DNA adduct formation by o-phenylphenol metabolite in vivo and in vitro.

[U-14C]o-Phenylphenol (OPP) was found to bind covalently to calf thymus DNA during a 60 min incubation in the presence of microsomes, but not in their absence, indicating that metabolic conversion of the parent compound, OPP, to an activated form is essential. Postlabeling analysis with bladder DNA of rats fed a diet containing 2% OPP for 13 weeks revealed one major adduct on TLC. In an in vitro postlabeling experiment with calf thymus DNA, both of the major metabolites of OPP, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), formed adducts, but no adducts were observed with OPP. The chemical structure responsible for adduct formation is thought to be the PHQ semiquinone radical intermediate formed during interconversion between PHQ and PBQ. When the oligonucleotides, pd(A)12-18, pd(C)12-18, pd(G)12-18 and pd(T)12-18, were used in vitro, only pd(G)12-18 gave TLC-detectable adducts on treatment with PHQ and PBQ. The covalent binding appears to be rather specific to guanine residues. These results suggest that covalent binding of the OPP metabolite is one of the underlying events in OPP-induced carcinogenesis in rats.     

32P-Postlabelling analysis of the DNA adducts formed by aristolochic acid I and II

We report the quantitation of DNA adducts in target and non-targetorgans of male Wistar rats treated orally with five daily doses(10 mg/kg body wt) aristolochic acid I (AAI) or aristolochicacid II (AAII), the major components of the herbal drug aristolochicacid, a forestomach carcinogen In the rat. DNA adducts weredetected and analysed using the nuclease P1-enhanced variationof the Randerath 32 postlabeiling assay. The highest level ofDNA adducts formed was by AAI inthe target organ, forestomach(330 ± 30 adducts/10⁸ nucleotides), but high levels werealso observed in a non-target tissue, the glandular stomach(180 ± 15). Lower amounts of adducts were detected inliver, kidney and urinary bladder epltheliuin. With AAII thebinding Levels were generally lower than the AAII, the highestLevel of adducts being detected in kidney (80 ± 20 adducts/10⁸nucleotides) and lower levels in liver, stomach and urinarybladder epithelia. Adduct patterns similar to those in vivowere observed in two new in vitro assays. Rat faecal bacteriawere shown to be able to activate AM and AAII to reactive species,which were trapped with exogenous calf thymus DNA and analysedby postlabelling. llncuhatlon of AM and AAII in explanted ratstomach held in short-term organ culture resulted In DNA adductformation in the epithelia of both forestomach and glandularstomach. To assign the recently characterized in vitro nucleosideadducts of AII to the bisphosphate derivatives, a new ion-pairH]PLC procedure on a reversed-phase column was developed. Bymonitoring Cerenkov radiation on-line, a good separation ofAII adducts was observed, demonstrating that adducts formedin vivo were chromatographically indistinguishable with thoseformed in vitro, and previously characterized as an aristolactammoiety bound covalently to the exocydlic amino groups of deoxyadenosineand deoxyguanosine.     

A cyclophosphamide/DNA phosphoester adduct formed in vitro and in vivo

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and 32P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10(5) and 1/3 x 10(7) nucleotides, respectively.     

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.      

DNA adduct formation and unscheduled DNA synthesis in rat esophagus in vivo after treatment with N-methyl-N-nitrosourea

An in vivo method for assessment of DNA adduct formation and unscheduled DNA synthesis (UDS) in the esophagus of rats was devised. Small ventral incisions were made in the neck and upper abdomen regions of 6 week old F344 rats and ligation of the esophagus with thread at the two extreme ends performed to make an esophageal pouch. For the DNA adduct formation study, a solution (0.5 ml) containing various concentrations of N-[3H]methyl-N-nitrosourea ([3H]MNU) was injected into the pouch. DNA binding levels were calculated from radioactivity of the isolated DNA and dose-dependent DNA adduct formation could be detected 2 h after the treatment with MNU. By HPLC analysis, both 7-methylguanine (7-mGua) and O6-methylguanine (O6-mGua) adducts were identified in the esophageal DNA, the ratio of 7-mGua/O6-mGua being 5.7-12:1. For UDS measurement, a solution containing MNU plus [3H]thymidine (200 microCi/ml) was similarly injected into the pouch. UDS was dose-dependently demonstrated as silver grains over the nuclei of the epithelial cells by autoradiography. The results thus showed that MNU, when injected into the esophageal lumen, can penetrate the surface mucosa, react with the epithelial cell DNA and induce DNA adduct formation and DNA repair synthesis dose-dependently.     

