Article pubs.acs.org/est
Mutagenic Effects of Perfluorooctanesulfonic Acid in gpt Delta Transgenic System Are Mediated by Hydrogen Peroxide Yichen Wang,† Xuefeng Zhang,‡ Meimei Wang,† Yiyi Cao,§ Xinan Wang,† Yun Liu,† Juan Wang,† Jing Wang,‡ Lijun Wu,† Tom K. Hei,∥ Yang Luan,*,§ and An Xu*,† †
Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences and Anhui Province, Hefei, Anhui 230031, P. R. China ‡ Jiangsu Tripod Preclinical Research Laboratories, Pukou Economic Development Zone, 9# Xinglong Road, Nanjing, China § Hongqiao International Institute of Medicine, Shanghai Tongren Hospital and Faculty of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P. R. China ∥ Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York City, New York 10027, United States ABSTRACT: Perfluorooctane sulfate (PFOS), a persistent organic pollutant, has recently been closely linked with an increased risk of tumorigenesis. However, PFOS has yielded negative results in various tests of genotoxicity. The present study aimed to investigate the mutagenic response to PFOS in the gpt delta transgenic mouse mutation system and to illustrate the contribution of hydrogen peroxide (H2O2) to PFOS genotoxicity. Mutations at the redBA/gam loci were determined by Spi− assay both in vitro and in vivo. DNA damage was measured by phosphorylated histone H2AX (γ-H2AX) and mouse bone marrow micronucleus (MN) testing. Our data showed that PFOS induced concentration-dependent increases in γ-H2AX foci and in mutation frequencies at redBA/gam loci in transgenic mouse embryonic fibroblast cells, which were confirmed by the formation of MNs in the bone marrow and the observations of mutation induction in the livers of gpt delta transgenic mice. Concurrent treatment with catalase, an efficient H2O2 scavenger, significantly decreased the formation of γ-H2AX foci and the mutation yields induced by PFOS. In addition, the generation of H2O2 was found to be closely related to the abnormal peroxisomal β-oxidation caused by PFOS. These finding might provide new mechanistical information about genotoxic effects of PFOS.
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INTRODUCTION
Epidemiological studies of populations with occupational and nonoccupational exposure to PFOS have shown discrepancies regarding the association between exposure to this compound and an increased risk of cancer in humans. A few positive observations have indicated that PFOS exposure is closely related to the incidence of cancers of the prostate, kidney, testis, and thyroid.8 Chronic exposure to PFOS and its carboxylic acid analog PFOA has been reported to increase the potential risk of tumorigenesis in rodents.9−11 Chromosomal aberrations and genome instability are thought to be among the earliest cellular responses caused by environmental carcinogens, which ultimately lead to an increased risk of carcinogenesis initiation and progression. However, the effects of PFOS have been examined using various genotoxicity tests and the results have shown that it does not directly induce DNA damage.12,13 The
Perfluoroalkyl chemicals (PFCs) are man-made, organofluorine compounds that have been used worldwide in the manufacture of surfactants, paints, fabrics, and waterproofing products over the past several decades. Perfluorooctane sulfate (PFOS) and perfluooctanoic acid (PFOA) are two of the main PFC-based end products generated by biological metabolism and environmental degradation.1 Due to its prolonged persistence in the environment and its bioaccumulation through the food chain, PFOS has been listed as a persistent organic pollutant by the Stockholm Convention.2,3 National Health and Nutrition Examination Survey (NHANES), which showed that the serum concentrations of PFOS ranged from 10 μM (p < 0.05). The mutation frequencies in cells treated with 20 μM PFOS were 3.2-fold higher than background. These results indicated that PFOS was mutagenic in mammalian cells. Effect of H2O2 Scavenger on DSBs and Mutation. Due to its structural similarity with endogenous octanoic acid, exposure to PFOS has been reported to result in the upregulation of the enzymes of peroxisomal fatty acid β-oxidation, which might stimulate the generation of H2O2 as a byproduct.28 To detect intercellular ROS induced by PFOS, MEF cells were treated with CM-H2DCFDA, which diffused into the cells and was oxidized by ROS to a fluorescent form. As shown in Figure 3A, the florescence level in cells exposed to 20 μM PFOS was significantly higher than that of the controls, indicating that PFOS stimulated intercellular oxidative stress. To examine the
Figure 2. Mutagenic potential of PFOS at redBA and gam loci in transgenic MEF cells by Spi− mutation assay. Cells were treated with 0, 1, 5, 10, and 20 μM PFOS for 24 h. Error bars indicate ± standard deviation. *p < 0.05, compared with untreated controls.
