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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. [ABSTRACT FROM AUTHOR]

Inhalation of carbon black nanoparticles (CBNPs) can impair lung tissue and cause DNA damage, but the epigenetic mechanism responsible for these effects is still unclear. We explored the role of circular RNAs (circRNAs) in DNA damage induced by CBNPs in the lung. Human bronchial epithelial cell lines (16HBE and BEAS-2B) were treated with 0, 5, 10, 20, 40, or 80 μg/ml CBNPs for 24, 48, and 72 h, and BALB/c mice were exposed to 8 and 80 μg/d CBNPs for 14 days to establish in vitro and vivo models of CBNP exposure, respectively. We found that CBNPs caused DNA double-strand breaks in the lung. Using high-throughput sequencing and quantitative real-time PCR to identify CBNP-related circRNAs, we identified a novel circRNA (circ_0089282) that was overexpressed in the CBNP-exposed group. We used gain-/loss-of-function approaches, RNA pulldown assays, and silver staining to explore the regulatory function of circ_0089282 and its interactions with targeted proteins. We found that circ_0089282 interference could increase CBNP-induced DNA damage, whereas overexpression resulted in the opposite. Circ_0089282 could directly bind to the fused in sarcoma (FUS) protein and positively regulate downstream DNA repair protein DNA ligase 4 (LIG4) through FUS. This regulatory effect of circRNA on DNA damage via promotion of LIG4 illustrated the interactions between genetics and epigenetics in toxicology. [ABSTRACT FROM AUTHOR]

Carbon black and cadmium (Cd) are important components of atmospheric particulate matter and cigarette smoke that are closely associated with the occurrence and development of lung diseases. Carbon black, particularly carbon black nanoparticles (CBNPs), can easily adsorbs metals and cause severe lung damage and even cell death. Therefore, this study aimed to explore the mechanisms underlying the combined toxicity of CBNPs and Cd. We found that the combined exposure to CBNPs and Cd promoted significantly greater autophagosome formation and ferroptosis (increased malonaldehyde (MDA), reactive oxygen species (ROS), and divalent iron ions (Fe2+) levels and altered ferroptosis-related proteins) compared with single exposure in both 16HBE cells (human bronchial epithelioid cells) and mouse lung tissues. The levels of ferroptosis proteins, transferrin receptor protein 1 (TFRC) and glutathione peroxidase 4 (GPX4), were restored by CBNPs-Cd exposure following treatment with a 3-MA inhibitor. Additionally, under CBNPs-Cd exposure, circPSEN1 overexpression inhibited increases in the autophagy proteins microtubule-associated protein 1 light chain 3 (LC3II/I) and sequestosome-1 (P62). Moreover, increases in TFRC and Fe2+, and decreases in GPX4were inhibited. Knockdown of circPSEN1 reversed these effects. circPSEN1 interacts with autophagy-related gene 5 (ATG5) protein and upregulates nuclear receptor coactivator 4 (NCOA4), the co-interacting protein of ATG5, thereby degrading ferritin heavy chain 1 (FTH1) and increasing Fe2+ in 16HBE cells. These results indicated that the combined exposure to CBNPs and Cd promoted the binding of circPSEN1 to ATG5, thereby increasing autophagosome synthesis and ATG5-NCOA4-FTH1 axis activation, ultimately inducing autophagy-dependent ferroptosis in 16HBE cells and mouse lung tissues. This study provides novel insights into the toxic effects of CBNPs and Cd in mixed pollutants. [Display omitted] • Carbon black and cadmium co-exposure induce autophagy-dependent ferroptosis. • CircPSEN1 interacts with ATG5 protein to facilitate autophagosome formation. • CircPSEN1 activates ATG5-NCOA4-FTH1 axis leading to ferroptosis. • Ferroptosis induced by carbon black and cadmium co-exposure may be synergistic effect. [ABSTRACT FROM AUTHOR]

