Arsenic exposure induced renal fibrosis via regulation of mitochondrial dynamics and the NLRP3-TGF-ß1/SMAD signaling pathway.

Environmental arsenic exposure is one of the major global public health problems. Studies have shown that arsenic exposure can cause renal fibrosis, but the underlying mechanism is still unclear. Integrating the in vivo and in vitro models, this study investigated the potential molecular pathways for arsenic-induced renal fibrosis. In this study, SD rats were treated with 0, 5, 25, 50, and 100 mg/L NaAsO2 for 8 weeks via drinking water, and HK2 cells were treated with different doses of NaAsO2 for 48 h. The in vivo results showed that arsenic content in the rats' kidneys increased as the dose increased. Body weight decreased and kidney coefficient increased at 100 mg/L. As a response to the elevated NaAsO2 dose, inflammatory cell infiltration, renal tubular injury, glomerular atrophy, tubulointerstitial hemorrhage, and fibrosis became more obvious indicated by HE and Masson staining. The kidney transcriptome profiles further supported the protein-protein interactions involved in NaAsO2-induced renal fibrosis. The in vivo results, in together with the in vitro experiments, have revealed that exposure to NaAsO2 disturbed mitochondrial dynamics, promoted mitophagy, activated inflammation and the TGF-ß1/SMAD signaling pathway, and finally resulted in fibrosis. In summary, arsenic exposure contributed to renal fibrosis via regulating the mitochondrial dynamics and the NLRP3-TGF-ß1/SMAD signaling axis. This study presented an adverse outcome pathway for the development of renal fibrosis due to arsenic exposure through drinking water.

Integrative proteomics and metabolomics analysis of non-observable acute effect level PM2.5 induced accumulative effects in AC16 cells.

Chronic exposure to very low ambient PM2.5 has been linked to cardiovascular risks in epidemiological observation, which also brought doubts on its safety threshold. In this study, we approached this question by chronic exposure of AC16 to the non-observable acute effect level (NOAEL) PM2.5 5 µg/mL and its positive reference 50 µg/mL, respectively. The doses were respectively defined on the cell viabilities >95% (p = 0.354) and >90% (p = 0.004) when treated acutely (24 h). To mimic the long-term exposure, AC16 was cultured from the 1st to 30th generations and treated with PM2.5 24 h in every three generations. The integration of proteomic and metabolomic analysis was applied, and 212 proteins and 172 metabolites were significantly altered during the experiments. The NOAEL PM2.5 induced both dose- and time-dependent disruption, which showed the dynamic cellular proteomic response and oxidation accumulation, the main metabolomics changes were ribonucleotide, amino acid, and lipid metabolism that have involved in stressed gene expression, and starving for energy metabolism and lipid oxidation. In summary, these pathways interacted with the monotonically increasing oxidative stress and led to the accumulated damage in AC16 and implied that the safe threshold of PM2.5 may be non-existent when a long-term exposure occurred.

Arsenic exposure caused male infertility indicated by testis and sperm metabolic dysfunction in SD rats.

Arsenic containment is one of the most severe environmental problems. It has been reported that arsenic exposure could cause male reproductive damage. However, the evidence chain from sodium arsenite (NaAsO2) exposure to adverse male fertility outcomes has not been completed by molecular events. In this study, adult male rats were exposed to NaAsO2 for eight weeks via drinking water for verifying their reproductive capacity by checking the phenotypes of testis damage, sperm quality, and female pregnancy rate. H&E staining indicated testicular cells had atrophied, and necrosis was observed under transmission electron microscopy. Sperm viability tended to decrease, and sperm malformation increased. Notably, metabolites in the testes and sperm showed substantial disruption, especially sperm metabolites. The pregnancy rate tests showed that arsenic decreased male rats' reproduction, with some adverse outcomes of the increased numbers of unpregnant females. However, the fetal crown-rump length remained unaltered, indicating that the pregnancy rate was impacted by arsenic exposure but not fetal growth. On arsenic toxicometabolomics analysis, docosahexaenoic acid (DHA) in sperm was the clearest metabolic sign to correlate with the unpregnant rate. In summary, arsenic exposure can cause male infertility via the injured sperm, which results in decreased female pregnancy. The DHA information may imply the dietary intervention for improving sperm quality. Although the fetal growth of the successful pregnancy has not been affected, the changes in epigenetic phenotypes carried by sperms still need to be verified.

