Inhibition of Benzo[a]pyrene-induced DNA Adduct in Buccal Cells of Smokers by Black Raspberry Lozenges.

Using LC-MS/MS analysis we previously showed for the first time (Carcinogenesis 43:746-753, 2022) that levels of DNA damage-induced by benzo[a]pyrene (B[a]P), an oral carcinogen and tobacco smoke (TS) constituent, were significantly higher in buccal cells of smokers than those in non-smokers; these results suggest the potential contribution of B[a]P in the development of oral squamous cell carcinoma (OSCC) in humans. Treating cancers, including OSCC at late stages even with improved targeted therapies, continues to be a major challenge. Thus interception/prevention remains a preferable approach for OSCC management and control. In previous preclinical studies we and others demonstrated the protective effects of black raspberry (BRB) against carcinogen-induced DNA damage and OSCC. Thus, to translate preclinical findings we tested the hypothesis, in a Phase 0 clinical study, that BRB administration reduces DNA damage induced by B[a]P in buccal cells of smokers. After enrolling 27 smokers, baseline buccal cells were collected before the administration of BRB lozenges (5/day for 8 weeks, 1 gm BRB powder/lozenge) at baseline, at the middle and the end of BRB administration. The last samples were collected at four weeks after BRB cessation (washout period). B[a]P-induced DNA damage (BPDE-N2-dG) was evaluated by LC-MS/MS. BRB administration resulted in a significant reduction in DNA damage: 26.3% at the midpoint (p = 0.01506) compared to baseline, 36.1% at the end of BRB administration (p = 0.00355), and 16.6% after BRB cessation (p = 0.007586). Our results suggest the potential benefits of BRB as a chemopreventive agent against the development of TS-initiated OSCC.

Sulforaphane inhibits the migration and invasion of BPDE-induced lung adenocarcinoma cells by regulating NLRP12.

This study aims to explore the impact and underlying mechanism of sulforaphane (SFN) intervention on the migration and invasion of lung adenocarcinoma induced by 7, 8-dihydroxy-9, 10-epoxy-benzo (a) pyrene (BPDE). Human lung adenocarcinoma A549 cells were exposed to varying concentrations of BPDE (0.25, 0.50, and 1.00 µM) and subsequently treated with 5 µM SFN. Cell viability was determined using CCK8 assay, while migration and invasion were assessed using Transwell assays. Lentivirus transfection was employed to establish NLRP12 overexpressing A549 cells. ELISA was utilized to quantify IL-33, CXCL12, and CXCL13 levels in the supernatant, while quantitative real-time PCR (qRT-PCR) and Western Blot were used to analyze the expression of NLRP12 and key factors associated with canonical and non-canonical NF-κB pathways. Results indicated an increase in migratory and invasive capabilities, concurrent with heightened expression of IL-33, CXCL12, CXCL13, and factors associated with both canonical and non-canonical NF-κB pathways. Moreover, mRNA and protein levels of NLRP12 were decreased in BPDE-stimulated A549 cells. Subsequent SFN intervention attenuated BPDE-induced migration and invasion of A549 cells. Lentivirus-mediated NLRP12 overexpression not only reversed the observed phenotype in BPDE-induced cells but also led to a reduction in the expression of critical factors associated with both canonical and non-canonical NF-κB pathways. Collectively, we found that SFN could inhibit BPDE-induced migration and invasion of A549 cells by upregulating NLRP12, thereby influencing both canonical and non-canonical NF-κB pathways.

B(a)P induces ovarian granulosa cell apoptosis via TRAF2-NFκB-Caspase1 axis during early pregnancy.