role of H2O2 induced by PFOS in PFOS-induced cytotoxicity and genotoxicity, MEF cells were treated with cell permeable catalase (catalase-PEG), which was able to catalyze the decomposition of H2O2 and protect cells from oxidative damage. We had observed that the cellular viability was dramatically decreased to 26.8% in the presence of 25 μM PFOS; this could be rescued to 59.7% by concurrent treatment with 800 U/mL catalase (Figure 3B). Similarly, in the presence of catalase, the percentages of DSB positive cells following exposure to 20 μM PFOS were decreased from 33.5 to 16.5%, as shown in Figure 3C. The mutation yields induced by 20 μM PFOS were significantly suppressed from 18.06 ± 3.35 to 11.03 ± 1.05 per 106 recovered plaques with coexposure of PFOS and catalase (Figure 3D). These results demonstrated that PFOSinduced cellular genotoxicity was mediated by the induction of H2O2. Generation of H2O2 through Abnormal Peroxisomal β-Oxidation Induced by PFOS. Peroxisomes play an important role in fatty acids β-oxidation in mammalian cells, which might result in the generation of H2O2 as a reaction byproduct. The process of peroxisomal β-oxidation H2O2 production is catalyzed by acyl-CoA oxidase (Acox1). We found that the mRNA expression of Acox1 was slightly increased by 1 μM PFOS as compared with that of the control group; however, 20 μM PFOS stimulated a 1.3- and 1.5-fold increase of Acox1 gene expression at 12 and 24 h, respectively (Figure 4A). The peroxisome proliferator-activated receptor (PPAR) superfamily (PPARα, β, γ), which regulates peroxisomal fatty acids β-oxidation, constitutes the genes upstream of Acox1. As shown in Figure 4B, after treatment with 20 μM PFOS for 12 h, the mRNA expression levels of PPARα, PPARβ, and PPARγ were increased by 2.4, 2.4, and 2.0-fold as compared to the control group, respectively. Furthermore, the number of immunofluorescent foci of PMP70 (a peroxisome marker) was greatly increased by 20 μM PFOS exposure (Figure 4C), which was consistent with the protein expression levels (Figure 4D). As shown in Figure 5A, the fluorescence intensities of the lipid droplets were significantly increased in the cells exposed to 20 μM PFOS as compared with those of the untreated cells as visualized under confocal microscopy, which was consistent with the quantification by flow cytometry. To determine the role of lipid β-oxidation in PFOS-induced H2O2 formation and DNA damage, BHT was chosen as a lipid peroxidation inhibitor. As shown in Figure 5B, BHT partly suppressed 6297
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Figure 3. Effect of an H2O2 scavenger on PFOS-induced cytotoxicity and genotoxicity. The levels of PFOS-induced reactive oxygen species in transgenic MEF cells were detected by the free radical probe CM-H2DCFDA (A). Cell viability was measured after exposure to 25 μM PFOS or 25 μM PFOS with 800 U/mL catalase-PEG for 24 h (B). Following cell exposure to either 20 μM PFOS or 20 μM PFOS with 800 U/mL catalase-PEG for 24 h, the levels of DSB-positive cells and mutation frequencies were examined by γ-H2AX immunofluorescence (C) and Spi− assays (D), respectively. Quantification of positive DSB cells was obtained from data pooled from three independent experiments. In each experiment, at least 500 cells were counted. Scale bar, 100 μm. Error bars indicate ± standard deviation.
