Polycyclic aromatic hydrocarbon and its adducts in peripheral blood: Gene and environment interaction among Chinese population.

BACKGROUND: Benzo(a)pyrene (B[a]P) is the most widely concerned polycyclic aromatic hydrocarbons (PAHs), which metabolizes benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) in vivo to produce carcinogenic effect on the body. Currently, there is limited research on the role of the variation of metabolic enzymes in this process. METHODS: We carried out a study including 752 participants, measured the concentrations of 16 kinds PAHs in both particle and gaseous phases, urinary PAHs metabolites, leukocyte BPDE-DNA adduct and serum BPDE- Albumin (BPDE-Alb) adduct, and calculated daily intake dose (DID) to assess the cumulative exposure of PAHs. We conducted single nucleotide polymorphism sites (SNPs) of metabolic enzymes, explored the exposure-response relationship between the levels of exposure and BPDE adducts using multiple linear regression models. RESULT: Our results indicated that an interquartile range (IQR) increase in B[a]P, PAHs, BaPeq, 1-hydroxypyrene (1-OHP), 1-hydroxynaphthalene (1-OHNap) and 2-hydroxynaphthalene (2-OHNap) were associated with 26.53 %, 24.24 %, 28.15 %, 39.15 %, 12.85 % and 14.09 % increase in leukocyte BPDE-DNA adduct (all P < 0.05). However, there was no significant correlation between exposure with serum BPDE-Alb adduct (P > 0.05). Besides, we also found the polymorphism of CYP1A1(Gly45Asp), CYP2C9 (Ile359Leu), and UGT1A1(downstream) may affect BPDE adducts level. CONCLUSION: Our results indicated that leukocyte BPDE-DNA adduct could better reflect the exposure to PAHs. Furthermore, the polymorphism of CYP1A1, CYP2C9 and UGT1A1affected the content of BPDE adducts.

The dual effects of Benzo(a)pyrene/Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide on DNA Methylation.

Benzo(a)pyrene (BaP) is one of the most thoroughly studied polycyclic aromatic hydrocarbons(PAHs) and a widespread organic pollutant in various areas of human life. Its teratogenic, immunotoxic and carcinogenic effects on organisms are well documented and widely recognized by researchers. In the body, BaP is enzymatically converted to form a more active benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). BaP/BPDE has the potential to trigger gene mutations, influence epigenetic modifications and cause damage to cellular structures, ultimately contributing to disease onset and progression. However, there are different points of view when studying epigenetics using BaP/BPDE. On the one hand, it is claimed in cancer research that BaP/BPDE contributes to gene hypermethylation and, in particular, induces the hypermethylation of tumor's suppressor gene promoters, leading to gene silencing and subsequent cancer development. Conversely, studies in human and animal populations suggest that exposure to BaP results in genome-wide DNA hypomethylation, potentially leading to adverse outcomes in inflammatory diseases. This apparent contradiction has not been summarized in research for almost four decades. This article presents a comprehensive review of the current literature on the influence of BaP/BPDE on DNA methylation regulation. It demonstrates that BaP/BPDE exerts a dual-phase regulatory effect on methylation, which is influenced by factors such as the concentration and duration of BaP/BPDE exposure, experimental models and detection methods used in various studies. Acute/high concentration exposure to BaP/BPDE often results in global demethylation of DNA, which is associated with inhibition of DNA methyltransferase 1 (DNMT1) after exposure. At certain specific gene loci (e.g., RAR-ß), BPDE can form DNA adducts, recruiting DNMT3 and leading to hypermethylation at specific sites. By integrating these different mechanisms, our goal is to unravel the patterns and regulations of BaP/BPDE-induced DNA methylation changes and provide insights into future precision therapies targeting epigenetics.

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.

Benzo[a]pyrene evokes epithelial-mesenchymal transition and pulmonary fibrosis through AhR-mediated Nrf2-p62 signaling.

