Dose Effects of Ammonium Perfluorooctanoate on Lipoprotein Metabolism in APOE*3-Leiden.CETP Mice

Abstract

Epidemiological studies have reported positive associations between serum perfluorooctanoic acid (PFOA) and total and non-high-density lipoprotein cholesterol (non-HDL-C) although the magnitude of effect of PFOA on cholesterol lacks consistency. The objectives of this study were to evaluate the effect of PFOA on plasma cholesterol and triglyceride metabolism at various plasma PFOA concentrations relevant to humans, and to elucidate the mechanisms using APOE*3-Leiden.CETP mice, a model with a human-like lipoprotein metabolism. APOE*3-Leiden.CETP mice were fed a Western-type diet with PFOA (10, 300, 30 000 ng/g/d) for 4-6 weeks. PFOA exposure did not alter plasma lipids in the 10 and 300 ng/g/d dietary PFOA dose groups. At 30 000 ng/g/d, PFOA decreased plasma triglycerides (TG), total cholesterol (TC), and non-HDL-C, whereas HDL-C was increased. The plasma lipid alterations could be explained by decreased very low-density lipoprotein (VLDL) production and increased VLDL clearance by the liver through increased lipoprotein lipase activity. The concomitant increase in HDL-C was mediated by decreased cholesteryl ester transfer activity and changes in gene expression of proteins involved in HDL metabolism. Hepatic gene expression and pathway analysis confirmed the changes in lipoprotein metabolism that were mediated for a major part through activation of the peroxisome proliferator-activated receptor (PPAR)α. Our data confirmed the findings from a phase 1 clinical trial in humans that demonstrated high serum or plasma PFOA levels resulted in lower cholesterol levels. The study findings do not show an increase in cholesterol at environmental or occupational levels of PFOA exposure, thereby indicating these findings are associative rather than causal.

Keywords: cholesterol; lipoproteins; non-HDL cholesterol; perfluorooctanoic acid (PFOA); peroxisome proliferator-activated receptor (PPAR)α; triglycerides.

Figure 1.

Study set-up. At the end of the 4-week run-in period (t = week 0), mice were randomized into 4 groups based on age, body weight, and baseline plasma TC, TG, and HDL-C levels measured at the end of the run-in period. Upon randomization, mice were either fed with Western-type diet alone (control group) or Western-type diet containing ammonium PFOA at 10, 300, or 30 000 ng/g/d (n = 8 mice per dose group). Abbreviations: BW, body weight; CETP, cholesteryl ester transfer protein; FFA, free fatty acids; FI, food intake; HDL-C, high-density lipoprotein cholesterol; HL, hepatic lipase; LPL, lipoprotein lipase; MRI, magnetic resonance imaging; TC, total cholesterol; TG, triglycerides; VLDL, very-low-density lipoprotein; WTD, Western-type diet.

Figure 2.

PFOA at low dose does not alter plasma lipids. Mice received a Western-type diet without or with 10, 300 or 30 000 ng/g/d PFOA. At baseline (t0) and after 6 weeks of exposure (t6), 4-h fasted blood was taken and plasma was assayed for TG (A), TC (B), non-HDL-C (C), and HDL-C (D). After 4 weeks of intervention, cholesterol distribution over lipoproteins was determined by FPLC in group-wise pooled plasma (E). Data are presented as means + SD (n = 8 per group). ***p < .001 as compared with the control group. Abbreviations: FPLC, fast protein liquid chromatography; HDL-C, high-density lipoprotein cholesterol; VLDL/LDL, (very) low-density lipoprotein.

Figure 3.

PFOA at high dose increases HDL-C by reducing CETP activity. Mice received a Western-type diet without or with PFOA in 3 different doses, 10, 300 and 30 000 ng/g/d. After 6 weeks of PFOA exposure in experiment 1, CETP activity was determined (A), and the activity in mice of the control group was compared with the activity in human plasma samples (B). Data are represented as mean + SD (n = 6–8 mice per group and n = 4 human plasma samples). **p < .01 as compared with the control group and ***p < .001 as compared with control APOE*3-Leiden.CETP mice. Abbreviations: CETP, cholesteryl ester transfer protein.

Figure 4.

PFOA at high dose decreases VLDL-TG production and ApoB synthesis. Mice received a Western-type diet without or with 10, 300 or 30 000 ng/g/d PFOA. After 6 weeks, 4-h fasted mice of experiment 1 were injected with Tran³⁵S-label and Triton after which blood samples were drawn up to 90 min. Plasma VLDL-TGs (A) were plotted and used to calculate the rate of TG production (B) from the slope of the individual curves. Ninety minutes after Triton injection plasma was used to isolate VLDL by ultracentrifugation, and the rate of de novo ApoB synthesis was determined (C). The TG production per ApoB was then calculated (D). Data are represented as mean ± SD (n = 6–8 per group). ***p < .001 as compared with the control group. Abbreviations: ApoB, apolipoprotein B; TG, triglycerides; VLDL, very low-density lipoproteins.

Figure 5.

PFOA at high dose increases VLDL clearance mainly due to increased hepatic uptake. Mice received a Western-type diet without or with 10, 300 or 30 000 ng/g/d PFOA. After 4 weeks, 4-h fasted mice of experiment 2 were injected with glycerol tri[3H]-oleate ([3H]-TO) and [14C]-cholesteryl oleate ([14C]-CO)-labeled VLDL-like particles. [3H]-TO plasma decay was plotted (A) and used to calculate the rate of [3H]-TO uptake (B). Clearance of [3H]-TO in individual organs was determined (C). [14C]-CO plasma decay was plotted (D) and used to calculate the rate of [14C]-CO uptake (E). Clearance of [14C]-CO in individual organs was determined (F). Data are represented as mean ± SD (n = 6–7 per group).*p < .05, **p < .01, and ***p < .001 as compared with the control group. Abbreviations: BAT, brown adipose tissue; muscle, femoral muscle; sWAT, subcutaneous white adipose tissue; VLDL, very low-density lipoproteins.

Figure 6.

PFOA at high dose increases LPL activity. Mice received a Western-type diet without or with 10, 300 or 30 000 ng/g/d PFOA. After 5 weeks, 4-h fasted mice of experiment 1 were injected with heparin (0.1 U/g body weight) and postheparin plasma was collected. Plasma was incubated with a [3H]-TO-containing substrate mixture in the absence or presence of 1M NaCl, to estimate both the HL and LPL activity. Data are represented as mean + SD (n = 5–8 per group). *p < .05 as compared with the control group. Abbreviations: FFA, free fatty acids; HL, hepatic lipase; LPL, lipoprotein lipase; NaCl, sodium chloride.

Figure 7.

PFOA at high dose induces hepatic hypertrophy. Mice received a Western-type diet without or with 10, 300 or 30 000 ng/g/d PFOA. After 4 weeks of PFOA exposure in experiment 2, livers were histologically analyzed for macrovesicular and microvesicular steatosis, hypertrophy and the number of inflammatory aggregates per mm² (A), and hepatic lipid content per mg liver protein was measured (B). After 5 weeks of PFOA exposure in experiment 1, feces were collected per cage during a 48–72 h period at 2 consecutive time points, in which bile acid excretion (C) and neutral sterol excretion (D) were determined using gas chromatography. Data are represented as mean + SD (n = 6–7 per group and n = 6–8 collection points per group). *p < .05 and ***p < .001 as compared with the control group.