Accumulated oxidative stress risk in HUVECs by chronic exposure to non-observable acute effect levels of PM2.5.

Few studies have reported the accumulation of non-observable acute effect (NOAE) of PM2.5, especially exposure to the NOAE doses (NOAEDs) of PM2.5 in chronic way. To address this issue, HUVECs were cultured from the 1st to 30th generations (G1 to G30) and treated by the NOAED PM2.5 once every three passages. The generational changes of oxidative damage markers, inflammatory factors, and cell adhesion molecules (CAMs) were monitored in HUVECs at G6, G12, G18, G24, and G30, and proteomes at G18 and G30, respectively. The oxidative damages monotonically accumulated with exposure time elongation and PM2.5 dose increases. Similar to the oxidative trends, VCAM1 and ICAM1 significantly and dose-dependently increased at G30. However, many inflammatory factors altered with complex patterns to respond the NOAEDs' PM2.5. Proteomic results demonstrated most proteins expressed stably, and the generational proteome alterations were more apparent than the NOAEDs' PM2.5 induced ones. The PM2.5-related proteins varied much, but only few can cross the doses and generations. These observations suggested that the proteins changed holistically rather than individually. In summary, SOD1, SUMO2, and H3F3A may initiate HUVECs responses to PM2.5, and then broadcast and accumulate the NOAE via DNA repair, immune response, and glycolysis.

Joint effect of indoor size-fractioned particulate matters and black carbon on cardiopulmonary function and relevant metabolic mechanism: A panel study among school children.

Indoor particulate matter (PM) and black carbon (BC) are associated with adverse cardiopulmonary effect. However, the cumulative and interactive effects of the mixture of size-fractioned PMs and BC on cardiopulmonary function are not well understood, and the underlying biological mechanisms remain unclear. This repeated-measure study was conducted to assess the joint cardiopulmonary effect and metabolic mechanisms of multiple-size particles and BC among 46 children. PM0.5, PM1, PM2.5, PM5, PM10 and BC were monitored for 5 weekdays. Cardiorespiratory function measurements and urine samples collection were conducted three times. Untargeted-metabolomics and meet-in-metabolite approach were applied to mechanism investigation. Bayesian machine kernel regression was adopted to analyze associations among PMs, cardiopulmonary function and metabolites. Lung function and heart rate variability significantly decreased with the increased PMs and BC co-exposure (p < 0.05). The effective particles were BC, PM1-2.5 and PM0.5 in turn. No interaction effects of different particles on cardiopulmonary function were observed at different lag days. BC-related glucose and fatty acid increase, and PM1-2.5-related branched-chain amino acid degradation were primarily observed. Other metabolisms were successively disturbed. The greatest joint effects of PMs and BC on metabolism were mainly at lag0 and lag01 day. They occurred earlier than the strongest effects on cardiopulmonary function, which were at lag01 and lag02 day. BC, PM1-2.5 and PM0.5 were mainly associated with cardiorespiratory indices by disturbing amino acids, glucose, lipid, isoflavone and purine metabolism. Mitochondrial productivity and antioxidation reduction are pivotal to the relevant metabolic alterations. More attention should be paid to BC and smaller-size PMs to control indoor PM pollution and its adverse effect on children.

Chronic low-level perfluorooctane sulfonate (PFOS) exposure promotes testicular steroidogenesis through enhanced histone acetylation.