Benzo(a)pyrene [B(a)P] is an environmental endocrine disruptor with reproductive toxicity. The corpus luteum (CL) of the ovary plays an important role in embryo implantation and pregnancy maintenance. Our previous studies have shown that B(a)P exposure affects embryo implantation and endometrial decidualization in mouse, but its effects and mechanisms on CL function remain unclear. In this study, we explore the mechanism of ovarian toxicity of B(a)P using a pregnant mouse model and an in vitro model of human ovarian granulosa cells (GCs) KGN. Pregnant mice were gavaged with corn oil or 0.2 mg/kg.bw B(a)P from pregnant day 1 (D1) to D7, while KGN cells were treated with DMSO, 1.0IU/mL hCG, or 1.0IU/mL hCG plus benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a B(a)P metabolite. Our findings revealed that B(a)P exposure damaged embryo implantation and reduced estrogen and progesterone levels in early pregnant mice. Additionally, in vitro, BPDE impaired luteinization in KGN cells. We observed that B(a)P/BPDE promoted oxidative stress (OS) and inflammation, leading to apoptosis rather than pyroptosis in ovaries and luteinized KGN cells. This apoptotic response was mediated by the activation of inflammatory Caspase1 through the cleavage of BID. Furthermore, B(a)P/BPDE inhibited TRAF2 expression and suppressed NFκB signaling pathway activation. The administration of VX-765 to inhibit the Caspase1 activation, over-expression of TRAF2 using TRAF2-pcDNA3.1 (+) plasmid, and BetA-induced activation of NFκB signaling pathway successfully alleviated BPDE-induced apoptosis and cellular dysfunction in luteinized KGN cells. These findings were further confirmed in the KGN cell treated with H2O2 and NAC. In conclusion, this study elucidated that B(a)P/BPDE induces apoptosis rather than pyroptosis in GCs via TRAF2-NFκB-Caspase1 during early pregnancy, and highlighting OS as the primary contributor to B(a)P/BPDE-induced ovarian toxicity. Our results unveil a novel role of TRAF2-NFκB-Caspase1 in B(a)P-induced apoptosis and broaden the understanding of mechanisms underlying unexplained luteal phase deficiency.

Environmental BaP/BPDE suppressed trophoblast cell invasion/migration and induced miscarriage by down-regulating lnc-HZ01/MEST/VIM axis.

Environmental benzo(a)pyrene (BaP) and itsmetabolite benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE), classic endocrine disrupting chemical and persistent organic pollutant, could cause miscarriage. However, the detailed mechanisms are still largely unclear and should be further explored. In this study, we discovered that exposure of trophoblast cells with BPDE could suppressed cell invasion/migration by inhibiting MEST/VIM (Vimentin) pathway. Moreover, BPDE exposure also increased lnc-HZ01 expression level, which further inhibited MEST/VIM pathway and then suppressed invasion/migration. Knockdown of lnc-HZ01 or overexpression of MEST could efficiently rescue invasion/migration of BPDE-exposed Swan 71 cells. Furthermore, lnc-HZ01 was highly expressed and MEST/VIM were lowly expressed in recurrent miscarriage (RM) villous tissues compared with healthy control (HC) group. Finally, we also found that BaP exposure inhibited murine Mest/Vim pathway in placental tissues and induced miscarriage in BaP-exposed mice. Therefore, the regulatory mechanisms were similar in BPDE-exposed human trophoblast cells, RM villous tissues, and placental tissues of BaP-exposed mice with miscarriage, building a bridge to connect BaP/BPDE exposure, invasion/migration, and miscarriage. This study provided novel insights in the toxicological effects and molecular mechanisms of BaP/BPDE-induced miscarriage, which is helpful for better elucidating the toxicological risks of BaP/BPDE on female reproduction.

Effects of polycyclic aromatic hydrocarbons (PAHs) on pregnancy, placenta, and placental trophoblasts.

Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic pollutants that are carcinogenic, mutagenic, endocrine-toxic, and immunotoxic. PAHs can be found in maternal and fetal blood and in the placenta during pregnancy. They may thus affect placental and fetal development. Therefore, the exposure levels and toxic effects of PAHs in the placenta deserve further study and discussion. This review aims to summarize current knowledge on the effects of PAHs and their metabolites on pregnancy and birth outcomes and on placental trophoblast cells. A growing number of epidemiological studies detected PAH-DNA adducts as well as the 16 high-priority PAHs in the human placenta and showed that placental PAH exposure is associated with adverse fetal outcomes. Trophoblasts are important cells in the placenta and are involved in placental development and function. In vitro studies have shown that exposure to either PAH mixtures, benzo(a)pyrene (BaP) or BaP metabolite benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) affected trophoblast cell viability, differentiation, migration, and invasion through various signaling pathways. Furthermore, similar effects of BPDE on trophoblast cells could also be observed in BaP-treated mouse models and were related to miscarriage. Although the current data show that PAHs may affect placental trophoblast cells and pregnancy outcomes, further studies (population studies, in vitro studies, and animal studies) are necessary to show the specific effects of different PAHs on placental trophoblasts and pregnancy outcomes.

