Here, we report on the occurrence of a novel perfluoroalkyl ether carboxylic acid, ammonium perfluoro-2-[(propoxy)propoxy]-1-propanoate (HFPO-TA), in surface water and common carp (Cyprinus carpio) collected from the Xiaoqing River and in residents residing near a fluoropolymer production plant in Huantai County, China. Compared with the levels upstream of the Xiaoqing River, HFPO-TA concentrations (5200–68500 ng/L) were approximately 120–1600-times higher downstream after receiving fluoropolymer plant effluent from a tributary. The riverine discharge of HFPO-TA was estimated to be 4.6 t/yr, accounting for 22% of total PFAS discharge. In the wild common carp collected downstream from the point source, HFPO-TA was detected in the blood (median: 1510 ng/mL), liver (587 ng/g ww), and muscle (118 ng/g ww). The log BCFblood of HFPO-TA (2.18) was significantly higher than that of PFOA (1.93). Detectable levels of HFPO-TA were also found in the sera of residents (median: 2.93 ng/mL). This is the first report on the environmental occurrence and bioaccumulation of this novel chemical. Our results indicate an emerging usage of HFPO-TA in the fluoropolymer manufacturing industry and raise concerns about the toxicity and potential health risks of HFPO-TA to aquatic organisms and humans.

Perfluorocarboxylic acids (PFCAs) have been widely used in consumer and industrial products, such as food packaging, and found in the blood of both humans and wildlife. Although studies showed a high tendency toward biological accumulation and a variety of toxic effects for PFCAs, the mechanistic aspects of their toxicity remain unknown. In present study, we investigated the dosage-dependent metabonomic and transcriptomic responses of male rats to the exposure to perfluorododecanoic acid (PFDoA) over 110 days. Our NMR-based metabonomics results for both liver tissues and serum demonstrated that PFDoA exposure led to hepatic lipidosis, which was characterized by a severe elevation in hepatic triglycerides and a decline in serum lipoprotein levels. The results from transcriptomic changes induced by PFDoA corroborated these results with changes in gene transcript levels associated with fatty acid homeostasis. These results demonstrate that PFDoA induces hepatic steatosis via perturbations to fatty acid uptake, lipogenesis, and fatty acid oxidation. Several serum metabolites exhibited dose-dependences, providing thorough descriptions of changes induced by PFDoA exposure. These observations yielded novel insights regarding the toxicological mechanism of PFCAs at the systems level.Keywords: hepatotoxicology; metabonomics; perfluorododecanoic acid; systems biology; toxicogenomics;

Perfluorooctanoic acid (PFOA) has been shown to cause hepatotoxicity and other toxicological effects. Though PPARα activation by PFOA in the liver has been well accepted as an important mechanism of PFOA-induced hepatotoxicity, several pieces of evidence have shown that the hepatotoxic effects of PFOA may not be fully explained by PPARα activation. In this study, we observed autophagosome accumulation in mouse livers as well as HepG2 cells after PFOA exposure. Further in vitro study revealed that the accumulation of autophagosomes was not caused by autophagic flux stimulation. In addition, we observed that PFOA exposure affected the proteolytic activity of HepG2 cells while significant dysfunction of lysosomes was not detected. Quantitative proteomic analysis of crude lysosomal fractions from HepG2 cells treated with PFOA revealed that 54 differentially expressed proteins were related to autophagy or vesicular trafficking and fusion. The proteomic results were further validated in the cells in vitro and livers in vivo after PFOA exposure, which implied potential dysfunction at the late stage of autophagy. However, in HepG2 cells, it seemed that further inhibition of autophagy did not significantly alter the effects of PFOA on cell viability. Although these findings demonstrate that PFOA blocked autophagy and disturbed intracellular vesicle fusion in the liver, the changes in autophagy were observed only at high cytotoxic concentrations of PFOA, suggesting that autophagy may not be a primary target or mode of toxicity. Furthermore, since altered liver autophagy was not observed at concentrations of PFOA associated with human exposures, the relevance of these findings must be questioned.

•Endocytosis and blood-testis barrier proteins were changed after PFOA exposure.•Seventeen miRNAs were differentially expressed in testes after PFOA exposure.•MiRNAs might play a role in gene regulation in testes after PFOA exposure.CLTA is a potential target of miR-133b-3p.Perfluorooctanoic acid (PFOA) is an abundant perfluoroalkyl substance widely applied in industrial and consumer products. Among its potential health hazards, testicular toxicity is of major concern. To explore the potential effect of miRNA on post-translational regulation after PFOA exposure, changes in miRNAs were detected via miRNA array. Seventeen miRNAs were differentially expressed (eight upregulated, nine downregulated) in male mouse testes after exposure to 5 mg/kg/d of PFOA for 28 d (> 1.5-fold and P < 0.05 compared with the control). Eight of these miRNAs were further selected for TaqMan qPCR analysis. Proteomic profile analysis indicated that many changed proteins after PFOA treatment, including intersectin 1 (ITSN1), serine protease inhibitor A3K (Serpina3k), and apolipoprotein a1 (APOA1), were involved in endocytosis and blood-testis barrier (BTB) processes. These changes were further verified by immunohistochemical and Western blot analyses. Endocytosis-related genes were selected for qPCR analysis, with many found to be significantly changed after PFOA treatment, including epidermal growth factor receptor pathway substrate 8 (Eps8), Eps15, cortactin, cofilin, espin, vinculin, and zyxin. We further predicted the potential interaction between changed miRNAs and proteins, which indicated that miRNAs might play a role in the post-translational regulation of gene expression after PFOA treatment in mouse testes. Among them, miR-133b-3p/clathrin light chain A (CLTA) was selected and verified in vitro by transfection and luciferase activity assay. Results showed that PFOA exposure affects endocytosis in mouse testes and that CLTA is a potential target of miR-133b-3p.

•Cell viability and intracellular oxidant stress were not sensitive for PFOA exposure.•Transcriptome profile identified 177 changed transcripts after PFOA exposure.•Acot1 was the most sensitive DEG transcript.•Acot1 is a sensitive indicator for PPARα activation after PFOA exposure.Perfluorooctanoic acid (PFOA) is one of the most commonly detected and persistent perfluoroalkyl substances (PFASs) found in the environment. We found that cell viability and intracellular oxidant stress increased in primary rat hepatocytes exposed to PFOA (100 μM PFOA, 24 h), and mitochondrial superoxide increased from 6.25 μM PFOA treatment group. To screen for sensitive indicators in mRNA level, we investigated global transcriptome profile alteration after PFOA exposure using RNA-sequencing (RNA-seq) in primary rat hepatocytes, and identified 177 gene transcripts (158 upregulated, 19 downregulated) as significantly changed after exposure to 100 μM of PFOA for 24 h (fold change ≥ 2, FDR < 0.05). Quantitative PCR (qPCR) and RNA-fluorescence in situ hybridization (RNA-FISH) assays were conducted after PFOA treatment at various doses (0, 0.4, 1.56, 6.25, 25, and 100 μM) and times (6, 12, 18, 24, 48, and 96 h). Acot1 transcripts increased significantly in the 100 μM PFOA group (4500-fold) after 24 h of exposure, and increased remarkably for all time points (24, 48, 72 and 96 h) after exposure to 6.25 μM. Acot1 also responded to lower PFOA doses, with a significant increase found after exposure to 0.4 μM for 96 h. These results imply Acot1 could serve as a sensitive indicator for PPARα activation after PFOA exposure in primary rat hepatocytes.

