Real-ambient PM2.5 induced corneal epithelial barrier disruption through Wnt/ß-catenin signaling.

Continued daily exposure to fine particulate matter (PM2.5) is linked to increasing risks of ocular surface diseases. However, further study is needed to understand how real-ambient PM2.5 disrupts the barrier function of the corneal epithelial layers and its underlying mechanism. In our study, we utilized a real-ambient PM2.5 exposure system to investigate its effects on the corneal epithelial barrier in C57BL/6Jmice over 4 and 8 weeks. The mean concentration of PM2.5 in the exposure chambers over 8 weeks was 140.18 µg/m3. Following 4 and 8 weeks of continuous PM2.5 exposure, we observed disorganized cellular arrangements in the corneal epithelium of mice. Moreover, PM2.5 exposure led to a significant loss of microvilli on the surface of corneal epithelial cells and noticeable disconnections among epithelial cell layers. Subsequent in vitro analysis revealed that 100 µg/mL PM2.5 activated the Wnt/ß-catenin signaling pathway in corneal epithelium, resulting in decreased expression 1.81 fold and 2.25 fold of E-cadherin and ZO-1, respectively, ultimately impairing the corneal epithelial barrier function. Our findings provide the knowledge base for promoting eye health in the context of atmospheric pollution.

Studying the Effects of Inhaled Environmental Pollutants on Olfactory Function in Mice.

Olfactory impairment is a significant public health problem and independently predicts the risk of neurodegenerative diseases. Inhaled environmental pollutants exposure may impair olfaction; thereby, there is an urgent need for methods to evaluate the effects of inhaled environmental pollutants exposure on olfaction. Mice are ideal models for olfactory experiments because of their highly developed olfactory system and behavioral characteristics. To assess the effects of inhaled environmental pollutants exposure on olfactory function in mice, a detailed buried food test and social odor discrimination experiment is provided, including the experiment preparation, the selection and construction of experimental facilities, the testing process, and indexes of time. Meanwhile, timekeeping equipment, operational details, and the experimental environment are discussed to ensure the success of the assay. Zinc sulfate is used as the treatment to demonstrate the feasibility of the experimental approach. The protocol provides a simple and clear operational process for assessing the effects of inhaled environmental pollutants on olfactory function in mice.

Impaired GPX4 activity elicits ferroptosis in alveolar type II cells promoting PHMG-induced pulmonary fibrosis development.

Inhaling polyhexamethylene guanidine (PHMG) aerosol, a broad-spectrum disinfectant, can lead to severe pulmonary fibrosis. Ferroptosis, a form of programmed cell death triggered by iron-dependent lipid peroxidation, is believed to play a role in the chemical-induced pulmonary injury. This study aimed to investigate the mechanism of ferroptosis in the progression of PHMG-induced pulmonary fibrosis. C57BL/6 J mice and the alveolar type II cell line MLE-12 were used to evaluate the toxicity of PHMG in vivo and in vitro, respectively. The findings indicated that iron deposition was observed in PHMG induced pulmonary fibrosis mouse model and ferroptosis related genes have changed after 8 weeks PHMG exposure. Additionally, there were disturbances in the antioxidant system and mitochondrial damage in MLE-12 cells following a 12-hour treatment with PHMG. Furthermore, the study observed an increase in lipid peroxidation and a decrease in GPX4 activity in MLE-12 cells after exposure to PHMG. Moreover, pretreatment with the ferroptosis inhibitors Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lip-1) not only restored the antioxidant system and GPX4 activity but also mitigated lipid peroxidation. Current data exhibit the role of ferroptosis pathway in PHMG-induced pulmonary fibrosis and provide a potential target for future treatment.

Diesel exhaust exposure induced squamous metaplasia of corneal epithelium via yes-associated protein activation.

