ROLE OF INTERMEDIARY BIOMARKERS IN DETERMINING THE ANTICANCER EFFICACY OF MARINE COMPOUNDS

In the present study, two of the probable an umor marine compounds, manzamine A and sarcophine, were screened using benzo[a]pyrene (BP)-derived DNA adduct formation in MCF-7 cells as intermediary biomarker. Briefly, MCF-7 cells were treated with the compounds for 24 h followed by treatment with BP (0.5 μM). After 24h incubation, cellular DNA was isolated and analyzed for BP-derived DNA adducts by ³²P-postlabeling technique. Manzamine A and sarcophine increased the BP-DNA adducts by 2 to 4-folds. Further, manzamine A (50 μM) substantially down regulated the expression of p53 while sarcophine (50 μM) slightly induced the level of p21. The residual DNA repair ability was almost completely abolished by manzamine A while sarcophine was ineffective. Based on our preliminary results, these compounds may be classified as potential genotoxic.     

Effects of solvent on DNA adduct formation in skin and lung of CD1 mice exposed cutaneously to benzo(a)pyrene

The effect of solvent polarity and lipophilicity on DNA adduct formation by polycyclic aromatic hydrocarbons in skin and lung has been studied in CD1 mice exposed cutaneously in vivo to benzo(a)pyrene (∼0.01–7.0 μg/animal) in either tetrahydrofuran or n-dodecane. The nature and amounts of DNA adducts, measured as 7R,8S,9R-trihydroxy-10S-(N²-deoxyguanosyl-3′-phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene, in relation to exposure dose and treatment regime was determined by ³²P-postlabelling. In skin DNA there was a linear relationship between exposure dose and adduct formation with both solvents, though the amount of adduct formed was significantly lower from treatment with benzo(a)pyrene in n-dodecane than in tetrahydrofuran. The amounts of adducts measured in skin DNA ranged from 67 amol adducts/μg DNA at the lowest exposure dose of benzo(a)pyrene in n-dodecane to 3.5 fmol adducts/μg DNA (1 adduct in 5 × 10⁷ nucleotides to 1 adduct in 9 × 10⁵ nucleotides) at the highest dose. In tetrahydrofuran the corresponding levels were 89 amol adducts/μg DNA (1 adduct in 3 × 10⁷ nucleotides) to 16.9 fmol adducts/μg DNA (1 adduct in 2 × 10⁵ nucleotides). DNA adducts could not be detected in lung tissue following cutaneous treatment of animals with benzo(a)pyrene in n-dodecane. Cutaneous treatment of animals with benzo(a)pyrene in tetrahydrofuran, however, resulted in adducts in lung DNA at a level of 88 amol/μg DNA from exposures only at the highest dose (6.72 μg/animal). The difference in octanol-water partition coefficient, log P_ow between n-dodecane compared to tetrahydrofuran is considered to be the most likely reason for the reduction in the bioavailability of benzo(a)pyrene and/or its metabolites and hence the degree of genotoxicity in tissues. The results suggest that other paraffinic hydrocarbon solvents may moderate the genotoxicity of polycyclic aromatic hydrocarbons in vivo. The assessment of the genotoxicity in vivo of mixtures of compounds should be carried out on complete mixtures of substances of interest in order to take account of these possible antagonistic or synergistic effects. Received: 10 February 1999 / Accepted: 10 May 1999     

DNA adducts in carp exposed to artificial diesel-2 oil slicks

In attempts to mimic field exposure, oil slicks prepared from diesel-2 oil/water emulsions were poured onto the surface of water in tanks prepared fresh every day and liver DNA adducts were analyzed by ³²P-postlabeling in carp free-swimming in these tanks. ‘Clusters’ of lipophilic DNA adducts were detected, with five major and numerus minor adducts. Essentially a similar adduct pattern was found in the liver DNA of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to > 3000 amol/μg DNA at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 (≈ 10,000 amol/μg DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive DNA damage in carp in vivo and the damage accumulates proportionately with time of exposure.     

