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Bulky DNA damages block transcription and compromise genome integrity and function. The cellular response to these damages includes global transcription shutdown. Still, active transcription is necessary for transcription-coupled repair and for induction of damage-response genes. To uncover common features of a general bulky DNA damage response, and to identify response-related transcripts that are expressed despite damage, we performed a systematic RNA-seq study comparing the transcriptional response to three independent damage-inducing agents: UV, the chemotherapy cisplatin, and benzo[a]pyrene, a component of cigarette smoke. Reduction in gene expression after damage was associated with higher damage rates, longer gene length, and low GC content. We identified genes with relatively higher expression after all three damage treatments, including NR4A2 , a potential novel damage-response transcription factor. Up-regulated genes exhibit higher exon content that is associated with preferential repair, which could enable rapid damage removal and transcription restoration. The attenuated response to BPDE highlights that not all bulky damages elicit the same response. These findings frame gene architecture as a major determinant of the transcriptional response that is hardwired into the human genome., (© 2024 Merav et al.)

Environmental carcinogen benzo(a)pyrene (BaP) is a representative compound of polycyclic aromatic hydrocarbons (PAHs). BaP is strongly associated with prostate carcinogenesis. However, the molecular mechanism of BaP in development of prostate carcinoma remains largely unknown. The aim of this study was to investigate the effect and mechanism of BaP on the development in prostate cancer. PC-3 cells were exposed to different concentrations of BaP for 24, 48, 72 h, respectively. We analyzed the effect of BaP on PC-3 cell viability, cell cycle, DNA strand breaks, mutagenic activity, and migration. The expression of associated regulatory genes and the effect of JAK2/STAT3 signaling were also measured to explore the relationships among BaP metabolism, the JAK2/STAT3 pathway and proliferative activity in PC-3 cells. We observed significant effects on proliferation, DNA strand breaks and mutagenic activity after BaP exposure in PC-3 cells, and inhibitors of CYP1 and the AhR transcription factor α -naphthoflavone (ANF) and CH223191 treatment clearly reduced both cell survival and mutagenesis associated with BaP exposure. Reduction in G0-G1 phase population and elevation in S phase were observed after BaP exposure. Migratory cells for PC-3 were significantly increased. The results were further confirmed by the expression of mRNA levels in the significant increments of Snail, Slug, MMP-9, CYP1A1, CYP1B1, CycilnD1, CDK4 and significant reduction of E-cadherin. Significant enhancements were found in the expression of JAK2, STAT3 after BaP treatment. Additionally, activator IL-6 significantly enhanced the effect of BaP on cell survival, mutagenic activity, Cyclin D1, CDK4, Snail, and JAK2/STAT3 expression in PC-3 cells. Significant reductions in cell survival, mutagenic activity, Cyclin D1, CDK4, Snail, and JAK2/STAT3 expression were found after inhibitor AG490, ANF and CHJ223191 treatment. These findings reveal that BaP enhances the proliferative and mutagenic activity via JAK2-STAT3 pathway in PC-3 cells, and provide the additional evidence to understand the crucial role of BaP in prostate cancer carcinogenesis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Over-activation of microglia disrupts the physiological homeostasis of the brain, and induces inflammatory response and other processes which are implicated in neurodegenerative diseases. Therefore, theoretically, suppression of neuroinflammation would slow the progression of neurodegenerative disease. In this study, we investigated the possible protective effects of Ferulic acid (FA) against benzo(a)pyrene (BaP)-induced microglial activation using BV2 cells as the model system. Exposure of BV2 cells to BaP (10 μM) significantly increased DNA damage and the production of pro-inflammatory mediators, including nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), reactive oxygen species (ROS), malondialdehyde (MDA), and cytokines (interleukins-1β and -6). On the other hand, when BaP-treated BV2 cells were further incubated with FA (10, 20, 40, or 80 mg/mL) for another 24 h, a significant reduction in BaP-induced DNA damage and the release of multiple pro-inflammatory and cytotoxic factors (including interleukin-1β, interleukin-6, NO, and ROS) was observed in a dose-dependent manner. Further study revealed that the microglial NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) signaling pathway was involved in the protective effect of FA. Taken together, these results suggested that FA suppressed BaP-induced toxicity in microglia, and thus may exert neuroprotective effects by inhibiting microglia-mediated pro-inflammatory response., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Benzo [a]pyrene (BaP) have been demonstrated to induce lung cancer risk in humans and many different animal models, with aberrant gene methylation as one of the epigenetic errors; however, the molecular mechanisms remain unclear. Here, we used three types of human lung-derived cells with BaP exposure as a model and attempted to investigate the long non-coding RNA (lncRNA) H19/S-adenosylhomocysteine hydrolase (SAHH) pathway that regulates gene methylation in vitro exposure to BaP. Results showed that compared to the controls, BaP-treated cells H19 expressions were increased in a dose- and time-dependent manner, whereas SAHH protein expressions were decreased. Indeed, H19 binds to and attenuates SAHH expressions and activity, and this interaction will be enhanced by BaP. However, suppression of H19 exaggerates SAHH protein expression and activity exposed to BaP. Although BaP-treated cells H19 single knockdown expectedly increased long interspersed nuclear elements-1 (LINE-1) methylation and inhibited benzo [a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) -DNA adducts formation with altering SAHH protein expressions and activity, the double knockdown restored methylation to the control level and exacerbated BPDE-DNA adducts formation. Overall, our results uncover a H19/SAHH circuit involving gene-methylation alterations by carcinogen BaP., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We used the marine bivalve (Mytilus galloprovincialis) to assess a range of biological or biomarker responses following exposure to a model-engineered nanoparticle, C 60 fullerene, either alone or in combination with a model polycyclic aromatic hydrocarbon, benzo(α)pyrene [B(α)P]. An integrated biomarker approach was used that included: (i) determination of 'clearance rates' (a physiological indicator at individual level), (ii) histopathological alterations (at tissue level), (iii) DNA strand breaks using the comet assay (at cellular level) and (iv) transcriptional alterations of p53 (anti-oncogene) and ras (oncogene) determined by real-time quantitative polymerase chain reaction (at the molecular/genetic level). In addition, total glutathione in the digestive gland was measured as a proxy for oxidative stress. Here, we report that mussels showed no significant changes in 'clearance rates' after 1 day exposure, however significant increases in 'clearance rates' were found following exposure for 3 days. Histopathology on selected organs (i.e. gills, digestive glands, adductor muscles and mantles) showed increased occurrence of abnormalities in all tissues types, although not all the exposed organisms showed these abnormalities. Significantly, increased levels of DNA strand breaks were found after exposure for 3-days in most individuals tested. In addition, a significant induction for p53 and ras expression was observed in a tissue and chemical-specific pattern, although large amounts of inter-individual variability, compared with other biomarkers, were clearly apparent. Overall, biological responses at different levels showed variable sensitivity, with DNA strand breaks and gene expression alterations exhibiting higher sensitivities. Furthermore, the observed genotoxic responses were reversible after a recovery period, suggesting the ability of mussels to cope with the toxicants C 60 and/or B(α)P under our experimental conditions. Overall, in this comprehensive study, we have demonstrated mussels as a suitable model marine invertebrate species to study the potential detrimental effects induced by possible genotoxicants and toxicants, either alone or in combinations at different levels of biological organisation (i.e. molecular to individual levels)., (© The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Gene expression changes induced by carcinogens may identify differences in molecular function between target and non-target organs. Target organs for benzo[a]pyrene (BaP) carcinogenicity in mice (lung, spleen and forestomach) and three non-target organs (liver, colon and glandular stomach) were investigated for DNA adducts by 32P-postlabelling, for gene expression changes by cDNA microarray and for miRNA expression changes by miRNA microarray after exposure of animals to BaP., Results: BaP-DNA adduct formation occurred in all six organs at levels that did not distinguish between target and non-target. cDNA microarray analysis showed a variety of genes modulated significantly by BaP in the six organs and the overall gene expression patterns were tissue specific. Gene ontology analysis also revealed that BaP-induced bioactivities were tissue specific; eight genes (Tubb5, Fos, Cdh1, Cyp1a1, Apc, Myc, Ctnnb1 and Cav) showed significant expression difference between three target and three non-target organs. Additionally, several gene expression changes, such as in Trp53 activation and Stat3 activity suggested some similarities in molecular mechanisms in two target organs (lung and spleen), which were not found in the other four organs. Changes in miRNA expression were generally tissue specific, involving, in total, 21/54 miRNAs significantly up- or down-regulated., Conclusions: Altogether, these findings showed that DNA adduct levels and early gene expression changes did not fully distinguish target from non-target organs. However, mechanisms related to early changes in p53, Stat3 and Wnt/β-catenin pathways may play roles in defining BaP organotropism.

