Nuclease P1-enhanced 32P-postlabeling Research Articles

Genotoxicity of Organic Extracts of House Dust from Shanxi, China

Indoor combustion of solid fuel such as coal may generate respirable particles containing polycyclic aromatic hydrocarbons (PAH) that may adhere to settled dust. Dust might therefore present a major source of PAH exposure in humans. This study evaluated the in vitro and in vivo genotoxicity of PAH mixtures extracted from house dust samples. Four dust samples (E1–4) were collected from houses in Shanxi, China, where coal is heavily used for heating and cooking. For comparison, a coal sample was also collected from one of the houses and included in the analyses. The samples were extracted with methylene chloride:acetone (95:5 v/v), dried, and redissolved in appropriate solvents for assessment in genotoxicity assays. Samples were evaluated for their ability to induce point mutations in bacteria and DNA adducts in vivo. DNA adduct levels were analyzed by nuclease P1-enhanced 32P-postlabeling. PAH were quantified using gas chromatography/mass spectrometry. Based on chemical analysis, sample E1 had the highest concentration by sampling area of benzo[a]pyrene (BaP) (181 μg/m2) and total PAH (10100 μg/m2). However, based on the microbial genotoxicity assay, sample E3, with the highest carcinogenic PAH/total PAH ratio (26%), produced the greatest number of revertants. In mice, administration of the extract of coal induced more adducts (9.81 adducts per 109 nucleotides) than dust extracts. The results of this study confirm the presence of genotoxic chemicals in residential dust. Inhalation of respirable particles containing similar mixtures of PAH represents a cancer risk for humans.

Naturally occurring coumarins inhibit 7,12-dimethylbenz[ a ]anthracene DNA adduct formation in mouse mammary gland

Naturally occurring coumarins (NOCs) are anti-carcinogenic in the mouse skin model. To characterize the chemopreventive potential of NOCs against breast cancer, we first examined their effects on 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adduct formation in mouse mammary gland. We hypothesized that those NOCs that both inhibited cytochrome P450 1A1/1B1 and induced hepatic glutathione S-transferases (GSTs) would be the most effective in blocking DMBA-DNA adduct formation in mouse mammary gland. To address this hypothesis, simple coumarins (e.g. coumarin and limettin, which induced mouse hepatic GSTs but had little effect on P4501A1/1B1) and linear furanocoumarins (e.g. imperatorin and isopimpinellin, which induced hepatic GSTs and were potent inhibitors of P4501A1/1B1) were compared. Mice were pretreated with NOCs (150 mg/kg body wt, by gavage) prior to either a single dose of DMBA (50 microg) or multiple doses of DMBA (20 microg daily for 3 and 6 weeks). Mammary DMBA-DNA adduct formation was quantitated by the nuclease P1-enhanced 32P-postlabeling assay. With the single dose of DMBA, coumarin, limettin, imperatorin and isopimpinellin inhibited DMBA-DNA adduct formation by 50, 41, 79 and 88%, respectively. Coumarin, limettin and imperatorin blocked DMBA-DNA adduct formation by 36, 60, and 66% at 3 weeks, and by 0, 49 and 55% at 6 weeks of DMBA dosing, respectively. In a 6 week dose-response study of select NOCs and 7,8-benzoflavone (a potent P4501 inhibitor that had little effect on GSTs), DMBA-DNA adduct formation was inhibited by 0, 43 and 24% in the limettin groups; by 26, 26 and 69% in the isopimpinellin groups; and by 80, 96 and 97% in the 7,8- benzoflavone groups at 35, 70 and 150 mg/kg, respectively. Taken together, these results suggest that linear furanocoumarins had a greater inhibitory effect on DMBA-DNA adduct formation in mouse mammary glands compared with simple coumarins, and that the predominant effect may be P4501 inhibition.

