m6A-methylated Lonp1 drives mitochondrial proteostasis stress to induce testicular pyroptosis upon environmental cadmium exposure.

Cadmium (Cd) is a widely distributed typical environmental pollutant and one of the most toxic heavy metals. It is well-known that environmental Cd causes testicular damage by inducing classic types of cell death such as cell apoptosis and necrosis. However, as a new type of cell death, the role and mechanism of pyroptosis in Cd-induced testicular injury remain unclear. In the current study, we used environmental Cd to generate a murine model with testicular injury and AIM2-dependent pyroptosis. Based on the model, we found that increased cytoplasmic mitochondrial DNA (mtDNA), activated mitochondrial proteostasis stress occurred in Cd-exposed testes. We used ethidium bromide to generate mtDNA-deficient testicular germ cells and further confirmed that increased cytoplasmic mtDNA promoted AIM2-dependent pyroptosis in Cd-exposed cells. Uracil-DNA glycosylase UNG1 overexpression indicated that environmental Cd blocked UNG-dependent repairment of damaged mtDNA to drive the process in which mtDNA releases to cytoplasm in the cells. Interestingly, we found that environmental Cd activated mitochondrial proteostasis stress by up-regulating protein expression of LONP1 in testes. Testicular specific LONP1-knockdown significantly reversed Cd-induced UNG1 protein degradation and AIM2-dependent pyroptosis in mouse testes. In addition, environmental Cd significantly enhanced the m6A modification of Lonp1 mRNA and its stability in testicular germ cells. Knockdown of IGF2BP1, a reader of m6A modification, reversed Cd-induced upregulation of LONP1 protein expression and pyroptosis activation in testicular germ cells. Collectively, environmental Cd induces m6A modification of Lonp1 mRNA to activate mitochondrial proteostasis stress, increase cytoplasmic mtDNA content, and trigger AIM2-dependent pyroptosis in mouse testes. These findings suggest that mitochondrial proteostasis stress is a potential target for the prevention of testicular injury.

Environmental cadmium inhibits testicular testosterone synthesis via Parkin-dependent MFN1 degradation.

Low testosterone (T) levels are associated with many common diseases, such as obesity, male infertility, depression, and cardiovascular disease. It is well known that environmental cadmium (Cd) exposure can induce T decline, but the exact mechanism remains unclear. We established a murine model in which Cd exposure induced testicular T decline. Based on the model, we found Cd caused mitochondrial fusion disorder and Parkin mitochondrial translocation in mouse testes. MFN1 overexpression confirmed that MFN1-dependent mitochondrial fusion disorder mediated the Cd-induced T synthesis suppression in Leydig cells. Further data confirmed Cd induced the decrease of MFN1 protein by increasing ubiquitin degradation. Testicular specific Parkin knockdown confirmed Cd induced the ubiquitin-dependent degradation of MFN1 protein through promoting Parkin mitochondrial translocation in mouse testes. Expectedly, testicular specific Parkin knockdown also mitigated testicular T decline. Mito-TEMPO, a targeted inhibitor for mitochondrial reactive oxygen species (mtROS), alleviated Cd-caused Parkin mitochondrial translocation and mitochondrial fusion disorder. As above, Parkin mitochondrial translocation induced mitochondrial fusion disorder and the following T synthesis repression in Cd-exposed Leydig cells. Collectively, our study elucidates a novel mechanism through which Cd induces T decline and provides a new treatment strategy for patients with androgen disorders.

Multigenerational paternal obesity enhances the susceptibility to male subfertility in offspring via Wt1 N6-methyladenosine modification.

There is strong evidence that obesity is a risk factor for poor semen quality. However, the effects of multigenerational paternal obesity on the susceptibility to cadmium (a reproductive toxicant)-induced spermatogenesis disorders in offspring remain unknown. Here, we show that, in mice, spermatogenesis and retinoic acid levels become progressively lower as the number of generations exposed to a high-fat diet increase. Furthermore, exposing several generations of mice to a high fat diet results in a decrease in the expression of Wt1, a transcription factor upstream of the enzymes that synthesize retinoic acid. These effects can be rescued by injecting adeno-associated virus 9-Wt1 into the mouse testes of the offspring. Additionally, multigenerational paternal high-fat diet progressively increases METTL3 and Wt1 N6-methyladenosine levels in the testes of offspring mice. Mechanistically, treating the fathers with STM2457, a METTL3 inhibitor, restores obesity-reduced sperm count, and decreases Wt1 N6-methyladenosine level in the mouse testes of the offspring. A case-controlled study shows that human donors who are overweight or obese exhibit elevated N6-methyladenosine levels in sperm and decreased sperm concentration. Collectively, these results indicate that multigenerational paternal obesity enhances the susceptibility of the offspring to spermatogenesis disorders by increasing METTL3-mediated Wt1 N6-methyladenosine modification.

