Formation of cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde

Acrolein reacted with deoxyguanosine at pH 7 and 37 degrees to give three major products, Adducts 1 to 3, which were separated by high-performance liquid chromatography. They were identified by their ultraviolet, mass, and nuclear magnetic resonance spectra, by the spectra of the corresponding guanine derivatives, and by chemical transformations. Adducts 1 and 2 were two rapidly equilibrating diastereomers of 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-6- hydroxypyrimido [1,2-a]purine-10(3H)one, and Adduct 3 was 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8- hydroxypyrimido [1,2-a]purine-10(3H)one. Adducts 1 and 2 were formed by Michael addition of N-1 of deoxyguanosine to C-3 of acrolein, followed by ring closure between N2 of deoxyguanosine and C-1 of acrolein. Adduct 3 was formed by ring closure in the opposite direction. Adduct 3 was analogous to the major crotonaldehyde-deoxyguanosine adducts which were previously characterized. Adduct 3 (0.2 mmol/mol DNA-P) or the corresponding crotonaldehyde adduct (0.03 mmol/mol DNA-P) was formed when either acrolein or crotonaldehyde was allowed to react with DNA at pH 7 and 37 degrees. These results demonstrate that cyclic 1,N2-propanodeoxyguanosine adducts are formed by reaction of acrolein and crotonaldehyde with DNA.     

Formation of cyclic 1,N2-propanodeoxyguanosine and thymidine adducts in the reaction of the mutagen 2-bromoacrolein with calf thymus DNA

The interaction of the mutagen 2-bromoacrolein (2BA) with DNA and thymidine was studied in vitro by reaction of [3-3H]2BA with thymidine, RNA, single-stranded DNA, and double-stranded DNA in phosphate buffer (pH 7.4). After purification of the nucleic acids, they were incubated at alkaline pH to convert any (hydroxybromo)propano(deoxy)-guanosine adducts to their dihydroxy analogues. After acid or enzymatic hydrolysis, the hydrolysates were analyzed by reversed-phase high-performance liquid chromatography. At a concentration of 1.6 mM, the fraction of 2BA that became covalently bound to DNA was 2.3% of the amount added. Only 3% of the radioactivity bound to DNA after extensive purification could be accounted for as cyclic 1,N2-(6,7-dihydroxy)-propanoguanine adducts. More 2BA became covalently bound to single-stranded DNA and RNA as compared with double-stranded DNA. However, high-performance liquid chromatographic analyses showed that formation of cyclic 1,N2-(6,7-dihydroxy)propanoguanine adducts was also a minor reaction with these macromolecules. Because these data showed that other type(s) of reaction(s) are more important in the reaction of 2BA with nucleic acids, we have investigated the reaction of 2BA with other nucleosides. It was found that 2BA reacted well with thymidine in vitro, and the major product was identified by 500 MHz 1H and 75.43 MHz 13C nuclear magnetic resonance and thermospray mass spectrometry as 3-(2"-bromo-3"-oxopropyl)thymidine. This adduct was unstable and decomposed upon storage. After enzymatic hydrolysis of [3H]2BA-modified double-stranded DNA and subsequent analysis of the hydrolysate by high-performance liquid chromatography, 22% of the covalently bound radioactivity to DNA coeluted with decomposition products of the 3-(bromooxypropyl)thymidine adduct. This indicates that reaction of 2BA with this nucleotide in DNA is a major reaction.     

Detection of 1,N(2)-propanodeoxyguanosine adducts in DNA of Fischer 344 rats by an adapted (32)P-post-labeling technique after per os application of crotonaldehyde

Crotonaldehyde is an important industrial chemical to which humans and animals are ubiquitously exposed. The main intake occurs via food, tobacco smoke and possibly also via beverages. Estimation of intake via the different routes is difficult since the data available on exposure are inconsistent. Crotonaldehyde is genotoxic, mutagenic and carcinogenic and forms 1,N(2)-propanodeoxyguanosine adducts as the main DNA adducts. We have developed a (32)P-post-labeling method for these adducts based on nuclease P1 enrichment and polyethyleneimine-cellulose TLC which allows reliable detection with a detection limit of 3 adducts/10(9) nucleotides, a labeling efficiency of 80-90% and a recovery of 38%. Using this method we found crotonaldehyde adducts in different organs of Fischer 344 rats after a single gavage of high doses of 300 and 200 mg/kg body wt in the range 0.3-3.2 +/- 0.4 adducts/10(8) nucleotides and after repeated gavage of low doses of 10 and 1 mg/kg body wt (five times a week for 6 weeks) 6.2 +/- 0.2 and 2.0 +/- 0.4 adducts/10(8)nucleotides, but not in untreated animals nor in calf thymus DNA not treated with crotonaldehyde. In contrast to our results, Chung and co-workers found adducts in tissue of untreated Fischer 344 rats. This discrepancy could depend on the different methods used but also on differences in exposure of the animals via food or due to animal housing, etc.     