32P-Postlabeling analysis of lipophilic DNA adducts resulting from interaction with (±)-3-hydroxy- trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-benzo[ a]pyrene

Bay-region diol epoxides are considered the putative ultimate carcinogens of polynuclear aromatic hydrocarbons. However, the results of studies on tumorigenesis and DNA binding of benzo[ a]pyrene (BP) and its bay-region diol epoxide, (+)- trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene [(+)- anti-BPDE] suggest that, in addition to anti-BPDE, other reactive metabolite(s) of BP may also be involved in BP-induced carcinogenesis. Recent studies have demonstrated that 3-hydroxy- trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene ( anti-BPTE) is another highly reactive metabolite of BP. In order to identify syn- and anti-BPTE-derived DNA adducts and their base selectivity, we synthesized both compounds by two different methods and reacted in vitro with calf thymus DNA and individual nucleotides. The resultant adducts were analyzed by nuclease P1-enhanced 32P-postlabeling. Anti-BPTE produced three major and several minor adducts with DNA; dAp and dGp were the preferred substrates, while dCp and dTp were the least reactive. In contrast, syn-BPTE produced two major adducts each with DNA and dGp; dAp generated only one adduct. Co-chromatography of anti-BPTE-derived DNA adducts with those of mononucleotide adducts revealed that the major adducts in DNA were guanine derived. Further, co-chromatographic results revealed that the anti-BPTE-DNA adducts were distinctly different from that of anti-BPDE-DNA adducts. These observations indicate that both syn- and anti-BPTE can react with DNA bases and these DNA adducts may also contribute to BP-induced carcinogenesis.

Glutathione S-transferase Mu (GST Mu) deficiency and DNA adducts in lymphocytes of smokers

The effect of smoking on DNA adduct formation in lymphocytes was analysed in individuals with low (deficient) and high (non-deficient) glutathione S-transferase (class Mu) activity. DNA adduct levels in lymphocytes were determined by the highly sensitive nuclease P1-enhanced 32P-postlabeling assay. The lymphocyte DNA adducts/10 8 nucleotides of smokers deficient in glutathione S-transferase Mu activity ( n=12) were significantly higher than those of smokers non-deficient ( n=9) in glutathione S-transferase Mu activity. The DNA adduct levels of the lymphocytes inversely correlated with glutathione S-transferase Mu activity. A correlation was found between DNA adduct levels and daily cigarette consumption. Results of the present study suggest that individuals deficient in glutathione S-transferase Mu activity may be at greater risk of DNA damage.

Inhibition of 5,9‐dimethyldibenzo[c,g]carbazole‐DNA adduct formation in mouse liver by pretreatment with cytochrome P4501A inducers in vivo

Mice of the XVIInc/Z and DBA/2N strains, which are responsive and nonresponsive, respectively, to the aryl hydrocarbon (Ah) receptor, were treated with the hepatocarcinogen 5,9-dimethyldibenzo[c,g]carbazole and their livers were examined by nuclease P1-enhanced 32P-postlabeling for the levels of DNA adducts formed. Pretreatment at the doses usually reported in the literature with the cytochrome P4501A (CYP1A) inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), β-naphthoflavone (BNF), and isosafrole modulated DNA adduction. In XVIInc/Z mice, DNA adduction was totally inhibited by TCDD (a CYP1A1/1A2 inducer), BNF (a CYP1A1/1A2 inducer), and isosafrole (a CYP1A2 inducer). In DBA/2N mice, in which DNA adduction was also inhibited by TCDD, about 25% of the DNA adduct levels persisted after pretreatment with BNF (not a CYP1A1/1A2 inducer in this strain) or isosafrole (a CYP1A2 inducer in this strain). The increase (in all cases less than twofold) in the levels of the phase-II drug-metabolizing enzymes glutathione S-transferase and uridine diphospho-glucuronyltransferase after treatment with inducers cannot explain the total disappearance of DNA adducts. Assays of 5-bromo-2′-deoxyuridine incorporation did not show any induction of DNA synthesis which could explain the decrease in adducts. These results suggest that in vivo 1) increases in CYP1A enzymes by inducers are not correlated with enhanced levels of certain DNA adducts; and 2) phase-II drug metabolizing enzymes are not the main cellular protection pathway for detoxification. An additional mechanism, perhaps also induced by the Ah receptor but highly dependent on the dose of inducer, could be involved in parallel to multidrug resistance (mdr); further experiments are needed to identify this process used by the cell to enhance its protection against toxic or genotoxic effects. Environ. Mol. Mutagen. 32: 314–324, 1998 © 1998 Wiley-Liss, Inc.

