Synthesis and analysis of DNA adducts of arylamines

Atylamines and nitroarenes are very important environmental and occupational pollutants. Genotoxic effects of arylamines are believed to be initiated by the formation of DNA adducts. DNA adducts of arylamines have been found in experimental animals and in exposed humans, and are predominantly formed with the carbon 8 of 2'-deoxyguanosine. Reference standards are necessary to develop methods for the quantification of DNA-adducts. Therefore, we have synthesized the 2'-deoxyguanosin-8-yI adducts of 2-methylaniline, 2-chloroaniline, 4-chloroaniline, 2,4dimethylaniline, and 2,6-dimethylaniline. The products were characterized by ¹H-NMR, ¹³C-NMR, MS and UV. The corresponding 2'-deoxyguanosine-3' -monophosphate adducts were synthesized for the quantification of DNA adducts by the ³²P-postlabelling technique. A GC-MS method was developed for the analysis of the new adducts as an alternative to the ³²P-postlabelling. DNA was spiked with the synthesized adducts and treated with 0.3 m NaOH overnight at 110 °C in the presence of a deuterated internal standard. We observed up to 80% recovery from about 1 adduct in 10⁸ to 1 in 10⁵ nucleotides.     

Enhanced stimulation of chromosomal translocations by radiomimetic DNA damaging agents and camptothecin in Saccharomyces cerevisiae rad9 checkpoint mutants

Several chemical mutagens and carcinogens, including polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs, are adsorbed to the surface of diesel exhaust particles (DEP). DEP can induce formation of reactive oxygen species and cause oxidative DNA damage as well as bulky carcinogen DNA adducts. Lung tissue is a target organ for DEP induced cancer following inhalation. Recent studies have provided evidence that the lung is also a target organ for DNA damage and cancer after oral exposure to other complex mixtures of PAHs. The genotoxic effect of oral administration of DEP was investigated, in terms of markers of DNA damage, mutations and repair, in the lung of Big Blue^® rats fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. There was no significant increase in the mutation frequency in the cII gene. However, an increase of DNA damage measured as DNA strand breaks (comet assay) and bulky DNA adducts (³²P post labeling) was observed. The level of DNA strand breaks increased significantly at all dose levels while the level of DNA adducts increased significantly only at the intermediate dose levels. Similarly, the number of oxidized DNA bases measured as endonuclease III and fapyguanine glycosylase (FPG) sensitive sites increased at the intermediate dose levels. The induction of DNA damage by DEP exposure did not increase the expression of the repair genes OGG1 and ERCC1 at the mRNA level. The present study indicates that the lung is a target organ for primary DNA damage following oral exposure to DEP. DNA damage was induced following exposure to relatively low levels of DEP, but under the conditions used in the present experiment DNA damage did not result in an increased mutation rate.     

Circulating 4-hydroxynonenal-protein thioether adducts assessed by gas chromatography-mass spectrometry are increased with disease progression and aging in spontaneously hypertensive rats

Oxidative stress has been implicated in numerous degenerative diseases of aging, including heart diseases. However, there is still a need to identify biomarkers of oxidative stress-related events, such as protein modification by the lipid peroxidation product 4-hydroxynonenal (HNE) in these diseases in humans. The objective of this study was to assess if circulating levels of HNE-protein adducts (i) can be assessed with precision by GCMS and (ii) vary with disease progression and aging in a model of cardiomyopathy that displays enhanced oxidative stress, namely the spontaneously hypertensive rats (SHR). We modified a previously published isotope dilution GCMS method that quantifies HNE and its inactive metabolite, 1,4-dihydroxynonene (DHN), bound to thiol proteins following treatment with NaB(2)H(4) and Raney nickel, to increase its sensitivity (20-fold), precision, and robustness. Levels of these adducts were measured in blood and plasma collected from SHR and control Wistar rats at 7, 15, 22, and 30 weeks of age. Levels of protein-bound HNE, which were quantitated with good precision in the nanomolar range in blood, but not in plasma, were significantly increased by disease (SHR) and age (P < 0.0001 for both). Compared to Wistar rats, SHR showed greater blood levels of HNE-protein adducts at 22 and 30 weeks. Levels of protein-bound DHN, which were detected in blood and in plasma, were not affected by disease or age. Collectively, the results of this study conducted in an animal model of cardiomyopathy demonstrate that changes in blood HNE-protein thioether adducts with disease progression and aging can be assessed with good precision by the described GCMS method. This method may prove to be useful in evaluating the occurrence and impact of oxidative stress-related events involving bioactive HNE in heart diseases and aging in humans.     

