BP-induced DNA Damage Research Articles

Mixture effects of food-relevant polycyclic aromatic hydrocarbons on the activation of nuclear receptors and gene expression, benzo[a]pyrene metabolite profile and DNA damage in HepaRG cells

Carcinogenic benzo[a]pyrene (BP) and other non-carcinogenic polycyclic aromatic hydrocarbons (PAH) like fluoranthene (FA) and pyrene (PYR) occur as food contaminants. Molecular effects of BP, FA and PYR in human liver cells were investigated using mixtures occurring in grilled meat. Activation of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) was investigated along with target gene expression. Mixture effects on BP metabolite profile and DNA-damaging potential were studied as biological downstream effects.Compared to BP, FA and PYR activated the AHR only weakly. Mixtures were less efficient than BP. Analysis of CYP1A1 expression showed synergistic induction after co-exposure in HepaRG cells. FA and PYR were strong CAR agonists, whereas BP was less potent. Mixtures containing BP caused a strong decrease of CAR transactivation in line with lower CYP2B6 expression. The BP metabolite profile and BP-induced DNA damage were only weakly affected.PAH mixtures modulate AHR, CAR activation and their target genes. However, these mixture effects appear not to be reflected at the level of downstream events like BP metabolite formation or BP-induced DNA damage. Our study clearly shows that endpoints at all biological levels should be considered for mixture evaluation, instead of drawing conclusions exclusively based on early molecular events.

Inhibition of Ku70 in a high-glucose environment aggravates bupivacaine-induced dorsal root ganglion neurotoxicity

BackgroundBupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression. MethodsWe examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50 mM glucose, and the cells were then treated with BP for 3 h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting. ResultsCompared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions. ConclusionThese data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70.

Gas Chromatography-Mass Spectrometry Analysis of Ulva fasciata (Green Seaweed) Extract and Evaluation of Its Cytoprotective and Antigenotoxic Effects.

The chemical composition and biological properties of Ulva fasciata aqueous-ethanolic extract were examined. Five components were identified in one fraction prepared from the extract by gas chromatography-mass spectrometry, and palmitic acid and its ethyl ester accounted for 76% of the total identified components. Furthermore, we assessed the extract's antioxidant properties by using the DPPH, ABTS, and lipid peroxidation assays and found that the extract had a moderate scavenging effect. In an experiment involving preexposition and coexposition of the extract (1–500 µg/mL) and benzo[a]pyrene (BP), the extract was found to be nontoxic to C9 cells in culture and to inhibit the cytotoxicity induced by BP. As BP is biotransformed by CYP1A and CYP2B subfamilies, we explored the possible interaction of the extract with these enzymes. The extract (25–50 µg/mL) inhibited CYP1A1 activity in rat liver microsomes. Analysis of the inhibition kinetics revealed a mixed-type inhibitory effect on CYP1A1 supersome. The effects of the extract on BP-induced DNA damage and hepatic CYP activity in mice were also investigated. Micronuclei induction by BP and liver CYP1A1/2 activities significantly decreased in animals treated with the extract. The results suggest that Ulva fasciata aqueous-ethanolic extract inhibits BP bioactivation and it may be a potential chemopreventive agent.

Punicalagin and ellagic acid demonstrate antimutagenic activity and inhibition of benzo[a]pyrene induced DNA adducts.

Punicalagin (PC) is an ellagitannin found in the fruit peel of Punica granatum. We have demonstrated antioxidant and antigenotoxic properties of Punica granatum and showed that PC and ellagic acid (EA) are its major constituents. In this study, we demonstrate the antimutagenic potential, inhibition of BP-induced DNA damage, and antiproliferative activity of PC and EA. Incubation of BP with rat liver microsomes, appropriate cofactors, and DNA in the presence of vehicle or PC and EA showed significant inhibition of the resultant DNA adducts, with essentially complete inhibition (97%) at 40 μM by PC and 77% inhibition by EA. Antimutagenicity was tested by Ames test. PC and EA dose-dependently and markedly antagonized the effect of tested mutagens, sodium azide, methyl methanesulfonate, benzo[a]pyrene, and 2-aminoflourine, with maximum inhibition of mutagenicity up to 90 percent. Almost all the doses tested (50–500 μM) exhibited significant antimutagenicity. A profound antiproliferative effect on human lung cancer cells was also shown with PC and EA. Together, our data show that PC and EA are pomegranate bioactives responsible for inhibition of BP-induced DNA adducts and strong antimutagenic, antiproliferative activities. However, these compounds are to be evaluated in suitable animal model to assess their therapeutic efficacy against cancer.

