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Increased industrial offshore activities in northern waters raise the question of impact of polycyclic aromatic hydrocarbons (PAHs) on key Arctic marine species. One of these is the ecologically important polar cod (Boreogadus saida), which is the primary food source for Arctic marine mammals and seabirds. In the present work, we have conducted the first comprehensive proteomics study with this species by exploring the effects of dietary PAH exposure on the hepatic proteome, using benzo[a]pyrene (BaP) as a PAH model-compound. Functional annotation and pathway analyses of the proteins affected by BaP revealed a concerted cellular response for handling and adopting to its exposure, involving numerous interconnected signalling pathways and metabolic processes. In accordance with BaP being a strong aryl hydrocarbon receptor (Ahr) agonist, a prominent activation of the canonical Ahr signalling pathway was observed, including upregulation of Ahr target proteins like cytochrome P450 enzymes and microsomal glutathione transferase. Furthermore, cellular pathways for handling oxidative stress, protein misfolding and degradation, as well as endoplasmic reticulum stress and calcium homeostasis, were also activated by BaP, possibly as a result of the formation of harmful and redox reactive BaP metabolites via phase I metabolism. Activation of proteins that participate in the acute-phase response was also observed, suggesting prevalent tissue- and cellular damage that triggers the immune system and inflammatory responses. Our results at the protein level aligns well with previous analyses on the effects of BaP on the polar cod liver transcriptome and support that exposure to BaP and structural similar PAHs can cause adverse effects on polar cod physiology. Although more data is required for demonstrating how these molecular responses propagate to higher levels of biological organisation, increased knowledge about the initial cellular and molecular mechanisms that induce toxicity is a key-step towards a mechanistically informed impact assessment of PAH pollutants in the Arctic., Competing Interests: Declaration of competing interest The author JD Rasinger declares that he is currently employed with the European Food Safety Authority (EFSA) in the Food Ingredients and Packaging Unit (FIP), which provides scientific and administrative support to the Panel on Food Additives and Flavourings. However, the present article is published under the sole responsibility of the author JD Rasinger and may not be considered as an EFSA scientific output. The positions and opinions presented in this article are those of the author alone and are not intended to represent the views/any official position of EFSA. To know about the views or scientific outputs of EFSA, please consult its website. The author's main contributions to this article were made before joining EFSA when still employed at the Institute of Marine Research. The author A Goksøyr is a major shareholder in Biosense Laboratories AS, supplier of the Atlantic cod anti-Cyp1a polyclonal antibody., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Benzo(a)pyrene (BaP), a pervasive environmental pollutant with endocrine-disrupting properties, has been associated with detrimental effects on pregnancy. During early pregnancy, the endometrial decidualization process is critical for embryo implantation. Abnormal decidualization can lead to implantation failure, aberrant placental formation, and pregnancy loss. We previously revealed that BaP exposure impaired decidualization and implantation in mice, yet the underlying mechanisms remained elusive. Autophagy, a cellular mechanism pivotal for energy and material recycling, contributes to the decidualization process. The chemokine C-X-C motif chemokine ligand 12 (CXCL12), secreted by endometrium stromal cells (ESCs), is involved in regulating endometrial decidualization and autophagy. Therefore, this study aimed to explore the hypothesis that BaP disrupts the decidualization process by interfering with autophagic pathways via the CXCL12/CXCR4 axis during early pregnancy. We found that BaP inhibited CXCL12/CXCR4 expression, and induced autophagy by promoting autophagosome formation, which in turn impaired the decidualization in early pregnant mice uterus and decidual stromal cells (DSCs). Using autophagy inhibitors 3-methyladenine and chloroquine in combination with BaP to treat DSCs, successfully weakened BaP-induced autophagy, and relieved decidual injury. Additionally, activation of CXCL12/CXCR4 by recombinant protein CXCL12 attenuated BaP-induced autophagy, inhibited the PI3K/AKT signal activation caused by BaP, and partly rescued the expression of decidualization-related genes. In summary, this study demonstrates that BaP induces autophagy in DSCs by inhibiting the CXCL12/CXCR4 axis, leading to damage in endometrial decidualization during early pregnancy. The findings provide a critical chemokine-mediated regulatory mechanism involved in embryo implantation and contribute valuable knowledge to the reproductive toxicology of BaP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mutagenicity testing is a component of the hazard assessment of industrial chemicals, biocides, and pesticides. Mutations induced by test substances can be detected by in vitro and in vivo methods that have been adopted as OECD Test Guidelines. One of these in vivo methods is the Transgenic Rodent Assay (TGRA), OECD test guideline no. 488. An analogous in vitro TGRA has been described, but experience with this test method is limited. In this study, six in vivo TGRA positive mutagens were tested in the in vitro TGRA based on primary MutaMouse hepatocytes. In addition to the functional read-out of the lacZ reporter gene, induced mutations were analysed by next-generation sequencing (NGS). Five of the six in vivo TGRA positive mutagens (N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), mitomycin C (MMC), benzo[a]pyrene (B[a]P), and azathioprine (AZA), but not cyproterone acetate) mutated the lacZ gene in vitro. NGS identified mutations which matched the mutagenic mechanisms described in the literature. The alkylating agent ENU induced a greater proportion of A:T to T:A transversions than did the other alkylating agent, EMS, whereas EMS increased smaller deletions (1-4 bp). G:C to T:A transversions accounted for the majority of mutations identified after treatments with MMC and B[a]P, both of which form monoadducts at the guanine N2 position. AZA induced mainly G:C to A:T transitions, explained by the structural similarity of one of its metabolites to guanine. An increased proportion of mid-size changes (0.3-2.5 kb) was detected only for the crosslinking mutagen MMC. The in vitro TGRA based on primary MutaMouse hepatocytes is a promising in vitro assay for the assessment of mutation induction, reflecting many aspects of the corresponding in vivo TGRA and allowing for mutation spectra analysis to evaluate the induced mutations., Competing Interests: Declaration of Competing Interest Alina Göpfert, Claudia Rülker, Michael Eichenlaub, Bogdan Tokovenko, Martina Dammann, Dorothee Funk-Weyer, Naveed Honarvar and Robert Landsiedel are employees of BASF SE, a chemical company which may use the assay to develop and register commercial products., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Persistent organic pollutants (POPs) are easily accumulated in organisms due to its high lipophilicity. Bivalves are still one of the marine invertebrates with high species diversity despite experiencing with environmental pollution. However, studies investigating the adaptation mechanisms of bivalves towards POPs have been lacking. In this study, we chose benzo[a]pyrene (B[a]P), a typical POPs, to investigate the accumulation and excretion mechanism of clam Ruditapes philippinarum towards B[a]P. Laser confocal microscopy images of ovaries and testes confirmed significant colocalization between lipid droplets and B[a]P. However, B[a]P in the testes might mainly excrete with the excretory fluid. Furthermore, lipidomics results indicated that the upregulated TGs were TGs with 48-58 total carbon atoms and 6-12 double bonds in acyl side chains in ovaries, while the upregulated TGs mainly distributed in 48-58 total carbon atoms and 4-14 double bonds in acyl side chains in testes. In ovaries, gene expression of lipid metabolism and phospholipid metabolism basically presented an upward trend at Pro and Mat stages, while entirely performed a downregulation trend at Gro stage. Testes had the opposite regulation with that. Additionally, B[a]P prompted the excretion ability of female clams, but exhibited a complex effect in the male. This study provides a scientific basis for bivalve toxicology and healthy aquaculture, which is of great significance for marine ecological environment protection and aquatic product safety of China., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Benzo[a]pyrene (BaP), a major harmful component in PM2.5, is widely present in automobile emissions and atmospheric pollution. BaP exposure directly targets the lungs, often resulting in acute lung injury (ALI). However, comprehensive metabolic and transcriptomic profiles related to BaP-induced ALI remain unexplored. To simulate BaP-induced lung injury, we performed intratracheal instillation of BaP. To investigate how BaP exposure affects lung transcriptome and metabolic profiles, we used RNA sequencing and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). We aimed to understand the underlying mechanisms of BaP-induced lung damage. Metabolomics analyses indicated that in BaP-exposed animals, most fatty acids, carbohydrates, and steroids were significantly reduced, whereas most amino acids and organic acids remained unchanged. Analysis of transcriptomics data showed that fatty acid synthesis decreased and fatty acid oxidation increased, suggesting that lipid breakdown occurs after BaP