Phthalates are a large family of ubiquitous environmental pollutants suspected of being endocrine disruptors. Epidemiological studies have associated phthalate metabolites with decreased reproductive parameters and linked phthalate exposure with the level of urinary 5-methyl-2′-deoxycytidine (5mdC, a product of methylated DNA). In this study, adult male mice were exposed to 450 mg di-isobutyl phthalate (DiBP)/(kg·day) via dietary exposure for 28 days. Mono-isobutyl phthalate (MiBP, the urinary metabolite) and reproductive function parameters were determined. The levels of 5mdC and 5-hydroxymethyl-2′-deoxycytidine (5hmdC) were measured in urine to evaluate if their contents were also altered by DiBP exposure in this animal model. Results showed that DiBP exposure led to a significant increase in the urinary 5mdC level and significant decreases in sperm concentration and motility in the epididymis, accompanied with reduced testosterone levels and down-regulation of the P450 cholesterol side-chain cleavage enzyme (P450scc) gene in the mice testes. Our findings indicated that exposure to DiBP increased the urinary 5mdC levels, which supported our recent epidemiological study about the associations of urinary 5mdC with phthalate exposure in the male human population. In addition, DiBP exposure impaired male reproductive function, possibly by disturbing testosterone levels; P450scc might be a major steroidogenic enzyme targeted by DiBP or other phthalates.Download high-res image (109KB)Download full-size image

•Maternal exposure to DiBP decreased offspring serum and testis testosterone levels.•DiBP decreased expression of 3β-HSD and P450scc in PD21 pups and PD80 adults.•Pre- and postnatal DiBP exposure impaired adult sperm concentration and motility.Phthalates are a large family of ubiquitous environmental chemicals suspected of being endocrine disruptors, with exposure to these chemicals during prenatal and postnatal development possibly resulting in reproductive disorders. Di-isobutyl phthalate (DiBP) is widely used in consumer and industrial products, and although its exposure in the general population has increased in recent years, the mechanisms behind DiBP-induced reproductive disorders in male offspring remain unclear. Here, pregnant mice were exposed to 0 or 450 mg/kg bw/day DiBP via diet from gestation day (GD) 0 to GD21. Until postnatal day 21 (PD21), half of the exposed pups were also exposed to DiBP by lactation (TT), while the rest were not (TC). Half of each group were sacrificed on PD21, with the remaining mice fed a normal diet until PD80 (TCC and TTC, respectively). Reproductive toxicological parameters such as relative organ weights and testosterone levels were determined in male offspring on PD21 and PD80 and sperm quality was tested on PD80. Maternal exposure (pregnancy and lactation) led to decreased serum and testis testosterone concentrations, accompanied by decreased expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 family 11 subfamily A member 1 (CYP11A1) in PD21 pups and PD80 adults. Furthermore, the TTC group showed decreased epididymis sperm concentration and motility. Taken together, DiBP exposure in early life (prenatal and postnatal) impaired male reproductive function in later life, possibly by interfering with testosterone levels and CYP11A1, which might be a major steroidogenic enzyme targeted by DiBP or other phthalates.

•The amount of PCP found decreased in the order quail kidney > liver > muscle.•Quail exposed to PCP showed significant increase in CYP450 gene expression.•PCP triggered the antioxidant system in the quail liver.•Transcription factors had preventive effects on PCP caused oxidative damage.Pentachlorophenol (PCP), an extensively used pesticide and biocide, is of critical environmental concern due to its toxicity and recalcitrance to degradation. In this study, the effect of PCP on induction of transcription factors, cytochrome P450 (CYP450) genes, and the antioxidative enzyme system were investigated in the quail liver. A total of 60 (4- to 6-week-old) male quails (Coturnix japonica) were administered 0, 0.05, 0.5, and 5 mg/kg/d PCP orally for 42 d. Following exposure, both absolute and relative liver weights were significantly lower than those of the control. Using gas chromatography-mass spectrometry, PCP accumulation was, from highest to lowest, kidney > liver > muscle for all exposure groups. The expressions of CYP1A5, CYP1B1, CYP2C18, nuclear translocator 1 (ARNT1), and aryl hydrocarbon receptor 1 (AHR1) were induced after PCP treatment, and increases were found in the activities of hepatic superoxide dismutase (SOD) and catalase (CAT), and the content of hepatic malondialdehyde (MDA). In addition, exposure to PCP induced an increase in liver 8-hydroxydeoxyguanosine (8-OHdG) and significantly elevated ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and 7-ethoxycoumarin-O-deethylase (ECOD) activity, but decreased that of glutathione peroxidase (GSH-Px), benzyloxyresorufin O-debenzylase (BROD), pentoxyresorufin O-depentylase (PROD), and erythromycin N-demethylase (END). No significant responses were observed for benzyloxy-trifluoromethyl-coumarin (BFC). The protein level of liver nuclear factor κB (NF-κB) was higher, whereas that of nuclear factor E2-related factor 2 (Nrf2) was lower for exposed quail. These results suggest that PCP affects quail oxidative stress by modulating CYP450 enzymes and nuclear transcription factors.

Per- and polyfluoroalkyl substances (PFASs) may cross the placental barrier and lead to fetal exposure. However, little is known about the factors that influence maternal-fetal transfer of these chemicals. PFAS concentrations were analyzed in 100 paired samples of human maternal sera collected in each trimester and cord sera at delivery; these samples were collected in Wuhan, China, 2014. Linear regression was used to estimate associations of transfer efficiencies with factors. Chlorinated polyfluorinated ether sulfonates (Cl-PFAESs, 6:2 and 8:2) were frequently detected (>99%) in maternal and cord sera. A significant decline in PFAS levels during the three trimesters was observed. A U-shape trend for transfer efficiency with increasing chain length was observed for both carboxylates and sulfonates. Higher transfer efficiencies of PFASs were associated with advancing maternal age, higher education, and lower glomerular filtration rate (GFR). Cord serum albumin was a positive factors for higher transfer efficiency (increased 1.1–4.1% per 1g/L albumin), whereas maternal serum albumin tended to reduce transfer efficiency (decreased 2.4–4.3% per 1g/L albumin). Our results suggest that exposure to Cl-PFAESs may be widespread in China. The transfer efficiencies among different PFASs were structure-dependent. Physiological factors (e.g., GFR and serum albumin) were observed for the first time to play critical roles in PFAS placental transfer.