Benzo [a]pyrene (B [a]P) is a widespread environmental chemical pollutant that has been linked to the development of various diseases. However, the specific mechanism of action remains unclear. In this study, human bronchial epithelial 16HBE and BEAS-2B cells were exposed to B [a]P at 0–32 μM to assess the DNA-damaging effects. B [a]P exposure resulted in elevated expression of γ-H2AX, a marker of DNA damage. The m6A RNA methylation assay showed that B [a]P exposure increased the extent of m6A modification and the demethylase ALKBH5 played an integral role in this process. Moreover, the results of the comet assay and Western blot analysis showed an increase in m6A modification mediated by ALKBH5 that promoted DNA damage. Furthermore, the participation of a novel circular RNA, circ_0003552, was assessed by high-throughput sequencing under the condition of high m6A modification induced by B [a]P exposure. In subsequent functional studies, an interference/overexpression system was created to confirm that circ_0003552 participated in regulation of DNA damage. Mechanistically, circ_0003552 had an m6A binding site that could regulate its generation. This study is the first to report that B [a]P upregulated circ_0003552 through m6A modification, thereby promoting DNA damage. These findings revealed that epigenetics played a key role in environmental carcinogen-induced DNA damage, and the quantitative changes it brought might provide an early biomarker for future medical studies of genetic-related diseases and a new platform for investigations of the interaction between epigenetics and genetics. [Display omitted] • B [a]P induces DNA damage in bronchial epithelial cells. • B [a]P upregulates the degree of m6A modification by suppressing ALKBH5 expression. • Both ALKBH5 and circ_0003552 can regulate the B [a]P-induced DNA damage. • M6A modification promotes circ_0003552 production through the m6A binding site. [ABSTRACT FROM AUTHOR]

The health hazards of nanoparticles of neodymium oxide (NPs-Nd 2 O 3) have aroused public concern in recent years. Exposure to NPs-Nd 2 O 3 can change the level of reactive oxygen species (ROS) that cause DNA damage and alter whole transcriptome expression profiles for micro (mi)RNA, circular (circ)RNA, long noncoding (lnc)RNA, and mRNA. However, there have been no reports to our knowledge about the role of circRNAs in DNA damage caused by NPs-Nd 2 O 3. In our study, we analyzed the circRNA expression profile of human bronchial epithelial cells(16 HBE)exposed to 40 μg/ml NPs-Nd 2 O 3. Our results indicated that exposure produced 1025 up-regulated and 890 down-regulated circRNAs. Real-time quantitative polymerase chain reaction (qRT-PCR) was applied to verify some of the significantly changed circRNAs and demonstrated that circ_009773 was apparently down-regulated. Through exploration of its host gene function, we found that circ_009773 may be related to DNA damage. Functional experiments found that circ_009773 regulated NPs-Nd 2 O 3 -induced DNA damage in 16HBE cells. A circ_009773-associated competing endogenous (ce)RNA network was constructed based on one differentially expressed (DE) circRNA, 74 DE miRNAs and 208 DE mRNAs. Module analysis identified hub genes related to DNA damage and repair and a protein-protein interaction (PPI) network was created. • NPs-Nd 2 O 3 can induces cytotoxicity in 16HBE cells. • The downregulated circ_009773 promotes NPs-Nd 2 O 3 -induced DNA damage in 16HBE cells. • A competing endogenous RNA (ceRNA) network of circRNA-miRNA-mRNA was constructed in NPs-Nd 2 O 3 -induced DNA damage 16HBE cells. [ABSTRACT FROM AUTHOR]

[Display omitted] Hexavalent chromium [Cr (VI)] is an important environmental pollutant and may cause lung injury when inhaled into the human body. Cr (VI) is genotoxic and can cause DNA damage, although the underlying epigenetic mechanisms remain unclear. To simulate the real-life workplace exposure to Cr (VI), we used a novel exposure dose calculation method. We evaluated the effect of Cr (VI) on DNA damage in human bronchial epithelial cells (16HBE and BEAS-2B) by calculating the equivalent real-time exposure dose of Cr (VI) (0 to 10 μM) in an environmental population. Comet experiments and olive tail moment measurements revealed increased DNA damage in cells exposed to Cr (VI). Cr (VI) treatment increased nuclear γ-H2AX foci and γ-H2AX protein expression, and caused DNA damage in the lung tissues of mice. An effective Cr (VI) dose (6 μM) was determined and used for cell treatment. Cr (VI) exposure upregulated circ_0008657, and knockdown of circ_0008657 decreased Cr (VI)-induced DNA damage, whereas circ_0008657 overexpression had the opposite effect. Mechanistically, we found that circ_0008657 binds to microRNA (miR)-203a-3p and subsequently regulates ATM serine/threonine kinase (ATM), a key protein involved in homologous recombination repair downstream of miR-203a-3p, thereby regulating DNA damage induced by Cr (VI). The present findings suggest that circ_0008657 competitively binds to miR-203a-3p to activate the ATM pathway and regulate the DNA damage response after environmental chemical exposure in vivo and in vitro. [ABSTRACT FROM AUTHOR]

