A sensitive immunoslot-blot assay for detection of malondialdehyde-deoxyguanosine in human DNA

As part of a large programme on food risk assessment, we have become Interested in the endogenous production of genotoxic agents from dietary precursors. Malondialdehyde (MDA), a product of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in bacterial and mammalian systems. MDA reacts with DNA, and the major adduct (M1-dG) has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to large numbers of individuals or a large variety of samples. Often, only a few micrograms of DNA from human tissues are available for analysis, and a very sensitive assay is needed to detect background levels of M1-dG in very small amounts of DNA. In this paper, we describe the development of an immunoslot-blot (ISB) assay for the measurement of M1-dG in 1 microgram of DNA. The limit of detection of the assay is about 5 adducts per 10(8) bases. The advantages of ISB over other assays for DNA adduct detection, such as the possibility of analysing 1 microgram DNA per sample and the fact that it is less time-consuming and laborious, mean that it can be more easily used for routine analysis of large numbers of samples in biomonitoring. A series of human samples was analysed, and levels of 0.3-6.43 M1-dG per 10(7) normal bases were detected in 42 gastric biopsy samples and 0.7-16.65 M1-dG per 10(7) normal bases in 28 samples of leukocyte DNA. In an initial study in five human volunteers on standardized diets, the levels of M1-dG in leukocyte DNA changed in relation to meat, vegetable and tea intake.     

Detection of malondialdehyde DNA adducts in human colorectal mucosa: relationship with diet and the presence of adenomas.

Colorectal biopsies from normal mucosa of participants in the United Kingdom Flexible Sigmoidoscopy Trial and European Prospective Investigation on Cancer (EPIC; n = 162) were analyzed for the presence of malondialdehyde-deoxyguanosine (M(1)-dG), a DNA adduct derived from lipid peroxidation. The aim was to investigate whether dietary factors can modulate M(1)-dG levels and whether M(1)-dG in normal mucosa is a risk factor for colorectal adenomas. Samples were analyzed using a sensitive immunoblot blot assay. This study has shown for the first time that M(1)-dG is present in human colorectal tissue. M(1)-dG levels ranged from undetectable (n = 13) to 12.23 per 10(7) total bases. Mean levels were 4.3 +/- 3 and 4.6 +/- 2.9 per 10(7) total bases in men and women, respectively. In men, there were positive associations of adduct levels with height and age, and inverse associations with body mass index. Legumes, fruit, salad, and whole meal bread were inversely associated with M(1)-dG adducts, whereas consumption of offal, white meat, beer, and alcohol were positively associated with elevated levels. In women, there was an inverse association of the adduct with the ratio of polyunsaturated:saturated fatty acids (P = 0.019) and a weak positive correlation with saturated fat (P < 0.061). When levels of adducts were compared in individuals with and without adenomas, there was a trend for higher levels in individuals presenting with adenomas especially in the highest category of M(1)-dG adducts (P < 0.005).     

Levels of malondialdehyde-deoxyguanosine in the gastric mucosa: relationship with lipid peroxidation, ascorbic acid, and Helicobacter pylori.

