Benzo[a]pyrene Diol Epoxide Research Articles

Unique DNA Polymerase kappa Interactome Suggests Novel Cellular Functions

Translesion DNA synthesis (TLS) polymerases evolved to tolerate DNA damage that bypasses DNA lesions, thus preventing genomic instability. Multiple TLS polymerases exist with different damage tolerance capabilities, since they have low fidelity their access to the replication fork must be regulated to minimize mutations. The current paradigm is that a combination of kinetic partitioning and protein‐protein interactions are used to regulate TLS polymerase activity. A major knowledge‐gap in elucidating the roles of these polymerases is that it is difficult to identify which polymerase is active in a specific situation. We designed and synthesized a novel nucleotide analog N2‐benzyl‐ 2′‐deoxyguanosine (EBndG) that is highly selective toward DNA polymerase kappa (Pol κ), a Y family TLS polymerase. Pol k can bypass bulky lesions in the minor groove generated by a metabolite benzo[a]pyrene diolepoxide (BPDE), an environmental carcinogen. Although Pol κ has been identified to have multiple cellular roles, the mechanisms regulating its different cellular activities are unknown.To interrogate the identity of proteins surrounding the Pol κ active sites, we performed an extensive study using modified iPOND (isolation of proteins on nascent DNA), called iPoKD (isolation of proteins on Pol kappa synthesized DNA). Human cell lines were treated with BPDE, subsequently 5‐ethynyl‐2′‐deoxyuridine (EdU) or EBndG was added and proteins bound to the DNA containing EdU and EBndG were analyzed by mass spectrometry. In addition, we performed quantitative analysis of the Pol κ active sites interactome using iPoKD followed by iTRAQ (isobaric tags for relative and absolute quantitation). Our data identified DNA replication and repair proteins previously identified with EdU pull‐downs; and interestingly enrichment of RNA binding, ribosome biogenesis, nucleolar proteins and transcriptional repressive complex(es) associated with EBndG pull‐downs. Chromatin modifiers, histone chaperones and histone variants are identified suggesting changes in chromatin structure that facilitates Pol κ‐mediated DNA lesion bypass, repair and restorative process. identification of unique proteins associated with EBndG‐containing DNA, suggests novel roles for Pol κ’s activity in the cell.Using super‐resolution confocal microscopy, Pol κ activity is identified in the nucleolus after BPDE damage. EBndG is observed in the nucleolar DNA and Pol κ 's activity regulated by the canonical polycomb‐complex recruited by the PARylation of PARP1. BPDE lead to transcriptional stress and repression that is gradually recovered. We are investigating whether Pol κ maintains ribosomal DNA integrity after BPDE damage and is required for TLS DNA synthesis or repair. Pol κ active site associated candidate proteins are being validated using CRISPR‐Cas9 knockout or siRNA knockdown strategies. These data will provide first insight into Pol κ’s core interactome, it's regulation, chromatin surrounding Pol κ active sites and its novel cellular roles.

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 1.00μmol/L BPDE for 24h 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-1β were elevated, and the expression levels of IL-1β, 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-1β, 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.

Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide induces ferroptosis in neuroblastoma cells through redox imbalance.