exposure. Additionally, there were increases in oxidative stress system activity and decreases in immune system function. Finally, BaP altered mitochondrial, lipid, immune system, and fatty acid pathways, as indicated by pathway enrichment analyses. These results show that BaP substantially affects metabolic and inflammatory responses, enhancing the broader understanding of the underlying mechanisms of ALI after BaP exposure., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The deleterious health impacts of polycyclic aromatic hydrocarbons (PAHs) on the population have been extensively substantiated and acknowledged. Mounting evidence underscores that PAH exposure is closely linked to an elevated risk of mental disorders, particularly in populations experiencing occupational and high-level exposure. In this study, we aimed to investigate the mechanisms underlying anxiety-like behaviors induced by different dosages of PAHs, with a concentrated focus on brain region-specific metabolic alterations in mice using various metabolomics approaches. Male C57BL/6 mice were exposed to benzo[a]pyrene (B[a]P), a typical PAH, through gavage at occupational exposure and EPA toxicologically relevant dosages (2.0 and 20.0 mg/kg/day) for 21 days, respectively. Behavioral assessments revealed that occupational exposure to B[a]P induced anxiety-like behaviors in C57BL/6 mice. Meanwhile, elevated serum norepinephrine and corticotropin-releasing hormone further confirmed the anxiety-inducing effects of B[a]P exposure. Metabolomics analysis uncovered dysregulation across various metabolic pathways following B[a]P exposure, encompassing brain neurotransmitter, organic acid, amino acid, lipid, fatty acid, and cholesterol. Anxiety levels and lipid metabolic abnormalities were notably exacerbated at the higher dosage, despite being only a 10-fold increase. Of particular significance, a decrease in lysophosphatidic acid (LPA) and lysophosphatidylserine (LPS) emerged as pivotal indicators of B[a]P neurotoxicity. Spatial-resolved metabolomics further demonstrated distinctive lipid and metabolite profiles across different brain subregions after exposure to B[a]P. Remarkably, alterations were specifically observed in the anxiety-related brain regions, such as the hippocampus, cortex, white matter, and thalamus, varying with exposure dosages. These findings underscore the significance of brain metabolic abnormalities in the development of mental disorders triggered by B[a]P exposure and highlight the need for establishing precise exposure limits of B[a]P to safeguard public mental health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Toxicant-induced behavioural changes provide important insights into environmental toxicity, particularly in vulnerable tropical marine habitats. However, ecotoxicological knowledge of organisms in these environments is insufficient. We aimed to develop innovative and cost-effective ecotoxicology methods using Parhyale hawaiensis as a tropical model organism. Adult P. hawaiensis were exposed to aqueous benzo[a]pyrene (BaP) (2 μM) and dietary BaP (50, 250, or 1250 μg BaP/g diet). Survival (24 to 96 h) and behavioural responses (21d) to foraging, reproduction, and predator avoidance were studied. Aqueous and dietary exposures to benzo[a]pyrene (BaP) did not affect survival but induced significant immobility with effective concentration (EC 50  ± SE, 96 h at 11.89 ± 1.19 μM). Relative to the control group, aqueous exposure to 2 μM and dietary exposure to 250 and 1250 μg BaP/g feed resulted in statistically significant behavioural changes. These included a 55-76 % reduction in feeding rates, 133 % increase in chemosensation time, 60-122 % drop in moulting frequency, 200 % delay in precopulatory activity, 50-83 % decrease in geotactic activity, and 300-400 % increase in phototactic activity (all significant at p ≤ 0.05). The methods developed in this study are cost-effective, sensitive, and readily integrated into other endpoint analyses, reinforcing the potential of P. hawaiensis as a tropical ecotoxicology model for detecting toxicant-induced behavioural responses and enhancing marine risk assessments., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ibrahim Lawan reports financial support was provided by Petroleum Technology Development Fund (PTDF) of Nigeria. Ibrahim Lawan reports a relationship with Petroleum Technology Development Fund (PTDF) of Nigeria that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Road traffic is a major contributor to air pollution and consequently negatively affects human health. Car pollution originates both from exhaust emissions (EE) and non-exhaust emissions (NEE, such as tire and brake wear particles, erosion of road surfaces and resuspension of road dust). While the toxicity of EE and NEE has been characterized separately, their combined effects are poorly documented. However, we are constantly exposed to a mixture of pollutants and their interactions should not be neglected as they may significantly impact their toxicological profile resulting in additive, synergistic or antagonistic effects. To fill this gap, we investigated in vitro the combined toxicity of exhaust gases and benzo[a]pyrene (representative of EE) and tire particles (representative of NEE). Macrophages from the RAW264.7 cell line were exposed for 24 h to tire particles (TP) of variable size (6-113 µm), alone or in combination with exhaust gases (CO 2 , CO, NO, NO 2 ) and benzo[a]pyrene (B[a]P) as an archetype of polycyclic aromatic hydrocarbon (PAH). The cell response was assessed in terms of cytotoxicity, proinflammatory response and oxidative stress. TP, gases and B[a]P, alone or in combination triggered neither cytotoxicity nor oxidative stress. On the contrary, a proinflammatory response was elicited with two different profiles depending on the size of the TP: TNF-α production was either slightly (with the finest TP) or strongly (with coarse TP) increased in the presence of gases and B[a]P, suggesting that the effects of TP, gases and B[a]P were either additive or synergistic, depending on TP size., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests, Abderrahmane Bouredji reports financial support was provided by The French Agency for Ecological Transition. Bogdan Muresan reports a relationship with Michelin that includes: funding grants. Jeremie Pourchez reports a relationship with Michelin that includes: funding grants. Valerie Forest reports a relationship with Michelin that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon from incomplete combustion, builds up in coastal regions through river runoff, atmospheric deposition, and coastal activities. BaP's lipophilicity and stability lead to persistent environmental impacts due to its resistance to degradation. The economically valuable golden cuttlefish, Sepia esculenta, often spawns and hatches in shallow waters, making it prone to BaP exposure. This study employs transcriptomic analysis to initially investigate juvenile golden cuttlefish's response to BaP. The results indicate that BaP exposure significantly affects various physiological and molecular functions of the juveniles, particularly affecting pathways related to immune and inflammatory responses, metabolic regulation, and nervous system functions. Functional enrichment and PPI network analyses identified key genes such as HSGALT-like, ASAH1-like, and GTL-like in the BaP response. These genes exhibited a suppressive trend during short-term exposure, indicating that BaP exposure may influence lipid metabolism, energy conversion, and digestive functions at the genetic level, which could further disrupt the overall physiological state and developmental processes of juvenile golden cuttlefish. The study offers novel insights into BaP's effects on juvenile golden cuttlefish and marine life, aiding marine ecosystem and biodiversity conservation., Competing Interests: Declaration of competing interest The corresponding author stated that all authors reported no potential conflicts., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In vitro models based on permanent fish liver cell lines have proven to be versatile tools for examining chemical biotransformation and toxicity. However, their in vivo relevance remains uncertain due to their potentially de-differentiated phenotype. Here, we investigate whether a 3D cell culture environment can restore hepatocyte-like properties of the Rainbow trout liver cell line RTL-W1. Utilizing ultralow attachment (ULA) microwell plates, we achieved controlled sizing and extended culture (3 weeks) of spheroidal aggregate cultures (spheroids). RTL-W1 cells within the spheroids remained viable and metabolically active, as confirmed by the CellTiter-Glo 3D assay. Transmission electron microscopy revealed that spheroids exhibit tissue-like arrangements, such as interdigitations, cell-cell junctions, and endo- or exocytic activity at the cell-cell interface. They also displayed ultrastructural characteristics typical of metabolically active cells/hepatocytes, including abundant endoplasmic reticulum (ER), Golgi apparatus, and mitochondria. RT-qPCR analysis showed upregulation of genes involved in xenobiotic and endogenous (lipid) metabolism in 3D cultures over time. Notably, for several genes, especially cyp1a, expression levels were significantly higher in spheroids than in monolayers cultured for the same duration. This was corroborated at the enzyme level by increased Cyp1a-dependent catalytic activity (EROD). Interestingly, increased Cyp1a expression did not lead to heightened susceptibility to benzo[a]pyrene toxicity, which requires bioactivation. However, RTL-W1 3D and 2D cell cultures exhibited differential susceptibility to toxicity from other model chemicals, such as the surfactant SDS and the metal copper (Cu). These findings support the hypothesis that RTL-W1 cells can re-differentiate to a hepatocyte-like phenotype when cultured in a 3D configuration and may exhibit distinct biological