• B[ a ]P induces DNA damage response (DDR) in bronchial epithelial cells. • B[ a ]P upregulates circ_0057504 through suppressing DNMT3A-mediated DNA methylation. • Circ_0057504 promotes B[ a ]P-induced DDR via modulating NONO-SFPQ complex formation. Benzo[ a ]pyrene (B[ a ]P) is a class I carcinogen and hazardous environmental pollutant with genetic toxicity. Understanding the molecular mechanisms underlying genetic deterioration and epigenetic alterations induced by environmental contaminants may contribute to the early detection and prevention of cancer. However, the role and regulatory mechanisms of circular RNAs (circRNAs) in the B[ a ]P-induced DNA damage response (DDR) have not been elucidated. In this study, human bronchial epithelial cell lines (16HBE and BEAS-2B) were exposed to various concentrations of B[ a ]P, and BALB/c mice were treated with B[ a ]P intranasally. B[ a ]P exposure was found to induce DNA damage and upregulate circular RNA hsa_circ_0057504 (circ_0057504) expression in vitro and in vivo. In addition, B[ a ]P upregulated TMEM194B mRNA and circ_0057504 expression through inhibition of DNA methyltransferase 3 alpha (DNMT3A) expression in vitro. Modulation (overexpression or knockdown) of circ_0057504 expression levels using a lentiviral system in human bronchial epithelial cells revealed that circ_0057504 promoted B[ a ]P-induced DNA damage. RNA pull-down and western blot assays showed that circ_0057504 interacted with non-POU domain-containing octamer-binding (NONO) and splicing factor proline and glutamine rich (SFPQ) proteins and regulated formation of the NONO-SFPQ protein complex. Thus, our findings indicate that circ_0057504 acts as a novel regulator of DNA damage in human bronchial epithelial cells exposed to B[ a ]P. The current study reveals novel insights into the role of circRNAs in the regulation of genetic damage, and describes the effect and regulatory mechanisms of circ_0057504 on B[ a ]P genotoxicity. [ABSTRACT FROM AUTHOR]

Exposure to high concentrations of copper is associated with pulmonary inflammation and chronic respiratory disease (CRD). Epigenetic modulation of noncoding RNAs contributes to the development of several CRDs. It is unknown whether epigenetic modulation is involved in copper mediated pulmonary inflammation and CRD. We conducted a case-control study of 101 CRD cases and 161 control subjects in Shijiazhuang, China, and evaluated circRNAs and cytokine levels (IL-6 and IL-8) by qPCR and ELISA. Urinary copper concentration was determined by inductively coupled plasma mass spectrometry. Linear mixed models and generalized linear mixed models were used to assess the associations of circRNAs with CRD, urinary copper, and cytokines. We exposed the human bronchial epithelial cell line, 16HBE, to copper and assessed the functional role of a circRNA, circ_0008882, by RNA overexpression. Cellular location of circ_0008882 was assessed by separation of nuclear and cytoplasmic RNAs. Nine circRNAs were associated with an increased risk for CRDs, while the relative expression of circ_0008882 was decreased after copper exposure in vitro and in vivo. Copper exposure stimulated 16HBE cells to release proinflammatory IL-6 and IL-8. The release of the cytokines was inhibited by overexpression of circ_0008882. These results suggest a role for circ_0008882 in the regulation of CRD associated inflammation following copper exposure. [Display omitted] • Circular RNA, circ_0008882, is related to chronic respiratory disease. • Expression of circ_0008882 decreases after copper exposure in vitro and in vivo. • IL-6 and IL-8, induced by copper, are inhibited by overexpression of circ_0008882. [ABSTRACT FROM AUTHOR]