Inhibition of 5,9-dimethyldibenzo[c,g]carbazole-DNA adduct formation in mouse liver by pretreatment with cytochrome P4501A inducers in vivo

Mice of the XVIInc/Z and DBA/2N strains, which are responsive and nonresponsive, respectively, to the aryl hydrocarbon (Ah) receptor, were treated with the hepatocarcinogen 5,9-dimethyldibenzo[c,g]carbazole and their livers were examined by nuclease P1-enhanced 32P-postlabeling for the levels of DNA adducts formed. Pretreatment at the doses usually reported in the literature with the cytochrome P4501A (CYP1A) inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (BNF), and isosafrole modulated DNA adduction. In XVIInc/Z mice, DNA adduction was totally inhibited by TCDD (a CYP1A1/1A2 inducer), BNF (a CYP1A1/1A2 inducer), and isosafrole (a CYP1A2 inducer). In DBA/2N mice, in which DNA adduction was also inhibited by TCDD, about 25% of the DNA adduct levels persisted after pretreatment with BNF (not a CYP1A1/1A2 inducer in this strain) or isosafrole (a CYP1A2 inducer in this strain). The increase (in all cases less than twofold) in the levels of the phase-II drug-metabolizing enzymes glutathione S-transferase and uridine diphospho-glucuronyltransferase after treatment with inducers cannot explain the total disappearance of DNA adducts. Assays of 5-bromo-2'-deoxyuridine incorporation did not show any induction of DNA synthesis which could explain the decrease in adducts. These results suggest that in vivo 1) increases in CYP1A enzymes by inducers are not correlated with enhanced levels of certain DNA adducts; and 2) phase-II drug metabolizing enzymes are not the main cellular protection pathway for detoxification. An additional mechanism, perhaps also induced by the Ah receptor but highly dependent on the dose of inducer, could be involved in parallel to multidrug resistance (mdr); further experiments are needed to identify this process used by the cell to enhance its protection against toxic or genotoxic effects.

Microsome-mediated bioactivation of dibenzo[a,l]pyrene and identification of DNA adducts by 32P-postlabeling.

Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60-fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.

Inhibition of sulfotransferase affecting in vivo genotoxicity and DNA adducts induced by safrole in rat liver.

The effect of pretreatment with pentachlorophenol (PCP), a known inhibitor of sulfotransferases, on the induction of chromosomal aberrations, sister chromatid exchanges (SCEs), replicative DNA synthesis (RDS), and the formation of DNA adducts was studied in the liver of rats treated with safrole (1-allyl-3,4-methylenedioxy-benzene). Rats were given a single oral dose (1,000 mg/kg body weight) or 5 repeated doses (500 mg/kg body weight) of safrole, with or without intraperitoneal pretreatment with PCP (10 mg/kg body weight). Hepatocytes were isolated 24 hr after administration of safrole and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor to test for chromosomal aberrations and SCEs. For examination of RDS, hepatocytes were incubated in Williams' medium E containing 5-bromo-2'-deoxyuridine. Safrole-DNA adducts were detected by a nuclease P1-enhanced 32P-postlabeling assay. A single dose of safrole induced significant SCEs and RDS, while chromosomal aberrations were induced by 5 repeated doses. Two major and 2 minor DNA adducts were detected by both a single dose and 5 repeated doses. PCP significantly decreased safrole-induced cytogenetic effects and RDS, and caused a decrease in DNA adducts formed by safrole. These results suggest that safrole is capable of inducing SCEs, chromosomal aberrations, and RDS in the rat liver in vivo and that these effects may be induced by the sulfuric acid ester metabolite that can bind DNA.