The OECD has developed test guidelines (TG) to identify agents with genotoxic effects. The in vivo alkaline single cell gel electrophoresis (SCGE) assay is currently being prepared to become such a TG. The performance of a combined SCGE/Pig-a gene mutation study was evaluated with the prototypical genotoxicant benzo[a]pyrene (BaP) at an exposure level known to induce germ cell mutation. We aimed to better understand (i) the strengths and weaknesses of the two methods applied in blood and their potential to predict germ cell mutagenicity, and (ii) the involvement of reactive oxygen species (ROS) following in vivo BaP-exposure. To explore the involvement of ROS on BaP genotoxicity, we utilised a mouse model deficient in a DNA glycosylase. Specifically, C57BL/6 mice (Ogg1(+/+) and Ogg1(-/-)) were treated for three consecutive days with 50 mg BaP/kg/day. DNA damage in nucleated blood cells was measured four hours after the last treatment with the SCGE assay, with and without formamidopyrimidine DNA glycosylase (Fpg). Pig-a mutant phenotype blood erythrocytes were analysed two and four weeks after treatment. BaP-induced DNA lesions were not significantly increased in either version of the SCGE assay. The phenotypic mutation frequencies for immature and mature erythrocytes were significantly increased after two weeks. These effects were not affected by genotype, suggesting oxidative damage may have a minor role in BaP genotoxicity, at least in the acute exposure situation studied here. While both assays are promising tools for risk assessment, these results highlight the necessity of understanding the limitations regarding each assay's ability to detect chemicals' genotoxic potential., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Although both arsenic trioxide (As2O3) and benzo(a)pyrene (BaP) are well-established human carcinogens, the interaction between As2O3 and BaP is synergistic or antagonistic remains controversial in terms of the existing studies. In addition, the mechanisms responsible for the combined effects are still unclear. In this study, we examined the potential interactive effects between As2O3 (1, 5, and 10 μM) and BaP (5, 10, and 20 μM) in cultured A549 cells by treating with BaP and As2O3 alone or in combination at various concentrations for 24 h. The single and combined effects of As2O3 and BaP on the cytotoxicity, DNA/chromosomal damage, and oxidative stress were examined by using tetrazolium (3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) dye colorimetric assay, colony formation assay, fluorescence probe, chemical colorimetry, comet assay as well as micronucleus test. Our results showed that As2O3 synergistically enhanced the cytotoxicity, genotoxicity, and level of oxidative stress induced by BaP at various tested concentrations. Also, our experimental results showed that intracellular glutathione (GSH) contents were increased by various doses of BaP, but single or cotreatment with As2O3 significantly decreased the GSH level in the cells at all tested concentrations. Taken together, our results suggest that As2O3 may exert its synergistic cyto- and genotoxic effects with BaP mainly via elevated intracellular reactive oxygen species and reduced GSH contents and superoxide dismutase activities, thus promoting high level of oxidative stress, which may be a pivotal mechanism underlying As2O3 cocarcinogenic action.

Ubiquitylation of histones plays a pivotal role in DNA repair. The ubiquitin ligase Ring2 was recently shown to be the dominant ubiquitin ligase of histone H2A. In a series of experiments using the human bronchial epithelia cells (16HBE) and small interfering RNA (siRNA)-Ring2 cells exposed to benzo(a)pyrene (BaP), we measured dynamic changes in the levels of DNA damage, expressions of ubiquitinated histone H2A, and nucleotide excision repair (NER) subunit xeroderma pigmentosum (XP) groups A, C, and F (XPA, XPC, XPF). We found that in vitro exposure to BaP increased DNA damage in a time- and dose-dependent manner in 16HBE and siRNA-Ring2 cells. The results show that although decrease of Ring2 causes DNA hypersensitivity to BaP, the levels of XPA, XPC, and XPF were not affected. These results indicated that Ring2 may effect the DNA repair through other pathways but not through the expressions of NER protein., (© 2013 Wiley Periodicals, Inc.)

Objective: To identify the available phytochemicals and carotenoids in the selected green algae and evaluate the potential genotoxic/antigenotoxic effect using lymphocytes., Methods: Organic solvent extracts of Chlorococcum humicola (C. humicola) were used for the phytochemical analysis. The available carotenoids were assessed by HPLC, and LC-MS analysis. The genotoxicity was induced by the benzo(a)pyrene in the lymphocyte culture, the genotoxic and antigenotoxic effects of algal carotenoids with and without genotoxic inducer were evaluated by chromosomal aberration (CA), sister chromatid exchange (SCE) and micronucleus assay (MN)., Results: The results of the analysis showed that the algae were rich in carotenoids and fatty acids. In the total carotenoids lutein, β-carotene and α-carotene were found to be present in higher concentration. The frequency of CA and SCE increased by benzo(a)pyrene were significantly decreased by the carotenoids (P<0.05 for CA, P<0.001 for SCE). The MN frequencies of the cells were significantly decreased by the treatment with carotenoids when compared with the positive controls (P<0.05)., Conclusions: The findings of the present study demonstrate that, the green algae C. humicola is a rich source of bioactive compounds especially carotenoids which effectively fight against environmental genotoxic agents, the carotenoids itself is not a genotoxic substance and should be further considered for its beneficial effects.

