Potential Application of Benzo(a)Pyrene-Associated Adducts (Globin or Lipid) as Blood Biomarkers for Target Organ Exposure and Human Risk Assessment

In order to investigate the potential application of blood biomarkers as surrogate indicators of carcinogen–adduct formation in target-specific tissues, temporal formation of benzo[a]pyrene (BaP)-associated DNA adducts, protein adducts, or lipid damage in target tissues such as lung, liver, and kidney was compared with globin adduct formation or plasma lipid damage in blood after continuous intraperitoneal (ip) injection of [³H]BaP into female ICR mice for 7 d. Following treatment with [³H]BaP, formation of [³H]BaP–DNA or –protein adducts in lung, liver, and kidney increased linearly, and persisted thereafter. This finding was similar to the observed effects on globin adduct formation and plasma lipid damage in blood. The lungs contained a higher level of DNA adducts than liver or kidneys during the treatment period. Further, the rate of cumulative adduct formation in lung was markedly greater than that in liver. Treatment with a single dose of [³H]BaP indicated that BaP–globin adduct formation and BaP–lipid damage in blood reached a peak 48 h after treatment. Overall, globin adduct formation and lipid damage in blood were significantly correlated with DNA adduct formation in the target tissues. These data suggest that peripheral blood biomarkers, such as BaP–globin adduct formation or BaP–lipid damage, may be useful for prediction of target tissue-specific DNA adduct formation, and for risk assessment after exposure.     

DNA damage induced in mouse tissues by organic wood preserving waste extracts as assayed by 32P-postlabeling

 Numerous wood preserving waste (WPW) sites in the United States pose genotoxic hazards. WPWs consist of complex mixtures containing toxic, including genotoxic, compounds which are derived from the preservatives coal tar creosote and pentachlorophenol (PCP) and other polychlorinated aromatics. The genotoxicity of WPW extracts, which has not been tested in mammals, cannot be evaluated on the basis of data for individual components because of possible compound interactions. Therefore, whole extracts need to be assayed. ³²P-postlabeling represents a powerful tool to determine DNA adduct formation by complex genotoxic mixtures, such as cigarette smoke, diesel exhaust, and coke oven and foundry emissions in experimental animals and humans. In the present study, a mouse bioassay was used in combination with ³²P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6×10⁶ DNA nucleotides in skin (both extracts) and one adduct in 3.2×10⁷ or 1.2×10⁷ DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the ³²P-labeled derivative of the reaction product of 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) with N ² of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. The benzo[a]pyrene adduct levels in the five tissues correlated linearly with total adduct levels and thus represented a surrogate for the latter. Overall, the results suggest that DNA adducts in mouse tissues, as analyzed by ³²P-postlabeling, are suitable biomarkers and dosimeters of the genotoxicity of WPW extracts. Received: 26 September 1995/Accepted: 7 December 1995     

Detection and quantitation of benzo[a]pyrene-derived DNA adducts in mouse liver by liquid chromatography-tandem mass spectrometry: comparison with 32P-postlabeling

The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.     

Tissue differences,dose-response relationship and persistence of DNA adducts in blue mussels (Mytilus edulis L) exposed to benzo[a]pyrene