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 mumol/kg) either alone or in combination with PCP (75 mumol/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 microM) strongly inhibited (by 21-97%) mEH activity in a dose-dependent manner, the IC50 being 35 microM. DCNP was ineffective as a mEH inactivator. PCP also inhibited purified equine liver GST activity, with an IC50 of 23.5 microM. 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.

Metabolic activation of benzo[ a]pyrene in two fetal mouse hepatocyte lines: induction of DNA adducts and micronuclei

We have studied the metabolic competence of two non-transformed epithelial-like cell lines derived from fetal mouse liver, C 6 and C 2.8, to activate the promutagen benzo[ a]pyrene by measuring both the induction of DNA adducts through the nuclease P1-enhanced 32P-postlabeling assay and the formation of micronuclei. The pattern and level of DNA adducts detected in C 6 and C 2.8 cells treated with benzo[ a]pyrene were compared with those obtained in human peripheral blood lymphocytes treated with the same compound and with [ 3H]anti-benzo[ a]pyrene diolepoxide. In both the cell lines and in human lymphocytes we observed a consistent induction of distinct DNA adducts. In C 6 and C 2.8 cells, the most evident adduct showed a position similar to that of the main adduct induced by [ 3H]-anti-benzo [a] pyrene diolepoxide in human lymphocytes. In addition, benzo[ a]pyrene caused a significant increase of micronucleated C 6 and C 2.8 cells, whereas the frequency of micronuclei did not increase in CHO cells treated, for comparison, in the same way.

Effects of a single dose of the cytochrome P450 inducer, β-naphthoflavone, on hepatic and renal covalent DNA modifications (I-compounds)

I-compounds are age-dependent covalent DNA modifications, which occur in rodent tissues without known carcinogen exposure. A number of studies from our laboratory indicate that I-compounds may serve as biomarkers of carcinogenesis. Recently, we demonstrated significant lowering of liver I-compound levels in rats that were exposed to different cytochrome P450 inducers. In order to gain further mechanistic insights into the possible relationship between P450 induction and I-compound reduction, female Sprague-Dawley rats were administered a single dose of the CYP1 Al inducer, β-naphthoflavone (BNF) (80 mg/kg), in com oil (CO) (2 ml/kg) or CO only (2 ml/kg) as vehicle control. Liver and kidney microsomal P450 contents and P450-related enzyme activities and DNA I-compounds were determined at 4, 24, and 48 h after treatment. Liver and kidney I-compounds were analyzed by nuclease P1-enhanced 32P-postlabeling. DNA synthesis was determined by measuring [ 3H]methylthymidine incorporation. Liver and kidney microsomal P450 contents were elevated by BNF at 24 and 48 h. Ethoxyresorufin-O-deethylase (EROD) and methoxyresorufm- O-demethylase (MROD) were significantly elevated at all time points, with the former displaying a much higher extent of induction. BNF treatment resulted in significant diminution of the levels of several individual and total I-compounds in liver at 48 h, but few effects were seen at the earlier time-points. Kidney I-compounds were also markedly affected by BNF at 48 h, albeit to a lesser extent than in liver. In both tissues, P450 induction preceded I-compound reduction. Taken together, the results of this investigation demonstrate significant diminution of I-compound levels by a single dose of BNF, a CYP1 A1 inducer, in a time-dependent manner, suggesting the participation of a specific biochemical process, possibly involving CYP1A1, in the metabolic regulation of these endogenous DNA adducts.