Benzo[a]pyrene (BP) DNA adduct formation in DNA repair-deficient p53 haploinsufficient [Xpa(-/-)p53(+/-)] and wild-type mice fed BP and BP plus chlorophyllin for 28 days

We have evaluated DNA damage (DNA adduct formation) after feeding benzo[a]pyrene (BP) to wild-type (WT) and cancer-susceptible Xpa(-/-)p53(+/-) mice deficient in nucleotide excision repair and haploinsufficient for the tumor suppressor p53. DNA damage was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS), which measures r7,t8,t9-trihydroxy-c-10-(N (2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG), and a chemiluminescence immunoassay (CIA), using anti-r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum, which measures both BPdG and the other stable BP-DNA adducts. When mice were fed 100 ppm BP for 28 days, BP-induced DNA damage measured in esophagus, liver and lung was typically higher in Xpa(-/-)p53(+/-) mice, compared with WT mice. This result is consistent with the previously observed tumor susceptibility of Xpa(-/-)p53(+/-) mice. BPdG, the major DNA adduct associated with tumorigenicity, was the primary DNA adduct formed in esophagus (a target tissue in the mouse), whereas total BP-DNA adducts predominated in higher levels in the liver (a non-target tissue in the mouse). In an attempt to lower BP-induced DNA damage, we fed the WT and Xpa(-/-)p53(+/-) mice 0.3% chlorophyllin (CHL) in the BP-containing diet for 28 days. The addition of CHL resulted in an increase of BP-DNA adducts in esophagus, liver and lung of WT mice, a lowering of BPdG in esophagi of WT mice and livers of Xpa(-/-)p53(+/-) mice and an increase of BPdG in livers of WT mice. Therefore, the addition of CHL to a BP-containing diet showed a lack of consistent chemoprotective effect, indicating that oral CHL administration may not reduce PAH-DNA adduct levels consistently in human organs.

Abstract 3458: Benzo[a]pyrene (BP)-induced DNA damage is elevated in mice lacking Xeroderma pigmentosum complementation group A (Xpa) and heterozygous for the tumor suppressor gene p53

Abstract In developing countries, esophageal cancer is the third most common cancer in men. We have employed a mouse model of esophageal cancer using DNA repair deficient mice. Unlike their wild type counterparts, C57BL/6J mice lacking Xpa and heterozygous for p53 [Xpa(−/−) p53(+/−)] have previously been shown to develop forestomach and esophageal tumors when fed BP (Hoogervorst et al., Carcinogenesis, 2003). Two methodologies were employed to examine BP-induced steady-state levels of DNA damage in esophagi of male mice fed 0 or 100 ppm BP for 4 weeks. HPLC, in conjunction with tandem mass spectrometry (HPLC-MS/MS), was used to measure 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) and a chemiluminiscence immunoassay (CIA), using antiserum elicited against r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA was used to evaluate BP-DNA adducts. In the esophagi of Xpa(−/−) p53(+/−) mice, there were 38.7 ± 4.8 BPdG adducts/108 nucleotides (mean ± SE, n=5), as measured by HPLC-MS/MS, while in the wild type mice there were 28.0 ± 1.8 BPdG adducts/108 nucleotides (mean ± SE, n=5). Furthermore, complete loss of p53 resulted in the greatest accumulation of BPdG adducts, with 58.1 ± 8.6 BPdG adducts/108 nucleotides (n=5) in Xpa(−/−) p53(−/−) mice (p<0.05 vs. wild type mice). Esophageal BP-DNA adduct values, as measured by CIA, were 23.3 ± 1.7 adducts/108 nucleotides (n=3) in the Xpa(−/−) p53(+/−) mice, compared to 14.6 ± 3.0 adducts/108 nucleotides (n=3) in the wild type mice. Since similar adduct levels were determined using both HPLC-MS/MS and CIA, this indicates that BPdG is the major adduct resulting from BP in mouse esophagus. The higher levels of DNA damage in DNA repair-deficient p53-deficient mice, compared to their wild type counterparts, may contribute to the enhanced susceptibility of these mice to esophageal tumor induction by BP. Currently we are examining the role of chlorophyllin, a chemopreventive agent, in reducing BP-induced DNA damage levels and tumor formation in this model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3458.