Benzo[a]pyrene (BaP) and its metabolic end product benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE), are known toxic environmental pollutants. This study aimed to analyze whether sub-chronic BPDE exposure initiated pulmonary fibrosis and the potential mechanisms. In this work, male C57BL6/J mice were exposed to BPDE by dynamic inhalation exposure for 8 weeks. Our results indicated that sub-chronic BPDE exposure evoked pulmonary fibrosis and epithelial-mesenchymal transition (EMT) in mice. Both in vivo and in vitro, BPDE exposure promoted nuclear translocation of Snail. Further experiments indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) and p62 were upregulated in BPDE-exposed alveolar epithelial cells. Moreover, Nrf2 siRNA transfection evidently attenuated BPDE-induced p62 upregulation. Besides, p62 shRNA inhibited BPDE-incurred Snail nuclear translocation and EMT. Mechanically, BPDE facilitated physical interaction between p62 and Snail in the nucleus, then repressed Snail protein degradation by p62-dependent autophagy-lysosome pathway, and finally upregulated transcriptional activity of Snail. Additionally, aryl hydrocarbon receptor (AhR) was activated in BPDE-treated alveolar epithelial cells. Dual-luciferase assay indicated activating AhR could bind to Nrf2 gene promoter. Moreover, pretreatment with CH223191 or α-naphthoflavone (α-NF), AhR antagonists, inhibited BPDE-activated Nrf2-p62 signaling, and alleviated BPDE-induced EMT and pulmonary fibrosis in mice. Taken together, AhR-mediated Nrf2-p62 signaling contributes to BaP-induced EMT and pulmonary fibrosis.

Particulate polycyclic aromatic hydrocarbons and metals, DNA methylation and DNA methyltransferase among middle-school students in China.

The main components of particulate matter (PM) had been reported to change DNA methylation levels. However, the mixed effect of PM and its constituents on DNA methylation and the underlying mechanism in children has not been well characterized. To investigate the association between single or mixture exposures and global DNA methylation or DNA methyltransferases (DNMTs), 273 children were recruited (110 in low-exposed area and 163 in high-exposed area) in China. Serum benzo[a]pyridin-7,8-dihydroglycol-9, 10-epoxide (BPDE)-albumin adduct and urinary metals were determined as exposure markers. The global DNA methylation (% 5mC) and the mRNA expression of DNMT1, and DNMT3A were measured. The linear regression, quantile-based g-computation (QGC), and mediation analyses were performed to investigate the effects of individual and mixture exposure. We found that significantly lower levels of % 5mC (P < 0.001) and the mRNA expression of DNMT3A in high-PM exposed group (P = 0.031). After adjustment for age, gender, BMI z-score, detecting status of urinary cotinine, serum folate, and white blood cells, urinary arsenic (As) was negatively correlated with the % 5mC. One IQR increase in urinary As (19.97 µmol/mol creatinine) was associated with a 11.06 % decrease in % 5mC (P = 0.026). Serum BPDE-albumin adduct and urinary cadmium (Cd) were negatively correlated with the levels of DNMT1 and DNMT3A (P < 0.05). Mixture exposure was negatively associated with expression of DNMT3A in QGC analysis (ß: -0.19, P < 0.001). Mixture exposure was significantly associated with decreased % 5mC in the children with non-detected cotinine or normal serum folate (P < 0.05), which the most contributors were PAHs and As. The mediated effect of hypomethylation through DNMT1 or DNMT3A pathway was not observed. Our findings indicated that individual and mixture exposure PAHs and metal components had negative associations with global DNA methylation and decreased DNMT3A expression significantly in school-age individuals.

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

OBJECTIVE: To investigate the link between systemic lupus erythematosus (SLE) incidence and exposure to environmental polycyclic aromatic hydrocarbons (PAH). METHODS: A 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. RESULTS: Diverse 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. CONCLUSIONS: Environmental PAH, particularly lifestyle-related, are significant, yet under-recognized, risk factors for SLE. STATEMENT OF ENVIRONMENTAL IMPLICATION: We 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.

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.

