Increased DNA Adduct Formation Research Articles

Geographical approach analysis of the impact of air pollution on newborn intrauterine growth and cord blood DNA damage in Mexico City

Abstract Background Few epidemiologic studies have focused on the specific source of ambient air pollution and adverse health effects in early life. Here, we investigated whether air pollutants from different emission sources were associated with decreased birth anthropometry parameters and increased DNA adduct formation in mother-child pairs residing in the Mexico City Metropolitan Area (MCMA). Methods This cross-sectional study included 190 pregnant women recruited during their last trimester of pregnancy from two hospitals at MCMA, and a Modeling Emissions Inventory (MEI) to calculate exposure to ambient air pollutants from different emissions sources (area, point, mobile, and natural) for two geographical buffers 250 and 750 m radii around the participants households. Results Contaminants were positively correlated with umbilical cord blood (UCB) adducts, but not with maternal blood (MB) adducts. PM10 emissions (area and point sources, overall emissions), PM2.5 (point sources), volatile organic compounds (VOC), total organic compounds (TOC) from point sources were positively correlated with UCB adducts. Air pollutants emitted from natural sources were correlated with a decrease in MB and UCB adducts. PM10 and PM2.5 were correlated (p < 0.05) with a decrease in birth weight (BW), birth length (BL) and gestational age at term (GA). In multivariate analyses adjusted for potential confounders, PM10 was associated with an increase in UCB adducts. PM10 and PM2.5 from overall emissions were associated with a decrease in BW, BL and GA at term. Impact Results suggested higher susceptibility of newborns compared to mothers to damage related to ambient air pollution. PMs are associated with birth anthropometry parameters and DNA damage in adjusted models, highlighting the need for more strict regulation of PM emissions.

Force-Dependent Intercalative Bulky DNA Adduct Formation Detected by Single-Molecule Stretching.

Quantitatively analyzing the binding topology and reactivity is essential for understanding the cytotoxic or tumorigenic activities of bulky DNA adducts formed by chemotherapeutic drugs or carcinogens. Biochemical methods require purification of DNA and discontinuous steps to digest or label the adducts and thus have difficulties in identifying the binding topology and are not suitable for detecting unstable adducts. Herein, we used a single-molecule stretching assay to characterize the number of intercalative adducts, the formation kinetics, and the mechanical properties of intercalative DNA adducts based on measuring adduct-induced DNA elongation. We analyzed various reactive conditions, including formaldehyde-mediated anthracycline-DNA adducts, UV light-catalyzed psoralen-DNA adducts, and liver S9 fraction-catalyzed aflatoxin B1-DNA adducts. We showed that adduct formation abilities are correlated with the noncovalent intercalation binding ability. External forces on double-stranded DNA increased the intercalation of ligands and can result in a 1.8- to 5.3-fold increase in DNA adduct formation.

DNA Adduct Assessment During Antihormonal Treatment of Perianal Gland Tumors With Tamoxifen in Male Dogs.

Determination of DNA adduct count was performed in mononuclear cells during antihormonal treatment of perianal gland tumors. Eight- to fifteen-year-old dogs with carcinoma (CAR Group; N=5), epithelioma (EPI Group; N=16) or adenoma (ADE Group; N=24) were used. The control group suffered from perineal hernia or rectal diverticulum (CTR Group; N=25). Blood was collected at baseline, and at one and six months after the beginning of the anti-hormonal treatment with tamoxifen (1 mg/kg of body weight). DNA adduct count was determined using autoradiography. At baseline, DNA adduct count reached the highest value in the CTR Group, and the lowest in the EPI Group (p<0.05). Six-month-long therapy with tamoxifen resulted in a significant increase in the DNA adduct count by 78.7%, 221.5% and 198.3% in the ADE, EPI and CAR groups, respectively (p<0.05). Increased DNA adduct formation after long-term administration of tamoxifen shows its genotoxicity.

Regulation of Chromatin Assembly and Cell Transformation by Formaldehyde Exposure in Human Cells.

