Critical role of PPAR-alpha in perfluorooctanoic acid- and perfluorodecanoic acid-induced downregulation of Oatp uptake transporters in mouse livers

Abstract

Perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) have been detected globally in wildlife and humans. Data from a gene array indicate that PFOA decreases organic anion transporting polypeptides (Oatps) in liver. Na(+)-taurocholate cotransporting polypeptide (Ntcp) and Oatp1a1, 1a4, and 1b2 are major transporters responsible for uptake of bile acids (BAs) and other organic compounds into liver. The purpose of the present study was to determine the effects of two perfluorinated fatty acids, PFOA and PFDA, on mRNA and protein expression of hepatic uptake transporters Oatps and Ntcp, and to determine the underlying regulatory mechanisms by using peroxisome proliferator-activated receptor alpha (PPAR-alpha), constitutive androstane receptor, pregnane-X receptor, NF-E2-related factor 2, and farnesoid X receptor-null mouse models. After 2 days following a single i.p. administration, PFOA did not alter serum BA concentrations, but PFDA increased serum BA concentrations 300%. Furthermore, PFOA decreased mRNA and protein expression of Oatp1a1, 1a4, and 1b2, but not Ntcp in mouse liver. In contrast, PFDA decreased mRNA and protein expression of all four transporters, and decreased the mRNA expression in a dose-dependent manner, with the decrease of Oatp1a4 occurring at lower doses than the other three transporters. Multiple mechanisms are likely involved in the down-regulation of mouse Oatps and Ntcp by PFDA. By using the various transcription factor-null mice, PPAR-alpha was shown to play a central role in the down-regulation of Oatp1a1, 1a4, 1b2, and Ntcp by PFDA. The current studies provide important insight into understanding the mechanisms by which PFDA regulate the expression of hepatic uptake transporters. In conclusion, PFOA and PFDA decrease mouse liver uptake transporters primarily via activation of PPAR-alpha.

FIG. 1.

Effects of PFOA and PFDA on total BA concentrations in mouse serum. BA concentration in serum from adult male C57BL/6 WT mice 2 days after a single i.p. administration of control (propylene glycol:water; vol/vol, 1:1), PFOA (40 mg/kg), or PFDA (80 mg/kg). The data are presented as mean ± SEM (n = 5 mice). Asterisks (*) represent a statistical difference (p < 0.05) between control and treatment group.

FIG. 2.

Effects of PFOA and PFDA on Oatp1a1, 1a4, and 1b2, as well as Ntcp protein expression in mouse livers. Adult male C57BL/6 WT mice (n = 5 per treatment) were given a single i.p. administration of control (propylene glycol:water; vol/vol, 1:1), PFOA (40 mg/kg), or PFDA (80 mg/kg). Two days after, mouse livers were collected. Total membrane protein was isolated from mouse livers. Two of five protein samples from each treatment were analyzed by Western blots. (a) Protein levels of Oatp1a1, 1a4, and 1b2, as well as Ntcp in mouse liver membranes were analyzed by Western blotting. (b) Protein levels of Oatp1a1, 1a4, and 1b2, as well as Ntcp in mouse liver membranes are expressed as ratio of each transporter to β-actin protein levels per 75 μg total membrane protein. Data are presented as mean ± SEM. Asterisks indicate statistically significant differences between control and PFOA or PFDA-treated male mice (p < 0.05).

FIG. 3.

Effects of PFOA and PFDA on mRNA expression of Oatp1a1, 1a4, and 1b2, as well as Ntcp in mouse livers. Adult male C57BL/6 WT mice (n = 5 per treatment) were given a single i.p. administration of control (propylene glycol:water; vol/vol, 1:1), PFOA (40 mg/kg), or PFDA (80 mg/kg). Two days later, mouse livers were collected. Total RNA from mouse livers (n = 5 per treatment) was analyzed by the bDNA assay for the mRNA expression of each transporter. The data are reported as RLUs per 10 μg of total RNA, and are presented as mean ± SEM. Asterisks indicate statistically significant differences between control and PFOA or PFDA-treated male mice (p < 0.05).

FIG. 4.

Effects of various doses of PFDA on Oatp1a1, 1a4, and 1b2, as well as Ntcp mRNA expression in mouse livers. Adult male C57BL/6 WT mice were administered PFDA at a single i.p. dose (0.5, 1.0, 10, 20, 40, or 80 mg/kg of body weight), whereas control mice were treated i.p. with the vehicle, propylene glycerol:water (1:1, vol/vol). After 2 days, livers were collected. Total RNA samples from treated male mouse livers (n = 4) were analyzed by the bDNA assay. The data are reported as RLUs per 10 μg of total RNA, and are presented as mean ± SEM. Asterisks indicate statistically significant differences between control and PFDA-treated male mice (p < 0.05).

FIG. 5.

