m6A contributes to a pro-survival state in GC-2 cells by facilitating DNA damage repair: Novel perspectives on the mechanism underlying DEHP genotoxicity in male germ cells.

Di(2-ethyl-hexyl) phthalate (DEHP), an environmental endocrine disruptor, can destroy the sperm genomic integrity and impairs spermatogenesis. N6-methyladenosine (m6A) is involved in the cellular effects of DEHP. However, the genotoxic effect of DEHP on spermatocytes and the possible role of m6A in this process remain unclear. This study demonstrated that m6A alleviates DEHP genotoxicity in GC-2 cells. In DEHP-treated mice, DNA double-strand breaks (DSBs) were induced in the testis and spermatocytes. To further explore the molecular mechanism of DEHP genotoxicity on spermatocytes, GC-2 cells were exposed to DEHP. DEHP produced distinct genotoxicity and caused DSBs, which led to the inhibition of DNA synthesis and cell cycle arrest. The DNA damage response (DDR) was initiated to repair the DSBs induced by environmentally relevant levels of DEHP (100 µM and 200 µM). During this process, METTL3 upregulated m6A, which facilitated the DDR via stabilizing the DNA damage repair factors (Rad51 and Xrcc5) mRNA to maintain the pro-survival state. Moreover, Mettl3 knockdown partially inhibited DDR. Interestingly, high-dose DEHP (400 µM and 600 µM) directly induced apoptosis rather than the pro-survival state. Altogether: METTL3-mediated m6A participates in maintaining the pro-survival state by upregulating DDR, providing guidance for mitigating the genotoxicity of environment-related level DEHP exposure.

N6-methyladenosine modification on Hmbox1 is related to telomere dysfunction in DEHP-induced male reproductive injury.

AIMS: Di (2-ethylhexyl) phthalate (DEHP), as an environmental endocrine-disrupting chemical (EDC), can induce male reproductive injury. N6-methyladenosine (m6A) plays a vital role in environmental exposure-induced diseases by regulating gene expression. Therefore, we aim to investigate the role of m6A in DEHP-induced reproductive injury. MAIN METHODS: We established an in vivo model of mice exposed to DEHP to explore the effect of DEHP on reproductive injury and m6A. To further explore the molecular mechanism of DEHP toxicity, we built a model of GC-2 cells exposed to mono-(2-ethylhexyl) phthalate (MEHP) in vitro and further silenced Mettl3 in GC-2cells. Besides, we also conducted MeRIP-qPCR and RIP assays to identify the target genes for m6A modification. KEY FINDINGS: DEHP induced testicular injury and senescence. And telomeres shortening, reduced levels of telomere repeat-binding factor 1 (TRF1), TRF2, protection of telomeres 1 (POT1), and telomerase reverse transcriptase (TERT) can be observed in DEHP-treated testes. MEHP also induced GC-2 cellular senescence and telomere dysfunction. Besides, increased m6A mediated by METTL3 stabilized homeobox containing 1 (Hmbox1) in an m6A-dependent manner in MEHP-exposed GC-2 cells. Mettl3 knockdown led to lower m6A modification and reduced Hmbox1 stability, resulting in further shortening of telomere length. SIGNIFICANCE: our work uncovered that DEHP led to male reproductive injury by telomere dysfunction and m6A modified Hmbox1 contributed to maintaining telomere homeostasis in this process, suggesting that accurate regulation of m6A modification level by drugs has potential value in the treatment of DEHP-induced male reproductive injury.

Nonylphenol inhibited HIF-1alpha regulated aerobic glycolysis and induced ROS mediated apoptosis in rat Sertoli cells.