As a widespread environmental pollutant, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-induced neurotoxicity has received increasing attention. Studies have shown that BPDE-induced neurodegeneration is due partly to neuronal apoptosis. Unlike apoptosis, ferroptosis is a non-apoptotic form of programmed cell death, but its specific role in the neurotoxicity of BPDE remains unclear. In this work, we investigated the ferroptosis in BPDE-induced cell death in human neuroblastoma cell line SH-SY5Y using a specific pharmacological inhibitor. Lipid peroxides, malondialdehyde production, glutathione / glutathione peroxidase activity, superoxide dismutase activity, and iron content were evaluated. Consistent with previous studies, our data showed that 0.5 μM BPDE poisoning for 24 hr could induce cell apoptosis and that cell survival could be improved by using apoptosis inhibitors. But with prolonged exposure time (72 hr) or increased exposure dose (1.0 μM), we have elucidated and validated that BPDE triggered ferroptosis in human SH-SY5Y cells. We also revealed that suppression of ferroptosis by specific inhibitors, ferrostatin-1 and deferoxamine, significantly rescued the phenotypes of ferroptosis induced by BPDE. BPDE downregulated Nrf2 and its target genes related to redox regulation, GPX4 and SLC7A11, but upregulated HO-1. Our results first demonstrated that BPDE caused cytotoxic effects on cell death via apoptosis and ferroptosis. Most notably, long-term environmental exposure to BPDE becomes a concern due to ferroptosis. Redox imbalance is controlled by the Nrf2, SLC7A11, and HO-1, through which lipid peroxides and ferrous ion accumulation cause ferroptosis after BPDE treatment. These findings highlight that targeting ferroptosis could serve as an effective protective strategy for neurotoxicity of BPDE.

Povezanost polimorfizama SOD2 i GST gena sa rizikom za nastanak i prognozom papilarnog karcinoma bubrežnog parenhima

Introduction: Redox imbalance is an important factor in both carcinogenesis and progression of renal cell carcinoma. Numerous studies are focused on finding potential biomarkers that can aid in early detection, as well as in monitoring disease progression. Among the candidates there are genes coding for antioxidant enzymes - superoxide dismutase 2 (SOD2) and glutathione S -transferase (GST). Aim: This study aims to assess the role of SOD2 and GST genes polymorphisms as risk biomarkers for papillary renal cell carcinoma (pRCC), along with their impact on the survival of these patients. Material and methods: This study included 39 patients and 336 controls. The following polymorphisms were determined by appropriate PCR methods: SOD2 (rs4880), GSTA1 C69T, GSTM1, GSTT1, and GSTP1 (rs1695) . ELISA method was used to measure 8-hydroxy-2'-deoxyguanosine (8-OHdG) and benzo(a)pyrene diol epoxide (BPDE)-DNA adducts plasma level. The effect of the polymorphisms on postoperative prognosis was examined using the available survival data. Results: There was no significant difference in the distribution of SOD2, GSTA1, GSTM1, and GSTT1 gene variants between patients and controls (p > 0.05). However GSTP1 variant (GSTP1 * IleVal + ValVal) genotype was statistically significantly more frequent in patients compared to controls (p < 0.05). Similarly, carriers of GSTP1 variant genotype were at significantly higher risk of developing carcinoma compared to carriers of GSTP1 reference genotype (OR = 16.103, 95% IP = 2.036 - 127.398). There was no association between the level of both 8-OHdG and BPDE-DNA adducts, and different genotypes (p > 0.05). The investigated polymorphisms did not show any prognostic significance (p > 0.05). Conclusion: These results indicate that the GSTP1 variant genotype was related to an increased risk of papillary renal cell carcinoma development. In order to fully understand the effect of investigated polymorphisms as a potential risk and prognostic biomarkers of this cancer, further research with a bigger sample size and longer follow-up are required.

Urban grey spaces are associated with increased allergy in the general population

