Deoxyribonucleic Acid Adducts Research Articles

Heterodinuclear Ru-Pt Complexes Bridged with 2,3-Bis(pyridyl)pyrazinyl Ligands: Studies on Kinetics, Deoxyribonucleic Acid/Bovine Serum Albumin Binding and Cleavage, In Vitro Cytotoxicity, and In Vivo Toxicity on Zebrafish Embryo Activities.

Di- and poly-homo/heteronuclear complexes have great potential as anticancer drugs. Here, we report their reactivity, deoxyribonucleic acid (DNA)/bovine serum albumin (BSA) binding and cleavage interactions, in vitro cytotoxicity, and in vivo zebrafish embryo toxicity of [(phen)2Ru(μ-L)PtCl2]2+ (phen = 1,10-phenanthroline and L = 2,3-bis(2-pyridyl)pyrazine, bpp, C1; 2,3-bis(2-pyridyl)quinoxaline, bpq, C2ial; 2,3-bis(2-pyridyl)benzo[g]quinoxaline, bbq, C3) anticancer prodrugs. The substitution reactivity increases from C1 to C3 owing to an increase in the π-conjugation on the bridging chelate which facilitates π-back bonding. As a result, the electrophilicity index on the C3 complex increases than that on the complex C2 followed by C1 which leads to higher rates of substitution and thus the reactivity order follows C1 < C2 < C3. The coordination of Ru at one end of each of the complexes enhances water solubility. Moreover, the charge addition of the two metal ions increases their reactivity toward substitution in addition to ensuring electrostatic interactions at target sites such as the DNA/BSA. Spectroscopic (UV–vis absorption and fluorescence quenching) titration and viscosity measurement results of the interactions of C1/2/3 with CT-DNA established the formation of stable, nonconvent C1/2/3-DNA adducts with DNA most likely via the intercalative binding mode. Furthermore, studies with BSA showed a good binding affinity of these complexes owing to hydrophobic interactions with the coordinated ligands. The interactions of these complexes with DNA/BSA are in line with the reactivity trend, and all these experimental findings were further supported by molecular docking analysis. In vitro MTT cytotoxic activities on human breast cancer cell line MCF-7 revealed that all the complexes have high cytotoxicity activity (IC50 > 9 μM); furthermore, the selectivity index and SI values were higher (>3). Complex C3 showed the highest cytotoxicity with IC50 = 3.1 μM and SI value (5.55) against MCF7 cell lines and these values were comparable to those of the cisplatin (IC50 and SI values are 5.0 μM and 4.02, respectively). In vivo toxicological assessments on zebrafish embryos revealed that all the Ru–Pt complexes (CI/2/3) have poor embryo acute toxic effects over 96 h postfertilization, hpf with LC50 > 65.2 μM. The complex C3 has shown the lowest embryo toxicity (LC50 = 148.8 μM), which is comparable to that of commercial cisplatin (LC50 = 181.1 μM). Based on the cytotoxicity results, complexes C2 and C3 could be considered for further development as chemotherapeutic agents against MCF breast cancer cells.

Advances in Our Understanding of the Molecular Mechanisms of Action of Cisplatin in Cancer Therapy.

Cisplatin and other platinum-based chemotherapeutic drugs have been used extensively for the treatment of human cancers such as bladder, blood, breast, cervical, esophageal, head and neck, lung, ovarian, testicular cancers, and sarcoma. Cisplatin is commonly administered intravenously as a first-line chemotherapy for patients suffering from various malignancies. Upon absorption into the cancer cell, cisplatin interacts with cellular macromolecules and exerts its cytotoxic effects through a series of biochemical mechanisms by binding to Deoxyribonucleic acid (DNA) and forming intra-strand DNA adducts leading to the inhibition of DNA synthesis and cell growth. Its primary molecular mechanism of action has been associated with the induction of both intrinsic and extrinsic pathways of apoptosis resulting from the production of reactive oxygen species through lipid peroxidation, activation of various signal transduction pathways, induction of p53 signaling and cell cycle arrest, upregulation of pro-apoptotic genes/proteins, and down-regulation of proto-oncogenes and anti-apoptotic genes/proteins. Despite great clinical outcomes, many studies have reported substantial side effects associated with cisplatin monotherapy, while others have shown substantial drug resistance in some cancer patients. Hence, new formulations and several combinational therapies with other drugs have been tested for the purpose of improving the clinical utility of cisplatin. Therefore, this review provides a comprehensive understanding of its molecular mechanisms of action in cancer therapy and discusses the therapeutic approaches to overcome cisplatin resistance and side effects.

