Diol Epoxide Research Articles

Exploring the Mechanisms of Yishen Tongluo Decoction on Repairing DNA Damage in Mouse Spermatogonia Cells Based on Whole Transcriptome Sequencing.

The aim of this study was to investigate the potential mechanism through which Yishen Tongluo decoction (YSTL) repairs DNA damage caused by benzo(a)pyrene diol epoxide (BPDE) in mouse spermatocytes (GC-2). The GC-2 cells were divided randomly into the control group, BPDE group, and low-, medium-, and high-dose YSTL groups of YSTL decoction. A comet assay was used to detect the DNA fragment index (DFI) of cells in each group. Based on the DFI results, whole transcriptome sequencing was conducted, followed by trend analysis, gene ontology (GO) enrichment analysis, kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, and ceRNA network analysis. Compared with the control group, the BPDE group reported a significant increase in the DNA fragmentation index (DFI) (p < .05). Compared with the BPDE group, the low-, high- and medium-dose YSTL groups had a significantly reduced DFI (p < .05). Whole-transcriptome sequencing revealed seven differentially expressed circRNAs, 203 differentially expressed miRNAs, and 3,662 differentially expressed mRNAs between the control group and the BPDE group. There was a total of 12 differentially expressed circRNAs, 204 miRNAs, and 2150 mRNAs between the BPDE group and the traditional Chinese medicine group. The pathways involved include DNA repair pathway, nucleotide excision repair pathway, base excision repair pathway, etc. The ceRNA network reported that Hmga2 was the core protein involved, novel_cir_000117 and mmu-miR-466c-3p were located upstream of Hmga2, and they were regulatory factors associated with Hmga2. Finally, we conclude that YSTL decoction may repair sperm DNA damage caused by BPDE through the novel_cir_000117-mmu-miR-466c-3p-Hmga2 pathway.

Environmental exposure to polycyclic aromatic hydrocarbons: An underestimated risk factor for systemic lupus erythematosus onset and progression

ObjectiveTo investigate the link between systemic lupus erythematosus (SLE) incidence and exposure to environmental polycyclic aromatic hydrocarbons (PAH). MethodsA case-control study (ChiCTR2000038187) involving 316 SLE patients and 851 healthy controls (HCs) was executed. Environmental exposure was assessed via a questionnaire, stratified by gender and age (females <35 and ≥35 years, males). Blood samples collected from 89 HCs, 85 inactive, and 95 active SLE patients were used to measure serum benzo[a]pyrene diol epoxide -albumin (BPDE-Alb) adducts and PAH concentrations, indicating long-term and short-term exposure respectively. Intergroup comparisons and statistical analyses were conducted using R version 4.3.1. ResultsDiverse patterns were found in how environmental factors affect SLE onset across different demographics. Lifestyle exposure factors were found to be a stronger determinant of SLE onset than occupational exposure factors in women under 35. Indoor air pollution had a significant impact on SLE incidence, potentially comparable to outdoor air pollution. Lifestyle-related PAH exposure had a greater impact on SLE than occupational PAH exposure. PAH exposure levels progressively increase from HCs to inactive and active SLE patients. Active SLE patients show markedly higher BPDE-Alb levels than HCs. ConclusionsEnvironmental PAH, particularly lifestyle-related, are significant, yet under-recognized, risk factors for SLE. Statement of environmental implicationWe examined the relationship between exposure to environmental polycyclic aromatic hydrocarbons (PAH) and the incidence of systemic lupus erythematosus (SLE). PAH, prevalent in sources such as cigarette smoke, air pollution, and charred food, pose significant health hazards. This study is the first to investigate specific PAH exposure levels in SLE patients. We determined actual PAH exposure levels in both SLE patients and healthy individuals and indicated that long-term PAH exposure biomarker is more reliable for evaluating exposure in non-occupationally exposed groups like SLE, compared to short-term markers. These findings provide valuable insights for future research on similar non-occupationally exposed populations.

Circ0087385 promotes DNA damage in benzo(a)pyrene-induced lung cancer development by upregulating CYP1A1.