Rapid decreases in indigenous covalent DNA modifications (I-compounds) of male Fischer-344 rat liver DNA by diquat treatment

I-compounds are indigenously appearing covalent DNA modifications that can be detected by 32P-postlabeling assay in tissues of normal animals without known exposure to any carcinogens or toxins. Although these compounds have not been structurally identified, indirect evidence from earlier work suggested the possibility of involvement of molecular fragments derived from lipid peroxides. Diquat is a herbicide that stimulates lipid peroxidation and massive intrahepatic oxidant stress through redox cycling-mediated generation of reactive oxygen species. In the present study, we examined the effects of diquat on hepatic I-compounds of male Fischer-344 rats. Two groups of rats, approximately 14 weeks and 8 weeks old, were given a hepatotoxic dose (0.1 mmol/kg) of diquat or equal volumes of saline, i.p. Two and 6 h later plasma alanine aminotransferase (ALT) activities were measured and hepatic DNA I-compound levels were examined by nuclease P1-enhanced 32P-postlabeling. Elevated ALT activities were observed in some animals in both groups, at both time points, but considerable inter-animal variation was seen. A total of 15–16 I-compound fractions were measured in control and in diquat-treated animals, but no extra spots indicative of treatment-induced adducts were detected. Despite the qualitative similarities, the quantities of individual I-compounds were markedly decreased at 2 h in diquat-treated animals of both age groups. In 14 week old rats the hepatic I-compound contents were decreased at 2 h by 22–59%, which was statstically significant (ANOVA, P < 0.05) for all of the 9 polar I-compound fractions and none of the non-polar fractions. Eleven I-spots from this group showed significant negative linear correlations ( P < 0.05) with ALT values. In 8 week old rats treated with diquat a 22–43% depletion in I-compound contents was statistically significant for 4 of the 7 non-polar and 2 of the 8 polar adduct fractions, but there was no significant correlation of I-compound contents with ALT values at the 2 h time point. By 6 h most of the I-spot levels had returned to normal or above normal values in both groups of animals. While most I-spots from 14 week old rats did not correlate with ALT levels at 6 h, two I-spots displayed positive correlations in the 8 week group. Overall, the susceptibility to diquat-associated DNA alterations appeared to differ with age. Decreases in hepatic I-compound contents of diquat-treated Fischer-344 rats and the absence of additional I-compounds in these animals were not consistent with the hypothesis that I-compounds are derived from lipid peroxide fragments. However, the rapid but transient decrease in I-compound levels is notable because of similar trends previously observed during a number of procarcinogenic experimental manipulations.

Lack of DNA adduct formation in mice treated with benzene

The potential of benzene to produce DNA adducts in B6C3F1 mice was investigated by the nuclease P1-enhanced 32P-postlabeling assay. The mice were daily treated i.p. with 500 mg/kg benzene in olive oil for 4 days, and the liver, mammary gland, and bone marrow were collected 24 h after the last treatment. Thin-layer chromatograms obtained with treated-tissue DNA specimens were qualitatively identical to those from corresponding olive oil-treated (control) tissue DNA. Quantitative evaluations revealed that there was no treatment-related increase in radioactivity on the chromatograms at or near the locations where the major in vitro adducts of phenol, hydroquinone and benzoquinone migrated. Benzene treatment, however, resulted in a decrease in the levels of certain endogenous adducts, the biological significance of which is unknown. Our results indicate that benzene treatment does not produce detectable levels of aromatic DNA adducts in mouse tissues.

Analysis of cigarette-smoke-induced DNA adducts by butanol extraction and nuclease P1-enhanced 32P-postlabeling in human lymphocytes and granulocytes.