PFOS-induced intracellular ROS. The percentages of 20 μM PFOS-induced DSB positive cells were decreased from 38 to 18.5% with concurrent treatment with BHT (Figure 5C). These results suggested that PFOS could induce the generation of H2O2 by the disruption of peroxisomal fatty acid β-oxidation in cells. Mutation Induction and Micronucleus Formation in gpt Delta Transgenic Mice Exposed to PFOS. Liver and bone marrow were reported to be two primary sites of PFOS accumulation in mice after oral exposure.29 As shown in Figure 6A and B, PFOS exposure induced a dose-dependent increase of ALT and ALP serum levels. The serum ALT and ALP levels in mice exposed to 4 mg/kg PFOS were 2.62- and 3.2-fold higher than those in the in the vehicle control group, indicating that PFOS exposure induced liver injury. The Spi− mutation frequencies in the liver were 0.78, 2.2, and 6.81 per 106 recovered plaques at the doses of 1.5, 4, and 10 mg/kg, respectively. The Spi− mutation frequency in the vehicle control group in the livers was 0.67 ± 0.94 × 10−6, which was
much lower than that observed in vitro. This might be due to the efficiency of the in vivo DNA repair system. In addition, the MN frequencies were increased by 1.52- and 1.43-fold when the concentrations of PFOS treatment were 4 and 10 mg/kg, respectively (Figure 6D). These results provided further evidence that PFOS was mutagenic in vivo.
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DISCUSSION Epidemiological and toxicological studies on PFOS have been primarily focused on the associated impaired fertility and disruption in endocrine hormone homeostasis.30,31 Genetic mutations such as gene deletion, point mutations, and insertions have been well-documented to play an important role in the initiation and development of carcinogenesis.32 However, the in vitro and in vivo mutagenicity of PFOS is still largely undetermined. A number of genotoxicity tests, such as micronucleus, comet assay, Ames test, and hprt gene mutation assay, were commonly used to predict rodent carcinogenicity in vitro. DNA damage 6298
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Figure 4. PFOS induced abnormal peroxisomal β-oxidation. Acox-1 mRNA expression levels of transgenic MEF cells treated with 1 or 20 μM PFOS for 12 or 24 h measured by real time reverse transcription-polymerase chain reaction (A). PPARα, β, and γ mRNA expression levels of transgenic MEF cells treated with 1 or 20 μM PFOS for 12 h (B). After PFOS exposure for 24 h, peroxisome proliferation levels were examined by immunofluorescence (C) and Western blot (D) detection of the PMP70 protein, a peroxisome marker. Red dots and blue spots represent PMP70 protein and nuclei, respectively. Scale bar, 10 μm. Error bars indicate ± standard deviation. *p < 0.05, compared with untreated controls.