Background:Formaldehyde (FA) is an environmental and occupational chemical carcinogen. Recent studies have shown that exogenous FA causes only a modest increase in DNA adduct formation compared with the amount of adducts formed by endogenous FA, raising the possibility that epigenetic mechanisms may contribute to FA-mediated carcinogenicity.Objectives:We investigated the effects of FA exposure on histone modifications and chromatin assembly. We also examined the role of defective chromatin assembly in FA-mediated transcription and cell transformation.Methods:Cellular fractionation and Western blot analysis were used to measure the levels of histone modifications in human bronchial epithelial BEAS-2B cells and human nasal RPMI2650 cells in the presence of FA. Chromatin immunoprecipitation (ChIP) and micrococcal nuclease (MNase) digest assays were performed to examine the changes in chromatin assembly and accessibility after FA exposure. RNA sequencing (RNA-seq) and real-time polymerase chain reaction (PCR) were used to examine transcriptional dysregulation. Finally, anchorage-independent cell growth ability was tested by soft agar assay following FA exposure.Results:Exposure to FA dramatically decreased the acetylation of the N-terminal tails of cytosolic histones. These modifications are important for histone nuclear import and subsequent chromatin assembly. Histone proteins were depleted in both the chromatin fraction and at most of the genomic loci tested following FA exposure, suggesting that FA compromises chromatin assembly. Moreover, FA increased chromatin accessibility and altered the expression of hundreds of cancer-related genes. Knockdown of the histone H3.3 gene (an H3 variant), which mimics inhibition of chromatin assembly, facilitated FA-mediated anchorage-independent cell growth.Conclusions:We propose that the inhibition of chromatin assembly represents a novel mechanism of cell transformation induced by the environmental and occupational chemical carcinogen FA. https://doi.org/10.1289/EHP1275

Effects of Intravenous Benzo[a]Pyrene Dose Administration on Levels of Exposure Biomarkers, DNA Adducts, and Gene Expression in Rats

The effects of benzo[a]pyrene (BaP) administration on biomarkers of exposure and early effects were studied in male Sprague-Dawley rats intravenously injected with doses of 0.4, 4, 10, or 40 μmol BaP/kg . Blood, tissues, and excreta were collected 8 and 24 h posttreatment. BaP and several of its metabolites were simultaneously measured in blood, tissues and excreta by ultra-high-performance liquid chromatography (UHPLC)/fluorescence. DNA adducts of BaP diol epoxide (BaPDE) in lungs were quantified using an ultrasensitive immunoassay with chemiluminescence detection. Expression of selected genes in lungs of treated rats (lung RNA) compared to control rats was also assessed by quantitative real-time polymerase chain reaction. There was a dose-dependent increase in blood, tissue, and excreted levels of BaP metabolites. At 8 and 24 h postinjection, BaP and hydroxyBaP were found in higher concentrations in blood and tissues compared to other analytes. However, diolBaP were excreted in greater amounts in urine and apparently more rapidly than hydroxyBaP. Mean percentages (± SD) of injected dose excreted in urine as 4,5-diolBaP during the 0–8 h and 0–24 h period posttreatment were 0.16 ± 0.027% and 0.14 ± 0.083%, respectively. Corresponding values for 3-OHBaP were 0.0045 ± 0.0009% and 0.026 ± 0.014%. BaP-diones were not detectable in blood, tissues, and excreta; 7,8-diolBaP and BaPtetrol were found to be minor metabolites. There was also a dose-dependent increase in DNA adduct formation in lung. Analysis of gene expression further showed a modulation of Cyp1a1, Cyp1b1, Nqo1, Nrf2, Fos, and Ahr expression at 10- and 40-μmol/kg doses, but not at the lower doses. This study provided a better assessment of the influence of absorbed BaP doses on biological levels of diolBaP and OHBaP exposure biomarkers and association of the latter with early biological alterations, such as DNA adducts and gene expression.

In vivo validation of DNA adduct formation by estragole in rats predicted by physiologically based biodynamic modelling

Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in human.