responses upon exposure to xenobiotics. Overall, this study advances our understanding of the potential of cell line-derived 3D in vitro models for research and providing more physiologically relevant data for regulatory contexts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Infertility has gradually become a global health concern, and evidence suggests that exposure to environmental endocrine-disrupting chemicals (EDCs) represent one of the key causes of infertility. Benzo(a)pyrene (BaP) is a typical EDC that is widespread in the environment. Previous studies have detected BaP in human urine, semen, cervical mucus, oocytes and follicular fluid, resulting in reduced fertility and irreversible reproductive damage. However, the mechanisms underlying the effects of gestational BaP exposure on offspring fertility in male mice have not been fully explored. In this study, pregnant mice were administered BaP at doses of 0, 5, 10 and 20 mg/kg/day via gavage from Days 7.5 to 12.5 of gestation. The results revealed that BaP exposure during pregnancy disrupted the structural integrity of testicular tissue, causing a disorganized arrangement of spermatogenic cells, compromised sperm quality, elevated levels of histone modifications and increased apoptosis in the testicular tissue of F1 male mice. Furthermore, oxidative stress was also increased in the testicular tissue of F1 male mice. BaP activated the AhR/ERα signaling pathway, affected H3K4me3 expression and induced apoptosis in testicular tissue. AhR and Cyp1a1 were overexpressed, and the expression of key molecules in the antioxidant pathway, including Keap1 and Nrf2, was reduced. The combined effects of these molecules led to apoptosis in testicular tissues, damaging and compromising sperm quality. This impairment in testicular cells further contributed to compromised testicular tissues, ultimately impacting the reproductive health of F1 male mice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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-β), 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mitochondria are essential for various physiological functions in astrocytes in the brain, such as maintaining ion and pH homeostasis, regulating neurotransmission, and modulating neuroinflammation. Mitophagy, a form of autophagy specific to mitochondria, is essential for ensuring mitochondrial quality and function. Benzo[a]pyrene (BaP) accumulates in the brain, and exposure to it is recognized as an environmental risk factor for neurodegenerative diseases. However, while the toxic mechanisms of BaP have been investigated in neurons, their effects on astrocytes-the most prevalent glial cells in the brain-are not clearly understood. Therefore, this study aims to investigate the toxic effects of exposure to BaP on mitochondria in primary astrocytes. Fluorescent probes and genetically encoded indicators were utilized to visualize mitochondrial morphology and physiology, and regulatory factors involved in mitochondrial morphology and mitophagy were assessed. Additionally, the mitochondrial respiration rate was measured in BaP-exposed astrocytes. BaP exposure resulted in mitochondrial enlargement owing to the suppression of mitochondrial fission factors. Furthermore, BaP-exposed astrocytes demonstrated reduced mitophagy and exhibited aberrant mitochondrial function and physiology, such as altered mitochondrial respiration rates, increased mitochondrial superoxide, disrupted mitochondrial membrane potential, and dysregulated mitochondrial Ca 2+ . These findings offer insights into the underlying toxic mechanisms of BaP exposure in neurodegenerative diseases by inducing aberrant mitophagy and mitochondrial dysfunction in astrocytes., Competing Interests: Declaration of Competing Interest The authors report that there are no competing interests to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) widely present in the environment, but their effect on cerebrovascular development has been rarely reported. In this study, dechorionated zebrafish embryos at 24 hpf were exposed to benzo(a)pyrene (BaP) at 0.5, 5 and 50 nM for 48 h, cerebrovascular density showed a significant reduction in the 5 and 50 nM groups. The expression of aryl hydrocarbon receptor (AhR) was significantly increased. Transcriptomic analysis showed that the pathway of positive regulation of vascular development was down-regulated and the pathway of inflammation response was up-regulated. The transcription of main genes related to vascular development, such as vegf, bmper, cdh5, f3b, itgb1 and prkd1, was down-regulated. Addition of AhR-specific inhibitor CH233191 in the 50 nM BaP group rescued cerebrovascular developmental defects and down-regulation of relative genes, suggesting that BaP-induced cerebrovascular defects was AhR-dependent. The cerebrovascular defects were persistent into adult fish raised in clean water, showing that the relative area of vascular network, the length of vessels per unit area and the number of vascular junctions per unit area were significantly decreased in the 50 nM group. Supplementation of berberine (BBR), a naturally derived medicine from a Chinese medicinal herb, alleviated BaP-induced cerebrovascular defects, accompanied by the restoration of altered expression of AhR and relative genes, which might be due to that BBR promoted BaP elimination via enhancing detoxification enzyme activities, suggesting that BBR could be a potential agent in the prevention of cerebrovascular developmental defects caused by PAHs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Exposure to polycyclic aromatic hydrocarbons (PAHs), ubiquitously environmental contaminant, leads to the development of major toxic effects on human health, such as carcinogenic and immunosuppressive alterations reported for the most studied PAH, i.e., benzo(a)pyrene (B(a)P). In order to assess the risk associated with this exposure, it is necessary to have predictive biomarkers. Thus, extracellular vesicles (EVs) and their microRNA (miRNA) contents, have recently been proposed as potentially interesting biomarkers in Toxicology. Our study here explores the use of vesicles secreted and found in blood fluids, and their miRNAs, as biomarkers of exposure to B(a)P alone and within a realistic occupational mixture. We isolated EVs from primary human cultured blood mononuclear cells (PBMCs) and rat plasma after PAH exposure and reported an increased EV production by B(a)P, used either alone or in the mixture, in vitro and in vivo. We then investigated the association of this EV release with the blood concentration of the 7,8,9,10-hydroxy (tetrol)-B(a)P reactive metabolite, in rats. By performing RNA-sequencing (RNA-seq) of miRNAs in PBMC-derived EVs, we analyzed miRNA profiles and demonstrated the regulation of the expression of miR-342-3p upon B(a)P exposure. We then validated B(a)P-induced changes of miR-342-3p expression in vivo in rat plasma-derived EVs. Overall, our study highlights the feasibility of using EVs and their miRNA contents, as biomarkers of PAH exposure and discusses their potential in environmental Toxicology., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lydie SPARFEL reports financial support was provided by National Agency for Food Environmental and Occupational Health and Safety. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Micro-nano plastics have been reported as important carriers of polycyclic aromatic hydrocarbons (PAHs) for long-distance migration in the environment. However, the combined toxicity from long-term chronic exposure beyond the vehicle-release mechanism remains elusive. In this study, we investigated the synergistic action of Benzo[a]pyrene (BaP) and Polystyrene nanoparticles (PS) in Caenorhabditis elegans (C. elegans) as a combined exposure model with environmental concentrations. We found that the combined exposure to BaP and PS, as opposed to single exposures at low concentrations, significantly shortened the lifespan of C. elegans, leading to the occurrence of multiple senescence phenotypes. Multi-omics data indicated that the combined exposure to BaP and PS is associated with the disruption of glutathione homeostasis. Consequently, the accumulated reactive oxygen species (ROS) cannot be effectively cleared, which is highly correlated with mitochondrial dysfunction. Moreover, the increase in ROS promoted lipid peroxidation in C. elegans and downregulated Ferritin-1 (Ftn-1), resulting in ferroptosis and ultimately accelerating the aging process of C. elegans. Collectively, our study provides a new perspective to explain the long-term compound toxicity caused by BaP and PS at real-world exposure concentrations., Competing Interests: Declaration of competing interest The authors declare no competing financial interests or personal relationship that could potentially influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The global interest in edible insects as sustainable protein sources raises concerns about the bioaccumulation of contaminants, including polycyclic aromatic hydrocarbons (PAHs), to problematic levels. Understanding the accumulation dynamics of PAHs in edible insects is highly relevant due to the widespread sources and toxicological profiles; however, the bioaccumulative potential of PAHs in edible insects is unexplored. This study examined the uptake and elimination dynamics of benzo(a)pyrene (B(a)P), a representative and carcinogenic PAH, in yellow mealworm larvae (YMW, Tenebrio molitor). Larvae were exposed to feeding substrate with varying B(a)P concentrations (0.03, 0.3, and 3 mg kg -1 ), and uptake (21 days in B(a)P-contaminated substrate) and elimination (21 days in B(a)P-free substrate) kinetics were subsequently assessed. The results showed that YMW can eliminate B(a)P, revealing dose-dependent B(a)P bioaccumulation in these insects. Larvae fed on a substrate with 0.03 mg kg -1 accumulated B(a)P over 21 days, presenting values of 0.049 (Standard deviation - 0.011) mg kg -1 and a kinetic-based (BAF kinetic ) of 1.93 g substrate g organism -1 , exceeding the EU regulatory limits for food. However, with a B(a)P half-life (DT 50 ) of 4.19 days in the larvae, an EU legislation safety criterion was met after a 13-day depuration period in clean substrate. Larvae exposed to substrates with 0.3 and 3 mg kg -1 showed B(a)P accumulation, with BAF kinetic values of 3.27 and 2.09 g substrate g organism -1 , respectively, not meeting the current legal standards for food consumption at the end of the exposure to B(a)P. Although the B(a)P half-life values after 35 days were 4.30 and 10.22 days (DT 50s ), the larvae retained B(a)P levels exceeding permitted food safety limits. These findings highlight a significant oversight in regulating PAHs in animal feed and the need for comprehensive safety evaluations of PAH hazards in edible insects for improved PAH feeding guidelines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Recently, the abundance of environmental microplastics (MPs) has become a global paramount concern. Besides the danger of MPs for biota due to their tiny size, these minute particles may act as vectors of other pollutants. This study focused on evaluating the toxicity of environmentally relevant concentrations of MPs (10 and 50 mg/kg sediment) and benzo[a]pyrene (B[a]P, 1 µg/kg sediment), alone and in mixture, for 3 and 7 days in marine polychaete Hediste diversicolor, selected as a benthic bioindicator model. The exposure period was sufficient to confirm the bioaccumulation of both contaminants in seaworms, as well as the potential capacity of plastic particles to adsorb and vehiculate the B[a]P. Interestingly, increase of acidic mucus production was observed in seaworm tissues, indicative of a defense response. The activation of oxidative system pathways was demonstrated as a strategy to prevent lipid peroxidation. Furthermore, the comprehensive Nuclear Magnetic Resonance (NMR)-based metabolomics revealed significant disorders in amino acids metabolism, osmoregulatory process, energetic components, and oxidative stress related elements. Overall, these findings proved the possible synergic harmful effect of MPs and B[a]P even in small concentrations, which increases the concern about their long-term presence in marine ecosystems, and consequently their transfer and repercussions on marine fauna., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To address two current issues in evaluating the toxicity of microplastics (MPs) namely, conflicting results due to species specificity and the ecological irrelevance of laboratory data, this study conducted a 10-day exposure experiment using a microalgal community comprising three symbiotic species. The experiment involved virgin and Benzo[a]pyrene-spiked micron-scale fibers and fragments made of polyethylene terephthalate (PET) and polypropylene (PP). The results showed that, from a physiological perspective, environmentally relevant concentrations of micron-scale MPs decreased saccharide accumulation in microalgal cells, as confirmed by ultrastructural observations. MPs may increase cellular energy consumption by obstructing cellular motility, interfering with nutrient uptake, and causing sustained oxidative stress. Additionally, MPs and adsorbed B[a]P induced DNA damage in microalgae, potentially further disrupting cellular energy metabolism. Ecologically, MPs altered the species abundance in microalgal communities, suggesting they could weaken the ecological functions of these communities as producers and affect ecosystem diversity and stability. This study marks a significant advancement from traditional single-species toxicity experiments to community-level assessments, providing essential insights for ecological risk assessment of microplastics and guiding future mechanistic studies utilizing multi-omics analysis., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shuo Cheng reports financial support was provided by Japan Society for the Promotion of Science. Shuo Cheng reports a relationship with East China Normal University, Shanghai, China that includes: paid expert testimony. We with this apply for recusal. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Benzo[a]pyrene (BaP) is associated with the development of lung cancer, but the underlying mechanism has not been completely clarified. Here, we used 10 μM BaP to induce malignant transformation of human bronchial epithelial BEAS-2B cells, named BEAS-2B-T. Results indicated that BaP (6.25, 12.5 and 25 μM) treatment significantly promoted the migration and invasion of BEAS-2B-T cells. Meanwhile, BaP exposure inhibited ferroptosis in BEAS-2B-T, ferroptosis-related indexes Fe 2+ , malondialdehyde (MDA), lipid peroxidation (LPO) and reactive oxygen species (ROS) decreased significantly. The protein level of ferroptosis-related molecule transferrin receptor (TFRC) decreased significantly, while solute carrier family 7 membrane 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) increased significantly. The intervention of ferroptosis dramatically effected the migration and invasion of BEAS-2B-T induced by BaP. Furthermore, the expression of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) was markedly increased after BaP exposure. YTHDF1 knockdown inhibited BEAS-2B-T migration and invasion by promoting ferroptosis. In the meantime, the contents of Fe 2+ , MDA, LPO and ROS increased significantly, TFRC was markedly increased, and SLC7A11, FTH1, and GPX4 were markedly decreased. Moreover, overexpression of YTHDF1 promoted BEAS-2B-T migration and invasion by inhibiting ferroptosis. Importantly, knockdown of YTHDF1 promoted ferroptosis and reduced BEAS-2B-T migration and invasion during BaP exposure, and overexpression of YTHDF1 increased migration and invasion of BEAS-2B-T by inhibiting ferroptosis during BaP exposure. RNA immunoprecipitation assays indicated that the binding of YTHDF1 to SLC7A11 and FTH1 markedly increased after YTHDF1 overexpression. Therefore, we concluded that BaP promotes the malignant progression of BEAS-2B-T cells through YTHDF1 upregulating SLC7A11 and FTH1 to inhibit ferroptosis. This study reveals new epigenetic and ferroptosis markers for preventing and treating lung cancer induced by environmental carcinogens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs), notably benzo[a]pyrene (BaP), are environmental contaminants with multiple adverse ecological implications. Numerous studies have suggested the use of BaP biodegradation using various bacterial strains to remove BaP from the environment. This study investigates the BaP biodegradation capability of Pigmentiphaga kullae strain KIT-003, isolated from the Nak-dong River (South Korea) under specific environmental conditions. The optimum conditions of biodegradation were found to be pH 7.0, 35°C, and a salinity of 0 %. GC-MS analysis suggested alternative pathways by which KIT-003 produced catechol from BaP through several intermediate metabolites, including 4-formylchrysene-5-carboxylic acid, 5,6-dihydro-5,6-dihydroxychrysene-5-carboxylic acid (isomer: 3,4-dihydro-3,4-dihydroxychrysene-4-carboxylic acid), naphthalene-1,2-dicarboxylic acid, and 2-hydroxy-1-naphthoic acid. Proteomic profiles indicated upregulation of enzymes associated with aromatic compound degradation, such as nahAc and nahB, and of those integral to the tricarboxylic acid cycle, reflecting the strain's adaptability to and degradation of BaP. Lipidomic analysis of KIT-003 demonstrated that BaP exposure induced an accumulation of glycerolipids such as diacylglycerol and triacylglycerol, indicating their crucial role in bacterial adaptation mechanisms under BaP stress. This study provides significant scientific knowledge regarding the intricate mechanisms involved in BaP degradation by microorganisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon compound that is generated during combustion processes, and is present in various substances such as foods, tobacco smoke, and burning emissions. BaP is extensively acknowledged as a highly carcinogenic substance to induce multiple forms of cancer, such as lung cancer, skin cancer, and stomach cancer. Recently it is shown to adversely affect the reproductive system. Nevertheless, the potential toxicity of BaP on oocyte quality remains unclear. In this study, we established a BaP exposure model via mouse oral gavage and found that BaP exposure resulted in a notable decrease in the ovarian weight, number of GV oocytes in ovarian, and oocyte maturation competence. BaP exposure caused ribosomal dysfunction, characterized by a decrease in the expression of RPS3 and HPG in oocytes. BaP exposure also caused abnormal distribution of the endoplasmic reticulum (ER) and induced ER stress, as indicated by increased expression of GRP78. Besides, the Golgi apparatus exhibited an abnormal localization pattern, which was confirmed by the GM130 localization. Disruption of vesicle transport processes was observed by the abnormal expression and localization of Rab10. Additionally, an enhanced lysosome and LC3 fluorescence intensity indicated the occurrence of protein degradation in oocytes. In summary, our results suggested that BaP exposure disrupted the distribution and functioning of organelles, consequently affecting the developmental competence of mouse oocytes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sexual dimorphism in immune responses is an essential factor in environmental adaptation. However, the mechanisms involved remain obscure owing to the scarcity of data from sex-role-reversed species in stressed conditions. Benzo[a]pyrene (BaP) is one of the most pervasive and carcinogenic organic pollutants in coastal environments. In this study, we evaluated the potential effects on renal immunotoxicity of the sex-role-reversed lined seahorse (Hippocampus erectus) toward environmental concentrations BaP exposure. Our results discovered the presence of different energy-immunity trade-off strategies adopted by female