Nonylphenol (NP) is an endocrine disruptor with reproductive toxicity, which can induce apoptosis of Sertoli cells (SCs). SCs have a high aerobic glycolytic flux to ensure sufficient lactate for germ cells as central energy metabolite, and hypoxia-inducible factors 1alpha (HIF-1α) is a major regulator of glycolysis. This study aimed to investigate whether NP can alter HIF-1α-regulated aerobic glycolysis metabolism and thus induce apoptosis in rat SCs. The results revealed that cell viability, intracellular and extracellular lactate levels, the expression of Hk2, Ldha and Mct4, and the protein levels of HIF-1α, HK2, LDHA and MCT4 were decreased significantly when rat SCs exposed to 20 and 30 µM NP for 24 h. Compared with the 30 µM NP group, the protein levels of HIF-1α, HK2 and LDHA, the expression of Hk2 and Ldha and intracellular lactate levels were increased in 30 µM NP and 125 µM cobalt chloride (CoCl2, inhibitor of HIF-1α proteasome-mediated degradation) co-treated group. Furthermore, the elevation of reactive oxygen species (ROS) and apoptosis induced by 30 µM NP were also reversed. In summary, exposure to NP inhibited the ability of SCs to produce and secrete lactate. Meanwhile, NP exposure could lead to a decrease in HIF-1α thereby inhibiting aerobic glycolysis in rat SCs, disrupting intracellular homeostasis and further inducing ROS-mediated apoptosis. This research is the first to explore the NP toxicity on SCs function with respect to nutrition support to germ cells, and provide new evidence on the inhibition of aerobic glycolysis inducing ROS-mediated apoptosis in SCs.

Steatosis induced by nonylphenol in HepG2 cells and the intervention effect of curcumin.

Non-alcoholic fatty liver disease (NAFLD) has increasingly become a serious public health problem. There is growing evidence that nonylphenol (NP) exposure may cause steatosis, but the underlying mechanism is not fully understood. Curcumin (CUR) improves NAFLD-related lipid metabolism disorders and oxidative stress, but its preventive and therapeutic effects on NP-induced steatosis have not been reported. The objective of this investigation was to determine the capability and potential mechanism of NP to induce steatosis in vitro and the intervention of curcumin. HepG2 cells were treated with 0 µM, 20 µM, 30 µM, 40 µM NP for 24 h. Lipid droplets accumulated significantly in HepG2 cells after NP treatment, and the concentration of triglyceride (TG) and total cholesterol (T-CHO) increased significantly. Simultaneously, lipogenesis gene expression was up-regulated significantly, fatty acid oxidation (FAO) gene expression was significantly down-regulated, and reactive oxygen species (ROS) were overproduced. Meanwhile, the expression of p-AMPK/AMPK in the AMPK/mTOR signaling pathway was significantly down-regulated and the expression of p-mTOR/mTOR was markedly up-regulated. However, blocking ROS production with N-acetyl-L-cysteine (NAC) can reverse these phenomena. In addition, our study found that curcumin effectively ameliorated the effects of NP-induced steatosis. Our study indicates that NP can induce steatosis in HepG2 cells, and may be implicated in inhibiting the ROS-dependent AMPK/mTOR pathway, and that curcumin ameliorates the NAFLD-like changes induced by NP in HepG2 cells.

Toxicokinetics and distribution in female rats after chronic nonylphenol exposure.

Our previous studies have shown that continuous exposure to nonylphenol (NP) may cause female reproductive toxicity even at low doses. To better understand this toxic effect, the aim of this study was to investigate the basic characteristics of the disposal kinetics of NP under a chronic exposure scenario to simulate human exposure. Female rats were exposed to NP at three dose levels (50-, 500-, and 10,000 µg kg-1 bw day-1, low, medium, and high dose, respectively) by gavage daily for 17 weeks. Ultrahigh-performance liquid chromatography-tandem mass spectrometry was used to detect NP in rat sera and tissues. The results suggested that a two extravascular compartment model was found to better match the actual serum metabolic behavior of NP. Compared with the high-dose group, the NP absorption in the low-dose group was relatively efficient, the clearance rate was slower, and the residual amount of NP was greater. NP was found mostly in the uterus, adipose and brain tissues and to a lesser degree, in the liver, kidney, and ovary. The results indicated that the extensive organ distribution may cause corresponding toxicity even at relatively low doses.