A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes

Estragole has been shown to be hepatocarcinogenic in rodent species at high-dose levels. Translation of these results into the likelihood of formation of DNA adducts, mutation, and ultimately cancer upon more realistic low-dose exposures remains a challenge. Recently we have developed physiologically based biokinetic (PBBK) models for rat and human predicting bioactivation of estragole. These PBBK models, however, predict only kinetic characteristics. The present study describes the extension of the PBBK model to a so-called physiologically based biodynamic (PBBD) model predicting in vivo DNA adduct formation of estragole in rat liver. This PBBD model was developed using in vitro data on DNA adduct formation in rat primary hepatocytes exposed to 1′-hydroxyestragole. The model was extended by linking the area under the curve for 1′-hydroxyestragole formation predicted by the PBBK model to the area under the curve for 1′-hydroxyestragole in the in vitro experiments. The outcome of the PBBD model revealed a linear increase in DNA adduct formation with increasing estragole doses up to 100 mg/kg bw. Although DNA adduct formation of genotoxic carcinogens is generally seen as a biomarker of exposure rather than a biomarker of response, the PBBD model now developed is one step closer to the ultimate toxic effect of estragole than the PBBK model described previously. Comparison of the PBBD model outcome to available data showed that the model adequately predicts the dose-dependent level of DNA adduct formation. The PBBD model predicts DNA adduct formation at low levels of exposure up to a dose level showing to cause cancer in rodent bioassays, providing a proof of principle for modeling a toxicodynamic in vivo endpoint on the basis of solely in vitro experimental data.

Therapeutic Targeting of ATP7B in Ovarian Carcinoma

Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

Inhibition of gut glutathione transport by a breast cancer carcinogen

Abstract Introduction: Oral intake of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) causes breast cancer in the rat and is associated with a marked decrease in gut glutathione (GSH) efflux to the liver. We hypothesized GSH efflux is decreased by a block in either gut GSH production and/or GSH release leading to decreased liver detoxification of the carcinogen, facilitating adduct formation and carcinogenesis. Methods: GSH transport in jejunal basolateral membrane (JBLM) vesicles was investigated in female pubertal rats gavaged with 100 mg/kg DMBA (n = 15) or control (n = 15). Rats were pair-fed pre-defined chow and sacrificed one week post DMBA. Vesicles were prepared using Percoll differential centrifugation. Na+-dependent amino acid transport systems, A, ASC/B(0), and B(0,+) were examined using the inhibitors Me-AIB and Arginine. Transport velocity was determined by measuring 3H-GSH uptake. Blood and gut mucosal GSH were measured using spectrophotometric assay. Results: A significant decrease in Na+ dependent ASC/B(0) gut GSH transport velocity (42%) was seen in the DMBA group (21.89 +/− 2.63 pmol/mgprotein/min vs 35.52 +/− 4.05 pmol/mgprotein/min in controls, p Conclusions: One mechanism by which the carcinogen DMBA facilitates breast cancer induction may be through a significant decrease in gut GSH release secondary to an inhibition of the GSH ASC/B(0) transport system which would lead to increased DNA adduct formation and/or decreased carcinogen detoxification in the liver.

Cytochrome P4501B1 Expression in Normal Breast Tissue

Polycyclic aromatic hydrocarbons (PAHs) are metabolically activated to ultimate carcinogens by the cytochrome P-450 isozymes CYP1A1 and CYP1B1. High levels of these enzymes may result in increased DNA adduct formation and cancer initiation. We investigated whether expression of CYP1B1 in breast tissue varies to a similar extent as has been shown for CYP1A1 and whether increased CYP1B1 expression could constitute a risk factor for breast cancer. Expression of CYP1B1 and CYP1A1 was measured in a collection of 75 nontumor epithelial breast tissue specimens from breast cancer patients ( n = 36) and from cancer-free individuals ( n = 39). Using a semiquantitative reverse transcription (RT)-polymerase chain reaction (PCR) assay, CYP1B1 and CYP1A1 expression levels relative to the constantly expressed g - actin gene were determined. In this study, we found 300-fold and 1,000-fold interindividual variation in expression for CYP1B1 and CYP1A1 , respectively. The mean CYP1B1 transcript level in normal breast tissue was 70% higher in mastectomy patients compared with cancer-free individuals ( p = .0473). These data suggest that CYP1B1 may play a role in breast cancer etiology, particularly in women exposed to high levels of CYP1B1 substrates such as PAHs.

