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.     

A comparison of two ultrasensitive methods for measuring 1,N6-etheno-2'- deoxyadenosine and 3,N4-etheno-2'-deoxycytidine in cellular DNA

1,N⁶-Ethenodeoxyadenosine (edA) and 3,N⁴-ethenodeoxycytidine(edC) are two mutagenic adducts associated with exposure toethyl carbamate (urethane) and vinyl chloride. We have recentlydeveloped two ultrasensitive methods for determining the moleculardose of these adducts in cellular DNA. In both methods, purifiedDNA was first enzymatically digested to 2c-deoxyribonucleotide3c-monophosphates. Etheno-modified nucleotides were then separatedfrom normal nucleotides in one of two ways: either by reversephase, ion-pair HPLC coupled with 260 nm UV detection, or byimmunoaffinity chromatography using reusable microcolumns containingspecific monoclonal antibodies coupled to Protein A–Sepharose.Fractions enriched for the adducted nucleotides were labeledusing T4 polynucleotide kinase and [³²P]ATP, and individualnucleotides were subsequently resolved by two-dimensional TLC,visualized by autoradiography, and quantified by liquid scintillationcounting. When used to analyze the same sample of etheno-modifiedcalf thymus DNA, both assays produced similar results. However,when both methods were used to analyze rat liver DNA ‘spiked’with known amounts of etheno nucleotide standards, the immunoaffinity/³²PTLC procedure proved to be more sensitive and more reproduciblethan the HPLC/³²P TLC method: while the detection limit of theimmunoaffinity/³²P TLC technique was < 4 etheno adducts/10⁹parent deoxynucleotides, the HPLC/³²P TLC method often failedto detect adducts at concentrations <2/108. In other experiments,the immunoaffinity/³²P TLC method was used to demonstrate formationof edA and edC in cells treated with vinyl chloride monomer.Because of its exquisite sensitivity, the immunoaffinity/³²PTLC method promises to be extremely useful for measuring bothbackground and induced levels of etheno adducts, making it possibleto examine the role of these adducts in inducing mutations and/orcarcinogenesis.     

Immunohistochemical detection of 1,N(6)-ethenodeoxyadenosine, a promutagenic DNA adduct, in liver of rats exposed to vinyl chloride or an iron overload

Etheno adducts in DNA bases are formed from exogenous agents such as vinyl chloride and urethane, but also via endogenous lipid peroxidation products like trans-4-hydroxy-2-nonenal. An immunohistochemical method was developed to localize the promutagenic 1,N(6)-ethenodeoxyadenosine DNA adduct in liver of rats exposed to vinyl chloride or an iron overload with or without carbon tetrachloride. Six monoclonal antibodies, previously produced through collaborative efforts, were screened for their optimal adduct recognition and low background formation. The antibody generated by clone EM-A-4 was found to be most suitable. Semi-quantitative image analysis of relative pixel intensity showed approximately 1.5 times higher adduct levels (P < 0.05) in the livers of rats treated with vinyl chloride or an iron overload when compared with untreated controls. Significantly elevated adduct levels persisted in vinyl chloride-treated rat liver 14 days after cessation of exposure, suggesting that this adduct is not rapidly eliminated from rat liver DNA. Using the new immunohistochemical method it is possible to visualize this promutagenic etheno-DNA adduct that may play a role in oxidative stress and lipid peroxidation-induced DNA damage in carcinogenesis.     

Molecular dosimetry and repair of N(2),3-ethenoguanine in rats exposed to vinyl chloride

