*Joint senior authorship
Perfluorooctane sulfonate (PFOS) is a persistent pollutant that exerts toxicity and induces cardiogenesis in humans and animals. Yet, the effect of PFOS exposure on cardiac toxicity in adult rats has, to our knowledge, not been reported and the mechanism still remains unknown. The present study aimed to investigate the toxicity of PFOS on rat hearts and any associated mechanisms. Rats were exposed to 0 (control), 1 and 10 mg/kg PFOS every other day for 14 days. Body weight and heart weight were recorded. The serum levels of lactic dehydrogenase (LDH), creatine kinase (CK), creatine kinase-isoenzyme-MB (CK-MB) and cardiac troponin-T (cTn-T) in heart tissues were measured using biochemical assays. TUNEL staining and western blotting were applied to analyze levels of apoptosis in rat hearts. Pathological assessment and immunohistochemistry analysis of heart tissues were used to evaluate the levels of PFOS-induced cardiotoxicity and inflammatory infiltration. PFOS exposure at the dosage of 10 mg/kg significantly increased the percentage of heart to body weight; however, it did not alter the body weight. At 10 mg/kg, PFOS significantly increased expression levels of myocardial injury markers, such as cTn-T, LDH, CK and CK-MB, while 1 mg/kg PFOS upregulated the expression level of cTn-T in rats. Notably, cardiac fibrosis and myocardiac hypertrophy appeared in the 10 mg/kg PFOS group. In addition, TUNEL-positive cells were significantly increased by exposure to 10 mg/kg PFOS in rat heart tissues. The protein expressions profiles of p53 and Bax were also significantly upregulated in the 10 mg/kg PFOS group. Inflammatory infiltration, detected by anaylzing expression levels of IL-1β and TNF-α, was significantly raised by 10 mg/kg PFOS exposure. In conclusion, these results demonstrated that 10 mg/kg PFOS-induced cardiac toxicity in rats, which was associated with an increase in apoptosis and the expression of proinflammatory cytokines.
Perfluorooctane sulfonate (PFOS) is a degradation product of perfluorinated compounds and is characterized by widespread use and environmental stability (
As previously reported, PFOS involves several toxic effects in the cardiovascular (
Apoptosis is a type of programmed cell death that participates in various pathological events (
The current study aimed to explore whether PFOS exposure would induce heart impairment and a degree of pathological change in rats. Moreover, the level of apoptosis and inflammatory infiltration in cardiomyocytes was investigated to provide evidence for further research on PFOS-induced cardiac toxicology.
Animal experiments in the current study were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (
A total of 48 male Sprague Dawley (SD) rats (220±5 g) in 8-week old were purchased from the Laboratory Animal Center of Zhejiang Province (Hangzhou, China) and bred in house. All rats were housed in a specific pathogen free facility under a 12-h light/dark cycle in an ambient temperature of 22-26˚C and relative humidity of ~55%. Animals were fed standard laboratory rat chow. Animals were provided food and water
SD rats were divided into three groups (each, n=6) that received 0 (control), 1 and 10 mg/kg PFOS (Sigma Aldrich; Merck KGaA). The rats were intraperitoneally (I.P) injected at 1 cm to the left of the midline of the lower abdomen with PFOS every other day for 14 days. The dose, period and drug-delivery way of PFOS was selected based on previous reports (
Changes of body weight in each group were measured every other day until the 14th day. At the end of the PFOS treatment, the rats were sacrificed and hearts were quickly moved, carefully blotted dry and weighed. The percentage of heart weight to body weight was calculated as follow: (heart weight/body weight) x100%.
Blood samples were obtained from the abdominal aorta and left to stand at room temperature for 1 h, and then at 4˚C for 2 h, followed by 3,000 x g centrifugation for 10 min at 4˚C. The supernatant was used for serum lactic dehydrogenase (cat. no. 201902; LDH), creatine kinase (cat. no. 201823; CK) and creatine kinase-isoenzyme-MB (cat. no. 201811; CK-MB) measurements. The collected heart tissues were homogenized and centrifuged at 3,200 x g for 30 min at 4˚C. The supernatant was harvested for cardiac troponin-T (cat. no. 201904; cTn-T) measurement. All biomarkers were determined using commercial ELISA kits (BD Biosciences).