In the current investigation, the ameliorative effect of green tea (GT) and white tea (WT) against benzo(a)pyrene (BaP) induced oxidative stress and DNA damage has been studied in the livers and lungs of Balb/c mice. A single dose of BaP (125 mg/kg, b.w. orally) increased the levels of lipid peroxidation (LPO) and decreased endogenous antioxidants such as superoxide dismutase (SOD), glutahione reductase (GR), catalase (CAT), and glutathione (GSH) significantly. Pretreatment with GT and WT for 35 days before a single dose of BaP elevated the decreased activity of GR, SOD, and CAT in liver tissue and also tended to normalize the levels of GSH and LPO in both hepatic and pulmonary tissues. The percentage of DNA in comet tail and 8-hydroxy-2'-deoxyguanosine levels reflected the decreasing pattern of DNA damage from the BaP-treated group to the groups that received pretreatment with GT and WT. Our study concludes that both GT and WT are effective in combating BaP induced oxidative insult and DNA damage. However, WT was found to be more protective than GT with respect to CAT (only in the liver), percentage of DNA in comet tail (only in the lungs), GST activity, and GSH content in both the tissues.

Benzo[a]pyrene (BaP) is cytotoxic and/or genotoxic to lung, stomach and skin tissue in the body. However, the effect of BaP on cervical tissue remains unclear. The present study detected DNA damage and the expression of the p53 gene in BaP-induced cervical tissue in female mice. Animals were intraperitoneally injected and orally gavaged with BaP at the doses of 2.5, 5, and 10mg/kg twice a week for 14 weeks. The single-cell gel electrophoresis (SCGE) assay was used to detect the DNA damage. Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to detect the expression of p53 protein and p53 mRNA, respectively. The results showed that BaP induced a significant and dose-dependent increase of the number of cells with DNA damaged and the tail length as well as Comet tail moment in cervical tissue. The expression level of p53 protein and mRNA was increased. The results demonstrate that BaP may show toxic effect on the cervix by increasing DNA damage and the expression of the p53 gene., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.

Several teratogenic agents, including ionizing radiation and xenobiotics such as phenytoin, benzo[a]pyrene, thalidomide, and methamphetamine, can initiate the formation of reactive oxygen species (ROS) that oxidatively damage cellular macromolecules including DNA. Oxidative DNA damage, and particularly the most prevalent 8-oxoguanine lesion, may adversely affect development, likely via alterations in gene transcription rather than via a mutational mechanism. Contributions from oxidative DNA damage do not exclude roles for alternative mechanisms of initiation like receptor-mediated processes or the formation of covalent xenobiotic-macromolecular adducts, damage to other macromolecular targets like proteins and lipids, and other effects of ROS like altered signal transduction. Even in the absence of teratogen exposure, endogenous developmental oxidative stress can have embryopathic consequences in the absence of key pathways for detoxifying ROS or repairing DNA damage. Critical proteins in pathways for DNA damage detection/repair signaling, like p53 and ataxia telangiectasia mutated, and DNA repair itself, like oxoguanine glycosylase 1 and Cockayne syndrome B, can often, but not always, protect the embryo from ROS-initiating teratogens. Protection may be variably dependent upon such factors as the nature of the teratogen and its concentration within the embryo, the stage of development, the species, strain, gender, target tissue and cell type, among other factors., ((c) 2010 Wiley-Liss, Inc.)

Aim of the Study: Although traditional herbal medicines are widely used in Ethiopia, no information is available on their potential genotoxicity. In the present study, hydroalcoholic extracts of Glinus lotoides, Plumbago zeylanica, Rumex steudelii and Thymus schimperi were evaluated for their DNA damaging effects using the comet assay., Material and Methods: Mouse lymphoma L5178Y cells were exposed to different concentrations of the extracts for 3h with and without metabolic activation (S9-mix) using 4-nitroquinoline-N-oxide and benzo(a)pyrene as positive controls, and vehicles as negative controls., Results: In the absence of S9, all extracts were found to induce significant DNA damage without affecting the cell viability. T. schimperi and R. steudelii were the most potent DNA-damaging extracts, and G. lotoides and P. zeylanica the least potent. The addition of S9 had different effects on the DNA damage induced by the extracts: it lowered the DNA damaging effect of P. zeylanica, did not affect the DNA damaging effect of T. schimperi, and increased the DNA damaging effects of R. steudelii and G. lotoides., Conclusion: The findings of the present study suggest that all extracts evaluated have a genotoxic potential in vitro which needs to be substantiated by further studies.

Anthocyanins, belonging to the flavonoid family of phytochemicals, have received attention as agents that may have potential in preventing chronic diseases such as cardiovascular diseases and certain cancers. In the present study, an anthocyanin-rich extract from Concord grapes [referred to as Concord grape extract (CGE)] and the anthocyanin delphinidin were evaluated for their capacity to inhibit DNA adduct formation due to the environmental carcinogen benzo[a]pyrene (BP) in MCF-10F cells, a noncancerous, immortalized human breast epithelial cell line. CGE at 10 and 20 microg/mL and delphinidin at 0.6 microM concentrations significantly inhibited BP-DNA adduct formation. This was associated with a significant increase in activities of the phase II detoxification enzymes glutathione S-transferase and NAD(P)H:quinone reductase 1. In addition, these grape components also suppressed reactive oxygen species (ROS) formation, but did not induce antioxidant response element-dependent transcription. Taken together, these data suggest that CGE and a component grape anthocyanin have breast cancer chemopreventive potential due in part to their capacity to block carcinogen-DNA adduct formation, modulate activities of carcinogen-metabolizing enzymes, and suppress ROS in these noncancerous human breast cells.

Background: Induction of DNA double strand breaks and alterations in the repair of these breaks is implicated in breast carcinogenesis. Prior studies have demonstrated that peripheral blood mononuclear cells (PBMC) from breast cancer patients exhibit increased numbers of DNA strand breaks after exposure to ionizing radiation, but these studies did not specifically measure DNA double strand breaks and it is not known whether chemical carcinogens produce similar effects., Materials and Methods: PBMC from 32 women undergoing breast surgery were genotyped at nine loci of seven DNA repair genes. DNA double strand break repair was measured using the neutral comet assay after exposure to ionizing radiation (0.5 Gy) or bioactivated benzo[a]pyrene (B[a]P, 5 microM., Results: PBMC from breast cancer patients showed higher levels of residual DNA double strand breaks 30 min after exposure to radiation than PBMC from patients with benign breast disease (1.40 times baseline [95% confidence intervals [CI] 1.29-1.51] versus 1.24 times baseline [95% CI 1.15-1.33], respectively, P = 0.04). The response to B[a]P trended in the same direction, but did not reach statistical significance. The MGMT K178R variant genotype was associated with improved DNA double strand break repair in PBMC exposed to B[a]P., Conclusions: Reduced repair of radiation-induced DNA double strand breaks in PBMC is a robust biomarker of breast cancer risk. Reduced DNA repair capacity may have a genetic component even in sporadic breast cancer.

The involvement of oxidative and nitrosative stress mechanisms in several biological and pathological processes including aging, cancer, cardiovascular and neurodegenerative diseases has continued to fuel suggestions that processes can potentially be modulated by treatment with free-radical scavengers and antioxidant. The fermented papaya preparation (FPP) derived from Carica papaya Linn was investigated for its ability to modulate oxidative DNA damage due to H2O2 in rat pheochromocytoma (PC12) cells and protection of brain oxidative damage in hypertensive rats. Cells pre-treated with FPP (50 microg/ml) prior to incubation with H2O2 had significantly increased viability and sustenance of morphology and shape. The human hepatoma (HepG2) cells exposed to H2O2 (50 microM) showed an olive tail moment of 10.56 +/- 1.44 compared to 1.37 +/- 0.29 of the solvent control. A significant reduction (P < or = 0.05) of DNA damage was observed at concentrations > or = 10 microg/ml FPP, with 50 microg/ml FPP reducing the genotoxic effect of H2O2 by about 1.5-fold compared to only H2O2 exposed cells.