Baltic Sea blue mussels (Mytilus edulis) were experimentally exposed to the genotoxic model substance benzo[a]pyrene (B[a]P) to study DNA adduct formation. The specific aims were (a). to examine where in the mussels the DNA adducts were formed, in gills or digestive gland; (b). to study the dose-response relationship between B[a]P exposure and DNA adduct formation; and (c). to examine the persistence of the formed adducts. A Scope for growth (SFG) study was also run to compare physiological responses of the mussels with the degree of DNA adduct formation. In an initial dose-response experiment, the mussels were exposed to 0, 5, 50, and 100 microg/l of tritium labelled B[a]P under semi-static conditions for 4 days, and thereafter the bioaccumulation of B[a]P and DNA adduct formation in different tissues was determined using liquid scintillation counting and 32P-postlabelling analysis, respectively. In a following exposure-depuration experiment, mussels were exposed to 17 microg/l of radiolabelled B[a]P under semi-static conditions for 6 days. B[a]P accumulation and DNA adduct formation were determined during the exposure, and B[a]P elimination and persistence of DNA adducts were studied during 28 days of depuration in uncontaminated water. The results revealed large tissue differences in DNA adduct formation. DNA adduct levels were not elevated in the digestive gland of the mussels at any exposure concentration (0-100 microg/l), even though the highest B[a]P tissue concentrations were found in the digestive gland (1.0+/-0.1 mg B[a]P/g tissue dry wt at 100 microg/l, mean+/-SE, n=12). DNA adducts were on the other hand formed in the gills, with the highest levels found in mussels exposed to 50 and 100 microg B[a]P/l, and a dose dependent increase in adduct levels (from 1.6 to 5.9 nmol adducts/mol nucleotides) from 0 to 50 microg B[a]P/l. In gills, DNA adduct levels increased with time during the 6-day exposure period in the exposure-depuration experiment, and then persisted for at least 2 weeks after exposure cessation while B[a]P tissue levels exhibited a rapid decrease (half-life of 8 days). No significant differences were observed in SFG between the control and exposed groups. Since DNA adducts exhibited a relatively high persistence in gills compared to B[a]P tissue concentrations, they seem to be a more integrated measure of genotoxic exposure than only chemical analysis of the contaminant bioaccumulation. The results also suggest that if using analysis of DNA adducts in M. edulis for monitoring purposes, analysis of gills in addition to the more commonly used digestive gland should be taken into consideration.     

Hydroxychavicol modulates benzo[a]pyrene-induced genotoxicity through induction of dihydrodiol dehydrogenase

Areca quid (AQ) chewing and smoking have synergistic potential in the development of oral squamous cell carcinoma (OSCC). In Taiwan, fresh Piper betle inflorescence is uniquely added to AQ, and hydroxychavicol (HC) is the major phenolic component of P. betle inflorescence. This study investigated whether HC modulates cigarette carcinogen benzo[a]pyrene (B[a]P)-mediated toxic effects. Pretreatment of HC and followed by B[a]P challenge resulted in higher cytotoxicity and HPRT gene mutation frequency (P < 0.05). However, this treatment protocol resulted in decreased bulky B[a]P-DNA adduct levels as demonstrated by 32P-postlabeling technique (P < 0.05). Western blotting analysis indicated that HC pretreatment induced the expression of cyclooxygenase-2 (COX-2) and dihydrodiol dehydrogenase (DDH). COX-2 is know to participate in the B[a]P-DNA adduct formation, while DDH has been shown to divert B[a]P-diol to B[a]P-7,8-quinone and the generation of reactive oxygen species (ROS). Using flow cytometry, this study demonstrated the increased production of 8-oxoguanine (P < 0.001). Overall, the results suggest that HC-induced DDH is more important than site-by-site up-regulation of COX-2 in B[a]P-induced cytotoxicity and HPRT gene mutation. Furthermore, DDH-mediated oxidative DNA damage and not B[a]P-DNA adduct formation may be involved in the HC and B[a]P-induced toxic effects.     

32P-postlabeling analysis of DNA adduction in mouse skin following topical administration of (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene.

32P-Postlabeling was employed for analysis of DNA adducts produced in mouse skin following topical administration of enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). Deoxynucleoside 3'-monophosphates were isolated by digestion of epidermal DNA with micrococcal endonuclease and spleen phosphodiesterase and phosphorylated with [gamma-32P]ATP. 32P-Labeled deoxynucleoside 3',5'-bisphosphate adducts to diastereomeric benzo[a]pyrene dihydrodiol epoxides (BPDE) were separated by four-directional thin-layer chromatography on poly(ethylenimine)-cellulose plates using a recently described solvent system [Reddy, A. P., Pruess-Schwartz, D., and Marnett, L. J. (1992) Chem. Res. Toxicol. (preceding paper in this issue)]. When (+)-BP-7,8-diol was topically administered, a major adduct spot was detected that cochromatographed with a standard produced by reaction of 7(S),8(R)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-syn-BPDE] with DNA. The level of this adduct increased in a dose- and time-dependent fashion and was elevated in animals pretreated with beta-naphthoflavone. Relatively small amounts of radioactivity cochromatographed with standards of deoxynucleoside 3',5'-bisphosphate adducts derived from 7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE]. Following topical administration of (-)-BP-7,8-diol, a single adduct spot was detected that cochromatographed with a standard of the major deoxyguanosine adduct derived from 7(R),8(S)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. The stereochemistry of epoxidation of the enantiomers of BP-7,8-diol indicates that cytochrome P-450 catalyzes the terminal activation step of benzo[a]pyrene activation to an ultimate carcinogen in mouse skin, a target organ for its carcinogenic activity.     