Although the DNA adducts of vinyl chloride (VC) have been well characterized, previous studies have used single concentrations of VC that are well above contemporary human exposures. This study examined the exposure response to VC in male Sprague Dawley rats with respect to the molecular dose of the promutagenic DNA adduct N(2),3-ethenoguanine (N(2),3-epsilonG). Adult rats were exposed by inhalation to 0, 10, 100, or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week). Weanling rats were similarly exposed for 5 days. The amount of N(2),3-epsilonG in hepatocyte (HEP) and nonparenchymal cell (NPC) fractions obtained from the liver was measured with a sensitive immunoaffinity/gas chromatography/high-resolution mass spectrometry assay. Endogenous N(2),3-epsilonG was present in HEPs and NPCs from all unexposed rats. The exposure response to VC in each group and cell population was supralinear, with a linear increase from 0 to 100 ppm, and a plateau between 100 and 1100 ppm. There was no statistically significant difference in N(2),3-epsilonG concentrations between HEPs and NPCs in any adult exposure group, which suggests that factors other than adduct concentrations contribute to the particular susceptibility of NPCs to VC-induced carcinogenesis. The accumulation of N(2),3-epsilonG with respect to time was nearly linear in rats exposed to 600 ppm VC for 1, 2, 4, or 8 weeks (4 h/day, 5 days/week), and no repair of N(2),3-epsilonG was detected in rats exposed to VC for 4 weeks and allowed to recover for 1 week. N(2),3-epsilonG concentrations in HEPs from weanling rats were 2-3-fold greater than those in adult rats exposed for the same time. Higher adduct concentrations in young rats may contribute to their greater susceptibility to VC-induced hepatic angiosarcoma as well as their particular susceptibility to hepatocellular carcinoma. The molecular dosimetry of N(2),3-epsilonG in liver appears to be a sensitive and informative biomarker of genotoxic effect after exposure to VC. N(2),3-epsilonG was the predominant etheno adduct measured in vivo after exposure to VC, and the saturable nature of VC metabolism was reflected in its molecular dose. The relationships between endogenous N(2),3-epsilonG and that formed by low exposures to VC were demonstrated. Conclusions drawn from these exposures may be more relevant for risk assessment purposes than those drawn from high exposures where activation, detoxication, and repair pathways may be saturated or otherwise perturbed. These data are well suited for consideration in future risk assessments of VC that incorporate nontumor mode of action data.     

1,N6-Ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in liver DNA from humans and untreated rodents detected by immunoaffinity/32P-postlabelling

The etheno-bridged exocyclic DNA adducts 1,N⁶-ethenodeoxyadenosine(     

DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1, 2, 3-trichloropropane

1, 2, 3-Trichloropropane (TCP) is a multispecies, multisitecarcinogen which has been found to be an environmental contaminantIn this study, we have characterized and measured DNA adductsformed in vivo following exposure to TCP. [¹⁴C]TCP was administeredto male B6C3F1 mice and Fischer-344 rats by gavage at dosesused in the NTP carcinogenesis bioassay. Both target and nontargetorgans were examined for the formation of DNA adducts. Adductswere hydrolyzed from DNA by neutral thermal or mild acid hydrolysis,isolated by HPLC, and detected and quanti-tated by measurementof radioactivity. The HPLC elution profile of radioactivitysuggested that one major DNA adduct was formed. To characterizethis adduct, larger yields were induced in rats by intraperitonealadministration of TCP (300 mg/kg). The DNA adduct was isolatedby HPLC based on coelution with the radiolabeled adduct, andcompared to previously identified adducts. The isolated adductcoeluted with S-[1-(hydroxymethyl)-2-(N⁷-guanyl)-ethyljglutathione,an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray massspectrometry suggested that the TCP-induced adduct and the DBCP-derivedadduct were identical. The ¹⁴C-labeled DNA adduct was distributedwidely among the organs examined. Adduct levels varied dependingon species, organ, and dose. In rat organs, adduct concentrationsfor the low dose ranged from 0.8 to 6.6 µmol per mol guanineand from 7.1 to 47.6 µmol per mol guanine for the highdose. In the mouse, adduct yields ranged from 0.32 to 28.1 µmolper mol guanine for the low dose and from 12.2 to 208.1 µmolper mol guanine for the high dose. The relationship betweenDNA adduct formation and organ-specific tumorigenesis was unclear.Although relatively high concentrations of DNA adducts weredetected in target organs, several nontarget sites also containedhigh adduct levels. Our data suggest that factors in additionto adduct formation may be important in TCP-induced carcinogenesis.     

Ras proto-oncogene activation in liver and lung tumors from B6C3F1 mice exposed chronically to methylene chloride