Objective: To investigate the expressions of JWA gene and heat shock protein 70 (hsp70) in human embryonic lung (cccHPF-1) cells after exposure to activated benzo(a)pyrene (B(a)P), and to explore the role and the possible mechanism of JWA gene involved in B(a)P-induced DNA damage and repair., Methods: The models of DNA damage of ccc-HPF-1 cells were established by treatment of cells with B(a)P plus S9 at 10 to 100 micromol/L for 3 hours with or without 24 hours recovery for DNA repairing. The DNA damage was detected by single cell gel electrophoresis (SCGE) assay (comet assay). And the immuno-blotting assay was used for detecting expressions of JWA and hsp70., Results: JWA expression was actively modulated by B(a)P exposure. The expressions of both JWA and hsp70 were increased greatly at 50 micromol/L and 100 micromol/L B(a)P treated cells and maintained at over expressed levels treated by 10-100 micromol/L B(a)P during the restored time. In addition, JWA expression pattern was similar to that of hsp70., Conclusion: JWA is determined in this study by its functioning as an effective environmental responsive gene and actively participating in the signal pathways of DNA damage and repair which might be associated with excision repair.

Polkappa is one of many DNA polymerases involved in translesion DNA synthesis (TLS). It belongs to the Y-family of polymerases along with Poleta, Poliota and hREV1. Unlike Poleta encoded by the xeroderma pigmentosum variant (XPV) gene, Polkappa is unable to bypass UV-induced DNA damage in vitro, but it is able to bypass benzo[a]pyrene (B[a]P)-adducted guanines accurately and efficiently. In an attempt to identify factor(s) targeting Polkappa to its cognate DNA lesion(s), we searched for Polkappa-interacting proteins by using the yeast two-hybrid assay. We found that Polkappa interacts with a C-terminal region of hREV1. Poleta and Poliota were also found to interact with the same region of hREV1. The interaction between Polkappa and hREV1 was confirmed by pull-down and co-immunoprecipitation assays. The C-terminal region of hREV1 is known to interact with hREV7, a non-catalytic subunit of Polzeta that is another structurally unrelated TLS enzyme, and we show that Polkappa and hREV7 bind to the same C-terminal region of hREV1. Thus, our results suggest that hREV1 plays a pivotal role in the multi-enzyme, multi-step process of translesion DNA synthesis., (Copyright Blackwell Publishing Limited)

The aim of this study was to investigate the chemoprotective effects of mustard sprouts on benzo(a)pyrene [B(a)P]-induced DNA damage in the single cell gel electrophoresis (SCGE)/Hep G2 assay. This model combines the advantages of the SCGE assay with that of human-derived cells that possess inducible phase I and phase II enzymes. Treatment of the cells with small amounts of mustard juice (0.1-1.25 microl/ml) and B(a)P reduced the genotoxic effect of the carcinogen in a dose-dependent manner. Contrary to the results with the juice, unexpected synergistic effects were observed with allyl isothiocyanate (AITC, 0.3 microM), a breakdown product of sinigrin, which is contained in black mustard and many other cruciferous vegetables. Although these concentrations of AITC did not cause DNA damage per se, pronounced dose-dependent DNA damage was seen with higher concentrations of AITC (>or= 25 microM). In parallel with the comet assays, also enzyme measurements were carried out which showed that exposure of the cells to mustard juice (2.0 microl/ml) causes a moderate induction of ethoxyresorufin-O-deethylase, and more pronounced (approximately 2-fold) increase of the activity of glutathione-S-transferase. In conclusion, our findings indicate that i) mustard juice is highly protective against B(a)P-induced DNA damage in human derived cells and ii) that induction of detoxifying enzymes may account for its chemoprotective properties. iii) Furthermore, our findings show that the effects of crude juice can not be explained by its allyl isothiocyanate contents., (Copyright 2003 Wiley-Liss, Inc.)

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by 32P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or beta-naphthoflavone (beta-NF) or digestive glands from mussels. The beta-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10(8) nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the 32P-postlabelled BaP 7,8-diol, 9,10-epoxide-N2-guanine adduct. Fewer adducts (P < 0.05) were generated by BaP-induced microsomes (9.4-30.6 adducts per 108 nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10(8) nucleotides). Co-incubation with 500 microM alpha-naphthoflavone (alpha-NF) resulted in 97-99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.

This study examined the effect of exogenous benzo[ a ]pyrene (BaP), an important constituent of cigarette smoke, on cultured bovine retinal pigment epithelial (RPE) cells. Evidence is presented for its metabolic conversion into benzo[ a ]pyrene diol epoxide (BPDE) and the consequent formation of potentially cytotoxic nucleobase adducts in DNA. Cultured RPE cells were treated with BaP at concentrations in the range of 0-100 microM. The presence of BaP was found to cause inhibition of cell growth and replication. BaP induced the expression of a phase I drug metabolizing enzyme which was identified as cytochrome P450 1A1 (CYP 1A1) by RT-PCR and by Western blotting. Coincident with the increased expression of CYP 1A1, covalent adducts between the mutagenic metabolite BPDE and DNA could be detected within RPE cells by immunocytochemical staining. Additional support for their formation was afforded by nuclease P1 enhanced (32)P-postlabelling assays on cellular DNA. Single-cell gel electrophoresis (comet) assays showed that exposure of RPE cells to BaP rendered them markedly more susceptible to DNA damage induced by broad band UVB or blue light laser irradiation. In the case of UVB, this is consistent with the photosensitization of DNA cleavage by nucleobase adducts of BPDE. Collectively, these findings imply that BaP has a significant impact on RPE cell pathophysiology and suggest mechanisms whereby exposure to cigarette smoke might cause RPE dysfunction and cell death, thus possibly contributing to degenerative disorders of the retina., (Copyright 2002 Elsevier Science Ltd.)

Differences in the incidence of prostate cancer (CaP) amongst different migrant populations point to causative agents of dietary and/or environmental origin. Prostate tissues were obtained following transurethral resection of the prostate (TURP) or radical retropubic prostatectomy. After surgery, TURP-derived or tumour-adjacent tissue fragments were minced in warm PFMR-4A medium (37 degrees C) and suspensions pipetted into collagen-coated petri dishes. Non-adherent material was removed by washing with fresh medium after 12 h. Adhered cells subsequently reacted positively with monoclonal antibodies to prostate specific antigen (PSA). PSA was also detected in the medium. The genotoxicities of the chemical carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), its N-hydroxy metabolite (N-OH-PhIP) and benzo[a]pyrene (B[a]P) in adherent cell populations from different donors (n=8) were examined. Cells were treated in suspension for 30 min at 37 degrees C in the presence of the DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C). DNA single-strand breaks were detected in cells by the alkaline single cell-gel electrophoresis ('Comet') assay and quantified by measuring comet tail length (CTL) in microm. All three carcinogens induced dose-related increases in CTLs (P<0.0001) in cells from four donors 24 h post-seeding. However, in cells from a further two donors the genotoxic effects of PhIP, N-OH-PhIP and B[a]P were much less apparent after 48 h than after 24 h in culture. After 96 h in culture, cells from these donors appeared to be resistant to the comet-forming activity of the compounds. However, B[a]P-DNA adducts were still measurable by (32)P-postlabelling for up to 14 days following a 24-h exposure to 50 microM B[a]P in adhered cells from another two donors. This study shows that primary cultures of cells derived from the prostate can activate members of two classes of chemical carcinogens. Further development may provide a robust model system in which to investigate the aetiology of CaP.