Increasing environmental genotoxic chemicals have been shown to induce epigenetic alterations. However, the interaction between genetics and epigenetics in chemical carcinogenesis is still not fully understood. Here, we constructed an in vitro human lung carcinogenesis model (16HBE-T) by treating human bronchial epithelial cells with a typical significant carcinogen benzo(a)pyrene (BaP). We identified a novel circular RNA, circ0087385, which was overexpressed in 16HBE-T and human lung cancer cell lines, as well as in lung cancer tissues and serum exosomes from lung cancer patients. The upregulated circ0087385 after exposure to BaP promoted DNA damage in the early stage of chemical carcinogenesis and affected the cell cycle, proliferation, and apoptosis of the malignantly transformed cells. Overexpression of circ0087385 enhanced the expression of cytochrome P450 1A1 (CYP1A1), which is crucial for metabolically activating BaP. Interfering with circ0087385 or CYP1A1 reduced the levels of ultimate carcinogen benzo(a)pyrene diol epoxide (BPDE) and BPDE-DNA adducts. Interfering with CYP1A1 partially reversed the DNA damage induced by high expression of circ0087385, as well as decreased the level of BPDE and BPDE-DNA adducts. These findings provide novel insights into the interaction between epigenetics and genetics in chemical carcinogenesis which are crucial for understanding the epigenetic and genetic toxicity of chemicals.

A Practical Site-specific Method for the Detection of Bulky DNA Damages

Helix-distorting DNA damages block RNA and DNA polymerase, compromising cell function and fate. In human cells, these damages are removed primarily by nucleotide excision repair (NER). Here, we describe damage-sensing PCR (dsPCR), a PCR-based method for the detection of these DNA damages. Exposure to DNA damaging agents results in lower PCR signal in comparison to non-damaged DNA, and repair is measured as the restoration of PCR signal over time. We show that the method successfully detects damages induced by ultraviolet (UV) radiation, by the carcinogenic component of cigarette smoke benzo[a]pyrene diol epoxide (BPDE) and by the chemotherapeutic drug cisplatin. Damage removal measured by dsPCR in a heterochromatic region is less efficient than in a transcribed and accessible region. Furthermore, lower repair is measured in repair-deficient knock-out cells. This straight-forward method could be applied by non-DNA repair experts to study the involvement of their gene-of-interest in repair. Furthermore, this method is fully amenable for high-throughput screening of DNA repair activity.

Characterizing Benzo[a]pyrene Adducts in Transfer RNAs Using Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS).

The activated forms of the environmental pollutant benzo[a]pyrene (B[a]P), such as benzo[a]pyrene diol epoxide (BPDE), are known to cause damage to genomic DNA and proteins. However, the impact of BPDE on ribonucleic acid (RNA) remains unclear. To understand the full spectrum of potential BPDE-RNA adducts formed, we reacted ribonucleoside standards with BPDE and characterized the reaction products using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To understand the potential types of adducts that could form with biological RNAs, eukaryotic transfer RNAs (tRNAs) were also reacted with BPDE. The isolation and analysis of the modified and adducted ribonucleosides using LC-MS/MS revealed several BPDE derivatives of post-transcriptional modifications. The approach outlined in this work enables the identification of RNA adducts from BPDE, which can pave the way for understanding the potential impacts of such adducts on the higher-order structure and function of modified RNAs.

The medicinal mushroom Ganoderma lucidum prevents lung tumorigenesis induced by tobacco smoke carcinogens.

Ganoderma lucidum (GL), commonly known as "Lingzhi", is a well-known medicinal mushroom with antioxidant and anti-cancer activity. This study examined the effects of a commercial GL product (GLSF) containing the spore and fruiting body in a 30:8 ratio on tobacco smoke carcinogen-induced lung toxicity and carcinogenesis. The potential chemopreventive effect of GLSF was evaluated in vitro and in vivo. The non-tumorous human bronchial epithelial cells (BEAS-2B cells) were treated with GLSF extract (0.025 and 0.05mg/mL), which significantly blocked malignant transformation induced by benzo[a]pyrene diol epoxide (BPDE) in a dose-dependent manner. To confirm its anti-carcinogenic activity in vivo, the mice were pre-treated with GLSF (2.0g/kg of body weight) or curcumin (100mg/kg of body weight) by oral gavage daily for 7days and then exposed to a single dose of benzo[a]pyrene (B[a]P) (125mg/kg of body weight). The GLSF-treated mice showed a significant reduction in B[a]P-induced lung toxicity, as indicated by decreased lactate dehydrogenase activity, malondialdehyde levels, inflammatory cell infiltration, and improved lung histopathology. We next determined the chemopreventive activity of GLSF in mice which were exposed to two weekly doses of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 100mg/kg, on the 1st and 8thdays) and fed with control or a modified diet containing GLSF (2.0g/kg) or metformin (250mg/kg) for 33weeks. The GLSF and metformin treatments blocked NNK-induced lung tumor development by decreasing the lung weight, tumor area, and tumor burden compared to the mice exposed to NNK only. GLSF treatment also attenuated the expression of inflammatory, angiogenic, and apoptotic markers in lung tumors. Therefore, GLSF may be used for ameliorating tobacco smoke carcinogens-induced lung toxicity and carcinogenesis.