Methylene chloride has been the subject of recent toxicologicaland carcinogenesis studies because of significant human exposureand widespread use in industrial processing, food preparationand agriculture. In this study, liver and lung tumors, inducedin female B6C3F1 mice by inhalation of 2000 p.p.m. methylenechloride (6 h/day, 5 days/week continuous exposure), were examinedfor the presence of activated rasproto-oncogenes. DNA was isolatedfrom 49 spontaneous and 50 methylene chloride-induced livertumors and screened by oligonucleotide hybridization of PCRamplified H-ras gene fragments for codon 61 mutations. In thechemically induced tumors, 38 mutations were detected, 16 Cto A transversions in base 1, 16 A to G transitions in base2 and 6 A to T transversions in base 2. This mutation profilewas similar to that identified for the H-ras gene in the spontaneousliver tumors and suggests that methylene chloride acts in liverby promoting cells with spontaneous lesions. Tumors in whichH-ras codon 61 mutations were not detected were examined forthe presence of transforming genes by the nude mouse tumorigenicityassay. Except for activated K-ras genes detected in DNA fromtwo methylene chloride induced tumors and one spontaneous tumor,no other transforming genes were identified. DNA from 54 lungtumors was screened by direct sequencing of PCR amplified DNAfragments of the K-ras gene for first and second exon mutations,and 12 mutations were identified, 5 in exon one and 7 in exon2. The low number of spontaneous tumors available in this studylimits the interpretation of the data, and thus the frequencyand spectrum of K-ras activation in the methylene chloride inducedtumors was not significantly different from that in the sevenspontaneous tumors analyzed. Since K-ras activation was notdetected in 80% of the tumors, the nude mouse tumorigenicityassay was used to examine the lung tumors for the presence ofother transforming genes. At present no transforming genes otherthan ras genes were identified in either liver or lung tumors.     

ACCELERATED PAPER: Formation and accumulation of DNA ethenobases in adult Sprague-Dawley rats exposed to vinyl chloride

DNA ethenbases are promutagenic lesions formed by carcinogenssuch as vinyl chloride (VC). Their formation was investigatedin 9-week old, male Sprague-Dawley rats exposed to 500 p.p.m.VC by inhalation (4 h/day, 5 days/ week) for 1, 2, 4 or 8 weeksand in 7- and 14-week old, matched control animals. 1, N⁶Ethenoadenine(A) and 3, N⁴-ethenocytosine (eC) deoxyribonucleotides wereanalysed by immunoaffinity Purification and 32P-postlabelling.This postabelling method was compared with a radio-immunoassaymethod, which yielded similar results. Back-ground levels ofethenobases were found in DNA from the liver, lungs, kidneysand circulating lymphocytes of unexposed, control rats. In theliver, the following back-ground molar ratios of ethenobaseto parent base in DNA were detected (mean valuesx10–8):A/A, 0.04–0.05; C/C, 0.06–0.07. In the lungs, kidneysand circluating lympho-cytes, background levels of A and C rangedfrom 1.7 to 4.2x10–8 and from respectively. Followinga 5-day exposure to VC, a significant increase of A and D wasmeasured in hepatic DNA from rats sacrificed immediately aftertreatment. Further, a dose-dependent increase of both ethenoadducts was observed in liver DNA of VC-treated rats. Comparedto the 5-day exposure, 4-fold higher levels of A and C wereobserved in the liver of animals after 8 weeks of exposure.In contrast, there was an accumulation of C but not of A inlungs and kidneys. In circulating lymphocytes, no significantincrease of ethenobase levels above control values was observedafter 2 months of exposure to VC. Both etheno adducts were foundto be pesistent in liver DNA, after 2 months following the terminationof VC exposure. These results further support the notion thatDNA ethenogenesis. are critical lesions in VC-induced carcinogenesis.The possible contribution of lipid peroxidation products thatalso yield ethenobases, on the formation and persistence ofthese DNA adducts, remains to be clarified.     

Differential induction of N(2),3-ethenoguanine in rat brain and liver after exposure to vinyl chloride