Effects of nonylphenol induced oxidative stress on apoptosis and autophagy in rat ovarian granulosa cells.

Nonylphenol (NP) is a kind of environmental endocrine disruptors which is generally recognized to cause female reproductive toxicity, but its basic mechanism has not been fully elucidated. In this study, granulosa cells (GCs) were treated with 0-70 µM NP for 24 h, the cell viability of GCs was reduced significantly, as well as increased cell apoptosis with G2/M arrest. Furthermore, NP significantly induced autophagy and the production of reactive oxygen species (ROS). However, these phenomenons were inhibited by blocking the production of ROS with N-Acetyl-l-cysteine (NAC) administration. Intriguingly, the inhibition of autophagy with 3-Methyladenine (3-MA) could enhance the apoptosis induced by NP. Moreover, the down regulating of p-Akt/Akt, p-mTOR/mTOR and subsequent up-regulation of p-AMPK/AMPK induced by NP can be rescued by pretreatment of NAC. Our findings suggested that NP promotes rat ovarian GCs apoptosis and autophagy simultaneously, which may involve the activation of ROS-dependent Akt/AMPK/mTOR pathway. Whatever, the activation of autophagy is likely to develop a protective mechanism to improve the apoptosis of rat ovarian GCs induced by NP.

Determination of urinary carnitine levels as a potential indicator of uterine fibroids caused by nonylphenol exposure.

Our previous studies have shown that uterine fibroids are associated with nonylphenol (NP) exposure, and the changes of carnitines in critical reproductive tissues and body fluids could be used to indicate the female reproductive toxicity caused by NP exposure. In this work, on the basis of further clarifying the correlation between NP exposure level and uterine fibroids, the possibility of the urinary carnitine levels as a potential indicator of uterine fibroids caused by NP exposure was discussed. The urine samples were collected from 84 female volunteers: the control group of 34 healthy women without gynecological disease and 50 uterine fibroids patients, respectively. Methods were respectively established for the determination of NP and eight carnitines in human urine samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the NP level of uterine fibroids group was significantly higher than that of control group (P = 0.002), indicating that NP exposure was an important environmental factor in the occurrence of uterine fibroids. It was further found that in urine samples of the uterine fibroids group, the levels of L-Carnitine (C0), L-Acetyl-carnitine (C2), L-Octanoyl-carnitine (C8), Tetradecanoyl-carnitine (C14), Oleoyl-carnitine (C18:1) and Linoleoyl-carnitine (C18:2) had obviously increased compared with those in the control group (P < 0.001; < 0.001; < 0.001; = 0.003; < 0.001; = 0.010). The concentrations of L-Hexanoyl-carnitine (C6) and L-Palmitoyl-carnitine (C16) in the uterine fibroids group were also higher than those in the control group, although the difference was not statistically significant (P > 0.05). The results suggested that the changes in urinary carnitine levels might be a potential indicator to help to warn of the risk of uterine fibroids caused by NP exposure at the early stage.

Zinc supplement ameliorates phthalates-induced reproductive toxicity in male rats.