Atmospheric pollution has been demonstrated to be associated with ocular surface diseases characterized by corneal epithelial damage, including impaired barrier function and squamous metaplasia. However, the specific mechanisms underlying the impact of atmospheric pollution on corneal damage are still unknow. To address this gap in knowledge, we conducted a study using a whole-body exposure system to investigate the detrimental effects of traffic-related air pollution, specifically diesel exhaust (DE), on corneal epithelium in C57BL/6 mice over a 28-day period. Following DE exposure, the pathological alterations in corneal epithelium, including significant increase in corneal thickness and epithelial stratification, were observed in mice. Additionally, exposure to DE was also shown to disrupt the barrier functions of corneal epithelium, leading to excessive proliferation of basal cells and even causing squamous metaplasia in corneal epithelium. Further studies have found that the activation of yes-associated protein (YAP), characterized by nuclear translocation, may play a significant role in DE-induced corneal squamous metaplasia. In vitro assays confirmed that DE exposure triggered the YAP/ß-catenin pathway, resulting in squamous metaplasia and destruction of barrier functions. These findings provide the preliminary evidence that YAP activation is one of the mechanisms of the damage to corneal epithelium caused by traffic-related air pollution. These findings contribute to the knowledge base for promoting eye health in the context of atmospheric pollution.

Spatio-temporal distribution of cadmium levels in Chinese population and its potential risk factors.

Cadmium (Cd), a ubiquitous heavy metal, exists in numerous environmental matrices and has severe adverse effects on various human organs and tissues. This research evaluates blood and urine Cd levels in the Chinese population through data mining using Monte Carlo simulation (MCS). A total of 168 scientific studies (120 on urine and 48 on blood) published between January 1980 and December 2020, reflecting a population of 109,743 individuals in China, were included in the study. The results indicate that the blood and urine Cd levels in the Chinese population exhibited a peak from 1990 to 1995 and remained stable after 1995, averaging 1.21 µg/L of blood Cd (BCd) and 0.61 µg/L of urine Cd (UCd). The spatial trend of Cd levels varied significantly. Shandong, Zhejiang, Heilongjiang, and Guangdong provinces were identified as the top provinces with high Cd levels, which were related to factors such as tobacco sales, E-waste amounts, and contaminated rice. Additionally, the study highlights that BCd concentrations are highest among preschool-aged individuals, whereas school-age and adolescent groups exhibit the lowest levels. However, no significant difference existed among the different age groups. Males showed significantly higher Cd levels than females in the general population. Moreover, exposure to smoking, drinking, and staple food preferences had an impact on Cd levels. Furthermore, this comprehensive study, using biological monitoring and data mining, provides valuable information on Cd pollution levels in the Chinese population. It presents a statistical analysis that can aid decision-makers in implementing effective measures to control potential Cd pollution and improve the health of vulnerable populations.

Distribution and potential risk factors of bisphenol a in serum and urine among Chinese from 2004 to 2019.

Background: Bisphenol A (BPA) is an oil-derived, large-market volume chemical with endocrine disrupting properties and reproductive toxicity. Moreover, BPA is frequently used in food contact materials, has been extensively researched recently, and widespread exposure in the general population has been reported worldwide. However, national information on BPA levels in general Chinese people is lacking. Methods: This study collected and analyzed 145 (104 in urine and 41 in serum) research articles published between 2004 and 2021 to reflect the BPA internal exposure levels in Chinese populations. The Monte Carlo simulation method is employed to analyze and estimate the data in order to rectify the deviation caused by a skewed distribution. Results: Data on BPA concentrations in urine and serum were collected from 2006 to 2019 and 2004 to 2019, respectively. Urinary BPA concentrations did not vary significantly until 2017, with the highest concentration occurring from 2018 to 2019 (2.90 ng/mL). The serum BPA concentration decreased to the nadir of 1.07 ng/mL in 2011 and gradually increased to 2.54 ng/mL. Nationally, 18 provinces were studied, with Guangdong (3.50 ng/mL), Zhejiang (2.57 ng/mL), and Fujian (2.15 ng/mL) having the highest urine BPA levels. Serum BPA was investigated in 15 provinces; Jiangsu (9.14 ng/mL) and Shandong (5.80 ng/mL) were relatively high. The results also indicated that males' urine and serum BPA levels were higher than females, while the BPA levels in children were also higher than in adults (p < 0.001). Furthermore, the volume of garbage disposal (r = 0.39, p < 0.05), household sewage (r = 0.34, p < 0.05), and waste incineration content (r = 0.35, p < 0.05) exhibited a strong positive connection with urine BPA levels in Chinese individuals. Conclusion: Despite using a data consolidation approach, our study found that the Chinese population was exposed to significant amounts of BPA, and males having a higher level than females. Besides, the levels of BPA exposure are influenced by the volume of garbage disposal, household sewage, and waste incineration content.

