Ruina Cui

Sex Differences in Transcriptional Expression of FABPs in Zebrafish Liver after Chronic Perfluorononanoic Acid Exposure

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.

Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50μM PFOA for 48h and 96h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50-100μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200-400μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

Proteomic analysis of cell proliferation in a human hepatic cell line (HL-7702) induced by perfluorooctane sulfonate using iTRAQ.

Perfluorooctane sulfonate (PFOS) is a commonly used and widely distributed perfluorinated compound proven to cause adverse health outcomes. However, how PFOS affects liver cell proliferation is not well understood. In this experiment, we exposed a human liver cell line (HL-7702) to 50 μM PFOS for 48 h and 96 h. We identified 52 differentially expressed proteins using a quantitative proteomic approach. Among them, 27 were associated with cell proliferation, including hepatoma-derived growth factor (Hdgf) and proliferation biomarkers Mk167 (Ki67) and Top2α. Results from MTT, cell counting, and cell cycle analysis showed low-dose PFOS (<200 μM) stimulated HL-7702 cell viability at 48 h and 96 h, reduced the G0/G1 percentage, and increased the S+G2/M percentage. Moreover, levels of Cyclin D1, Cyclin E2, Cyclin A2, Cyclin B1 and their partner Cdks were elevated, and the expression of regulating proteins like c-Myc, p53, p21 waf/cip1 and Myt1, as well as the phosphorylation levels of p-Wee1(S642), p-Chk1(S345) and p-Chk2(T68), were disturbed. We hypothesized that low-dose PFOS stimulated HL-7702 proliferation by driving cells into G1 through elevating cyclins/cdks expression, and by promoting cell cycle progression through altering other regulating proteins. This research will shed light on the mechanisms behind PFOS-mediated human hepatotoxicity.

Phosphoproteome analysis reveals an important role for glycogen synthase kinase-3 in perfluorododecanoic acid-induced rat liver toxicity.

Perfluorododecanoic acid (PFDoA) is a member of the perfluoroalkyl acid (PFAA) family and has broad applications and a wide distribution in the environment. Here, we used TiO(2)-based phosphopeptide enrichment coupled with LC-MS/MS analysis to identify phosphopeptides in rat livers that were influenced by PFDoA treatment. We identified a total of 1443 unique phosphopeptides from among 769 phosphoproteins identified in normal and PFDoA-treated rat livers, 849 unique phosphorylation sites were also identified. Of these sites, 143 were considered to be novel phosphorylation sites. Many phosphoproteins were found to be associated with hepatic injuries and diseases, such as hepatotoxicity, regeneration, fatty liver, neoplasms and carcinoma. Furthermore, 25 of the identified phosphoproteins were found to be related to glycogen synthase kinase-3 (GSK3), either directly or indirectly. Western blot and qPCR results suggested that chronic PFDoA exposure inhibited insulin signal pathways and that inhibition of GSK3 might contribute to the observed increases of lipid levels in the liver.

RNA-sequencing analysis reveals the hepatotoxic mechanism of perfluoroalkyl alternatives, HFPO2 and HFPO4, following exposure in mice.

The toxicological impact of traditional perfluoroalkyl chemicals has led to the elimination and restriction of these substances. However, many novel perfluoroalkyl alternatives remain unregulated and little is known about their potential effects on environmental and human health. Daily administration of two alternative perfluoroalkyl substances, HFPO2 and HFPO4 (1 mg kg−1 body weight), for 28 days resulted in hepatomegaly and hepatic histopathological injury in mice, particularly in the HFPO4 group. We generated and compared high‐throughput RNA‐sequencing data from hepatic tissues in control and treatment group mice to clarify the mechanism of HFPO2 and HFPO4 hepatotoxicity. We identified 146 (101 upregulated, 45 downregulated) and 1295 (716 upregulated, 579 downregulated) hepatic transcripts that exhibited statistically significant changes (fold change ≥2 or ≤0.5, false discovery rate < 0.05) after HFPO2 and HFPO4 treatment, respectively. Among them, 111 (82 upregulated, 29 downregulated) transcripts were changed in both groups, and lipid metabolism associated genes were dominant. Thus, similar to their popular predecessors, HFPO2 and HFPO4 exposure exerted hepatic effects, including hepatomegaly and injury, and altered lipid metabolism gene levels in the liver, though HFPO4 exerted greater hepatotoxicity than HFPO2. The unregulated use of these emerging perfluoroalkyl alternatives may affect environmental and human health, and their biological effects need further exploration. Copyright

Inhibition effects of perfluoroalkyl acids on progesterone production in mLTC-1

Perfluoroalkyl substances (PFASs) are a class of fluorine substituted carboxylic acid, sulfonic acid and alcohol, structurally similar to their corresponding parent compounds. Previous study demonstrated the potential endocrine disruption and reproductive toxicity of perfluorooctane sulfonic acid and perfluorooctanoic acid, two dominant PFASs in animals and humans. We explored the relationship between eleven perfluoroalkyl acids (PFAAs) with different carbon chain length and their ability to inhibit progesterone production in mouse Leydig tumor cells (mLTC-1). We found an obvious dose-response relationship between progesterone inhibition rate and PFAA exposure concentration in mLTC-1. The relative inhibition rate of progesterone by PFAAs was linearly related to the carbon chain length and molar refractivity of PFAAs. Mitochondrial membrane potential (MMP) decreased after PFAA exposure at the half-maximal inhibitory effect concentration (IC50) of progesterone production in mLTC-1, while the reactive oxygen species (ROS) content increased significantly. These results imply that the inhibition effect of PFAAs on progesterone production might be due, in part, to ROS damage and the decrease in MMP in mLTC-1.