Differences in gene expression and benzo[a]pyrene-induced DNA adduct formation in the liver of three strains of female mice with identical AhRb2 genotype treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin and/or benzo[a]pyrene

To search for genes whose products modify aryl hydrocarbon receptor (AhR)-dependent toxicity caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), gene expression profiles in the liver were surveyed using microarrays 24 h after the administration of TCDD to three strains of female mice, BALB/cAnN (BALB), C3H/HeN (C3H) and CBA/JN (CBA) all of identical AhR genotype. The BALB/cAnN strain had a more marked induction of a number of glutathione S-transferase (GST) sub-families, particularly the GSTmicro gene family, compared with the other two strains. To assess the effects of GSTs induction to metabolize carcinogens, TCDD (40 microg kg(-1)) was administered to BALB and CBA strains, followed 24 h later by an i.p. injection of low or high dose of benzo[a]pyrene (B[a]P, 50 or 200 mg kg(-1)). The 32P-postlabelling analysis showed that administration of TCDD alone failed to induce DNA adduct formation in both BALB and CBA strain mouse livers. The low dose of B[a]P alone produced DNA adduct in the liver of both strains to a similar extent. Treatment with TCDD 24 h before the low dose of B[a]P suppressed the formation of B[a]P-induced DNA-adduct more markedly in the BALB strain compared with the CBA strain. Taken together, these findings show that TCDD treatment causes strain-specific alterations in gene expression and B[a]P-induced DNA adduct formation in the liver of female mice of the same AhRb2 genotype. Furthermore, it suggests that TCDD-treated female mice of the BALB strain may have genes whose products modify the toxicity of B[a]P as evidenced by TCDD-induced alterations in B[a]P-DNA adduct formation.     

Assessment of the protective effects of selected dietary anticarcinogens against DNA damage and cytogenetic effects induced by benzo[a]pyrene in C57BL/6J mice

The protective action in C57BL/6 J mice from orally administered ellagic acid (EA), benzyl isothiocyanate (BITC), an extract of epigallocatechins (Tegreen®) as well as chlorophyllin (CHL) against benzo[a]pyrene (B[a]P)-induced DNA damage and cytogenetic effects was investigated. In pilot experiment the comet assay indicated protective effects for all compounds, while such activity was confined to EA and CH with respect to B[a]P-DNA adducts and micronuclei. EA and CH were chosen for the main study where the levels of DNA adducts in liver after injection of 30 mg B[a]P/kg b.w. did not differ from those found for animals exposed to B[a]P and treated with the protective substances. In leukocytes no significant protective effect of CHL was detected while a 2-fold increase of adduct concentrations was observed after co-administration of EA. In the comet assay CHL or EA caused a 3-fold decrease of SSB, and a 2-fold decrease of FPG sites in comparison to animals treated with B[a]P. CHL or EA showed a significant protective effect against B[a]P-induced MN in polychromatic erythrocytes in bone marrow. In contrast, flow cytometry measurements in peripheral blood indicated the MN frequency after treatment with CHL or EA almost twice as high as that recorded for B[a]P alone.     

Formation and persistence of DNA adducts of anticancer drug ellipticine in rats

Ellipticine is an antineoplastic agent, whose mode of antitumor and/or toxic side effects is based on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochromes P450 and peroxidases. We investigated the formation and persistence of DNA adducts generated in rat, the animal model mimicking the bioactivation of ellipticine in human. Using (32)P-postlabeling, ellipticine-DNA adducts were found in liver, kidney, lung, spleen, heart and brain of female and male rats exposed to ellipticine (4, 40 and 80 mg/kg body weight, i.p.). The two major adducts were identical to the deoxyguanosine adducts generated in DNA by 13-hydroxy- and 12-hydroxyellipticine in vitro as confirmed by HPLC of the isolated adducts. At four post-treatment times (2 days, 2, 10 and 32 weeks) DNA adducts in rats treated with 80 mg/kg of ellipticine were analyzed in each tissue to study their long-term persistence. In all organs maximal adduct levels were found 2 days after administration. At all time points highest total adduct levels were in liver (402 adducts/10(8) nucleotides after 2 days and 3.6 adducts/10(8) nucleotides after 32 weeks), kidney and lung followed by spleen, heart and brain. Total adduct levels decreased over time to 0.8-8.3% of the initial levels till the latest time point and showed a biphasic profile, a rapid loss during the first 2 weeks was followed by a much slower decline till 32 weeks. These results, the first characterization of persistence of ellipticine-DNA adducts in vivo, are necessary to evaluate genotoxic side effects of ellipticine.     