Polyhexamethylene guanidine accelerates the macrophage foamy formation mediated pulmonary fibrosis.

Polyhexamethylene guanidine (PHMG) is manufactured and applied extensively due to its superior disinfectant capabilities. However, the inhalatory exposure to PHMG aerosols is increasingly recognized as a potential instigator of pulmonary fibrosis, prompting an urgent call for elucidation of the underlying pathophysiological mechanisms. Within this context, alveolar macrophages play a pivotal role in the primary immune defense in the respiratory tract. Dysregulated lipid metabolism within alveolar macrophages leads to the accumulation of foam cells, a process that is intimately linked with the pathogenesis of pulmonary fibrosis. Therefore, this study examines PHMG's effects on alveolar macrophage foaminess and its underlying mechanisms. We conducted a 3-week inhalation exposure followed by a 3-week recovery period in C57BL/6 J mice using a whole-body exposure system equipped with a disinfection aerosol generator (WESDAG). The presence of lipid-laden alveolar macrophages and downregulation of pulmonary tissue lipid transport proteins ABCA1 and ABCG1 were observed in mice. In cell culture models involving lipid-loaded macrophages, we demonstrated that PHMG promotes foam cell formation by inhibiting lipid efflux in mouse alveolar macrophages. Furthermore, PHMG-induced foam cells were found to promote an increase in the release of TGF-ß1, fibronectin deposition, and collagen remodeling. In vivo interventions were subsequently implemented on mice exposed to PHMG aerosols, aiming to restore macrophage lipid efflux function. Remarkably, this intervention demonstrated the potential to retard the progression of pulmonary fibrosis. In conclusion, this study underscores the pivotal role of macrophage foaming in the pathogenesis of PHMG disinfectants-induced pulmonary fibrosis. Moreover, it provides compelling evidence to suggest that the regulation of macrophage efflux function holds promise for mitigating the progression of pulmonary fibrosis, thereby offering novel insights into the mechanisms underlying inhaled PHMG disinfectants-induced pulmonary fibrosis.

Inhaled tire-wear microplastic particles induced pulmonary fibrotic injury via epithelial cytoskeleton rearrangement.

Tire wear microplastic particles (TWMPs) are emerging microplastic pollutants that have gained increasing attention lately. However, the health effect of inhaled airborne TWMPs has never been explored before and may already be included in particulate matter morbidity and mortality. Here, we endeavored to address the preliminary study of TWMP inhalation-induced pulmonary toxic effects and its epigenetic mechanisms in C57BL/6 mice. As a result, restricted ventilatory dysfunction and fibrotic pathological changes were observed in TWMP-treaded mice. Further research found that attenuation of miR-1a-3p plays an important role in TWMP-induced lung injury. Results from in vitro study confirmed that cytoskeleton regulatory gene twinfilin-1 was one of the target genes of miR-1a-3p, and involved in cytoskeleton rearrangement caused by TWMP exposure. Mechanistically, miR-1a-3p inhibited the F-actin formation by targeting cytoskeletal regulatory proteins twinfilin-1, leading to TWMP-induced pulmonary fibrotic injury. While we are in the very early stages of explaining the role of epigenetics in TWMP-induced lung injury, the potential for the use of epigenetic marks as biomarkers is high and discoveries made in this field will likely bring us closer to better understanding this crucial mechanism.

Circulatory metabolites trigger ex vivo arterial endothelial cell dysfunction in population chronically exposed to diesel exhaust.