Carcinogen DNA adducts and the risk of colon cancer: case-control study

Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case-control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by ³²P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 10¹⁰ nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 10¹⁰ nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individual's internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833-15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.     

Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk

The methodology applied for DNA adducts in humans has become more reliable in recent years, allowing to detect even background carcinogenic adduct levels in environmentally exposed persons. Particularly, combinations of the various methods now allow the elucidation of specific adduct structures with detection limits of 1 adduct in 10⁸ unmodified nucleotides or even lower. The quantification of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in human tissues and cells has been achieved with a number of highly sensitive techniques: immunoassays and immunocytochemistry using polyclonal or monoclonal antisera specific for DNA adducts or modified DNA, the ³²P-postlabelling assay, and adduct identification using physicochemical instrumentation. The results summarized in this review show that PAH-DNA adducts have been detected in a variety of human tissues, including target organs of PAH- and tobacco-associated cancers. Although dosimetry has not always been precise, a large number of data now clearly show that lowering exposure to carcinogenic PAH results in decreasing PAH-DNA adduct levels. In most studies, however, bulk DNA of a certain tissue or cell type has been examined, and there were relatively few studies in which mutations as a consequence of DNA damage at specific genes have been investigated. Promising as these biomarker studies seem for epidemiology and health surveillance, future biomonitoring and molecular epidemiological studies should be directed to combine several endpoint measurements: i.e., adduct formation (preferably at specific sites), mutational spectra in cancer-relevant genes, and genetic markers of (cancer) susceptibility in a number of cancer-predisposing genes.     

苦参碱对大鼠乳腺癌细胞荷瘤生长及其炎性因子与免疫功能的影响

目的：探讨苦参碱对大鼠乳腺癌细胞荷瘤大鼠生长及其炎性因子与免疫功能的影响。方法：雌性Wistar大鼠60只,随机分为对照组（n=10）,乳腺癌细胞荷瘤大鼠建模组（n=50）。建模大鼠再随机分为5组（n=10）：模型组、苦参碱低（50 mg/kg）、中（100 mg/kg）、高（200 mg/kg）剂量组和香菇多糖（200 mg/kg）组。除对照组外,每只大鼠右腋皮下接种0.4 ml Walker 256乳腺癌细胞悬液（5×10⁷ cells/ml）。按10 ml/kg的体积腹腔注射给药,2次/日,连续14 d。14 d后麻醉大鼠,腹主动脉取血,取瘤组织称取质量,计算抑瘤率;检测大鼠外周血IL-2、IFN-γ、IL-6、IL-10、TGF-β、CD3⁺、CD4⁺、CD8⁺、IgG、IgM、IgA水平。结果：苦参碱低、中、高剂量组和香菇多糖组大鼠瘤块平均质量分别为（4.99±0.93）g、（4.52±0.92）g、（4.22±1.18）g、（4.52±0.92）g,与模型组（6.62±1.20）g比较均明显降低（P<0.01）。苦参碱各剂量组与香菇多糖组比较,瘤块平均质量无明显差异（P>0.05）。苦参碱低、中、高剂量组和香菇多糖组抑瘤率分别为24.6%、31.7%、36.3%和27.9%,组间无统计学差异。模型组大鼠IL-2、IFN-γ、CD8⁺较对照组明显降低（P<0.01）;而IL-6、IL-10、TGF-β、CD3⁺、CD4⁺、IgG、IgM、IgA较对照组明显升高（P<0.01）。苦参碱低、中、高剂量组和香菇多糖组IL-2、IFN-γ、CD8+较模型组显著升高（P<0.01,P<0.05）;而IL-6、IL-10、TGF-β、CD3⁺、CD4⁺、IgG、IgM、IgA较模型组明显降低（P<0.01,P<0.05）。与香菇多糖组比较,苦参碱低、中剂量组IgM、IgA明显升高（P<0.01,P<0.05）;高剂量组IL-2和IFN-γ、IgA水平明显升高（P<0.01,P<0.05）;低剂量组IFN-γ水平明显降低（P<0.05）;高剂量组IL-10、CD4⁺水平明显降低（P<0.01,P<0.05）。结论：苦参碱具有明显的抑制肿瘤生长的作用,其抑瘤机制与其提高细胞和体液免疫、减轻炎症反应有关。     

Diesel exhaust particles cause increased levels of DNA deletions after transplacental exposure in mice

Diesel exhaust particles (DEP) are a major source of air-borne pollution and are linked to increased risk of disease including lung cancer. Here we investigated effects of exposure to DEP on the frequency of DNA deletions, levels of oxidative DNA damage and DNA adduct formation during embryonic development in mice. Pregnant dams were orally exposed to various doses of DEP (500, 250, 125, 62.5, 31.25 mg/kg/day) at embryonic days 10.5–15.5. We determined the frequency of 70 kb DNA deletions spanning exons 6–18 at the p^un allele that results in black-pigmented spots in the unpigmented retinal pigment epithelium in the eyes of p^un/p^un offspring mice. DEP caused a significant increase in the frequency of DNA deletions. Levels of 8-OH deoxyguanosine indicating oxidative DNA damage were within the limits of the unexposed mouse embryos. ³³P post-labeling analysis revealed very low levels of DNA adducts in the embryo tissue. Thus, transplacental exposure to DEP resulted in a significant increase in the frequency of DNA deletions in the mouse fetus and such genetic alterations in the offspring may have pathological consequences later in life.     