An antimutagenic activity of fluphenazine, todralazine, anthocyanins and alkylresorcinols was established in a battery of short-term cytogenetic tests. One of the possible mechanisms of their antimutagenic action could be an increase in apoptotic elimination of heavily-damaged cells from a culture. In this paper we provide data on quantitative estimation of the antimutagens' impact on apoptosis in lymphocyte cultures exposed in the G(0)-phase to genotoxic agents: hydrogen peroxide (0.2mM, 20 min.) or benzo[a]pyrene (40 microM, 90 min.), and then cultured for 36 hrs in the presence of a lectin (PHA-M, 1% v/v) and each of the tested antimutagens. Apoptosis was estimated by means of microscopic examination of cell smears stained with a mixture of fluorochromes (ethidium bromide/acridine orange) as well as of the results of DNA separation with the field inversion gel electrophoresis. By microscopic examination we assessed that the frequencies of cells exhibiting morphological features of apoptosis considerably increased in the cultures containing the antimutagens. The FIGE separation of DNA from those cultures proved that the DNA content in the 30-50 kb domain was markedly elevated, as compared with the control cultures that did not contain antimutagens. It was established in the regression analysis that the apoptosis-enhancing effect significantly depended on the concentration of each tested antimutagen in a culture medium. However, marked differences of apoptosis-enhancing potency were noticed among the four antimutagens. The multicriterial analysis proved that the apoptosis-enhancing effects of fluphenazine and also, to a smaller extent, by alkylresorcinols, were many times stronger than those of anthocyanins and of todralazine. The results suggest that the enhancement of apoptosis by fluphenazine and by alkylresorcinols can explain a major part of their antimutagenic activity, whereas in the case of anthocyanins and of todralazine other mechanisms of antimutagenic action should be sought for.

Immunoglobulins G (lgG) specific for benzo[a]pyrene-DNA adducts were elicited in Swiss mice by repeated subcutaneous injections of a high molecular weight benzo[a]pyrene-DNA conjugate-adjuvant mix. The immunization procedure resulted in the production of specific antibodies against adducted benzo[a]pyrene B[a]P in all treated animals. One week after completion of the immunization procedure, groups of ten immunized and ten non immunized female mice were treated by single intraperitoneal injection with two different doses of B[a]P. The mice were sacrificed 48 hours after treatment, and both liver and bone marrow cells were isolated for subsequent determinations of DNA binding and micronucleus induction, respectively. Covalent benzo[a]pyrene adducts in liver DNA were detected by competitive ELISA and the incidence of micronucleated polychromatic erythrocytes was evaluated by scoring one thousand cells per animal. The determination of DNA adducts in liver revealed significantly (p < 0.05) lower levels of B[a]P adducts in immunized mice compared to non-immunized animals at both doses, whereas no significant difference was observed between controls. Administration of benzo[a]pyrene produced moderate, dose-related increases in the incidence of micronucleated polychromatic erythrocytes in all treated groups, with no significant difference between immunized and non-immunized mice. The decrease of covalent DNA adducts in the liver of immunized mice suggests that the specific humoral immunity elicited by repeated carcinogen exposure may act as a relevant modulating factor in chemical carcinogenesis.

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

During previous studies, we found that mdm2 mRNA levels were elevated in benzo[a]pyrene (BaP, a polycyclic aryl hydrocarbon)-treated cells under conditions of DNA damage-induced cell cycle arrest (Vaziri, C., and Faller, D. V. (1997) J. Biol. Chem. 272, 2762-2769). We have identified potential aryl-hydrocarbon receptor-binding sites in the mdm2 promoter. However, we show that induction of mdm2 mRNA by BaP is entirely dependent upon aryl-hydrocarbon-induced genotoxicity and does not involve direct aryl-hydrocarbon receptor-mediated transcriptional activation of the mdm2 gene. Heterologous mdm2 promoter-reporter constructs containing p53-response elements were not responsive to BaP treatment. Therefore the p53-response elements in the mdm2 promoter are insufficient to confer DNA damage-dependent expression of mdm2. Furthermore, mdm2 transcripts were induced by BaP in p53 null cells from transgenic mice (although both basal and BaP-induced mdm2 expression levels were reduced in these cells relative to p53(+/+) cultures). These data show that p53-mediated mechanisms cannot account for BaP/DNA damage-induced mdm2 expression. Mdm2 promoter-reporter gene assays and nuclear run-off analyses of nascent mdm2 transcripts showed that transcriptional induction was unable to account for the large changes in mdm2 transcript levels following BaP treatment. However, mdm2 mRNA half-life measurements showed stabilization of the mdm2 transcript (from approximately 1 h to >4 h) in response to BaP. To our knowledge, this is the first report of control of mdm2 at the post-transcriptional level and in a p53-independent manner. Transient ectopic expression of mdm2 strongly augmented aryl-hydrocarbon-induced apoptosis, demonstrating that mdm2 levels can have a profound effect on the cellular response to DNA damage. Overall, our results suggest a potentially important link between DNA damage signaling and RNA stability that may be relevant to cell cycle regulation, tumor suppression, and environmental carcinogenesis.

A plant polysaccharide, Aloe gel extract, was reported to have an inhibitory effect on benzo[a]pyrene (B[a]P)-DNA adduct formation in vitro and in vivo. Hence, chemopreventive effects of plant polysaccharides [Aloe barbadensis Miller (APS), Lentinus edodes (LPS), Ganoderma lucidum (GPS) and Coriolus versicolor (CPS)] were compared using in vitro short-term screening methods associated with both initiation and promotion processes in carcinogenesis. In B[a]P-DNA adduct formation, APS (180 micrograms/ml) was the most effective in inhibition of B[a]P binding to DNA in mouse liver cells. Oxidative DNA damage (by 8-hydroxydeoxyguanosine) was significantly decreased by APS (180 micrograms/ml) and CPS (180 micrograms/ml). In induction of glutathione S-transferase activity, GPS was found to be the most effective among plant polysaccharides. In screening anti-tumor promoting effects, APS (180 micrograms/ml) significantly inhibited phorbol myristic acetate (PMA)-induced ornithine decarboxylase activity in Balb/3T3 cells. In addition, APS significantly inhibited PMA-induced tyrosine kinase activity in human leukemic cells. APS and CPS significantly inhibited superoxide anion formation. These results suggest that some plant polysaccharides produced both anti-genotoxic and anti-tumor promoting activities in in vitro models and, therefore, might be considered as potential agents for cancer chemoprevention.