Perfluorooctane sulfonate (PFOS), an artificial perfluorinated compound, has been associated with male reproductive disorders. Histone modifications are important epigenetic mediators; however, the impact of PFOS exposure on testicular steroidogenesis through histone modification regulations remains to be elucidated. In this study, we examined the roles of histone modifications in regulating steroid hormone production in male rats chronically exposed to low-level PFOS. The results indicate that PFOS exposure significantly up-regulated the expressions of StAR, CYP11A1 and 3ß-HSD, while CYP17A1 and 17ß-HSD were down-regulated, thus contributing to the elevated progesterone and testosterone levels. Furthermore, PFOS significantly increased the histones H3K9me2, H3K9ac and H3K18ac while reduced H3K9me3 in rat testis. It is known that histone modifications are closely involved in gene transcription. Therefore, to investigate the association between histone modifications and steroidogenic gene regulation, the levels of these histone marks were further measured in steroidogenic gene promoter regions by ChIP. It was found that H3K18ac was augmented in Cyp11a1 promoter, and H3K9ac was increased in Hsd3b after PFOS exposure, which is proposed to result in the activation of CYP11A1 and 3ß-HSD, respectively. To sum up, chronic low-level PFOS exposure activated key steroidogenic gene expression through enhancing histone acetylation (H3K9ac and H3K18ac), ultimately stimulating steroid hormone biosynthesis in rat testis.

Environmental doses of arsenic exposure are associated with increased reproductive-age male urinary hormone excretion and in vitro Leydig cell steroidogenesis.

Humans are ubiquitously exposed to arsenic from multiple sources, and chronic arsenic exposure may be associated with male reproductive health. Although association regarding arsenic exposure and sex hormone secretion in blood has been reported, sex hormone excretion in urine studies is lacking. Urinary sex hormone excretion has emerged as a complementary strategy to evaluate gonadal function. Herein, we determined the associations between environmental exposure to arsenic and urinary sex hormone elimination and in vitro Leydig cell steroidogenesis. Concentrations of arsenic and testosterone (T), estradiol (E2) and progesterone (P) in repeated urine samples were determined among 451 reproductive-age males. Moreover, an in vitro Leydig cell MLTC-1 steroidogenesis experiment was designed to simulate real-world scenarios of low human exposure. Multivariable linear regression models were used to assess the associations of urinary arsenic levels with urinary hormones. Urinary arsenic concentrations were positively associated with urinary sex hormone (T, E2, and P) levels. An in vitro test further demonstrated that a population-based environmental exposure range (0.01-5 µM) of arsenic induced Leydig cell steroidogenesis potency. Our results indicate that low-dose arsenic exposure exhibits an endocrine disrupting effect by stimulating Leydig cell steroidogenesis and accelerating urinary steroid excretion, which extends previous knowledge of the inverse association of high-dose arsenic exposure with sexual steroid production that is assumed to be anti-androgen.

Freeze-Thaw Pretreatment Can Improve Efficiency of Bacterial DNA Extraction From Meconium.

Although the presence of live microbes in utero remains under debate, newborn gastrointestinal bacteria are undoubtedly important to infant health. Measuring bacteria in meconium is an ideal strategy to understand this issue; however, the low efficiency of bacterial DNA extraction from meconium has limited its utilization. This study aims to improve the efficiency of bacterial DNA extraction from meconium, which generally has low levels of microflora but high levels of PCR inhibitors in the viscous matrix. The research was approved by the ethical committee of the Xiamen Maternity and Child Health Care Hospital, Xiamen, China. All the mothers delivered naturally, and their newborns were healthy. Meconium samples passed by the newborns within 24 h were collected. Each sample was scraped off of a sterile diaper, transferred to a 5-ml sterile tube, and stored at -80°C. For the assay, a freeze-thawing sample preparation protocol was designed, in which a meconium-InhibitEX buffer mixture was intentionally frozen 1-3 times at -20°C, -80°C, and (or) in liquid nitrogen. Then, DNA was extracted using a commercial kit and sequenced by 16S rDNA to verify the enhanced bacterial DNA extraction efficiency. Ultimately, we observed the following: (1) About 30 mg lyophilized meconium was the optimal amount for DNA extraction. (2) Freezing treatment for 6 h improved DNA extraction at -20°C. (3) DNA extraction efficiency was significantly higher with the immediate thaw strategy than with gradient thawing at -20°C, -80°C, and in liquid nitrogen. (4) Among the conditions of -20°C, -80°C, and liquid nitrogen, -20°C was the best freezing condition for both improving DNA extraction efficiency and preserving microbial species diversity in meconium, while liquid nitrogen was the worst condition. (5) Three freeze-thaw cycles could markedly enhance DNA extraction efficiency and preserve the species diversity of meconium microflora. We developed a feasible freeze-thaw pretreatment protocol to improve the extraction of microbial DNA from meconium, which may be beneficial for newborn bacterial colonization studies.