Production and Immunochemical Characterization of a High-Affinity Monoclonal Antibody Specific for 7-(Benzo[a]pyren-6-yl)guanine (Bp-6-N7GUA), a Depurinating DNA Adduct of Benzo[a]pyrene

Abstract Molecular dosimetry of depurinating DNA adducts of benzo[a]pyrene (BP) offers a promising new approach to determining risk of PAH-induced cancer. Depurinating adducts are the predominant form of BP-induced DNA damage, are excreted in urine and, importantly, are linked to cancer initiation. We have produced a monoclonal antibody (MAb) with high specific affinity for BP-6-N7Gua, a major depurinating DNA adduct of BP, and have developed a sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA). The I50S (quantity producing 50% inhibition of MAb binding in the ELISA) of selected BP adducts and metabolites were determined. The results indicated 1) a high degree of discrimination between BP-6-N7Gua (I50=750 fmol) and BP (I50=900,000 fmol), 2) high affinity of the MAb for BP-6-N7Ade (I50=1,500 fmol), another major depurinating DNA adduct, and 3) specific structural requirements for MAb-adduct binding. In addition, the competitive ELISA provided an accurate determination of BP-6-N7Gu...

P53 protein expression is correlated with benzo[a]pyrene-DNA adducts in carcinoma cell lines.

p53 inhibits cell cycle progression and DNA damaging cytostatics induce p53 protein expression, indicating that p53 responds to DNA damage. We have measured benzo[a]pyrene (BP)-induced DNA damage in association with p53 expression. The most relevant DNA adducts for carcinogenesis, benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts, were measured by synchronous fluorescence spectrophotometry and p53 immunohistochemistry using polyclonal antibody CM1, which detects both wild-type and mutated forms of p53. Activation of BP in A-549 lung carcinoma and MCF-7 breast adenocarcinoma cell lines containing wild-type p53 was followed by an increase in p53 protein expression. alpha-Naphthoflavone, an inhibitor of cytochrome P450 (CYP)1A1, decreased both the formation of diolepoxide metabolites and the p53 response. The cell lines not able to activate BP, A-427 and SK-LU-1 (both human lung carcinomas), SK-MES-1 (human lung squamous carcinoma) and human fibroblasts, did not show any increase in p53 immunohistochemistry. The OVCAR-3 ovarian adenocarcinoma cell line, containing a mutation in exon 7 of p53, and the SK-LU-1 cell line expressed very high levels of p53 protein before BP treatment and no increase in p53 immunohistochemistry was seen. These findings indicate that p53 protein is part of the response of the cells to BP-induced DNA damage.

In vitro studies on chemoprotective effect of purnark against benzo(a)pyrene-induced chromosomal damage in human lymphocytes.

Human lymphocytes were used as an assay system to test chemopreventive activity of natural products. Purnark, a mixture of extracts of turmeric, betel leaf and catechu, was tested for its chemoprotective activity against BP induced DNA damage. Sister chromatid exchange and micronuclei were used as markers to assess the protective activity of Purnark. Purnark gave 50-60 % protection against BP induced SCEs and micronuclei. Purnark at 1000μg dose did not show any genotoxicity.

Sister chromatid exchange induction by benzo(a)pryene in cultured peripheral blood lymphocytes of lung cancer patients and healthy individuals with or without familial history of neoplasms.

The inducibility of sister chromatid exchanges (SCEs) by benzo(a)pyrene (BP) was studied in cultured peripheral blood lymphocytes of 15 untreated lung cancer patients and 25 healthy persons including 11 high- and 14 low-cancer-risk individuals tentatively classified by the familial history of lung cancer and other neoplasms. The baseline SCE frequency in cultured lymphocytes was significantly high in lung cancer patients, as compared with all healthy persons or low-cancer-risk individuals. Following exposure to BP, the lymphocytes of lung-cancer patients and high-cancer-risk individuals exhibited significantly greater SCE yields than those of persons at low risk, although no significant difference was observed in the lymphocyte SCE yields when the levels of lung cancer patients were compared with those of all healthy persons. A comparison of the net SCE increase (delta SCE) in BP-exposed lymphocytes among the study groups, however, revealed a significant difference in delta SCE values only between high- and low-cancer-risk individuals. The present findings on both the observed SCE yields and delta SCE values suggest that lymphocytes of high-risk individuals may be more susceptible to BP-induced DNA damage than those of persons at low risk, and that such a chromosomal hypersensitivity to genotoxins may be associated with a high risk of neoplasms.