The activation of SIRT1 ameliorates BPDE-induced inflammatory damage in BEAS-2B cells via HMGB1/TLR4/NF-κB pathway.

Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), the metabolite of environmental pollutant benzo(a)pyrene (B(a)P) could induce pulmonary toxicity and inflammation. SIRT1, an NAD+ -dependent histone deacetylase, is known to regulate inflammation in the occurrence and development of various diseases, but its effects on BPDE-induced acute lung injury are still unknown. The present study aimed to explore the role of SIRT1 in BPDE-induced acute lung injury. Here, human bronchial epithelial (HBE) cells (BEAS-2B) cells were stimulated with BPDE at different concentrations (0.50, 0.75, and 1.00 µmol/L) for 24 h, we found that the levels of cytokines in the supernatant were increased and the expression of SIRT1 in cells was down-regulated, at the same time, BPDE stimulation up-regulated the protein expression of HMGB1, TLR4, and p-NF-κBp65 in BEAS-2B cells. Then the activator and inhibitor of SIRT1 were used before BPDE exposure, it was shown that the activation of SIRT1 significantly attenuated the levels of inflammatory cytokines and HMGB1, and reduced the expression of HMGB1, AC-HMGB1, TLR4, and p-NF-κBp65 protein; while these results were reversed by the inhibition of SIRT1. This study revealed that the SIRT1 activation may protect against BPDE-induced inflammatory damage in BEAS-2B cells by regulating the HMGB1/TLR4/NF-κB pathway.

Novel lncRNA-HZ04 promotes BPDE-induced human trophoblast cell apoptosis and miscarriage by upregulating IP3 R1 /CaMKII/SGCB pathway by competitively binding with miR-hz04.

Normal pregnancy is essential for human reproduction. However, BaP (benzo(a)pyrene) and its metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) could cause dysfunctions of human trophoblast cells and might further induce miscarriage. Yet, the underlying mechanisms remain largely unknown. Herein, we identified a novel upregulated lnc-HZ04 and a novel downregulated miR-hz04 in villous tissues of unexplained recurrent miscarriage (RM) relative to those in healthy control tissues and also in BPDE-treated human trophoblast cells. Lnc-HZ04 directly and specifically bound with miR-hz04, diminished the reduction effects of miR-hz04 on IP3 R1 mRNA expression level and on IP3 R1 mRNA stability, and then activated the Ca2+ -mediated IP3 R1 /p-CaMKII/SGCB pathway, which further promoted trophoblast cell apoptosis. The miR-hz04 target site on lnc-HZ04 played crucial roles in these regulations. In normal trophoblast, relatively less lnc-HZ04 and more miR-hz04 suppressed this apoptosis pathway and gave normal pregnancy. After exposure to BPDE or in RM tissues, p53 was upregulated, which might promote p53-mediated lnc-HZ04 transcription. Relatively more lnc-HZ04 and less miR-hz04 activated this apoptosis pathway and might further induce miscarriage. BaP could also induce mice miscarriage by upregulating its corresponding murine apoptosis pathway. Therefore, BPDE-induced apoptosis of human trophoblast cells was associated with the occurrence of miscarriage. This work discovered the regulation roles of lnc-HZ04 and miR-hz04 and provided scientific and clinical understanding of the occurrence of unexplained miscarriage.

Lnc-HZ08 regulates BPDE-induced trophoblast cell dysfunctions by promoting PI3K ubiquitin degradation and is associated with miscarriage.