Although vinyl chloride (VC) clearly induces hepatic angiosarcoma in humans and rodents, a causal association with brain tumors has not been definitively established with the available epidemiological and experimental evidence. Because VC acts by genotoxic mechanisms, DNA adduct formation is thought to be a sensitive biomarker of early events in carcinogenesis. Adult male Sprague Dawley rats were exposed to 0 or 1100 ppm VC for 1 or 4 weeks (6 h/day, 5 days/week) by inhalation. Male weanlings were similarly exposed for 5 days. Another group of male adults was exposed to 1100 ppm [(13)C(2)]VC in a nose-only inhalation apparatus for 5 days (6 h/day). A sensitive gas chromatography high-resolution mass spectrometry assay was used to measure the major promutagenic DNA adduct, N(2),3-ethenoguanine (N(2),3-epsilonG), in rat brain and hepatocyte (HEP) DNA. The respective concentrations of N(2),3-epsilonG in control rat brain DNA at 1 and 4 weeks were 5.0 +/- 0.9 and 5.6 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. There was no change in N(2),3-epsilonG in adult rat brain after exposure to 1100 ppm VC for 1 or 4 weeks. In HEPs from the same animals, these adduct concentrations increased from 5.5 +/- 1.4 to 55 +/- 2.0 N(2),3-epsilonG/10(8) unmodified guanine after a 1-week exposure and from 3.0 +/- 0.3 to 110 +/- 20 N(2),3-epsilonG/10(8) unmodified guanine after a 4-week exposure. When weanlings were exposed to 1100 ppm VC for 5 days, there was a statistically significant (P = 0.04) increase in N(2),3-epsilonG in brain from 1.5 +/- 0.2 to 4.4 +/- 1.1 N(2),3-epsilonG/10(8) unmodified guanine. Weanlings exposed to 1100 ppm VC had an even greater increase in N(2),3-epsilonG in HEPs from 1.6 +/- 0.1 to 97 +/- 5.0 N(2),3-epsilonG/10(8) unmodified guanine. [(13)C(2)]N(2),3-epsilonG was not detected in brain DNA from adult rats exposed to 1100 ppm [(13)C(2)]VC for 5 days but was present in HEP DNA at 55 +/- 4.0 [(13)C(2)]N(2),3-epsilonG/10(8) unmodified guanine. The concentrations of the endogenous adduct in both organs were unchanged after this exposure. 7-(Oxoethyl)guanine (OEG), the major DNA adduct formed by VC, was reduced to 7-(2-hydroxyethyl)guanine and measured by liquid chromatography-electrospray ionization-tandom mass spectrometry in brain and HEP DNA from rats exposed to 1100 ppm VC for 1 week. Whereas 4.0 +/- 0.8 OEG/10(6) unmodified guanine were present in HEP DNA from VC-exposed rats, no adducts were detectable in brain DNA (detection limit, 0.3 OEG/10(6) unmodified guanine). These findings indicate that the genotoxic metabolite of VC is not formed in or transported to adult rat brain. Thus, it is unlikely that N(2),3-epsilonG or other VC-induced promutagenic DNA adducts play a significant role in initiating carcinogenesis in adult rat brain after exposure to VC. The data for weanling rats are less clear. Whereas a small increase in N(2),3-epsilonG in the brains of weanlings was found after exposure to 1100 ppm VC, the resulting adduct concentration was similar to that measured in unexposed adults. Future exposures of weanling rats to the stable isotopically labeled compound will be necessary to conclusively determine whether this increase was due to VC.     

Immimological titration of 3-N-acetyl-hydroxyamino-4, 6-dimethyldipyrido(l, 2-a: 3', 2'-d)imidazole-rat liver DNA adducts

Antibodies to N-(guanosin-8-yI)-3-N-acetylamino-4, 6-di-methyldipyrido(l,2-a:3', 2'-d)imidazole were elicited in rabbits by immunizationwith a conjugate formed between this compound and bovine serumalbumin. The specificity of the antibodies was studied by radioimmunoassay.These antibodies were used to titrate the adducts formed inliver DNA of rats treated with 3-N-acetyl-hyoYoxyamino-4, 6-dimethyldipyrido,2-a: 3', 2'-d)iinidazole, a supposed metabolite of the mutagenicamine 3-amino-4, 6-dimethyldipyrido(l, 2-a:3', 2'-d)imidazofe(Glu-P-3). It is found that Gtu-P-3 residues are covalentlybound to the C8 of guanine in acetylated form (70%) and deacetylatedform (30%).     

Detection of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine by immunoaffinity/32P-post-labelling in liver and lung DNA of mice treated with ethylcarbamate (urethane) or its metabolites

The capacity of the chemical carcinogen ethyl carbamate (EC,urethane) and its metabolites vinyl carbamate (VC) and vinylcarbamate epoxide (VCO) to form ethenobases was studied in liverand lung DNA of 12-day-old and adult CD-1, B6C3F1, C3H/HeJ andC57BL/6J mice. Following single and multiple doses of EC, VCor VCO, the formation of 1,N⁶-ethenodeoxyadenosine (     

SHORT COMMUNICATION: 1, N6-Ethenoadenine and 3, N4-ethenocytosine are excised by separate human DNA glycosylases