A unique circ_0067716/EIF4A3 double-negative feedback loop impacts malignant transformation of human bronchial epithelial cells induced by benzo(a)pyrene.

Benzo(a)pyrene as a pervasive environmental contaminant is characterized by its substantial genotoxicity, and epidemiological investigations have established a correlation between benzo(a)pyrene exposure and the susceptibility to human lung cancer. Notably, much research has focused on the link between epigenetic alterations and lung cancer induced by chemicals, although circRNAs are also emerging as relevant contributors to the carcinogenic process of benzo(a)pyrene. In this study, we identified circ_0067716 as being significantly upregulated in response to stress injury and downregulated during malignant transformation induced by benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) in human bronchial epithelial cells. The observed differential expression of circ_0067716 in cells treated with BPDE for varying durations suggests a strong correlation between this circRNA and BPDE exposure. The tissue samples of lung cancer patients also suggest that a lower circ_0067716 expression is associated with BPDE-DNA adduct levels. Remarkably, we demonstrate that EIF4A3, located in the nucleus, interacts with the flanking sequences of circ_0067716 and inhibits its biogenesis. Conversely, circ_0067716 is capable of sequestering EIF4A3 in the cytoplasm, thereby preventing its translocation into the nucleus. EIF4A3 and circ_0067716 can form a double-negative feedback loop that could be affected by BPDE. During the initial phase of BPDE exposure, the expression of circ_0067716 was increased in response to stress injury, resulting in cell apoptosis through the involvement of miR-324-5p/DRAM1/BAX axis. Subsequently, as cellular adaptation progressed, long-term induction due to BPDE exposure led to an elevated EIF4A3 and a reduced circ_0067716 expression, which facilitated the proliferation of cells by stabilizing the PI3K/AKT pathway. Thus, our current study describes the effects of circ_0067716 on the genotoxicity and carcinogenesis induced by benzo(a)pyrene and puts forwards to the possible regulatory mechanism on the occurrence of smoking-related lung cancer, providing a unique insight based on epigenetics.

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.

Mediation of association between benzo[a]pyrene exposure and lung cancer risk by plasma microRNAs: A Chinese case-control study.

Epidemiologic studies have reported the positive relationship of benzo[a]pyrene (BaP) exposure with the risk of lung cancer. However, the mechanisms underlying the relationship is still unclear. Plasma microRNA (miRNA) is a typical epigenetic biomarker that was linked to environment exposure and lung cancer development. We aimed to reveal the mediation effect of plasma miRNAs on BaP-related lung cancer. We designed a lung cancer case-control study including 136 lung cancer patients and 136 controls, and measured the adducts of benzo[a]pyrene diol epoxide-albumin (BPDE-Alb) and sequenced miRNA profiles in plasma. The relationships between BPDE-Alb adducts, normalized miRNA levels and the risk of lung cancer were assessed by linear regression models. The mediation effects of miRNAs on BaP-related lung cancer were investigated. A total of 190 plasma miRNAs were significantly related to lung cancer status at Bonferroni adjusted P < 0.05, among which 57 miRNAs showed different levels with |fold change| > 2 between plasma samples before and after tumor resection surgery at Bonferroni adjusted P < 0.05. Especially, among the 57 lung cancer-associated miRNAs, BPDE-Alb adducts were significantly related to miR-17-3p, miR-20a-3p, miR-135a-5p, miR-374a-5p, miR-374b-5p, miR-423-5p and miR-664a-5p, which could in turn mediate a separate 42.2%, 33.0%, 57.5%, 36.4%, 48.8%, 32.5% and 38.2% of the relationship of BPDE-Alb adducts with the risk of lung cancer. Our results provide non-invasion biomarker candidates for lung cancer, and highlight miRNAs dysregulation as a potential intermediate mechanism by which BaP exposure lead to lung tumorigenesis.

The immunotoxicity mechanism of hemocytes in Chlamys farreri incubated with noradrenaline and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide alone or in combination.

Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) is the active intermediate metabolite of benzo[a]pyrene (B[a]P) and is considered the ultimate immunotoxicant. The neuroendocrine immunoregulatory network of bivalves is affected under pollutant stress. Besides, bivalves are frequently affected by pollutants in marine environments, yet the combined effects of neuroendocrine factors and detoxification metabolites on bivalves under pollutant stress and the signal pathways that mediate this immunoregulation are not well understood. Therefore, we incubated the hemocytes of Chlamys farreri with the neuroendocrine factor noradrenaline (NA) and the B[a]P detoxification metabolite BPDE, alone or in combination, to examine the immunotoxic effects of NA and BPDE on the hemocytes in C. farreri. Furthermore, the effects of NA and BPDE on the hemocyte signal transduction pathway were investigated by assessing potential downstream targets. The results revealed that NA and BPDE, alone or in combination, resulted in a significant decrease in phagocytic activity, bacteriolytic activity and the total hemocyte count. In addition, the immunotoxicity induced by BPDE was further exacerbated by co-treatment with NA, and the two showed synergistic effects. Analysis of signaling pathway factors showed that NA activated G proteins by binding to α-AR, which transmitted information to the Ca2+-NF-κB signaling pathway to regulate the expression of phagocytosis-associated proteins and regulated cytokinesis through the cAMP signaling pathway. BPDE could activate PTK and affect phagocytosis and cytotoxicity proteins through Ca2+-NF-κB signal pathway, also affect the regulation of phagocytosis and cytotoxicity by inhibiting the AC-cAMP-PKA pathway to down-regulate the expression of NF-κB and CREB. In addition, BPDE and NA may affect the immunity of hemocytes by down-regulating phagocytosis-related proteins through inhibition of the lectin pathway, while regulating the expression of cytotoxicity-related proteins through the C-type lectin. In summary, immune parameters were suppressed through Ca2+ and cAMP dependent pathways exposed to BPDE and the immunosuppressive effects were enhanced by the neuroendocrine factor NA.

BaP/BPDE suppressed endothelial cell angiogenesis to induce miscarriage by promoting MARCHF1/GPX4-mediated ferroptosis.

Environmental benzo(a)pyrene (BaP) and its ultimate metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) are universal and inevitable persistent organic pollutants and endocrine disrupting chemicals. Angiogenesis in placental decidua plays a pivotal role in healthy pregnancy. Ferroptosis is a newly identified and iron-dependent cell death mode. However, till now, BaP/BPDE exposure, ferroptosis, defective angiogenesis, and miscarriage have never been correlated; and their regulatory mechanisms have been rarely explored. In this study, we used assays with BPDE-exposed HUVECs (human umbilical vein endothelial cells), decidual tissues and serum samples collected from unexplained recurrent miscarriage and their matched healthy control groups, and placental tissues of BaP-exposed mouse miscarriage model. We found that BaP/BPDE exposure caused ferroptosis and then directly suppressed angiogenesis and eventually induced miscarriage. In mechanism, BaP/BPDE exposure up-regulated free Fe2+ level and promoted lipid peroxidation and also up-regulated MARCHF1 (a novel E3 ligase of GPX4) level to promote the ubiquitination degradation of GPX4, both of which resulted in HUVEC ferroptosis. Furthermore, we also found that GPX4 protein down-regulated the protein levels of VEGFA and ANG-1, two key proteins function for angiogenesis, and thus suppressed HUVEC angiogenesis. In turn, supplement with GPX4 could suppress ferroptosis, recover angiogenesis, and alleviate miscarriage. Moreover, the levels of free Fe2+ and VEGFA in serum might predict the risk of miscarriage. Overall, this study uncovered the crosstalk among BaP/BPDE exposure, ferroptosis, angiogenesis, and miscarriage, discovering novel toxicological effects of BaP/BPDE on human reproductive health. This study also warned the public to avoid exposure to polycyclic aromatic hydrocarbons during pregnancy to effectively prevent adverse pregnancy outcomes.

BPDE-DNA adduct formation and alterations of mRNA, protein, and DNA methylation of CYP1A1, GSTP1, and GSTM1 induced by benzo[a]pyrene and the intervention of aspirin in mice.