observed in the bone marrow of gpt delta transgenic mice, which was consistent with previous observations in rats.37 It has been reported that the mutagenic potential PFOS was negative in S. typhimurium stains by bacterial reverse mutation assay (Ames test). However, there is less information on the mutagenicity of PFOS in mammalian cells and in vivo. Thus, a comprehensive in vitro and in vivo assessment on the mutagenicity of PFOS should be considered as an integral part of genotoxicity investigation. The use of transgenic mouse systems carrying bacterial reporter genes lacI/lacZ/cII, such as Big Blue mice and Muta Mouse, provides a promising opportunity for short-term mutagenicity analysis in vivo.38,39 Although the coding sizes of lacI, lacZ, and cII are different, they show very similar high levels of spontaneous mutation frequencies in vivo. The spontaneous mutation frequencies of these genes are generally in the 10−5 range in most tissues. Furthermore, the Big Blue mice and Muta Mouse mutation assay are generally limited to small sequence alterations, such as point mutations, and small deletions and insertions. To efficiently recover gene deletions and decrease spontaneous mutation levels in vivo, the gpt delta transgenic mice system
induced by PFOS have shown discrepancies in both comet assay and micronucleus test,16,17,33,34 which might be due to the nature of the assay, used doses, exposure time, and different cell models. Among the various types of DNA damage, DSBs are highly toxic lesions that can drive genetic instability, mutations, and cell death. One of the earliest cellular responses to DSBs is the generation of γ-H2AX on chromatin flanking the break sites. γ-H2AX foci might play an essential role in the accumulation of chromatin remodeling and DNA repair proteins such as NBS1, 53BP1, and MDC1 at the DSB sites.35 Our data showed that the accumulation of γ-H2AX foci and the expression level of γ-H2AX protein were significantly increased by PFOS in MEF cells, suggesting that PFOS was genotoxic and could induce chromosomal aberration in vitro. Nonhomologous end-joining is the predominant mechanism of DSB repair in eukaryotes and is often prone to error, which might lead to the amplification or loss of chromosomal material. At the late stage of DSB repair processes, if, for example, the deleted chromosomal regions remain in the cytoplasm, these events are likely to lead to MN formation.36 In this study, slight induction of MN formation by PFOS was 6299
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Figure 5. BHT, a lipid peroxidation inhibitor, suppressed PFOS-induced ROS and DSBs. Cells exposure to 1 or 20 μM PFOS for 24 h were fixed and stained with Nile red. The fluorescence was viewed using a confocal microscope. Quantification of Nile red fluorescence in cells by flow cytometry; over 10 000 cells were examined (A). Following cell exposure to either 20 μM PFOS or 20 μM PFOS with 20 μM BHT for 24 h, the level of intracellular ROS (B) and percentage of DSB-positive cells (C) were examined. Scale bar, 50 μm. Error bars indicate ± standard deviation.
was established by integrating multiple copies of λEG10 phage DNA containing the redBA and gam genes into each chromosome 17 of C57BL/6J mice; the spontaneous mutation frequencies in these repeats is 0.5−1.5 × 10−6,.40−42 Because
the wild-type λ phage is restricted by the P2 lysogenes in host cells (sensitive to P2 interference or Spi) and only mutant λ phages that are deficient in the functions of both the red and gam genes can avoid P2 interference (Spi−), this system permits 6300
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Figure 6. PFOS induced liver damage and genotoxicity in vivo. The gpt delta transgenic mice were exposed to PFOS at doses of 0, 1.5, 4, and 10 mg/ kg/day for 28 days. Each group contained six mice (n = 6). The ALT and ALP activities of the gpt delta transgenic mice are shown in (A) and (B), respectively. The mutation frequencies in the livers of the gpt delta transgenic mice after PFOS exposure were measured by Spi− assay (C). The frequencies of micronucleated polychromatic erythrocytes (MNPCEs) were measured by a bone marrow micronucleus assay (D). The MNPCE frequencies were calculated from 2000 scored PCEs per sample. Error bars indicate ± standard deviation. *p < 0.05, compared with the untreated controls.