We previously reported our finding that human cells containglycosylase activity toward all four etheno bases formed inDNA by chloroacetaldehyde and related bi-functional aldehydes.By enzyme purification, including FPLC, we isolated two separateglycosylase activities for 1,N⁶-ethenoadenine (     

Development of monoclonal antibodies specific for 1,N2-ethenodeoxyguanosine and N2,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde

Monoclonal antibodies specific for N²3-ethenodeoxyguanosine(N²,3-dGuo) and 1,N²-ethenodeoxyguanosine (1,N²-dGuo) were developed.In a competitive ELISA, 50% inhibition of binding of the N²,3-dGuospecific antibody (ETH1) was achieved with 18 fmol of N²,3-dGuo.Fifty per cent inhibition of the 1,N²-dGuo-specific antibody(ETH2) required 11 pmol 1,N²-dGuo. Immunoassays for N²,3-dGuoand 1,N²-dGuo in single-stranded DNA were developed using theseantibodies. The immunoassays could detect as little as 48 fmolof N²,3-dGuo or 340 fmol 1,N²-dGuo in 25 µg of singlestranded DNA. These assays and previously developed immunoassaysfor 1,N⁶-ethenodeoxyadenosine (1,N⁶-dAdo) and 3,N⁴-ethenodeoxycytidine(3,N⁴-dCyd) were used to measure etheno adduct levels in DNAof cells exposed to chloroacetaldehyde. The cells used wereV79 cells with an inactivated hprt gene and a single copy ofthe bacterial gpt gene (G12 cells). The most abundant ethenoadduct was 1,N⁶-dAdo, followed by 3,N⁴-dCyd and N²,3-dGuo. 1,N²-dGuowas not detected in chloroacetaldehyde-treated G12 cells. Chloroacetaldehydewas also shown to be mutagenic in these same cells.     

Induction of cytochrome P450 1B1 in lung, liver and kidney of rats exposed to diesel exhaust.

We have shown previously that diesel exhaust particle (DEP) extracts (DEPE) and 1-nitropyrene were genotoxically activated by human cytochrome P450 1B1 in SOS/umu assay. In this study, the in vivo induction of P450 family 1 enzymes in rats by exposure to diesel exhaust was investigated with regard to mRNA levels, P450 enzyme content, drug oxidation activities in the microsomes and umu gene expression of typical P450 substrates and DEPE itself catalyzed by the microsomes. Male Fischer 344 rats (4 weeks old) were exposed to 0.3 and 3.0 mg/m(3) DEP for 12 h per day for 4 weeks; the former dose corresponded to the typical daily airborne particle concentration. The levels of mRNA of rat P450 1B1 and P450 1A1 in the lung and liver were significantly increased 1.1-1.4-fold by exposure to 0.3 mg/m(3) DEP. Diesel exhaust particle extracts induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of a functional P450 system and were further activated by human recombinant P450 1B1. Using an O-acetyltransferase overexpressing Salmonella strain, genotoxic activation of P450 1B1 marker chemicals (1-nitropyrene, 1-aminopyrene and DEPE) by lung, liver and kidney microsomes was increased 1.7-4.2-, 1.4-1.5- and 1.0-1.3-fold, respectively, by exposure to 0.3 mg/m(3) DEP. Activation of 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1; marker for P450 1A1) by lung microsomes and the P450 1A2 content in liver microsomes were slightly increased by exposure to 3.0 mg/m(3) DEP. This is the first report to suggest that typical daily contaminant levels (0.3 mg particle/m(3)) of diesel exhaust can induce P450 1B1 in rats and that the induced P450 1B1 may catalyze the genotoxic activation of DEP.     