Helicobacter pylori infection is associated with elevated gastric mucosal concentrations of the lipid peroxidation product malondialdehyde and reduced gastric juice vitamin C concentrations. Malondialdehyde can react with DNA bases to form the mutagenic adduct malondialdehyde-deoxyguanosine (M(1)-dG). We aimed to determine gastric mucosal levels of M(1)-dG in relation to H. pylori infection and malondialdehyde and vitamin C concentrations. Patients (n = 124) attending for endoscopy were studied. Levels of antral mucosal M(1)-dG were determined using a sensitive immunoslot-blot technique; antral mucosal malondialdehyde was determined by thiobarbituric acid extraction, and gastric juice and antral mucosal ascorbic acid and total vitamin C were determined by high-performance liquid chromatography. Sixty-four H. pylori-positive patients received eradication therapy, and endoscopy was repeated at 6 and 12 months. Levels of M(1)-dG did not differ between subjects with H. pylori gastritis (n = 85) and those with normal mucosa without H. pylori infection (n = 39; 56.6 versus 60.1 adducts/10(8) bases) and were unaffected by age or smoking habits. Malondialdehyde levels were higher (123.7 versus 82.5 pmol/g; P < 0.001), gastric juice ascorbic acid was lower (5.7 versus 15.0 micromol/ml; P < 0.001), and antral mucosal ascorbic acid was unchanged (48.0 versus 42.7 micromol/g) in H. pylori gastritis compared with normal mucosa. Multiple regression analysis revealed that M(1)-dG increased significantly with increasing levels of malondialdehyde, antral ascorbic acid, and total antral vitamin C. M(1)-dG levels were unchanged 6 months (63.3 versus 87.0 adducts/10(8) bases; P = 0.24; n = 38) and 12 months (66.7 versus 77.5 adducts/10(8) bases; P = 0.8; n = 13) after successful eradication of H. pylori. M(1)-dG thus is detectable in gastric mucosa, but is not affected directly by H. pylori.     

Comparative analysis of 8-oxo-2' -deoxyguanosine in DNA by 32P- and 33P- postlabeling and electrochemical detection

8-Oxo-2'-deoxyguanosine (8-oxo-dG) is emerging as a useful marker for oxidative DNA damage. Reported basal levels determined by 32P- postlabeling (PPL) method were 10-fold or more higher than those obtained with HPLC/electrochemical detection (ECD). This discrepancy was investigated. In commercial calf thymus DNA, levels of 4 +/- 1 and 64 +/- 14 8-oxo-dG per 10(6) 2'-deoxynucleosides (dN) were measured by the standard HPLC/ECD and PPL methods, respectively. DNA digestion by micrococcal nuclease/spleen phosphodiesterase and nuclease P1 (as used in the standard PPL method), followed by ECD analysis resulted in a level of 8 +/- 3. In calf thymus DNA spiked with chemically synthesized 8-oxo-dGp to give an increment of 9 8-oxo-dG/10(6) dN, the added standard produced a significant increase with HPLC/ECD but not PPL. After spiking the DNA with 90 8-oxo-dG/10(6) dN, the added 8-oxo-dGp was detectable also with PPL, with a labeling efficiency of 65%. In order to investigate the role of ionizing radiation from 32P for the higher 8-oxo-dG levels in PPL, incubation times and amounts of radioactivity in the phosphorylation reaction with commercial dGp were increased, and external irradiation of commercial dG with 32P was investigated. All modifications resulted in higher values of 8-oxo-dG measured, but the effect was not large enough to fully explain the discrepancy between PPL and HPLC/ECD. Using [gamma-33P]ATP instead of [gamma-32P]ATP or adding [33P]phosphate to a 32P-PPL assay resulted in even higher levels of 8-oxo-dG measured. The increase in 8-oxo-dG levels during the PPL workup is attributed to the presence and oxidation of unmodified dGp in the reaction mixture. For a determination of true basal levels, the PPL method will have to be modified, including the removal of dGp prior to the phosphorylation reaction.      

Expression of cytochromes P450 and glutathione S-transferases in human prostate, and the potential for activation of heterocyclic amine carcinogens via acetyl-coA-, PAPS- and ATP-dependent pathways