Backgroundthe built environment in urban areas may have side effects on children's respiratory health, whilst less is known for adulthood. Aimto assess the association between increasing exposure to grey spaces and allergic status in an adult general population sample. Methods2070 subjects (age range 15–84 yrs), living in Pisa/Cascina, Italy, were investigated in 1991–93 through a questionnaire on health status and risk factors, skin prick test (SPT), serum Immunoglobulins E (IgE), and serum antibodies to benzo(a)pyrene diol epoxide (BPDE)-DNA adducts.Land-cover exposure within a 1000 m buffer from each subject's home address was assessed through the CORINE Land Cover program (CLC 1990) within the FP7/HEALS project (2013–2018). Participants' residential addresses were geocoded and the proportion of surrounding grey spaces was calculated.Through logistic regression models, adjusting for potential confounding factors, the effect of a 10% increase in grey spaces exposure on allergic biomarkers/conditions was assessed; the relationship with serum antibodies to BPDE-DNA adducts positivity was also analyzed. ResultsA 10% increase in grey spaces coverage was associated with a higher probability of having SPT positivity (OR 1.07, 95% CI 1.02-1.13), seasonal SPT positivity (OR 1.12, 1.05-1.19), polysensitization (OR 1.11, 1.04-1.19), allergic rhinitis (OR 1.10, 1.04-1.17), co-presence of SPT positivity and asthma/allergic rhinitis (OR 1.16, 1.08-1.25), asthma/allergic rhinitis (OR 1.06, 1.00-1.12), presence of serum antibodies to BPDE-DNA adducts positivity (OR 1.07, 1.01-1.14). Conclusionsgrey spaces have adverse effects on allergic status and are related to a biomarker of polycyclic aromatic hydrocarbons exposure in adulthood. Thus, they may be used as a proxy of urban environmental exposure.

KIF11, a plus end-directed kinesin, as a key gene in benzo(a)pyrene-induced non-small cell lung cancer

Evidence indicates that Benzo(a)pyrenediol-epoxide (BPDE) can damage lung cells, resulting in carcinogenesis with complex mechanisms. We aimed to explore the genes and pathway variations in this process. First, the key gene was screened out and identified through data mining, and then, it was in turn validated by bioinformatics analysis and experimental methods. Consequently, 106 up-regulated and 260 down-regulated differentially expressed genes were yielded, which were enriched in various pathways, such as Cell cycle, and p53 signaling pathway. Then, KIF11 was identified as the key gene. Overexpression of KIF11 in lung cancer had a correlation with advanced pathological grade, advanced T stage, and presence of lymph node metastasis, which predicted poor prognosis. In summary, the present study revealed that KIF11 might be a key gene in the tumorigenesis of BPDE-related lung cancer, raising the possibility of KIF11 as a target for BPDE-induced lung cancer prevention and therapy.

Malignant transformation of human bronchial epithelial cells induced by benzo [a] pyrene suggests a negative feedback of TP53 to PPP1R13L via binding a possible enhancer element

Previous studies have shown that PPP1R13L as an inhibitor of apoptosis protease TP53 can lead to abnormal cell proliferation and carcinogenesis, however, the function of PPP1R13L was complicated and the interaction between TP53 and PPP1R13L needs to be further explored. In the present study, a malignant transformation model of human bronchial epithelial cells induced by benzo (a) pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) was established to observe the regulatory patterns between TP53 and PPP1R13L during carcinogenesis. In vitro experiments including CRISPR-Cas9 editing, RNA silence, Co-Immunoprecipitation and Chromatin Immunoprecipitation were applied to discuss their interactive effects. Additionally, TCGA data profile and our clinical samples of lung cancer were also used to analyze their relationship at the transcriptome level. Interestingly, we found that the mRNA and protein level of TP53 and PPP1R13L fluctuated as a wave in BPDE-induced malignant transformation under wild-type TP53 genetic background. Our results have also demonstrated that PPP1R13L acts as an inhibitor of TP53, while TP53 can regulate PPP1R13L via binding a possible enhancer of the first intron of PPP1R13L gene. Likewise, TCGA data and clinical samples have identified that in the case of TP53 mutation, TP53 expression was negatively correlated with PPP1R13L, while in the case of TP53 wild-type, TP53 expression was not correlated with PPP1R13L. It suggested that there existed a negative feedback of wild-type TP53 to PPP1R13L, which reminded a unique implication during chemical carcinogenesis.