Benzo[a]pyrene (B[a]P), one typical environmental pollutant, the toxicity mechanisms, and potential prevention remain perplexing. Available evidence suggests cytochrome P450 1A1 (CYP1A1) and glutathione S-transferases (GSTs) metabolize B[a]P, resulting in metabolic activation and detoxification of B[a]P. This study aimed to reveal the impact of B[a]P exposure on trans-7,8-diol-anti-9,10-epoxide DNA (BPDE-DNA) adduct formation, level of CYP1A1, glutathione S-transferase pi (GSTP1) and glutathione S-transferase mu1 (GSTM1) mRNA, protein and DNA methylation in mice, and the potential prevention of aspirin (ASP). This study firstly determined the BPDE-DNA adduct formation in an acute toxicity test of a large dose in mice induced by B[a]P, which subsequently detected CYP1A1, GSTP1, and GSTM1 at levels of mRNA, protein, and DNA methylation in the organs of mice in a subacute toxicity test at appropriate doses and the potential prevention of ASP, using the methods of real-time quantitative PCR (QPCR), western blotting, and real-time methylation-specific PCR (MSP), respectively. The results verified that B[a]P induced the formation of BPDE-DNA adduct in all the organs of mice in an acute toxicity test, and the order of concentration of which was lung > kidney > liver > brain. In a subacute toxicity test, following B[a]P treatment, mice showed a dose-dependent slowdown in body weight gain and abnormalities in behavioral and cognitive function and which were alleviated by ASP co-treatment. Compared to the controls, following B[a]P treatment, CYP1A1 was significantly induced in all organs in mice at mRNA level (P < 0.05), was suppressed in the lung and cerebrum of mice at protein level, and inhibited at DNA methylation level in the liver, lung, and cerebrum, whereas GSTP1 and GSTM1 at mRNA, protein, and DNA methylation levels showed organ-specific changes in mice following B[a]P treatment, which was generally alleviated by ASP intervention. In conclusion, B[a]P induced BPDE-DNA adduct formation in all organs in mice and altered the mRNA, protein, and DNA methylation levels in CYP1A1, GSTP1, and GSTM1 in an organ-dependent pattern, which could be related to the organ toxicity and mechanism of B[a]P. ASP intervention may be an effective measure to prevent B[a]P toxicity. The findings provide scientific evidence for further study on the organ toxicity and mechanisms of B[a]P.

Potential molecular mechanism underlying the harmed haemopoiesis upon Benzo[a]pyrene exposure in Chlamys farreri.

Benzo[a]pyrene (B[a]P), a ubiquitous contamination in the marine environments, has the potential to impact the immune response of bivalves by affecting the hemocyte parameters, especially total hemocyte count (THC). THC is mainly determined by haematopoietic mechanisms and apoptosis of hemocytes. Many studies have found that B[a]P can influence the proliferation and differentiation of hemocytes. However, the link between the toxic mechanisms of haematopoietic and environmental pollutants is not explicitly stated. This study is to investigate the toxic effects of B[a]P on haematopoietic mechanisms in C. farreri. Through the tissue expression distribution experiment and EDU assay, gill is identified as a potential haematopoietic tissue in C. farreri. Subsequently, the scallops were exposed to B[a]P (0.05, 0.5, 5 µg/L) for 1d, 3d, 6d, 10d and 15d. Then BPDE content, DNA damage, gene expression of haematopoietic factors and haematopoietic related pathways were determined in gill and hemocytes. The results showed that the expression of CDK2 was significantly decreased under B[a]P exposure through three pathways: RYR/IP3-calcium, BPDE-CHK1 and Notch pathway, resulting in cell cycle arrest. In addition, B[a]P also significantly reduced the number of proliferating hemocytes by affecting the Wnt pathway. Meanwhile, B[a]P can significantly increase the content of ROS, causing a downregulation of FOXO gene expression. The gene expression of Notch pathway and ERK pathway was also detected. The present study suggested that B[a]P disturbed differentiation by multiple pathways. Furthermore, the expression of SOX11 and CD9 were significantly decreased, which directly indicated that differentiation of hemocytes was disturbed. In addition, phagocytosis, phenoloxidase activity and THC were also significant decreased. In summary, the impairment of haematopoietic activity in C. farreri further causes immunotoxicity under B[a]P exposure. This study will improve our understanding of the immunotoxicity mechanism of bivalve under B[a]P exposure.