Dietary factors appear to be involved in the high incidence of prostate cancer in "Westernized" countries, implicating dietary carcinogens such as heterocyclic amines (HAs) in the initiation of prostate carcinogenesis. We examined 24 human prostate samples with respect to their potential for activation and detoxification of HAs and the presence of DNA adducts formed in vivo. Cytochromes P450 1B1, 3A4 and 3A5 were expressed at low levels (<0.1-6.2 pmol/mg microsomal protein). N-Acetyltransferase (NAT) activities, using p-aminobenzoic acid (NAT1) and sulfamethazine (NAT2) as substrates, were <5-5,500 and <5-43 pmol/min/mg cytosolic protein, respectively. Glutathione S-transferases (GSTs) P1, M2 and M3 were expressed at 0.038-1.284, 0.005-0.126 and 0.010-0.270 microg/mg cytosolic protein, respectively; GSTM1 was expressed in all GSTM1-positive samples (0.012-0.291 microg/mg cytosolic protein); and GSTA1 was expressed at low levels (<0.01-0.11 microg/mg cytosolic protein). Binding of N-hydroxy-PhIP to DNA in vitro occurred primarily by an AcCoA-dependent process (<1-54 pmol/mg/DNA), PAPS- and ATP-dependent binding being <1-7 pmol/mg DNA. In vivo, putative PhIP- or 4-aminobiphenyl-DNA adducts were found in 4 samples (0.4-0.8 adducts/10(8) bases); putative hydrophobic adducts were found in 6 samples (8-64 adducts/10(8) bases). Thus, the prostate appears to have low potential for N-hydroxylation of HAs but greater potential for activation of N-hydroxy HAs to genotoxic N-acetoxy esters. The prostate has potential for GSTP1-dependent detoxification of ATP-activated N-hydroxy-PhIP but little potential for detoxification of N-acetoxy-PhIP by GSTA1. However, there were no significant correlations between expression/activities and DNA adducts formed in vitro or in vivo, DNA adducts in vivo possibly reflecting carcinogen exposure.     

High DNA damage by benzo[a]pyrene 7,8-diol-9,10-epoxide in bronchial epithelial cells from patients with lung cancer: comparison with lung parenchyma

In the present study, the level of benzo[a]pyrene 7,8-diol-9,10-epoxide-N(2)-deoxyguanosine (BPDE-N(2)-dG) in normal bronchial epithelial cells from non-cancerous bronchus of 22 lung cancer subjects was evaluated and compared to the lung parenchyma. We found very high formation of BPDE-N(2)-dG adduct in samples corresponding to a pure preparation of bronchial epithelial cells with 4-fold interindividual differences in the DNA adduct levels in the range of 36.5-175.4 BPDE-N(2)-dG adducts/10(8) nucleotides in smokers (mean: 84.7+/-38.4; n = 13) and 3-fold differences in the range of 19.7-62.4 in non-smokers (mean: 37.6+/-22.2; n = 3). DNA isolated from the bronchial tissue consisting of bronchial lining epithelium with adjacent lamina propria showed significantly lower BPDE-N(2)-dG formation (P < 0.001) in the range of 0.4-4.2 BPDE-N(2)-dG adducts/10(8) nucleotides (mean: 1.8+/-0.56; n = 6). This difference is clearly related to the procedure used to prepare the bronchial tissue samples leading to the presence of different types of cells. Eight samples from the normal parenchyma did not show measurable adducts, the other 14 samples showed 50-fold variation (mean: 1.7+/-1.5; range 0.1-5.2 adducts/10(8) nucleotides; n = 14). There were considerably higher adduct levels in pure bronchial epithelial cells than in parenchymal tissue (75.8+/-38.8 vs 0.9+/-1.5 adducts/10(8) nucleotides) (P < 0.0002) BPDE-N(2)-dG adduct concentrate almost exclusively in bronchial epithelial cells. The adduct values obtained in bronchial epithelial cells could be considered as 'critical' for the initiation of human lung cancer. The high formation of BPDE-N(2)-dG adducts in bronchial epithelial cells and investigations showing that the profile of mutations induced by BPDE in these cells is similar to that seen in the p53 gene isolated from human lung tumors implicates benzo[a]pyrene as important carcinogen in tobacco-induced lung cancer in human beings.     

Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients

We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.     

Comparison between high-performance liquid chromatography and enzyme-linked immunosorbent assay for the determination of 8-hydroxy-2'-deoxyguanosine in human urine.