Hemoglobin and DNA adducts in rats exposed to 2-nitrotoluene

2-Nitrotoluene (2NT) is an important commercial chemical intermediate. A recent National Toxicology Programme (NTP)-study demonstrated clear evidence of carcinogenic activity of 2NT in rats. In the present study male WELS-Fohm rats were dosed chronically with 2NT, 5 days a week for 12 weeks. Hemoglobin (Hb) adducts and hepatic DNA adducts were analyzed. After mild base treatment of Hb, 2-methylaniline (2MA) was released and quantified using gas chromatography/mass spectrometry. 2'-Deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts of 2MA were found in hepatic DNA using electrospray-mass spectrometry (ESI-MS/MS). The dG adduct found in vivo did not co-elute with N-(2'-deoxyguanosine-8-yl)-2-methylaniline which is the expected adduct for arylamines. The dG adduct detected in the dosed rats was not present in calf thymus-DNA (ct-DNA) modified in vitro with N-acetoxy-2MA. The dA adduct detected in rats was a very minor product in ct-DNA modified in vitro. The dG and dA adducts found in the 2NT-dosed rats increased with the dose. The same increase was seen for the Hb adduct levels measured in the same animals. The increase of DNA and Hb adduct levels were supralinear. There was a very strong linear relationship between the level of dG-2MA adducts and dA-2MA adducts in hepatic DNA from rats administered 2NT over the whole dose range studied (r(2) = 0.9). A strong linear relationship also existed between the level of dG-2MA or dA-2MA adducts, in hepatic DNA, and Hb adducts, over the whole dose range (r(2) > or = 0.9). Thus, there was strong evidence to support the notion that Hb adducts were an effective surrogate marker for the hepatic DNA damage of rats chronically administered 2NT.     

Hemoglobin and DNA adducts in rats exposed to 2-nitrotoluene

The authors wish to amend the DNA hydrolysis paragraph     

Decreased repair activities of 1,N(6)-ethenoadenine and 3,N(4)-ethenocytosine in lung adenocarcinoma patients

To assess the role of oxidative stress and lipid peroxidation (LPO) in the pathogenesis of lung cancer, we measured the levels of 1,N(6)-ethenoadenine (epsilonA) and 3,N(4)-ethenocytosine (epsilonC) in the DNA by immunoaffinity/(32)P postlabeling (33 cases). We also measured the capacity for epsilonA and epsilonC repair (by the nicking assay) in normal and tumor lung tissues, as well as in blood leukocytes of lung cancer patients (56 cases). Repair activities for epsilonA and epsilonC were also assayed in leukocytes of healthy volunteers, matched with cancer patients for age, sex, and smoking habit (25 individuals). Up to 10-fold variations among individuals were observed both in adducts level and repair activities. No differences in epsilonA and epsilonC levels between tumor and nonaffected lung tissues were recorded. However, leukocytes accumulated a significantly higher number of DNA adducts than the lung tissues. Repair activities for both epsilonA and epsilonC were significantly higher in tumor than in normal lung tissue. No significant differences in epsilonA and epsilonC repair activities were associated with age, sex, or smoking habit. However, a significant difference in repair capacity was observed between two histological types of lung cancer, squamous cell carcinoma (SQ) and adenocarcinoma (AD). In individuals suffering from lung AD, epsilonA- and epsilonC-repair activities in normal lung and blood leukocytes were significantly lower than in SQ patients. Moreover, in nonaffected lung tissue of AD patients, the ratio epsilonA/epsilonC adducts was lower than in SQ patients. Differences have also been found between epsilonA and epsilonC repair activities of cancer patients and healthy volunteers. Repair capacity for epsilonA was significantly lower in blood leukocytes of lung cancer patients than in leukocytes of healthy volunteers (P = 0.012). This difference was even larger between healthy volunteers and patients developing inflammation-related AD (P = 0.00033). Repair activities for epsilonC were the same in leukocytes of healthy controls, all lung cancer patients, and SQ patients. However, individuals with ADs revealed significantly lower epsilonC-repair activity (P = 0.013). These results suggest that oxidative stress-mediated lipid peroxidation might contribute to induction and/or progression of lung cancer. Decreased activity of base excision repair pathway for epsilonA and epsilonC is associated particularly with inflammation-related lung AD.     

Inhibition of 2-acetylaminofluorene oxidations by monoclonal antibodies specific to 3-methylcholanthrene-induced rat liver cytochrome P450

The inhibitory effects of ten monoclonal antibodies specificfor methylcholanthrene (MC)-induced P₄₅₀ were determined onboth C- and N-hydroxylations of 2-acetylaminofluorene (AAF)by liver microsomes from rats pretreated with MC, phenobarbitalas well as untreated animals. The inhibitory effects of theseantibodies were also determined on AAF hydroxylations by purifiedrat liver MC-P₄₅₀. The inhibition patterns of AAF hydroxylationswith both microsomes and the purified P₄₅₀ indicate that theformation of 7-OH-, 5-OH-, 3-OH-, 1-OH- and N-OH-AAF was catalyzedby the same unique isoenzyme or at least isoenzymes of commonantigenic determinants.