In order to increase the understanding of the factors responsible for causing human colon cancer, a technique was developed to detect genotoxic effects of chemicals in human colon cells. Risk factors suspected to be associated with the aetiology of human colon cancer were subsequently investigated: the method is based on the measurement of DNA damage in primary cells freshly isolated from human colon biopsies with the single cell microgel ectrophoresis technique ('Comet Assay'). 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methyl-3H-imidazo[4,5f]quinoline (IQ), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), dinitrosocaffeidine (DNC) lithocholic acid (LCA), hydrogen peroxide (H2O2) and benzo[a]pyrene (B[a]P) were investigated for their genotoxic and cytotoxic effects following 30 min incubation with colon cells of human, and for comparative purposes also of the rat colon. The nitrosamides (MNNG, DNC) were very genotoxic in human colon cells. MNNG was more genotoxic in human than in rat colon cells. In contrast, the rat colon carcinogens PhIP and IQ were not genotoxic in human colon cells. PhIP did induce DNA damage in rat colon cells, which correlates to its capacity of inducing tumors in this animal tissue. LCA was toxic (rat > human) and concomitantly caused DNA damage in higher concentrations. The widespread contaminant B[a]P was not genotoxic in colon cells of either species using this system. H2O2 was found to be a potent genotoxic agent to both rat and human colon cells (human > rat). In summary, those compounds chosen as representatives of endogenously formed risk factors (MNNG, H2O2, LCA) have a higher toxic and/or genotoxic potency in human colon tissue than in rat colon. They are also more effective in this system than the contaminants tested so far (B[a]P, PhIP, IQ). The newly developed technique is rapid and yields relevant results. It is a novel and useful approach to assess different chemical compounds for genotoxic activities in tumour target tissues of the human.

Crocetin is a major component in the fruit of Gardenia jasminoides Ellis, a Chinese herbal medicine. In the work, we investigate the protective action and mechanism against benzo(a)pyrene [B(a)P]-induced genotoxicity and neoplastic transformation with a non-toxic dose of crocetin (0.01-0.10 mM) for 1 hour prior to the administration of 0.1 mM B(a)P. B(a)P genotoxicity was inhibited significantly by crocetin in a dose responsive manner. Pretreating C3H10T1/2 cells with crocetin (0.1 mM) also caused a decrease in the covalent binding of B(a)P-diol-epoxide to DNA, to about half that of cells without crocetin treatment. Crocetin also inhibited B(a)P-induced transformations. When the culture was treated with crocetin (0.01, 0.05 and 0.10 mM) for 7 days, the transformation frequencies were lower than that of the culture without crocetin treatment. Furthermore, pretreating cells with crocetin (0.01-0.10 mM) also caused an increase in the activity of GSH S-transferase (GST) to 18-71% that of the cells without crocetin treatment. These results suggest that the inhibition by crocetin of B(a)P-induced genotoxicity and neoplastic transformation in C3H10T1/2 cells is due to a mechanism that increases the activity of GST and decreases the formation of a B(a)P-DNA adduct.

In a comparative study, benzo[a]pyrene (BaP), cyclophosphamide (CP), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and tetrachloroethylene (PER) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) test and the sister-chromatid exchange (SCE) test with human blood cells. MNNG as well as S9 mix activated BaP- and CP-induced DNA effects in both tests in a dose-dependent manner. While the range of concentrations which induced DNA migration or SCE was the same for MNNG and for BaP, much higher CP concentrations were necessary for a positive response in the SCG test than in the SCE test. PER was tested in the absence and in the presence of S9 mix and neither induced DNA migration nor increased SCE frequencies. In these experiments, a clear cytotoxic effect of PER was observed. To investigate a possible influence of DNA repair on the effects in the SCG test, cells were treated for 2 h and further incubated for 1 h after removal of the test substance. This procedure caused a clear decrease in induced DNA migration in experiments with BaP and CP, whereas no reduction was found with MNNG. This modified protocol did not lead to the detection of DNA effects after treatment with PER. The results indicate that the SCG test responds to various DNA lesions and does not seem to be sensitive to non-genotoxic cell killing. Its sensitivity obviously depends on the type(s) of induced DNA lesions and the effects can be modified by DNA repair processes in a complex manner. For the detection of genotoxic properties of chemicals with the in vitro SCG test, a single evaluation at the end of the exposure period seems to be sufficient.

Piperine, a major component of black pepper and long peppers, has been reported previously to have an effect on the activation and deactivation of some exogenous substances. In the present study, piperine was found to promote DNA damage and cytotoxicity induced by benzo[a]pyrene (B[a]P) in cultured V-79 lung fibroblast cells. The V-79 cells were treated with a non-toxic dose of piperine (1-20 microM) plus 10 microM B[a]P, or pretreated with piperine for 30 min or 2 hr prior to the administration of 10 microM B[a]P. B[a]P cytotoxicity was potentiated significantly by piperine under each experimental condition. The relative plating efficiency (RPE) was 71% when V-79 cells were exposed to 10 microM B[a]P alone. When the culture was exposed to B[a]P plus piperine or pretreated with piperine for 30 min prior to the administration of B[a]P, the RPE values were 63 and 44% (P < 0.001), respectively. Pretreatment with piperine for 2 hr had no significant effect (P > 0.05). Furthermore, the lowest activities (P < 0.05) of glutathione S-transferase (GST) and uridine diphosphate glucuronyl transferase (UDP-GTase) of piperine-treated V-79 cells occurred 30 min to 1 hr after the piperine pretreatment. Pretreatment of V-79 cells with piperine also caused an increase in the covalent binding of B[a]P-diol-epoxide to DNA, 2.3 times greater than that of the V-79 cells without piperine treatment. These results suggest that the promotion by piperine of B[a]P-induced cytotoxicity in V-79 lung fibroblast cells is due to mechanisms that decrease the activities of GST and UDP-GTase and increase the formation of a B[a]P-DNA adduct.

Syrian golden hamsters are much more susceptible than Wistar rats to the induction of tracheal tumors by benzo[a]pyrene (B[a]P). To investigate whether this difference is reflected in the pattern of DNA adduct induction and removal, tracheas from either species were isolated and exposed to B[a]P (5 micrograms/ml) in organ culture. At various time-points B[a]P-DNA adducts were quantified by 32P-postlabeling; unscheduled DNA synthesis (UDS) and cell proliferation were determined by [3H]thymidine incorporation during the 18 h before sampling. In an induction-repair experiment tracheas were exposed to B[a]P for 2 days, and cultured for another 4 days without B[a]P. After 2 days of exposure total B[a]P-DNA adduct levels were 10 times higher in hamster compared to rat tracheas. In hamster tracheas one major adduct was formed (95%), namely the adduct between (+)-anti-benzo[a]pyrene diolepoxide and deoxyguanosine (BPDE-N2dG). In rat tracheas BPDE-N2dG comprised approximately 60% of the total B[a]P-DNA adduct level. The other major adduct found in rat tracheas is probably derived from interaction of syn-BPDE and deoxyadenosine. During exposure to B[a]P in hamsters the adduct level increased to 36 +/- 19 adducts/10(6) nucleotides (add/10(6)n) on day 2. Two days after removal of B[a]P the B[a]P-DNA adduct level had decreased to 60% of that on day 2; there was no further decrease in the B[a]P-DNA adduct level, despite considerable cell proliferation at the end of the 6 day culture period. UDS increased during exposure to B[a]P and decreased after removal of B[a]P. In rats removal of B[a]P did not lead to a decrease in the B[a]P-DNA adduct level, which agreed with the observed absence of UDS. In a second experiment tracheas were exposed to B[a]P continuously for 15 days. In hamster tracheas the total B[a]P-DNA adduct level increased from 11 +/- 0.7 add/10(6)n after 1 day of exposure to 105 +/- 2 add/10(6)n after 15 days; also UDS increased with increasing exposure until day 11. Cell proliferation was low at the end of the culture period. In rat tracheas no progressive increase in the B[a]P-DNA adduct level was seen, UDS was not increased and cell proliferation had increased significantly at the end of the exposure period. The extent of adduct induction in the trachea of the two species corresponded with the different susceptibilities to B[a]P-induced tumor formation.