In an earlier study, we analyzed the aromatic DNA adducts separated from lymphocytes and granulocytes of smokers and nonsmokers using the nuclease P1-enhanced 32P-postlabeling assay. Here we compare the butanol extraction and nuclease P1-enhanced procedure on the same kind of samples. The DNA adducts of 42 per 10(8) nucleotides from smokers' lymphocytes were statistically higher (p < 0.05) than those of 11 from nonsmokers', when analyzed by the nuclease P1 treatment, but not by the 1-butanol extraction. The radioactivity obtained from the DNA digests on the TLC plates was lower in butanol-treated DNA samples when compared to those of nuclease P1 digestion. Lymphocytes appear to be a suitable test tissue for determining aromatic carcinogen exposure when detecting smoking-related DNA adducts by the nuclease P1-enhanced 32P-postlabeling analysis.

Detection of carcinogen-DNA adducts in human fetal tissues by the 32P-postlabeling procedure.

Tobacco smoke contains a number of genotoxic compounds that are metabolized to their biologically active forms that subsequently react with cellular DNA to form covalently bound carcinogen-DNA adducts. Several analytical procedures have been developed to detect these adducts in human tissues. Using the nuclease P1-enhanced 32P-postlabeling procedure for bulky adducts, we have detected at least 24 adducts in DNA isolated from placenta and umbilical cord DNA. Adducts were detected in both smokers and nonsmokers, but the relative adduct level (RAL) was significantly higher in smokers (42.8, 8 cases) than in nonsmokers (19.7, 11 cases). The origin of the adducts in nonsmokers remains unknown. The adduct levels in artery DNA were significantly lower than in the vein and the placenta, and a paired nonparametric analysis showed a significant association between the adduct levels in the three tissues. Our results show a maternal transfer of carcinogens present in cigarette smoke to fetal tissues and show that the tissues can metabolize the carcinogens to their DNA binding metabolites. The presence of adducts in fetal tissues may be indicative of genomic damage and may predispose the individual for the development of a serious disease later in life.

A comparison of DNA adduct formation in white blood cells and internal organs of mice exposed to benzo[ a]pyrene, dibenzo[ c, g]carbazole, safrole and cigarette smoke condensate

Measurement of tissue/cell DNA adducts represents a suitable monitor of carcinogens exposure because the majority of chemical mutagens/carcinogens react with DNA, forming covalent adducts, a key event in the initiation of chemical carcinogenesis. Investigations of DNA-adduct formation in vivo in white blood cells (WBC) versus target tissues, i.e. internal organs for most carcinogens, is expected to yield useful information about the suitability of WBC for biomonitoring and risk assessment. For this purpose, female ICR mice were given 0.4 mmole/kg benzo[ a]pyrene (BP), 0.045 mmole/kg dibenzo[ c, g]carbazole (DBC) or 2.47 mmole/kg safrole by oral gavage or 4 daily doses (equivalent to 3 cigarettes) of cigarette-smoke condensate (CSC) by topical application. At 24 h after dosing, DNA adducts were detected by a nuclease P1-enhanced 32P-postlabeling assay [M.V. Reddy and K. Randerath, Carcinogenesis, 7 (1986) 1543] in WBC and internal tissues treated with individual carcinogens, while CSC treatment elicited aromatic adducts in most tissues but not in WBC. Adduct patterns of WBC DNA were qualitatively similar to those of internal organs, but adduct amounts varied. BP, a systemic carcinogen, bound nearly as much to WBC DNA as to target-tissue DNA samples; whereas the liver carcinogens, DBC and safrole, bound to WBC DNA considerably less (22- and 51-fold, respectively) compared with liver DNA. The number of adducts in 10 7 nucleotides of WBC, liver, lung, kidney and spleen DNA, respectively, were: 2, 5, 3, 2 and 3 with BP; 6, 131, 6, 14 and 4 with DBC; 5, 238, 3, 5 and 0.6 with safrole. For CSC, these values were 0, 1 and 0.02 in WBC, lung and spleen, respectively. Our results show that carcinogen binding to WBC DNA does not reflect binding to target-tissue DNA in a quantitative sense for the carcinogens studied except for BP,