Whole-body hyperthermia (41.8°C) combined with bimonthly oxaliplatin, high-dose leucovorin and 5-fluorouracil 48-hour continuous infusion in pretreated metastatic colorectal cancer:a phase II study

BackgroundSecond- and third-line treatments remain a challenge in advanced colorectal cancer. Studies of bimonthly regimens of high-dose leucovorin (LV) and 5-fluorouracil (5-FU) by continuous infusion combined with oxaliplatin (L-OHP) have shown encouraging response rates in patients not responding to a bimonthly LV/5-FU regimen. Hyperthermic enhancement of L-OHP efficiency by increased DNA adduct formation has been demonstrated in vitro. This study was designed to address feasibility, toxicity and efficacy issues of whole-body hyperthermia (WBH) as an adjunct to L-OHP/LV/5-FU in pretreated patients after progression to first- and second-line treatments with LV/5-FU by continuous infusion and irinotecan. Patients and methodsForty-four patients with advanced colorectal cancer, who had progressed during or within 3 months after completion of chemotherapy with LV/5-FU 24-h infusion (LV/5-FU24h) (eight patients) or irinotecan combined with or after LV/5-FU24h (36 patients), were treated with L-OHP 85 mg/m2, 2-h intravenous (i.v.) infusion, followed by LV 200 mg/m2, 1-h i.v. infusion, and 5-FU 3 g/m2, 48-h continuous infusion. Every second cycle of the biweekly regimen was combined with WBH, thus allowing a comparison of toxicity with and without WBH in the same patient. Whole-body hyperthermia was administered by a humidified radiant heat device. The target temperature of 41.8°C was maintained for 60 min. L-OHP (2-h infusion) was started at a core body temperature of 39°C. ResultsAll patients could be evaluated for toxicity, and 41 patients were evaluable for response. A total of 273 L-OHP-containing regimens were administered, 130 with and 143 without WBH. Hyperthermic treatment combined with L-OHP/LV/5-FU showed no unexpected toxicities. WHO grade 3 toxicities were rare and evenly balanced between cycles given with or without WBH. One early death occurred due to sepsis and tumor lysis. The overall response rate was 20%, with two complete and six partial responses. Twenty-three patients (56%) had stable disease and nine patients (22%) progressive disease. With a median observation time of 70 weeks, the median time to progression was 21 weeks [95% confidence interval (CI) 17–25 weeks] and the median survival was 50 weeks (95% CI 39–61 weeks) from the start of therapy. ConclusionsThis trial suggests some advantage of combining L-OHP/LV/5-FU with WBH. Results compare favorably with the activity of similar regimens without WBH in less extensively pretreated patients. These data support further evaluation and warrant phase III studies.

Cytochrome P450 (CYP1A) induction and DNA adducts in a rat hepatoma cell line (Fao), exposed to environmentally relevant concentrations of organic compounds, singly and in combinations

Cytochrome P4501A (CYP1A) induction and DNA adduct formation were evaluated in the rat hepatoma cell line Fao, as biomarkers of exposure to organic compounds. Cells were exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) or 3,3′,4,4′-tetrachlorobiphenyl (TCB), and to combinations of B[a]P and TCB. Both B[a]P and TCB induced CYP1A proteins in a concentration-dependent relationship, up to concentrations of 10 and 1 μM, respectively, detected by Western blotting. DNA adducts, analyzed by 32P-postlabeling, were found at the highest concentrations of B[a]P (1 and 10 μM). No adducts were found in cells exposed to 0.1 μM TCB alone. The cotreatment of TCB and B[a]P indicated an increase in DNA adduct formation, compared with B[a]P, but no further induction of CYP1A protein compared with TCB alone. This study suggests that Western blotting and 32P-postlabeling might be suitable methods for detecting CYP1A protein induction and DNA adducts, respectively, after exposure to environmentally relevant concentrations of organic compounds.

