with cellular proteins in target organs. The protocol included experimental verification of a previously proposed algorithm for adjustment of reference values for urinary 1-OHP with exposure to different mixtures of polycyclic aromatic hydrocarbons in a rat model. Hence, the relationships between ETB in liver, lung, and heart as well as the BaPDE-haemoglobin adducts level on the one hand, and urinary/faecal 1-OHP or urinary/faecal 3-hydroxybenzo( a)pyrene (3-OHBaP) on the other hand have been examined. Male Sprague-Dawley rats received intraperitoneally, once daily for 10 consecutive days, binary mixtures of benzo( a)pyrene (BaP) and pyrene (P) in three different exposure scenarios corresponding to BaP/P ratios of 0.2, 1 and 5, with three doses of BaP (2, 6 and 20 mg/kg) for each scenario. The ETB levels were measured as the ultimate analyte benzo( a)pyrene tetrol (BaPTeT) obtained after mild acid hydrolysis of BaPDE adducts with proteins. It was experimentally confirmed that: (1) urinary 1-OHP is a reliable biomarker linked to the ETB in tissues that are targets for carcinogenicity, such as lung, for the BaP/P ratios of 0.2 and 1 (linear regression p=0.0099 and 0.0293, respectively); (2) urinary 3-OHBaP is correlated with the BaPDE-haemoglobin adducts for all three exposure scenarios ( p=0.0011 for BaP/P=0.2, p<0.0001 for BaP/P=1 and p=0.0099 for BaP/P=5). The experimental relationship between ETB and urinary 1-OHP was used to interpolate biological limit values for the urinary metabolite assuming three arbitrary critical levels of ETB. These were compared with the values calculated from the algorithm using the BaP/P ratio 1 mixture as a reference. The ratios of calculated to observed values varied from 1.0 to 1.6 for the BaP/P 0.2 mixture, and from 1.9 to 3.0 for the BaP/P 5 mixture. The results obtained in the present study indicate that the algorithm mentioned above applies well for two of the three exposure scenarios corresponding to realistic occupational BaP/P ratios of 0.2 and 1. This suggests that, using ETB as an endpoint, the proposed algorithm will reasonably predict the critical value of urinary 1-OHP for mixtures having different BaP/P ratios. Stronger linear relationships between ETB in all chosen tissues and 1-OHP or 3-OHBaP excretion were obtained with urinary metabolites than with their faecal analogues. Thus urinary 1-OHP and 3-OHBaP are more reliable biomarkers in biological monitoring strategies.