Scientific rationale of hospital discharge as a sustainable source of irrigation water: Detection, phytological assessment and toxicity verification

Hospital discharge (HD) represents the primary point source of pharmaceutical components that may constitute not only a sharp risk of phytotoxic effects in food crops, but leaving behind the toxicity implications to the environment. The present work has been oriented to the scientific assessment of pharmaceutical compounds in HD. The phenological, physiological, and morphological changes, oxidative stress biomarkers, antioxidative responses, bioaccumulation and bioconcentration factor in relation to different concentrations of HD-induced irrigation in Ipomoea aquatica were evaluated; while the cytotoxicity and genotoxicity on human hepatocellular carcinoma (HepG2) cell were assessed. Acetaminophen, metoprolol, atenolol, enalapril, and metformin were predominantly detected in the raw HD and bioaccumulated in I. aquatica. Concentration-dependent phenological inhibition, chlorophylls reductions, morphological distortions, reactive oxygen species generation, and antioxidant enzymes upregulation were recorded in the HD-irrigated I. aquatica. Drastic reductions in cell viability and increase of tail damage and tail moment indicated the metabolic alterations of mitochondrial function, and deoxyribonucleic acid (DNA) damage via oxidative stress and DNA adducts formation in the HD-treated groups. The diluted HD at 5% and 10 % could be feasibly reused for food crops irrigation, verified by the negligible phytotoxic implications in the plant model, bioaccumulation, and cytotoxic and genotoxic effects in HepG2 cell.

Effect of the GSTM1 genotype on the biomarkers of exposure to polycyclic aromatic hydrocarbons: Meta-analysis.

The role of glutathione S-transferase Mu 1 (GSTM1) in the biomonitoring of polycyclic aromatic hydrocarbons (PAHs) is not clear. Our purpose has been to evaluate the influence of GSTM1 genotypes on 1-hydroxypyrene (1-OHP), deoxyribonucleic acid (DNA) adducts, and micronucleus frequency in both occupational and non-occupational populations of null and active GSTM1 carriers. We conducted a meta-analysis on 25 articles that met our strict inclusion criteria (11 studies on 1-OHP, 9 on DNA adducts, and 5 on the micronucleus frequency). In the case of occupationally exposed workers, micronucleus frequency was only significantly higher in the null GSTM1 carriers than in the active GSTM1 carriers. In the non-occupationally exposed general population, 1-OHP and micronucleus frequency were significantly higher in the null GSTM1 carriers. The results of Egger's test and funnel plot analysis indicated no significant publication bias. In conclusion, GSTM1 genotypes may affect the urinary 1-OHP in the non-occupationally exposed general population, and micronucleus frequency in both occupational workers and non-occupational population. Int J Occup Med Environ Health 2017;30(2):177-201.

Sperm quality and DNA integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons.

The objective of this study was to assess sperm quality and deoxyribonucleic acid (DNA) integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs) as compared to control subjects. The coke oven workers (N = 52) and administrative staff (N = 35) of a steel plant served as the exposed and control groups, respectively. Exposure to PAHs was assessed by measuring 1-hydroxypyren. Analysis of sperm quality (concentration, motility, vitality, and morphology) was performed simultaneously with sperm DNA integrity analysis, including DNA fragmentation, denaturation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). A questionnaire was conducted to collect demographic and potential confounding data. The coke oven workers had lower percentages of sperm motility, vitality and normal morphology than the control group, but the difference was not significant. For DNA integrity, the coke oven workers had significantly higher concentrations of bulky DNA adducts and 8-oxo-dGuo than the control subjects (p = 0.009 and p = 0.048, respectively). However, DNA fragmentation percentages did not significantly increase as compared to those in the subjects from the control group (p = 0.232). There was no correlation between sperm quality parameters and DNA integrity indicators. Occupational exposure of the coke oven workers to PAHs was associated with decreased sperm DNA integrity. Int J Occup Med Environ Health 2016;29(6):915-926.