Evidence of involvement of multiple sites of metabolism in the in vivo covalent binding of dibenzo[a,h]pyrene to DNA.

The in vivo formation of dibenzo[a,h]pyrene-DNA adducts in mouse skin was assessed by laser-excited fluorescence spectroscopy at 77 and 4.2 K. Two adducts were identified with fluorescence origin bands at approximately 383.5 and 407.2 nm, and these were shown to possess pyrene and benzo[a]pyrene (B[a]P) chromophores, respectively. Both DNA-bound chromophores displayed considerable electron-phonon coupling and likely assume a highly base-stacked or quasi-intercalated configuration within DNA duplexes. The presence of B[a]P and pyrene aromatic systems indicates that two-electron or monooxygenation metabolism occurred on either the a or h benzo moieties (which are equivalent) in the former case, and on both these rings in the latter case. The presence of two adduct species agrees with 32P-postlabeling analysis of the DNA, which showed the presence of two major adducts in both thin-layer and high-performance liquid chromatographic separations.     

Cytochrome P450 (CYP1A) induction and DNA adducts in a rat hepatoma cell line (Fao), exposed to environmentally relevant concentrations of organic compounds, singly and in combinations

Cytochrome P4501A (CYP1A) induction and DNA adduct formation were evaluated in the rat hepatoma cell line Fao, as biomarkers of exposure to organic compounds. Cells were exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) or 3,3',4,4'-tetrachlorobiphenyl (TCB), and to combinations of B[a]P and TCB. Both B[a]P and TCB induced CYP1A proteins in a concentration-dependent relationship, up to concentrations of 10 and 1 μM, respectively, detected by Western blotting. DNA adducts, analyzed by (32)P-postlabeling, were found at the highest concentrations of B[a]P (1 and 10 μM). No adducts were found in cells exposed to 0.1 μM TCB alone. The cotreatment of TCB and B[a]P indicated an increase in DNA adduct formation, compared with B[a]P, but no further induction of CYP1A protein compared with TCB alone. This study suggests that Western blotting and (32)P-postlabeling might be suitable methods for detecting CYP1A protein induction and DNA adducts, respectively, after exposure to environmentally relevant concentrations of organic compounds.     

细胞色素P450调节剂对DNA加合物形成的影响

人羊膜上皮细胞ＦＬ系分别接触ａ－萘黄酮（０．６ｍｍｏｌ·Ｌ￣（－１））β－萘黄酮（２０ｐｍｏｌ·Ｌ￣（－１））２４ｈ后，再用苯并（ａ）芘［Ｂ（ａ）Ｐ，１０ｕｍｏｌ·Ｌ￣（－１）］处理２４ｈ，用３２Ｐ后标记技术测定以Ｂ（ａ）－ＤＮＡ加合物。结果发现，阳性对照组，ａ－萘黄酮预处理组及β－萘黄酮预处理组加合物的量分别为（加合物个数／１０’个核苷酸）：４．７±０．２（１００％），１．８±０．９（３８．３％），１６．０±２．２（３４０．１％）．该实验结果直接显示了纳胞色素Ｐ４５０调节剂对肿瘤发生影响的作用水平。亦为药物对致癌物代谢影响的研究提供了一种方法．     

Formation of benzo[a]pyrene metabolite-nucleoside adducts in isolated perfused rat and mouse liver and in mouse lung slices.