Persistence and reversibility of arsenic-induced gut microbiome and metabolome shifts in male rats after 30-days recovery duration.

The metabolites of gut microbiome are important host-health regulating factors and can be interrupted when the host is exposed to environmental pollutant via ingestion route. Arsenic contaminated drinking water is one of the most serious environmental health problems worldwide. Therefore, the arsenic-induced alterations of gut microbiome and metabolome, especially the persistence and reversibility of the alterations after the long-term arsenic exposure will be interesting to know. In this study, we investigated the relationship between gut microbiota and metabolites in male rats both after the 30-days arsenic treatment and 30-days recovery duration. The composition and diversity of gut microbiota were affected significantly by the treatment, but they presented partial improvement in recovery duration. Moreover, arsenic exposure induced the significant changes of 73 metabolites, which involved in the metabolism of glycerophospholipid, linoleic acid, as well as the biosynthesis of phenylalanine, tyrosine and tryptophan. Although it had a persistent effect, the restoration of glycerophospholipid metabolism was observed in the 30-days recovery. Integration analysis further correlated the arsenic impacting microbes with some important differential metabolites. Lactobacillus associated with the decreases of phosphatidylethanolamine(34:1), 16alpha-hydroxydehydroepiandrosterone 3-sulfate, seryltryptophan and alanyltyrosine in recovery duration. Lactobacillus strains have potential to work as protective agents against arsenic toxicity by restoring perturbed glycerophospholipid metabolism. In summary, arsenic significantly disrupted gut microbiome and metabolome, but the disruptions are reversible to some extent after a 30-days recovery.

Cardiorespiratory Effects of Indoor Ozone Exposure Associated with Changes in Metabolic Profiles among Children: A Repeated-Measure Panel Study.

Ozone is one of the major gaseous pollutants associated with short-term adverse cardiopulmonary effects, even at concentrations below the current indoor air quality limits. However, the underlying biological mechanisms of cardiorespiratory changes with exposure to ozone remain unclear. To further explore molecular linkages between indoor ozone exposure and relevant cardiorespiratory effects, a repeated-measure panel study including 46 schoolchildren was conducted and real-time exposure measurements including ozone were performed inside classrooms every weekday during the study period. Repeated health measurements and urine sample collection were conducted in each participant. Ultra-high-performance liquid chromatography/tandem mass spectrometry and meet-in-metabolite approach were used in metabolomics analysis. Methods including mixed-effect models were adopted to identify metabolites associated with ozone exposure or health indices. Nine metabolites were found to be associated with ozone after mixed-effect model analysis, which are mainly involved in amino acid and bile acid metabolism. Boys may have a greater decrease in bile acid and RNA related metabolites. Four of the nine ozone-related metabolites were also associated with cardiorespiratory function indices. Furthermore, 26.67% of the positive association between ozone and heart rate was mediated by cholestane-3,7,12,25-tetrol-3-glucuronide. Exposure to ozone below the current indoor standards was associated with the deteriorated cardiovascular function by disturbing bile acid and endogenous nitric oxide-related oxidation and inflammation, and associated with the exacerbated airway inflammation by reducing GPx-related anti-oxidation. The results provide metabolic evidence of the cardiorespiratory effects of indoor ozone exposure. Indoor ozone pollution should be controlled further, and more attention should be paid to preventing its adverse health effects, especially in children.

Real-world particulate matters induce lung toxicity in rats fed with a high-fat diet: Evidence of histone modifications.