Carcinogen-induced DNA structural distortion differences in the RAS gene isoforms; the importance of local sequence

BackgroundLocal sequence context is known to have an impact on the mutational pattern seen in cancer. The RAS genes and a smoking carcinogen, Benzo[a]pyrene diol epoxide (BPDE), have been utilised to explore these context effects. BPDE is known to form an adduct at the guanines in a number of RAS gene sites, KRAS codons 12, 13 and 14, NRAS codon 12, and HRAS codons 12 and 14.ResultsMolecular modelling techniques, along with multivariate analysis, have been utilised to determine the sequence influenced differences between BPDE-adducted RAS gene sequences as well as the local distortion caused by the adducts.ConclusionsWe conclude that G:C > T:A mutations at KRAS codon 12 in the tumours of lung cancer patients (who smoke), proposed to be predominantly caused by BPDE, are due to the effect of the interaction methyl group at the C5 position of the thymine base in the KRAS sequence with the BPDE carcinogen investigated causing increased distortion. We further suggest methylated cytosine would have a similar effect, showing the importance of methylation in cancer development.

The mechanism of apoptosis of Chlamys farreri hemocytes under benzopyrene stress in vitro

Hemocytes are critical to the immune defense system of bivalves, and polycyclic aromatic hydrocarbons (PAHs) can mediate the immunity of bivalves by affecting the apoptosis of hemocytes. However, the underlying mechanism is still unclear. Chlamys farreri, as an important economic bivalve, was selected as the research subject for this experimentation. The hemocytes were exposed to typical PAHs–benzopyrene (B[a]P) in vitro to explore the apoptosis mechanism through detecting oxidative stress and oxidative damage-related indicators, apoptosis pathway factors, and apoptosis rate within 24 h. The results showed that the reactive oxygen species (ROS) and benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) content in hemocytes increased significantly under B[a]P exposure, while antioxidant genes, glutathione peroxidase content and total antioxidant capacity all showed a trend of first rising and subsequent falling. B[a]P also caused serious damage to DNA and lysosomal membrane stability. The proapoptotic factors genes in the mitochondrial apoptosis pathway were significantly up-regulated, and the anti-apoptotic gene Bcl-2 was significantly down-regulated. Besides, mitochondrial membrane potential stability was significantly reduced and caspase 9 enzyme activity was significantly improved with the B[a]P stimulation. The factors of death receptor pathway were also significantly up-regulated by B[a]P. Moreover, the expression levels of Mitogen-Activated Protein Kinases were also induced. The gene expression and enzyme activity of the caspase 3 and the apoptosis rate were significantly increased under B[a]P exposure. In conclusion, these results indicated that ROS was induced by B[a]P, and further triggered the oxidative stress and oxidative damage in hemocytes. B[a]P induced hemocyte apoptosis was mediated by both mitochondrial apoptosis pathway and death receptor apoptosis, and the activation of mitochondrial apoptosis pathway was affected by ROS. In addition, BPDE and MAPKs may play important roles in the B[a]P-mediated apoptosis pathway. This study deepens understanding of the apoptosis pathway and the immunotoxicity mechanism in bivalves hemocytes stimulated by persistent organic pollutants.

Benzo[a]pyrene Cytotoxicity Tolerance in Testicular Sertoli Cells Involves Aryl-hydrocarbon Receptor and Cytochrome P450 1A1 Expression Deficiencies.