Crystalline silica-exposed human lung epithelial cells presented enhanced anchorage-independent growth with upregulated expression of BRD4 and EZH2 in autocrine and paracrine manners.

Crystalline silica-induced inflammation possibly facilitates carcinogenesis. Here, we investigated its effect on lung epithelium damage. We prepared conditioned media of immortalized human bronchial epithelial cell lines (hereinafter bronchial cell lines) NL20, BEAS-2B, and 16HBE14o- pre-exposed to crystalline silica (autocrine crystalline silica conditioned medium), a phorbol myristate acetate-differentiated THP-1 macrophage line, and VA13 fibroblast line pre-exposed to crystalline silica (paracrine crystalline silica conditioned medium). As cigarette smoking imposes a combined effect on crystalline silica-induced carcinogenesis, a conditioned medium was also prepared using the tobacco carcinogen benzo[a]pyrene diol epoxide. Crystalline silica-exposed and growth-suppressed bronchial cell lines exhibited enhanced anchorage-independent growth in autocrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium compared with that in unexposed control conditioned medium. Crystalline silica-exposed nonadherent bronchial cell lines in autocrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium showed increased expression of cyclin A2, cdc2, and c-Myc, and of epigenetic regulators and enhancers, BRD4 and EZH2. Paracrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium also accelerated the growth of crystalline silica-exposed nonadherent bronchial cell lines. Culture supernatants of nonadherent NL20 and BEAS-2B in crystalline silica and benzo[a]pyrene diol epoxide conditioned medium had higher EGF concentrations, whereas those of nonadherent 16HBE14o- had higher TNF-α levels. Recombinant human EGF and TNF-α promoted anchorage-independent growth in all lines. Treatment with EGF and TNF-α neutralizing antibodies inhibited cell growth in crystalline silica conditioned medium. Recombinant human TNF-α induced BRD4 and EZH2 expression in nonadherent 16HBE14o-. The expression of γH2AX occasionally increased despite PARP1 upregulation in crystalline silica-exposed nonadherent lines with crystalline silica and benzo[a]pyrene diol epoxide conditioned medium. Collectively, crystalline silica- and benzo[a]pyrene diol epoxide-induced inflammatory microenvironments comprising upregulated EGF or TNF-α expression may promote crystalline silica-damaged nonadherent bronchial cell proliferation and oncogenic protein expression despite occasional γH2AX upregulation. Thus, carcinogenesis may be cooperatively aggravated by crystalline silica-induced inflammation and genotoxicity.

Global Discovery and Temporal Changes of Human Albumin Modifications by Pan-Protein Adductomics: Initial Application to Air Pollution Exposure