Responses of Human Cells to PAH-Induced DNA Damage

Benzo[ a ]pyrene (B[ a ]P) and dibenzo[ a,l ]pyrene (DB[ a,l ]P) induce cytochrome P450 (CYP) CYP1A1 and CYP1B1, which metabolize these polycyclic aromatic hydrocarbons (PAHs) into DNA-binding species. In order to detail roles of CYP1A1 and CYP1B1 in activation of DB[ a,l ]P to the diol epoxide, we here report the inhibition of CYP1A1 in human MCF-7 cells with phosphorodiamidate morpholino antisense oligomers (morpholinos). PAH-DNA adduct formation was also determined after treatment with morpholinos and B[ a ]P or DB[ a,l ]P. p53 is involved in DNA repair, cell cycle arrest, and apoptosis. Cells with normal p53 protein arrest in the G1 phase of the cell cycle on exposure to DNA-damaging agents (presumably allowing the cell sufficient time to repair damaged DNA prior to replication). Previous studies in human MCF-7 cells indicate that cells with PAH-DNA adducts escape cell cycle arrest and accumulate in the S phase. In the present study the effect of PAH-DNA adducts on the cell cycle were observed in human diploid fibroblasts (HDF). We found that treatment of HDF with the diol epoxide of DB[ a,l ]P causes cell cycle arrest in G 1 . An increase in DNA adduct formation with increase in concentration of dibenzo[ a,l ]pyrene diol epoxide {( m )- anti -DB[ a,l ]PDE} was also observed.

Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A: role of CYP 2C9 and microsomal glutathione-S-transferase.

Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by (32)P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9.

Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A: Role of CYP 2C9 and microsomal glutathione-S-transferase

Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by 32P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9. Environ. Mol. Mutagen. 35:123–131, 2000 © 2000 Wiley-Liss, Inc.

Nickel(II) increases the sensitivity of V79 Chinese hamster cells towards cisplatin and transplatin by interference with distinct steps of DNA repair.

Nickel compounds are carcinogenic to humans and to experimental animals. In contrast to their weak mutagenicity, they have been shown previously to increase UV-induced cytotoxicity and mutagenicity and to interfere with the repair of UV-induced DNA lesions by disrupting DNA-protein interactions involved in DNA damage recognition. In the present study we applied cisplatin, transplatin and mitomycin C to investigate whether these enhancing effects and DNA repair inhibition are also relevant for other DNA damaging agents. Nickel(II) at non-cytotoxic concentrations of 50 microM and higher caused a pronounced increase in cisplatin-, transplatin- and mitomycin C-induced cytotoxicity, which was neither due to an altered uptake of cisplatin or transplatin nor to an increase in DNA adduct formation. However, nickel(II) inhibited the repair of cisplatin- and transplatin-induced DNA lesions. In combination with transplatin, it decreased the incision frequency, indicating that the DNA damage recognition/incision step during nucleotide excision repair is affected in general by nickel(II). In support of this, concentrations as low as 10 microM nickel(II) decreased binding of the xeroderma pigmentosum complementation group A protein to a cisplatin-damaged oligonucleotide. When combined with cisplatin, the incision frequency was affected only marginally, while nickel(II) led to a marked accumulation of DNA strand breaks, indicating an inhibition of the polymerization/ligation step of the repair process. This effect may be explained by interference with the repair of DNA-DNA interstrand crosslinks induced by cisplatin. Our results suggest that nickel(II) at non-cytotoxic concentrations inhibits nucleotide excision repair and possibly crosslink repair by interference with distinct steps of the respective repair pathways.

Role of Hydrogen Peroxide in the Formation of DNA Adducts in HL-60 Cells Treated with Benzene Metabolites

We have investigated the influence of peroxides on DNA adduct formation in HL-60 cells treated with polyphenolic metabolites of benzene. Treatment of HL-60 cells with 50 μM hydroquinone (HQ), 500 μM catechol (CAT) or 200 μM 1,2,4-benzenetriol (BT) resulted in adduct levels of 0.27, 0.21 and 0.21 × 10−7, respectively. Addition of 50–250 μM H2O2or 250 μM cumene hydroperoxide to HL-60 cells increased DNA adduct formation 2.7–10-fold following treatment with HQ or CAT but had no effect on adduct formation by BT. Treatment of HL-60 cells with the combinations of HQ plus either BT or phorbol myristate acetate (PMA) potentiated DNA adduct formation by 2.5–4-fold. Significant elevations of cellular H2O2levels occurred after treatment of HL-60 cells with either PMA, CAT or BT. These results indicate that cellular levels of H2O2regulate the peroxidase dependent activation of benzene metabolites to form DNA adducts.