Benzo[a]pyrene (B[a]P) is a potent carcinogen and is classified as an endocrine-disrupting chemical. In mammalian testes, Sertoli cells support spermatogenesis. Therefore, if these cells are negatively affected by exposure to xenotoxic chemicals, spermatogenesis can be seriously disrupted. In this context, we evaluated whether mouse testicular TM4 Sertoli cells are susceptible to the induction of cytotoxicity-mediated cell death after exposure to B[a] P in vitro. In the present study, while B[a]P and B[a]P-7,8-diol were not able to induce cell death, exposure to BPDE resulted in cell death. BPDE-induced cell death is accompanied by the activation of caspase-3 and caspase-7. Depolarization of the mitochondrial membrane and cytochrome c release from mitochondria were observed in benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated cells. These results indicate that TM4 cells are susceptible to apoptosis in a caspase-dependent manner. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that aryl hydrocarbon receptor (AhR) expression was almost undetectable in TM4 cells and that its expression was not altered after B[a]P treatment. This indicates that TM4 cells are nearly AhR-deficient. In TM4 cells, the CYP1A1 protein and its activity were not present. From these results, it is clear that AhR may be a prerequisite for CYP1A1 expression in TM4 cells. Therefore, TM4 cells can be referred to as CYP1A1-deficient cells. Thus, TM4 Sertoli cells are believed to have a rigid and protective cellular machinery against genotoxic agents. In conclusion, it is suggested that tolerance to B[a]P cytotoxicity is associated with insufficient AhR and CYP1A1 expression in testicular Sertoli cells.

Error-prone bypass patch by a low-fidelity variant of DNA polymerase zeta in human cells

DNA polymerase ζ (Pol ζ) is a specialized Pol that is involved in translesion DNA synthesis (TLS), in particular, in the extension of primer DNA after bypassing DNA lesions. Previously, we established human cells that express a variant form of Pol ζ with an amino acid change of leucine 2618 to methionine (L2618M) in the catalytic subunit REV3L (DNA Repair, 45, 34–43, 2016). This amino acid change made the cells more sensitive to the mutagenicity of benzo[a]pyrene diol epoxide (BPDE). In this study, we embedded BPDE-N2-guanine at a defined position in the supF gene on the shuttle plasmid and introduced it to REV3 L2618M cells or the wild-type (WT) cells to examine how far Pol ζ L2618M extends the primer DNA after bypassing the lesion. The adduct induced primarily G to T and G to C at the adducted site in both cell lines, but generated additional sequence changes such as base substitutions, deletions and additions in the extension patch much more often in REV3 L2618M cells than in the WT cells. Mutations in the extension patch in REV3 L2618M cells occurred most often within 10 bps from the adducted site. Then, the number of mutations gradually decreased and no mutations were observed between 30 and 40 bps from the lesion. We concluded that human Pol ζ L2618M and perhaps WT Pol ζ extend the primer DNA up to approximately 30 bps from the lesion in vivo. The possibility of involvement of Pol ζ L2618M in the insertion step of TLS is discussed.

Benzo[a]pyrene and Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide induced locomotor and reproductive senescence and altered biochemical parameters of oxidative damage in Canton-S Drosophila melanogaster.

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) commonly found in cigarette smoke, automobile exhaust fumes, grilled meat, and smoked food among others. Exposure to B[a]P is associated with a range of toxic effects including developmental, neurological, oxidative, inflammatory, mutagenic, carcinogenic and mortal. Efficient and more affordable experimental models like Drosophila melanogaster could provide more insight into the mechanism of PAH toxicity and help develop new strategies for prevention, diagnosis and treatment of PAH-related conditions. In this study, we examined the induction of some biochemical changes along with mortality and functional senescence by B[a]P and its metabolite, benzo[a]pyrene- 7,8-dihydrodiol-910-epoxide (BPDE) in the Canton-S strain of Drosophila melanogaster, with the aim to establish an alternative assay medium for B[a]P toxicity in flies. Flies were exposed to 2-200 μM of B[a]P and 1-10 μM of BPDE through diet for a seven-day survival assay followed by a four-day treatment to determine the effects of the compounds on negative geotaxis, fecundity and some biochemical parameters of oxidative damage. BPDE significantly reduced the survival rate of flies along the 7 days of exposure whereas B[a]P did not cause any significant change in the survival rate of flies. B[a]P and BPDE significantly reduced the climbing ability of flies after 4 days of exposure. Rate of emergence of flies significantly reduced at 10-200 μM of B[a]P and 5-10 μM of BPDE. Both compounds caused various levels of alterations in the values of reduced glutathione (GSH), total thiol (T-SH), glutathione-S-transferase (GST), catalase (CAT), hydrogen peroxide (H2O2), nitric oxide (NO) and acetylcholinesterase (AChE) of the flies. The compounds also exhibited high binding affinities and molecular interactions with the active site amino acid residues of Drosophila GST and the inhibitor binding site of Drosophila AChE in an in silico molecular docking analysis, with BPDE forming stable hydrogen bonds with AChE. Hence, the Canton-S strain of Drosophila melanogaster could offer a simple and affordable assay medium to study B[a]P toxicity.