Increasing evidences have shown that pregnant women might miscarry after exposure with environmental BaP (benzo(a)pyrene). Additionally, BPDE (benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide), the ultimate metabolite of BaP, could induce dysfunctions of human trophoblast cells. However, it is rarely correlated between miscarriage and trophoblast dysfunctions. Moreover, their underlying mechanisms are still largely unidentified. In this study, a novel lncRNA (long non-coding RNA), lnc-HZ08, was identified to be highly expressed in human recurrent miscarriage (RM) tissues and in BPDE-treated human trophoblast cells. Lnc-HZ08 acts as a RNA scaffold to interact with both PI3K and its ubiquitin ligase CBL (Cbl proto-oncogene), enhances their protein interactions, and promotes PI3K ubiquitin degradation. In RM tissues and BPDE-treated trophoblast cells, DNA methylation level in lnc-HZ08 promoter region was reduced, which promotes estrogen receptor 1 (ER)-mediated lnc-HZ08 transcription. Subsequently, this upregulated lnc-HZ08 downregulated PI3K level, suppressed PI3K/p-AKT/p-P21/CDK2 pathway, and thus weakened proliferation, migration, and invasion of human trophoblast cells, which further induces miscarriage. These results may provide novel scientific and clinical insights in the occurrence of unexplained miscarriage. A novel lncRNA (lnc-HZ08) regulates the functions of human trophoblast cells and affects miscarriage. Lnc-HZ08 promotes PI3K ubiquitin degradation by enhancing CBL and PI3K interactions, downregulates PI3K/p-AKT/p-P21/CDK2 pathway, and weakens proliferation, migration, and invasion of trophoblast cells. BPDE exposure reduces the DNA methylation level in lnc-HZ08 promoter region and promotes estrogen receptor 1 (ER)-mediated lnc-HZ08 transcription. The suppressed PI3K/p-AKT/p-P21/CDK2 pathway regulated by increased lnc-HZ08 is associated with miscarriage. These results provide novel insights in the occurrence of unexplained miscarriage. Graphical Headlights • Lnc-HZ08 downregulates PI3K/p-AKT/p-P21/CDK2 pathway to suppress proliferation, migration, and invasion of human trophoblast cells, and affects miscarriage. • Lnc-HZ08 acts as a RNA scaffold to enhance the protein interaction of PI3K and its ubiquitin ligase CBL, which increases PI3K ubiquitination and degradation. • Lnc-HZ08 transcription is associated with DNA methylation on its promoter region and transcription factor ER.

Lnc-HZ05 regulates BPDE-inhibited human trophoblast cell proliferation and affects the occurrence of miscarriage by directly binding with miR-hz05.

Approximately 15-25% pregnant women end with miscarriage in the world. Environmental BaP (benzo(a)pyrene) and its terminal metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) may result in the dysfunctions of trophoblast cells, which might further lead to RM (recurrent miscarriage). However, potential mechanisms remain unelucidated. In this work, we identified a novel lnc-HZ05 highly expressed and a novel miR-hz05 lowly expressed in both trophoblast cells exposed to BPDE and human RM tissues. MiR-hz05 reduces FOXO3a mRNA level by weakening its mRNA stability. Lnc-HZ05 increases the expression of FOXO3a by acting as a ceRNA for miR-hz05, and then increases P21 level and reduces CDK2 level. Thus, cell cycle is arrested at G0/G1 phase and trophoblast proliferation is inhibited. Lnc-HZ05 harboring wild-type binding site for miR-hz05, but not its mutant site, could upregulate FOXO3a expression. In normal trophoblast cells, relatively less lnc-HZ05 and more miR-hz05 activate FOXO3a/P21/CDK2 pathway and promote trophoblast proliferation, giving normal pregnancy. In RM tissues and BPDE-treated human trophoblast cells, lnc-HZ05 is increased and miR-hz05 is reduced, both of which suppress this pathway and inhibit cell proliferation, and finally lead to miscarriage. Thus, lnc-HZ05 and miR-hz05 simultaneously regulate cell cycle and proliferation of BPDE-exposed trophoblast cells and miscarriage, providing new perspectives and clinical understandings in the occurrence of unexplained miscarriage.

Environmental BPDE induced human trophoblast cell apoptosis by up-regulating lnc-HZ01/p53 positive feedback loop.