Due to their structural similarities, 6:2 fluorotelomer sulfonic acid (6:2 FTSA) and 6:2 fluorotelomer carboxylic acid (6:2 FTCA) are often used as alternatives to perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), respectively. With limited health risk data and 6:2 FTSA detection in water and sludge, the toxicity of these chemicals is of growing concern. Here, adult male mice were exposed with 5 mg/kg/day of 6:2 FTCA or 6:2 FTSA for 28 days to investigate their hepatotoxicological effects. In contrast to 6:2 FTCA, 6:2 FTSA was detected at high and very high levels in serum and liver, respectively, demonstrating bioaccumulation potential and slow elimination. Furthermore, 6:2 FTSA induced liver weight increase, inflammation, and necrosis, whereas 6:2 FTCA caused no obvious liver injury, with fewer significantly altered genes detected compared with that of 6:2 FTSA (39 vs. 412). Although PFOA and PFOS commonly activate peroxisome proliferator-activated receptor α (PPARα), 6:2 FTSA induced an increase in PPARγ and related proteins, but not in lipid metabolism-related genes such as PPARα. Our results showed that 6:2 FTCA and 6:2 FTSA exhibited weak and moderate hepatotoxicity, respectively, compared with that reported for legacies PFOA and PFOS.

•6:2 FTCA can induce developmental toxicity in zebrafish embryos.•6:2 FTCA caused cardiac toxicity and reduced heart rate.•6:2 FTCA can affect erythrocyte formation and differentiation.Saturated fluorotelomer carboxylic acids (FTCAs) are intermediates in the degradation of fluorotelomer alcohols (FTOHs) to perfluorinated carboxylic acids (PFCAs). Recent studies have detected FTCAs in precipitation, surface waters, and wildlife, but few studies have focused on their toxicity. In this study, zebrafish embryos were exposed to different concentrations of 6:2 FTCA (0, 4, 8, and 12 mg/L) from 6 to 120 h post-fertilization (hpf) to investigate its developmental toxicity. Results showed that 6:2 FTCA exposure decreased the hatching and survival percentages, reduced the heart rate, and increased the malformation of zebrafish embryos. The median lethal concentration of 6:2 FTCA was 7.33 mg/L at 120 hpf, which was lower than that of perfluorooctanoic acid (PFOA), thus indicating higher toxicity for zebrafish. The most common developmental malformation was pericardial edema, which appeared in the 8 and 12 mg/L 6:2 FTCA-exposed embryos from 60 hpf. Using o-dianisidine staining, we found that the hemoglobin content in embryos was reduced in a concentration-dependent manner after 6:2 FTCA exposure at 72 hpf. Based on quantitative real-time polymerase chain reaction (q-RT-PCR) and whole-mount in situ hybridization, the transcriptional levels of hemoglobin markers (hbae1, hbbe1, and hbae3) were down-regulated at 48 and 72 hpf, even though no observed malformation appeared in zebrafish at 48 hpf. Moreover, 6:2 FTCA exposure decreased the protein level of gata1, a principal early erythrocytic marker, in Tg (gata1:DsRed) transgenic zebrafish at 72 hpf. We analyzed the transcriptional level of other erythrocyte-related genes using q-RT-PCR assay. For heme formation, the transcription of alas2, which encodes the key enzyme for heme biosynthesis, was down-regulated after 6:2 FTCA exposure, whereas the transcription of ho-1, which is related to heme degradation, was up-regulated at 48 and 72 hpf. The transcriptional patterns of gata1 and gata2, which are related to erythroid differentiation, differed. At 48 hpf, the mRNA level of gata2 was significantly increased, whereas that of gata1 exhibited no significant changes in any treatment group. At 72 hpf, the expressions of both were down-regulated in a concentration-dependent manner. Taken together, 6:2 FTCA exposure decreased the erythrocyte number and disrupted erythroid differentiation during zebrafish embryonic development. Our results suggest that 6:2 FTCA can cause developmental toxicity in zebrafish embryos, and that FTCAs exhibit greater toxicity than that of PFCAs.

Perfluorododecanoic acid (PFDoA) is a ubiquitous environmental pollutant known to cause hepatocellular hypertrophy; however, the mechanisms of hepatotoxicity remain poorly understood. In this study, male rats were exposed to 0, 0.05, 0.2 and 0.5 mg/kg/day of PFDoA for 110 days. After two-dimensional differential gel electrophoresis and MALDI-TOF/TOF analysis, 73 differentially expressed proteins involved in lipid metabolism, inflammation, stress response and other functions were successfully identified. Among them, six significantly changed proteins (CTE1, MTE1, HADHA, ECH1, ALDH2 and CPS1) were found to be regulated by peroxisome proliferator-activated receptor alpha (PPARα). The anti-oxidant enzyme activity assays of superoxide dismutase and glutathione peroxidase and the content of thiobarbituric acid-reactive substances in the liver implied that PFDoA caused oxidative stress. The mRNA levels of PPARα in rat primary hepatocytes were knocked down by lentivirus-mediated RNAi. Furthermore, targeted protein levels of CTE1 and MTE1 were down-regulated, while those of HADHA, ALDH2 and CPS1 were up-regulated. After PFDoA exposure, however, the targeted protein levels of CTE1 and ALDH2 increased compared with those of the knockdown untreated group. The reactive oxygen species (ROS) content in rat hepatocytes assayed by flow cytometry significantly increased in the PPARα knockdown groups, consistent with the PPARα antagonist GW6471- and agonist WY14643-treated groups. These results strongly suggested that PPARα played an important role in suppressing ROS content in hepatocytes following PFDoA exposure.

Perfluorooctanoic acid (PFOA) is correlated with male reproductive dysfunction in animals and humans, but the underlying mechanisms for this remain unknown. To explore the potential reproductive toxicity of PFOA, we studied blood–testis barrier (BTB) damage using in vivo and in vitro models. Male mice were gavage-administered PFOA (0–20 mg/kg/d) for 28 consecutive days, and breeding capacity and permeability of the Sertoli cell-based BTB were estimated. Primary Sertoli cells (SCs) were exposed to PFOA (0–500 μM) for 48 h, and transepithelial electrical resistance (TER) was assessed. Furthermore, BTB-associated protein expression, TNFα content, and phosphorylation and protein levels of the mitogen-activated protein kinase (MAPK) pathway were detected. An apparent decrease in mated and pregnant females per male mouse as well as litter weight was observed. Marked BTB damage was evidenced by increased red biotin fluorescence in the lumen tubular of the testes and the decrease in TER in SCs in vitro. The protein levels of claudin-11, connexin-43, N-cadherin, β-catenin, and occludin were significantly decreased in the testes and also in the SCs in vitro except for N-cadherin and β-catenin. TNFα content showed a dose-dependent increase in the testes and a dose- and time-dependent increase in the SCs, with the p-p38/p38 MAPK ratio also increasing in testes and SCs after PFOA exposure. Moreover, PFOA altered expressions of claudin-11, connexin-43, TNFα, and p-p38 MAPK were recovered 48 h after PFOA removal in the SCs. The SCs appeared to be target to PFOA, and the disruption of the BTB may be crucial to PFOA-induced reproductive dysfunction in mice.

Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans.

Perfluoroalkyl chemicals (PFASs) are a class of highly stable man-made compounds, and their toxicological impacts are currently of worldwide concern. Administration of perfluorononanoic acid (PFNA), a perfluorocarboxylic acid (PFCA) with a nine carbon backbone, resulted in dose-dependent hepatomegaly in mice (0, 0.2, 1, and 5 mg/kg body weight, once a day for 14 days) and an increase in hepatic triglycerides (TG) and total cholesterol (TCHO) in the median dose group as well as serum transaminases in the high dose group. Using isobaric tags for relative and absolute quantitation (iTRAQ), we identified 108 (80 up-regulated, 28 down-regulated) and 342 hepatic proteins (179 up-regulated, 163 down-regulated) that exhibited statistically significant changes (at least a 1.2-fold alteration and P < 0.05) in the 1 and 5 mg/kg/d PFNA treatment groups, respectively. Sixty-six proteins (54 up-regulated, 12 down-regulated) significantly changed in both of the two treatment groups. Among these 54 up-regulated proteins, most were proteins related to the lipid metabolism process (31 proteins). The mRNA analysis results further suggested that PFNA exposure not only resulted in a fatty acid oxidation effect but also activated mouse liver genes involved in fatty acid and cholesterol synthesis. Additionally, three (2 down-regulated, 1 up-regulated) and 30 (14 down-regulated, 16 up-regulated) microRNAs (miRNAs) exhibited at least a 2-fold alteration (P < 0.05) in the 1 and 5 mg/kg/d PFNA treatment groups, respectively, Three miRNAs (up-regulated: miR-34a; down-regulated: miR-362-3p and miR-338-3p) significantly changed in both of the two treatment groups. The repression effect of miR-34a on fucosyltransferase 8 (Fut8) and lactate dehydrogenase (Ldha) was confirmed by luciferase activity assay and Western blot analysis. The results implied that PFNA exerted a hepatic effect, at least partially, by miRNAs mediated post-translational protein repression.

Perfluoroalkyl acids are widely used in numerous industrial and commercial applications due to their unique physical and chemical characteristics. Although perfluorooctanoic acid (PFOA) is associated with hepatomegaly through peroxisome proliferator-activated receptor α (PPARα) activation, liver fat accumulation and changes in gene expression related to fatty acid metabolism could still be found in PPARα-null mice exposed to PFOA. To explore the potential effects of PFOA on sterol regulatory element-binding proteins (SREBPs) activity, male mice were dosed with either Milli-Q water or PFOA at doses of 0.08, 0.31, 1.25, 5, and 20 mg/kg/day by gavage for 28 days. Liver total cholesterol concentrations and PFOA contents showed a dose-dependent decrease and increase, respectively. Transcriptional activity of PPARα and SREBPs was significantly enhanced in livers. Protein expression analyzed by Western blotting showed that PFOA exposure stimulated SREBP maturation. Furthermore, proteins blocked SREBP precursor transport, insulin-induced gene 1 (INSIG1) and INSIG2 proteins, as well as a protein-mediated nuclear SREBP proteolysis, F-box and WD-40 domain protein 7, decreased in mouse liver exposed to PFOA. The expression levels of the miR-183-96-182 cluster, which is possibly involved in a regulatory loop intermediated by SREBPs maturation, were also increased in the mouse liver after PFOA exposure. We also observed that PFOA induced lipid content and PPARα in Hepa 1-6 cells after exposure to PFOA for 72 h but SREBPs were not activated in vitro. These results demonstrated that SREBPs were maturated by activating the miR-183-96-182 cluster-SREBP regulatory loop in PFOA-exposed mouse liver.

Perfluorooctanoic acid (PFOA) is a ubiquitous environmental pollutant suspected of being an endocrine disruptor; however, mechanisms of male reproductive disorders induced by PFOA are poorly understood. In this study, male mice were exposed to 0, 0.31, 1.25, 5, and 20 mg PFOA/kg/day by oral gavage for 28 days. PFOA significantly damaged the seminiferous tubules and reduced testosterone and progesterone levels in the testis in a dose-dependent manner. Furthermore, PFOA exposure reduced sperm quality. We identified 93 differentially expressed proteins between the control and the 5 mg/kg/d PFOA treated mice using a quantitative proteomic approach. Among them, insulin like-factor 3 (INSL3) and cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1) as Leydig-cell-specific markers were significantly decreased. We examined in detail the expression patterns of CYP11A1 and associated genes involved in steroidogenesis in the mouse testis. PFOA inhibited the mRNA and protein levels of CYP11A1 and the mRNA levels of 17β-hydroxysteroid dehydrogenase (17β-HSD) in a dose-dependent manner. Moreover, in vitro study showed the reduction in progesterone levels was accompanied by decreased expression of CYP11A1 in cAMP-stimulated mLTC-1 cells. Our findings indicate that PFOA exposure can impair male reproductive function, possibly by disturbing testosterone levels, and CPY11A1 may be a major steroidogenic enzyme targeted by PFOA.

Perfluoroalkyl chemicals (PFCs) are stable man-made compounds with many industrial and commercial uses. Concern has been raised that they may exert deleterious effects, especially on lipid regulation. We aimed to assess exposure to perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and seven other PFCs in occupational workers from a fluorochemical plant and nearby community residents, and to investigate the association between PFOA and serum biomarkers. Serum biomarkers included not only biochemical parameters, such as lipids and enzymes, but also circulating microRNAs (miRNAs). Samples were analyzed by high-pressure liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Circulating miRNA levels were detected by quantitative polymerase chain reaction (PCR). Analyses were conducted by correlation and linear regression. We detected PFOS, PFOA, perfluorohexane sulfonate (PFHxS), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) in all samples. The median levels of serum PFOA and PFOS were 284.34 ng/mL and 34.16 ng/mL in residents and 1635.96 ng/mL and 33.46 ng/mL in occupational participants, respectively. To our knowledge, we found for the first time that PFOA was negatively associated with high-density lipoprotein cholesterol (HDL-C) in workers using linear regression after adjusting for potential confounders. Circulating miR-26b and miR-199a-3p were elevated with serum concentration of PFOA. Although the limitations of small sample size and the cross-sectional nature of the current study constrained causal inferences, the observed associations between PFOA and these serum biomarkers warrant further study.