Factors modulating the formation of DNA adduct by aflatoxin B1 in vitro

Forty-two compounds belonging to various chemical groups havebeen tested for their ability to suppress formation of aflatoxinB₁-DNA adduct mediated by microsome in vitro. While many ofthese compounds have either marginal or no modulating effect,some have been identified as effective inhibitors. The stronginhibition of DNA adduct formation by retinoids (retinol, retinal,retinoic acid and retinyl acetate), riboflavin, riboflavin 5'-phosphate,flavin adenine dinudeotide, Cu²⁺, 7,8-benzoflavone, disulfiram,butylated hydroxyanisole, butylated hydroxytoluene and phenothiazinesuggests that these agents may have potential anticardnogenicactivity against aflatoxin B₁.     

Exceptionally long‐term persistence of DNA adducts formed by carcinogenic aristolochic acid I in renal tissue from patients with aristolochic acid nephropathy

Aristolochic acid (AA) causes aristolochic acid nephropathy (AAN), first described in women in Belgium accidently prescribed Aristolochia fangchi in a slimming treatment, and also Balkan endemic nephropathy (BEN), through probable dietary contamination with Aristolochia clematitis seeds. Both nephropathies have a high risk of urothelial cancer, with AA being the causative agent. In tissues of AAN and BEN patients, a distinct DNA adduct, 7‐(deoxyadenosin‐N⁶‐yl)‐aristolactam I (dA‐AAI), has been detected. DNA adducts can be removed through DNA repair, they can result in mutations through erroneous DNA replication or they can cause cell death. The dA‐AAI adduct induces AT to TA transversions in the tumor‐suppressor TP53 gene in experimental systems, matching TP53 mutations observed in urothelial tumors from AAN cancer cases. Using thin‐layer chromatography ³²P‐postlabeling and mass spectrometric analysis we report the detection of dA‐AAI in renal DNA from 11 Belgian AAN patients over 20 years after exposure to AA had ceased. Our results showed that dA‐AAI is an established biomarker of AA exposure, and that this biomarker can be demonstrated to be persistent decades after a distinct AA exposure. Further, the persistence of dA‐AAI adducts appears to be a critical determinant for the AA mutational fingerprint frequently found in oncogenes and tumor suppressor genes recently identified by whole genome sequencing of AA‐associated urothelial tumors. The potential for exposure to AA worldwide is high; the unprecedented long‐term persistence of dA‐AAI provides a useful long‐term biomarker of exposure and attests to the role of AA in human urothelial malignancy.     

32P-postlabeling analysis of peroxisome proliferator-DNA adduct formation in rat liver in vivo and hepatocytes in vitro

Hepatocarcinogenic peroxisome proliferators, clofibrate, ciprofibrate, Wy-14643 or di(2-ethylhexyl)phthalate, were administered once daily by gavage to groups of three male F344 rats for 3 days and the rats were killed 2 h after the last dose. The DNA isolated from the livers was analyzed for possible carcinogen-DNA adducts, by a most sensitive 32P-postlabeling technique which can detect one adduct in 10(10) nucleotides. No adducts were detected by this assay in the DNA isolated from the livers of rats treated with any of the peroxisome proliferators. Adducts were also not found in the DNA of hepatocytes exposed in vitro to these peroxisome proliferators for 4 h in primary suspension cultures. Failure to detect peroxisome proliferator DNA adducts in hepatocytes under in vivo and in vitro conditions supports the contention that formation of a peroxisome proliferator-DNA adduct is not an essential step in the carcinogenesis by this novel class of carcinogens.     

Effects of monoterpenoids on in vivo DMBA-DNA adduct formation and on phase I hepatic metabolizing enzymes