Human trophoblast cell apoptosis may induce miscarriage. Trophoblast cells are sensitive to environmental BaP-7,8-dihydrodiol-9,10-epoxide (BPDE). However, how BPDE induces human trophoblast cell apoptosis is still largely elusive. In this work, we used BPDE-treated human trophoblast cells and villous tissues collected from recurrent miscarriage and health control groups to explore the underlying mechanism of BPDE-induced human trophoblast cell apoptosis. Continued with our recent work, we found that lncRNA HZ01 (lnc-HZ01) could induce human trophoblast cell apoptosis. In mechanism, lnc-HZ01 up-regulated p53 expression level by suppressing its MDM2-mediated proteasomal degradation. Meanwhile, we found that p53 acted as lnc-HZ01 transcription factor and promoted lnc-HZ01 transcription. Thus, lnc-HZ01 and p53 composed a positive feedback loop in human trophoblast cells. In normal trophoblast cells, relatively low levels of lnc-HZ01 and p53 suppressed p53/caspase-3 apoptosis pathway, giving normal pregnancy. Upon BPDE exposure, BPDE up-regulated the expression levels of lnc-HZ01 and p53, triggered this positive feedback loop, activated the p53/caspase-3 apoptosis pathway, and then induced miscarriage. Collectively, we discovered new mechanism by which lnc-HZ01 regulated BPDE-induced human trophoblast cell apoptosis, providing scientific basis for the diagnosis and treatment of unexplained recurrent miscarriage.

Endoplasmic reticulum stress regulates pyroptosis in BPDE-induced BEAS-2B cells.

Benzo(a)pyrene(B(a)P), as the main representative of polycyclic aromatic hydrocarbons, can promote inflammation and many chronic pulmonary diseases. However, the underlying mechanism of Benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-induced human bronchial epithelial cell pyroptosis related to endoplasmic reticulum stress (ERS) has not been elucidated. This study focused on the effects of BPDE on ERS and pyroptosis in human bronchial epithelial cells (BEAS-2B), and explored the relationship between ERS and pyroptosis. BEAS-2B cells were stimulated with 0.50, 0.75, and 1.00 µmol/L BPDE for 24 h to detect ERS and pyroptosis. After inhibition of ERS with 4-phenylbutyrate (4-PBA), pyroptosis of BEAS-2B cells was tested. The results showed that BPDE decreased the cell viability, changed the morphological structure of endoplasmic reticulum and increased the expression levels of GRP78 and p-PERK. After BPDE treatment, the cell membrane was damaged and incomplete under transmission electron microscope; Hoechst 33342/PI fluorescence staining showed that the number of PI-positive cells was enhanced. The expression levels of GSDMD-N, cleaved-caspase 1, and cleaved-IL-1ß were elevated, and the expression levels of IL-1ß, IL-18, and NLRP3 protein were improved. In BPDE combined with 4-PBA intervention group, the rate of PI-positive cells was reduced, the expression levels of GRP78, GSDMD-N, and cleaved-caspase 1 were decreased, and the expression levels of IL-1ß, IL-18, and NLRP3 were decreased. In conclusion, BPDE could induce ERS and pyroptosis in BEAS-2B cells, and ERS may promote the occurrence of BPDE-induced pyroptosis.

Novel lnc-HZ03 and miR-hz03 promote BPDE-induced human trophoblastic cell apoptosis and induce miscarriage by upregulating p53/SAT1 pathway.