Role of 1,N2-propanodeoxyguanosine adducts as endogenous DNA lesions in rodents and humans

Results obtained in a number of studies in vitro and in vivo support the hypothesis that short- and long-chain enals and their epoxides derived from oxidized polyunsaturated fatty acids are potential endogenous sources of cyclic propano and etheno DNA adducts. We previously reviewed the evidence from some of these studies. Here, we describe the results of our more recent studies on the role of 1,N2-propanodeoxyguanosine adducts as endogenous DNA lesions. These studies include: the detection of distinct patterns of such adducts in various tissues of different species; the detection of long-chain trans-4-hydroxynonenal-derived deoxyguanosine adducts in vivo; the specificity of the formation of enal-derived propano adducts from omega-3 and omega-6 polyunsaturated fatty acids; and the detection of acrolein- and crotonaldehyde-derived adducts in human oral tissue DNA and their increased levels in smokers. Taken together, these studies further strengthen the hypothesis that enals produced by lipid peroxidation are the primary source for cyclic propano adducts in vivo, but these results cannot rule out the possible contribution of environmental and other sources. The mutagenicity of enals and their epoxides and the results of site-specific mutagenesis studies indicate that the cyclic adducts are potential promutagenic lesions; however, only circumstantial evidence is currently available for their role in carcinogenesis.     

Detection of exocyclic guanine adducts in hydrolysates of hepatic DNA of rats treated with N-nitrosopyrrolidine and in calf thymus DNA reacted with alpha-acetoxy-N-nitrosopyrrolidine

This report describes the isolation and characterization of DNA adducts formed in vitro from alpha-acetoxy-N-nitrosopyrrolidine and in rats treated with the hepatocarcinogen N-nitrosopyrrolidine. Esterase-catalyzed hydrolysis of alpha-acetoxy-N-nitrosopyrrolidine in the presence of calf thymus DNA, followed by neutral thermal hydrolysis of the DNA, resulted in formation of three previously unknown Adducts 1-3. They were isolated and characterized by their UV, mass, and proton magnetic resonance spectra as the exocyclic 7,8-guanine adducts 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purine-4(3H)-one (Adduct 1), and cis- and trans-2-amino-7,8-dihydro-8-hydroxy-6-methyl-3H-pyrrolo[2,1-f] purine-4(6H)-one (Adducts 2 and 3). Adduct 1 was formed by addition of 4-oxobutyl diazohydroxide, or a related carbonium ion, to the 7 and 8 positions of guanine. Adducts 2 and 3 resulted from Michael addition of 2-butenal to the 7 and 8 positions of guanine. Esterase-catalyzed hydrolysis of alpha-acetoxy-N-nitrosopyrrolidine in the presence of DNA also produced the exocyclic 1,N2-propanodeoxyguanosine Adducts 4a and 4b which we have previously described. Neutral thermal hydrolysates of hepatic DNA isolated from rats treated with N-nitrosopyrrolidine contained a fluorescent adduct, as previously reported (E.J. Hunt and R.C. Shank, Biochem. Biophys. Res. Commun., 104: 1343, 1982). This fluorescent adduct was shown to be identical to Adduct 1. Adducts 2, 3, 4a, and 4b were not detected in hepatic DNA hydrolysates from these animals. The results of this study provide the first example of a structurally characterized DNA adduct formed in vivo from a cyclic nitrosamine and support the alpha-hydroxylation hypothesis of cyclic nitrosamine metabolic activation.     

Detection of 1,N2-propanodeoxyguanosine adducts of 2-hexenal in organs of Fischer 344 rats by a 32P-post-labeling technique.