DNA adducts and oxidative DNA damage induced by organic extracts from PM2.5 in an acellular assay

The genotoxic activities of complex mixtures of organic extracts from the urban air particles collected in various localities of the Czech Republic, which differed in the extent and sources of air pollution, were compared. For this purpose, PM2.5 particles were collected by high volume samplers in the most polluted area of the Czech Republic - Ostrava region (localities Bartovice, Poruba and Karvina) and in the locality exhibiting a low level of air pollution - Trebon - a small town in the non-industrial region of Southern Bohemia. To prepare extractable organic matter (EOM), PM2.5 particles were extracted by dichloromethane and c-PAHs contents in the EOMs were determined. As markers of genotoxic potential, DNA adduct levels and oxidative DNA damage (8-oxo-7,8-dihydro-2′-deoxyguanosine, 8-oxodG, levels) induced by EOMs in an acellular assay of calf thymus DNA coupled with ³²P-postlabeling (DNA adducts) and ELISA (8-oxodG) in the presence and absence of microsomal S9 fraction were employed. Twofold higher DNA adduct levels (17.20 adducts/10⁸ nucleotides/m³ vs. 8.49 adducts/10⁸ nucleotides/m³) were induced by EOM from Ostrava-Bartovice (immediate proximity of heavy industry) compared with that from Ostrava-Poruba (mostly traffic emissions). Oxidative DNA damage induced by EOM from Ostrava-Bartovice was more than fourfold higher than damage induced by EOM from Trebon (8-oxodG/10⁸ dG/m³: 0.131 vs. 0.030 for Ostrava-Bartovice vs. Trebon, respectively). Since PM2.5 particles collected in various localities differ with respect to their c-PAHs content, and c-PAHs significantly contribute to genotoxicity (DNA adduct levels), we suggest that monitoring of PM2.5 levels is not a sufficient basis to assess genotoxicity of respirable aerosols. It seems likely that the industrial emissions prevailing in Ostrava-Bartovice represent a substantially higher genotoxic risk than mostly traffic-related emissions in Ostrava-Poruba. B[a]P and c-PAH contents in EOMs are the most important factors relating to their genotoxic potential.     

Distribution and DNA adduct formation of radiolabeled methylenediphenyl-4,4′-diisocyanate (MDI) in the rat after topical treatment

Female Wistar rats were treated topically with [¹⁴C]methylenediphenyl-4,4′-diisocyanate (MDI) in acetone on the back. Fecal excretion of radioactivity amounted to 20% of the administered radioactivity within 24 h. Urinary excretion was below 1%. About 10% of the radioactivity was retained at the site of application. Epidermal nuclear protein exhibited very high specific radioactivities; ³²P-postlabeling analysis did not reveal isocyanate-DNA adducts. In liver, lung and kidney, nuclear protein radioactivity was much lower than in the epidermis. DNA radioactivity in liver was at the limit of detection. Conversion to the units of the Covalent Binding Index, CBI=(μmol adduct/mol DNA nucleotide) per (mmol chemical administered/kg body weight) resulted in a value of <0.1. In comparison with genotoxic carcinogens, this upper bound value is indicative of a very weak maximum possible systemic genotoxic potency of topically administered MDI.     

Bioavailability of the genotoxic components in coal tar contaminated soils in Fischer 344 rats.

The effect of chemical aging on the bioavailability and subsequent genotoxicity of coal tar (CT)-contaminated soils was evaluated in a 17-day feeding study using Fischer 344 male rats. Rats consumed a control diet or diets amended with soil, 0.35% CT, or soil freshly prepared or aged for 9 months with 0.35% CT. Mild treatment-related microscopic lesions in liver tissue and elevated enzyme levels in serum were detected in all CT treatment groups. The (32)P-postlabeling assay was employed to determine DNA adduct formation in treated animals. All CT treatment groups induced DNA adducts in both the liver and lung. Adduct levels were 3-fold higher in lung DNA compared to hepatic DNA. After correcting adduct levels for total ingested polycyclic aromatic hydrocarbons (PAHs), a significant decrease (p < 0.05) in adduct levels was observed in both CT/soil treatment groups compared to CT control in liver and lung DNA. Adduct profiles of (32)P-postlabeled hepatic and lung DNA displayed several nonpolar DNA adducts that comigrated with PAH-adducted calf thymus DNA standards as determined through both thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). These results suggest that soil, but not aging of contaminants in soil, decreases the bioavailability of genotoxic components in CT, as evidenced by DNA adduct analysis.     