and male seahorses during BaP exposure. BaP induced more severe renal damage in female seahorses in a concentration-dependent manner. BaP biotransformation and detoxification in seahorses resemble those in mammals. Benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BPDE) and 9-hydroxybenzo[a]pyrene (9-OH-BaP) formed DNA adducts and disrupted Ca 2+ homeostasis may together attribute the renal immunotoxicity. Sexual dimorphisms in detoxification of both BPDE and 9-OH-BaP, and in regulation of Ca 2+ , autophagy and inflammation, mainly determined the extent of renal damage. Moreover, the mechanism of sex hormones regulated sexual dimorphism in immune responses needs to be further elucidated. Collectively, these findings contribute to the understanding of sexual dimorphism in the immunotoxicity induced by BaP exposure in seahorses, which may attribute to the dramatic decline in the biodiversity of the genus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Benzo(a)pyrene [B(a)P] is an environmental endocrine disruptor with reproductive toxicity. The corpus luteum (CL) of the ovary plays an important role in embryo implantation and pregnancy maintenance. Our previous studies have shown that B(a)P exposure affects embryo implantation and endometrial decidualization in mouse, but its effects and mechanisms on CL function remain unclear. In this study, we explore the mechanism of ovarian toxicity of B(a)P using a pregnant mouse model and an in vitro model of human ovarian granulosa cells (GCs) KGN. Pregnant mice were gavaged with corn oil or 0.2 mg/kg.bw B(a)P from pregnant day 1 (D1) to D7, while KGN cells were treated with DMSO, 1.0IU/mL hCG, or 1.0IU/mL hCG plus benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a B(a)P metabolite. Our findings revealed that B(a)P exposure damaged embryo implantation and reduced estrogen and progesterone levels in early pregnant mice. Additionally, in vitro, BPDE impaired luteinization in KGN cells. We observed that B(a)P/BPDE promoted oxidative stress (OS) and inflammation, leading to apoptosis rather than pyroptosis in ovaries and luteinized KGN cells. This apoptotic response was mediated by the activation of inflammatory Caspase1 through the cleavage of BID. Furthermore, B(a)P/BPDE inhibited TRAF2 expression and suppressed NFκB signaling pathway activation. The administration of VX-765 to inhibit the Caspase1 activation, over-expression of TRAF2 using TRAF2-pcDNA3.1 (+) plasmid, and BetA-induced activation of NFκB signaling pathway successfully alleviated BPDE-induced apoptosis and cellular dysfunction in luteinized KGN cells. These findings were further confirmed in the KGN cell treated with H 2 O 2 and NAC. In conclusion, this study elucidated that B(a)P/BPDE induces apoptosis rather than pyroptosis in GCs via TRAF2-NFκB-Caspase1 during early pregnancy, and highlighting OS as the primary contributor to B(a)P/BPDE-induced ovarian toxicity. Our results unveil a novel role of TRAF2-NFκB-Caspase1 in B(a)P-induced apoptosis and broaden the understanding of mechanisms underlying unexplained luteal phase deficiency., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Cultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1., Methods: Hand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software., Results: BaP (0.05-1μM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2-4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44-47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation., Discussion: First, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microplastics (MPs) and benzo[a]pyrene (B[a]P) are prevalent environmental pollutants. Numerous studies have extensively reported their individual adverse effects on organisms. However, the combined effects and mechanisms of exposure in mammals remain unknown. Thus, this study aims to investigate the potential effects of oral administration of 0.5μm polystyrene (PS) MPs (1 mg/mL or 5 mg/mL), B[a]P (1 mg/mL or 5 mg/mL) and combined (1 mg/mL or 5 mg/mL) on 64 male SD rats by gavage method over 6-weeks. The results demonstrate that the liver histopathological examination showed that the liver lobules in the combined (5 mg/kg) group had blurred and loose boundaries, liver cord morphological disorders, and significant steatosis. The levels of AST, ALT, TC, and TG in the combined dose groups were significantly higher than those in the other groups, the combined (5 mg/kg) group had the lowest levels of antioxidant enzymes and the highest levels of oxidants. The expression of Nrf2 was lowest and the expression of P38, NF-κB, and TNF-α was highest in the combined (5 mg/kg) group. In conclusion, these findings indicate that the combination of PSMPs and B[a]P can cause the highest levels of oxidative stress and elicit markedly enhanced toxic effects, which cause severe liver damage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The relationship of exposure to benzo[a]pyrene (BaP) with lung cancer risk has been firmly established, but whether this association could be modified by other environmental or genetic factors remains to be explored. To investigate whether and how zinc (Zn) and genetic predisposition modify the association between BaP and lung cancer, we performed a case-cohort study with a 5.4-year median follow-up duration, comprising a representative subcohort of 1399 participants and 359 incident lung cancer cases. The baseline concentrations of benzo[a]pyrene diol epoxide-albumin adduct (BPDE-Alb) and Zn were quantified. We also genotyped the participants and computed the polygenic risk score (PRS) for lung cancer. Our findings indicated that elevated BPDE-Alb and PRS were linked to increased lung cancer risk, with the HR (95%CI) of 1.54 (1.36, 1.74) per SD increment in ln-transformed BPDE-Alb and 1.27 (1.14, 1.41) per SD increment in PRS, but high plasma Zn level was linked to a lower lung cancer risk [HR (95%CI)=0.77 (0.66, 0.91) per SD increment in ln-transformed Zn]. There was evidence of effect modification by Zn on BaP-lung cancer association (P for multiplicative interaction = 0.008). As Zn concentrations increased from the lowest to the highest tertile, the lung cancer risk per SD increment in ln-transformed BPDE-Alb decreased from 2.07 (1.48, 2.89) to 1.33 (0.90, 1.95). Additionally, we observed a significant synergistic interaction of BPDE-Alb and PRS [RERI (95%CI) = 0.85 (0.03, 1.67)], with 42% of the incident lung cancer cases among individuals with high BPDE-Alb and high PRS attributable to their additive effect [AP (95%CI) = 0.42 (0.14, 0.69)]. This study provided the first prospective epidemiological evidence that Zn has protective effect against BaP-induced lung tumorigenesis, whereas high genetic risk can enhance the harmful effect of BaP. These findings may provide novel insight into the environment-environment and environment-gene interaction underlying lung cancer development, which may help to develop prevention and intervention strategies to manage BaP-induced lung cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

PIWI-interacting RNAs (piRNAs) is an emerging class of small non-coding RNAs that has been recently reported to have functions in infertility, tumorigenesis, and multiple diseases in humans. Previously, 5 toxicity pathways were proposed from hundreds of toxicological studies that underlie BaP-induced lung injuries, and a "Bottom-up" approach was established to identify small non-coding RNAs that drive BaP-induced pulmonary effects by investigating the activation of these pathways in vitro, and the expression of the candidate microRNAs were validated in tissues of patients with lung diseases from publications. Here in this study, we employed the "Bottom-up" approach to identifying the roles of piRNAs and further validated the mechanisms in vivo using mouse acute lung injury model. Specifically, by non-coding RNA profiling in in vitro BaP exposure, a total of 3 suppressed piRNAs that regulate 5 toxicity pathways were proposed, including piR-004153 targeting CYP1A1, FGFR1, ITGA5, IL6R, NGRF, and SDHA, piR-020326 targeting CDK6, and piR-020388 targeting RASD1. Animal experiments demonstrated that tail vein injection of respective formulated agomir-piRNAs prior to BaP exposure could all alleviate acute lung injury that was shown by histopathological and biochemical evidences. Immunohistochemical evaluation focusing on NF-kB and Bcl-2 levels showed that exogenous piRNAs protect against BaP-induced inflammation and apoptosis, which further support that the inhibition of the 3 piRNAs had an important impact on BaP-induced lung injuries. This mechanism-driven, endpoint-supported result once again confirmed the plausibility and efficiency of the approach integrating in silico, in vitro, and in vivo evidences for the purpose of identifying key molecules., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Benzo(a)pyrene (BaP) can be detected in the human placenta. However, little is known about the effects of BaP exposure on different placental cells under various conditions. In this study, we aimed to investigate the effects of BaP on mitochondrial function, pyrin domain-containing protein 3 (NLRP3) inflammasome, and apoptosis in three human trophoblast cell lines under normoxia, hypoxia, and inflammatory conditions. JEG-3, BeWo, and HTR-8/SVneo cell lines were exposed to BaP under normoxia, hypoxia, or inflammatory conditions for 24 h. After treatment, we evaluated cell viability, apoptosis, aryl hydrocarbon receptor (AhR) protein and cytochrome P450 (CYP) gene expression, mitochondrial function, including mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨm), intracellular adenosine triphosphate (iATP), and extracellular ATP (eATP), nitric oxide (NO), NLPR3 inflammasome proteins, and interleukin (IL)-1β. We found that BaP upregulated the expression of AhR or CYP genes to varying degrees in all three cell lines. Exposure to BaP alone increased ΔΨm in all cell lines but decreased NO in BeWo and HTR-8/SVneo, iATP