2-Hexenal is an alpha,beta-unsaturated carbonyl compound which is mutagenic, genotoxic and forms cyclic 1,N2-propanodeoxyguanosine adducts like similar propenals for which carcinogenicity was shown, e.g. acrolein or crotonaldehyde. Since humans have a permanent intake of 2-hexenal via vegetarian food this genotoxic compound is considered to play a role in human carcinogenicity. The data base is, however, presently not sufficient for a cancer risk assessment. To date no long term carcinogenicity study on 2-hexenal has been published. Detection of respective DNA adducts of this substance in animals or humans could allow cancer risk assessment. Therefore, we have developed a 32P-post-labeling technique based on nuclease P1 enrichment and TLC separation of the labeled adducts. The respective adducts are stable over a wide pH range from pH 4 to pH 11 and relatively stable against nuclease P1. The detection limit was 0.03 adducts per 10(6) nucleotides and the recovery was 10%. With this method we have shown in vivo formation of 1,N 2-propanodeoxyguanosine adducts of 2-hexenal for the first time and found the respective DNA adducts in different organs of Fischer 344 rats after gavage of 500, 200 and 50 mg 2-hexenal/kg body wt. No adducts could be detected in the organs of untreated rats. There is a clear dependence of the adduct level and the CBI (covalent binding index) on the dose. The CBI of 2-hexenal calculated on the basis of our adduct levels is extremely low (0.06). Since intake of 2-hexenal via fruit and vegetables is very low the cancer risk from 2-hexenal intake via food must also be considered as very low according to a first raw estimation on the basis of CBI and intake. The situation deserves, however, a more precise risk assessment in the future.     

Increased formation of hepatic N2-ethylidene-2'-deoxyguanosine DNA adducts in aldehyde dehydrogenase 2-knockout mice treated with ethanol

N2-ethylidene-2'-deoxyguanosine (N2-ethylidene-dG) is a major DNA adduct induced by acetaldehyde. Although it is unstable in the nucleoside form, it is relatively stable when present in DNA. In this study, we analyzed three acetaldehyde-derived DNA adducts, N2-ethylidene-dG, N2-ethyl-2'-deoxyguanosine (N2-Et-dG) and alpha-methyl-gamma-hydroxy-1,N2-propano-2'-deoxyguanosine (alpha-Me-gamma-OH-PdG) in the liver DNA of aldehyde dehydrogenase (Aldh)-2-knockout mice to determine the influence of alcohol consumption and the Aldh2 genotype on the levels of DNA damage. In control Aldh2+/+ mice, the level of N2-ethylidene-dG adduct in liver DNA was 1.9 +/- 0.7 adducts per 10(7) bases and was not significantly different than that of Aldh2+/- and -/- mice. In alcohol-fed mice (20% ethanol for 5 weeks), the adduct levels of Aldh2+/+, +/- and -/- mice were 7.9 +/- 1.8, 23.3 +/- 4.0 and 79.9 +/- 14.2 adducts per 10(7) bases, respectively, and indicated that adduct level was alcohol and Aldh2 genotype dependent. In contrast, an alcohol- or Aldh2 genotype-dependent increase was not observed for alpha-Me-gamma-OH-PdG, and N2-Et-dG was not detected in any of the analyzed samples. In conclusion, the risk of formation of N2-ethylidene-dG in model animal liver in vivo is significantly higher in the Aldh2-deficient population and these results may contribute to our understanding of in vivo adduct formation in humans.     

Development of a monoclonal antibody-based immunoassay for cyclic DNA adducts resulting from exposure to crotonaldehyde

In order to develop an immunoassay for DNA modifications resulting from exposure to crotonaldehyde, monoclonal antibodies specific for the 8R,6R- and 8S,6S-stereoisomers of 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxy-6 - methylpyrimido[1,2-a]purine-10(3H)one were produced. These cyclic 1,N2-propanodeoxyguanosines are formed in DNA exposed to crotonaldehyde in vitro. Three of the four antibodies were most specific for one stereoisomer while the fourth was most specific for the other stereoisomer. Fifty % inhibition of binding in an enzyme-linked immunoabsorbent assay using two of these antibodies and capable of detecting 0.5 mumol of 1,N2-propanodeoxyguanosine per mol of deoxyguanosine was developed. The method was validated by comparison to results obtained with fluorescence assay.     