BACKGROUND: Chronic exposure to diesel exhaust has a causal link to cardiovascular diseases in various environmental and occupational settings. Arterial endothelial cell function plays an important role in ensuring proper maintenance of cardiovascular homeostasis and the endothelial cell dysfunction by circulatory inflammation is a hallmark in cardiovascular diseases. Acute exposure to diesel exhaust in controlled exposure studies leads to artery endothelial cells dysfunction in previous study, however the effect of chronic exposure remains unknown. RESULTS: We applied an ex vivo endothelial biosensor assay for serum samples from 133 diesel engine testers (DETs) and 126 non-DETs with the aim of identifying evidence of increased risk for cardiovascular diseases. Environmental monitoring suggested that DETs were exposed to high levels of diesel exhaust aerosol (282.3 µg/m3 PM2.5 and 135.2 µg/m3 elemental carbon). Surprisingly, chronic diesel exhaust exposure was associated with a pro-inflammatory phenotype in the ex vivo endothelial cell model, in a dose-dependent manner with CCL5 and VCAM as most affected genes. This dysfunction was not mediated by reduction in circulatory pro-inflammatory factors but significantly associated with a reduction in circulatory metabolites cGMP and an increase in primary DNA damage in leucocyte in a dose-dependent manner, which also explained a large magnitude of association between diesel exhaust exposure and ex vivo endothelial biosensor response. Exogenous cGMP addition experiment further confirmed the induction of ex vivo biosensor gene expressions in endothelial cells treated with physiologically relevant levels of metabolites cGMP. CONCLUSION: Serum-borne bioactivity caused the arterial endothelial cell dysfunction may attribute to the circulatory metabolites based on the ex vivo biosensor assay. The reduced cGMP and increased polycyclic aromatic hydrocarbons metabolites-induced cyto/geno-toxic play important role in the endothelial cell dysfunction of workers chronic exposure to diesel exhaust.

Olfactory bulb microglia activation mediated neuronal death in real-ambient particulate matter exposure mice with depression-like behaviors.

Growing evidence has indicated that air pollution is associated with depression, and damage of olfactory bulb (OB) is regarded as an early marker for depression. However, the toxicity of fine particulate matter (PM2.5) on OB and underlying mechanisms remains to be elucidated. In our study, a real-ambient PM2.5 exposure system was applied to explore the effects of PM2.5 on OB in C57BL/6 mice for 4 or 8 weeks. After 8 weeks exposure, the mice emerged potential depressive-like responses with reduction and disorder of cells in olfactory bulb tissues. Apoptosis and ultra-microstructure analysis indicated that the real-ambient PM2.5 exposure caused the neuronal death of OB. The immunofluorescence observation and KEGG pathway analysis revealed the real-ambient PM2.5 exposure induced microglia activation along with tumor necrosis factor α (TNFα)-mediated signaling enriched in OB of mice with depression-like behaviors. Moreover, results from ex vivo biosensor assay exhibited that PM2.5 might trigger systemic inflammation with increased levels of various proinflammatory factors to activate microglia. Further in vitro co-culture model identified that the PM2.5 evoked microglia cells activation with TNFα secretion and induced neuronal cells apoptosis via classical caspase3 signaling. Our findings provide new insights that PM2.5 induced microglia activation characterized by the release of TNFα to cause neurotoxicity either by direct action or by circulatory inflammation, resulting in OB damage, which may play a critical role in early diagnosis and pathogenic mechanisms for PM2.5 to cause depression.

Human airway organoids as 3D in vitro models for a toxicity assessment of emerging inhaled pollutants: Tire wear particles.