We have previously demonstrated the anticarcinogenic effects of monocyclic monoterpenes such as limonene when given during the initiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary cancer in Wistar-Furth (WF) rats. Here we investigated the possible mechanisms for this chemoprevention activity including limonene's effects on DMBA-DNA adduct formation and hepatic metabolism of DMBA. Twenty-four hours after carcinogen administration, there were approximately 50% of the total DMBA-DNA adducts found in control animals formed in the liver, spleen, kidney and lung of limonene-fed animals. While circulating levels of DMBA and/or its metabolites were not different in control and limonene-fed rats, there was a 2.3-fold increase in DMBA and/or DMBA-derived metabolites in the urine of the limonene-fed animals. Studies of the effects of limonene and sobrerol, a hydroxylated monocyclic monoterpenoid with increased chemoprevention activity, on phase I metabolizing enzymes revealed that these terpenoids modulated cytochrome P450 (CYP) and epoxide hydratase (EH) activity. The 5% limonene diet increased total CYP to the same extent as phenobarbital (PB) treatment when compared to control, while 1% sobrerol (isoeffective in chemoprevention to 5% limonene) did not. However, both 5% limonene and 1% sobrerol diets greatly increased the levels of microsomal EH protein and associated hydrating activities towards benzo[a]pyrene 4,5-oxide when compared to control and PB treatment. These changes also modified the rate and regioselectivity of in vitro microsomal DMBA metabolism when compared to PB treatment or control. Identification of the specific isoforms of CYP induced by these terpenoids was performed using antibodies to CYP isozymes in Western blot analysis and inhibition studies of microsomal DMBA metabolism. Five per cent limonene was more effective than 1% sobrerol at increasing the levels of members of the CYP2B and 2C families but was equally effective at increasing EH. Furthermore, both terpenoid diets caused increased formation of the proximate carcinogen, DMBA 3,4-dihydrodiol. While these terpene-induced changes in hepatic CYP and EH do not explain the anticarcinogenic mechanism of these chemopreventive agents, or the ability of limonene systemically to reduce DMBA-DNA binding, they do reveal novel and selective induction mechanisms of hepatic enzymes.     

5-Aminolevulinic acid mediates the in vivo and in vitro formation of 8-hydroxy-2'-deoxyguanosine in DNA

5-Aminolevulinic acid (ALA), a heme precursor accumulated inchemical and inborn porphyrias, may behave as an endogenouspro-oxidant In chronically treated rats (40 mg ALA/kg body wtevery 2 days for 15 days) the steady-state level of 8-hydroxy-2'-deoxyguanosine(8-OHdG) in liver DNA (94.5 ± 233 residues/106 dG) was4.5 times higher than in non-treated rats (21 ± 7.5 residues/10⁶ dG). In vitro exposure of calf thymus DNA to ALA (0.05-5mM) in the presence of 10 uM Fe²+ caused the formation of 8-OHdG.The amount of 8-OHdG rose from 135 ± 15 residues/10⁶dG in the control system to 1140 ± 150 residues/106 dGafter incubation with 5 mM ALA and 10 µM Fe²1. Diethylenetriaminepentaaceticacid (5 mM) or mannitol (100 mM) inhibited the formation of8-OHdG by 63 and 69% respectively, evidencing the involvementof both H2O2 and HO in this process. Hydrogen peroxide (100µM) or Fe²+ alone did not cause DNA oxidation. The presentdata support the hypothesis that ALA-generated reactive oxygenspecies can oxidize DNA and may be involved in the developmentof primary liver cell carcinoma in individuals with symptomaticacute intermittent porphyria.     

DNA adduct formation by secondary metabolites of cyclopenta[cd]pyrene in vitro.

In this study, calf thymus DNA was reacted in vitro with cyclopenta[cd]pyrene 3,4-epoxide (CPPE) or its metabolites, 3,4-dihydroCPP-3,4-diol (CPP-3,4-diol) and 4-hydroxy-3,4-dihydroCPP (4-OH-DCPP), activated with sulfotransferase. The adducts formed were analyzed by HPLC with fluorescence detection following enzymatic digestion of DNA to deoxynucleosides. We have shown previously that the major CPPE-reacted DNA adducts are cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4-dihydroCPP. Sulfotransferase activation of trans-CPP-3,4-diol yielded two adducts that were identical to the products resulting from the reaction of CPPE with DNA, while cis-CPP-3,4-diol gave very low covalent binding. Two adducts formed by sulfotransferase activation of 4-OH-DCPP were identical to the products of the reaction of synthetic 4-NaO3S-O-DCPP or sulfotransferase-activated 4-OH-DCPP with deoxyguanosine. These results indicate that guanine is the predominant site of CPP adduct formation in DNA, and that the 4-hydroxy-3-dGuo adducts can arise by reaction of DNA with either CPPE or sulfotransferase-activated trans-CPP-3,4-diol.     

