Carbon black nanoparticles and cadmium co-exposure aggravates bronchial epithelial cells inflammation via autophagy-lysosome pathway.

Carbon black nanoparticles (CBNPs) and cadmium (Cd) are major components of various air pollutants and cigarette smoke. Autophagy and inflammation both play critical roles in understanding the toxicity of particles and their components, as well as maintaining body homeostasis. However, the effects and mechanisms of CBNPs and Cd (CBNPs-Cd) co-exposure on the human respiratory system remain unclear. In this study, a CBNPs-Cd exposure model was constructed to explore the respiratory toxicity and combined mechanism of these chemicals on the autophagy-lysosome pathway in the context of respiratory inflammation. Co-exposure of CBNPs and Cd significantly increased the number of autophagosomes and lysosomes in human bronchial epithelial cells (16HBE) and mouse lung tissues compared to the control group, as well as the groups exposed to CBNPs and Cd alone. Autophagic markers, LC3II and P62 proteins, were up-regulated in 16HBE cells and mouse lung tissues after CBNPs-Cd co-exposure. However, treatment with Cq inhibitor (an indicator of lysosomal acid environment) resulted in a substantial decreased co-localization fluorescence of LC3 and lysosomes in the CBNPs-Cd combination group compared with the CBNPs-Cd single and control groups. No difference in LAMP1 protein expression was observed among the exposed groups. Adding 3 MA alleviated inflammatory responses, while applying the Baf-A1 inhibitor aggravated inflammation both in vitro and in vivo following CBNPs-Cd co-exposure. Factorial analysis showed no interaction between CBNPs and Cd in their effects on 16HBE cells. We demonstrated that co-exposure to CBNPs-Cd increases the synthesis of autophagosomes and regulates the acidic environment of lysosomes, thereby inhibiting autophagy-lysosome fusion and enhancing the inflammatory response in both 16HBE cells and mouse lung. These findings provide evidence for a comprehensive understanding of the interaction between CBNPs and Cd in mixed pollutants, as well as for the prevention and control of occupational exposure to these two chemicals.

Role of circPSEN1 in carbon black and cadmium co-exposure induced autophagy-dependent ferroptosis in respiratory epithelial cells.

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.

Circ_0008657 regulates lung DNA damage induced by hexavalent chromium through the miR-203a-3p/ATM axis.

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.

Benzo[a]pyrene-induced up-regulation of circ_0003552 via ALKBH5-mediated m6A modification promotes DNA damage in human bronchial epithelial cells.

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.