Three-dimensional (3D) structured organoids have become increasingly promising and effective in vitro models, and there is an urgent need for reliable models to assess health effects of inhaled pollutants on the human airway. In our study, we conducted a toxicity assessment of human airway organoids (hAOs) for tire wear particles (TWPs) as an emerging inhaled pollutant. We induced primary human bronchial epithelial cells (HBECs) to generated human airway organoids, which recapitulated the key features of human airway epithelial cells including basal cells, ciliated cells, goblet cells, and club cells. TWPs generated from the wearing of tire treads were considered a major source of emerging inhaled road traffic-derived non-exhaust particles, but their health effect on the lungs is poorly understood. We used human airway organoids to assess the toxicology of tire wear particles on the human airway. In an exposure study, the inhibitory effect of TWPs on the growth of human airway organoids was observed. TWPs induced significant cell apoptosis and oxidative stress in a dose-dependent manner. From the qPCR analysis, TWPs significantly up-regulated the expression pf genes involved in the inflammation response. Additionally, the exposure of TWPs reduced SCGB1A1 gene expression associated with the function of the club cell and KRT5 gene expression related to the function of basal cells. In conclusion, this was first study using human airway organoids for a toxicological assessment of TWPs, and our findings revealed that human airway organoids provide an evaluation model of inhaled pollutants potentially affecting the lungs.

Comparison of the mechanisms of estrogen disrupting effects between triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP).

As the typical aryl-organophosphate flame retardants (OPFRs), triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) were reported to be estrogen disruptors. However, estrogen receptor α (ERα) binding experiments could not explain their biological effects. In this study, their action on ERα, G protein-coupled estrogen receptor (GPER) and the synthesis of 17ß-estradiol (E2) were investigated using in vitro assays and molecular docking. The results showed that TPhP acted as an ERα agonist and recruited steroid receptor co-activator 1 (SRC1) and 3 (SRC3), which was found for the first time. Unlike TPhP, TDCIPP acted as an ERα antagonist. However, both TPhP and TDCIPP activated the estrogen pathway by GPER in SKBR3 cells which were lack of ERα. Although molecular docking results revealed that both TPhP and TDCIPP could dock into ERα and GPER, their substituent groups and combination mode might affect the receptor activation. In addition, by using estrogen biosynthesis assay in H295R cells, both of TPhP and TDCIPP were found to promote E2 synthesis and E2/T ratio involving their different alteration on levels of progesterone, testosterone and estrone, and expression of various key genes. Our data proposed estrogen-disrupting mechanism frameworks of TPhP and TDCIPP. Moreover, our results will contribute to future construction of adverse outcome pathway (AOP) framework of endocrine disruptors.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of immunity in THP-1-derived macrophages and the possible mechanisms.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known immunotoxic environmental pollutant. However, most immunotoxicology studies of TCDD were based on the animal models and the inner mechanisms have just focused on a few genes/proteins. In this study, the immune functions of THP-1-derived macrophages was measured with in-vitro bioassays after 24-h exposure of TCDD including environmentally relevant concentrations. RNA-seq and Weighted Gene Co-expression Network Analysis were used to characterize the immunotoxicity molecular mechanisms. Our study is the first report on the TCDD-induced effects of cell adhesion, morphology, and multiple cytokines/chemokines production on THP-1 macrophages. After TCDD treatment, we observed an inhibited cell adherence, probably attributed to the suppressed mRNA levels of adhesion molecules ICAM-1, VCAM-1 and CD11b, and a decrease in cell pseudopodia and expression of F-actin. The inflammatory cytokines TNF-α, IL-10 and other 8 cytokines/chemokines regulating granulocytes/T cells and angiogenesis were disrupted by TCDD. Alternative splicing event was found to be a sensitive target for TCDD. Using WGCNA, we identified 10 hub genes (TNF, SRC, FGF2, PTGS2, CDH2, GNG11, BDNF, WNT5A, CXCR5 and RUNX2) highly relevant to these observed phenotypes, suggesting AhR less important in the effects TCDD have on THP-1 macrophages than in other cells. Our findings broaden the understanding of TCDD immunotoxicity on macrophages and provide new potential targets for clarifying the molecular mechanisms.

Tricresyl phosphate isomers exert estrogenic effects via G protein-coupled estrogen receptor-mediated pathways.