Assessing personal exposure to environmental toxicants is a critical challenge for predicting disease risk. Previously, using human serum albumin (HSA)-based biomonitoring, we reported dosimetric relationships between adducts at HSA Cys34 and ambient air pollutant levels (Smith et al., Chem. Res. Toxicol. 2021, 34, 1183). These results provided the foundation to explore modifications at other sites in HSA to reveal novel adducts of complex exposures. Thus, the Pan-Protein Adductomics (PPA) technology reported here is the next step toward an unbiased, comprehensive characterization of the HSA adductome. The PPA workflow requires <2 μL serum/plasma and uses nanoflow-liquid chromatography, gas-phase fractionation, and overlapping-window data-independent acquisition high-resolution tandem mass spectrometry. PPA analysis of albumin from nonsmoking women exposed to high levels of air pollution uncovered 68 unique location-specific modifications (LSMs) across 21 HSA residues. While nearly half were located at Cys34 (33 LSMs), 35 were detected on other residues, including Lys, His, Tyr, Ser, Met, and Arg. HSA adduct relative abundances spanned a ∼400 000-fold range and included putative products of exogenous (SO2, benzene, phycoerythrobilin) and endogenous (oxidation, lipid peroxidation, glycation, carbamylation) origin, as well as 24 modifications without annotations. PPA quantification revealed statistically significant changes in LSM levels across the 84 days of monitoring (∼3 HSA lifetimes) in the following putative adducts: Cys34 trioxidation, β-methylthiolation, benzaldehyde, and benzene diol epoxide; Met329 oxidation; Arg145 dioxidation; and unannotated Cys34 and His146 adducts. Notably, the PPA workflow can be extended to any protein. Pan-Protein Adductomics is a novel and powerful strategy for untargeted global exploration of protein modifications.

Biodegradation of benzo(a)pyrene by Pseudomonas strains, isolated from petroleum refinery effluent: Degradation, inhibition kinetics and metabolic pathway

Benzo(a)pyrene, a five-ring polyaromatic hydrocarbon, originating from coal tar, crude oil, tobacco, grilled foods, car exhaust etc, is highly persistent in the environment. It has been classified as a Group I carcinogen, as on its ingestion in human body, diol epoxide metabolites are generated, which bind to DNA causing mutations and eventual cancer. Among various removal methods, bioremediation is most preferred as it is a sustainable approach resulting in complete mineralization of benzo(a)pyrene. Therefore, in this study, biodegradation of benzo(a)pyrene was performed by two strains of Pseudomonas, i. e WDE11 and WD23, isolated from refinery effluent. Maximum benzo(a)pyrene tolerance was 250 mg/L and 225 mg/L against Pseudomonas sp. WD23 and Pseudomonas sp. WDE11 correspondingly. Degradation rate constants varied between 0.0468 and 0.0513/day at 50 mg/L with half-life values between 13.5 and 14.3 days as per first order kinetics, while for 100 mg/L, the respective values varied between 0.006 and 0.007 L/mg. day and 15.28–16.67 days, as per second order kinetics. The maximum specific growth rate of strains WDE11 and WD23 was 0.3512/day and 0.38/day accordingly, while concentrations over 75 mg/L had an inhibitory effect on growth. Major degradation metabolites were identified as dihydroxy-pyrene, naphthalene-1,2-dicarboxylic acid, salicylic acid, and oxalic acid, indicating benzo(a)pyrene was degraded via pyrene intermediates by salicylate pathway through catechol meta-cleavage. The substantial activity of the catechol 2,3 dioxygenase enzyme was noted during the benzo(a)pyrene metabolism by both strains with minimal catechol 1,2 dioxygenase activity. This study demonstrates the exceptional potential of indigenous Pseudomonas strains in complete metabolism of benzo(a)pyrene.

The β-Blocker Carvedilol Prevents Benzo(a)pyrene-Induced Lung Toxicity, Inflammation and Carcinogenesis.

The current study evaluated the effects of the β-blocker carvedilol on benzo(a)pyrene (B(a)P) and its active metabolite benzo(a)pyrene diol epoxide (BPDE)-induced lung toxicity, inflammation and carcinogenesis and explored the potential mechanisms. Carvedilol blocked the BPDE-induced malignant transformation of human bronchial epithelial cells BEAS-2B. In BEAS-2B cells, B(a)P strongly activated ELK-1, a transcription factor regulating serum response element (SRE) signaling, which was attenuated by carvedilol. Carvedilol also inhibited the B(a)P-induced AhR/xenobiotic responsive element (XRE) and mRNA expression of CYP1A1 and attenuated B(a)P-induced NF-κB activation. In a B(a)P-induced acute lung toxicity model in CD-1/IGS mice, pretreatment with carvedilol for 7 days before B(a)P exposure effectively inhibited the B(a)P-induced plasma levels of lactate dehydrogenase and malondialdehyde, inflammatory cell infiltration and histopathologic abnormalities in the lung, and upregulated the expression of GADD45α, caspase-3 and COX-2 in the lung. In a B(a)P-induced lung carcinogenesis model in A/J mice, carvedilol treatment for 20 weeks did not affect body weight but significantly attenuated tumor multiplicity and volume. These data reveal a previously unexplored role of carvedilol in preventing B(a)P-induced lung inflammation and carcinogenesis by inhibiting the cross-talk of the oncogenic transcription factors ELK-1, AhR and NF-κB.