The formation of benzo[a]pyrene metabolite-nucleoside adducts in perfused rat and mouse liver and in mouse lung slices was studied by Sephadex LH20 chromatography. In liver from β-naphthoflavone-pretreated rats, four different deoxyribonucleoside complexes were observed; these are tentatively attributed to DNA modification by the 7,8-diol-9, 10-epoxide(s), secondary metabolites of benzo[a]pyrene quinones, the 4,5-oxide, and secondary metabolites of benzo[a]pyrene phenols. The diol-epoxide-deoxyribonucleoside adduct was also detected in perfused liver and in lung slices from 3-methylcholanthrene-treated genetically responsive C57BL/6N mice, whereas no adducts were detectable in such samples from 3-methylcholan-threne-treated genetically nonresponsive DBA/2N mice. In perfused liver of phenobarbital-pretreated rats, the 4,5-oxide-deoxyribonucleoside adduct was present. These results suggest that some of the benzo[a]pyrene metabolite-nucleoside complexes generated by microsomes and deproteinized DNA in vitro also occur in the intact rodent liver and lung tissues.Furthermore, complexes with the diol-epoxide(s) were observed with RNA from perfused liver of β-naphthoflavone-treated, but not from untreated or phenobarbital-treated rats. Complexes between ribonucleoside(s) and the diol-epoxide(s) were also found in perfusedliver or lung slices from genetically responsive but not from genetically nonresponsive mice.     

Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers

This study was designed to investigate the molecular mechanism underlying the chemopreventive effects of methionine on benzo[a]pyrene (B[a]P)-DNA adducts formation in HepG2 cells. Methionine significantly inhibited B[a]P-DNA adduct formation in HepG2 cells. Methionine significantly decreased the cellular uptake of [³H] B[a]P, but increased the cellular discharge of [³H] B[a]P from HepG2 cells into the media. B[a]P significantly lowered total cellular glutathione (GSH) level, but co-cultured with B[a]P and methionine, gradually attenuated intracellular GSH levels in a concentration-dependent manner, which was markedly higher at 20-500 μM methionine. The cellular proteins of treated cells were resolved by 2D-polyacrylamide gel electrophoresis. Proteomic profiles showed that phase II enzymes such as glutathione S-transferase (GST) omega-1, GSTM3, glyoxalase I (GLO1) and superoxide dismutase (SOD) were down-regulated by B[a]P treatment, whereas cathepsin B (CTSB), Rho GDP-dissociation inhibitor alpha (Rho-GDP-DIA), histamine N-methyltransferase (HNMT), spermidine synthase (SRM) and arginase-1 (ARG1) were up-regulated by B[a]P. B[a]P and methionine treatments, GST omega-1, GSTM3, GLO1 and SOD were significantly enhanced compared to B[a]P alone. Similarly, methionine was effective in diminishing the B[a]P-induced up-regulation of CTSB, Rho-GDP-DIA, HNMT, SRM and ARG1. Our data suggests that methionine might exert a chemoprotective effect on B[a]P-DNA adduct formation by attenuating intracellular GSH levels, blocking the uptake of B[a]P into cells, or by altering expression of proteins involved in DNA adduct formation.     

DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas. Potential use for genotoxicant biomonitoring of fresh water ecosystems

The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a 32P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 microg g(-1) dry weight) in comparison to individuals from the reference site (0.053 microg g(-1) dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10(8) nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 microg g(-1) dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[a]pyrene led to a dose-dependent accumulation of B[a]P (up to 7063 microg g(-1) dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10(8) nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced 32P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in zebra mussels could be a suitable biomarker to monitor PAH-exposure and evaluate genotoxicity in fresh water ecosystems.     

Ovarian susceptibility to benzo[a]pyrene: tissue burden of metabolites and DNA adducts in F-344 rats

Exposure to environmental toxicants has been implicated as one of the causative factors for infertility in mammals. The objective of this study was to determine the amount of ingested benzo[a]pyrene (BaP), an environmental toxicant that reaches the reproductive tissues (internal dose) subsequent to a single acute exposure. Toward this end, the concentrations of BaP reactive metabolites and BaP-DNA adducts were measured throughout the course of BaP's residence in the body. Ten-week-old female Fischer-344 rats weighing approximately 220 g were administered 5 mg BaP/kg body weight orally. 1, 7, 14, 2,1 and 28 d post BaP exposure, BaP parent compound and metabolites from plasma, ovaries, and liver tissues were extracted using liquid-liquid extraction. The extracts were analyzed by reverse-phase highperformance liquid chromatography (HPLC). DNA was isolated and analyzed for BaP-induced DNA adducts by (32)P-postlabeling method. The BaP total metabolite concentrations in plasma, ovaries, and liver showed a gradual decrease from d 1 to 28 post BaP administration. The BaP-DNA adducts concentrations in ovaries and liver tissues from the treatment group demonstrated a trend similar to that observed for metabolites. Ovaries showed greater concentrations of DNA adducts compared to liver. However, with an increase in time post cessation of exposure, the adduct concentrations in liver tissue started declining rapidly, from d 1 to 28. For ovaries, the adduct concentrations demonstrated a significant decline from d 1 to 7 and a gradual fall thereafter. A concordance between BaP reactive metabolite levels and adduct concentrations indicates that the bioavailability of reactive metabolites determines the binding with DNA and consequently the formation and persistence of adducts in an acute exposure regimen.     