Formation and persistence of a DNA adduct in rodents treated with N-nitrosopyrrolidine

The rate of formation and the persistence of an exocyclic guanine adduct formed in DNA of rodents treated with various doses of N-nitrosopyrrolidine (NPYR) have been determined. NPYR is hepatocarcinogenic to the rat and forms a covalent adduct in liver DNA; this adduct was recently identified as 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2, 1-f]purine-4[3H]-one. Dose-dependent amounts of adduct formed in liver, kidney and lung DNA of rats, hamsters and mice given oral doses (56-900 mg/kg body wt) of NPYR. The persistence of the adduct in DNA after administration of low doses of NPYR to rats was greatest in the target organ, i.e. the liver; at high doses of NPYR, adduct levels in DNA changed little over a period of at least 72 h. In the hamster, in which NPYR is carcinogenic to the lung but apparently not the liver, the adduct level in liver DNA was an order of magnitude greater than in lung or kidney DNA for a dose of NPYR of 225 or 900 mg/kg body wt; persistence of the adduct in lung DNA was only slightly longer than in liver DNA. The formation and persistence of the 7,8-pyridoguanine adduct in the rat appeared to be consistent with the organotropy of this carcinogen, but this was not true for the hamster, a species that seems to be more resistant to induction of liver and kidney cancer by this carcinogen. Imidazole, an inhibitor of microsomal amine oxidase, and disulfiram, an inhibitor of aldehyde dehydrogenase, decreased metabolic activation of NPYR to an alkylating intermediate; inducers and inhibitors of cytochrome P450 monooxygenases had little effect on the metabolic activation of NPYR to an alkylating agent.     

Formation of cyclic deoxyguanosine adducts in Chinese hamster ovary cells by acrolein and crotonaldehyde

Acrolein and crotonaldehyde are alpha,beta-unsaturated carbonyl compounds that form 1,N2-propanodeoxyguanosine adducts when reacted with DNA in vitro. These compounds are mutagenic in Salmonella, and crotonaldehyde is tumorigenic in rats. This study used immunoassay and 32P-postlabeling methods to determine if acrolein and crotonaldehyde form these adducts in cultured mammalian cells. Adduct levels were highest in Chinese hamster ovary cells exposed to acrolein (1 mM) with 162 mumol adduct/mol deoxyguanosine. Crotonaldehyde (10 mM) formed adduct at a level of 75 mumol/mol deoxyguanosine. 32P-Postlabeling analysis confirmed the presence of adducts in crotonaldehyde-treated cells. Persistence studies showed that adduct levels were unchanged if the cells were cultured for 6 h before DNA isolation. Mutagenicity studies were performed to determine the biological consequences of these adducts. Mutations were not observed due to the toxicity of the compounds.     

DNA adducts, mutant frequencies and mutation spectra in lambda lacZ transgenic mice treated with N-nitrosodimethylamine

Groups of lambda lacZ transgenic mice were treated i.p. with N- nitrosodimethylamine (NDMA) as single doses of 5 mg/kg or 10 mg/kg or as 10 daily doses of 1 mg/kg and changes in DNA N7- or O6-methylguanine or the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) were followed for up to 14 days in various tissues. Adduct induction in the liver exceeded by at least one order of magnitude than observed in the next nearest target tissue (lung), and was approximately linearly related to dose, except for O6-methylguanine after the first dose of 1 mg/kg which was lower than expected. Substantial induction of lambda lacZ mutagenesis was observed only in the liver, where the mutant frequency was already maximal within 7 days after 5 mg/kg NDMA and remained unchanged thereafter up to 49 days. Small but marginally significant increases in mutant frequency were consistently observed in the spleen after all three modes of treatment. A lack of proportionality between mutation induction and the administered dose or the corresponding adduct levels was observed, probably reflecting the importance of toxicity-related cell proliferation caused by NDMA at higher doses. Twenty eight days after a dose of 10 mg/kg (causing a 3.6- fold increase in mutant frequency), NDMA was found to increase the frequency of GC-->AT mutations (with a concomitant shift of their preferential location from CpG sites to GpG sites), which made up approximately 60% of the induced mutations. Surprisingly, NDMA also caused a significant increase in deletions of a few (up to 11) base- pairs (22%).      