People are inevitably exposed to phthalates (PEs) ubiquitously existing in environment. Our previous studies, simulating the actual situations of people exposure to PEs, have shown that the sub-chronic exposure to low-doses PEs mixture (MIXPs) impaired reproductive function in male rats. Zinc is an important element in maintaining male reproductive functions. However, it is still unknown whether zinc supplement could mitigate PEs-induced male reproductive toxicity or not with sub-chronic low-dose mixture exposure. This study aimed to explore the effect of zinc supplement on the reproductive toxicity caused by sub-chronic MIXPs exposure (160 mg/(kg•body weight)/d, for 90 days) in male rats, and further to reveal the underlying mechanisms. Testosterone (T), FSH and LH in serum, early toxicity indicators in urine, PIWI proteins (PIWIL1 and PIWIL2) expression in testes and pathological examination were performed for toxicity evaluation. Steroidogenic proteins (17ß-HSD, StAR, CYP17A1, P450scc and SRD5A) were measured for mechanisms of exploration. The results indicated that zinc supplement could inhibit the T, LH, FSH level decreases in serum, abolish the effect of 5 early toxicity indicators' levels in urine, restrain the alteration of PIWI proteins expression and improve the constructional injury of testes. These effects might be relevant with the suppressed alteration of the expression of steroidogenic proteins induced by MIXPs in rat testicular cells. This work may offer further insights into reducing health risks of MIXPs exposure.

A nanofiber mat prepared from sulfonated polyaniline for solid-phase extraction of fluoroquinolones from water and biological fluids prior to their quantitation by UPLC-MS/MS.

A bifunctional sulfonated polyaniline nanofiber mat (NFM) was synthesized by oxidative polymerization and by using polyacrylonitrile nanofiber mat (NFM) as the template. The adsorption capacity of the NFM for fluoroquinolones (FQs) is distinctly improved and the adsorption was a spontaneous process. According to theoretical calculations, hydrogen bonding and ion-exchange interaction are the two major kinds of interaction mechanisms between adsorbent and FQs. The adsorption and desorption properties for FQs were systematically evaluated by adsorption isotherms and by thermodynamic and kinetic studies. The results indicate that the NFM is a viable sorbent for FQs because of rapid mass transfer and good adsorption/desorption efficiency. The NFM is re-usable for at least 20 cycles without decline in performance. Following desorption of the FQs with 10% (V/V) formic acid/acetonitrile, they were quantified by UPLC with MS/MS detection. The sorbent was applied to the solid phase extraction of the FQs norfloxacin, ciprofloxacin, ofloxacin, enrofloxacin, danofloxacin, pefloxacin, marbofloxacin, lomefloxacin and difloxacin from water and biological fluids. Figures of merit include (a) low limits of detection (0.5-1.5 ng L-1 for water, 0.016-0.052 µg L-1 for urine and serum), (b) high recoveries from spiked samples (82.3%-109.4%) with low relative standard deviations (1.1%-12.3%); (c) short extraction times (2 min), and (d) low adsorbent dosage (4 mg). Graphical abstractSchematic representation of a bi-functional sulfonated polyaniline nanofiber mat (NFM) for solid phase extraction (SPE) of fluoroquinolones (FQs) in water, urine and serum.

Evaluation of toxicokinetics of nonylphenol in the adult female Sprague-Dawley rats using a physiologically based toxicokinetic model.

The physiologically based toxicokinetic (PBTK) model was firstly developed in female rats to quantitatively evaluate toxicokinetics of nonylphenol (NP). Changes in NP serum concentrations over time of single oral NP administration experiments in Sprague-Dawley rats and literature data were collected to establish and calibrate the PBTK model in the SimBiology framework. The calibrated model predicted the serum and tissue NP concentrations of repeat oral NP administration for model evaluation. NP concentrations in serum and tissues (liver, kidneys, adipose, brain, uterus and ovaries) were quantified using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The model output of the time course data (values are within the standard deviation defined for each data point) indicated proximity of predictions to reality. The coefficients of determination r2 were all greater than 0.9, and the root mean squared error (RMSE) were within 0.177-2.027, which means the model predicted and observed serum NP concentrations were in excellent agreement. The results indicated that the model could contribute to a simplification of the future exposure risk assessments of NP in a more realistic scenario and provided a better understanding of the disposition process of NP in human.

Chronic low-dose exposure of nonylphenol alters energy homeostasis in the reproductive system of female rats.

