| This study aims to use in vivo mouse model and in vitro gastrointestinal methods to study the bioavailability and bioaccessibility of perfluorooctanoic acid (PFOA) in different foods and effect of food components on their bioavailability. In addition, systematically study the liver toxicity of two representative perfluorinated acid substances, PFOA and perfluorohexane sulfonic acid (PFHxS), in male and female mice, and further use proteomic approach to study the molecular mechanisms of their toxicity. The main results were as follows:Food is a major source of human exposure to PFOA, thus reducing its bioavailability in food could lower the exposure to PFOA. An in vivo mouse model and three in vitro methods (unified BARGE method-UBM, physiologically based extraction test-PBET, and in vitro digestion method-IVD) were used to determine the relative bioavailability and bioaccessibility of PFOA in the presence of 17 foods. PFOA was mixed with foods of different nutritional compositions and fed to mice over a 7-d period. PFOA relative bioavailability was determined by comparing PFOA accumulation in the liver following PFOA exposure via food to that in water. PFOA bioavailability relative to water ranged from 4.30±0.80 to 69.0±11.9% and was negatively correlated with lipid content (r=0.76). This was possibly due to competitive sorption of free fatty acids with PFOA onto transporters on intestine epithelial cells. Besides, cations in the gastrointestinal tract, such as Ca2+ and Mg2+, are capable of complexing PFOA and partition to the chyme phase. On the other hand, when assessed using in vitro assays, PFOA bioaccessibility varied with methods, being 8.7～73%(UBM),9.8～99%(PBET) and 21～114%(IVD). PFOA bioaccessibility was negatively correlated with lipid content when assessed using UBM (r=0.82); however, a poor correlation with food composition was observed for PBET and IVD (r= 0.01～0.50). When in vivo and in vitro data were compared, a strong correlation was observed for UBM (r=0.79), but poor relationships were observed for PBET and IVD (r= 0.11～0.22). This was probably because the higher lipolysis ability and presence of Ca2+ and Mg2+ in its gastrointestinal fluid of UBM resulted in a lower potential to form stable micelles compared to PBET and IVD. These results indicated that PFOA relative bioavailability was mainly affected by lipid content in foods, and UBM has the potential to predict PFOA bioavailability in food samples.Previous studies have shown that, following the exposure, PFOA mainly accumulates in liver and blood, and causes hepatic toxicity. We studied hepatotoxicity of male and female Balb/c mice after exposing to PFOA at 0.05,0.5, and 2.5 mg/kg/d for 28 d on a body weight basis. After 28 d exposure, only the mice dosed with 2.5 mg/kg/d PFOA showed body weight decline. However, all treated mice except for the 0.05 mg/kg/d males showed significant liver weight increase, which notes the hepatohydrophy. The distribution test also showed accumulation of PFOA in the liver and blood for female and male mice exposed to 0.05 mg/kg/d PFOA, the final serum PFOA concentrations were 0.97 and 1.20 mg/L, comparable to occupational workers and nearby residents in Changshu Fluoro-Chemistry Industrial Zone. Histology and substructure diagnosis then proved two major symptoms occurred in PFOA-treated mice liver, peroxisome proliferation and apoptosis, besides, lipids accumulation was observed in 0.05 mg/kg/d PFOA treated mice. The peroxisome proliferater property of PFOA has been well studied before, while its ability to induce apoptosis has not observed in vivo until recently, so we further tested the ability of PFOA to induce mitochondrial transmembrane potential (mtΔΨ) dissipation and Caspase-9 activity change, the two major events of mitochondria-induced apoptosis. In all treated mice other than male 0.05 mg/kg/d, the mtΔΨ dissipated cell proportion as well as Caspase-9 activity showed significantly upregulation than control mice. Thus PFOA induced hepatic apoptosis in vivo through mitochondria signal pathway, the same as observed previously in vitro in human HepG2 cell line, and therefore requires further investigation.PFHxS is another PFASs widely detected in human beings, however, theie toxicity is unclear. We conducted a parallel experiment using PFHxS. The results showed that PFHxS is stonger in causing mice body weight loss than PFOA, but weaker in hepatic hydrophy. PFHxS also accumulates in liver and blood. However, the exposure level of mice after 28 d was much higher than general population. The toxicity of PFHxS was much weaker than PFOA, only in a high dose did PFHxS cause peroxisome proliferation and apoptosis. The dose to cause these toxic effects requires a serum concentration much higher than observed in production workers, so there is little human hazards posed by PFHxS.Proteome assay was then conducted to study the molecular mechanism of PFOA-induced mice liver apoptosis. The proteome profile showed that PPARa was not activated in femal 0.05 mg/kg/d mice, since all proteins from this signal pathway showed no significant change, and semi-quatification PCR also proved no activation of three PPAR isoforms. On the other hand, proapoptotic proteins PDCD5 and CYC were all significantly upregulated, which were correlated with p53 signal pathway. Western blot assay confirmed the iTRAQ results. Under PFOA exposure, especially in low level for female mice, p53 instead of PPARa pathway stimulated cell apoptosis. Further experiments showed PFOA induced 8-OHdG upregulation in dose-dependent way, and a gender difference was observed. Female mice treated with 0.05 mg/kg/d PFOA showed elevation of 8-OHdG in hepatocytes, which indicated the oxydize damage caused DNA damage, leading to carcinogenesis. As PFOA concentration in serum of 0.05 mg/kg/d mice was comparable to those in blood of occupational workers and nearby residents, considering the conservation of p53 between human and mice, this alternative pathway was a more realistic toxicity mode in human beings exposed to PFOA, especially for women. |
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