p53 protein expression has been shown to increase in response to DNA damage in cell culture systems. We have studied p53 expression and benzo[a]pyrene (B[a]P)-induced DNA-damage in the form of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts as measured by synchronous fluorescence spectrophotometry (SFS) in B[a]P-treated C57BL/6 mouse skin. Polyclonal murine antibody CM5, which is comparable to human CM1, detecting both wild-type and mutated protein, was used. BPDE-DNA adducts reached their maximum at 24 h after all dosage regimens, but were very well detectable also at 12 and 48 h after the treatment, while no adducts were measurable at 1 week and thereafter. p53 expression was seen in 9/17 (53%) skin samples from mouse treated with 500 microgram of B[a]P 12-48 h after the treatment, while all 25 (100%) cases of similarly treated mouse skins were negative after 30 weeks of the treatment. Only one positive sample of total 11 was found among mice treated with repeated 62.5 micrograms doses and this was 24 h after the last treatment. After one 62.5 micrograms dose all mice were negative. This is the first report of an association of p53 protein with DNA damage in vivo and gives support for the putative function of p53 in cellular defense machinery towards chemical damage.

Several lines of evidence indicate the important role of mutation in the process of carcinogenesis, however our understanding of the factors that influence the formation of mutation is still limited. Recent developments in the analysis of gene mutations at selectable loci in cultured mammalian cells has allowed to accumulate a significant amount of information on the type and distribution of mutations induced by a variety of chemical and physical agents. The purpose of this review is to summarize the data relative to chemical carcinogens and to discuss how their mechanism of interaction with DNA and the repair processes active on the induced lesions affect mutational spectra.

A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studies seeking to investigate associations between DNA repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the suggestion that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum is manifested as a result of the reduced capacity of patients' cells to repair DNA damaged by UV-mimetic agents. For the assay, damaged, nonreplicating, recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (cat) reporter gene is introduced into lymphocytes by using a DEAE-dextran/DNA complex short-term transfection conditions. Excision repair of the damaged bacterial cat gene is monitored proportionately as a function of reactivated CAT enzyme activity following a 40-h repair/expression incubation period. The validity of the approach was indicated by the ability of the assay to discriminate xeroderma pigmentosum virus-transformed lymphocyte cell lines of both severe (complementation groups A and D) and moderate (complementation group C) excision repair deficiencies from repair-proficient cell lines. Similar results were observed when a mitogen-stimulated peripheral blood lymphocyte culture from an xeroderma pigmentosum A patient was assayed concurrently with mitogen-stimulated peripheral blood lymphocytes obtained from healthy individuals. Adaptation of this DNA repair assay as a field test in a pilot-tested select group of basal cell carcinoma patients and cancer-free controls led to the preliminary identification of a specific subset at risk for this disease as a consequence of significant reduction to the repair of photochemically (UV)-damaged plasmid DNA.

It was the aim of this study to compare the formation of DNA adducts in different human white blood cells. Lymphocytes and monocytes were isolated from peripheral blood by density centrifugation and by a monocyte-specific antibody linked to magnetic beads. DNA adducts were determined by 32P-postlabelling. After in vitro incubation with benzo[a]-pyrene (B[a]P) we found DNA adducts in monocytes, but not in unstimulated blood lymphocytes. Only after growth stimulation by phytohaemagglutinin did lymphocytes show an adduct pattern similar to monocytes. In contrast, B[a]P-7,8-dihydro-9,10-diolepoxide, an activated intermediate of B[a]P metabolism leads to similar DNA adducts in both cell types. When lymphocytes were incubated in the presence of B[a]P together with monocytes, then after subsequent separation lymphocytes exhibited most of the adducts found in monocytes, though to a lower extent. Our results suggest that unstimulated blood lymphocytes--which are unable to activate metabolically promutagens like B[a]P--receive the genotoxic material to form DNA adducts from other blood cells or from cells in the vessel lining. We conclude that the amount and the pattern of DNA adducts formed in whole white blood cells may be influenced considerably by a variable proportion of monocytes.

The ability of sodium bisulfite to induce morphological transformation and mutagenesis of cultured Syrian hamster embryo cells was examined. Treatment of the cells at neutral pH for 15 min with 5-20 mM sodium bisulfite resulted in a dose-dependent increase in cell transformation but no induction of gene mutations measured at two genetic loci. Treatment of the cells for 24 h increased the level of transformation, but also failed to induce chromosome aberrations, aneuploidy or DNA strand breaks in the cells. The only positive response for a DNA alteration was an increase in sister chromatid exchanges, but this effect was observed only with the longer exposures and not with the 15 min exposure, which also transformed the cells. Possible mechanisms for bisulfite-induced cell transformation are discussed.

In Chinese hamster ovary (CHO) cells, benzo[a]pyrene induces both persistent and transient lesions that are detected by alkaline sucrose gradient sedimentation analysis (ASG sites). The transient lesions disappear within 15 min while the persistent lesions can be detected for several hours following treatment. Although the persistent ASG sites are believed to be repaired by excision repair, the process responsible for the disappearance of the transient ASG sites is unknown. To determine the contribution of excision repair to the removal of these transient lesions, CHO cells were treated with benzo[a]pyrene (B(a)P) in the presence of the inhibitors of excision repair, araC and novobiocin. The results indicate that: (1) araC inhibits the removal of persistent, but not the transient B(a)P-induced ASG sites; (2) novobiocin, a putative inhibitor of the incision step of DNA excision repair, reduced the number of lesions detected immediately following treatment, indicating that many of these lesions may represent single-strand discontinuities generated during repair; and (3) the lesions detected in the presence of novobiocin disappear rapidly following treatment. Based on these results, we concluded that B(a)P-induced transient ASG sites are repaired by a process other than excision repair.

The effects of benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on cell growth and DNA damage were determined in primary cultures of rat tracheal epithelial cells dissociated from rat tracheas. Cell cultures treated with 25 microM BAP for 24 h or 200 microM HCHO for 90 min did not have a marked reduction in cell growth. However, their combined treatment reduced cell growth by 60% of control when cultures were exposed to BAP followed by HCHO as well as the reverse order. None of these treatments significantly decreased cell viability as judged by dye exclusion, nor did they enhance cell terminal differentiation as measured by cornified envelope formation. Alkaline elution analysis of DNA damage detected both DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) as a result of HCHO treatment, whereas BAP treatment caused only SSB. While HCHO-induced SSB were repaired within 2 h, BAP-induced SSB were detected 3 days after treatment. Combined treatment of cell cultures with BAP followed by HCHO resulted in more SSB than was obtained from either agent alone, but less DPC than was detected from HCHO alone. The increased number of SSB obtained from this combined treatment may be related to the marked enhancement of carcinogenesis observed in earlier in vivo-in vitro studies.