8-Hydroxy-2'-deoxyguanosine (8-OH-dG), which has been regarded as a potential marker of oxidative DNA damage induced by reactive oxygen species, was measured in human urine by a commercial ELISA using a monoclonal antibody N45.1 and by high-performance liquid chromatography (HPLC) coupled to an electrochemical detector (HPLC-ECD) to evaluate whether the ELISA system is applicable to human monitoring studies. The urine samples were collected from 120 healthy men ages 18-58 in a steel-manufacturing company. A good correlation (r = 0.833; P < 0.0001) was observed between the two methods on HPLC-purified 8-OH-dG fractions from 23 urine samples. The mean value (+/-SD) of 8-OH-dG (microg/g creatinine) was 5.47 (+/-2.97) by HPLC-ECD assay and 5.50 (+/-2.36) by ELISA. However, the correlation (r) between the two methods on 120 original urine samples was 0.460 [P < 0.001; mean value (+/-SD) of 8-OH-dG (microg/g creatinine) was 4.46 (+/-2.03) by the HPLC assay and 9.33 (+/-3.23) by ELISA]. ELISA estimates were about 2-fold higher than the HPLC estimates on original urine. For an unknown reason, 10% of the urine samples showed more than a 4-fold increase in value by ELISA. It is suggested that the ELISA system is applicable for comparative human monitoring studies. Prepurification of 'samples is required to determine the absolute value of 8-OH-dG in individual urine samples by ELISA.     

Quantitative immunoanalysis of promutagenic 8-hydroxy-2'-deoxyguanosine in oxidized DNA

The modified DNA base 8-hydroxyguanine has been implicated inspontaneous mutagenesis, carcinogenesis and cellular aging.Polyclonal antibodies specific for the 8-hydroxy-2'-deoxyguanosinemoiety in oxidized DNA were used for sensitive detection andquantitation of this biomarker of oxidative damage to cellularDNA. The analysis was performed with immunoslot blot assay (ISB)of oxidized DNA modified in vitro with methylene blue plus lightand upon H₂O₂ treatment of cultured human cells. The level of8-OHdG in DNA exposed to 90 and 120 min light in the presenceof 100 µM methylene blue showed 15.96 ± 2.4 and22.65 ± 3.65 pmol/µg DNA compared to 0.107 ±0.024 pmol/µg in commercial calf thymus DNA control. Inherentdamage, due to cellular endogenous oxidation of DNA, increasedsignificantly upon inhibition of catalase activity in humancells with 10 mM azide. The damage increased further on exposureof azide-treated cells to H₂O₂. The amounts of 8-OHdG followingtreatment of cells with 10 and 100 µM H₂O₂ were determinedto be 205 ± 42 and 333 ± 17.5 pmol/µg DNArespectively. Very low but quantifiable antibody binding wasseen with the ‘control unoxidized’ human nuclearDNA. This DNA, obtained under controlled conditions to restrictthe induction of 8-OHdG during isolation, provides a backgroundlevel of 0.022 ± 0.005 pmol 8-OHdG/µg DNA. Thequantitative assessment of 8-OHdG by ISB assay, with fmol sensitivityand direct analysis using unhydrolyzed DNA, should prove a highlyvaluable alternative to currently used approaches to detecting8-OHdG in enzymatic DNA hydrolysates.     