Formation of 7-(4-oxobutyl)guanine in hepatic DNA of rats treated with N-nitrosopyrrolidine.

We have reported previously the formation of two structurally distinct exocyclic guanine adducts (adducts 1 and 6) in liver DNA of F344 rats treated with N-nitrosopyrrolidine (NPYR). In this study, we detected and characterized a previously unidentified guanine adduct in liver DNA of NPYR-treated rats. The structure of this adduct was established as 7-(4-oxobutyl)guanine (adduct 2) by comparison with the synthetic standard and confirmed by NaBH4 reduction to 7-(4-hydroxybutyl)guanine. The level of adduct 2 in liver DNA of F344 rats treated with 450 mg/kg of NPYR by i.p. administration was 643 +/- 9 mumol/mol guanine, approximately one-third of the level of adduct 1. This study is the first to demonstrate the in vivo formation of a formylalkyl-substituted guanine adduct by a nitrosamine.     

SHORT COMMUNICATION: Detection of DNA adducts in the white blood cells of B6C3F1 mice treated with benzene

We have employed the P₁-enhanced ³²P-postlabeling procedureto detect the formation DNA of adducts in the white blood cells(WBC) of B6C3F1 mice treated by i.p. injection with benzene.Treatment twice a day with 440 mg/kg benzene for 1–7 daysresulted in the formation of one major (adduct 1) and one minor(adduct 2) DNA adduct in the WBCs of mice. The same DNA adductpattern was also found in the bone marrow (BM) of benzene treatedmice. The relative adduct levels were dependent upon both benzenedose from 100–440 mg/kg and treatment time from 1 to 7days. The relative adduct levels ranged between 0.11 and 1.33adducts in 10⁷ nucleotides for WBCs and 0.16–1.21 adductsin 107 nucleotides for BM. Following treatment with benzene,the levels of DNA adducts formed in WBCs were significantlycorrelated with the levels of DNA adducts formed in BM (r² =0.97, P <0.001). Our results suggest that measurement ofDNA adducts in WBCs may be an indicator of DNA adduct formationin BM following BZ exposure.     

Cancer risk assessment for the environmental mutagen and carcinogen crotonaldehyde on the basis of TD(50) and comparison with 1,N(2)-propanodeoxyguanosine adduct levels.

Humans are ubiquitously exposed to crotonaldehyde to a strongly varying extent, in particular, via food and alcoholic beverages. Like other alpha,beta-unsaturated carbonyl compounds, crotonaldehyde forms 1,N(2)-propanodeoxyguanosine adducts and is genotoxic, mutagenic, and carcinogenic. This study was designed to perform a cancer risk assessment on the basis of TD(50), which was available from a long-term cancer study with F-344 rats (F. L. Chung et al., Cancer Res., 46: 1285-1289, 1986), and the estimated daily intake via food and beverages. A relatively high cancer risk of 0.1-1 cancer incidence/10(3) humans was extrapolated on the basis of the TD(50) from the cancer study of Chung et al. for the estimated dietary intake and drinking wine. To compare the 1,N(2)-propanodeoxyguanosine DNA adduct levels of crotonaldehyde with the assessed cancer risk, we synthesized adduct standards and developed a (32)P-postlabeling method for DNA adducts of crotonaldehyde providing a detection limit of 3 adducts/10(9) nucleotides. Repeated gavages of 10 and 1 mg/kg were given to simulate the steady-state situation of the animal cancer study of Chung et al. and to estimate the adduct levels after intake of crotonaldehyde via food. The estimated adduct levels at these crotonaldehyde intakes were in the range of 3 adducts/10(9) nucleotides. The adducts persisted to a certain extent. The persistence is important for considering the steady-state situation after permanent intakes of crotonaldehyde via food. However, the adducts are repaired to some extent; 2 weeks after the last of repeated gavages, only 19% of the initial amount measured directly after the last gavage is left. According to our results, a steady-state concentration in the range of 3 adducts/10(9) nucleotides is responsible for the induction of cancer in the study of Chung et al., in the case that cancer from crotonaldehyde depends exclusively on the 1,N(2)-propanodeoxyguanosine adducts considered here. No propanodeoxyguanosine adducts of crotonaldehyde were found in the DNA of untreated animals in our studies.     