Tricresyl phosphates (TCPs), as representative aromatic organophosphate flame retardants (OPFRs), have received much attention due to their potential neurotoxicity and endocrine-disrupting effects. However, the role of estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) in their estrogen disrupting effects remains poorly understood. Therefore, in this study, three TCP isomers, tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) and tri-p-cresyl phosphate (TpCP), were examined for their activities on ERα by using two-hybrid yeast assay, and action on GPER by using Boyden chamber assay, cAMP production assay, calcium mobilization assay and molecular docking analysis. The results showed that three TCP isomers were found to act as ERα antagonists. Conversely, they had agonistic activity on GPER to promote GPER-mediated cell migration of MCF7 cells and SKBR3 cells. Both ToCP and TpCP activated GPER-mediated cAMP production and calcium mobilization, whereas TmCP had different mode of action, it only triggered GPER-mediated calcium mobilization, as evidenced by using the specific GPER inhibitor (G15) and GPER overexpressing experiments. Molecular docking further revealed that the way of interaction of TmCP and TpCP with GPER was different from that of ToCP with GPER, and higher activity of ToCP in activating GPER-mediated pathways might be associated with the alkyl substitution at the ortho position of the aromatic ring. Our results, for the first time, found a new target, GPER, for TCPs exerting their estrogen-disrupting effects, and demonstrated complex estrogen-disrupting effects of three TCP isomers involved their opposite activities toward ERα and GPER.

In vitro estrogen-disrupting effects of organophosphate flame retardants.

Organophosphate flame retardants (OPFRs), as substitutes for polybrominated diphenyl ethers (PBDEs), are frequently detected in the environment and biota due to their widespread use. Thus, there is a need to investigate their potential estrogen-disrupting effects and possible mechanisms of action in an effort to obtain a better risk assessment. In this study, we characterized the activities on estrogen receptor α (ERα) and the estrogen-disrupting potential of fourteen OPFRs, TMP, TEP, TPP, TnBP, TiBP, THP, TPhP, TCP, DPK, MDPP, IDPP, CDP, IPPDP and MPhP, using three in vitro assays representing different specific modes of action (MoAs). In the yeast two-hybrid assay, no OPFRs induced agonistic activity, but TiBP, DPK, TPhP, MDPP, CDP and IPPDP were shown to be hydrophobicity-dependent antagonists and to compete with E2 for binding to ERα. In the MVLN cell assay, TPhP was the only OPFR among the 14 tested that was able to activate ERα-estrogen responsive element (ERE) pathways. The results from the E-SCREEN assay showed that all tested OPFRs except TMP had estrogenic properties, and G protein-coupled receptor 30 (GPR30) was involved in the estrogenicity of eight OPFRs, TiBP, THP, TPhP, TCP, MDPP, IPPDP, CDP and MPhP. It was also found that in the E-SCREEN assay, the estrogenicity of alkyl-OPFRs but not aryl-OPFRs was closely correlated to hydrophobicity. Our research suggested that most OPFRs were estrogen disruptors, but their related mechanisms were complex and might involve ERα-mediated and/or ERα-independent pathways. Further in vitro studies concerning the estrogenic effects and involved mechanisms of OPFRs, as well as comprehensive evaluations of OPFRs including health and ecological assessments are needed to determine whether they are safe substitutes for PBDEs.

The steroid receptor coactivator 1 (SRC1) and 3 (SRC3) recruitment as a novel molecular initiating event of 4-n-nonylphenol in estrogen receptor α-mediated pathways.

Recently, the action of steroid receptor coactivators (SRCs) has been recognized to be an important molecular initiating event (MIE) in estrogenic adverse outcome pathways (AOPs). However, the role of SRCs in the molecular mechanisms of many highly concerned environmental estrogens remains poorly understood. In this study, the widely studied environmental estrogen, 4-n-nonylphenol (4-n-NP), was used as a typical pollutant to study SRCs recruitment in its estrogenic effects. In MCF7 cell proliferation (E-SCREEN) assay and MVLN cell assay, 4-n-NP showed significant estrogenic potency that involved an increase in estrogen receptor α (ERα), SRC1 and SRC3 transcript levels. Moreover, 4-n-NP was found to induce estrogen response element (ERE)-mediated activity via ERα in MVLN cells. To investigate the mechanism by which SRCs recruitment is induced by 4-n-NP-ERα, a coactivators recruitment assay was performed, and the results showed that 4-n-NP-ERα recruited both SRC1 and SRC3, whereas it failed to recruit SRC2. Similarly, it had no interaction with SRC2 in the ERα-SRC2 two-hybrid yeast assay. This is the first report to investigate the novel MIE of SRCs recruitment in 4-n-NP-ERα-induced estrogenicity. Overall, our results suggest that the action of 4-n-NP on estrogenic effects involves the following MIEs: the activation of ERα, the recruitment of SRC1 and SRC3, and the induction of ERE-mediated activity. The findings also provide valuable insights into the MIE associated with the different SRCs that are recruited in the adverse outcome pathways of environmental estrogens.