Heterocyclic aromatic amines induce DNA strand breaks and cell transformation

Heterocyclic aromatic amines (HAAs), formed during the cooking of foods, are known to induce tumours in rodent bioassays and may thus contribute to human cancer risk. We tested six HAAs in a morphological transformation assay and in three in vitro genotoxicity assays. The morphological transforming abilities of HAAs were tested, in the presence of rat-liver S9, in the C3H/M2 fibroblast cell line. Concentration levels of 50 microM 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 100 microM 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (4,8-DiMeIQx), 50 microM 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 100 microM 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 100 microM 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 15 microM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced maximum transformation potencies of 5.5, 6.6, 6.3, 5.2, 7.3 and 9.2 transformed foci per 10(4) surviving cells, respectively. Bacterial mutagenic activity was determined in the presence of rat-liver S9 using the Salmonella typhimurium reverse-mutation assay employing strain YG1019. Mutagenic potencies of 3800 revertants (revs)/ng with 8-MeIQx, 2900 revs/ng with 4,8-DiMeIQx, 3480 revs/ng with IQ, 1.6 revs/ng with AalphaC, 2.9 revs/ng with MeAalphaC and 5 revs/ng with PhIP were observed. Clastogenic activity in vitro was analysed by the micronucleus assay in metabolically competent MCL-5 cells. Dose-dependent induction of micronuclei was observed for all HAAs tested with 1-5.4% of cells containing micronuclei at 10 ng/ml. Micronucleus induction was in the order 4,8-DiMeIQx > 8-MeIQx > IQ > MeAalphaC > PhIP > AalphaC. DNA strand-breaking activity in MCL-5 cells was measured by the alkaline single cell-gel (comet) assay. The lowest effect doses for significant increases (P < or = 0.0007, Mann-Whitney test) in comet tail length (microm) were 45.5 microg/ml (200 microM) for PhIP, 90.9 microg/ml (410-510 microM) for 4,8-DiMeIQx, IQ, MeAalphaC and AalphaC, and 454.5 microg/ml (2130 microM) for 8-MeIQx. It is not yet clear which of these assays most accurately reflects the genotoxic potential to humans of compounds of this class of environmental carcinogens.     

Oxidative stress in humans: validation of biomarkers of DNA damage

Two studies have been performed to clarify the relationship between different markers of oxidative DNA damage commonly employed in molecular epidemiological studies. In the first, 8-Oxo-7,8-dihydroguanine (8-oxoGua) was induced in DNA of HeLa cells by treatment with different concentrations of photosensitizer Ro 19-8022 together with visible light. 8-OxoGua was estimated by the comet assay (alkaline single cell gel electrophoresis) with formamidopyrimidine DNA glycosylase and by HPLC with electrochemical detection. The dose-response curves indicate that the comet assay and HPLC are equally efficient at detecting induced damage. Background levels of 8-oxoGua in HeLa cells were 0.92 +/- 0.22 per 10(6) guanines by the comet assay and 2.09 +/- 0.13 per 10(6) guanines by HPLC. The second study was a small human trial, in which lymphocytes were collected for analysis of background levels of 8-oxoGua, as well as overnight and 24 h urine samples for measurement of excreted 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) by ELISA. The mean level of 8-oxoGua in lymphocytes was determined as 1.33 +/- 0.21 per 10(6) guanines by the comet assay and 3.72 +/- 1.06 per 10(6) guanines by HPLC. A strong correlation was seen between overnight and 24 h urinary 8-oxodGuo (r = 0.93, P < 0.01). Overnight urinary 8-oxodGuo concentrations correlated with 8-oxoGua in lymphocytes measured by HPLC (r = 0.85, P < 0.05) or by the comet assay (r = 0.86, P < 0.05), although individual values from HPLC and the comet assay did not correlate with each other. It is reasonable to assess oxidative stress by any of these methods.     

Immunological detection and visualization of 8-methoxypsoralen-DNA photoadducts

Monoclonal antibodies specific for DNA damaged by 8-methoxypsoralen (8-MOP) plus ultraviolet A (UVA) light were used to study adduct formation in human keratinocytes and mouse and rat skin in vivo. This antibody does not cross-react with nonmodified DNA or free 8-MOP. Sensitive competitive enzyme-linked immunosorbent assays with color or fluorescence endpoints were used to quantitate adducts on DNA isolated from treated keratinocytes or skin samples. Localization of 8-MOP-DNA adducts was studied by indirect immunofluorescence with fluorescein-conjugated anti-mouse-IgG antibodies. When cultured keratinocytes were treated with 8-MOP and UVA, immunofluorescence was localized in the nucleus. There was no fluorescence in untreated control cells or treated cells incubated with nonspecific serum. Comparison of intensity of immunofluorescence staining with quantitation of adduct levels by enzyme-linked immunosorbent assay indicated that the limit of sensitivity of the immunofluorescence technique is 9.0 fmol adduct/micrograms DNA or 2.9 adducts/10(6) nucleotides.     

Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts: validation by comparison with other methods,and use in human biomonitoring

A chemiluminescence immunoassay (CIA) utilizing antiserum elicited against DNA modified with (+/-)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]- pyrene (BPDE) has been developed and validated to study the formation of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human tissues. Advantages include a low limit of detection for 10b-(deoxyguanosin-N(2)-yl)-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG, approximately 1.5 adducts/10(9) nucleotides using 20 micro g DNA) and a high signal-to-noise ratio (> or =100). The CIA BPDE-DNA standard curve gave 50% inhibition at 0.60 +/- 0.08 fmol BPdG (mean +/- SE, n = 30), which was a 10-fold increase in sensitivity compared with the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Calf thymus DNA modified with [1,3-(3)H]BPDE was assayed by radiolabeling, (32)P-postlabeling, DELFIA and CIA, and all assays gave similar values. Liver DNAs from mice exposed to 0.5 and 1.0 mg [7,8-(3)H]benzo[a]pyrene (BP) were assayed by the same four assays and a dose-response was obtained with all assays. The BPDE-DNA CIA was further validated in MCL-5 cells exposed to 4 micro M BP for 24 h, where nuclear and mitochondrial DNA adduct levels were associated with an increase in DNA tail length measured by the Comet assay. Human peripheral blood cell (buffy coat) DNA samples (n = 43) obtained from 25 individuals who were either colorectal adenocarcinoma patients or controls were assayed by BPDE-DNA CIA. Three samples (7%) were non-detectable, and the remaining 40 samples had values between 0.71 and 2.21 PAH-DNA adducts/10(8) nucleotides. The intra-assay coefficient of variation (CV), for four wells on the same microtiter plate, was 1.85%. Sufficient DNA for two assays, on separate plates, was available for 38 of the 43 samples, and the PAH-DNA adduct values obtained were highly correlated (r(2) = 0.95). Coded duplicate DNA samples from 15 individuals were assayed four times gave an inter-assay CV of 13.8%.     

Aflatoxin B1 formamidopyrimidine adducts are preferentially repaired by the nucleotide excision repair pathway in vivo

Aflatoxin B(1) (AFB(1)), the most potent member of the aflatoxin family of hepatocarcinogens, upon metabolic activation reacts with DNA and forms a population of covalent adducts. The most prevalent adduct, 8,9-dihydro-8-(N(7)-guanyl-)-9-hydroxyaflatoxin (AFB(1)-N(7)-dG), as well as the AFB(1) formamidopyrimidine adduct (AFB(1)-FAPY), resulting from imidazole ring opening of the major adduct, are thought to be responsible for mutations caused by AFB(1). The AFB(1)-N(7)-dG lesions are rapidly removed in Escherichia coli and mammals, whereas the AFB(1)-FAPY lesions persist in mammalian cells, which along with the higher stability of this lesion suggests that AFB(1)-FAPY might significantly contribute to the observed toxicity and carcinogenicity of AFB(1) in higher organisms. Other workers have shown in vitro evidence that AFB(1)-FAPY lesions are substrates for both nucleotide excision repair (NER) and base excision repair (BER). The present study, done in vivo, utilized a modified host cell reactivation assay and showed that AFB(1)-FAPY lesions are preferentially repaired in E.coli by NER. Comparisons of repair in wild-type, NER-deficient (uvrA), BER-deficient (mutM) and NER/BER double mutant E.coli strains transformed with plasmids enriched for AFB(1)-N(7)-dG or AFB(1)-FAPY lesions indicate that both lesions are efficiently repaired by NER-proficient cells (both wild-type and BER-deficient strains). We have found that the level of activity of the reporter gene is significantly affected by the presence of either lesion in NER-deficient strains due to the lack of repair. This effect is similar in NER-deficient and NER/BER-deficient strains indicating that BER (specifically in the strains we investigated) does not contribute significantly to the repair of these lesions in vivo. Consistent with this finding, in vitro analysis of AFB(1)-FAPY adduct excision by purified MutM and its functional analog human 8-oxoguanine DNA glycosylase using site-specifically modified oligonucleotides indicates that this lesion is a poor substrate for both proteins compared with canonical substrates for these enzymes, such as 7,8-dihydro-8-oxoguanine and methylformamidopyrimidine.     