Dietary butyrylated high-amylose starch reduces azoxymethane-induced colonic O6-methylguanine adducts in rats as measured by immunohistochemistry and high-pressure liquid chromatography

O6-methyl guanine (O6MeG) adducts are major toxic, promutagenic, and procarcinogenic adducts involved in colorectal carcinogenesis. Resistant starch and its colonic metabolite butyrate are known to protect against oncogenesis in the colon. In this study, we hypothesized that a dietary intervention that specifically delivers butyrate to the large bowel (notably butyrylated high-amylose maize starch [HAMSB]) would reduce colonic levels of O6MeG in rats shortly after exposure to the deoxyribonucleic acid (DNA) alkylating agent azoxymethane (AOM) when compared with a low-amylose maize starch (LAMS). A further objective was to validate an immunohistochemistry (IHC) method for quantifying O6MeG against a high-performance liquid chromatography method using fluorescence and diode array detection. Rats were fed either LAMS or HAMSB diets for 4 weeks followed by a single injection of AOM or saline and killed 6 hours later. After AOM exposure, both IHC and high-performance liquid chromatography method using fluorescence and diode array detection measured a substantially increased quantity of DNA adducts in the colon (P<.001). Both techniques demonstrated equally that consumption of HAMSB provided a protective effect by reducing colonic adduct load compared with the LAMS diet (P<.05). In addition, IHC allowed visualization of the O6MeG distribution, where adduct load was reduced in the lower third of the crypt compartment in HAMSB-fed rats (P=.036). The apoptotic response to AOM was higher in the HAMSB-fed rats (P=.002). In conclusion, the reduction in O6MeG levels and enhancement of the apoptotic response to DNA damage in the colonic epithelium through consumption of HAMSB provide mechanistic insights into how HAMSB protects against colorectal tumorigenesis.

Oxaliplatin-induced acute thrombocytopenia

Oxaliplatin (1, 2-diamminocyclohexaneoxalato-platinum) is a novel platin analog, which is widely used in gastrointestinal malignancies. Platinum analogs damage cellular deoxyribonucleic acid (DNA) by leading covalent bifunctional DNA adducts with cellular DNA. Major side effects of oxaliplatin are neurotoxicity (peripheral neuropathy), myelosuppression with moderate thrombocytopenia and gastrointestinal toxicity (diarrhea). Thrombocytopenia might be related to myelosuppression and/or drug-induced immune thrombocytopenia (DIIT). In here, oxaliplatin-induced thrombocytopenia is discussed with review of the literature.

Identification and analysis of acetaldehyde induced deoxyribonucleic acid adducts by ultra performance liquid chromatography-tandem mass spectrometry

Identification and analysis of acetaldehyde induced deoxyribonucleic acid adducts by ultra performance liquid chromatography-tandem mass spectrometry

High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts

Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O6-methylguanine (O6-MeG), O6-carboxymethylguanine (O6-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation.