The biological activity of a series of nitropyrenes was assayed by measuring their ability to induce the asynchronous replication of viral DNA in rat fibroblasts transformed by a ts-a mutant of polyoma virus. Concentrations of 10-30 micrograms/ml of 1-nitropyrene (1-NP) induced viral replication, and this effect was enhanced by addition of rat-liver S9 microsomal fraction (300 micrograms/ml) to the culture medium. The response was less than that obtained with 0.1 micrograms/ml of the activated metabolite of benzo[a]pyrene (BP), BP trans-7,8-dihydrodiol-9,10 epoxide (anti) (BPDE). A series of di-, tri-, and tetra-nitropyrenes were also found to induce polyoma DNA replication, in the absence of exogenous microsomal activation, displaying strongly positive effects at 0.5-2.0 microgram/ml. Dose-response curves with 1,6-dinitropyrene (1,6-DNP) from 0.01 to 0.5 microgram/ml indicated that this compound was approximately equipotent with BPDE for induction of polyoma DNA synthesis. Studies of drug metabolism, DNA binding and DNA adduct formation indicate that 1,6-DNP is metabolized in this cell line, binds to DNA, and forms stable adducts. The level of DNA modification seen with 1,6-DNP is higher than that observed under comparable conditions with an equivalent dose of BPDE. These findings provide additional evidence that the nitropyrene class of compounds can exert biological effects in mammalian cells, and that the dinitropyrenes are more potent than 1-NP.

The nuclei of cells from regenerating rat liver were incubated with benzo(a)pyrene and the concentrations of the metabolites that covalently bound to DNA of different nuclear fractions were compared. It appeared that DNA associated with nuclear matrix (containing replicating DNA) is modified most intensively. The synchronized mouse embryo cells were incubated with benzo(a)pyrene during S phase and the levels of modifications in short and long single-stranded DNA fragments were compared. It has been observed that replicating DNA is represented in short fragments. These short DNA fragments were found to be modified by benzo(a)pyrene 4-9 times more intensively than total DNA. The possible mechanisms of both the increase in the number of DNA modifications in proliferating cells and the reason for the enhancement of carcinogenic effect on dividing cells are being discussed.

The confluent culture of hamster embryo cells was incubated with benzo(a)pyrene for 24 hours. Then the medium was replaced by maximal lacking both the serum and benzo(a)pyrene. The process of DNA repair was observed in four nuclear fractions according to two indexes: the disappearance of metabolites of benzo(a)pyrene covalently bound to DNA and the incorporation of 3H-thymidine to DNA in the period from I min to 72 hours. Hydroxyurea at the concentration of 5 mM was added 2-19 hours before 3H-thymidine. The highest concentration of benzo(a)pyrene metabolites was found in the DNA of nuclear matrix fraction throughout all the experiment. The initial concentration of 3H-thymidine right after its addition into the cell culture medium was the highest in DNA of nuclear matrix fraction and the lowest in DNA fraction soluble in the buffer with low ionic strength. Later on, the concentration of 3H-thymidine was decreased in matrix-bound fractions and increased in other fractions up to the total DNA level. The results suggest that the repair process requires joining of benzo(a)pyrene damaged DNA region to the nuclear matrix with the following reverse transition into the fraction where the fragment was initially located.

This study was designed to investigate the effects of four compounds that are shown to influence the cytochrome P450 system, on the metabolism of and DNA adduct formation by benzo[alpha]pyrene (BaP) in human skin epithelial cells in culture. Radiolabeled BaP was used in the metabolism studies, and the levels of metabolites in the ethylacetate extracts of the intracellular and extracellular fractions were determined by HPLC. Among the various metabolites detected BaP-7,8-diol was the only one that was an intermediate on the activation pathway of BaP to the ultimate carcinogen, BPDE I. Both BHA and 7,8-BF pretreatment significantly decreased intracellular production of BaP-7,8-diol compared to cultures treated with only radiolabeled BaP. MeBHA pretreatment greatly increased intracellular BaP-7,8-diol formation compared to BaP treated controls, while disulfiram pretreatment had no effect on the intracellular concentration. Cultures pretreated with BHA, 7,8-BF or disulfiram formed 30-40% less BPDE I-dG adducts than nonpretreated cultures, while cultures pretreated with MeBHA exhibited approximately 200% increase in the BPDE I-dG adduct formation. Thus, BHA and 7,8-BF act similarly in reducing BaP activation and adduct formation. Alternatively, MeBHA increased BaP activation and adduct formation in human keratinocyte cultures in vitro. Disulfiram pretreatment did not reduce BaP-7,8-diol formation, but decreased BPDE I-dG adducts. These studies indicate that modulators of the P450 system act in different fashions at the level of production of an oxygenated procarcinogen metabolite, altering the amount of specific carcinogen-dG adducts that lead to the expression of a transformed phenotype.

Bacteriophage T7 gene 4 protein is an essential component of the T7 DNA replication system, acting as both a primase and a helicase. The gene 4 protein has been shown to translocate along single-stranded DNA in the 5'----3' direction, using an energy source for this movement the hydrolysis of nucleoside 5'-triphosphates, preferably dTTP. Thus, measuring the rate and extent of dTTP hydrolysis provides a means to directly measure translocation. We have determined that the hydrolysis of dTTP by the gene 4 protein is strongly inhibited by the presence of benzo[a]pyrene (B[a]P) adducts on the DNA. Time course experiments on adduct-containing DNA show that after an initial burst of hydrolysis, which parallels what is observed on unmodified DNA, further hydrolysis abruptly ceases. Addition of excess unmodified DNA does not restore the hydrolysis activity. These data suggest that the gene 4 protein is blocked and sequestered on the DNA at the site of the adduct. This was confirmed by experiments in which gene 4 protein preferentially protected the radiolabeled adduct-containing DNA but not randomly labeled M13 DNA. The gene 4 protein bound to the B[a]P-modified DNA was isolated, and the complex was found only to contain dTTP. These results have been used to formulate a model for gene 4 protein translocation in which we speculate that the power stroke for unidirectional movement along the single-stranded DNA is the displacement of dTDP by dTTP. Finally, we observe a constant ratio of DNA synthesis to dTTP hydrolysis regardless of the number of B[a]P adducts in the template suggesting that a significant portion of the inhibition of DNA synthesis is a direct consequence of the inhibition of gene 4 translocation.

The effect of modifying the T7 promoter-containing plasmid pDR100 with aminofluorene (AF), acetylaminofluorene (AAF), and benzo[a]pyrene (B[a]P) adducts on RNA synthesis by the T7 RNA polymerase was determined. We found that increasing levels of each of the three adducts caused a progressive decline in RNA synthesis, but that the inhibition produced by benzo[a]pyrene adducts was substantially greater than that caused by either AF or AAF adducts. Polyacrylamide gel electrophoresis analysis confirmed that the B[a]P adducts more strongly inhibited chain elongation since their presence resulted in the production of RNA fragments having average chain lengths very close to that predicted if each adduct permanently blocked the polymerase during transcription. We have also determined the effect of each type of adduct on the initiation of transcription and show that the T7 RNA polymerase initiates as efficiently on both AF or AAF-containing templates as it does on unmodified DNA, while B[a]P adducts were found to increase in the number of chain starts.