正常食管上皮和食管鳞状细胞癌组织中丙二醛-DNA加合物含量

目的 研究正常食管上皮和食管癌组织中致癌性丙二醛－DNA加合物（M1－dG)含量,探讨DNA氧化损伤与食管癌发生和发展的关系。方法 所有组织标本均来自食管癌高发区河南林县,32例正常食管上皮标本来源于组织活检,30例食管癌组织标本来源于外科手术切除的食管癌。DNA中M1－dG加合物含量以^32P-后标记方法测定。结果 在全部正常食管上皮和癌组织DNA中均检测到M1－dG加合物,但其含量（中位数）在正常食管上皮中为3．4／10^8核苷酸（范围为1．7／10^8-55.4/10^8核苷酸）,远低于食管癌组织的14.1/10^8核苷酸（范围为1.4/10^8-59.0/10^8核苷酸）,差异有极显著性（P＜0．0001）。该加合物水平与受试者的性别、年龄、吸烟状态以及涉及酒精氧化产生自由基的细胞色素p4502E1基因多态性无明显相关。结论 脂质过氧化产生的丙二醛对DNA的损伤可在食管上皮中累积,在癌组织中达相当高的水平,提示M1－dG加合物可能是导致食管癌发生和发展的重要因素。     

A multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechanisms

We investigated the effects of smoking-induced oxidative stress in healthy volunteers (21 smokers versus 24 non-smokers) by quantifying various markers of oxidative DNA damage and repair, and antioxidative defense mechanisms. Lymphocytic 7-hydroxy-8-oxo-2'-deoxyguanosine (8-oxo-dG) levels measured by high performance liquid chromatography with electrochemical detection, were significantly lower in smokers as compared with non-smokers (38.6 +/- 5.2 versus 50.9 +/- 4.6/10(6) dG, P = 0.05). The levels of oxidized pyrimidine bases in lymphocytes of smokers quantified by the endonuclease III-modified comet assay were non-significantly lower than those of non-smokers (% DNA in tail: 13 +/- 3 versus 14 +/- 2; tail length: 69 +/- 13 versus 96 +/- 10; tail moment: 6416 +/- 1220 versus 7545 +/- 1234). Urinary excretion levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assessed by enzyme-linked immunosorbent assay did not differ significantly between smokers and non-smokers (197 +/- 31 versus 240 +/- 33 ng/body mass index, P = 0.3). Overall DNA repair activity expressed as unscheduled DNA synthesis in blood leukocytes, was not significantly different between smokers and non-smokers (2.9 +/- 0.3 versus 3.3 +/- 0.3, P = 0.4). Plasma antioxidative capacity measured by the Trolox equivalent antioxidant capacity assay was slightly higher in smokers as compared with non-smokers (440 +/- 16 versus 400 +/- 15 microM Trolox equivalent, P = 0.09), and it was significantly related to lymphocytic 8-oxo-dG levels (r = 0.4, P = 0.001). Genotyping of human 8-OH-dG glycosylase/apurinic lyase and glutathione S-transferase M1 showed that a polymorphism in either or both of the two genes does not affect any of the quantified biomarkers. We conclude that oxidative stress imposed by cigarette smoking has a low impact upon certain pathways involved in DNA damage and the antioxidative defense system.     