Effects of PFDA on mouse Oatp1a1, 1a4, and 1b2, as well as Ntcp mRNA expression in wild-type and PPAR-α-null mice. The mouse treatments are separated into 4 groups: PPAR^(+/+) + CONT, wild-type mice treated with the vehicle, propylene glycerol:water (1:1, vol/vol); PPAR^(+/+) + PFDA, wild-type mice treated with PFDA at dose 40 mg/kg of body weight; PPAR^(−/−) + CONT, PPAR-α-null mice treated with the vehicle; PPAR^(−/−) + PFDA, PPAR-α-null mice treated with PFDA at dose 40 mg/kg of body weight. The mice were given a single i.p. administration. After 2 days, livers were removed. Total RNA from treated mouse livers was analyzed by the bDNA assay. All data are expressed as mean ± SEM of four to five mice for each treatment. Asterisk indicates statistically significant difference between treated and control mice (p < 0.05). Single dagger (†) represents statistically significant differences (p < 0.05) between the vehicle-treated wild-type male mice and the vehicle-treated PPAR-α-null male mice.

FIG. 6.

Effects of PFDA on mouse Oatp1a1, 1a4, and 1b2, as well as Ntcp mRNA expression in wild-type and CAR-null mice. The mouse treatments are separated into four groups: CAR^(+/+)+CONT, wild-type mice treated with the vehicle, propylene glycerol:water (1:1, vol/vol); CAR^(+/+) + PFDA, wild-type mice treated with PFDA at dose 80 mg/kg of body weight; CAR^(−/−) + CONT, CAR-null mice treated with the vehicle; CAR^(−/−) + PFDA, CAR-null mice treated with PFDA at dose 80 mg/kg. The mice were given a single i.p. administration. After 2 days, livers were removed. Total RNA from treated mouse livers was analyzed by the bDNA assay. All data are expressed as mean ± SEM of five mice for each treatment. Asterisk indicates statistically significant difference between treated and control mice (p < 0.05).

FIG. 7.

Effects of PFDA on mouse Oatp1a1, 1a4, and 1b2, as well as Ntcp mRNA expression in wild-type and PXR-null mice. The mouse treatments are separated into four groups: PXR^(+/+) + CONT, wild-type mice treated with the vehicle, propylene glycerol:water (1:1, vol/vol); PXR^(+/+) + PFDA, wild-type mice treated with PFDA at dose 80 mg/kg of body weight; PXR^(−/−) + CONT, PXR-null mice treated with the vehicle; PXR^(−/−) + PFDA, PXR-null mice treated with PFDA at dose 80 mg/kg. The mice were given a single i.p. administration. After 2 days, livers were removed. Total RNA from untreated or treated mouse livers was analyzed by the bDNA assay. All data are expressed as mean ± SEM of five mice for each treatment. Asterisk indicates statistically significant difference between PFDA-treated and control mice (p < 0.05); single dagger (†) represents statistically significant differences (p < 0.05) between the vehicle-treated wild-type male mice and the vehicle-treated PXR-null male mice.

FIG. 8.

Effects of PFDA on mouse Oatp1a1, 1a4, and 1b2, as well as Ntcp mRNA expression in wild-type and Nrf2-null mice. The mouse treatments are separated into four groups: Nrf2^(+/+) + CONT, wild-type mice treated with the vehicle, propylene glycerol:water (1:1, vol/vol); Nrf2^(+/+) + PFDA, wild-type mice treated with PFDA at dose 80 mg/kg of body weight; Nrf2^(−/−) + CONT, Nrf2-null mice treated with the vehicle; Nrf2^(−/−) + PFDA, Nrf2-null mice treated with PFDA at dose 80 mg/kg. The mice were given a single i.p. administration. After 2 days, livers were removed. Total RNA from treated mouse livers was analyzed by the bDNA assay. All data are expressed as mean ± SEM of five mice for each treatment. Asterisk indicates statistically significant difference between treated and control mice (p < 0.05).

FIG. 9.

Effects of PFDA on Oatp1a1, 1a4, and 1b2, as well as Ntcp mRNA expression in wild-type and FXR-null mice. The black and the dark-latticed bars represent regulation of each transporter by vehicle treatment; the striated bars depict regulation of each transporter by PFDA treatment. Adult C57BL/6 wild-type or FXR-null male mice were treated with the vehicle, propylene glycerol:water (1:1, vol/vol) or PFDA at dose 80 mg/kg of body weight. The mice were treated for 2 days. Total RNA from untreated or treated mouse livers was analyzed by the bDNA assay. All data are expressed as mean ± SEM of five mice for each treatment. Asterisk indicates statistically significant difference between PFDA-treated and control mice (p < 0.05). Single dagger (†) represents statistically significant differences (p < 0.05) between the vehicle-treated wild-type male mice and the vehicle-treated FXR-null male mice.