Abstract A009: Effects of commonly prescribed β-blockers on tobacco carcinogen induced reactive oxygen species in human non-tumorigenic lung epithelial cells

Abstract Lung cancer is a detrimental disease to humans and tobacco smoking causes more than 80% of lung cancer deaths. Chemoprevention is currently the major strategy to prevent smoking related lung cancer deaths. Key components that contribute to tobacco carcinogenicity are benzo(a)pyrene [B(a)P] and its diol epoxide metabolite BPDE. The goal of this study is to evaluate the level of reactive oxygen species (ROS), a mediator for cellular DNA damage leading to cancer, when pre-treated with the β-blockers carvedilol and nebivolol, and the antioxidant resveratrol. B(a)P and BPDE were used to induce ROS in human lung epithelial cells (BEAS-2B). The non- fluorescent and non-polar 2’,7- dichlorofluorescein diacetate (DCFH-DA) probe was added to the cells. DCFH-DA was converted to DCFH in the cells by hydrolyzation of DCFH. ROS then induced the conversation of DCFH into 2,7-dichlorofluorescein (DCF). Florescence of DCF was measured by flow cytometry to detect ROS level. B(a)P at concentrations 10 and 100 μM failed to induce ROS while BPDE successfully induced ROS dose-dependently during our pilot study. As a result, different concentrations of BPDE(2 μM and 4 μM) were used to induce ROS. When BEAS-2B was pre-treated with resveratrol (50 μM), ROS was reduced by 42% compared to the cells treated with 2 μM BPDE alone. Additionally, there was a decrease in ROS when pre-treated with nebivolol and carvedilol (5 μM). Nebivolol reduced 52% and 64% of the ROS induced by BPDE 2 μM and 4 μM, respectively. Carvedilol reduced 31% and 10% of the ROS induced by BPDE 2 μM and 4 μM, respectively. These results suggest that some commonly used anti-hypertensive agents, such as the β-blockers , may block or reduce tobacco carcinogen induced ROS and thus can be used as chemopreventive agents for lung cancer. Citation Format: Jenny Lee, Ayaz Shahid, Ying Huang. Effects of commonly prescribed β-blockers on tobacco carcinogen induced reactive oxygen species in human non-tumorigenic lung epithelial cells [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A009.

Computational investigations of the excited state dynamics and quenching mechanisms of polycyclic aromatic hydrocarbon DNA adducts in solution

Synthetically modified fluorescent nucleotides (SFNs) are highly popular in a variety of experiments to explore biochemistry in molecular imaging, but the connection between their photodynamics and quenching mechanisms to their molecular structure remain relatively unstudied computationally. We combine various levels of theory, including classical force field dynamics and excited state quantum mechanic/molecular mechanic Born–Oppenheimer dynamics to characterize a set of polycyclic aromatic hydrocarbon based substituents bound to cytidine (dC) and guanine (dG) nucleobases. We specifically focus on perylene (P) bound to C5 and C6 of dC, and the naturally occurring benzo[a]pyrene diol epoxide (B[a]PDE) on dG. We find that specific angles of the connection points between them modulate mechanisms of intramolecular charge transfer, where an electron moves from P to dC and dG to B[a]PDE once an optimal angle is reached. Functionalization location and flexibility of the substituent affect access to these angles and, therefore, the amount of rapid charge transfer quenching of the fluorescence that we observe. This work demonstrates that the choice of functionalization location for SFNs changes the accessibility of charge transfer mechanisms via steric hindrance, and suggest that this feature can be applied for future tuning of fluorescence properties.