Normal pregnancy is essential for human reproduction. However, environmental BaP (benzo(a)pyrene) and its metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) induce dysfunctions of human trophoblastic cells, which could further result in miscarriage. Yet, the molecular mechanisms remain poorly understood. In this work, a novel lnc-HZ03 and a novel miR-hz03 were identified. Both lnc-HZ03 and miR-hz03 were highly expressed in human recurrent miscarriage villous tissues and in BPDE-exposed trophoblastic cells. Lnc-HZ03 and miR-hz03 upregulated each other, forming a positive feedback loop. MiR-hz03 could also upregulate p53 level by enhancing its mRNA stability. Both lnc-HZ03 and p53 mRNA contained the target site for miR-hz03 and could directly interact with miR-hz03. It was this target site instead of its mutant on lnc-HZ03 that regulated p53 expression. Subsequently, the upregulated p53 facilitated SAT1 transcription and enhanced SAT1-catalyzed spermine metabolism, which further resulted in trophoblastic cell apoptosis and induced miscarriage. All together, the p53/SAT1 pathway upregulated by lnc-HZ03 and miR-hz03 could promote BPDE-induced human trophoblastic cell apoptosis and the occurrence of miscarriage, shedding novel light on the causes of miscarriage. Graphical abstract Lnc-HZ03 and miR-hz03 regulate the occurrence of recurrent miscarriage (RM). In human trophoblastic cells, lnc-HZ03 upregulates miR-hz03 level. MiR-hz03 increases the RNA stability of lnc-HZ03 and p53 mRNA. P53 promotes SAT1 transcription and reduces its cellular spermine content, resulting in cell apoptosis. Under normal conditions, lnc-HZ03/miR-hz03 and p53/SAT1 pathways are downregulated, maintaining normal pregnancy. After exposure to BPDE, lnc-HZ03/miR-hz03 and p53/SAT1 pathways are upregulated and finally induce miscarriage.

Toxicological Effects of BPDE on Dysfunctions of Female Trophoblast Cells.

Polycyclic aromatic hydrocarbons (PAHs) are widely spread persistent environmental toxicants. Its typical representative benzo[a]pyrene (BaP) is a human carcinogen. BaP can pass through the placental barrier and is finally metabolized into benzo[a]pyren-7, 8-dihydrodiol-9, 10-epoxide (BPDE). BPDE can form DNA adducts, which directly affect the female reproductive health. Based on the special physiological functions of trophoblast cells and its important effect on normal pregnancy, this chapter describes the toxicity and molecular mechanism of BPDE-induced dysfunctions of trophoblast cells. By affecting the invasion, migration, apoptosis, proliferation, inflammation, and hormone secretion of trophoblast cells, BPDE causes diseases such as choriocarcinoma, intrauterine growth restriction, eclampsia, and abortion. In the end, it is expected to provide a scientific basis and prevention approach for women's reproductive health and decision-making basis for the formulation of environmental health standards.

Association among blood BPDE-DNA adduct, serum interleukin-8 (IL-8) and DNA strand breaks for children with pulmonary diseases.

Exposure to benzo[a]pyrene (B[a]P) may be a risk factor for pulmonary diseases. To investigate the correlations among B[a]P exposure level, DNA strand breaks and pulmonary inflammation, we recruited 83 children diagnosed with pulmonary diseases and 63 healthy children from Guangzhou, China. Results showed that the levels of Benzo[a]pyrene diol epoxide (BPDE) DNA adduct in blood and IL-8 in serum in case group were significantly higher than those in control group (p < 0.01). Moreover, levels of atmospheric B[a]P in case group was about twice of those in control group, which was consistent with the levels of BPDE-DNA adduct in blood. Significant positive correlations were observed among the levels of BPDE-DNA adduct, IL-8 and DNA strand breaks (p < 0.05). Our findings indicate that environmental air is an important exposure source of B[a]P and higher B[a]P exposure may contribute to the occurrence of pulmonary inflammation and lead to high health risks.

Modulatory Effects of Silymarin on Benzo[a]pyrene-Induced Hepatotoxicity.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon, is a group 1 carcinogen that introduces mutagenic DNA adducts into the genome. In this study, we investigated the molecular mechanisms underlying the involvement of silymarin in the reduction of DNA adduct formation by B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), induced by B[a]P. B[a]P exhibited toxicity in HepG2 cells, whereas co-treatment of the cells with B[a]P and silymarin reduced the formation of BPDE-DNA adducts, thereby increasing cell viability. Determination of the level of major B[a]P metabolites in the treated cells showed that BPDE levels were reduced by silymarin. Nuclear factor erythroid 2-related factor 2 (Nrf2) and pregnane X receptor (PXR) were found to be involved in the activation of detoxifying genes against B[a]P-mediated toxicity. Silymarin did not increase the expression of these major transcription factors, but greatly facilitated their nuclear translocation. In this manner, treatment of HepG2 cells with silymarin modulated detoxification enzymes through NRF2 and PXR to eliminate B[a]P metabolites. Knockdown of Nrf2 abolished the preventive effect of silymarin on BPDE-DNA adduct formation, indicating that activation of the Nrf2 pathway plays a key role in preventing B[a]P-induced genotoxicity. Our results suggest that silymarin has anti-genotoxic effects, as it prevents BPDE-DNA adduct formation by modulating the Nrf2 and PXR signaling pathways.