Formation of DNA adducts from oil-derived products analyzed by 32P-HPLC

The aim of this study was to investigate the genotoxic potential of DNA adducts and to compare DNA adduct levels and patterns in petroleum vacuum distillates, coal tar distillate, bitumen fume condensates, and related substances that have a wide range of boiling temperatures. An in vitro assay was used for DNA adduct analysis with human and rat S-9 liver extract metabolic activation followed by 32P-postlabeling and 32P-high-performance liquid chromatography (32p-HPLC). For petroleum distillates originating from one crude oil there was a correlation between in vitro DNA adduct formation and mutagenic index, which showed an increase with a distillation temperature of 250 degrees C and a peak around a distillation point of approximately 400 degrees C. At higher temperatures, the genotoxicity (DNA adducts and mutagenicity) rapidly declined to very low levels. Different petroleum products showed a more than 100-fold range in DNA adduct formation, with severely hydrotreated base oil and bitumen fume condensates being lowest. Coal tar distillates showed ten times higher levels of DNA adduct formation than the most potent petroleum distillate. A clustered DNA adduct pattern was seen over a wide distillation range after metabolic activation with liver extracts of rat or human origin. These clusters were eluted in a region where alkylated aromatic hydrocarbons could be expected. The DNA adduct patterns were similar for base oil and bitumen fume condensates, whereas coal tar distillates had a wider retention time range of the DNA adducts formed. Reference substances were tested in the same in vitro assay. Two- and three-ringed nonalkylated aromatics were rather low in genotoxicity, but some of the three- to four-ringed alkylated aromatics were very potent inducers of DNA adducts. Compounds with an amino functional group showed a 270-fold higher level of DNA adduct formation than the same structures with a nitro functional group. The most potent DNA adduct inducers of the 16 substances tested were, in increasing order, 9,10-dimethylanthracene, 7,12-dimethylbenz[a]anthracene and 9-vinylanthracene. Metabolic activation with human and rat liver extracts gave rise to the same DNA adduct clusters. When bioactivation with material from different human individuals was used, there was a significant correlation between the CYP 1A1 activity and the capacity to form DNA adducts. This pattern was also confirmed using the CYP 1A1 inhibitor ellipticine. The 32P-HPLC method was shown to be sensitive and reproducible, and it had the capacity to separate DNA adduct-forming substances when applied to a great variety of petroleum products.     

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.     

Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens

Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovo turkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using ³²P-postlabeling for DNA adducts. In ovo exposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had ³²P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity.     

P-postlabeling analysis of DNA adducts formed in vitro and in rat skin by methylenediphenyl-4,4'-diisocyanate (MDI)

The ³²P-postlabeling method was adapted for the detection of DNA adducts formed by methylenediphenyl-4,4'-diisocyanate (MDI). Incubation of the 3'-phosphates of the deoxyribosides of cytosine (C), adenine (A), guanine (G) and thymine (T) with MDI in Tris buffer resulted in the formation of 5, 7, 8, and 2 reaction products, respectively. Incubation of DNA with MDI resulted in detectable levels of 5, 2, and 1 adducts attributable to C, A, and G. Analysis of DNA isolated from the epidermis of rats treated dermally with 9 mg MDI showed an adduct pattern similar to the one seen in the in vitro DNA incubation. A total adduct level of 7 per 10⁸ nucleotides was measured, the limit of detection was 2 adducts per 10¹⁰ nucleotides. The data indicate that a minute fraction of MDI can reach DNA in vivo in a chemically reactive form. In comparison with the genotoxic skin carcinogen 7,12-dimethylbenz[a]anthracene on the other hand, the DNA-binding potency of MDI was more than 1000-fold lower.     

Pentachlorophenol enhances 9-hydroxybenzo [a] pyrene-induced hepatic DNA adduct formation in vivo and inhibits microsomal epoxide hydrolase and glutathione S-transferase activities in vitro: likely inhibition of epoxide detoxication by pentachlorophenol