Perfluorononanoic acid (PFNA), a nine carbon backbone of perfluorinated acids (PFAAs), has wide production applications and is found in environmental matrices as a contaminant. To understand the adverse effects of PFNA, adult male and female zebrafish were exposed to differing PFNA dosages (0, 0.01, 0.1, and 1.0 mg/L) for 180 days using a flow-through exposure system. Results showed body weight, body length, and hepatosomatic index (HSI) decreased in both sexes. The HPLC-MS/MS analysis found that PFNA concentrations were higher in male livers than in female livers with increasing significance in a dose-dependent manner. Total cholesterol levels increased in the livers of both sexes, whereas triglyceride (TG) levels increased in males and decreased in females. With the exception of FABP1b, the transcriptional expression levels of fatty acid binding proteins (FABPs) were up-regulated in males and down-regulated in females. A similar trend between sexes occurred for peroxisome proliferator-activated receptors (PPARs) and Ccaat-enhancer-binding proteins (C/EBPs), which may be the upstream regulatory elements of FABPs. The results indicated that PFNA exposure caused opposite adverse effects on liver TG levels between the sexes in zebrafish possibly due to the opposite expression of FABPs and its upstream genes.

Kupffer cells (KCs) have been demonstrated to play a role in the regulation of intra-hepatic lipid metabolism through the synthesis and secretion of biologically active products. The involvement of KCs in the disturbance of lipid metabolism that induced by perfluorononanoic acid (PFNA), a known agonist of the peroxisome proliferator-activated receptor alpha (PPARα), was investigated in this study. Rats were exposed to PFNA or PFNA combined with gadolinium chloride, an inhibitor of KCs, for 14 days. PFNA exposure dose-dependently increased absolute and relative liver weights, induced triglyceride accumulation, up-regulated the expression of both SERBP-1c and PPARα, and stimulated the release of TNFα and IL-1β. Inactivation of KCs markedly lowered TNFα and IL-1β level, enhanced PFNA-induced expression of PPARα and its target genes, and reduced liver triglyceride levels. In vitro, PFNA-induced expression of PPARα in primary cultured hepatocytes was suppressed by recombinant rat TNFα and IL-1β. However, inhibition of the NF-κB pathway prevented this. Transient transfection and promoter analysis further revealed that these two cytokines and NF-κB were coordinately involved in the suppression of PPARα promoter activity. Our data demonstrate that TNFα and IL-1β released from KCs following PFNA exposure can suppress the expression of PPARα via NF-κB pathway, which partially contribute to the evident accumulation of triglycerides in rat liver.

Concentrations of perfluorononanoate (PFNA) suggest an obvious increase in the environment, wildlife, and humans. However, the potential toxicity of PFNA still remains to be fully elucidated. Our present work is directed toward evaluating specific thyroid endpoints, and studying the long-term and the trans-generational effects of PFNA on zebrafish. Zebrafish (Danio rerio) were exposed to different concentrations of PFNA (0, 0.05, 0.1, 0.5, and 1 mg/l) from their early life stages (F0, 23 day post-fertilization dpf), and the exposure period lasted for 180 days. At the end of the exposure period, thyroid follicle histology and plasma thyroid hormone levels in male zebrafish were evaluated as direct endpoints for the specific thyroid toxicities, while gene expression relative to the hypothalamus–pituitary–thyroid axis was also investigated to study the underlying mechanisms. In addition, offspring embryos (F1) from the PFNA exposure parental zebrafish was reared in water either without PFNA or under continual exposure to PFNA for an additional 180 days to investigate effects of multi-generational exposures on the circulating T3 levels and thyroid-associated gene expression. Our results demonstrate significantly elevated plasma T3 levels were observed in both F0 and F1 adults, as well as PFNA-induced histological changes in the thyroid follicles of F0 male zebrafish. In the liver, the abundance of gene transcript encoding the protein transthyretin (TTR) was significantly induced, while the expression of UDP-glucuronosyltransferases in F0 adult males was inhibited. The induced thyroid-disrupting effects also demonstrated a trans-generational effect that was reflected by altered gene expression related to thyroid hormone (TH) synthesis and metabolism in F1 larvae. Our results provide the first evidence for the thyroid-disrupting effects of long-term PFNA exposure in zebrafish.

Recent reports indicate that decabromodiphenyl ethane (DBDPE) has become widespread in the environment. Yet databases regarding its bioavailability, biotransformation, and possible toxic effects to wildlife and humans are lacking. In this study, we investigated the bioconcentration and biotransformation of DBDPE after oral exposure and compared the results with those of decabrominated diphenyl ether (BDE-209) using rats as a model. Male rats were orally administrated with corn oil containing 100 mg/kg bw/day of DBDPE or BDE-209 for 90 days, after which the levels of DBDPE and BDE-209 in the liver, kidney, and adipose were measured. Biochemical parameters, including thyroid hormone levels, 13 clinical chemistry parameters, and the mRNA expression levels of certain enzymes were also monitored. Results showed DBDPE was found in all tissues with concentrations 3−5 orders of magnitude lower than BDE-209. At least seven unknown compounds were observed in the DBDPE-exposed rats, indicating that DBDPE biotransformation occurred in rats. These compounds were identified by comparing relative retention times and full-scan mass spectra of DBDPE debrominated products from a photolytic degradation experiment using GC/EI-MS and GC/ECNI-MS analysis. The results showed that debromination of DBDPE to lower brominated BDPEs were not the primary metabolic pathway observed in rats. Two of the metabolites were proposed tentatively as MeSO2-nona-BDPE and EtSO2-nona-BDPE using GC/EI-MS, but their structures require further confirmation by other techniques and authentic standards. In addition, evidence of a biological response to DBDPE and BDE-209 and their metabolites in rats are different. To our knowledge, these results are the first indications for the biotransformation of DBDPE in biota. Further studies are necessary to investigate the metabolites of DBDPE and their mechanisms of toxicities to assess the potential risks of DBDPE.

Perfluorinated compounds (PFCs) are used in a variety of industrial applications. We tested the hypothesis that, in Amur tigers (Panthera tigris altaica), captivity in industrialized areas increases PFC levels, potentially presenting a health risk to these animals. Serum samples were collected from 100 tigers from industrialized or nonindustrialized regions in China with nonpoint sources of PFCs. Mean concentrations of PFCs in these samples ranged from 1.57 ± 0.83 ng/mL in nonindustrial Hailin to 4.31 ± 2.90 ng/mL in industrial Beijing. PFC concentrations were significantly higher in tigers from the industrial city of Harbin than those from Hailin (p < 0.05). Perfluorooctanesulfonate (PFOS) was the most abundant PFC in all tigers and increased with age, regardless of industrial/nonindustrial background (p < 0.01). However, PFOS concentrations were 2−4 orders of magnitude less than the current no-observed-effect level. In addition, overall PFC levels in Amur tigers were low compared with various species living in other countries, consistent with the relatively short history of PFC use in China. These results are consistent with the hypothesis that captivity in industrialized areas increases PFC levels in Amur tigers. They also suggest that PFC accumulation will persist, and even increase, with continued use of PFCs in China.

Organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and brominated flame retardants (BFRs) were investigated in captive giant and red panda tissues from China. The total concentrations of OCPs, PCBs, and polybrominated diphenyl ethers (PBDEs) in tissues ranged from 16.3 to 888 ng/g lipid weight (lw), 24.8 to 854 ng/g lw, and 16.4 to 2158 ng/g lw, respectively. p,p’-DDE and β-HCH were major OCP contaminants. PCBs 99, 118, 153/132, 170, 180, and 209 were the major contributing congeners determined. Among PBDEs, congener BDE-209 was the most frequent and abundant, followed by BDE-206, BDE-208, BDE-207, BDE-203, BDE-47, and BDE-153. Decabromodiphenyl ethane (DeBDethane) was detected in 87 and 71% of the giant and red panda samples with concentrations up to 863 ng/g lw, respectively. The remarkable levels and dominance of BDE-209 and DeBDethane may relate to significant production, usage, or disposal of BFRs in China. The positive significant correlation between concentrations of PBDEs and PCBs in captive pandas may suggest that the exposure routes of PBDEs and PCBs to panda are similar. To our knowledge, this is the first report of the occurrence of DeBDethane in captive wildlife samples. Therefore, further studies are warranted to better understand DeBDethane production, transport, uptake, and toxicological effect.

Perfluorooctanoic acid (PFOA) is a ubiquitous contaminant that has been shown to lead to hepatoxicity and is implicated in the incidence of liver tumors in mammals. A number of previous studies have described the toxic effects of PFOA based on conventional toxicological indices and transcriptional data. However, little evidence on protein levels is available. To further our understanding of mechanisms of action and identify the potential protein biomarkers for PFOA exposure, two-dimensional electrophoresis coupled with mass spectrometry has been used to identify proteins differentially expressed in the livers of rare minnow (Gobiocypris rarus) following PFOA exposure of 3, 10, and 30 mg/L. After comparison of the protein profiles from treated and control groups, 34 and 48 protein spots were found altered in abundance (>2-fold) from males and females, respectively. Matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (TOF/TOF) analysis allowed the unambiguous identification of 25 spots, corresponding to 22 different proteins. These proteins were involved in intracellular fatty acid transport, oxidative stress, macromolecule catabolism, the cell cycle, maintenance of intracellular Ca2+ homeostasis, and mitochondrial function. In addition, marked gender differences in response to PFOA have been well-described from the comparison of the male and female protein profiles. Transcriptional analysis of nine mRNAs encoding proteins altered by PFOA in the proteome analysis was determined by real-time PCR. The consistent and discrepant results between mRNA and protein levels suggested that complicated regulatory mechanisms of gene expression were implicated in the response to PFOA exposure.

Recent advances in proteomics have provided an excellent opportunity to understand biological adaptation under complex environmental stress at the protein level. Gaobeidian Lake, located in Beijing, China, is characterized by complex environmental stresses by serving as both the effluent of a wastewater treatment plant and a coolant of a nearby thermal power plant. Liver is the primary organ of energy metabolism and xenobiotic detoxification. To further our understanding of how organisms that live in Gaobeidian Lake acclimatize themselves to these complex environmental stresses, hepatic protein expression patterns were examined in goldfish Carassius auratus that inhabit the lake. Huairou Reservoir, a drinking water source, was used as a reference site. Twenty four protein spots, which were differently expressed in the two sites, were further digested with trypsin and analyzed by matrix-assisted laser desorption/ionization (MALDI) tandem time of flight mass spectrometry (TOF/TOF). The expression of several energy metabolism and oxidative stress proteins, such as glutathione peroxidase (GPx), ferritin H3, and liver basic fatty acid-binding protein (Lb-FABP) were found to be altered in this stressful environment. In addition to the up-regulation of GPx translation, both the mRNA levels and enzymatic activity of GPx protein were elevated in goldfish living in Gaobeidian Lake. The expression of both peroxisome proliferator activated receptor (PPAR), one of the most important metabolism and stress regulation genes as well as cytochrome P450 1A1 (CYP1A1), a detoxification gene, was also detected by real-time PCR at the two sites. Increased expression levels of both PPAR-beta and CYP1A1 (P < 0.1) were observed in Gaobeidian Lake. Our study provides an integrative view of the expression levels of hepatic proteins and genes in goldfish under complex environmental stress that live in Gaobeidian Lake. Our results showed that anthropogenic environmental stresses in Gaobeidian Lake activated the regulation gene of lipid metabolism PPAR, elevated the lipid metabolism levels, and activated the anti-oxidative adaptation mechanism of organisms in the lake.

The toxicological effects of buprofezin, an insect growth regulator, on the fecundity, development, and pest control potential of the wolf spider Pirata piratoides (Schenkel) (Araneae: Lycosidae) were investigated in the laboratory. It was shown that buprofezin had low toxicity to P.piratoides and that the median lethal dosage (LD50) 48 h and 10% lethal dosage (LD10) after topical application for female spiders were 653 and 316 mg buprofezin/mg fresh weight of spider, respectively. Buprofezin significantly reduced the percent hatching of spiders’ eggs but had only a slight effect on egg production. No negative effects on the development and growth were observed. However, spider predation rates were strongly affected: Insecticide-treated females predated on fewer prey than the controls, and their predation rate did not recover even 5 days after insecticide application. This indicated that their pest control potential might be influenced by buprofezin, and the use of buprofezin in biological control of insects is discussed.

Gaobeidian Lake, located in Beijing, China, derives its water mainly from the effluent of the Gaobeidian Wastewater Treatment Plant, which is moderately polluted. Additionally, as this water is used as a coolant in the nearby thermal power plant, the water of this lake has an elevated temperature. To screen differential gene expression in Gaobeidian Lake, suppressive subtractive hybridization (SSH) methodology was performed on RNA in goldfish Carassius auratus hepatic tissues from the lake, using Huairou Reservoir as reference site. A total of 768 candidate clones were selected to perform differential screening. Of these, 264 clones were differentially expressed between the two sites, 124 of which were then subjected to DNA sequencing. Consequently, 36 different genes with known functions were obtained, and some of these differential genes were further confirmed by semiquantitative RT-PCR experiments. Many genes related to detoxification, stress and immune response, and metabolism, such as glutathione S-transferase (GST), cytochrome P450, family 2, subfamily b, polypeptide 10 (CYP2B10), CYP2X10, α-1-antitrypsin, and apolipoprotein A-I (Apo-AI), had higher expression levels in goldfish hepatic tissue from Gaobeidian Lake than those from the reference site. A set of nine genes with known functions were downregulated in Gaobeidian Lake compared to the reference site. The results provided evidence that organisms inhabiting Gaobeidian Lake were suffering a complex stress process and showing metabolism changes and disturbance of homeostasis.