Curcumin Ameliorates Benzo[a]pyrene-Induced DNA Damages in Stomach Tissues of Sprague-Dawley Rats.

Benzo[a]pyrene (BaP) is a well-known carcinogen formed during the cooking process. Although BaP exposure has been implicated as one of the risk factors for lung cancer in animals and humans, there are only limited data on BaP-induced gastrointestinal cancer. Therefore, this study investigated the protective effects of curcumin on BaP-induced DNA damage in rat stomach tissues. BaP (20 mg/kg/day) and curcumin (50, 100, or 200 mg/kg) were administered daily to Sprague-Dawley rats by oral gavage over 30 days. Curcumin was pre-administered before BaP exposure. All rats were euthanized, and liver, kidney, and stomach tissues were removed at 24 h after the last treatment. We observed that aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose levels were significantly reduced in rats treated with high dose co-administration of curcumin (200 mg/kg) compared to BaP alone. The expression levels of cytochrome P450 (CYP) 1A1 and CYP1B1 were significantly increased in the liver of rats treated with BaP. However, co-administration of curcumin (200 mg/kg) with BaP markedly reduced CYP1A1 expression in a dose-dependent manner. Furthermore, plasma levels of BaP-diolepoxide (BPDE) and BaP metabolites were significantly reduced by co-administration of curcumin (200 mg/kg). Additionally, co-administration of curcumin (200 mg/kg) with BaP significantly reduced the formation of BPDE-I-DNA and 8-hydroxydeoxy guanosine (8-OHdG) adducts in the liver, kidney, and stomach tissues. The inhibition of these adduct formations were more prominent in the stomach tissues than in the liver. Overall, our observations suggest that curcumin might inhibit BaP-induced gastrointestinal tumorigenesis and shows promise as a chemopreventive agent.

BPDE-induced genotoxicity: relationship between DNA adducts, mutagenicity in the in vitro PIG-A assay, and the transcriptional response to DNA damage in TK6 cells.

Benzo[a]pyrene is a known human carcinogen. As underlying mechanism, the induction of stable DNA adducts and mutations have been repeatedly demonstrated. Also, the activation of cellular stress response on the transcriptional level has been described. Nevertheless, the interrelationship between these different events is less well understood, especially at low, for human exposure relevant concentrations. Within the present study, we applied the reactive metabolite benzo[a]pyrene diolepoxide (BPDE) in the nanomolar, non-cytotoxic concentration range in human TK6 cells and quantified the induction and repair of stable DNA adducts at the N 2-position of guanine by HPLC with fluorescence detection. Significant levels of DNA lesions were detected even at the lowest concentration of 10 nM BPDE, with a linear increase up to 50 nM. Relative repair was similar at all damage levels, reaching about 30% after 8 h and 60% after 24 h. Mutation frequencies were quantified as GPI-deficient cells by the recently established in vitro PIG-A mutagenicity assay. Again, a linear dose-response-relationship in the before-mentioned concentration range was observed, also when plotting the number of GPI-deficient cells against the number of DNA adducts. Furthermore, we explored the time- and concentration-dependent DNA damage response on the transcriptional level via a high-throughput RT-qPCR technique by quantifying the impact of BPDE on the transcription of 95 genes comprising DNA damage response, DNA repair factors, oxidative stress response, cell cycle arrest, cell proliferation, and apoptosis. As expected, BPDE activated DNA damage signaling, p53 and AP-1 dependent signaling, oxidative stress response, and apoptosis. However, in contrast to DNA adducts and mutations, the onset of the transcriptional DNA damage response was restricted to higher concentrations, indicating that its respective activations require a certain level of DNA lesions. Altogether, the results indicate that in case of BPDE, DNA lesions and mutations were correlated at all concentrations, suggesting that repair is not complete even at low levels of DNA damage. Considering the ongoing discussion on potential thresholds also for genotoxic carcinogens, the results are of major relevance, both with respect to basic research as well as to risk assessment of chemical carcinogens.