 We recently reported that co-administration to female mice of tamoxifen or 4-hydroxytamoxifen (4-OH-tamoxifen) with pentachlorophenol (PCP), but not with 2,6-dichloro-4-nitrophenol (DNCP) results in strong intensification of a specific subgroup, termed group I, of tamoxifen-DNA adducts in female mouse liver. As both PCP and DCNP are sulfotransferase inhibitors, we concluded that the intensification of tamoxifen group I adducts is probably not due to inhibition of sulfation by these phenols of a tamoxifen metabolite. Since epoxide derivatives of 4-OH-tamoxifen are potential candidates involved in tamoxifen-induced DNA damage, the hypothesis was developed and tested that PCP inhibits epoxide detoxication. As 4-OH-tamoxifen metabolites were unavailable to us, we employed indirect approaches to test this hypothesis. In the first set of experiments we determined whether PCP would augment DNA adduct formation from the benzo[a]pyrene metabolite, 9-hydroxybenzo[a]pyrene (9-OH-BP), as 9-OH-BP-4,5-epoxide is known to be involved in the metabolic activation of this compound. Female mice were given a single i.p. dose of 9-OH-BP (50 μmol/kg) either alone or in combination with PCP (75 μmol/kg), and hepatic DNA adducts were measured 24 h later by nuclease P1-enhanced bisphosphate ³²P-postlabeling. Co-administration of PCP with 9-OH-BP resulted in a statistically significant 1.5- to 1.7-fold increase in 9-OH-BP adduct levels versus 9-OH-BP controls. In order to determine whether PCP inhibits the enzymatic detoxication of epoxides in vitro, in a second set of experiments, the effects of PCP on liver microsomal epoxide hydrolase (mEH) and purified equine liver glutathione S-transferase (GST) activities were studied using, respectively, styrene-7,8-oxide and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. Incubation of mouse liver microsomes with PCP (10–100 μM) strongly inhibited (by 21–97%) mEH activity in a dose-dependent manner, the IC₅₀ being 35 μM. DCNP was ineffective as a mEH inactivator. PCP also inhibited purified equine liver GST activity, with an IC₅₀ of 23.5 μM. Taken together, the results of this study strongly support the hypothesis that PCP inhibited enzymatic detoxication of epoxides in vivo and in vitro. By this mechanism PCP would lead to enhancement of DNA damage caused by 9-OH-BP, and possibly other drugs and their metabolites, which undergo epoxidation prior to DNA binding. Received: 28 November 1995/Accepted: 12 March 1996     

Lung DNA adducts detected in human smokers are unrelated to typical polyaromatic carcinogens

Several studies have reported the presence of DNA adducts derived from benzo(a)pyrene and other polyaromatics by 32P-postlabeling/TLC by measuring diagonal radioactive zones (DRZs) in lung tissues of human smokers. However, our experimental studies in rodent models, which used modified chromatographic conditions to obtain distinct adduct spots, suggested that cigarette smoke-related lipophilic DNA adducts may not be derived from polycyclic aromatic hydrocarbons (PAHs) or aromatic amines. In the present study, we have performed similar analysis of human lung tissues to study the chemical nature of DNA adducts. Fifty human lung tissues from cancer patients (ages 42-83 years) with active, ex-, or never-smoking status were analyzed for highly lipophilic DNA adducts by nuclease P1- and n-butanol enrichment-mediated 32P-postlabeling assay. All DNA samples yielded low to highly intense adduct DRZs when adducts were resolved by PEI-cellulose TLC in standard high-salt, high-urea solvents. Adduct burden ranged from 6.6 to 2930 per 10(10) nucleotides. However, when adducts were resolved in a different solvent system comprising of high-salt, high-urea in direction 3 and dilute ammonium hydroxide in direction 4, which retained adducts derived from PAHs and aromatic amines on the chromatograms, this yielded no detectable adducts from human lung DNAs. Furthermore, analysis of human lung DNAs mixed with reference adducted DNAs in multisolvent systems confirmed an absence of PAH- and aromatic amine-derived adducts in human smoker lung DNA. To determine the origin of cigarette smoke-associated DNA adducts, calf thymus DNA was incubated with formaldehyde and acetaldehyde, which are known to be present in cigarette smoke in significant quantities. Analysis of purified DNAs by 32P-postlabeling resulted in adduct DRZs in the aldehyde-modified DNAs when adducts were resolved in standard urea-containing solvents, but no adducts were detected when the ammonium hydroxide-based solvent was used, suggesting that even nonpolyaromatic electrophiles can result in adduct DRZs on the chromatograms similar to those from PAH metabolites. Taken together, our data demonstrate that cigarette smoke-associated lung DNA adducts appear on chromatograms as DRZs, consistent with the literature, but they are not related to PAHs and aromatic amines.     

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.     