DNA adduction by phenol, hydroquinone, or benzoquinone in vitro but not in vivo: nuclease P1-enhanced 32P-postlabeling of adducts as labeled nucleoside bisphosphates, dinucleotides and nucleoside monophosphates

The carcinogenicity of benzene has been considered to be in part mediated by its chemically reactive metabolic product benzoquinone (BQ), which is formed from the intermediary metabolites phenol and hydroquinone (HQ). We have evaluated the DNA-binding capability of these chemicals in vitro and in vivo by postlabeling. Treatment of rat Zymbal glands in culture with phenol and HQ or direct reaction of BQ with DNA produced DNA adducts, which were detectable by the nuclease P1-enhanced 32P-postlabeling assay as 5'-32P-labeled 3',5'-bisphosphate products. The enhancement of sensitivity in this assay is based on the previous finding that nuclease P1 hydrolyzes the phosphate attached to the 3' side of normal nucleotides but not the corresponding phosphate of most aromatic/bulky adducted nucleotides. Also based on this hydrolytic property of nuclease P1, we developed an additional sensitive procedure that permitted the detection of DNA lesions as 5'-32P-labeled products of dinucleotides, pXpN, or of nucleoside monophosphates, pX, where X and N indicate an adducted nucleoside and a normal nucleoside respectively. In the latter assay, adducted DNA was first digested with nuclease P1 and acid phosphatase to yield XpN and N. The latter were then 32P-labeled to yield [5'-32P] pXpN or 32P-labeled and treated with venom phosphodiesterase to obtain [5'-32P]pX. After optimization of enzymatic conditions, the modified nuclease P1 assay yielded adduct recoveries similar to those obtained by the bisphosphate assay for in vitro phenol-, HQ- and BQ-DNA adducts. Neither of the nuclease P1-enhanced postlabeling procedures showed exposure-specific adducts in vivo in the bone marrow, Zymbal gland, liver and spleen of female Sprague-Dawley rats at 24 h after the last of four single, daily p.o. doses of 75 mg/kg phenol or 150 mg/kg phenol/HQ (1:1). Our results show that phenol, HQ and BQ produce adducts in vitro, but corresponding adducts are not detected in vivo with phenol and phenol/HQ, even when measured by the standard and modified nuclease P1 postlabeling methods capable of detecting 1 adduct in 10(9-10) DNA bases.

32P‐assay of DNA adducts in white blood cells and placentas of pregnant women: Lack of residential wood combustion‐related adducts but presence of tissue‐specific endogenous adducts

Residential wood combustion (RWC), which has been increasingly used as a heating source, is of health concern because emissions from RWC contain carcinogenic polycyclic aromatic hydrocarbons (PAH). To assess health risk, the possible formation of PAH-DNA adducts in white blood cells (WBC) and placentas of nonsmoking women exposed to RWC smoke during pregnancy was measured by a nuclease P1-enhanced 32P-postlabeling assay having a sensitivity limit of one lesion per 10(9-10) DNA nucleotides. DNA samples isolated from 12 exposed specimens (8 WBC, 4 placentas) and 13 unexposed control specimens (8 WBC, 5 placentas) were hydrolyzed to mononucleotides, which were then 32P-labeled and separated by high-resolution thin-layer chromatography. Comparison of autoradiograms of exposed DNA samples with those of controls failed to show exposure-related adducts. All placental DNA maps exhibited one major (47 +/- 10%) and 12 minor extra spots, however, that were not seen on WBC-DNA maps. These derivatives corresponded to an average of 12 (+/- 6) modifications in 10(9) nucleotides. Similarly, WBC DNA showed four spots that were absent in placental DNA and were not quantified because of their low levels. Neither placental nor WBC DNA adducts coincided chromatographically with the product formed by the reaction of benzo(a)pyrene diol epoxide I with N2 of guanine in DNA. Although these results suggest that RWC smoke does not elicit detectable levels of aromatic DNA adducts in humans, lack of evaluation of RWC exposure levels leaves some uncertainty in this conclusion. The results, however, clearly show that placental and WBC DNA contain covalent modifications that are unrelated to RWC exposure. These DNA derivatives may be caused by ingestion and/or inhalation of, or skin contact with, low levels of environmental genotoxicants or may arise from endogenous electrophiles of as yet unknown origin.