Chronic Kidney Disease and Upper Tract Urothelial Carcinomas

Upper tract urothelial carcinomas (UTUC) have high prevalence rates in patients with chronic kidney disease (CKD), including dialysis patients and kidney transplant recipients. Analgesic nephropathy, Balkan endemic nephropathy, and aristolochic acid (AA) nephropathy share the common association with the development of CKD and UTUC. Genome studies allow identification of patients with genetic predisposition to Balkan endemic nephropathy and AA nephropathy. Furthermore, the identification of aristolactam deoxyribonucleic acid (DNA) adducts now provides robust evidence for AA exposure in UTUC patients, even in the absence of recallable exposure history. Diagnosis and treatment of UTUC in CKD patients are challenging. Clinical diagnosis of UTUCs in CKD patients with the available urological and imaging methods is much more difficult than in patients with normal renal function due to atrophic kidneys and poor excretory functions in these patients. High prevalence rates of contralateral upper tract and urinary bladder involvements of UC in CKD, either at presentation or recurrence, raise concerns of the necessity and techniques as well as perioperative risk of complete urinary tract exenteration. The standard treatment of radical nephroureterectomy with bladder cuff excision for UTUC patients may result in deterioration of renal function, rendering adjuvant chemotherapy unsuitable. This special issue collectively addresses these important topics regarding UTUC in CKD patients. The authors in one paper nominate three mutant genes (CELA1, HSPG2, and KCNK5) in Balkan endemic nephropathy patients, which are associated with angiogenesis. This finding suggests that angiogenesis might play an important role in the development of Balkan endemic nephropathy. Another paper shows an elevated risk of urothelial carcinomas (UC) in ESRD patients with the analysis of a national wide cohort in Taiwan. Female patients have 9–18 folds of increased risk of UC as compared to 4–14 folds in male ESRD patients, which may reflect female predominance in AA consumption. One paper discusses the challenges of UTUC diagnosis in CKD patients. It reviews the detection rate of UTUCs in dialysis patients and kidney transplant recipients with the use of a variety of imaging and urological methods in the literature. Combination use of urological and imaging methods has been suggested for diagnosing UTUCs in symptomatic dialysis patients and kidney transplant recipients. Another paper develops a predictive model for postoperative renal insufficiency in UTUC patients undergoing radical nephroureterectomy, which help in selecting eligible patients for cisplatin based adjuvant chemotherapy. Older age, lower estimated glomerular filtration rate before surgery, smaller tumor size, renal pelvis tumor, and absence of hydronephrosis or multifocal tumors are predictors for ineligibility for adjuvant chemotherapy. One paper describes modified incision complete urinary tract exenteration for UC in dialysis patients, providing short operative time, early oral feeding, and no major perioperative complications of this modified surgical procedure. Another paper reviews epidemiological evidence of AA exposure associated with occurrence of nephropathy and UC, including AA exposure scenarios in Belgium, Taiwan, and the Danube River as well as occupational exposure in Chinese herbalists. The identification of aristolactam DNA adducts in these patients further corroborates biological plausibility of AA exposure contributing to the occurrence of UTUC. Li-Jen Wang Joelle L. Nortier Bin Tean Teh Cheng-Keng Chuang Shen-Yang Lee

HspA1A facilitates DNA repair in human bronchial epithelial cells exposed to Benzo[a]pyrene and interacts with casein kinase 2

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant that induces deoxyribonucleic acid (DNA) damage. The inducible heat shock protein (HspA1A) can function as a molecular chaperone; however, its role in DNA repair remains largely unknown. In the present study, human bronchial epithelial cells (16HBE) stably transfected with plasmids carrying HspA1A gene or shRNAs against HspA1A were treated with BaP. DNA damage levels of the cells were evaluated by comet assay. Results suggest that HspA1A could protect cells against DNA damage and facilitate the decrease of DNA damage levels during the first 2h of DNA repair. DNA repair capacity (DRC) of Benzo(a)pyrene diol epoxide (BPDE)-DNA adducts was evaluated by host cell reactivation assay in the stable 16HBE cells transfected with luciferase reporter vector PCMVluc pretreated with BPDE. Compared with control cells, cells overexpressing HspA1A showed higher DRC (p < 0.01 at 10μM BPDE and p < 0.05 at 20μM BPDE, respectively), while knockdown of HspA1A inhibited DNA repair (p < 0.05 at 10μM BPDE). Moreover, casein kinase 2 (CK2) was shown to interact with HspA1A by mass spectrometry and co-immunoprecipitation assays. The two proteins were co-localized in the cell nucleus and perinuclear region during DNA repair, and were identified by confocal laser scanning microscope. In addition, cells overexpressing HspA1A showed an increased CK2 activity after BaP treatment compared with control cells (p < 0.01). Our results suggest that HspA1A facilitates DNA repair after BaP treatment. HspA1A also interacts with CK2 and enhances the kinase activities of CK2 during DNA repair.

Mass spectrometry of structurally modified DNA.