Comparative toxicity study of a novel non-ionic surfactant, vanillin ethoxylates, and nonylphenol ethoxylates in Chinese hamster ovary cells in vitro.

Surfactants such as alkylphenol polyethoxylates (APEOs) and nonylphenol ethoxylates (NPEOs) are commonly used worldwide, but the majority of these compounds, together with their metabolites, have been reported to induce severe biological toxicity. Here, we evaluated for the first time the cytotoxicity, genotoxicity and mitochondrial damage in Chinese hamster ovary (CHO-K1) cells caused by a novel non-ionic surfactant, vanillin ethoxylates (VAEOs), an alternative to APEOs. In parallel, the same in vitro bioassays were conducted on NPEOs along with their metabolic byproducts 4-nonylphenol (4-NP) and vanillin. The results showed that the cytotoxic potency order was NPEOs > 4-NP > VAEOs>vanillin using CCK-8 assays. Also, 4-NP showed potential direct DNA damage in SOS/umu tests, whereas NPEOs, VAEOs and vanillin showed no positive result with and without S9 addition. In addition, none of the test compounds showed obvious genotoxic effects with low olive tail moment value using comet assays. However, all test compounds were shown to cause mitochondrial impairment by increasing mitochondrial mass and decreasing mitochondrial membrane potential in a concentration-dependent manner. And further analysis of reactive oxygen species (ROS) and mitochondrial superoxide (MNSOD) measurement showed that mitochondrial impairment was induced by oxidative stress with intracellular ROS and MNSOD overproduction. It's worth noting that VAEOs and vanillin cause relative lower cytotoxic, genotoxic and mitochondrial damage effects than NPEOs and 4-NP, indicating that VAEOs have the potential to substitute NPEOs as suitable surfactants. Take together, this study elucidates the toxicity profiles of VAEOs and NPEOs relatively comprehensively, and further toxicity analyses are suggested in the population, community and ecosystem.

A comparison of endocrine disruption potential of nonylphenol ethoxylate, vanillin ethoxylate, 4-n-nonylphenol and vanillin in vitro.

The widely used surfactant nonylphenol ethoxylate (NPEO) and its raw material 4-n-nonylphenol (4-n-NP), as well as its degradation products, are recognized as endocrine disrupting chemicals. The USA Environmental Protection Agency (EPA) released an assessment that looked for safe alternatives to NPEO. Vanillin ethoxylate (VAEO) is a novel substitute for NPEO and is quite similar to NPEO in structure; there is a risk that it has similar endocrine disrupting effects to NPEO. However, their effects on various nuclear hormone receptors have not been thoroughly examined. In this study, the effects of NPEO, VAEO, 4-n-NP and Vanillin on the estrogen receptor α (ERα), androgen receptor (AR), thyroid hormone receptor (TR), retinoic X receptor ß (RXRß) and estrogen-related receptor γ (ERRγ) were determined and compared using a battery of recombined yeast strains expressing ß-galactosidase. The results showed that NPEO and 4-n-NP acted as significant antagonists of ER, AR, TR and ERRγ. In addition, 4-n-NP also had antagonistic activity toward RXRß. Moreover, VAEO was shown to be a very weak antagonist of TR and ERRγ, and Vanillin had no interaction with any nuclear receptors. For the first time, it was found that NPEO had AR, TR and ERRγ antagonistic effects and that 4-n-NP was an antagonist of RXRß. The in vitro data indicated that NPEO, 4-n-NP and VAEO have the potential to act as endocrine disruptors involving more than one nuclear hormone receptor, but VAEO has much lower endocrine disrupting potential than NPEO. Thus, it is critical to find safe substitutes for NPEO and a substitute of NPEO with structural analogues should be carried out with caution. Furthermore, to look for preferable alternatives for NPEO, more in vivo and in vitro studies of the alternatives concerning endocrine disruption are needed, especially in vitro studies need to involve various target points, not only focus on their effects on ER but also take other nuclear hormone receptor pathways into consideration.