Comparison of Serum Benzo(a)pyrene Diol Epoxide – Protein Adducts Level between Kretek Cigarette Smokers and Nonsmokers and the Related Factors

Background: Benzopyrene is a carcinogenic agent found in cigarette smoke. Benzo(a)pyrene diol epoxide (BPDE) is one of the benzopyrene metabolites. In this study, we investigated the level of serum BPDE in kretek cigarette smokers compared to non-smokers. Methods: A cross-sectional study which involved 32 “healthy” kretek cigarette smokers and 32 “healthy” nonsmokers were conducted. We collected the blood sample and the serum BPDE level was assayed using enzyme-linked immunosorbent assay kit. The BPDE serum level in kretek cigarette smokers was compared to the level in nonsmokers. Results: A total of 32 kretek smokers and 32 controls underwent an examination of the BPDE-protein adducts level. In the kretek smokers group, 59.4% were aged over 45 years and 56.3% have a high educational background, while in the control group, 87.5% were aged under 45 years and 75% have high educational backgrounds. The level of BPDE-protein adducts in the kretek smokers subject was 12.15 (8.87–33.55) ng/ml and the levels in the control group were 11.4 (3.87–13.27) ng/ml, P = 0.004. The factors which influence the levels BDPE-protein adducts in smokers cigarettes, as determined by multivariate analysis, were sucking pattern (P = 0.002) and the degree of addiction (P = 0.047). Conclusion: The serum BPDE-protein adducts level was higher in smokers compared to nonsmokers, and the sucking pattern and degree of addiction are the influencing factors.

Environmental Pollutant Benzo[a]pyrene Induces Recurrent Pregnancy Loss through Promoting Apoptosis and Suppressing Migration of Extravillous Trophoblast

Methods The implantation sites, fetus resorption, and abnormal fetuses were studied in pregnant mice treated with different doses of BaP by oral gavage from day 1 to day 10 of gestation. Additionally, apoptosis and related signaling pathway, and the migration and invasion of trophoblasts, were assessed before and after exposure of BPDE in Swan 71 trophoblast cell. Besides, the migration and invasion, and its related signaling pathway, were assessed in villi obtained from women. Results We observed a concentration-dependent incidence of abnormal murine fetuses, beginning with 0.1 mg/kg BaP; with a BaP concentration of 2 mg/kg, no fetuses developed. Correspondingly, a BPDE concentration-dependent apoptosis of human trophoblasts. Beginning with 0.5 μM BPDE exposure, Bax/Caspase-3 were increased and Bcl-2 decreased. Furthermore, BPDE also inhibited, in a dose-dependent manner, the migration of villous explants from elective abortion women, consistent with the reduced migration of villous explants from women with recurrent pregnancy loss (RPL), and reduced the cell immigration in Swan 71 trophoblasts, in a dose-dependent manner measured by transwell assays. Conclusions Our study results provide mechanistic insight to the effect of BPDE on trophoblast dysfunction through enhanced cell apoptosis and inhibited migration, providing further experimental evidence to the causative links between BaP exposure and PRL.

