Hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) alters maternal and fetal glucose and lipid metabolism and produces neonatal mortality, low birthweight, and hepatomegaly in the Sprague-Dawley rat

Abstract

Hexafluoropropylene oxide dimer acid (HFPO-DA or GenX) is an industrial replacement for the straight-chain perfluoroalkyl substance (PFAS), perfluorooctanoic acid (PFOA). Previously we reported maternal, fetal, and postnatal effects from gestation day (GD) 14-18 oral dosing in Sprague-Dawley rats. Here, we further evaluated the perinatal toxicity of HFPO-DA by orally dosing rat dams with 1-125 mg/kg/d (n = 4 litters per dose) from GD16-20 and with 10-250 mg/kg/d (n = 5) from GD8 - postnatal day (PND) 2. Effects of GD16-20 dosing were similar to those previously reported for GD14-18 dosing and included increased maternal liver weight, altered maternal serum lipid and thyroid hormone concentrations, and altered expression of peroxisome proliferator-activated receptor (PPAR) pathway genes in maternal and fetal livers. Dosing from GD8-PND2 produced similar effects as well as dose-responsive decreased pup birth weight (≥30 mg/kg), increased neonatal mortality (≥62.5 mg/kg), and increased pup liver weight (≥10 mg/kg). Histopathological evaluation of newborn pup livers indicated a marked reduction in glycogen stores and pups were hypoglycemic at birth. Quantitative gene expression analyses of F1 livers revealed significant alterations in genes related to glucose metabolism at birth and on GD20. Maternal serum and liver HFPO-DA concentrations were similar between dosing intervals, indicating rapid clearance, however dams dosed GD8 - PND2 had greater liver weight and gestational weight gain effects at lower doses than GD16-20 dosing, indicating the importance of exposure duration. Comparison of neonatal mortality dose-response curves between HFPO-DA and previously published perfluorooctane sulfonate (PFOS) data indicated that, based on serum concentration, the potency of these two PFAS are similar in the rat. Overall, HFPO-DA is a developmental toxicant in the rat and the spectrum of adverse effects is consistent with prior PFAS toxicity evaluations, such as PFOS and PFOA.

Keywords: Developmental toxicity; Glucose metabolism; In utero exposure; Low birthweight; PFAS; Peroxisome proliferator-activated receptor.

Figure 1.

Violin plots (heavy horizontal line=median, dashed horizontal lines=quartiles) of postnatal effects on pups exposed to maternal oral HFPO-DA from GD8-PND2. (A) Birthweight measured as average pup weight following completion of delivery on GD22/PND0. (B) Pup survival calculated as percent living on PND2 relative to number of liveborn pups. (C) Bodyweight (BW) on PND2 as litter average of all male and female pups. (D) Relative liver weight as average of one male and one female pup per litter on PND2. Asterisks represent significant difference compared to control (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

Figure 2.

Glycogen accumulation in newborn pup livers was graded by a Board Certified Veterinary Pathologist based on the percentage of hepatocytes containing glycogen. (A-D) Example photomicrographs of newborn pup liver sections stained with PAS from (A) control (grade 4), (B) control (grade 3), (C) exposure to 30 mg/kg HFPO-DA (grade 2), and (D) exposure to 250 mg/kg (grade 1). (E) Glycogen accumulation grade was significantly reduced in all dose groups. (F) Newborn pup serum glucose concentrations. Asterisks represent significant difference compared to control (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

Figure 3.

Violin plots (heavy horizontal line=median, dashed horizontal lines=quartiles) of serum cholesterol (A-C) and triglyceride (D-F) concentrations. (A,D) Newborn (PND0) concentrations following exposure from GD8 through delivery, (B,E) maternal concentrations from GD8-PND2 exposure, and (C,F) maternal concentrations from GD16–20 oral exposure to HFPO-DA. Asterisks represent significant difference compared to control (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001)

Figure 4.

Violin plots (heavy horizontal line=median, dashed horizontal lines=quartiles) of (A) maternal body weight gain and (B) relative liver weight normalized to control, and (C) serum HFPO-DA concentration, and (D) liver HFPO-DA concentration across two dosing intervals, GD16–20 (white bars) and GD8–22 or PND2 (shaded bars). Data for 1 and 3 mg/kg/d exposure during GD16–20 interval not shown (no significant difference from control). Asterisks represent significant difference compared to respective interval control (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). Hashes represent significant difference between intervals within a dose level (#, p<0.05; ##, p<0.01; ###, p<0.001; ####, p<0.0001).

Figure 5.

Heatmaps of significantly altered Glucose Metabolism array genes in fetal livers from oral maternal exposure to HFPO-DA from GD16–20. (A) Genes with ANOVA p<0.001 and (B) genes with ANOVA p<0.01. Cell values represent significant (pairwise p<0.05) fold induction as compared to control for that dose level. Legend indicates fold-induction compared with control (Ctl, fold=1) with darker shaded genes (with white text) beyond the scale of the legend (> 10-fold).

Figure 6.

Heatmaps of significantly altered Glucose Metabolism (A,C) and PPAR Signaling Pathway (B,D) array genes in newborn pup livers from oral maternal exposure to HFPO-DA from GD8 through delivery on GD22. (A,B) Genes with ANOVA p<0.001 and (C,D) genes with ANOVA p<0.01. Cell values represent significant (pairwise p<0.05) fold induction as compared to control for that dose level. Legend indicates fold-induction compared with control (ctl, fold=1) with darker shaded genes (with white text) beyond the scale of the legend (> 10-fold or < −10-fold).

Figure 7.

Maternal and fetal serum and liver HFPO-DA concentrations. (A) Log-linear regressions of HFPO-DA serum concentrations from GD14–18 (previously published in Conley et al. 2019) and GD16–20 exposure (current study). Maternal serum concentrations had a significant two-way ANOVA effect of dosing interval and are reported separately. Fetal serum concentrations were similar between intervals and are reported as combined. (B) Log-linear regressions of maternal (GD20), fetal (GD20), and neonatal (PND2 pup) liver HFPO-DA concentrations. GD20 fetal liver concentrations were not significantly different between male and female fetuses and are reported with sexes combined. PND2 liver concentrations had a significant two-way ANOVA affect of sex with female concentrations significantly lower than male concentrations, however both were ~10-fold lower than GD20 fetal liver concentrations.

Figure 8.

Dose response potency comparisons between HFPO-DA (current study) and PFOS (data from Lau et al. 2003 and Thibodeaux et al. 2003) for the effect of neonatal mortality following oral exposure to Sprague-Dawley rats during gestation. Dose response as functions of (A) oral dose, (B) serum concentration on mass/volume basis, and (C) serum concentration on molar basis. Table reports ED₅₀ or EC₅₀ parameter estimates and 95% confidence intervals. The two compounds vary by greater than 30-fold on an oral dose basis with PFOS considerably more potent than HFPO-DA; whereas, on a serum molar basis GenX is more potent with PFOS ~40% weaker.

Figure 9.

Violin plots (heavy horizontal line=median, dashed horizontal lines=quartiles) of margin of internal exposure ratios between the mean maternal rat serum concentrations from the present postnatal study and fluorochemical worker serum concentrations from a HFPO-DA manufacturing facility in Dordrecht, Netherlands (EPA HERO Database study ID: 4353920).