The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme cytochrome P450 1A1 in rat

Benzo[a]pyrene (BaP) is a human carcinogen requiring metabolic activation prior to reaction with DNA. Cytochrome P450 (CYP) 1A1 is the most important hepatic and intestinal enzyme in both BaP activation and detoxification. CYP1A2 is also capable of oxidizing BaP, but to a lesser extent. The induction of CYP1A1/2 by BaP and/or β-naphthoflavone in liver and small intestine of rats was investigated. Both BaP and β-naphthoflavone induced CYP1A expression and increased enzyme activities in both organs. Moreover, the induction of CYP1A enzyme activities resulted in an increase in formation of BaP–DNA adducts detected by ³²P-postlabeling in rat liver and in the distal part of small intestine in vivo. The increases in CYP1A enzyme activity were also associated with bioactivation of BaP and elevated BaP–DNA adduct levels in ex vivo incubations of microsomes of both organs with DNA and BaP. These findings indicate a stimulating effect of both compounds on BaP-induced carcinogenesis.     

Probenecid,an inhibitor of transmembrane organic anion transporters,alters tissue distribution of DNA adducts in 1-hydroxymethylpyrene-treated rats

1-Methylpyrene (1-MP), an abundant alkylated polycyclic aromatic hydrocarbon, is activated by side-chain hydroxylation to 1-hydroxymethylpyrene (1-HMP) and subsequent sulfo-conjugation to electrophilic 1-sulfooxymethylpyrene (1-SMP). In rats, this activation mainly occurs in liver. 1-SMP may react with hepatic DNA or be exported into the blood circulation to reach other tissues, in particular kidneys. Findings with recombinant cell lines suggest that renal 1-SMP uptake proceeds via organic anion transporters (OATs). Here, we tested the hypothesis that probenecid, a characteristic OAT inhibitor, interferes with kidney damage brought about by 1-SMP formed in rats. 1-HMP was administered intraperitoneally to 30 rats, half of which were co-treated with probenecid. The tissue distribution of DNA adducts was analyzed using ³²P-postlabeling and isotope dilution LC–MS/MS for the detection of the adducts N²-(1-methylpyrenyl)-2′-deoxyguanosine and N⁶-(1-methylpyrenyl)-2′-deoxyadenosine. In rats treated solely with 1-HMP, adduct levels in kidney tissue were about 3-fold and 8-fold higher than those in liver and lung, respectively. After co-treatment with probenecid, hepatic and pulmonary adduct levels were 12-fold and 4-fold elevated, respectively, whereas renal adduct levels were slightly lower compared to those of rats receiving 1-HMP alone. Moreover, serum levels of 1-SMP were increased 23-fold in animals pre-treated with probenecid. The differential effects on hepatic and pulmonary adduct levels suggest that not only renal OATs, but also additional anion transporters, e.g. those mediating the hepatic export of 1-SMP into the bile, were inhibited. Thus, transmembrane transport proteins play a crucial role in the distribution of reactive phase II metabolites, and thereby in tissue allocation of DNA adducts.     

Formation and removal of DNA adducts in Fischer-344 rats exposed to 2,4-diaminotoluene

  ³²P-Postlabeling was used to examine DNA adduct formation and removal in Fischer-344 rats exposed to the animal carcinogen 2,4-diaminotoluene (DAT). Adduct formation and persistence were compared between target (liver and mammary gland) and non-target organs (kidney and lung) to determine if possible differences could explain the observed organ specificity of DAT induced carcinogenesis. The effects of different exposure conditions on DNA adduct formation and removal were also examined by varying the concentration and frequency of compound administration. DAT produced three distinct DNA adducts. Among the organs examined, DNA binding was highest in the liver, with levels approximately 10 times greater than that of the mammary gland and up to 50 times greater than of the two nontarget sites. Despite the large differences in the initial extent of adduct formation, the persistence of adducts among sites was not significantly different. In the liver, there were dose-dependent differences in DNA adduct formation, but adduct removal following different dosages did not vary significantly. The effects of multiple administration on DNA adduct formation and removal were examined by treating rats with 5 mg/kg DAT daily for 10 consecutive days. Adduct yields from multiple treatment were greater than from a single 50 mg/kg exposure. The persistence of adducts following multiple treatment was also greater than after an equivalent single exposure. The results demonstrated organ-specific and dose-dependent differences in initial extent of DNA adduct formation, but no differences in adduct persistence. However, the results did suggest that adduct formation and persistence may change with repeated administration of DAT. Received: 13 April 1994 / Accepted: 4 May 1994     

Correlation of 32P-postlabelling-detection of DNA adducts in mouse skin in vivo with the polycyclic aromatic compound content and mutagenicity in Salmonella typhimurium of a range of oil products