Potential pathway and mechanisms underlining the immunotoxicity of benzo[a]pyrene to Chlamys farreri.

The long-distance migration of polycyclic aromatic hydrocarbons (PAHs) promotes their release into the marine environment, posing a serious threat to marine life. Studies have shown that PAHs have significant immunotoxicity effects on bivalves, but the exact mechanism of immunotoxicity remains unclear. This paper aims to investigate the effects of exposure to 0.4, 2, and 10 µg/L of benzo(a)pyrene (B[a]P) on the immunity of Chlamys farreri under environmental conditions, as well as the potential molecular mechanism. Multiple biomarkers, including phagocytosis rate, metabolites, neurotoxicity, oxidative stress, DNA damage, and apoptosis, were adopted to assess these effects. After exposure to 0.4, 2, and 10 µg/L B[a]P, obvious concentration-dependent immunotoxicity was observed, indicated by a decrease in the hemocyte index (total hemocyte count, phagocytosis rate, antibacterial and bacteriolytic activity). Analysis of the detoxification metabolic system in C. farreri revealed that B[a]P produced B[a]P-7,8-diol-9,10-epoxide (BPDE) through metabolism, which led to an increase in the expression of protein tyrosine kinase (PTK). In addition, the increased content of neurotransmitters (including acetylcholine, γ -aminobutyric acid, enkephalin, norepinephrine, dopamine, and serotonin) and related receptors implied that B[a]P might affect immunity through neuroendocrine system. The changes in signal pathway factors involved in immune regulation indicated that B[a]P interfered with Ca2+ and cAMP signal transduction via the BPDE-PTK pathway or neuroendocrine pathway, resulting in immunosuppression. Additionally, B[a]P induced the increase in reactive oxygen species (ROS) content and DNA damage, as well as an upregulation of key genes in the mitochondrial pathway and death receptor pathway, leading to the increase of apoptosis rate. Taken together, this study comprehensively investigated the detoxification metabolic system, neuroendocrine system, and cell apoptosis to explore the toxic mechanism of bivalves under B[a]P stress.

TERT transcription and translocation into mitochondria regulate benzo[a]pyrene/BPDE-induced senescence and mitochondrial damage in mouse spermatocytes.

Telomere and mitochondria may be the targets of Benzo[a]pyrene (BaP) -induced male reproductive damage, and further elucidation of the toxic molecular mechanisms is necessary. In this study, we used in vivo and in vitro exposure models to explore the molecular mechanisms of TERT regulation in BaP-induced telomere and mitochondrial damage in spermatocytes. The results showed that the treatment of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), the active metabolite of BaP, caused telomere dysfunction in mouse spermatocyte-derived GC-2 cells, resulting in S-phase arrest and increased senescence-associated secretory phenotype (SASP). These effects were significantly alleviated by telomerase agonist (ABG) pretreatment in GC-2 cells. SIRT1, FOXO3a, or c-MYC overexpressing GC-2 cell models were established to demonstrate that BPDE inhibited TERT transcriptional expression through the SIRT1/FOXO3a/c-MYC pathway, leading to telomere dysfunction. We also observed that BPDE induced mitochondrial compromise, including complex I damage, accompanied by reduced mitochondrial TERT expression. Based on this, we constructed wild-type TERT-overexpressing (OE-TERTwt) and mitochondria targeting TERT-overexpressing (OE-TERTmst) GC-2 cell models and found that OE-TERTmst GC-2 cells improved mitochondrial function better than OE-TERTwt GC-2 cells. Finally, ICR mice were given BaP by intragastric administration for 35 days, which verified the results of the in vitro study. The results shown that BaP exposure can lead to spermatogenesis disturbance, which is related to the telomere and mitochondrial damage in spermatocytes. In conclusion, our results suggest that BPDE causes telomere and mitochondrial damage in spermatocytes by inhibiting TERT transcription and mitochondrial TERT expression. This study elucidates the molecular mechanism of male reproductive toxicity due to environmental pollutant BaP, and also provides a new perspective for the exploration of interventions and protective measures against male reproductive damage by BaP.