A method for in vitro culture of rat Zymbal gland: use in mechanistic studies of benzene carcinogenesis in combination with 32P-postlabeling.

Zymbal glands were excised bilaterally from the ear ducts of female Sprague-Dawley rats (three/group), minced into approximately four fragments per gland, and transferred into a microtiter plate containing 1.5 mL per well of Waymouth's tissue culture medium supplemented with fetal calf serum, hydrocortisone, insulin, and gentamicin. After addition of a test compound or solvent vehicle, plates were incubated for 6, 24, 48, or 96 hr at 37 degrees C in a humidified atmosphere of 5% CO2 in air. Tissue in culture for 6 hr was histologically indistinguishable from the freshly excised tissue, while that in culture for 24, 48, and 96 hr showed a progressive deterioration often with necrosis and/or squamous metaplasia. More pronounced deterioration was noted in samples treated with 750 or 1500 micrograms/mL of benzene. Using a nuclease P1-enhanced 32P-postlabeling assay, aromatic DNA adducts were detected in cultured Zymbal glands exposed for 48 hr to benzene and its derivatives, as well as to 7,12-dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (AAF). Benzene produced very low levels of adducts (0.5 adducts per 10(9) nucleotides), whereas its congeners produced relatively high levels of adducts (50-2000 lesions per 10(9) nucleotides), which decreased in the order benzoquinone greater than hydroquinone greater than phenol greater than benzenetriol greater than catechol. Each adduct profile overall was characteristic for the compound studied, suggesting the formation of compound-specific electrophiles. AAF and DMBA adducts were identical to those formed in vivo in animals. Our results show that the Zymbal glands are capable of metabolizing different carcinogens to DNA-reactive intermediates, a process that may be causally associated with tumor formation in vivo in this organ.

32P analysis of DNA adducts in tissues of benzene-treated rats.

Solid tumors have been reported in the Zymbal gland, oral and nasal cavities, liver, and mammary gland of Sprague-Dawley rats following chronic, high-dose administration of benzene. The carcinogenic activity of benzene is thought to be caused by activation to toxic metabolites that can interact with DNA, forming covalent adducts. A nuclease P1-enhanced 32P-postlabeling assay, having a sensitivity limit of 1 adduct in 10(9-10) DNA nucleotides, was found suitable for measuring aromatic DNA adducts derived in vitro from catechol, benzenetriol (BT), phenol, hydroquinone (HQ), and benzoquinone (BQ), potential metabolites of benzene. When DNA specimens isolated from tissues of female Sprague-Dawley rats at 24 hr after an oral gavage dose of 200 to 500 mg/kg, 5 days/week, in olive oil (3 mL/kg) for 1 day, 1 week, 5 weeks, and 10 weeks were analyzed by the 32P-postlabeling procedure, no aromatic adducts were detected unequivocally with DNA samples of liver, kidney, bone marrow, and mammary gland. With Zymbal gland DNA, three weak spots at levels totaling four lesions per 10(9) DNA nucleotides were seen only after 10 weeks of treatment, and these adducts did not correspond chromatographically to major adducts in vitro from the above specified compounds. Consequently, this finding requires confirmatory experiments. This distinct adduct pattern may relate to tumor induction in this organ following benzene administration. Our results also indicate that DNA adducts derived from catechol, BT, phenol, HQ, and BQ are either not formed in vivo with benzene or formed at levels below the detection limit of 1 adduct per 10(9-10) DNA nucleotides.