[Analysis of O3 Pollution Characteristics, Formation Sensitivity, and Transport Impact in Southern Nanjing].

As one of the most important city clusters in China, surface ozone (O3) pollution in the Yangtze River Delta (YRD) Region has become a prominent air quality problem in recent years. Online observations of ambient O3, nitrogen oxides (NOx), and volatile organic compounds (VOCs) were conducted in southern Nanjing from July-September 2020 and April-May 2021. On this basis, the pollution characteristics of O3 were analyzed. The O3-VOCs-NOx sensitivity and the transport influence of its precursors were further discussed using models. The frequency statistics of the daily maximum 8-hour moving average (DMA-8h O3) and hourly concentration (O3-1h) showed that O3 pollution in southern Nanjing was more serious than that in urban areas. Three typical O3 pollution episodes were selected during the whole observation period, which included August 16th-27th, 2020; September 3rd-11th, 2020; and May 17th-25th, 2021. The O3-VOCs-NOx sensitivities in these three pollution episodes were analyzed using the observation-based model (OBM). The results of the relative incremental reactivity (RIR) and empirical kinetics modeling approach (EKMA) showed that the sensitivities of O3 formation in the three pollution episodes were in the NOx-limited regime, the transition regime, and the VOCs-limited regime, respectively. This difference in O3-VOCs-NOx sensitivity reflects that the site may have been influenced by transport. Therefore, the potential source contribution function (PSCF) and the concentration weighted trajectory (CWT) method were further used to analyze the potential source areas of NOx, VOCs, and O3 in these three pollution episodes. The results showed that there were obvious regional transport effects of NOx, VOCs, and O3 in these three pollution episodes. The location of potential sources differed in these three pollution episodes, which were affected by the eastern cities of the Lishui site; the urban area of Nanjing and eastern area of Anhui Province; and the urban area of Nanjing and the middle of the YRD Region, respectively. The impact of transport on O3 and its precursors also indicated the necessity of regional joint prevention and control of O3 pollution in the YRD Region.

[Transport Influence and Potential Sources of PM2.5 Pollution for Nanjing].

In this study, 24-hour backward trajectories of the air mass in Nanjing were calculated by using the HYSPLIT model with the NCEP global reanalysis data from March 2019 to February 2020. The backward trajectories combined with the hourly concentration data of PM2.5 were then utilized in the trajectory clustering analysis and potential pollution source analysis. The results showed that the average concentration of PM2.5 in Nanjing was(36±20) µg·m-3 during the study period, with 17 days exceeding the grade Ⅱ national ambient air quality standards (75 µg·m-3). PM2.5 concentration exhibited clear seasonal variation, with winter (49 µg·m-3)>spring (42 µg·m-3)>autumn (31 µg·m-3)>summer (24 µg·m-3). PM2.5 concentration was significantly positively correlated with surface air pressure but significantly negatively correlated with air temperature, relative humidity, precipitation, and wind speed. Based on the trajectories, seven transport routes were identified in spring, and six routes for the other seasons. The northwest and south-southeast routes in spring, southeast route in autumn, and southwest route in winter were the main pollution transport routes in each season, with the characteristics of short transport distance and slow air mass movement, indicating that local accumulation was one of the main reasons for the high value of PM2.5 in quiet and stable weather. The distance of the northwest route in winter was large, and the PM2.5 concentration was 58 µg·m-3, which was the 2nd highest concentration in all routes, indicating that the cities in the northeast of Anhui had a great transport influence on Nanjing PM2.5. The distribution of PSCF and CWT was relatively consistent, and the main potential source areas were mainly local and adjacent areas of Nanjing, indicating that PM2.5 control is needed to strengthen local control and carry out joint prevention and control with adjacent areas. Winter was most affected by transport, its main potential source area was located at the junction of northwest Nanjing and Chuzhou, and the main source origin was in Chuzhou; therefore joint prevention and control should be expanded to Anhui.