•Levels of urinary 5mdC and 5hmdC were measured in an epidemiological cohort.•Both 5mdC and 5hmdC in urine were positively associated with phthalate exposure.•5mdC was associated with below-reference sperm concentration, motility and morphology.•Sperm concentration was associated with the highest quartile of urinary 5hmdC.5-methyl-2′-deoxycytidine (5mdC) and 5-hydroxymethyl-2′-deoxycytidine (5hmdC), products of DNA methylation and hydroxymethylation processes, have been detected previously in human urine, but their associations with environmental chemicals or healthy outcomes are unclear. The present investigation explored the associations between urinary 5mdC and 5hmdC with phthalate exposure and semen quality. We assessed semen parameters including sperm concentration, motility, and morphology, before measuring urinary 5mdC, 5hmdC and 13 phthalate metabolites among 562 subfertile men from Nanjing, China. Urinary 5mdC and 5hmdC were positively associated with the levels of low molecular weight phthalate metabolites (Low-MWP), high molecular weight phthalate metabolites (High-MWP), and the sum of all phthalate metabolites (ΣPAEs), respectively. Urinary 5mdC was associated with below-reference sperm concentration (odds ratios for increasing quartiles = 1.0, 2.2, 3.0, 2.0; p for trend = 0.02), sperm motility (1.0, 1.1, 1.9, 1.3; p for trend = 0.05), and sperm morphology (1.0, 1.4, 2.3, 1.5; p for trend = 0.05). Sperm concentration was associated with the highest quartile of urinary 5hmdC [odds ratio = 1.9 (95% CI: 1.1, 3.6)]. Our findings showed significant associations between urinary 5mdC and 5hmdC with phthalate metabolites and semen parameters, which suggested urinary 5mdC and 5hmdC may be promising biomarkers in future epidemiological studies.

The accumulation of perfluorinated compounds (PFCs) in the sera of captive wildlife species Bengal tigers (Panthera tigris tigris) and African lions (Panthera leo Linnaeus) from Harbin Wildlife Park, Heilongjiang Province, in China were analyzed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Perfluorooctanesulfonate (PFOS) was the predominant contaminant with a mean serum concentration of 1.18 ng mL−1 in tigers and 2.69 ng mL−1 in lions. Perfluorononanoic acid (PFNA) was the second most prevalent contaminant in both species. The composition profiles of the tested PFCs differed between tigers and lions, and the percentages of perfluorooctanoic acid (PFOA) were greater in lions than in tigers, indicating different exposures and/or metabolic capabilities between the two species. Assessments of the risk of PFC contamination to the two species were obtained by comparing measured concentrations to points of departure or toxicity reference values (TRVs). Results suggest no risk of PFOS exposure or toxicity for the two species.

Pentachlorophenol (PCP), a priority pollutant due to its persistence and high toxicity, has been used worldwide as a pesticide and biocide. To understand the adverse effects of PCP, adult male white-rumped munias (Lonchura striata) were orally administrated commercial PCP mixed with corn oil at dosages of 0, 0.05, 0.5, and 5 mg/(kg·day) for 42 day. Gas chromatography–mass spectrometry (GC–MS) analysis found that PCP was preferentially accumulated in the kidney rather than in the liver and muscle in all exposure groups. To examine the function of CYP1A in pollutant metabolism, we isolated two full-length cDNA fragments (designated as CYP1A4 and CYP1A5) from L. striata liver using reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. PCP induced the expression of CYP1A5, although no obvious change was observed in CYP1A4 expression. Furthermore, PCP significantly elevated the activities of ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase and decreased the activity of benzyloxy-trifluoromethyl-coumarin, with no significant responses observed in benzyloxyresorufin O-debenzylase. PCP induced significant changes in antioxidant enzyme (superoxide dismutase and catalase) activities and malondialdehyde content, but decreased glutathione peroxidase (GSH-Px) and glutathione S-transferase activities and GSH content in the liver of L. striata. The present study demonstrated that PCP had hepatic toxic effects by affecting CYP1As and anti-oxidative status.Download high-res image (162KB)Download full-size image

Control of rodent populations is performed worldwide with coumarin derivatives, such as warfarin. After widespread use, their effect has been diminished by the rapid spread of resistant rodents. Warfarin resistance in Rattus loseas in Jiangmen and Zhanjiang City, Guangdong Province, was investigated by lethal feeding tests. Twenty-three of 30 R. loseas trapped in Jiangmen City were assayed as warfarin-resistant individuals, whereas only 1 of 30 rodents in Zhanjiang was resistant. These results emphasize the need for thorough resistance monitoring as a basis for adequate control measures to prevent the use of ineffective rodenticides in Jiangmen City. The resistance mechanism mainly involves VKORC1, the molecular target for coumarin drugs. VKORC1 mRNA expression in wild-caught resistant animals showed no difference compared with that in susceptible individuals. Mutation screening of VKORC1 was carried out and an Arg58Gly mutation was identified as the prevailing type in R. loseas from Jiangmen City, which may constitute the genetic basis of anticoagulation resistance in R. losea in this resistance region.

Perfluorononanoic acid (PFNA), a synthetic perfluorinated carboxylic acid and fluorosurfactant, is a known environmental contaminant found in people and wildlife. To understand the hepatotoxicity mechanism of PFNA, male zebrafish (n = 200) were exposed to differing concentrations of PFNA (0, 0.1, 0.5, and 1.0 mg/L) for 180 days. A two-dimensional difference gel electrophoresis (2-D DIGE) approach coupled with MALDI–TOF-MS/MS analysis was employed to detect and identify the differential expressed proteins. A total of 57 proteins were successfully identified and categorized into functional classes that included metabolism (amino acid metabolism, TCA cycle and pyruvate metabolism, gluconeogenesis and glycolysis, protein metabolism and modification, and nucleotides metabolism), structure and motility, stress and defense, signal transduction, and cell communication. Our proteomic analyses added new perspective to PFNA hepatotoxicity in zebrafish. Results regarding mRNA levels demonstrated that the involvement of peroxisome proliferator-activated receptors (PPARs) could not sufficiently explain the hepatotoxicity mechanism of PFAAs in zebrafish. The extensive protein variations indicated that multiple cellular pathways were involved in and suggested that multiple protein molecules should be simultaneously targeted as an effective strategy to counter PFNA toxicity. Other potential modes should be further investigated.

Rare minnow (Gobiocypris rarus) is a newly developed aquatic test organism that has been widely used in a range of studies of toxicological risk assessment by virtue of its higher sensitivity to xenobiotics. To describe extensively the transcripts expressed in the livers of adult rare minnow, we generated 6919 high-quality expressed sequence tags (ESTs) from a non-normalized cDNA library. After processing, a total of 1773 unigenes (unique genes) comprising 771 contigs (consensus sequences) and 1002 singlets were acquired. Based on the analysis by BLAST, 1512 unigenes (85%) had been identified and annotated. The result of functional classification reveals that the genes involved in the processes of general metabolism prevail in liver expressed genes. In addition, we compiled a potentially toxicology-related catalog comprising 262 unigenes that associated with metabolism of xenobiotics and adaptive responses. There are eleven groups referring to diverse functions in the catalog. This report provides the first set of genetic data for rare minnow which is of great value for further exploitation of this species in functional genomics and toxicogenomics, and sets a basis for the discovery of new molecular markers of exposure and for the production of the function-focused microarray.