Structural modifications of nucleobases within the deoxyribonucleic acid (DNA) of living cells can be induced as a result of actions of specialized DNA-modifying enzymes (creating epigenetic DNA modifications) or may result from exposure to reactive endogenous and exogenous electrophiles and oxidants (creating DNA adducts). Epigenetic DNA modifications such as 5-methylcytosine (MeC) are important regulators of cell function that influence chromatin structure and levels of gene expression. DNA methyltransferases (DMTs) catalyze the addition of the C-5 methyl group to cytosine nucleobases.1 DMTs preferentially recognize hypomethylated 5′-CG-3′ sequences, producing epigenetic modifications which preserve DNA methylation patterns. C-5 cytosine methylation controls gene expression by mediating the binding of specific proteins (methyl-CpG binding proteins) to MeCG sites, followed by the recruitment of histone-modifying enzymes that promote chromatin remodeling.2 Recent studies have discovered additional cytosine modifications, e.g. 5-hydroxymethyl-C, and 5-formyl-C, and 5-carboxyl-C; these modifications have been hypothesized to be demethylation intermediates or they may possess their own epigenetic functions within cells.3-5 In contrast to epigenetic modifications, chemical DNA damage including nucleobase alkylation, oxidation, deamination, and cross-linking occurs at a variety of sites, including the N-7, O-6, C-8, and N-2 of guanine; the N-1, N-3, and N-7 of adenine; the O-2 and O-4 of thymine; and the O-2 and N-4 of cytosine (Scheme 1 and Chart 1).6 Some carcinogens are inherently reactive towards DNA, while others must first be metabolically activated to electrophilic intermediates (e.g. epoxides, quinone methides, diazonium ions, and nitrenium ions), which subsequently bind to DNA producing nucleobase adducts (Figure 1).6 All living cells contain extensive DNA repair systems responsible for removing nucleobase lesions. If structurally modified DNA bases escape repair, they may induce base mispairing during DNA replication; thus, the chemical damage would be converted into permanent genetic damage (mutations).6 Accumulation of mutations in genes controlling cell growth, proliferation, programmed cell death, and cell differentiation is likely to cause cancer.7-10 Figure 1 Central role of DNA adducts in chemical carcinogenesis. Scheme 1 DNA sites frequently modified by carcinogens and their metabolites. Chart 1 Structures of representative DNA adducts Due to their central role in chemical carcinogenesis, DNA adducts are considered the true mechanism-based biomarkers of carcinogen exposure. The presence of DNA adducts within a given tissue can be correlated to the formation of reactive intermediates available for binding to DNA and other biomolecules.11 Unlike hemoglobin adducts that reflect a cumulative exposure to carcinogens over time, DNA adducts provide information on the burden of DNA damage within a given tissue at a specific time. Adducts can be used to quantify the capacity of DNA repair systems and to assess the potential for genetic damage as a result of faulty replication. By employing these measurements, DNA adduct levels have been utilized to set human exposure limits for industrial and environmental chemicals and also to identify individuals and populations at risk for developing cancer.11-14 The concentrations of epigenetically modified DNA bases in vivo are relatively high (e.g. four MeC per 100 of total nucleobases), however, the amounts of chemically induced DNA adducts in animal and human tissues can be quite low, in the range of 0.01 - 10 adducts per 108 normal nucleotides. Therefore, analytical methods used for quantifying carcinogen-DNA adducts must be ultra-sensitive, accurate, and specific, allowing the quantitation of low abundance DNA lesions in the presence of a large molar excess of normal nucleosides. Early studies of DNA damage utilized radiolabel-based assays such as 32P-postlabeling methods to measure adduct levels.15-18 Recent developments in mass spectrometry instrumentation have offered an alternative approach that provides both accurate and sensitive quantitation and structural information for the damaged bases, without the need for radioactivity.12 DNA adduct structure can be established using tandem mass spectrometry experiments, while mass spectrometry in combination with stable isotope labeled internal standards (isotope dilution HPLC-ESI-MS-MS or IDMS) is considered a golden standard for DNA adduct analysis due to its high specificity, sensitivity, and accurate quantification.14 Furthermore, mass spectrometry can be used for sequencing native and structurally modified DNA. The present review is devoted to the applications of mass spectrometry to DNA adduct and epigenetic DNA modification identification, screening, and quantitation. We will also discuss the use of MS based approaches to map the distribution of DNA modifications along DNA duplexes and to establish the biological consequences of DNA adduct formation in cells. Taken together, this article provides an overview of the contributions of mass spectrometry to the field of chemical carcinogenesis and epigenetics, with a primary focus on the new developments and recent advances in the field.