Effects of environmental air pollution on endogenous oxidative DNA damage in humans

Epidemiological studies conducted in metropolitan areas have demonstrated that exposure to environmental air pollution is associated with increases in mortality. Carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) are the major source of genotoxic activities of organic mixtures associated with respirable particulate matter, which is a constituent of environmental air pollution. In this study,we wanted to evaluate the relationship between exposure to these genotoxic compounds present in the air and endogenous oxidative DNA damage in three different human populations exposed to varying levels of environmental air pollution. As measures of oxidative DNA damage we have determined 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and cyclic pyrimidopurinone N-1,N² malondialdehyde-2′-deoxyguanosine (M₁dG) by the immunoslot blot assay from lymphocyte DNA of participating individuals. The level of endogenous oxidative DNA damage was significantly increased in individuals exposed to environmental air pollution compared to unexposed individuals from Kosice (8-oxodG adducts) and Sofia (M₁dG adducts). However, there was no significant difference in the level of endogenous oxidative DNA and exposure to environmental air pollution in individuals from Prague (8-oxodG and M₁dG adducts) and Kosice (M₁dG adducts). The average level of M₁dG adducts was significantly lower in unexposed and exposed individuals from Kosice compared to those from Prague and Sofia. The average level of 8-oxodG adducts was significantly higher in unexposed and exposed individuals from Kosice compared to those from Prague. A significant increasing trend according to the interaction of c-PAHs exposure and smoking status was observed in levels of 8-oxodG adducts in individuals from Kosice. However, no other relationship was observed for M₁dG and 8-oxodG adduct levels with regard to the smoking status and c-PAH exposure status of the individuals. The conclusion that can be made from this study is that environmental air pollution may alter the endogenous oxidative DNA damage levels in humans but the effect appears to be related to the country where the individuals reside. Genetic polymorphisms of the genes involved in metabolism and detoxification and also differences in the DNA repair capacity and antioxidant status of the individuals could be possible explanations for the variation observed in the level of endogenous oxidative DNA damage for the different populations.     

The path from mitochondrial ROS to aging runs through the mitochondrial permeability transition pore

Excessive production of mitochondrial reactive oxygen species (mROS) is strongly associated with mitochondrial and cellular oxidative damage, aging, and degenerative diseases. However, mROS also induces pathways of protection of mitochondria that slow aging, inhibit cell death, and increase lifespan. Recent studies show that the activation of the mitochondrial permeability transition pore (mPTP), which is triggered by mROS and mitochondrial calcium overloading, is enhanced in aged animals and humans and in aging‐related degenerative diseases. mPTP opening initiates further production and release of mROS that damage both mitochondrial and nuclear DNA, proteins, and phospholipids, and also releases matrix NAD that is hydrolyzed in the intermembrane space, thus contributing to the depletion of cellular NAD that accelerates aging. Oxidative damage to calcium transporters leads to calcium overload and more frequent opening of mPTP. Because aging enhances the opening of the mPTP and mPTP opening accelerates aging, we suggest that mPTP opening drives the progression of aging. Activation of the mPTP is regulated, directly and indirectly, not only by the mitochondrial protection pathways that are induced by mROS, but also by pro‐apoptotic signals that are induced by DNA damage. We suggest that the integration of these contrasting signals by the mPTP largely determines the rate of cell aging and the initiation of cell death, and thus animal lifespan. The suggestion that the control of mPTP activation is critical for the progression of aging can explain the conflicting and confusing evidence regarding the beneficial and deleterious effects of mROS on health and lifespan.     

DNA damage and repair in gastric cancer--a correlation with the hOGG1 and RAD51 genes polymorphisms

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by ³²P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N⁶-yl]-aristolactam I, 7-[deoxyadenosin-N⁶-yl]-aristolactam II and 7-[deoxyguanosin-N²-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10⁸ nucleotides in liver and 95–4598 adducts/10⁸ nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 × 10⁻⁶ in liver compared with the MFs of 78–1319 × 10⁻⁶ that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.