Perfluorononanoic acid (PFNA, C9), a synthetic perfluorinated chemical containing nine carbons, accumulates and is biomagnified through food webs. This compound has been detected in the serum of humans and wildlife and has the potential for reproductive interference. Few studies, however, have reported the effects of PFNA exposure on male reproduction. To determine this, male rats were orally dosed for 1, 3 and 5 mg/kg day PFNA or with vehicle for 14 days. In the present study, serum testosterone levels were decreased, while estradiol levels were increased dramatically in rats receiving 5 mg PFNA/kg day. Spermatogenic cells from rats that received 5 mg PFNA/kg day exhibited apoptotic features including crescent chromatin condensation and chromatin margination. Flow cytometric analysis and TUNEL assays revealed a dose-dependent increase of apoptotic cell numbers. In addition, expression of Fas and Bax mRNA levels were upregulated significantly, and Bcl-2 mRNA levels were downregulated markedly in the 3 and 5 mg/kg day groups. A dose-dependent increase in levels of active caspase-8 and no significant changes of active caspase-9 were observed. Our results indicate that PFNA exposure can lead to cell apoptosis in rat testis, and this apoptosis was probably associated with the Fas death receptor-dependent apoptotic pathway.

Perfluoroalkyl acid (PFAA)-induced apoptosis has been reported in many cell types. However, minimal information on its mode of action is available. This study explored the possible involvement of apoptotic signaling pathways in a nine-carbon-chain length PFAA–perfluorononanoic acid (PFNA)-induced splenocyte apoptosis. After a 14-day exposure to PFNA, rat spleens showed dose-dependent levels of apoptosis. The production of pro-inflammatory and anti-inflammatory cytokines was significantly increased and decreased, respectively. However, protein levels of tumor necrosis factor receptor 1 (TNFR1), fas-associated protein with death domain (FADD), caspase 8 and caspase 3, which are involved in inflammation-related and caspase-dependent apoptosis, were discordant. Peroxisome proliferator-activated receptors alpha (PPARα) and PPARγ genes expression was up-regulated in rats treated with 3 or 5 mg/kg/day of PFNA, and the level of hydrogen peroxide (H2O2) increased concurrently in rats treated with the highest dose. Moreover, superoxide dismutase (SOD) activity and Bcl-2 protein levels were dramatically decreased in spleens after treatment with 3 and 5 mg/kg/day of PFNA. However, protein levels of Bax were unchanged. Apoptosis-inducing factor (AIF), an initiator of caspase-independent apoptosis, was significantly increased in all PFNA-dosed rats. Thus, oxidative stress and the activation of a caspase-independent apoptotic signaling pathway contributed to PFNA-induced apoptosis in rat splenocytes.

Perfluorododecanoic acid (PFDoA), a synthetic perfluorinated chemical, has been detected in environmental matrices, wildlife, and human serum. Its potential health risk for humans and animals has raised public concern. However, the effects of chronic PFDoA exposure on male reproduction remain unknown. The aim of this study was to determine the effects of chronic PFDoA exposure (110 days) on testosterone biosynthesis and the expression of genes related to steroidogenesis in male rats. In this study, we examined the serum levels of sex hormones, growth hormone, and insulin in male rats. Testicular morphology and the expression of key genes and proteins in testosterone biosynthesis were also analyzed. Markedly decreased serum testosterone levels were recorded after 110 days of PFDoA exposure at 0.2 mg PFDoA/kg/day and 0.5 mg PFDoA/kg/day, and cast-off cells were observed in some seminiferous tubules in testes exposed to 0.5 mg PFDoA/kg/day. PFDoA exposure resulted in significantly decreased protein levels of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc), along with significantly reduced mRNA levels of insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR), and interleukin 1α (IL-1α) in rat testes at 0.2 mg/kg/day and 0.5 mg/kg/day. In addition, PFDoA exposure also affected the expression of some genes in the hypothalamo-neurohypophyseal system. However, PFDoA did not affect the expression of 5α-reductase, 3α-hydroxysteroid dehydrogenase, or aromatase in testis and liver. These findings demonstrate that chronic PFDoA exposure disrupts testicular steroidogenesis and expression of related genes in male rats. Multiple factors may be involved in the inhibition of testosterone by PFDoA.

Perfluorononanoic acid (PFNA) is a nine-carbon perfluoroalkyl acid widely used in industrial and domestic products. It is a persistent organic pollutant found in the environment as well as in the tissues of humans and wildlife. There is a concern that this chemical might be a developmental toxicant and teratogen in various ecosystems. In the present study, the toxic effects of PFNA were evaluated in zebrafish (Danio rerio) embryos. One hour post-fertilization embryos were treated with 0, 25, 50, 100, 200, 300, 350, and 400 μmol/L PFNA for 96 hr in 6-well plates. Developmental phenotypes and hatching rates were observed and recorded. Nineteen genes related to oxidative stress and lipid metabolism were examined using Quantitative RT-PCR and confirmed by whole mount in situ hybridization (WISH). Results showed that PFNA delayed the development of zebrafish embryos, reduced the hatching rate, and caused ventricular edema and malformation of the spine. In addition, the amount of reactive oxygen species in the embryo bodies increased significantly after exposure to PFNA compared with that of the control group. The Quantitative RT-PCR and WISH experiments demonstrated that mRNA expression of the lfabp and ucp2 genes increased significantly while that of sod1 and mt-nd1 decreased significantly after PFNA exposure. The mRNA expression levels of gpx1 and mt-atp6 decreased significantly in the high concentration group. However, the mRNA expression levels of both ppara and pparg did not show any significant variation after exposure. These findings suggest that PFNA affected the development of zebrafish embryos at relatively low concentrations.Download full-size image

Perfluorododecanoic acid (PFDoA), a ubiquitous contaminant detected in environmental matrices, wildlife, and human blood, has been shown to produce adverse effects on male reproduction in rats. The mechanism of action of PFDoA in testis, however, is not well understood. In the present study, male rats were orally exposed to PFDoA (0.02, 0.2, and 0.5 mg/kg/day for 110 days), and a two-dimensional gel electrophoresis (2-DE) approach was employed to investigate the alteration of protein expression in the testes. Decreased serum progesterone levels were observed. Matrix-assisted laser desorption/ionization (MALDI) tandem time of flight (TOF/TOF) mass spectrometry analysis allowed the unambiguous identification of 40 differentially expressed proteins. These proteins are mainly involved in mitochondrial respiration, oxidative stress, sperm activity, cytoskeleton and intracellular signal transduction. Furthermore, PFDoA led to decreases in activities of superoxide dismutase (SOD), mitochondrial H-ATPase, and cytochrome c oxidase as well as to an increase in lipid peroxidation in testes. Our results indicated that these proteins, which are involved in mitochondrial respiratory and antioxidative responses, play important roles in the inhibition of testicular steriodogenesis in response to PFDoA. Our data demonstrate that alterations of multiple pathways may be associated with the toxic effects of PFDoA on testes. SOD and H-ATPase subunit d may be sensitive to PFDoA exposure in testis.