Abstract A017: Lung cancer chemoprevention with the β-blocker carvedilol

Abstract Chronic exposure to carcinogens that are present in tobacco smoke is a causative factor for lung carcinogenesis. Although smoking cessation successfully reduced the prevalence of cigarette use in the US, lung cancer risk for current and former smokers remains high, for whom no effective chemopreventive agent currently exists. In the present study, carvedilol, an FDA-approved β-blocker was examined on lung carcinogenesis induced by the tobacco carcinogen benzo(a)pyrene [B(a)P] in vitro and in vivo. In non-tumorigenic human bronchial epithelial cell culture BEAS-2B, carvedilol inhibited benzo(a)pyrene diol epoxide (BPDE)-induced malignant transformation at non-toxic concentrations in a dose-dependent manner. Although β-adrenergic receptors (β-ARs) are expressed in BEAS-2B cells, the anticancer activity of carvedilol is independent of β-blockade since the non-β-blocking R-carvedilol enantiomer also blocked transformation while the β-blockers atenolol (β1-AR selective blocker), ICI-118,551 (β2-AR selective blocker), and propranolol (nonselective blocker) had no effect. Carvedilol’s anti-transformation activity is possibly mediated by aryl hydrocarbon receptor signaling because carvedilol, R- and S-carvedilol all inhibited B(a)P-activated xenobiotic responsive element (XRE) promoter and CYP1A1 mRNA expression. In a B(a)P-induced acute lung toxicity model in CD-1 mice, pretreatment with carvedilol, R- or S-carvedilol (20 mg/kg/day) for 7 days significantly attenuated increased plasma levels of lactate dehydrogenase and malondialdehyde, inflammatory cell infiltration, histopathologic changes, and overexpression of COX-2 in the lung. In a B(a)P-induced lung carcinogenesis model in A/J mice, carvedilol at 3.2 and 20 mg/kg significantly attenuated tumor multiplicity and burden, to a similar degree as the EGFR inhibitor gefitinib. Our study reveals a previously unexplored role for the FDA approved cardiovascular drug carvedilol in the prevention of tobacco carcinogen-associated lung cancer. Citation Format: Ayaz Shahid, Mengbing Chen, Carol Lin, Bradley T. Andresen, Cyrus Parsa, Robert Orlando, Ying Huang. Lung cancer chemoprevention with the β-blocker carvedilol [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A017.

Lung cancer risk in workers occupationally exposed to polycyclic aromatic hydrocarbons with emphasis on the role of DNA repair gene

ObjectiveWorkers in secondary aluminum production plants are occupationally exposed to polycyclic aromatic hydrocarbons (PAHs). We aimed to monitor the concentrations of PAHs in air and in serum of workers at two secondary aluminum production plants. We also investigated the potential risk of lung cancer development among PAHs exposed workers with emphasis on the role of A1AT mutation and APEX1 gene polymorphisms.MethodsThis study included 177 workers from administrative departments and production lines. Blood samples were obtained for estimation of benzo(a)pyrene diol epoxide albumin adduct (BPDE-Alb adduct), anti-Cyclin-B1 marker (CCNB1) and squamous cell carcinoma antigen (SCCAg). Genes’ polymorphism for human apurinic/apyrimidinic endonuclease (APEX1) and alpha-1-anti-trypsin (A1AT) gene mutation were detected.ResultsThere was a significant increase in the level of BPDE-Alb adduct among exposed workers in comparison to non-exposed group. Moreover, 41.67% of exposed workers in El Tebbin had BPDE-Alb adduct level ≥ 15 ng/ml versus 29.6% of workers in Helwan factory. There was a significant increase in tumor markers (SCCAg and CCNB1) among workers whose BPDE-Alb adduct ≥ 15 ng/ml. There was a significant increase in the level of BPDE-Alb adducts in exposed workers carrying homozygous APEX1 genotype Glu/Glu. Furthermore, exposed workers with the Glu/Glu genotype had high tumor markers levels. There was a significant increase in levels of BPDE-Alb adducts in workers carrying A1AT mutant allele. Moreover, workers with mutant A1AT genotype had significantly high tumor markers (SCCAg and CCNB1) levels.ConclusionTherefore, we conclude that aluminum workers may be at a potential risk of lung cancer development due to PAHs exposure. Although PAHs concentrations in air were within the permissible limits, yet evidence of DNA damage was present as expressed by high BPDE-albumin adduct level in exposed workers. Also, elevation of tumor markers (SCCAg and CCNB1) in exposed workers points to the importance of periodic biological monitoring of such workers to protect them from cancer risk.