Internal exposure to carcinogenic polycyclic aromatic hydrocarbons and DNA damage

In the August issue of the Archives, Kafferlein et al. report no association between occupational exposures to polycyclic aromatic hydrocarbons (PAHs) and deoxyribo nucleic acid (DNA) damage on the individual level (Kafferlein et al. 2012). Internal PAH exposure, assessed in a variety of occupations by means of urinary 3-hydroxy benzo[a]pyrene (3-OH-B[a]P) and 1and 2-naphtol ( P OHNaph), was unrelated with DNA strand break frequencies and the levels of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo), perhaps with the exception of a small group of converter workers where an uncertain correlation between P OHNaph and 8-oxo-dGuo was observed. Authors discuss reasons for this lack of association suggesting to focus on the individual exposure circumstances. These include possible different routes of exposure and co-exposures and a better understanding and assessment of the kinetics and dynamics of the endpoints. We report here the lack of correlations on the individual level in a group of coke oven non-smoker workers between other endpoints of exposure such as urinary 1-hydroxypyrene (1-OHP) and lymphocyte anti-benzo[a]pyrene diolepoxide DNA adduct (anti-B[a]PDE-DNA) and of genotoxic effect such as lymphocyte micronuclei (MN) frequency. Similar lack of correlations observed when using rather different endpoints call for a discussion on possible common explanations. Whereas co-exposures can be thoroughly investigated, we suspect that the kinetics and dynamics of both exposure and effect endpoints for PAHs are far to be understood. Meanwhile, we propose here that both the results of Kafferlein et al. 2012 and ours could be largely explained by the variable routes of exposure occurring among and within occupational settings.

DNA methylation differences in exposed workers and nearby residents of the Ma Ta Phut industrial estate, Rayong, Thailand

Adverse biological effects from airborne pollutants are a primary environmental concern in highly industrialized areas. Recent studies linked air pollution exposures with altered blood Deoxyribo-nucleic acid (DNA) methylation, but effects from industrial sources and underlying biological mechanisms are still largely unexplored. The Ma Ta Phut industrial estate (MIE) in Rayong, Thailand hosts one of the largest steel, oil refinery and petrochemical complexes in south-eastern Asia. We measured a panel of blood DNA methylation markers previously associated with air pollution exposures, including repeated elements [long interspersed nuclear element-1 (LINE-1) and Alu] and genes [p53, hypermethylated-in-cancer-1 (HIC1), p16 and interleukin-6 (IL-6)], in 67 MIE workers, 65 Ma Ta Phut residents and 45 rural controls. To evaluate the role of DNA damage and oxidation, we correlated DNA methylation measures with bulky DNA and 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M(1)dG) adducts. In covariate-adjusted models, MIE workers, compared with rural residents, showed lower LINE-1 (74.8% vs 78.0%; P < 0.001), p53 (8.0% vs 15.7%; P < 0.001) and IL-6 methylation (39.2% vs 45.0%; P = 0.027) and higher HIC1 methylation (22.2% vs 15.3%, P < 0.001). For all four markers, Ma Ta Phut residents exhibited methylation levels intermediate between MIE workers and rural controls (LINE-1, 75.7%, P < 0.001; p53, 9.0%, P < 0.001; IL-6, 39.8%, P = 0.041; HIC1, 17.8%, P = 0.05; all P-values vs rural controls). Bulky DNA adducts showed negative correlation with p53 methylation (P = 0.01). M(1)dG showed negative correlations with LINE-1 (P = 0.003) and IL-6 methylation (P = 0.05). Our findings indicate that industrial exposures may induce alterations of DNA methylation patterns detectable in blood leucocyte DNA. Correlation of DNA adducts with DNA hypomethylation suggests potential mediation by DNA damage.