Exposure to ambient particulate matters (PMs) has been associated with a variety of lung diseases, and high-fat diet (HFD) was reported to exacerbate PM-induced lung dysfunction. However, the underlying mechanisms for the combined effects of HFD and PM on lung functions remain poorly unraveled. By performing a comparative proteomic analysis, the current study investigated the global changes of histone post-translational modifications (PTMs) in rat lung exposed to long-term, real-world PMs. In result, after PM exposure the abundance of four individual histone PTMs (1 down-regulated and 3 up-regulated) and six combinatorial PTMs (1 down-regulated and 5 up-regulated) were significantly altered in HFD-fed rats while only one individual PTM was changed in rats with normal diet (ND) feeding. Histones H3K18ac, H4K8ac and H4K12ac were reported to be associated with DNA damage response, and we found that these PTMs were enhanced by PM in HFD-fed rats. Together with the elevated DNA damage levels in rat lungs following PM and HFD co-exposure, we demonstrate that PM exposure combined with HFD could induce lung injury through altering more histone modifications accompanied by DNA damage. Overall, these findings will augment our knowledge of the epigenetic mechanisms for pulmonary toxicity caused by ambient PM and HFD exposure.

Dynamic recovery after acute single fine particulate matter exposure in male mice: Effect on lipid deregulation and cardiovascular alterations.

Many studies have linked airborne fine particulate matter (PM2.5) exposure to cardiovascular diseases. We performed a time-series analysis to investigate whether the disruption of lipid metabolism recovered or lasted after acute PM2.5 exposure in mice. Targeted lipidomic analysis showed that four major plasma membrane phospholipids along with cholesterol esters (CE) were significantly altered on 7th post-exposure day (PED7), and the alteration reached a peak on PED14. On PED21, the phosphatidylcholine (PC) decrease was more marked than on PED14, and its resurgence was indirectly linked to triglyceride (TG) increase. Homocysteine (HCY), lactate dehydrogenase (LDH), and α-hydroxybutyrate dehydrogenase (α-HBDH) levels increased but glucose levels decreased markedly in a dose- and time-dependent manner throughout the experimental period. Network analysis showed that the lasting lipid deregulation on PED21 correlated to myocardial markers and glucose interruption, during which high-density lipoprotein cholesterol (HDL-C) decreased. The present data implied that the constructional membrane lipids were initially interrupted by PM2.5, and the subsequent rehabilitation resulted in the deregulation of storage lipids; the parallel myocardial and glucose effects may be enhanced by the lasting HDL-C lipid deregulation on PED21. These myocardial and lipidomic events were early indicators of cardiovascular risk, resulting from subsequent exposure to and accumulation of PM2.5.

Seminal plasma metabolites mediate the associations of multiple environmental pollutants with semen quality in Chinese men.

Environmental exposure to arsenic, phthalate esters (PAEs) and perfluorinated compounds (PFCs) has been associated with human semen quality. However, the epidemiological "black-box" of these associations remains poorly uncovered. In this study, based on the association analysis between arsenic, PAE and PFC exposure and semen quality parameters (i.e., semen volume, sperm concentration, sperm count, progressive motility, total motility and normal morphology) in a Chinese male population, we explored the seminal plasma metabolic signatures that may mediate the exposure-outcome relations by using the meet-in-metabolite-analysis (MIMA) approach. As a result, a negative association was found between DMA and sperm concentration, whereas MEHP and PFHxS were positively associated with sperm count and concentration, respectively. Metabolomics analysis revealed that sixteen and twenty-two seminal plasma metabolites were related to sperm concentration and count, respectively, and they are mainly involved in fatty acid, lipid and amino acid metabolism. Moreover, it was further indicated that eicosatetraenoate, carnitines and DHA may impact the inverse association between DMA and sperm concentration, while eicosatetraenoate, carnitines, DHA, PGB2 and tocotrienol are possible mediators of the positive association between PFHxS and sperm concentration. As these metabolic biomarkers are relevant to antioxidation and fatty acid ß-oxidation, we suggest that redox balance and energy generation shifts in seminal plasma are involved in the association of human semen quality with environmental DMA and PFHxS exposure.