in HTR-8/SVneo, and cell viability in JEG-3, without affecting apoptosis. Under hypoxic conditions, BaP did not increase the expression of AhR and CYP genes in JEG-3 cells but increased CYP gene expression in two others. Pro-inflammatory conditions did not affect the response of the 3 cell lines to BaP with respect to the expression of CYP genes and changes in the mitochondrial function and NLRP3 inflammasome proteins. In addition, in HTR-8/SVneo cells, BaP increased IL-1β secretion in the presence of hypoxia and poly(I:C). In conclusion, our results showed that BaP affected mitochondrial function in trophoblast cell lines by increasing ΔΨm. This increased ΔΨm may have rescued the trophoblast cells from activation of the NLRP3 inflammasome and apoptosis after BaP treatment. We also observed that different human trophoblast cell lines had cell type-dependent responses to BaP exposure under normoxia, hypoxia, or pro-inflammatory conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T.B.Z, A.B, P.V, C.C.V and J.J.S, declare that they are employees and equity holders at TwinStrand Biosciences, Inc. and are authors on one or more Duplex Sequencing-related patents., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aflatoxin B1 (AFB1) is the most toxic mycotoxin and a proven human carcinogen that requires metabolic activation, known by cytochrome P450 (CYP) 1A2 and 3A4. Previous evidence showed that AFB1 is activated by human recombinant CYP1A1 expressed in budding yeast. Yet, the toxicity, in particular the genotoxicity of the reactive metabolites formed from AFB1 remains unclear. Humans could be exposed to both AFB1 and benzo(a)pyrene (BaP) simultaneously, thus we were interested in their combined genotoxic effects subsequent to metabolic activation by CYP1A1. In this study, molecular docking of AFB1 to human CYP1A1 indicated that AFB1 is valid as a substrate. In the incubations with AFB1 in human CYP1A1-expressed microsomes, AFM1 as a marking metabolite of AFB1 was detected. Moreover, AFB1 induced micronucleus formation in a Chinese hamster V79-derived cell line and in a human lung epithelial BEAS-2B cell line, both expressing recombinant human CYP1A1, V79-hCYP1A1 and 2B-hCYP1A1 cells, respectively. Immunofluorescence of centromere protein B stained micronuclei was dominant in AFB1-treated BEAS-2B cells exposed to AFB1, suggesting an aneugenic effect. Moreover, AFB1 elevated the levels of ROS, 8-OHdG, AFB1-DNA adduct, and DNA breaks in 2B-hCYP1A1 cells, compared with those in the parental BEAS-2B cells. Meanwhile, AFB1 increased CYP1A1, RAD51, and γ-H2AX protein levels in 2B-hCYP1A1 cells, which were attenuated by the CYP1A1 inhibitor bergamottin. Co-exposure of AFB1 with BaP increased 8-OHdG, RAD51, and γ-H2AX levels (indicating DNA damage). In conclusion, AFB1 could be activated by human CYP1A1 for potent aneugenicity, which may be further enhanced by co-exposure to BaP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Exposure to benzo[a]pyrene (B[a]P) has been linked to lung injury and carcinogenesis. Airway epithelial cells express the B[a]P receptor AHR, so B[a]P is considered to mainly target airway epithelial cells, whereas its potential impact on alveolar cells remains inadequately explored. Metformin, a first-line drug for diabetes, has been shown to exert anti-inflammatory and tissue repair-promoting effects under various injurious conditions. Here, we explored the effect of chronic B[a]P exposure on alveolar cells and the impact of metformin on B[a]P-induced lung injury by examining the various parameters including lung histopathology, inflammation, fibrosis, and related signal pathway activation. MLKL knockout (Mlkl -/- ) and AT2-lineage tracing mice (Sftpc Cre-ERT2 ;LSL-tdTomato flox+/- ) were used to delineate the role of necroptosis in B[a]P-induced alveolar epithelial injury and repair. Mice receiving weekly administration of B[a]P for 6 weeks developed a significant alveolar damaging phenotype associated with pulmonary inflammation, fibrosis, and activation of the necroptotic cell death pathway. These effects were significantly relieved in MLKL null mice. Furthermore, metformin treatment, which were found to promote AMPK phosphorylation and inhibit RIPK3, as well as MLKL phosphorylation, also significantly alleviated B[a]P-induced necroptosis and lung injury phenotype. However, the protective efficacy of metformin was rendered much less effective in Mlkl null mice or by blocking the necroptotic pathway with RIPK3 inhibitor. Our findings unravel a potential protective efficacy of metformin in mitigating the detrimental effects of B[a]P exposure on lung health by inhibiting necroptosis and protecting AT2 cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no relevant financial or non-financial interests to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The effect of long term exposure to low concentrations of environmental pollutants on hepatic disorders is a major public health concern worldwide. Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants. In recent years, an increasing number of studies have focused on the deleterious effects of low concentrations of PAHs in the initiation or exacerbation of the progression of chronic liver disease. However, the underlying molecular mechanisms and effective intervention methods remain unclear. Here, we found that in hepatocytes, a low concentration of benzo(a)pyrene (B[a]P, an indicator of PAHs) chronic exposure continuously activated 14-3-3η via an epigenetic accumulation of DNA demethylation. As a "switch like" factor, 14-3-3η activated its downstream PI3K/Akt signal, which in turn promoted vascular endothelial growth factor (VEGF) production and secretion. As the characteristic fibrogenic paracrine factor regulated by B[a]P/14-3-3η, VEGF significantly induced the neovascularization and activation of hepatic stellate cells, leading to the development of hepatic fibrosis. Importantly, targeted 14-3-3η by using its specific inhibitor invented by our lab could prevent B[a]P-induced hepatic fibrosis, and could even reverse existent hepatic fibrosis caused by B[a]P. The present study not only revealed novel mechanisms, but also proposed an innovative approach for the targeted reversion/prevention of the harmful effects of exposure to PAHs on chronic liver disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Research on the mechanisms of reproductive toxicity caused by persistent organic pollutants (POPs) in marine animals has received significant attention. One group of typical POPs, called polycyclic aromatic hydrocarbons (PAHs), has been found to cause various reproductive toxicities in aquatic organisms, including epigenotoxicity, reproductive endocrine disruption, DNA damage effects and other reproductive toxicity, thereby affecting gonadal development. Interestingly, male aquatic animals are more susceptible to the disturbance and toxicity of environmental pollutants. However, current studies primarily focus on vertebrates, leaving a large gap in our understanding of the reproductive toxicity and mechanisms of PAHs interference in marine invertebrates. In this study, male Ruditapes philippinarum was used as an experimental subject to investigate reproduction-related indexes in clams under the stress of benzo[a]pyrene (B[a]P) at different concentrations (0, 0.8, 4 and 20 μg/L) during the proliferative, growth, maturity, and spawning period. We analyzed the molecular mechanisms of reproductive toxicity caused by PAHs in marine bivalves, specifically epigenotoxicity, reproductive endocrine disruption, and gonadal damage-apoptotic effect. The results suggest that DNA methylation plays a crucial role in mediating B[a]P-induced reproductive toxicity in male R. philippinarum. B[a]P may affect sex hormone levels, impede spermatogenesis and testis development in clams, by inhibiting the steroid hormone synthesis pathway and downregulating genes critical for cell proliferation, testis development, and spermatid expulsion. Moreover, the spermatids of male R. philippinarum were severely impaired under the B[a]P stress, leading to reduced reproductive performance in the clams. These findings contribute to a better understanding of the reproductive toxicity response of male marine invertebrates to POPs stress., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Luqing Pan reports financial support was provided by Shandong Provincial Natural Science Foundation., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Aryl hydrocarbon receptor (AHR) is one of the main mediators of the toxic effects of benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, a vast number of BaP- and TCDD-affected genes may suggest a more complex transcriptional regulatory network driving common adverse effects of these two chemicals. Unlike TCDD, BaP is rapidly metabolized in the liver, yielding products with a questionable ability to bind and activate AHR. In this study, we used transcriptomics data from the BaP- and TCCD-exposed human liver cell line HepG2, and performed differential eigengene network analysis to understand the correlation among genes and to untangle the common regulatory mechanism in the action of BaP and TCDD. The genes were grouped into 11 meta-modules with an overall preservation of 0.72 and were also segregated into three consensus time clusters: 12, 24, and 48 h. The analysis showed that the consensus genes in each time cluster were either directly regulated by the AHR or the AHR-TF interactions. Some TFs form a direct physical interaction with AHR such as ESR1, FOXA1, and E2F1, whereas others, including CTCF, RXRA, FOXO1, CEBPA, CEBPB, and TP53 show an indirect interaction with AHR. The analysis of biological processes (BPs) identified unique and common BPs in BaP and TCDD samples, with DNA damage response detected in all three time points. In summary, we identified a consensus transcriptional regulatory network common for BaP and TCDD consisting of direct AHR targets and AHR-TF