Sperm Rhoa m6A modification mediates intergenerational transmission of paternally acquired hippocampal neuronal senescence and cognitive deficits after combined exposure to environmental cadmium and high-fat diet in mice.

Little is currently known about the effect and mechanism of combined paternal environmental cadmium (Cd) and high-fat diet (HFD) on offspring cognitive ability. Here, using in vivo model, we found that combined paternal environmental Cd and HFD caused hippocampal neuronal senescence and cognitive deficits in offspring. MeRIP-seq revealed m6A level of Rhoa, a regulatory gene of cellular senescence, was significantly increased in combined environmental Cd and HFD-treated paternal sperm. Interestingly, combined paternal environmental Cd and HFD markedly enhanced Rhoa mRNA, its m6A and reader protein IGF2BP1 in offspring hippocampus. STM2457, the inhibitor of m6A modification, markedly mitigated paternal exposure-caused the elevation of hippocampal Rhoa m6A, neuronal senescence and cognitive deficits in offspring. In vitro experiments, Rhoa siR significantly reversed mouse hippocampal neuronal senescence. Igf2bp1 siR obviously reduced the level and stability of Rhoa in aging mouse hippocampal neuronal cells. In conclusion, combined paternal environmental Cd and HFD induce offspring hippocampal neuronal senescence and cognitive deficits by promoting IGF2BP1-mediated Rhoa stabilization in offspring hippocampus via elevating Rhoa m6A in paternal sperm.

Combination of high-fat diet and cadmium impairs testicular spermatogenesis in an m6A-YTHDF2-dependent manner.

Environmental cadmium (Cd) or high-fat diet (HFD) exposure alone are risk factors of male infertility. However, the effect and mechanism of co-exposure to HFD and Cd on sperm quality remain unclear. This study was aimed to explore the combined effects of HFD and Cd on spermatogenesis as well as its m6A-dependent mechanism in vivo and in vitro. As a result, co-exposure of HFD and Cd resulted in a significant decrease in the number of mature testicular seminiferous tubules and epididymis sperm quantity in mice, compared with Cd or HFD exposure alone. Correspondingly, the mRNAs expression of Smc3(spermatocytes marker), Acrv1(round spermatids marker) and Lzumo3(elongated spermatids marker) were downregulated in HFD and Cd group. Furthermore, combined exposure downregulated the expression of meiosis-related proteins (STRA8 and SYCP3), increased the m6A level of Stra8, and upregulated the expression of m6A-related proteins (METTL3 and YTHDF2) in mouse spermatocytes. Mechanistically, the above-mentioned impacts caused by co-exposure were markedly restored by Mettl3 siR and Ythdf2 siR. In addition, RNA stability assay showed that Ythdf2 siR obviously reversed co-exposure-increased Stra8 mRNA degradation rate in actinomycin-D-treated mouse spermatocytes. Meanwhile, excess ROS was observed in combined-exposure group, and a free radical scavenger N-tert-Butyl-α-phenylnitrone (PBN) attenuated co-exposure-upregulated expression of METTL3 and YTHDF2 in mouse spermatocytes. These results suggested that combination of HFD and Cd impaired spermatogenesis by degrading Stra8 in an m6A-YTHDF2-dependent manner via ROS activation.

[Scenario Simulation Study Constrained by the Ambient Air Quality Standards in Nanjing].

With the constraint that all six major pollutants in Nanjing must meet the air quality standards by 2030, on the basis of the 2015 emission inventory, the CMAQ air quality model was used to conduct PM2.5 sensitivity tests, and scenario analysis was used to predict the emission inventory and the air quality of four emission reduction scenarios were simulated. Finally, the total control index under the constraint of meeting the standards was obtained. The results show that primary particulate matter (PPM) reduction is the most effective at reducing the concentration of PM2.5 in the atmosphere, on the basis of emission reduction in surrounding areas, PPM emission reduction accounts for 88% of the total reduction of the annual average concentration of PM2.5, followed by NH3, NOx, SO2, and VOCs, which contribute to 10.3%, 5.5%, 3.2%, and 0.5%, respectively. Compared to 2015, the reduction ratios of the major pollutants are between 22% and 53%. Controlling the activity level is more effective for SO2, NH3 and CO emissions reduction, while there is still more opportunity for NOx and VOCs end treatment. When the emissions of SO2, NOx, PM10, PM2.5, BC, OC, CO, VOCs, and NH3 are controlled to 2.43×104, 8.47×104, 9.42×104, 3.74×104, 0.19×104, 0.30×104, 26.56×104, 13.08×104, and 1.50×104 t, respectively, it is expected that the levels of the six pollutants in Nanjing can meet the national ambient air quality level 2 standards.