Prediction of the combined effects of multiple estrogenic chemicals on MCF-7 human breast cancer cells and a preliminary molecular exploration of the estrogenic proliferative effects and related gene expression.

The environmental risks of environmental estrogens (EEs) are often assessed via the same mode of action in the concentration addition (CA) model, neglecting the complex combined mechanisms at the genetic level. In this study, the cell proliferation effects of estrone, 17α-ethinylestradiol, 17ß-estradiol, estriol, diethylstilbestrol, estradiol valerate, bisphenol A, 4-tert-octylphenol and 4-nonylphenol were determined individually using the CCK-8 method, and the proliferation effects of a multicomponent mixture of estrogenic chemicals mixed at equipotent concentrations using a fixed-ratio design were studied using estrogen-sensitive MCF-7 cells. Furthermore, transcription factors related to cell proliferation were analyzed using RT-PCR assays to explore the potential molecular mechanisms related to the estrogenic proliferative effects. The results showed that the estrogenic chemicals act together in an additive mode, and the combined proliferative effects could be predicted more accurately by the response addition model than the CA model with regard to their adverse outcomes. Furthermore, different signaling pathways were involved depending on the different mixtures. The RT-PCR analyses showed that different estrogens have distinct avidities and preferences for different estrogen receptors at the gene level. Furthermore, the results indicated that estrogenic mixtures increased ERα, PIK3CA, GPER, and PTEN levels and reduced Akt1 level to display combined estrogenicity. These findings indicated that the potential combined environmental risks were greater than those found in some specific assessment procedures based on a similar mode of action due to the diversity of environmental pollutions and their multiple unknown modes of action. Thus, more efforts are needed for mode-of-action-driven analyses at the molecular level. Furthermore, to more accurately predict and assess the individual responses in vivo from the cellular effects in vitro, more parameters and correction factors should be taken into consideration in the addition model.

Phenanthrene-Induced Apoptosis and Its Underlying Mechanism.

Phenanthrene (Phe) is one of the most abundant low-molecular-weight polycyclic aromatic hydrocarbons (PAHs). Widespread human and aquatic organism exposure to Phe has been reported, but the toxic effects of Phe and potential mechanisms are unclear. We focused on the chronic hepatotoxicity of Phe in adult Chinese rare minnows (Gobiocypris rarus) and the underlying mechanisms. The chronic effects of exposing Chinese rare minnows to 8.9, 82.3, or 510.0 µg/L Phe for 30 days were examined by histopathological observation, TUNEL assays, caspase activity assays, and gene expression profiles. The liver lesion frequency and hepatocyte apoptosis were increased in Phe-exposed groups. Caspase 9 and caspase 3 enzyme activity in liver tissues was markedly increased. The expression of miR-17/92 cluster members was significantly increased in the 82.3 and 510.0 µg/L groups. Moreover, the response of primary hepatocytes indicated a significant decrease in the mitochondrial membrane potential (MMP) after a 48 h exposure to Phe. Interestingly, miR-18a was significantly decreased in primary hepatocytes in all treatments. Moreover, molecular docking indicated that Phe might have the same binding domain as pri-miR-18a, forming pi-pi and pi-σ interactions with heterogeneous nuclear ribonucleoprotein (hnRNP) A1. Given the above, Phe caused liver lesions and induced hepatocyte apoptosis through the intrinsic apoptosis pathway, and the interaction of Phe with hnRNP A1 contributes to the suppression of miR-18a expression and hepatocyte apoptosis.