expression levels of the peroxisomal β-oxidation enzymes were found to be upregulated by more than 10-fold in response to PFOS exposure, while catalase, a key enzyme in the decomposition of H2O2, was changed only slightly. Here, the exogenously applied catalase efficiently suppressed the induction of DSBs and the mutation yields, suggesting that intracellular oxidative stress, especially that provided by H2O2, contributed substantially to PFOS-induced DNA damage. In the present study, we observed that PFOS exposure stimulated large amounts of lipid droplet formation, which was consistent with the previous toxicological studies that demonstrated that PFOS disturbed lipid metabolism leading to an excessive accumulation of triglycerides and fatty acids in adipocytes and the liver.45,46 As an energy source, fatty acids can be oxidized to energy products by β-oxidation in living cells. The enzymes of fatty acid oxidation in cells are not only located in the mitochondria but also in the peroxisome. The oxidative process performed in these two organelles is similar but not identical. The β-oxidation product in mitochondrial is FADH2; in contrast, peroxisomal β-oxidation passes electrons directly to oxygen producing H2O2, which is catalyzed by acylCoA oxidases. Acyl-CoA oxidases are the rate limiting enzymes of the peroxisomal β-oxidation and are encoded by Acox gene family.47 The expression levels of the Acox1 gene and the PPAR genes were up-regulated by PFOS exposure in transgenic MEF cells. As lipid sensors, PPARs form heterodimers with the retinoid X receptor (RXR) that bind to specific DNA sequences in regulatory regions and control the expression of target genes that function in lipid homeostasis for protecting cells from lipid
the efficient and quantitative detection of deletion mutations in any tissue by calculating the number of clear phage plaques. The present study showed that PFOS induced a concentrationdependent increase of mutation frequencies in transgenic MEF cells. Since our in vitro experiments were acute exposure, the highest concentration of PFOS used here was 20 μM, which was relatively higher than the concentrations in human serum. We chose the liver as a target tissue for mutation analysis since PFOS exposure has been reported to cause hepatotoxicity and alteration of the hepatic immune status.43 We found that hepatomegaly was also clearly apparent in the transgenic mice exposed to PFOS in the present study (data not show). The Spi− mutation frequencies in the liver were 0.78, 2.2, and 6.81 per 106 recovered plaques at the doses of 1.5, 4, and 10 mg/kg, respectively. The bone marrow MN frequencies were increased only 1.52- and 1.43-fold at the dose of 4 and 10 mg/kg. However, there was no statistically significant difference between PFOS exposed groups and the control, which might be due to the different responses to PFOS in different individuals and a more complete DNA repair system in vivo. Thus, it is of interest to clarify the underlying mechanism of PFOS-induced genotoxicity. There is evidence that PFOS-induced toxicity might be mediated by oxidative stress.44 Mitochondria-dependent ROS has been shown to play an important role in the mutagenicity of PFOA in mammalian cells. In addition to mitochondria, peroxisomes are additional important organelles which can generate ROS when stimulated by peroxisomal β-oxidation. Due to the structural similarity of PFOS to octanoic acid, the 6301
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RT-PCR real-time reverse transcription-polymerase chain reaction RXR retinoid X receptor TRITC tetraethyl rhodamine isothiocyanate SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +8663846590; e-mail:
[email protected]. *Phone: +86 551 65593336; e-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Major National Scientific Research Projects, 2014CB932002; Strategic Leading Science & Technology Program (B), XDB14030502; National Natural Science Foundation of China grants U1232144 and 30570442; Major/Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology 2014FXCX010 CASHIPS director's fund. We thank Dr. Takehiko Nohmi (National Institute of Health Sciences, NIHS, Japan) for providing the gpt delta transgenic mice. We thank Dr. Lubomir B. Smilenov (Columbia University, U.S.) for scientific suggestions.
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ABBREVIATIONS ABC ATP-binding cassette Acox1 acyl-CoA oxidase ALP alkaline phosphatase ALT alanine aminotransferase BHT butylated hydroxytoluene cDNA complementary DNA DMEM Dulbecco’s modified Eagle’s medium CM-H2DCFDA 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester DMSO dimethyl sulfoxide DSBs DNA double strand breaks FBS fetal bovine serum FITC fluorescein isothiocyanate MEF mouse embryonic fibroblast MN micronucleus MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide NIHS National Institute of Health Sciences NHANES National Health and Nutrition Examination Survey PBS phosphate buffered saline PEG polyethylene glycol PFCs perfluoroalkyl chemicals PFOA perfluooctanoic acid PFOS perfluorooctane sulfate PMP70 70 kDa peroxisomal membrane protein PPAR peroxisome proliferator-activated receptor PVDF polyvinylidine fluoride γ-H2AX phosphorylated histone H2AX ROS reactive oxygen species 6302
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DOI: 10.1021/acs.est.5b00530 Environ. Sci. Technol. 2015, 49, 6294−6303