Activation of 2-Aminofluorene by Prostaglandin Endoperoxide H Synthase 2

Prostaglandin endoperoxide H synthase is the key enzyme in the conversion of arachidonic acid to tissue prostanoids. Two isoforms of prostaglandin endoperoxide H synthase have been identified: PHS-1 is constitutively expressed in most tissues under normal physiological conditions and PHS-2 is expressed in response to inflammatory agents, tumor promoters, and other agents related to mitogenesis. Previous work demonstrated that PHS-1 can activate arylamine carcinogens. We report here that PHS-2 can also activate an arylamine carcinogen to form DNA adducts. This is shown by: (1) use of purified ovine PHS-2 to form DNA adducts; (2) increased DNA adduct formation, PHS-2 mRNA, and PHS-2 protein after treatment of HUVEC cells with the PHS-2 inducer PMA; and (3) transient expression of PHS-2 cDNA in COS-1 cells gave rise to both elevations of PHS-2 enzyme protein and DNA adduct formation. Finally, two PHS inhibitors, aspirin and indomethacin, showed significant inhibition of PHS-2-mediated DNA adduct formation.

DNA adduct measurements and tumor incidence during chronic carcinogen exposure in rodents.

In an attempt to elucidate the relationship between DNA adduct formation and tumorigenesis, DNA adducts were measured in the livers and bladders of mice during chronic exposure to several different doses of 2-acetylaminofluorene (2-AAF) and 4-aminobiphenyl (4-ABP). Continuous oral administration of these compounds for 4 weeks produced an increase in DNA adduct formation during the first 2 weeks, followed by a plateau, which presumably occurred because the rate of adduct removal offset the rate of adduct formation. The quantity of DNA adducts present at equilibrium correlated directly with the carcinogen concentration; therefore, when exposure was continued for 4 weeks, DNA adducts that reflected the plateau level at each dose could be expressed as a function of dose. Liver and bladder DNA adduct profiles thus obtained during administration of multiple doses of 2-AAF (to female mice) and 4-ABP (to male and female mice) were compared to profiles for tumor incidences obtained during lifetime exposures to the same doses. These experiments demonstrated similar profiles for DNA adduct formation and tumorigenesis in liver. In the bladder, DNA adducts were linear, but tumors only appeared at the higher doses in conjunction with cell proliferation. In addition to these aromatic amines, similar data are available for aflatoxin B1, diethylnitrosamine, and (methylnitrosamino)-1-(3-pyridyl)-1-butanone (also known as nicotine-derived nitrosoketone). Of the nine different biological situations (carcinogen/species/sex/organ) for which data are available, correlations between steady-state DNA adduct levels and tumorigenic response at the different doses were linear in five of the nine biological models.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Adducts Formed By Peroxidase Activation of Benzene Metabolites

Abstract DNA adduct formation was examined in HL-60 cells treated with the benzene metabolites p-benzoquinone (p-BQ), hydroquinone (HQ), catechol (CAT), and 1,2,4-benzenetriol (BT). p-BQ was 13-fold more effective at forming DNA adducts than HQ, which was 7-9-fold more effective than CAT and BT. The DNA adduct formed in human bone marrow (HBM) treated with HQ was the same as that in HL-60 cells. Combination treatment of HL-60 cells with HQ and either CAT or BT increased DNA adduct formation 2.2–6.4-fold. In vitro activation of HQ by myeloperoxidase produced the same DNA adduct in purified DNA as in HL-60 cells and HBM treated with HQ. Reaction of calf thymus DNA with p-BQ produced three adducts. The DNA adduct formed in HL-60 cells treated with p-BQ did not correspond to any of the principal adducts formed in DNA reacted with p-BQ. These results suggest that peroxidase enzymes activate benzene metabolites to form DNA adducts in HBM.