Black Raspberry Extract Enhances Glutathione Conjugation of the Fjord-Region Diol Epoxide Derived from the Tobacco Carcinogen Dibenzo[def,p]chrysene in Human Oral Cells.

In a series of previous studies we reported that black raspberry (BRB) powder inhibits dibenzo[a,l]pyrene (DBP)-induced DNA damage, mutagenesis, and oral squamous cell carcinoma (OSCC) development in mice. In the present study, using human oral leukoplakia (MSK-Leuk1) and squamous cell carcinoma (SCC1483) cells, we tested the hypothesis that BRB extract (BRBE) will enhance the synthesis of glutathione (GSH) and in turn increase GSH conjugation of the fjord-region DBP diol epoxide (DBPDE) derived from DBP leading to inhibition of DBP-induced DNA damage. The syntheses of DBPDE-GSH conjugate, DBPDE-dA adduct, and the corresponding isotope-labeled internal standards were performed; LC-MS/MS methods were used for their quantification. BRBE significantly (p < 0.05) increased cellular GSH by 31% and 13% at 6 and 24 h, respectively, in OSCC cells; in MSK-LeuK1 cells, the levels of GSH significantly (p < 0.05) increased by 55% and 22%, at 1 and 6 h. Since BRBE significantly enhanced the synthesis of GSH in both cell types, subsequent experiments were performed in MSK-Leuk1 cells. Western blot analysis was performed to determine the types of proteins involved in the synthesis of GSH. BRBE significantly (p < 0.05) increased the protein expression (2.5-fold) of the glutamate-cysteine ligase catalytic subunit (GCLC) but had no effect on the glutamate-cysteine ligase modifier subunit (GCLM) and glutathione synthetase (GSS). LC-MS/MS analysis showed that pretreatment of cells with BRBE followed by DBPDE significantly (p < 0.05) increased the levels of DBPDE-GSH conjugate (2.5-fold) and decreased DNA damage by 74% measured by assessing levels of DBPDE-dA adduct formation. Collectively, the results of this in vitro study clearly support our hypothesis, and the LC-MS/MS methods developed in the present study will be highly useful in testing the same hypothesis initially in our mouse model and ultimately in smokers.

Acute cytotoxicity test of PM2.5, NNK and BPDE in human normal bronchial epithelial cells: A comparison of a co-culture model containing macrophages and a mono-culture model

BackgroundBased on extensive research on cytotoxicity of exogenous compounds in vitro, it is essential to develop a cell model that better mimics environment in vivo to explore cytotoxic mechanisms of exogenous compounds. MethodsA co-culture system was established using a transwell system with Beas-2B and U937 cells. Cells were treated with fine particulate matter (PM2.5; 25, 50 and 100 μg/mL), nicotine-derived nitrosamine ketone (NNK; 50, 100 and 200 μg/mL) and benzo(a)pyrene diol epoxide (BPDE; 0.5, 2 and 8 μM) for 24 h. Cell proliferation, apoptosis and cell cycle, DNA damage were detected by CCK-8 and EdU, flow cytometry, and comet assay, respectively. Differentially expressed transcript and cytokine concentrations were determined by transcriptome sequencing and Cytokine Array, respectively. ResultsCompared with mono-culture, cell proliferation increased, apoptosis decreased, and DNA damage decreased in a dose-response relationship in co-culture. Gene expression profile was significantly different in co-culture, with significantly increased expression levels of 48 cytokines in co-culture. ConclusionCytotoxic damage to Beas-2B cells induced by exogenous carcinogens, including PM2.5, NNK and BPDE, was significantly reduced in a co-culture system compared with a mono-culture system. The mechanism may be related to changes in expression of cytokines, such as LIF, and activation of related pathways, such as TNF signaling pathway. Cytotoxic damage to Beas-2B induced by PM2.5, NNK and BPDE, was significantly reduced in co-culture. The mechanism may be related to changes in expression of cytokines and activation of related pathways. These findings provide new insights into cytotoxicity and experimental basis for safety evaluations of exogenous carcinogens.