Modulation by some natural carotenoids of DNA adduct formation by aflatoxin B1 in vitro

Nine natural carotenoids have been tested for their ability to modulate formation of DNA adduct by the carcinogen aflatoxin B1 (AFB1) in an in vitro reaction catalyzed by rat liver microsomes. Certain apo-carotenoids, which are precursors of vitamin A, were found to be very efficient in inhibiting the adduct formation. Some other carotenoids, which although have less pro-vitamin A activity, also showed similar inhibitory effect. The DNA adduct formation appeared to be modulated through the action of the carotenoids on microsomal enzymes, since an effective inhibition in each case was observed on the formation of the activated AFB1-metabolite. It is envisaged that such inhibition could form a basis by which natural carotenoids could counteract the carcinogenic action of AFB1.     

Detoxification of aristolochic acid I by O‐demethylation: Less nephrotoxicity and genotoxicity of aristolochic acid Ia in rodents

Ingestion of aristolochic acids (AA) contained in herbal remedies results in aristolochic acid nephropathy (AAN), which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA I and AA II, primary components in AA, have similar genotoxic potential, whereas only AA I shows severe renal toxicity in rodents. AA I is demethylated to form 8‐hydroxy‐aristolochic acid I (AA Ia) as a major metabolite. However, the nephrotoxicity and genotoxicity of AA Ia has not yet been determined. AA Ia was isolated from urine collected from rats treated with AA I and characterized by NMR and mass spectrometry. The purified AA Ia was administered intraperitoneally to C3H/He male mice for 9 days and its toxicity was compared with AA I. Using ³²P‐postlabeling/polyacrylamide gel electrophoresis, the level of AA Ia‐derived DNA adducts in renal cortex was ～70–110 times lower than that observed with AA I, indicating that AA Ia has only a limited genotoxicity. Supporting this result, when calf thymus DNA was reacted with AA Ia in a buffer containing zinc dust, the formation of AA Ia‐DNA adducts was two‐orders of magnitude lower than that of AA I. Histopathologic analysis revealed that unlike AA I, no significant changes were detected in the renal cortex of mice treated with AA Ia. Therefore, the contribution of AA Ia to renal toxicity is minimum. We conclude the metabolic pathway of converting AA I to AA Ia functions as the detoxification of AA I.     

Androgen manipulation alters oxidative DNA adduct levels in androgen-sensitive prostate cancer cells grown in vitro and in vivo

Intracellular reactive oxygen species (ROS) may cause oxidative DNA damage, resulting in the formation of adducts such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and the cyclic pyrimidopurinone N-1, N(2) malondialdehyde-2'-deoxyguanosine (M(1)dG). These adducts have been associated with carcinogenesis, genomic instability and clonal evolution. We tested two hypotheses in human prostate cancer cells grown in vitro and in a xenograft model: (1) treatment of androgen-sensitive cells with DHT increases levels of oxidative DNA adduct levels; (2) flutamide, a competitive androgen receptor antagonist, prevents DHT-induced changes. Levels of M(1)dG and 8-oxo-dG adducts were determined by immunoslot blot and liquid chromatography-tandem mass spectrometry. M(1)dG and 8-oxo-dG levels were significantly higher than control levels in LNCaP cells exposed to supra-physiological concentrations (25-100 nM) of DHT (both P<0.05 by ANOVA). Flutamide pre-treatment completely prevented this increase. In the xenograft model, tumour levels of M(1)dG were decreased by 46% (P=0.001 by Mann-Whitney Test) in flutamide-treated animals compared to controls. The changes demonstrated suggest that oxidative DNA adducts may serve as biomarkers of the efficacy of androgen manipulation in chemoprevention trials.     

Benzo(a)pyrene-DNA adduct formation and removal in mouse epidermis in vivo and in vitro: relationship of DNA binding to initiation of skin carcinogenesis