Improved high-performance liquid chromatography assay of doxorubicin: Detection of circulating aglycones in human plasma and comparison with thin-layer chromatography

Summary We compared doxorubicin and metabolite pharmacokinetic data obtained from thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) assay of plasma samples from six patients who had been treated with doxorubicin. Duplicate 1-ml samples were extracted with chloroform: isopropanol (1:1) and assayed using a sensitive HPLC system incorporating a dual pump gradient with tetrahydrofuran as the mobile phase and fluorescence detection. Duplicate 1-ml samples from the same specimens were assayed using a modification of a previously described TLC assay. Areas under the curve for doxorubicin by HPLC (3.36±2.30 M · h) and TLC (4.16±2.50 M · h) were not significantly different (P=0.5). Terminal half-life of doxorubicin by HPLC (28.0±6.98 h) and TLC (23.2±7.8) (P=0.29) and the calculated total-body clearances by HPLC (0.55±0.29 l/min) and TLC (0.45±0.23) (P=0.55) were not significantly different. Areas under the curve for doxorubicinol by HPLC (2.75±1.4 M · h) and TLC (2.53±7.1 M · h) (P=0.73) showed no significant differences. HPLC detected a mixed 7-deoxydoxorubicinol aglycone-doxorubicin aglycone peak, 7-deoxydoxorubicin aglycone, and two nonpolar, unidentified metabolites. TLC detected the following aglycone metabolites: doxorubicin aglycone, doxorubicinol aglycone, 7-deoxydoxorubicinol aglycone, an unidentified polar metabolite, and several unidentified nonpolar metabolites. From these data we conclude that HPLC and TLC detect concentrations of doxorubicin and doxorubicinol from human plasma equally well to concentrations of 7.0 nM (4 pmol injected doxorubicin). Aglycones do circulate in human plasma at concentrations above the detection limits of both assays. Doxorubicinol aglycone, which is detected by TLC but not by HPLC, may be formed from artifactual breakdown of doxorubicinol during TLC development. Unidentified nonpolar compounds seen on HPLC and TLC may represent further doxorubicin metabolism than previously described.     

Induction of DNA adducts and mutations in spleen, liver and lung of XPA- deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild- type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA- deficient mice.      

Lack of evidence for tamoxifen- and toremifene-DNA adducts in lymphocytes of treated patients

Tamoxifen (TAM) is used for the adjuvant treatment of women with breast cancer and has also been recommended as a chemopreventive agent. Among unwanted side effects, TAM was shown to increase endometrial cancer in treated women by mechanisms that are not yet clearly understood. We studied DNA adducts in lymphocytes of female breast cancer patients treated with TAM or toremifene (TOR), a TAM analogue and compared them with adducts formed by TAM in rat liver, where the drug induces tumours. DNA adducts were measured by TLC-(32)P-post-labelling assays. After TLC, all DNA samples including DNA from untreated healthy women showed a faint radioactive zone, where the positive control DNA adducts isolated from the liver of rats treated with TAM migrated. The relative adduct levels were calculated from the radioactivity present in this zone. Means +/- SD of adduct levels per 10(8) nucleotides (associated with this area) were for untreated volunteers (control) 1.83 +/- 1.41 (n = 13), for TAM treatment 2.17 +/- 3.04 (n = 25) and for TOR treatment 1.18 +/- 1.05 (n = 8). Most of the human samples were further analysed by HPLC after labelling with (32)P in order to compare adducts in human DNA with those in liver DNA isolated from TAM-treated rats. None of the human samples showed any peaks at retention times where putative TAM-DNA adducts were eluted. In conclusion, lymphocyte DNA from female patients treated at therapeutic levels did not show evidence of the formation of TAM- or TOR-DNA adducts.