Latent, sex-specific metabolic health effects in CD-1 mouse offspring exposed to PFOA or HFPO-DA (GenX) during gestation

Abstract

Background: Perfluorooctanoic acid (PFOA) is an environmental contaminant associated with adverse metabolic outcomes in developmentally exposed human populations and mouse models. Hexafluoropropylene oxide-dimer acid (HFPO-DA, commonly called GenX) has replaced PFOA in many industrial applications in the U.S. and Europe and has been measured in global water systems from <1 to 9350 ng/L HFPO-DA. Health effects data for GenX are lacking.

Objective: Determine the effects of gestational exposure to GenX on offspring weight gain trajectory, adult metabolic health, liver pathology and key adipose gene pathways in male and female CD-1 mice.

Methods: Daily oral doses of GenX (0.2, 1.0, 2.0 mg/kg), PFOA (0.1, 1.0 mg/kg), or vehicle control were administered to pregnant mice (gestation days 1.5-17.5). Offspring were fed a high- or low-fat diet (HFD or LFD) at weaning until necropsy at 6 or 18 weeks, and metabolic endpoints were measured over time. PFOA and GenX serum and urine concentrations, weight gain, serum lipid parameters, body mass composition, glucose tolerance, white adipose tissue gene expression, and liver histopathology were evaluated.

Results: Prenatal exposure to GenX led to its accumulation in the serum and urine of 5-day old pups (P = 0.007, P < 0.001), which was undetectable by weaning. By 18 weeks of age, male mice fed LFD in the 2.0 mg/kg GenX group displayed increased weight gain (P < 0.05), fat mass (P = 0.016), hepatocellular microvesicular fatty change (P = 0.015), and insulin sensitivity (P = 0.014) in comparison to control males fed LFD. Female mice fed HFD had a significant increase in hepatocyte single cell necrosis in 1.0 mg/kg GenX group (P = 0.022) and 1.0 mg/kg PFOA group (P = 0.003) compared to control HFD females. Both sexes were affected by gestational GenX exposure; however, the observed phenotype varied between sex with males displaying more characteristics of metabolic disease and females exhibiting liver damage in response to the gestational exposure.

Conclusions: Prenatal exposure to 1 mg/kg GenX and 1 mg/kg PFOA induces adverse metabolic outcomes in adult mice that are diet- and sex-dependent. GenX also accumulated in pup serum, suggesting that placental and potentially lactational transfer are important exposure routes for GenX.

Keywords: GenX; Gestational exposure; HFPO-DA; Metabolism; Microvesicular fatty change; PFAS; PFOA.

Fig. 1.. Study design and measured levels of PFOA and GenX in the serum and urine of developmentally exposed offspring at postnatal day (PND) 5.5.

(A) The study design encompassed about 22 weeks from start to finish. (B) PFOA or GenX concentrations in samples obtained from pooling serum from littermates (μg analyte/mL serum) and (C) PFOA or GenX concentration in samples obtained from pooling urine from littermates (μg analyte/mL urine) were determined by high performance liquid chromatography tandem mass spectrometry. Treatment group mean values are denoted with an “X” flanked above and below by error bars showing standard deviation and individual data points are shown as circles (N = 5 litters per group). Note: Vehicle control (VC) samples were quantified for PFOA and GenX. Vehicle control samples were run against a low concentration calibration curve with a limit of detection of 5 ng/mL. GenX and PFOA experimental samples were run against higher calibrations curve with a LOD of 100 ng/mL. All vehicle control samples were below the LOD of 5 ng/mL for both PFOA and GenX and all 0.1 mg/kg PFOA samples were below the LOD of 100 ng/mL for PFOA. Sufficient serum and urine sample quantities were achieved by pooling across pups within the same litter. Statistical comparisons across all treatment groups for serum and urine samples are shown in Supplemental Table 3.

Fig. 2.. Mixed model estimates of pup body weight (g) at PND 5.5.

Effect estimates are centered around the vehicle control group (y = 0) and show the model point estimate for each main effect (dose group and litter size) with 95% confidence intervals (CIs). *P < 0.05. **P < 0.01. ***P < 0.001. Beta estimate 95% confidence intervals do not overlap zero (mixed-effect model adjusting a priori for litter size as a fixed effect and the dam as a random effect, vehicle control as reference group). Adjusted estimates and 95% CIs are shown in Table S6. N = 4–19 offspring per litter with 9–10 total litters per group.

Fig. 3.. Mixed model estimates of weight gain (g) from PND 22 to 18 weeks of age.

(A) Low fat diet (LFD) male offspring, (B) high fat diet (HFD) male offspring, (C) LFD female offspring, and (D) HFD female offspring. Effect estimates are centered around the vehicle control group (y = 0) and show the model point estimate for each main effect (dose group) with 95% confidence intervals (CIs). *P < 0.05. **P < 0.01. ***P < 0.001. Beta estimate 95% confidence intervals do not overlap zero (mixed-effect model adjusting a priori for repeated measures of the same animals over time, vehicle control as reference group). Adjusted estimates and 95% CIs are shown in Table S13. N = 7–11 mice per group.

Fig. 4.. Body composition parameters at Week 17.

(A) Low fat diet (LFD) male offspring, (B) high fat diet (HFD) male offspring, (C) LFD female offspring, and (D) HFD female offspring grams of fat mass, fluid mass, and lean mass. Bars represent mean grams of mass per group calculated using LF90 Minispec Body Mass Analyzer. Additional data for males on LFD are shown in E) Relative fat mass expressed as percent total body mass, and F) Ratio of fat mass to lean mass. Circles represent individual data points within a treatment group (N = 7–11 mice/group) and horizontal dashes represent the group mean flanked by the standard deviation above and below in black error bars. *P < 0.05.

Fig. 5.. Examples of liver histopathology in mice at the 18-week timepoint.

(A) Liver from control male mouse fed LFD (20x). (B) Example of multifocal single cell hepatocyte necrosis in a liver from a 1.0 mg/kg/day GenX exposed male mouse fed HFD (40x). There are three foci of single cell necrosis with associated inflammatory cells (arrows). (C) Example of microvesicular fatty change in a liver from a 2.0 mg/kg/day GenX-exposed male mouse fed LFD (4x). Note the centrilobular and periportal bridging pattern (paler regions). (D) Higher magnification of panel C shows hepatocytes filled with numerous small lipid vacuoles giving a “foamy” appearance (40x). (E) Example of periportal macrovesicular fatty change from a female mouse gestationally exposed to 0.2 mg/kg/day GenX and fed HFD (20x). The affected hepatocytes contain large, well-defined rounded vacuoles, that displace the nucleus and cytoplasm to the periphery (arrows). Microvesicular fatty change (asterisk) and single cell necrosis (arrowhead) are also present. (F) Example of perivascular mixed cell infiltrates in a liver from a female mouse gestationally exposed to 0.2 mg/kg/day GenX and fed HFD (10x). Hematoxylin and eosin. LFD = low fat diet; HFD = high fat diet.

Fig. 6.. Heat map of white adipose tissue (WAT) mRNAs altered by PFOA or GenX at 18 weeks of age.

Messenger RNA expression was quantified by RT-qPCR using WAT from 18-week old mice prenatally exposed to either PFOA or GenX. Relative gene expression data are represented as mean fold change over VC in heatmap format, with red indicating an increase expression up to a 4-fold change and blue indicating a decrease in expression down to a 4-fold change, N = 4. Low fat and high fat denote the different diets. ANOVA with multiple comparisons Dunnett’s post hoc was performed on mean dCt values with statistical significance of *P < 0.1 and **P < 0.05.