MTHFR Polymorphisms and Carcinogen DNA Damage in the Lung

PP-30-157 Background/Aims: Methylenetetrahydrofolate reductase, MTHFR, is involved in the process of deoxyribonucleic acid (DNA) methylation and DNA synthesis, both implicated in carcinogenesis. Polymorphisms in MTHFR gene, C677T and A1298C, are associated with an elevated and/or decreased cancer risk, yet little is known about DNA damage in target lung tissue in human populations. Methods: DNA adducts in lung tissue and 5 polymorphic metabolic genotypes (GSTM1, GSTP1, GSTT1, NAT2, and MTHFR) were determined by using 32P-postlabeling assay and PCR techniques from 102 lung cancer patients undergoing resection for stage I-II lung cancer at Massachusetts General Hospital in Boston, MA. The independent effects of MTHFR polymorphisms on DNA lung adduct were assessed using multiple regression analysis, adjusting for covariates and other genotypes. Results: DNA lung adduct levels significantly increased by 64.4% (95% confidence interval [CI]: 3.0%–162.4%) for the 1298AC+CC genotype versus 1298AA and by 122.4% (95% CI: 1.7%–386.5%) for the combined heterozygous and homozygous variant genotypes versus 1298AA and 677CC, respectively. No association was seen with polymorphism of MTHFR C677T. After adjusting for potential confounders, as well as for other metabolic gene variants, the MTHFR 1298AC+CC had significantly enhanced levels of lung adducts by 109.9% (95% CI: 19.8%–267.8%). Conclusion: We found an association between increased DNA adduct levels with the MTHFR A1298C gene polymorphism, suggesting a potential effect of DNA methylation via MTHFR on DNA damage in the target lung tissue.

生体高分子の微小変化を通して見る生命現象 ―バイオマーカー探索のブレークスルーになりうるか？―

Biological macromolecules, such as deoxyribonucleic acid (DNA) and proteins, are exposed to higher chemical stresses during some physiological events such as increased oxidative stress from degenerative aging diseases and higher glucose stress in diabetes mellitus. Therefore, the resulting chemical modifications can provide significant information about biological events. Chemically modified DNA and abundant proteins have been analyzed by mass spectrometry, mainly for use as dosimeters of chemical exposure. However, quantitative analyses of bioactive peptides and proteins in specific diseases have relied almost exclusively on the use of immunoassay-based procedures with no concern for possible minute chemical modifications. Therefore, important information could be overlooked, or considerable misunderstandings could be created, if the modifications are not distinguished and if they alter protein functions such as phosphorylation. I believe that chemical modifications on biological macromolecules could play important roles, not only as diagnostic and therapeutic markers, but also as triggers that initiate some diseases. In this article, I first introduce my previous research on biomarkers such as DNA adducts and chemically modified proteins. I then present a novel “omics” strategy for biomarker discovery using minute chemical modifications on biological macromolecules.

Biological effects of the “Erika” oil spill on the common mussel(Mytilus edulis)

A 3-year survey was made of several biological markers in mussels (Mytilus edulis) exposed in situ to the oil that came ashore after the wreck of the Erika tanker on the Brittany (France) coast in December 1999. The mussel response was assessed using a set of 7 biomarkers, most of them related to the metabolism of organic contaminants. After a series of validation tests, data was evaluated for only 5 biomarkers: acetylcholinesterase (AChE), glutathione S-transferase (GST), catalase (CAT), malondialdehyde (MDA) and deoxyribonucleic acid (DNA) adducts. No signifi- cant reductions in GST or CAT levels were observed, levels of DNA adducts and MDA were high during the 6 months immediately following the accident and levels of AChE were significantly lower during the first year of the survey suggesting a general stress. A simple multivariate graphic method, the integrated biomarker response index, was used to combine 4 of the 5 validated biomarkers and quantify the degree of impact on mussels at different sites. The results show that mussel populations were affected by the oil spill only during the first year after the event.