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen

Using rat liver microsomal preparations and peroxidase enzymes,we have investigated the formation of DNA adducts by the antiestrogencompound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen(4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was used as a cofactor in microsomal activation of either4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levelsof 4.6 and 3.1x10^–8, respectively were detected by 32P-postlabeling.The DNA adduct produced by microsomal activation of 4-OH-TAMand TAM was the same. With cumene hydroperoxide (CuOOH) as thecofactor for the microsomal activation of either 4-OH-TAM orTAM, three to six DNA adducts were produced; the relative adductlevels were 8.0 and 20.6x10^–8, respectively. Comparisonof the DNA adduct patterns produced by 4-OH-TAM and TAM showedthat they were distinct However one of the DNA adducts (a) producedby microsomal activation of 4-OH-TAM using CuOOH was the sameas adduct a produced by microsomal activation of 4-OH-TAM withNADPH. Activation of 4-OH-TAM with horseradish peroxidase resultedin the formation of a single DNA adduct and a relative adductlevel of 20.7x10^–8. Rechromatography analysis of thisDNA adduct showed that it was identical to that produced bymicrosomal activation of 4-OH-TAM with NADPH and one of theadducts produced using CuOOH as the cofactor. Ten DNA adductsand a relative adduct level of 15.3x10^–8 were detectedin the liver of female Sprague-Dawley rats treated daily with20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liverof the treated animals was similar to that produced by microsomalactivation of TAM using CuOOH as the co-factor. The principalDNA adduct (no. 6) formed in the livers of rats treated withTAM was the same as the principal DNA adduct formed followingmicrosomal activation of TAM using CuOOH as a cofactor. TheDNA adduct formed following microsomal activation of eitherTAM or 4-OH-TAM using NADPH was also present as one of the adducts(1^*) formed in vivo following TAM treatment These studies demonstratethat 4-OH-TAM can be activated to form DNA adducts and thatit contributes to the formation of DNA adducts in the liverof rats treated with TAM.     

Formation and persistence of DNA adducts in organs of CD-1 mice treated with a tumorigenic dose of fluoranthene

Fluoranthene (FA) is tumorigenic to the lung when injected i.p.into CD-1 mice 1, 8 and 15 days after birth (Wang, J.-S. andBusby, W.F.Jr, Carcinogenesis, 14, 1871–1874, 1993). Levels,tissue distribution and persistence of FA-DNA adducts detectedby HPLC-³²P-postlabeling were investigated during the courseof lung tumorigenesis by FA.Anti-10b-N²-deoxyguanosin-1,2,3-trihydroxy-1,2,3,10b-tetrahydrofluoranthene(anti-FADE adduct) was consistently the major adduct in DNAsamples from lung, heart, liver and kidney of animals examinedat different time points from 2 h to 165 days after the lasttreatment with the tumorigenic dose (3.5 mg/mouse) of FA. Severalunidentified adducts were also detected. Lung, the target organfor FA tumorigenicity, contained higher levels of anti-FADEadduct than other tissues from 1–165 days after treatment.The anti-FADE adduct level decreased in a biphasic manner afterreaching maximum values at 2 h in heart and spleen plus thymusand 3 days in lungs, liver and kidneys. About 10% of the maximumamount of anti-FADE adduct remained in lung, liver and heart165 days after final FA treatment, at which time 44% of animalshad developed lung adenomas. Significant inter-litter variations,but no sex differences in adduct levels were observed. Theseresults indicated a positive correlation between anti-FADE adductlevel and persistence in relation to target organ specificityfor tumor formation.     

Molecular dosimetry of DNA adducts in C3H mice treated with glycidaldehyde.