Abstract A01: Environmentally prevalent polycyclic aromatic hydrocarbons elicit cocarcinogenic properties in human and mouse lung cells

Abstract Rationale: Polycyclic aromatic hydrocarbons (PAHs) are prevalent contaminants in our environment, workplace, and in first- and secondhand smoke from both cigarettes and marijuana. The toxicity of low molecular weight (LMW) PAHs compared to high molecular weight PAHs (e.g., benzo[a]pyrene, B[a]P) is not well studied in lung cells. Our overall hypothesis was that LMW PAHs act as cocarcinogens in the presence of a known carcinogen (B[a]P) in human and mouse lung epithelial cells. Gap junctions are commonly suppressed during tumor promotion, while DNA-adduct formation and micronuclei (MN) are observed during the initiation stage of cancer. We used these endpoints together as markers of carcinogenicity in Beas2B and C10 cells. Methods: Beas2B, a human bronchial epithelial cell line, and C10 cells, a nontumorigenic type II alveolar pneumocyte cell line, were used. LMW PAHs (1-methylanthracene, 1-MeA, and fluoranthene, Flthn) were applied in a 1:1 ratio mixture based on previous studies and B[a]P as a classic carcinogen. Cells were treated for 30 min.-24 h with the LMW PAH mixture, B[a]P, or both, and assessed for the following endpoints: cytotoxicity, genotoxicity (MN), apoptosis, proliferation, gap junctional intercellular communication (GJIC) by the lucifer yellow scalpel-loaded/dye-transfer assay, and DNA adducts of B[a]P by HPLC with fluorescence detection in C10 cells. Results: LMW PAHs are more cytotoxic in the human Beas2B cells than the mouse C10 cells. Significant toxicity is observed in Beas2B cells at 0.3 uM B[a]P combined with the LMW PAH mixture (0.1-1 uM), while no toxicity is observed in C10 cells at identical doses. No significant apoptosis was observed in Beas2B cells while significant increases in S-phase were observed in response to B[a]P combined with LMW PAH mixture compared to B[a]P alone. B[a]P in combination with the LMW PAH mixture significantly inhibited GJIC in C10 and Beas2B cells compared to B[a]P or the LMW PAH mixture alone, supporting a role for GJIC suppression in cancer development. B[a]P diol-epoxide (BPDE)-DNA adduct levels were also significantly induced in C10 cells at 1.0 uM B[a]P with the LMW PAH mixture at several doses (less than or equal to 1.0 uM) and significant increases in BPDE-DNA adduct formation compared to B[a]P alone were observed. Lastly, MN were also observed in both cell lines and significantly differed between B[a]P alone and in combination with the LMW PAH mixture. DNA adduct formation and genotoxicity coincided with GJIC inhibition. Conclusion: These studies provide evidence that LMW PAHs in combination with B[a]P can elicit increased carcinogenic potential in human and mouse cells. Support: Flight Attendant Medical Research Institute (CIA130022, AKB); the National Institute for Environmental Health Sciences (R15ES024893, AKB). Citation Format: Alison K. Bauer, Katelyn J. Siegrist, Sabine Plöttner, Thomas Brüning, Heiko U. Käfferlein. Environmentally prevalent polycyclic aromatic hydrocarbons elicit cocarcinogenic properties in human and mouse lung cells [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A01.

Metabolism and genotoxicity of polycyclic aromatic hydrocarbons in human skin explants: mixture effects and modulation by sunlight.

Cutaneous exposure to carcinogenic polycyclic aromatic hydrocarbons (PAH) occurs frequently in the industrialized workplace. In the present study, we addressed this topic in a series of experiments using human skin explants and organic extracts of relevant industrial products. PAH mixtures were applied topically in volumes containing either 10 or 1nmol B[a]P. We first observed that although mixtures were very efficient at inducing expression of CYP450 1A1, 1A2, and 1B1, formation of adducts of PAH metabolites to DNA, like those of benzo[a]pyrene diol epoxide (BPDE), was drastically reduced as the complexity of the surrounding matrix increased. Interestingly, observation of a nonlinear, dose-dependent response with the least complex mixture suggested the existence of a threshold for this inhibitory effect. We then investigated the impact of simulated sunlight (SSL) on the effects of PAH in skin. SSL was found to decrease the expression of CYP450 genes when applied either after or more efficiently before PAH treatment. Accordingly, the level of DNA-BPDE adducts was reduced in skin samples exposed to both PAH and SSL. The main conclusion of our work is that both increasing chemical complexity of the mixtures and co-exposure to UV radiation decreased the production of adducts between DNA and PAH metabolites. Such results must be taken into account in risk management.