An antiserum specific for the major benzo(a)pyrene (BP) adduct formed with deoxyguanosine in vivo has been used by enzyme-linked immunosorbent assay to monitor the formation and removal of DNA-bound products in BALB/c mouse epidermis exposed topically to initiating doses of BP and in BALB/c mouse keratinocytes exposed in vitro to BP or its activated derivatives. In mouse epidermal DNA, formation of antibody-recognizable products increased proportionally between doses of 50 and 250 nmol of BP, giving 2.3 to 6.0 fmol/micrograms of DNA, respectively, and reached a plateau of 10 to 11 fmol/micrograms of DNA at doses between 1000 and 1500 nmol. Antibody-recognizable adducts comprised roughly one-half of the total BP-DNA binding, since a 250-nmol dose of [3H]BP yielded 6 fmol/micrograms of DNA by enzyme-linked immunosorbent assay and 12.9 fmol/micrograms of DNA by radiolabeling. Removal of trans-(7R)-N2-(10-[7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]-yl)-deoxyguanosine adducts was monitored in epidermal DNA of mice exposed to 500 nmol of BP and, although no correction was approximated for DNA turnover in the skin, about one-half of the adducts formed by 24 hr were removed 3 days later, and only 10% remained at the end of a week. BP-DNA binding and removal were also studied in cultured mouse keratinocytes, where proliferating basal cells and terminally differentiating cells can be selectively studied by modulating the Ca2+ concentration of the medium. BP dose-response studies showed that, in cells of different maturation states, BP-DNA adduct levels were similar. Adduct formation greater than 10 to 11 fmol/micrograms (the highest obtained in vivo) was associated with extensive cytotoxicity and cell death. The kinetics of adduct removal was followed in culture under conditions in which dilution by DNA synthesis or cell loss could be monitored. Results of these experiments suggested that initial removal of BP-DNA adducts was more rapid in the differentiating population although, in both populations, 50% of the adduct was removed by 24 hr. The formation of foci resistant to Ca2+-induced terminal differentiation has been associated previously with carcinogen treatment in cultured keratinocytes. Exposure to BP or the antidiol-epoxide, at concentrations producing low cytotoxicity, yielded frequencies of differentiation-altered foci proportional to the dose of the compound used and to the number of DNA adducts formed.     

Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons

Understanding the kinetics of aromatic-DNA adducts in target tissues and white blood cells (WBC) would enhance the applicability of DNA adducts in WBC as surrogate source of DNA in biomonitoring studies. In the present study, rats were acutely exposed to benzo[a]pyrene (B[a]P; 10 mg/kg body wt) via intratracheal (i.t.), dermal and oral administration. DNA adducts were analyzed in relevant target organs and WBC by nuclease P1 enriched (32)P-post-labeling at 1, 2, 4, 11 and 21 days after exposure. Additionally, the internal dose was assessed by measurement of urinary excretion of 3-hydroxy-B[a]P (3-OH-B[a]P). Total B[a]P-DNA adduct levels in WBC were highest after i.t. and oral administration, whereas DNA adducts were hardly detectable after dermal exposure. Highest adduct levels were reached at 2 days after exposure. In lung tissue, DNA adduct levels reached maximal values at 2 days and were highest after i.t., oral and dermal exposure, respectively. DNA adduct levels were significantly lower in WBC as compared with lung. Nonetheless, overall B[a]P-DNA adduct levels in WBC were significantly correlated with those in lung. In target organs, highest DNA adduct levels were observed in skin after topical application, and lowest in stomach after oral administration of B[a]P. Furthermore, DNA adduct levels in WBC were correlated with DNA adduct levels in skin after dermal exposure and stomach after oral administration of B[a]P. Two-fold higher levels of 3-OH-B[a]P were excreted after i.t. administration of B[a]P as compared with dermal or oral exposure. Urinary 3-OH-B[a]P concentrations were correlated with DNA adduct levels at the site of B[a]P application. Overall, it can be concluded that aromatic-DNA adduct levels in WBC can be applied as a surrogate source of DNA for the site of application of B[a]P and reflect binding to lung DNA, independently of the exposure route.     

Bulky DNA adduct formation and risk of bladder cancer.

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with risk of bladder cancer and with increased bulky DNA adduct levels in several studies, mainly in smokers. We investigated the relation between bulky PAH-DNA adducts in peripheral blood mononuclear cells and bladder cancer in nonsmoking subjects from a large hospital-based case-control study in Spain. Additionally, we examined the association between DNA adduct formation and several air pollution proxies. The study comprised 76 nonsmoking cases and 76 individually matched controls by sex, region of residence, age, and smoking status (never, former). To maximize the relevance of the DNA adduct measurement as a proxy of PAH exposure, subjects selected had not changed residence, occupation, and major lifestyle factors during the last 10 years. Bulky DNA adducts were measured using the (32)P-postlabeling technique, nuclease P1 treatment. The percentage of detectable adducts was higher in controls (41%) than in cases (32%) with an odds ratio of 0.75 (95% confidence interval, 0.36-1.58). In an analysis limited to controls, a higher percentage of DNA adducts was found among those whose last residence was in a big city (50%) compared with those living in villages (19%; P = 0.04). No consistent associations were found for other markers of air pollution. In this study, among nonsmokers with stable environmental and lifestyle factors, bulky DNA adducts were not associated with bladder cancer risk. Results do not support an association of bladder cancer risk with low-level exposure to PAHs as measured through the formation of bulky DNA adducts in peripheral mononuclear cells.     