PARP1 protects from benzo[a]pyrene diol epoxide-induced replication stress and mutagenicity.

Poly(ADP-ribosyl)ation (PARylation) is a complex and reversible posttranslational modification catalyzed by poly(ADP-ribose)polymerases (PARPs), which orchestrates protein function and subcellular localization. The function of PARP1 in genotoxic stress response upon induction of oxidative DNA lesions and strand breaks is firmly established, but its role in the response to chemical-induced, bulky DNA adducts is understood incompletely. To address the role of PARP1 in the response to bulky DNA adducts, we treated human cancer cells with benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE), which represents the active metabolite of the environmental carcinogen benzo[a]pyrene [B(a)P], in nanomolar to low micromolar concentrations. Using a highly sensitive LC-MS/MS method, we revealed that BPDE induces cellular PAR formation in a time- and dose-dependent manner. Consistently, PARP1 activity significantly contributed to BPDE-induced genotoxic stress response. On one hand, PARP1 ablation rescued BPDE-induced NAD+ depletion and protected cells from BPDE-induced short-term toxicity. On the other hand, strong sensitization effects of PARP inhibition and PARP1 ablation were observed in long-term clonogenic survival assays. Furthermore, PARP1 ablation significantly affected BPDE-induced S- and G2-phase transitions. Together, these results point towards unresolved BPDE-DNA lesions triggering replicative stress. In line with this, BPDE exposure resulted in enhanced formation and persistence of DNA double-strand breaks in PARP1-deficient cells as evaluated by microscopic co-localization studies of 53BP1 and γH2A.X foci. Consistently, an HPRT mutation assay revealed that PARP inhibition potentiated the mutagenicity of BPDE. In conclusion, this study demonstrates a profound role of PARylation in BPDE-induced genotoxic stress response with significant functional consequences and potential relevance with regard to B[a]P-induced cancer risks.

Serum Level of Antibodies (IgG, IgM) Against Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA Adducts in Children Dermatologically Exposed to Coal Tar.

Crude coal tar (CCT) contains polycyclic aromatic hydrocarbons (PAHs). Benzo[a]pyrene (BaP) is metabolized into a highly reactive metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) that is able to bind to DNA and creates BPDE-DNA adducts. Adducted DNA becomes immunogenic and induces immune response by production of antibodies against BPDE-DNA adducts (Ab-BPDE-DNA). Circulating Ab-BPDE-DNA was proposed as potential biomarker of genotoxic exposure to BaP (PAHs). Goeckerman therapy (GT) of psoriasis uses dermal application of CCT ointment (PAHs). In presented study (children with psoriasis treated by GT; n = 19) the therapy significantly increased the level of Ab-BPDE-DNA (EI = 0.29/0.19-0.34 vs. 0.31/0.25-0.40; median/lower-upper quartile; p < 0.01). The results support the idea of Ab-BPDE-DNA level as a possible tentative indicator of exposure, effects and susceptibility of the organism to the exposure of BaP (PAHs).

Do biomarkers of exposure and effect correlate with internal exposure to PAHs in swine?

Humans are commonly exposed to polycyclic aromatic hydrocarbons (PAHs), a family of compounds present as mixtures in the environment. This study exposed swine to PAH mixtures in single and subacute dose regimens and collected liver and ileum tissue to measure cytochrome P450 mRNA expression and enzyme activity as biomarkers of exposure and DNA adducts and oxidized proteins as biomarkers of effect. Micronucleated reticulocytes were measured as systemic biomarkers of effect. Duration of exposure did not influence biomarkers of exposure, though exposure duration produced significant increases in DNA adducts and oxidative stress. Micronucleated reticulocyte numbers were not affected by exposure length.