targets. This analysis sheds new light on the common mechanism of action of a genotoxic (BaP) and non-genotoxic (TCDD) chemical in liver cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microplastics (MP) are harmful, causing stress in aquatic species and acting as carriers of hydrophobicity. In aquatic environments, benzo[α]pyrene (BaP) is an endocrine-disrupting chemical that accumulates in the body and causes toxic reactions in living organisms. We investigated the effects of single and combined microbead (MB) and BaP environments on goldfish antioxidant response and apoptosis. For 120 h, goldfish were exposed to single (MB10, MB100, and BaP5) and combined (MB10+BaP5 and MB100+BaP5) environments of 10 and 100 beads/L of 0.2 µm polystyrene MB and 5 µg/L BaP. We measured MB and BaP bioaccumulation as well as plasma parameters including ALT, AST, and glucose. The level of oxidative stress was determined by evaluating lipid peroxidation (LPO) and total antioxidant capacity (TAC) in plasma, as well as antioxidant-related genes for superoxide dismutase and catalase (SOD and CAT) and caspase-3 (Casp3) mRNA expression in liver tissue. The TUNEL assay was used to examine SOD in situ hybridization and apoptosis in goldfish livers. Except for the control group, plasma LPO levels increased at the end of the exposure period in all experimental groups. TAC increased up to 24 h of exposure and then maintained a similar level until the trial ended. SOD, CAT, and Casp3 mRNA expression increased substantially up to 120 h as the exposure concentration and time increased. The TUNEL assay revealed more signals and apoptotic signals in the combined exposure environments as a consequence of SOD in situ hybridization than in single exposure environments. These results suggest that combined exposure to toxic substances causes oxidative stress in organisms, which leads to apoptosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Air quality in Poland is among the lowest in Europe due to high emissions of harmful substances. This causes the development of diseases and leads to a high number of premature deaths. Particularly high pollution occurs in parts of urban areas. The most serious problem is unregulated emissions from buildings and vehicles. That is why it is so important to take action to improve air quality at the local level. The study assessed changes in the concentrations of NO 2 , O 3 , PM 10 , PM 2.5 and benzo(a)pyrene in 11 major Polish cities between 2005 and 2021. In 2021 average levels were: NO 2 - 25 μg/m 3 , O 3 - 45 μg/m 3 , PM 10 - 26 μg/m 3 , PM 2.5 -17 μg/m 3 , benzo(a)pyrene - 2.1 ng/m 3 . The highest exceedances of WHO standards over the studied period were for PM 2.5 , followed by NO 2 and PM 10 . The annual average levels fell by 17% for NO 2 and by 18% for PM 10 between 2005 and 2021, and by 34% for PM 2.5 and 27% for benzo(a)pyrene between 2010 and 2021. The most polluted cities are Kraków, Katowice and Łódź. The highest concentrations of pollutants typically occurred in 2006 and 2011, the lowest in 2020. Strategic documents and programmes that formulate objectives for reducing emissions and improving air quality were evaluated. Policy documents enable numerous measures to improve air quality. Plans are not always effectively implemented due to a lack of formal tools and financial resources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Benzo[a]pyrene (BaP) is a representative polycyclic aromatic hydrocarbon widely present in the environment. We previously reported that the aryl hydrocarbon receptor (AHR) mediates BaP-induced apoptosis and cardiac malformations in zebrafish embryos, but the underlying molecular mechanisms were unclear. Since BaP is a mutagenetic compound, we hypothesize that BaP induces apoptosis and heart defects via AHR-mediated DNA damage. In this study, zebrafish embryos were exposed to BaP at a concentration of 0.1 μM from 2 to 72 h post fertilization, either with or without inhibitors/agonists. AHR activity and levels of reactive oxygen species (ROS) were examined under a fluorescence microscope. mRNA expression levels were quantified by qPCR. DNA damage and apoptosis were detected by immunofluorescence. Our findings revealed that BaP exposure significantly increased BPDE-DNA adducts, mitochondrial damage, apoptosis and heart defects in zebrafish embryos. These effects were counteracted by inhibiting AHR/cyp1a1 using pharmaceutical inhibitors or genetic knockdown. Furthermore, we observed that spironolactone, an antagonist of nucleotide excision repair (NER), significantly enhanced BaP-induced BPDE-DNA adducts, mitochondrial damage, apoptosis and heart malformation rates. Conversely, SRT1720, a SIRT1 agonist, reduced the adverse effects of BaP. Supplementation with spironolactone also enhanced γ-H2AX signals in the heart of zebrafish embryos exposed to BaP. Additional experiments demonstrated that BaP suppressed the expression of SIRT1. We further established that AHR, when activated by BaP, directly inhibited SIRT1 transcription, leading to downregulation of XPC and XPA, which are essential NER genes involved in the recognition and verification steps of the NER process. Taken together, our results indicate that AHR mediates BaP-induced DNA damage in the heart of zebrafish embryos by inducing BPDE-DNA adduct formation via the AHR/Cyp1a1 signalling pathway, as well as suppressing NER via AHR-mediated inhibition of SIRT1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Benzo[a]pyrene (BaP) is commonly found in the environment as a result of incomplete combustion of organic materials and cigarette smoke. Epidemiological studies have consistently suggested that elderly smokers are at higher risk for small cell lung cancer (SCLC), with risks and clinical stages increasing with the intensity and duration of smoking. However, the underlying mechanism remains insufficiently investigated. Here, we established a positive correlation between smoking and BaP metabolite 3-hydroxybenzo[a]pyrene (3OH-BaP) in urine. The pooled standardized mean difference of urinary 3OH-BaP concentration for smokers versus nonsmokers was 5.18 (95 % CI 2.86-7.50). Clinical data suggested that smoking led to more lymph node metastasis, higher pathological N-stage, and worse overall survival in SCLC patients. We identified 75 genes that participate in BaP-associated cancer stemness of SCLC from Comparative Toxicogenomics Database and validated the expression of these candidate genes in SCLC patient samples. Protein kinase cAMP-activated catalytic subunit alpha (PRKACA) was found to be most upregulated in SCLC patients and in vitro experiments indicated that long-term exposure of SCLC cells to BaP, at the concentration equivalent to those detected in blood, increased PKA protein level. Further investigation revealed that PKA could directly interact with SOX2 and protect SOX2 from COP1-mediated ubiquitination and degradation. Upregulated SOX2 then contributed to the stemness and metastasis of SCLC cells while inhibition of aryl hydrocarbon receptor (AhR) signaling pathway abolished BaP induced PKA expression and downstream PKA/SOX2 axis. Our findings firstly pinpoint BaP exposure as a high-risk factor for SCLC and worse outcomes in patients, with the underlying mechanism being the activation of cancer stemness of SCLC via the AhR/PKA/SOX2 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Environmental and occupational exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with adverse health effects in humans. Uncertainty exists regarding the causation of urinary bladder cancer by benzo[a]pyrene (B[a]P) due to a lack of sufficient data. In this work, we focused on in-vitro DNA damage and the formation of micronuclei and chromosomal aberrations as predictors of cancer risk, applying a wide range of dosages and time periods to quantify the onset, intensity, and duration of the response. We chose two urothelial cell types to compare susceptibility and the ability to increase the malignity of a pre-existing bladder cancer: a cancer cell line (T24) and a pooled sample of primary urinary bladder epithelia cells (PUBEC) from pigs. The highest level of DNA damage assessed by comet assay was observed following 24-h treatment in both cell types, whereas PUBEC cells were clearly more susceptible. Even 4-h treatment induced DNA damage in PUBEC cells with benchmark doses of 0.0027 µM B[a]P and 0.00023 µM after 4-h and 24-h exposure, respectively. Nearly no effect was observed for periods of 48 h. The frequency of micronucleus formation increased more markedly in T24 cells, particularly with 24-h treatment. In PUBEC cells, 48-h exposure notably induced the formation of nucleoplasmic bridges and nuclear buds. Even though only one biological replicate was studied due to the sophisticated study design, our results give a strong indication of the potential of B[a]P to induce and increase malignity in human-relevant cell types., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Amino acids (AAs) are a class of important metabolites in metabolomics methodology that investigates metabolite changes in a cell, tissue, or organism for early diagnosis of diseases. Benzo[a]pyrene (BaP) is considered a priority contaminant by different environmental control agencies because it is a proven carcinogenic compound for humans. Therefore, it is important to evaluate the BaP interference in the metabolism of amino acids. In this work, a new amino acid extraction procedure (derivatized with propyl chloroformate/propanol) using functionalized magnetic carbon nanotubes was developed and optimized. A hybrid nanotube was used followed by desorption without heating, and excellent extraction of analytes was obtained. After exposure of Saccharomyces cerevisiae, the BaP concentration of 25.0 μmol L -1 caused changes in cell viability, indicating metabolic changes. A fast and efficient GC/MS method using a Phenomenex ZB-AAA column was optimized, enabling the determination of 16 AAs in yeasts