Subchronic reproductive effects of 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFAES), an alternative to PFOS, on adult male mice

With a similar structure to perfluorooctane sulfonate (PFOS), 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFAES) has been widely used as a mist suppressant in the chromium plating industry in China since the 1970s. After being disregarded for the past 30 years, 6:2 Cl-PFAES has now been detected in environmental matrices and human sera, suggesting potential health concerns. We carried out a subchronic exposure study to investigate the reproductive toxicity of 6:2 Cl-PFAES exposure (0, 0.04, 0.2, and 1.0 mg/kg/d body weight, 56 d) in adult male BALB/c mice. Results showed that relative epididymis and testis weights decreased in the 1.0 mg/kg/d group compared with the control. However, no changes were observed in the serum levels of testosterone, estradiol, follicle-stimulating hormone (FSH), or luteinizing hormone (LH), nor in the histopathological structure of the epididymis and testis and sperm count. In addition, 56 d of consecutive gavage of 1.0 mg/kg/d of 6:2 Cl-PFAES did not affect male mouse fertility. RNA sequencing showed that no genes were significantly altered in the testes after 6:2 Cl-PFAES exposure. Several testicular genes, which are sensitive to PFOS exposure, were also detected using Western blotting, and included steroidogenic proteins, STAR, CYP11A1, CYP17A1, and 3β-HSD and cell junction proteins, occludin, β-catenin, and connexin 43; however, none were changed after 6:2 Cl-PFAES exposure. Except for a decrease in the relative epididymis and testis weights in the 1.0 mg/kg/d group, 6:2 Cl-PFAES exposure for 56 d exerted no significant effect on the serum levels of reproductive hormones or the testicular mRNA profilesin adult male mice, implying a relative weak reproductive injury potential compared with that of PFOS.

Subchronic Hepatotoxicity Effects of 6:2 Chlorinated Polyfluorinated Ether Sulfonate (6:2 Cl-PFESA), a Novel Perfluorooctanesulfonate (PFOS) Alternative, on Adult Male Mice

The compound 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA), an alternative to perfluorooctanesulfonate (PFOS) in the metal-plating industry, has been widely detected in various environmental matrices. However, its hepatotoxicity has yet to be clarified. Here, male mice were exposed to 0.04, 0.2, or 1 mg/kg/day of 6:2 Cl-PFESA for 56 days. Results demonstrated that relative liver weight increased significantly in the 0.2 and 1 mg/kg/day 6:2 Cl-PFESA groups, whereas liver lipid accumulation increased in all 6:2 Cl-PFESA groups. Serum enzyme activities of alanine transaminase and alkaline phosphatase were increased. Serum triglycerides and low-density lipoprotein cholesterol both increased, whereas serum total cholesterol and high-density lipoprotein cholesterol decreased following 6:2 Cl-PFESA exposure. A total of 264 differentially expressed proteins (127 up-regulated and 137 down-regulated), mainly involved in lipid metabolism, xenobiotic metabolism, and ribosome biogenesis, were identified by quantitative proteomics. Bioinformatics analysis highlighted the de-regulation of PPAR and PXR, which may contribute to the hepatotoxicity of 6:2 Cl-PFESA. Additionally, 6:2 Cl-PFESA induced both cell apoptosis and proliferation in the mouse liver. Compared to the overt toxicity of PFOS, 6:2 Cl-PFESA exhibited more-serious hepatotoxicity. Thus, caution should be exercised in the application of 6:2 Cl-PFESA as a replacement alternative to PFOS in industrial areas.

Acot1 is a sensitive indicator for PPARα activation after perfluorooctanoic acid exposure in primary hepatocytes of Sprague-Dawley rats

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 (100μM PFOA, 24h), and mitochondrial superoxide increased from 6.25μM 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 100μM of PFOA for 24h (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 100μM) and times (6, 12, 18, 24, 48, and 96h). Acot1 transcripts increased significantly in the 100μM PFOA group (4500-fold) after 24h of exposure, and increased remarkably for all time points (24, 48, 72 and 96h) after exposure to 6.25μM. Acot1 also responded to lower PFOA doses, with a significant increase found after exposure to 0.4μM for 96h. These results imply Acot1 could serve as a sensitive indicator for PPARα activation after PFOA exposure in primary rat hepatocytes.