Differences in DNA adduct formation between monocytes and lymphocytes after in vivo incubation with benzo[a]pyrene

It was the aim of this study to compare the formation of DNA adducts in different human white blood cells. Lymphocytes and monocytes were isolated from peripheral blood by density centrifugation and by a monocyte-specific antibody linked to magnetic beads. DNA adducts were determined by 32P-postlabelling. After in vitro incubation with benzo[a]-pyrene (B[a]P) we found DNA adducts in monocytes, but not in unstimulated blood lymphocytes. Only after growth stimulation by phytohaemagglutinin did lymphocytes show an adduct pattern similar to monocytes. In contrast, B[a]P-7,8-dihydro-9,10-diolepoxide, an activated intermediate of B[a]P metabolism leads to similar DNA adducts in both cell types. When lymphocytes were incubated in the presence of B[a]P together with monocytes, then after subsequent separation lymphocytes exhibited most of the adducts found in monocytes, though to a lower extent. Our results suggest that unstimulated blood lymphocytes--which are unable to activate metabolically promutagens like B[a]P--receive the genotoxic material to form DNA adducts from other blood cells or from cells in the vessel lining. We conclude that the amount and the pattern of DNA adducts formed in whole white blood cells may be influenced considerably by a variable proportion of monocytes.     

DNA adduct formation by 7,12-dimethylbenz(a)anthracene in Syrian hamster cheek pouch epithelium in vivo

Studies examined the in vivo binding of radiolabeled 7,12-dimethylbenz(a)anthracene (DMBA) to hamster cheek pouch epithelial DNA. Adduct formation was studied as functions of [3H]DMBA dose and of the time after single [3H]DMBA applications in mineral oil. Total DMBA-DNA adduct formation was determined by DNA-bound 3H activity, and qualitative binding characteristics were further studied by high-pressure liquid chromatography. Adduct formation 24 h after single [3H]DMBA applications rapidly increased from DMBA concentrations of 0.05-5.0 micrograms. While binding also increased from DMBA concentrations of 5.0-50.0 micrograms, the variability in adduct formation at 50.0 micrograms was considerable. Adduct formation following single 5.0-micrograms [3H]DMBA applications rose slowly to a peak value of 76 pmol DMBA/mg DNA at 36 h. This level decreased very slowly in a biphasic manner through 240 h, at which time the adduct levels were 23% of maximum. Adduct levels of 1.5 pmol/mg DNA were measured as late as 5 wk after a single 5.0-micrograms [3H]DMBA application. Chromatographic analyses of the 24-, 36-, and 96-, and 240-h samples showed three major peaks which are likely to be 1,2,3,4-tetrahydro-3,4-dihydroxy-1,2-oxide-deoxyribonucleoside adducts. While these analyses were limited by the small amounts of radioactivity which could be retrieved from [3H]DMBA-treated pouches, the study suggests that the DMBA-induced hamster cheek pouch carcinoma may be useful in some molecular in vivo studies of chemical-DNA interactions in carcinogenesis.     

DNA adduct formation and assessment of aberrant crypt foci in vivo in the rat colon mucosa after treatment with N-methyl-N-nitrosourea

N-Nitroso-compound DNA adduct formation in vivo and occurrenceof aberrant crypt foci (ACF) were studied in the rat colon mucosaafter a single, local treatment with a carcinogen, N-methyl-N-nitrosourea(MNU), using a simple surgical approach. A segment of F344 ratcolon was ligated to make a pouch and injected with MNU solution.For the study of DNA adduct formation, the solution contained50 µCi of [³H]MNU. The results demonstrated that similarranges of carcinogen dose, i.e. 0.15x10^–2 –1.5x10^–2MMNU, could induce both DNA adduct formation and appearanceof ACF in the rat colon with both parameters showing a nearbylinear dose dependence. HPLC analysis revealed the DNA adductsto include both 7-methylguanine (7-mGua) and O⁶-methylguanine(O⁶-mGua) with the 7-mGua/O⁶-mGua ratio being 8.2–11.3:1in the system used. Assessment of ACF development from 4 to16 weeks after MNU treatment at a dose of 7.5x10^–2 M showedthe numbers to increase up to the 8th week, followed by a decreaseat weeks 12 and 16, when 40% of the ACF counted at the peaktime point were still present. The percentage of large ACF (