081 Modulation of the aryl hydrocarbon receptor by adipose tissue: Implications for skin carcinogenesis

The aryl hydrocarbon receptor(AhR) is a ligand-activated transcription factor that responds to chemical carcinogens. An endogenous AhR agonist, 6-Formylindolo[3,2-b]carbazole, is produced following ultraviolet light(UV) absorption by tryptophan. Epidemiological studies show a correlation between exposure to AhR agonists and non melanoma skin cancer(NMSC). As an example, AhR activation promotes metabolism of pro-carcinogens to carcinogens such as the metabolism of benzo(a)pyrene to benzo[a]pyrene diol epoxide(BPDE).

Facile Multi-gram Preparation of trans-7,8-Dihydro-7,8-dihydroxybenzo[a]pyrene, a Precursor of Benzopyrene Diol Epoxide

Facile Multi-gram Preparation of trans-7,8-Dihydro-7,8-dihydroxybenzo[a]pyrene, a Precursor of Benzopyrene Diol Epoxide

AHR gene expression and the polymorphism rs2066853 are associated with clinicopathological parameters in colorectal carcinoma

The relationship between red and processed meat and its risk towardcolorectal carcinoma (CRC) is not fully explored in literature. Polycyclic aromatic hydrocarbons (PAHs) are procarcinogenic molecules that are ingested with meat cooked at high temperatures. The metabolic conversion of PAHs to carcinogenic diol epoxides is in part mediated by the aryl hydrocarbon receptor (AhR)-dependent induction of CYP1A1. This study aims to examine the expression profiles and polymorphisms of the AHR (aryl hydrocarbon receptor) gene which is involved in the metabolic conversion of PAHs in patients with CRC. Genetic analysis was done in matched cancer and non-neoplastic tissues from 79 patients diagnosed with CRCs. Low AHR mRNA expression was associated with mucinous colorectal adenocarcinoma. Exon 10 of AHR showed that 27% of patients had the rs2066853 single-nucleotide polymorphism resulting in an arginine-to-lysine change at codon 554. This variant was significantly associated with a lower likelihood of perineural invasion, presence of synchronous cancer, and multiple colorectal polyps. Furthermore, rs2066853 individuals were significantly more likely to be of more advanced age and have a more favorable tumor grade and pathological stage. These results imply the pathogenic roles of AHR in PAH-associated colorectal carcinogenesis.

Detection of Benzo[a]pyrene Diol Epoxide Adducts to Histidine and Lysine in Serum Albumin In Vivo by High-Resolution-Tandem Mass Spectrometry.

Electrophilic diol epoxide metabolites are involved in the carcinogenicity of benzo[a]pyrene, one of the widely studied polycyclic aromatic hydrocarbons (PAHs). The exposure of humans to this PAH can be assessed by measuring stable blood protein adducts, such as to histidine and lysine in serum albumin, from their reactive metabolites. In this respect, measurement of the adducts originating from the genotoxic (+)-anti-benzo[a]pyrene diol epoxide is of interest. However, these are difficult to measure at such low levels as are expected in humans generally exposed to benzo[a]pyrene from air pollution and the diet. The analytical methods detecting PAH-biomarkers still suffer from low selectivity and/or detectability to enable generation of data for calculation of in vivo doses of specific stereoisomers, for evaluation of risk factors and assessing risk from exposures to PAH. Here, we suggest an analytical methodology based on high-pressure liquid chromatography (HPLC) coupled to high-resolution tandem mass spectrometry (MS) to lower the detection limits as well as to increase the selectivity with improvements in both chromatographic separation and mass determination. Method development was performed using serum albumin alkylated in vitro by benzo[a]pyrene diol epoxide isomers. The (+)-anti-benzo[a]pyrene diol epoxide adducts could be chromatographically resolved by using an HPLC column with a pentafluorophenyl stationary phase. Interferences were further diminished by the high mass accuracy and resolving power of Orbitrap MS. The achieved method detection limit for the (+)-anti-benzo[a]pyrene diol epoxide adduct to histidine was approximately 4 amol/mg serum albumin. This adduct as well as the adducts to histidine from (−)-anti- and (+/−)-syn-benzo[a]pyrene diol epoxide were quantified in the samples from benzo[a]pyrene-exposed mice. Corresponding adducts to lysine were also quantified. In human serum albumin, the anti-benzo[a]pyrene diol epoxide adducts to histidine were detected in only two out of twelve samples and at a level of approximately 0.1 fmol/mg.