Metabolism of ochratoxin A: absence of formation of genotoxic derivatives by human and rat enzymes

Ochratoxin A (OTA) is a potent renal carcinogen in male rats, although its mode of carcinogenicity is not known. The metabolism and covalent binding of OTA to DNA were investigated in vitro with cytochromes P450, glutathione S-transferases, prostaglandin H-synthase, and horseradish peroxidase. Incubation of OTA with rat or human liver microsomes fortified with NADPH resulted in formation of 4-(R)-hydroxyochratoxin A at low rates [10-25 pmol min(-1) (mg of protein)(-1)]. There was no evidence of OTA metabolism and glutathione conjugate formation with rat, mouse, or human kidney microsomes or postmitochondrial supernatants (S-9) [<5 pmol min(-1) (mg of protein)(-1)]. Recombinant human cytochromes P450 (P450) 1A1 and 3A4 formed 4-(R)-hydroxyochratoxin A at low rates [0.08 and 0.06 pmol min(-1) (pmol of P450)(-1), respectively]; no oxidation products of OTA were detected with recombinant human P450 1A2 or 2E1 or rat P450 1A2 or 2C11 [<0.02 pmol min(-1) (pmol of P450)(-1)]. Prostaglandin H-synthase produced small amounts of an apolar product [33 pmol min(-1) (mg of protein)(-1)], and OTA products were not formed with horseradish peroxidase. There was no evidence of DNA adduct formation when [(3)H]OTA was incubated with these enzyme systems in the presence of calf thymus DNA (<20 adducts/10(9) DNA bases); however, these enzymes catalyzed DNA adduct formation with the genotoxins aflatoxin B(1), 2-amino-3-methylimidazo[4,5-f]quinoline, benzo[a]pyrene, and pentachlorophenol. There was also no detectable [(3)H]OTA bound in vivo to kidney DNA of male Fischer-344 rats treated orally with [(3)H]OTA (1 mg/kg, 100 mCi/mmol, 24 h exposure, <2.7 adducts/10(9) DNA bases), based upon liquid scintillation counting. However, (32)P-postlabeling experiments did show evidence of DNA lesions with total adduct levels ranging from 31 to 71 adducts/10(9) DNA bases, while adducts in untreated rat kidney ranged from 6 to 24 adducts/10(9) DNA bases. These results do not support the premise that OTA or metabolically activated species covalently bind to DNA and suggest that the (32)P-postlabeled lesions are due to products derived from OTA-mediated cytotoxicity.     

A novel method for the isolation and identification of stable DNA adducts formed by Dibenzo[a,l]pyrene and Dibenzo[a,l]pyrene 11, 12-dihydrodiol 13,14-epoxides in vitro.

Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo[a,l]pyrene (DB[a,l]P) in vitro, which comprise about 84% of all the DNA adducts that are formed [Li, K.-M., et al. (1995) Biochemistry 34, 8043-8049]. To determine a complete adduct profile and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line-narrowing spectroscopy (FLNS) techniques. Calf thymus DNA, bound to either (+/-)-anti- or (+/-)-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l]P, was first digested to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase. The adducts were then separated by HPLC with an ion-pair column and identified by FLNS by using the spectra of standards for comparison. In reactions with (+/-)-anti-DB[a,l]PDE, three adducts, an anti-cis-DB[a,l]PDE-dGMP, an anti-trans-DB[a, l]PDE-dAMP, and an anti-cis-DB[a,l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (+/-)-syn-DB[a,l]PDE, a pair of syn-trans-DB[a,l]PDE-dGMP adducts as well as a syn-cis-DB[a, l]PDE-dGMP, a syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-trans-DB[a, l]PDE-dAMP adducts were identified. From the digest of microsome-activated DB[a,l]P-bound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGMP, a syn-trans-DB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cis-DB[a, l]PDE-dAMP adduct was identified only by (32)P-postlabeling. A total of five of the stable adducts formed by DB[a,l]P and nine of the stable adducts formed by DB[a,l]PDE in vitro have been identified. These adducts were also correlated to adduct spots in the (32)P-postlabeling method by cochromatography with standards. Approximately 93% of the stable adducts formed in reactions with (+/-)-anti-DB[a,l]PDE, 90% of adducts with (+/-)-syn-DB[a,l]PDE, and 85% of adducts formed with microsome-activated DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of stable Gua and Ade adducts were observed in the microsome-catalyzed binding of DB[a, l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (+/-)-anti- or (+/-)-syn-DB[a,l]PDE.     

Preparation, isolation, and characterization of Dibenzo[a,l]pyrene diol epoxide-deoxyribonucleoside monophosphate adducts by HPLC and fluorescence line-narrowing spectroscopy.

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon that has been identified in the environment. Earlier studies in our laboratory indicated that more than 80% of the DB[a,l]P-DNA adducts formed in vitro were depurinating adducts and that most of the stable adducts were formed from diol epoxide intermediates. To complete the profile of both stable and depurinating adducts of DB[a,l]P, we have synthesized standard adducts by reacting 3'-dAMP or 3'-dGMP with either (+/-)-anti- or (+/-)-syn-dibenzo[a,l]pyrene 11,12-dihydrodiol 13, 14-epoxide (DB[a,l]PDE). The adducts were separated by HPLC with an ion-pair column and were identified by fluorescence line-narrowing spectroscopy (FLNS). A total of six pairs of stereoisomers along with another stable DB[a,l]PDE-DNA adduct were successfully isolated and identified. Pairs of (+/-)-trans and (+/-)-cis isomers were expected to be formed from the reaction of anti-DB[a,l]PDE with either dAMP or dGMP. While we were able to identify two pairs of stereoisomeric (+/-)-syn-DB[a,l]PDE-dAMP (cis and trans) and two pairs of stereoisomeric (+/-)-anti-DB[a,l]PDE-dAMP (cis and trans) adducts, identification of all the stereoisomers of dGMP adducts proved to be impossible. A pair of (+/-)-syn-trans-DB[a,l]PDE-dGMP adducts, a pair of (+/-)-anti-cis-DB[a,l]PDE-dGMP adducts, and one syn-cis-DB[a,l]PDE-dGMP adduct were conclusively identified by FLNS. These standard adducts will be used to identify the stable DNA adducts formed by DB[a,l]P and DB[a,l]PDE in vitro and in vivo.     

Immunofluorescent detection of 8-oxo-dG and PAH bulky adducts in fish liver and mussel digestive gland

Development of methodologies to detect DNA damage induced by pollutants is of increasing concern in marine ecotoxicology. We previously described an immunoperoxidase method for revealing 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-dG) in marine organisms. In this work, the approach was extended to immunofluorescence detection and to the use of another antibody for polycyclic aromatic hydrocarbon (PAH)-DNA adducts. Specimens of European eel (Anguilla anguilla) and Mediterranean mussel (Mytilus galloprovincialis) were exposed to benzo(a)pyrene (B[a]P), as model chemical carcinogen to induce both oxidized bases and B[a]P diol-epoxide-deoxyguanosine adducts. Cryostat sections of liver and digestive gland from both species were immunostained, and DNA damage was semiquantitatively evaluated by an image analysis system. Compared to untreated organisms, B[a]P-exposed organisms exhibited increased levels of oxidative DNA damage; in eels, which rapidly metabolize PAHs, the occurrence of B[a]P-DNA adducts was also detected. The immunofluorescent assay maintained all the advantages previously reported for the immunoperoxidase protocol. Both methods were tested on the same eel specimens and the immunofluorescence method showed a greater extent of relative DNA damage and a higher sensitivity. Although field validation is being carried out, our results indicate the utility of antibodies to rapidly detect DNA alterations in aquatic organisms, and to investigate the risk associated with genotoxins in marine environment.