The isolated hearts were fixed in 4% paraformaldehyde for 24 h at room temperature and embedded in paraffin for histological analysis. Next, samples were cut into 5-µm sections and heated at 65˚C for 20 min. Slides were then deparaffinized with xylene and dehydrated in a grade series of ethanol through 70, 80, 90, 95 and 100%. The slices were stained with Masson for 10 min at room temperature to evaluate fibrosis and stained with wheat germ agglutinin (WGA) for 15 min at 37˚C to analyze myocardial hypertrophy. Images were captured (magnification, x200 or x400) under a light microscope (Leica Microsystems GmbH). The fibrosis area of heart tissues and the cardiomyocyte cross-sectional area were measured using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc.). The percentage of fibrotic areas were calculated.
For all groups, 5-µm sections were stained using the
For immunohistochemical assay, heart tissues were fixed in 4% paraformaldehyde for 24 h at room temperature and embedded in paraffin for immunohistochemical analysis. Sections were blocked using 5% goat serum (cat. no. G1209; Wuhan Boster Biological Technology, Ltd.) and incubated with specific primary antibodies overnight at 4˚C, such as IL-1β Rabbit mAb (1:800; cat. no. SRP8033; Sigma-Aldrich; Merck KGaA) and TNF-α Rabbit mAb (1:500; cat. no. ab6671, Abcam). The sections were washed with PBS three times, followed by staining with horseradish peroxidase-conjugated secondary anti-rabbit IgG (1:5,000; cat. no. GB23303; Wuhan Boster Biological Technology, Ltd.) for 2 h at room temperature and visualizing with substrate DAB. Images were obtained and captured (magnification, x200) using a fluorescent microscope (Leica Microsystems GmbH). The number of positive cells was analyzed by ImageJ analysis software (version 1.51; National Institutes of Health).
Protein from myocardial tissue was extracted using the cell and tissue total protein extraction kit (cat. no. KC415; Shanghai Kang Cheng Bioengineering Co., Ltd.). Protein concentration was quantified using the bicinchoninic acid (BCA) protein assay kit (cat. no. P0010; Beyotime Institute of Biotechnology) and was diluted to the same concentration with 5X loading buffer (cat. no. P0015L; Beyotime Institute of Biotechnology). A total of 50 µg protein was separated using 15% SDS-PAGE. Protein was transferred to polyvinylidene fluoride membrane and blocked in 5% skimmed milk in Tris-buffered saline for 90 min at room temperature. After blocking, the membranes were incubated overnight with primary antibodies against p53 mouse mAb (1:1,000; cat. no. 2524; Cell Signaling Technology, Inc.), anti-Bax antibody (1:800; cat. no. A00183; Wuhan Sanying Biotechnology) and GAPDH mouse mAb (1:3,000; cat. no. G3214; Bioworld Technology, Inc.) overnight at 4˚C. Following washing with Tris-buffered saline containing 1% Tween, membranes were incubated for 2 h with horseradish peroxidase-conjugated IgG (1:5,000, cat. no. GB23303; Wuhan Boster Biological Technology, Ltd.) secondary antibodies at room temperature for 2 h. The specific protein bands were visualized using an enhanced chemiluminescence detection kit (cat. no. 33021; Boster Biological Technology, Ltd.). The intensity of each band was quantified using Quantity One software version 6 (Bio-Rad Laboratories, Inc.).
Data are presented as mean ± standard deviation. Values from three groups were analyzed using one-way ANOVA followed by Student-Newman-Keuls multiple comparison post hoc test. Statistical analysis was performed in GraphPad Software (Prism Version 8.01; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
Body weight in 0 (control), 1 and 10 mg/kg PFOS groups were recorded every two days for 14 days. There was no significant difference in the body weight of rats among the three groups, though a slight reduction was observed between control and 10 mg/kg PFOS group (
In order to detect the toxic effect of PFOS on the heart, cTn-T, LDH, CK and CK-MB, which are markers for myocardial injury, were measured in rats. In the 1 mg/kg PFOS group, only the level of cTn-T in heart tissues was significantly elevated compared with control group (P<0.05;
To determine the toxicological effect of PFOS on rat hearts over a longer duration, PFOS was administrated every other day for 28 days to model sub-chronic exposure. The levels of cTn-T, LDH, CK and CK-MB were examined after 28 days of PFOS exposure. The 10 mg/kg PFOS group demonstrated significantly elevated levels of cTn-T, LDH, CK and CK-MB compared with the 0 mg/kg PFOS group (P<0.05;
To further determine the toxic influence of PFOS on heart tissues in rats, Masson and WGA staining were performed. The rats in the 1 mg/kg PFOS group exhibited no significant increase in cardiac fibrosis and hypertrophy when compared with the control group (P>0.05;
The cardiotoxicity effect of PFOS on apoptosis was explored using TUNEL staining. As presented in
The influence of PFOS on the cardiac inflammation in rats was investigated. Immunohistochemical staining for pro-inflammatory cytokine IL-1β indicated that the expression profile of IL-1β was significantly increased in the 10 mg/kg PFOS group compared with the control group (6.60±0.65 vs. 3.84±0.76 pg/mg protein; P<0.05;
The current study revealed several main findings. Firstly, exposure of PFOS at the dosage of 10 mg/kg caused myocardial damage in rats. Secondly, 10 mg/kg PFOS significantly increased cardiac fibrosis and myocardiac hypertrophy in rats. Finally, it was demonstrated that 10 mg/kg PFOS treatment upregulated myocardial apoptosis and expression levels of IL-1β and TNF-α in heart tissue of rats.
