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Leukotriene A(4) (LTA(4)), a reactive electrophilic intermediate formed during the biosynthesis of inflammation-related lipid mediators, has been found to bind covalently to DNA. The major DNA adducts formed by LTA(4) in vitro and human cells have been identified by mass spectrometry on the nucleoside level. Here we investigated whether the thin-layer chromatography (TLC) (32)P-postlabeling method is suitable for the detection of LTA(4)-DNA adducts. The reaction of individual deoxynucleoside 3'-monophosphates with LTA(4) in aqueous basic solution yielded numerous adduct spots when analyzed by the two enrichment procedures of the (32)P-postlabeling method-nuclease P1 digestion and butanol extraction. Highest LTA(4)-adduct levels were found with deoxyguanosine 3'-phosphate (around one adduct per 10(4) normal nucleotides). Under similar reaction conditions LTA(4) (25-320 microM) was incubated with calf thymus DNA, then DNA adduct patterns and levels were determined with the TLC (32)P-postlabeling method using both enrichment versions. The same DNA adduct pattern consisting of up to seven spots was observed with both enrichment versions. DNA adduct formation by LTA(4) was concentration-dependent with major adducts being derived from deoxyguanosine. When a human monocytic cell line (Mono Mac 6) was stimulated with arachidonic acid and calcium ionophore LTA(4)-DNA adducts were detected by (32)P-postlabeling. However, the level of these endogenously formed DNA adducts was close to the detection limit (3 +/- 2 adducts per 10(8) normal nucleotides). In summary, the TLC (32)P-postlabeling method is suitable for studying DNA adduct formation by LTA(4) and can be used for further investigations on the link between inflammation and cancer.

3-Nitrobenzanthrone (3-NBA) is an environmental pollutant and suspected human carcinogen found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter; human exposure to 3-NBA is likely to occur primarily via the respiratory tract. In our study female Sprague Dawley rats were treated by intratracheal instillation with a single dose of 0.2 or 2 mg/kg body weight of 3-NBA. Using the butanol enrichment version of the (32)P-postlabeling method, DNA adduct formation by 3-NBA 48 hr after intratracheal administration in different organs (lung, pancreas, kidney, urinary bladder, heart, small intestine and liver) and in blood was investigated. The same adduct pattern consisting of up to 5 DNA adduct spots was detected by thin layer chromatography in all tissues and blood and at both doses. Highest total adduct levels were found in lung and pancreas (350 +/- 139 and 620 +/- 370 adducts per 10(8) nucleotides for the high dose and 39 +/- 18 and 55 +/- 34 adducts per 10(8) nucleotides for the low dose, respectively) followed by kidney, urinary bladder, heart, small intestine and liver. Adduct levels were dose-dependent in all organs (approximately 10-fold difference between doses). It was demonstrated by high performance liquid chromatography (HPLC) that all 5 3-NBA-derived DNA adducts formed in rats after intratracheal instillation are identical to those formed by other routes of application and are, as previously shown, formed from reductive metabolites bound to purine bases. Although total adduct levels in the blood were much lower (41 +/- 27 and 9.5 +/- 1.9 adducts per 10(8) nucleotides for the high and low dose, respectively) than those found in the lung, they were related to dose and to the levels found in lung. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in several organs of the rat and an identical adduct pattern in DNA from blood. Therefore, 3-NBA-DNA adducts present in the blood are useful biomarkers for exposure to 3-NBA and may help to assess the effective biological dose in humans exposed to it.

We have previously shown that bisphenol A (BPA) is oxidized to bisphenol-o-quinone in the presence of activation system and that the chemical reaction of DNA or deoxyguanosine 3'-monophosphate (dGMP) with bisphenol-o-quinone produces adducts. In the present study, using the 32P-postlabeling technique, we have investigated the in vivo DNA adduct formation by BPA by examining covalent modification in DNA. Administration of a single or multiple dose of 200 mg/kg of BPA to CD1 male rats produced two major and several minor adducts in liver DNA. The two major in vivo adducts matched the adduct profile of DNA or dGMP-bisphenol-o-quinone. To determine how BPA may be converted to DNA-binding metabolites, adducts were examined after incubation of DNA with BPA in the presence of a microsomal activation system. The in vitro incubation of BPA with DNA in the presence of a microsomal activation system revealed one major adduct and several minor adducts. The formation of adducts in DNA by BPA in the presence of a microsomal activation system was drastically decreased by known inhibitors of cytochrome P450. Adduct formation in DNA when cumene hydroperoxide or NADPH was used as a cofactor showed adducts with similar chromatographic mobilities as those from the reaction of dGMP-bisphenol-o-quinone. These data demonstrate that BPA is capable of binding covalently to DNA. DNA binding can be inhibited by the inhibitors of cytochrome P450. One of the DNA-binding metabolite(s) both in vitro and in vivo may be bisphenol-o-quinone. Covalent modifications in DNA by in vivo exposure of BPA may be a factor in the induction of hepatotoxicity.

An examination has been made of the binding, both in vitro and in vivo, of the benzo(a)pyrene (BP) adduct to DNA components of differing sequence complexity. Annealing was performed at low renaturation temperatures in the presence of high concentrations of formamide to minimize hydrocarbon-induced depurination. BHK-DNA was modified in vitro using a tritiated derivative of the ultimate carcinogenic metabolite of BP, 7alpha,8beta-di-hydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE). Co-renaturation of this modfied DNA with [14C]-thymidine-labelled BHK-DNA demonstrated that the hydrocarbon adduct did not interfere with strand annealing and showed that the BP adduct was distributed randomly throughout all DNA sequence classes. However, when the DNA of cells in culture was modified by [3H]BP, following metabolic activation, and mixed with [14C]-thymidine-labelled DNA, a small but reproducible difference in the renaturation of the two labels was found. This difference in renaturation profiles was not due to base-compositional effects since a similar result was found when the alternate 14C-label was present in guanine bases, the principal site of BP modification. The small difference in the renaturation of the two radioactive labels indicated an enrichment of the hydrocarbon on the most rapidly-renaturing sequence components (the palindromic and highly repetitive sequences) where it amounted to between 19 and 64% increased modification.