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.

Impact of mobile phone-specific electromagnetic fields on DNA damage caused by occupationally relevant exposures: results of ex vivo experiments with peripheral blood mononuclear cells from different demographic groups.

The aim of this study was to investigate if age and body mass of humans have an impact on the DNA-damaging properties of high-frequency mobile phone-specific electromagnetic fields (HF-EMF, 1950 MHz, universal mobile telecommunications system, UMTS signal) and if this form of radiation has an impact on the genotoxic effects of occupationally relevant exposures. Pooled peripheral blood mononuclear cells (PBMC) from three groups [young normal weight, young obese (YO), and older age normal weight individuals] were exposed to different doses of HF-EMF (0.25, 0.5, and 1.0 W/kg specific absorption rate-SAR) and simultaneously or sequentially to different chemicals which cause DNA damage (CrO3, NiCl2, benzo[a]pyrene diol epoxide-BPDE, and 4-nitroquinoline 1-oxide-4NQO) via different molecular mechanisms. We found no difference in regard to the background values in the three groups but a significant increase of DNA damage (81% without and 36% with serum) in cells from old participants after radiation with 1.0 W/kg SAR 16 h. In combined treatment experiments we found no impact of the UMTS signal on chemically induced DNA damage in the different groups in general. However, a moderate decrease of DNA damage was seen in simultaneous treatment experiments with BPDE and 1.0 W/kg SAR in the YO group (decline 18%). Taken together our findings indicate that HF-EMF cause DNA damage in PBMC from older subjects (69.1 years). Furthermore, they show that the radiation does not increase induction of DNA damage by occupationally relevant chemicals.

BPDE exposure promotes trophoblast cell pyroptosis and induces miscarriage by up-regulating lnc-HZ14/ZBP1/NLRP3 axis.

Environmental Benzo(a)pyrene (BaP) and its ultimate metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) are typical persistent organic pollutants and endocrine disrupting chemicals. BaP/BPDE exposure might cause human trophoblast cell dysfunctions and induce miscarriage. However, the underlying mechanisms remain largely elusive. In this study, we found that BPDE exposure induced human trophoblast cell pyroptosis by up-regulating NLRP3/Caspase1/GSDMD pathway. We also identified that lnc-HZ14 was highly expressed in BPDE-exposed trophoblast cells and in recurrent miscarriage (RM) vs healthy control (HC) villous tissues. Lnc-HZ14 promoted trophoblast cell pyroptosis by promoting IRF1-mediated ZBP1 transcription, increasing METTL3-mediated m6A methylation on NLRP3 mRNA and its stability, and also enhancing ZBP1/NLRP3 protein interactions. Knockdown of lnc-HZ14/ZBP1/NLRP3 axis could efficiently alleviate BPDE-induced trophoblast cell pyroptosis. Higher level of pyroptosis, as indicated by the up-regulation of lnc-HZ14/ZBP1/NLRP3 axis, was found in RM vs HC villous tissues. In BaP-exposed mouse model, BaP exposure induced placental tissue pyroptosis and miscarriage by up-regulating murine Zbp1/Nlrp3 axis, and knockdown of Nlrp3 could efficiently reduce placenta pyroptosis and alleviate BaP-induced mouse miscarriage. Serum IL-1ß protein level might act as a promising indicator to predict the risk of miscarriage. These findings provided new insights into BaP/BPDE-induced trophoblast cell pyroptosis and miscarriage and might be helpful for further assessment of the toxicological effects of BaP/BPDE on the female reproduction.

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.