Maternal transfer, distribution, and metabolism of BDE-47 and its related hydroxylated, methoxylated analogs in zebrafish (Danio rerio).

OH-PBDEs have been reported to be more potent than the postulated precursor PBDEs or corresponding MeO-PBDEs. However, there are contradictory reports for transformation of these compounds in organisms, particularly, for biotransformation of OH-PBDEs and MeO-PBDEs, only one study reported transformation of 6-OH-BDE-47 and 6-MeO-BDE-47 in Japanese medaka. In present study zebrafish (Danio rerio) were exposed to BDE-47, 6-OH-BDE-47, 6-MeO-BDE-47, 2'-OH-BDE-28 and 2'-MeO-BDE-28 in the diet for 20 d. Concentrations of each exposed compound were measured in eggs collected on days 0, 5, 10, 15 or 20. After 20 d exposure, concentrations of precursor and biotransformation products in liver and liver-free residual carcass were measured by use of GC-MS/MS. Total mass of the five compounds in bodies of adults were: 2'-MeO-BDE-28 ∼ 6-MeO-BDE-47>BDE-47>2'-OH-BDE-28>6-OH-BDE-47. MeO-PBDEs were also accumulated more into parental fish body than in liver, while OH-PBDEs accumulated in liver more than in liver-free residual carcass. Concentrations in liver of males were greater than those of females. This result suggests sex-related differences in accumulation. Ratios between concentration in eggs and liver (E/L) were: 2.9, 1.7, 0.8, 0.4 and 0.1 for 6-MeO-BDE-47, BDE-47, 6-OH-BDE-47, 2'-MeO-BDE-28 and 2'-OH-BDE-28, respectively. This result suggests transfer from adult females to eggs. BDE-47 was not transformed into OH-PBDEs or MeO-PBDEs. Inter-conversions of 6-OH-BDE-47 and 6-MeO-BDE-47, 2'-OH-BDE-28 and 2'-MeO-BDE-28 were observed, with metabolite/precursor concentration ratios for 6-OH-BDE-47, 6-MeO-BDE-47, 2'-OH-BDE-28 and 2'-MeO-BDE-28 being 3.8%, 14.6%, 2.9% and 76.0%, respectively. Congener-specific differences were observed in distributions between liver and carcass, maternal transfer and transformation. The two MeO-PBDEs were accumulated into adults, transferred to eggs, and were transformed to the structural similar OH-PBDEs, which might be more toxic. BDE-47 was accumulated into adults and transferred from females to eggs, but not transformed to MeO-PBDEs and/or OH-PBDEs. Accumulation of OH-PBDEs into adults as well as rates of transformation of OH-PBDEs to MeO-PBDEs were all several orders of magnitude less. Thus, MeO-PBDEs are likely to present more of a risk in the environment.

Effects of perfluorinated compounds on development of zebrafish embryos.

Perfluorinated compounds (PFCs) have been widely used in industrial and consumer products and frequently detected in many environmental media. Potential reproductive effects of perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) have been reported in mice, rats and water birds. PFOS and PFOA were also confirmed developing toxicants towards zebrafish embryos; however, the reported effect concentrations were contradictory. Polyfluorinated alkylated phosphate ester surfactants (including FC807) are precursor of PFOS and PFOA; however, there is no published information about the effects of FC807 and PFNA on zebrafish embryos. Therefore, this study was conducted to determine the effects of these four PFCs on zebrafish embryos. Normal fertilized zebrafish embryos were selected to be exposed to several concentrations of PFOA, PFNA, PFOS or FC807 in 24-well cell culture plates. A digital camera was used to image morphological anomalies of embryos with a stereomicroscope. Embryos were observed through matching up to 96-h post-fertilization (hpf) and rates of survival and abnormalities recorded. PFCs caused lethality in a concentration-dependent manner with potential toxicity in the order of PFOS > FC807 > PFNA > PFOA based on 72-h LC(50). Forty-eight-hour post-fertilization pericardial edema and 72- or 96-hpf spine crooked malformation were all observed. PFOA, PFNA, PFOS and FC807 all caused structural abnormalities using early stages of development of zebrafish. The PFCs all retarded the development of zebrafish embryos. The toxicity of the PFCs was related to the length of the PFC chain and functional groups.