Polycyclic aromatic hydrocarbons in bays of the Rio de Janeiro state coast, SE - Brazil: Effects on catfishes

The Brazilian coast of the Rio de Janeiro State has bays of great economic, ecological and social importance. However, these ecosystems have been suffering intense anthropogenic influence, mainly due to the contamination by polycyclic aromatic hydrocarbons (PAHs) from urban-industrial activities. Moreover, PAHs are organic pollutants of high toxicity and carcinogenicity causing global concern to human and environmental health. This study evaluated on catfish (Genidens genidens) a set of key parameters (sex, morphometric traits, condition factor (K), PAH metabolites in gallbladder, frequency of micronucleus (MN) and erythrocytic nuclear abnormalities (ENA) in blood. In addition we also evaluated histopathological hepatic effects, Ethoxyresorufin-O-deethylase (EROD) activity and Benzo(a)pyrene diol epoxide (BPDE)-DNA adducts) in liver samples, in order to indicate the fish health status and environmental pollution levels of three main Bays (Guanabara, Sepetiba and Ilha Grande) of the Rio de Janeiro State, in the Southeast of Brazil. In general, the worst physical and metabolic conditions in catfishes were evidenced in Guanabara Bay, possibly indicating the highest level of contamination by PAHs. Contrary evidence was observed in Ilha Grande Bay, showing lower biological changes in G. genidens. However in Sepetiba Bay, the influence of PAHs contamination showed the highest hepatic lesions in catfishes, prevailing foci of cellular alterations, megalocytic hepatosis and hydropic vacuolations. The employability of a set of biomarkers on catfish was efficient for screening pollution for PAHs in tropical environments. This reinforces the need for effective actions of monitoring and conservation strategies of bays of the Rio de Janeiro State (Brazil), in order to ensure quality and health to both human and environment.

Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis

BACKGROUND: Escaping cell death pathways is an important event during carcinogenesis. We previously identified anti-TNFα-induced apoptosis (ATIA, also known as vasorin) as an antiapoptotic factor that suppresses reactive oxygen species (ROS) production. However, the role of vasorin in lung carcinogenesis has not been investigated. METHODS: Vasorin expression was examined in human lung cancer tissues with immunohistochemistry and database analysis. Genetic and pharmacological approaches were used to manipulate protein expression and autophagy activity in human bronchial epithelial cells (HBECs). ROS generation was measured with fluorescent indicator, apoptosis with release of lactate dehydrogenase, and cell transformation was assessed with colony formation in soft agar. RESULTS: Vasorin expression was increased in human lung cancer tissues and cell lines, which was inversely associated with lung cancer patient survival. Cigarette smoke extract (CSE) and benzo[a]pyrene diol epoxide (BPDE)–induced vasorin expression in HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation in association with enhanced ROS accumulation and autophagy. Scavenging ROS attenuated autophagy and cytotoxicity in vasorin knockdown cells, suggesting that vasorin potentiates transformation by impeding ROS-mediated CSE cytotoxicity and improving survival of the premalignant cells. Suppression of autophagy effectively inhibited CSE-induced apoptosis, suggesting that autophagy was pro-apoptotic in CSE-treated cells. Importantly, blocking autophagy strongly potentiated CSE-induced transformation. CONCLUSION: These results suggest that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation by suppressing autophagy-mediated apoptosis, which could be exploited for lung cancer prevention.

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

ABSTRACT 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.

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.