7H-BENZO[C]FLUORENE: A POTENT SYSTEMIC LUNG CARCINOGEN

7H-Benzo[c]fluorene (B[c]F) is a major lung deoxyribonucleic acid (DNA) adductor in mice treated with coal tar suggesting that B[c]F may be capable of inducing lung tumors. This study evaluated the tumorigenic potential of B[c]F in comparison to benzo[a]pyrene (B[a]P) using the A/J mouse model. Female A/J mice 7 weeks of age were administered B[c]F or B[a]P (100 mg/kg) by i.p. injection. These mice were fed Purina Rodent 2001 diet for the remainder of the study. Groups of mice were also fed diets containing B[c]F (397 or 27 μmol/kg diet) or B[a]P (397 μmol/kg diet). In addition, a mixture of 20 synthetic polycyclic aromatic hydrocarbons (PAHs) known to be present in coal tar was also fed to mice in the presence or absence of B[c]F. A basal gel diet system was used to administer hydrocarbons within the diet. Mice were maintained on control or adulterated diets for 260 days. B[c]F administered i.p. induced multiple lung tumors in 92% of the treated mice, with an average of 4.0 tumors per mouse. Similarly, B[a]P administered i.p. induced an average of 6.7 tumors per mouse in 90% of the treated mice. The highest level of lung tumor induction was observed in mice fed 397 μmol/kg diet B[c]F. A 100% tumor incidence and an average of 46 lung tumors per mouse was observed. In contrast, mice fed a diet containing 397 μmol/kg of B[a]P, had a 77% tumor incidence with an average of 1.4 tumors per mouse. Mice fed a 27 μmol/kg B[c]F diet, or the mixture of synthetic hydrocarbons with or without B[c]F resulted in tumor incidences and multiplicity not different from controls. Forestomach lesions were greatest in mice treated with B[a]P. Evaluation of chemical:DNA adduct formation in mice indicates that B[c]F is better than B[a]P in forming lung DNA adducts when fed to mice at 397 μmol/kg food. These results demonstrate that B[c]F is tumorigenic in lung of mice when administered by i.p. injection and in particular when fed to mice in the diet. These data strongly suggest that B[c]F is a systemic carcinogen that likely contributes to the potent mouse lung tumorigenicity previously demonstrated with coal tar when fed to mice.

On the reaction of mutagenic aflatoxin B 1 oxide and benz[ a]pyrene diol oxide with guanine residue in DNA double helix

Theoretical calculation was performed to study the binding mechanism of intercalators to deoxyribonucleic acid (DNA) base pairs. The structures of intercalating complexes and ligand–DNA adducts of mutagenic aflatoxin B 1 8,9-oxide ( 1) and benz[ a]pyrene diol oxide ( 2) and the reactivity for S N2 type nucleophilic oxirane ring opening for 2 were investigated. Calculation clarified the following points: 1. The atomic contacts between two reaction sites (nucleophilic N2 or N7 nitrogen lone pair of guanine residue in DNA base pairs and electrophilic carbon of three-membered oxirane ring in mutagenic 1 and 2) are considerably short, suggesting that intercalation makes two reaction sites closer and consequently promotes the covalent binding of mutagens to guanine base. 2. In the adducts of 1 and 2 with DNA double helix, large planar coumarin ring of 1 exists in the major groove of double helix and planar aromatic ring system of 2 in minor groove of it. The regularly layered structure of B type DNA double helix is not distorted in the adduct of 1, however, the double helix is considerably distorted in the adduct of 2. 3. Two mutagens probably intercalate into DNA base pairs and make stable complexes which are followed by transition structures (TSs) for covalent binding. Judging from the activation energy (Δ E ≠) estimated to be 47.7 kcal/mol, the reactivity for covalent binding is considerably low, implying that intercalation must play an important role as precedent phenomena to covalent binding between bases and intercalators. Calculation suggests that the reactivity for S N2 type nucleophilic reaction itself (the stability of TS for covalent binding) is not so high, and therefore, the potency for mutagenesis might be attributing to the formation of stable (and consequently) long standing intercalating complex and stable ligand–DNA adduct.

Biomarkers and their use in occupational medicine.

Biomarkers of exposure, parent chemicals, metabolites, and also hemoglobin and DNA (deoxyribonucleic acid) adducts, even urinary mutagenicity, have been used successfully to identify exposed persons, follow-up exposure, and quantitatively assess exposure. Some have been validated as indicators of health risk. The avenues for future biomarkers of exposure are in the development of robust, simple, and inexpensive semiquantitative methods for daily use on every worker for assuring that no (excessive) exposure takes place. Some biomarkers of effect have also been well validated and are widely used in routine monitoring activities. However, with the exception of cholinesterase inhibitors, biomarkers of effect offer little advantage over the analysis of the chemical itself. Their use will be limited to carefully planned ad hoc studies. Biomarkers of susceptibility currently have no practical application in worker health protection. Biomarker research should concentrate on validating methods, in terms of the prediction of health effects, and on elucidating exposure-biomarker concentration relationships.