The in vivo genotoxic activities in mouse skin of the dimethyl sulphoxide (DMSO) extracts of a range of oil products [residual aromatic extract; untreated heavy paraffinic distillate aromatic extract; mildly refined light naphthenic base oil; bitumen (vacuum residue); high viscosity index base oil obtained by catalytic hydrogenation] were evaluated by ³²P-postlabelling DNA analysis. The results of quantitative ³²P-postlabelling analyses of epidermal DNA from mice treated with the DMSO extracts showed linear relationships with the total polycyclic aromatic compound (PAC) contents, determined by the Institute of Petroleum method IP 346 and also the 3–6 ring PAC contents, measured by on-line liquid-liquid extraction using flow injection analysis. The ³²P-postlabelling data also showed a linear relationship with the mutagenicity indices of these oil products determined in S. typhimurium TA98 using the modified Ames Salmonella microsome test. The in vivo genotoxicity of the DMSO extracts from the oil products was low, judged by ³²P-postlabelling analysis of DNA adducts measured in epidermal DNA of treated mouse skin, and ranging from 2 to 723 attomole/μg DNA per mg oil product. The in vivo ³²P-postlabelling data from this study are consistent with these materials expressing low genotoxicity in mouse skin in vivo. The DMSO extraction procedure coupled with ³²P-postlabelling DNA analysis is useful for ranking the relative genotoxic potency in vivo of a wide range of oil products. In general the trend observed is similar to rankings based on physicochemical measurements of␣total PAC contents or 3–6 ring PAC contents of the oil products. Received: 11 February 1998 / Accepted: 2 April 1998     

DNA adduct formation in precision-cut rat liver and lung slices exposed to benzo[a]pyrene.

Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.     

3,2′-Dimethyl-4-aminobiphenyl-DNA Adduct Formation in Tumor Target and Nontarget Organs of Rapid and Slow Acetylator Syrian Hamsters Congenic at theNAT2Locus

DNA adduct formation is an important step in initiation of the carcinogenic process. 3,2′-Dimethyl-4-aminobiphenyl (DMABP) is a well-documented multiorgan carcinogenic aromatic amine in rodents. In the present study, DMABP-DNA adduct levels were measured in rapid (Bio. 82.73/H-Pat^r) and slow (Bio. 82.73/H-Pat^s) acetylator Syrian hamsters congenic at theNAT2locus following a single injection of 33 or 100 mg/kg body wt DMABP. Two DNA adducts,N-(deoxyguanosin-8-yl)-DMABP and 5-(deoxyguanosin-N²-yl)-DMABP, were identified and quantitated by³²P-postlabeling assay. After injection of 33 mg/kg, DMABP-DNA adducts were detected in urinary bladder at 6, 18, 24, and 48 hr with adduct levels increasing up to 48 hr postinjection. DMABP-DNA adducts were not detected in liver, colon, and heart. After injection of 100 mg/kg, DMABP-DNA adducts were detected in urinary bladder, liver, prostate, colon, and heart at 48 hr postinjection. DMABP-DNA adduct levels were significantly higher in urinary bladder (primary tumor target organ) than in the other organs of both rapid and slow acetylator congenic hamsters.N-(deoxyguanosin-8-yl)-DMABP levels were significantly higher in liver and prostate than in colon and heart of rapid and slow acetylator congenic hamsters, whereas 5-(deoxyguanosin-N²-yl)-DMABP levels were significantly higher in prostate than in colon and heart of rapid and slow acetylator congenic hamsters. DMABP-DNA adduct levels in each tissue examined did not differ significantly between rapid and slow acetylator hamsters following either 33 or 100 mg/kg injection. The tissue-dependent differences in DMABP-DNA adduct levels observed in the Syrian hamster differ from those reported in the rat and are consistent with previous studies that show DMABP induces primarily urinary bladder tumors in the Syrian hamster.     

Detection of DNA Adducts by 32P-Postlabeling and Multifraction Contact-Transfer Thin-Layer Chromatography

³²P-Postlabeling is a sensitive method for detecting DNA adducts.Large bulky adducts, particularly from polycyclic aromatic compounds,are readily detected using this technique. Detection of smallmodifications, such as methylations, has often required specificadditional enrichment procedures prior to ³²P-postlabeling.We report the use of a single analytical procedure that candetect DNA adducts of a wide range of sizes and hydrophobicities(exemplified by adducts produced with methyl methanesulfonate,diepoxybutane, styrene oxide, or benzo[a]- pyrene). This ³²P-postlabeling/thin-layerchromatography procedure is particularly useful when examiningthe potential of novel compounds or their metabolites to formDNA adducts.