exposed or not to BaP. A comparison of AA concentrations obtained in the two experimental groups showed that glycine (Gly), serine (Ser), phenylalanine (Phe), proline (Pro), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), tyrosine (Tyr), and leucine (Leu) statistically differentiated, after subsequent application of ANOVA with Bonferroni post-hoc test, with a confidence level of 95%. This amino acid pathway analysis confirmed previous studies that revealed the potential of these AAs as toxicity biomarker candidates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Oxidative stress is implicated in numerous diseases, with benzo(α)pyrene (BaP) known for causing substantial oxidative damage. Bifidobacterium longum (B. longum) is recognized as an antioxidant bacterium for certain hosts, yet its influence on oxidative damages instigated by BaP remains undetermined. In our study, we introduced various strains of Caenorhabditis elegans (C. elegans) to BaP to trigger oxidative stress, subsequently treating them with different forms of B. longum to evaluate its protective effects. Additionally, we explored the role of daf-16 in this context. Our findings indicated that in wild-type N2 C. elegans, B. longum-even in the form of inactivated bacteria or bacterial ultrasonic lysates (BULs)-significantly extended lifespan. BaP exposure notably decreased lifespan, superoxide dismutase (SOD) activity, and motility, while simultaneously down-regulating the expression of reactive oxygen species (ROS)-associated genes (sod-3, sek-1, cat-1) and daf-16 downstream genes (sod-3, ctl-2). However, it significantly increased the ROS level, malondialdehyde (MDA) content, and lipofuscin accumulation and up-regulated another daf-16 downstream gene (clk-1) (P <0.05). Interestingly, when further treated with B. longum peptide-1 (BLP-1), opposite effects were observed, and all the aforementioned indices changed significantly. In the case of RNAi (daf-16) C. elegans, BaP exposure significantly shortened the lifespan (P <0.05), which was only slightly prolonged upon further treatment with BLP-1. Furthermore, the expression of daf-16 downstream genes showed minor alterations in RNAi C. elegans upon treatment with either BaP or BLP-1. In conclusion, our findings suggest that B. longum acts as a probiotic for C. elegans. BLP-1 was shown to safeguard C. elegans from numerous oxidative damages induced by BaP, but these protective effects were contingent upon the daf-16 gene., (© 2023. The Author(s), under exclusive licence to Cell Stress Society International.)

Benzo(a)pyrene was sparsely studied for its early respiratory impairment. The non-canonical ligand WNT5A play a role in pneumonopathy, while its function during benzo(a)pyrene-induced adverse effects were largely unexplored. Individual benzo(a)pyrene, plasma WNT5A, and spirometry 24-hour change for 87 residents from Wuhan-Zhuhai cohort were determined to analyze potential role of WNT5A in benzo(a)pyrene-induced lung function alternation. Normal bronchial epithelial cell lines were employed to verify the role of WNT5A after benzo(a)pyrene treatment. RNA sequencing was adopted to screen for benzo(a)pyrene-related circulating microRNAs and differentially expressed microRNAs between benzo(a)pyrene-induced cells and controls. The most potent microRNA was selected for functional experiments and target gene validation, and their mechanistic link with WNT5A-mediated non-canonical Wnt signaling was characterized through rescue assays. We found significant associations between increased benzo(a)pyrene and reduced 24-hour changes of FEF 50% and FEF 75% , as well as increased WNT5A. The benzo(a)pyrene-induced inflammation and epithelial-mesenchymal transition in BEAS-2B and 16HBE cells were attenuated by WNT5A silencing. hsa-miR-122-5p was significantly and positively associated with benzo(a)pyrene and elevated after benzo(a)pyrene induction, and exerted its effect by downregulating target gene TP53. Functionally, WNT5A participates in benzo(a)pyrene-induced lung epithelial injury via non-canonical Wnt signaling modulated by hsa-miR-122-5p/TP53 axis, showing great potential as a preventive and therapeutic target., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Nanoplastics and polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in soil environments. In order to objectively evaluate the toxic interaction between polystyrene nanoplastics (PS NPs) and benzo [a] pyrene (BaP), oxidative damage at the level of earthworm cells and biomacromolecules was investigated by experiments combined with molecular dynamics simulation. Studies on cells reveal that PS NPs and BaP had synergistic toxicity when it came to causing oxidative stress. Cellular reactive oxygen species (ROS) levels under combined pollutant exposure were 24% and 19% higher, respectively than when PS NPs and BaP were exposed alone (compared to the blank group). In addition, BaP and PS NPs inhibited the ability of CAT to decompose H 2 O 2 by affecting the structure of the proximal amino acid Tyr 357 in the active center of CAT, which exacerbated oxidative stress to a certain extent. Therefore, the synergistic toxic effect of BaP and PS NPs is due to the mutual complement of the two to the induction of protein structural looseness, and the strengthening of the stability of the conjugate (CAT-BaP-PS) under the weak interaction. This work provides a new perspective and approach on how to talk about the toxicity of combined pollutants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Inhaled nanoparticles (NPs) can deposit in alveoli where they interact with the pulmonary surfactant (PS) and potentially induce toxicity. Although nano-bio interactions are influenced by the physicochemical properties of NPs, isolated NPs used in previous studies cannot accurately represent those found in atmosphere. Here we used molecular dynamics simulations to investigate the interplay between two types of NPs associated with benzo[a]pyrene (BaP) at the PS film. Silicon NPs (SiNPs), regardless of aggregation and adsorption, directly penetrated through the PS film with minimal disturbance. Meanwhile, BaPs adsorbed on SiNPs were rapidly solubilized by PS, increasing the BaP's bioaccessibility in alveoli. Carbon NPs (CNPs) showed aggregation and adsorption-dependent effects on the PS film. Compared to isolated CNPs, which extracted PS to form biomolecular coronas, aggregated CNPs caused more pronounced PS disruption, especially around irregularly shaped edges. SiNPs in mixture exacerbated the PS perturbation by piercing PS film around the site of CNP interactions. BaPs adsorbed on CNPs were less solubilized and suppressed PS extraction, but aggravated biophysical inhibition by prompting film collapse under compression. These results suggest that for proper assessment of inhalation toxicity of airborne NPs, it is imperative to consider their heterogeneous aggregation and adsorption of pollutants under atmospheric conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study aims to compare differential effects of continuous and pulsed BaP exposures on metabolism and antioxidant defense in the liver of large yellow croaker. Fish were subjected to BaP for 4 days and 36 days in three exposure regimes with the same time-averaged concentration of BaP: 4 μg/L BaP continuously, 8 μg/L BaP for 24 h every other day or 16 μg/L BaP for 24 h every 4 days. Our results showed that compared to pulsed BaP exposures, continuous BaP exposure reduced BaP metabolism (CYP1A, CYP3A and AHR transcriptional expressions, GSH content, GSH/GSSG ratio, EROD and GST activities) and antioxidant defense (T-SOD activity) on day 4, resulting to the increases in MDA and PC contents, indicating that continuous BaP exposure induced more severe oxidative damage during the early stage of exposure. But continuous BaP exposure reduced MDA and PC contents by improving BaP metabolism and antioxidant defense during the late stage of exposure. CYP1B transcriptional expression and CAT activity were unsuitable biomarkers of both continuous and pulsed BaP exposures. In conclusion, our results demonstrated differential effects of continuous and pulsed exposures on BaP metabolism and antioxidant responses, which were depend on exposure duration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Humans are exposed to the common carcinogen benzo[a]pyrene (BaP) by ingesting contaminated foods and water or inhaling polluted air. Given the enriched lipids and reduced antioxidative properties in the brain and the accumulation of BaP in the brain due to its high lipophilicity, the brain is susceptible to BaP-induced toxicity. Exposure to BaP leads to impairments in learning and memory, increased anxiety behavior, and neuronal death. It induces protein dysfunctions in neuronal compartments that play essential roles in neuronal activity or physiology. However, the neurotoxicity of BaP on presynaptic terminals, which is crucial to neurotransmission by releasing synaptic vesicles that contain neurotransmitters, has not yet been investigated. In the present study, we investigated the toxicity of BaP at presynaptic terminals in living hippocampal neurons. These neurons were sourced from transgenic mice pups (postnatal 1-day, a total of 12 pups, equal numbers for each sex) that endogenously express synaptic vesicle-fused pHluorin, which is a green fluorescent protein that enables monitoring of synaptic vesicle dynamics. We observed that BaP suppressed synaptic vesicle exocytosis by inhibiting presynaptic Ca 2+ entry via P/Q-type Ca 2+ channels. Together with molecular docking simulation, we speculate that BaP and metabolites may bind to the P/Q Ca 2+ channels. These results suggest the toxic mechanism of BaP exposure-induced abnormal behavior that provides a basis to evaluate the risk assessment of BaP-induced neurotoxicity., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or nonfinancial interests to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)