The formation of DNA adducts in the skin of male C3H mice treated cutaneously with glycidaldehyde (2 or 10 mg/animal) in acetone has been investigated by HPLC coupled with fluorescence detection and 32P-postlabelling analysis. Following a 24 h exposure period, epidermal DNA was isolated from treated dorsal skin and enzymically digested to nucleoside-3'-monophosphates. HPLC-32P-postlabelling analysis of the DNA hydrolysate indicated that a single major cyclic adduct was formed from the reaction of glycidaldehyde with deoxyadenosine residues in mouse skin DNA. This adduct was identified as 3-beta-D-deoxyribofuranosyl-7-(hydroxymethyl)-3H- imidazo[2,1-i]purine-3'-monophosphate by comparison with a synthetic standard. This adduct was stable, strongly fluorescent and readily detected by HPLC with fluorescence detection. There was no evidence for the formation of deoxyguanosine adducts in epidermal DNA of treated animals. Glycidaldehyde also reacted with calf thymus DNA in vitro at pH 7.0 to give the same deoxyadenosine adduct observed in vivo. At pH 10, however, this was a relatively minor product and the major adduct was 5,9-dihydro-7-(hydroxymethyl)-9- oxo-3-beta-D-deoxyribofuranosyl-3H-imidazo[1,2-a]purine-3'- monophosphate formed by the initial reaction of glycidaldehyde with deoxyguanosine residues.     

Detection of DNA adducts in HL-60 cells treated with hydroquinone and p-benzoquinone by 32P-postlabeling

We have examined DNA adduct formation and cytotoxicity in HL-60 cells treated with either hydroquinone (HQ) or p-benzoquinone (p-BQ). Treatment of HL-60 cells with either HQ or p-BQ produced the same DNA adduct. The DNA adduct level varied from 0.05 to 10 adducts per 10(7) nucleotides as a function of treatment time and concentration for both compounds. To achieve the same DNA adduct level required higher concentrations and longer treatment times with HQ compared to p-BQ. p-BQ was also more cytotoxic to HL-60 cells than HQ. Reaction of calf thymus DNA with a p-BQ/HQ mixture produced five adducts as detected by 32P-postlabeling. Two isomers of (hydroxy)-1,N2-benzetheno-2'- deoxyguanosine-3'-phosphate were isolated from the reaction of 2'-deoxyguanosine-3'-phosphate with a p-BQ/HQ mixture and one of the isomers was identified as adduct no. 1 of the DNA reaction. The DNA adduct formed in HL-60 cells treated with HQ or p-BQ did not correspond to any of the principal adducts formed in DNA reacted with p-BQ/HQ. This result suggests that cellular mechanisms modify DNA adduct formation by HQ and p-BQ.     

Detection of 1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine occurring endogenously in DNA

1,N6-Etheno-2'-deoxyadenosine (epsilon dA) and 3,N4-etheno-2'-deoxycytidine (epsilon dC) are DNA adducts formed by a number of genotoxic chemicals, including vinyl chloride. They are also formed endogenously in tissue DNA, probably from a reactive metabolite of lipid peroxidation. Both the qualitative and quantitative detection of endogenous adducts is important in order to place adduct formation by chemicals such as vinyl chloride in the context of this natural background level. Methods with sufficient sensitivity are therefore being developed to measure the natural background of epsilon dA and epsilon dC adducts. We have developed a high-performance liquid chromatography (HPLC)-32P-postlabelling method to measure epsilon dA and epsilon dC at alkylation frequencies of 1 adduct in 10(7)-10(8) nucleotides in 10-microgram samples of DNA. In HPLC-32P-postlabelling analysis of liver DNA from control Wistar rats, epsilon dA and epsilon dC were determined at levels of 1 adduct in 8.1 x 10(7) and 1 adduct in 1.8 x 10(7) nucleotides, respectively. The levels of epsilon dA and epsilon dC measured in liver DNA of animals exposed orally to five daily doses of 50 mg/kg body weight vinyl chloride were found by this method to be 1 adduct in 2.9 x 10(7) and 1 adduct in 1.4 x 10(7) nucleotides, respectively. In contrast, in a direct labelling study, radiolabelled epsilon dA and epsilon dC were not detected in liver DNA of rats exposed for 6 h by nose-only inhalation to [1,2-14C]vinyl chloride at up to 45 ppm v/v. Immunochemical procedures are also being developed for recognizing etheno adducts. Thus, a monoclonal antibody raised to protein conjugates of epsilon dC showed high selectivity in the recognition of this DNA adduct. When the antibody was immobilized on a solid support and used in an immunoenrichment procedure to purify epsilon dC from a large excess of normal nucleotides, one epsilon dC adduct from about 10(8) normal nucleotides could be resolved. Coupling the immunoaffinity enrichment procedure with capillary zone electrophoresis permitted the detection of approximately one epsilon dC adduct in 3 x 10(6) nucleotides.