Nonylphenol (NP) as a confirmed endocrine disrupt chemical that causes reproductive and developmental toxicity. Previous studies focused only on short-term, high-dose exposure in vivo, or in vitro on female reproductive toxicity, which cannot accurately simulate the real human exposure scenario. The present study aims to explore NP toxicity and the underlying mechanisms of chronic low-dose NP exposure (500 µg/kg·bw/day, for 8 weeks) in the reproductive system of female rats. The results indicated that NP exposure caused female reproductive toxicity, including alterations in serum 17ß-estradiol (E2) levels, endometria hyperplasia, altered oogenesis and significant changes in the metabolic profile observed in urine, serum, uterus and ovary. Furthermore, expression of the energy-sensitive proteins carnitine palmitoyltransferase I (CPTI), adenosine 5'-monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma (PPAR-γ) were found to be down-regulated in uterus under NP exposure, which suggested the impaired fatty acid oxidation. Accordingly, a comprehensive metabolomics study in key reproductive tissues and body fluids revealed that 12 metabolites were associated with energy metabolism as potential biomarkers for the evaluation of low toxicity at early stages, with L-carnitines being the most representative ones. The present findings provide evidence that chronic low-dose NP exposure can significantly disrupt energy homeostasis in females, thus offering further insights into NP reproductive toxicity.

Combined effects of simultaneous exposure to six phthalates and emulsifier glycerol monosterate on male reproductive system in rats.

Human beings are inevitably exposed to ubiquitous phthalate esters (PEs), and simultaneously ingesting high quantities of food emulsifiers via daily diet. Glycerin monostearate (GMS) is a widely used food emulsifier. The purposes of this study were to investigate the combined effects between the mixture of six frequently used PEs (MIXPs) and GMS on male rat reproductive system, and further to explore the underlying mechanisms. Male rats were orally administered either sodium carboxymethyl cellulose as controls or MIXPs at three different low-doses with or without GMS (200mg/kg/d) by gavage. The 15-week exposure of MIXPs caused male reproductive toxicity in a dose- and time-dependent manner, including the decrease of serum testosterone and morphological damage of testis. Metabonomics analyses of urine and Western blotting analyses of steroidogenic proteins (StAR, P450scc, CYP17A1, 17ß-HSD and P450arom) indicated that MIXPs exposure down-regulated the expression of steroidogenic proteins, and might alter androgen metabolism. The results also showed that the presence of GMS exacerbated the toxicities of MIXPs to male rat reproductive system. These findings suggest that food emulsifier GMS could enhance the toxic effects of MIXPs on male hormone biosynthesis.

Nanofibers mat-based solid-phase extraction method for the pretreatment of urine samples and its application in the primary study on the disposition of nonylphenol after long-term low-level exposure in rats.

A method was established for the analysis of nonylphenol (NP) in rat urine samples based on a solid-phase extraction (SPE) procedure with an amino functionalized polyacrylonitrile nanofibers mat (NH2-PAN NFSM) as sorbent coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The calibration curves prepared in three different days showed good linearity over a wide range of NP concentrations from 0.1 to 100.0ng/mL. It was remarkable that the proposed NH2-PAN NFsM based SPE method showed superior extraction efficiency with the consumption of only 4mg of sorbent and 500µL of eluent. The eluent without any further concentration was directly analyzed by HPLC-MS/MS. As a result, a simple and effective sample preparation was achieved. In addition, the notable lower detection limit (LOD) of 0.03ng/mL revealed the excellent sensitivity of the proposed method in comparison with that in literatures. The recoveries ranged from 85.0% to 114.8% with the relative standard deviations (RSDs) ranging from 7.5% to 13.7%, which were better than or comparable to those from the published methods, suggesting high accuracy of the proposed method. The proposed method was applied in primary study on the disposition of nonylphenol after long-term low-level exposure in rats, providing information for health risk assessment on the real scenarios of NP exposure. NH2-PAN NFsM shows great potential as a novel SPE sorbent for the analysis of biological samples.