PFOS, a type of fluorine-saturated eight-carbon compound, is a persistent, bioaccumulative and organic pollutant as a result of its ubiquitous distribution and extreme stability (
The cardiovascular toxicity of PFOS has been rudimentarily studied
Apoptosis is an important process in various human diseases and has been implicated in PFOS toxicity (
Increasing evidence has demonstrated that inflammation serves a notable role in Polyfluoroalkyl chemical-induced toxicity (
Previous studies have reported that the exposure of female rats to perfluorooctane sulfonate increased the estrogen receptor α (ERα) expression, suggesting that PFOS acts as estrogenic compounds to activate ERα (
In summary, the current study demonstrated that PFOS exposure caused pathological changes, reflected by cardiac fibrosis and myocardial hypertrophy in the hearts of adult rats, which was possibly associated with an increase in apoptosis and proinflammatory cytokines, such as IL-1β and TNF-α. The present study provided preliminary data for further study of cardiovascular system subjected to a PFOS challenge.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JY and MG designed the current study and wrote the manuscript. DX, LL and LT performed experiments and analyzed data. DX and JY confirmed the authenticity of all the raw data. All authors have read and approved the final manuscript.
Procedures of animal experiments were approved by The Ethics Committee of Laboratory Animal Care and Welfare, Zhejiang Academy of Medical Sciences (Zhejiang, China).
Not applicable.
The authors declare that they have no competing interests.
Illustration of experimental schedule and general indexes of rats. (A) Experimental schedule schematic. Sprague-Dawley rats received 0, 1 and 10 mg/kg perfluorooctane sulfonate (
PFOS induces myocardial injury in rats. Markers of myocardial damage in cardiac tissues and serum were measured. (A) Expression levels of CTn-T in the cardiac tissues were determined. Contents of (B) LDH, (C) CK and (D) CK-MB in rat serum was measured. n=5. *P<0.05 as indicated. PFOS, perfluorooctane sulfonate; cTn-T, cardiac troponin-T; LDH, lactic dehydrogenase; CK, creatine kinase; MB, isoenzyme-MB.
PFOS is associated with cardiac fibrosis and myocardiac hypertrophy in rats. (A) Masson staining and (B) wheat germ agglutinin staining of the rat hearts exposed to different doses of PFOS. Statistical graph of the (C) fibrosis area and the (D) cardiomyocyte cross-sectional area. White arrows point out Masson-positive regions (A magnification, x400; scale bars, 50 µm. B magnification, x200, scale bars, 100 µm). n=5. *P<0.05. PFOS, perfluorooctane sulfonate.
PFOS is associated with myocardial apoptosis in rats. (A) Representative images stained with TUNEL (red) and Dapi (blue). White arrows indicate TUNEL-positive cells (scale bar, 50 µm; magnification, x400). (B) Quantitative analysis of the percentage of TUNEL-positive cells in rat hearts (n=5). (C) Western blotting of p53 and Bax in rat heart. (D) Quantitative analysis of the effect of PFOS treatment effects on p53 and Bax (n=3). *P<0.05 as indicated. PFOS, perfluorooctane sulfonate.
PFOS is associated with inflammatory infiltration in rat heart tissues. (A) Immunohistochemistry staining demonstrated the effect of different doses of PFOS on protein levels of IL-1β and TNF-α (magnification, x200; scale bars, 100 µm). Protein expression levels of (B) IL-1β and (C) TNF-α were quantitatively analyzed. n=5. *P<0.05 as indicated. PFOS, perfluorooctane sulfonate.