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Evaluation of Maternal, Embryo, and Placental Effects in CD-1 Mice following Gestational Exposure to Perfluorooctanoic Acid (PFOA) or Hexafluoropropylene Oxide Dimer Acid (HFPO-DA or GenX)

Bevin E. Blake, Harlie A. Cope, Samantha M. Hall, Robert D. Keys, Beth W. Mahler, James McCord, Brittany Scott, Heather M. Stapleton, Mark J. Strynar, Susan A. Elmore, Suzanne E. Fenton
Environmental Health Perspectives(2020) DOI: https://doi.org/10.1289/EHP6233 PMID: 32074459 PMCID: PMC7064328
DOI: https://doi.org/10.22427/NTP-DATA-021-00006-0001-0000-0

Publication

Abstract

Background: Perfluorooctanoic acid (PFOA) is a perfluoroalkyl substance (PFAS) associated with adverse pregnancy outcomes in mice and humans, but little is known regarding one of its replacements, hexafluoropropylene oxide dimer acid (HFPO-DA, referred to here as GenX), both of which have been reported as contaminants in drinking water.

Objectives: We compared the toxicity of PFOA and GenX in pregnant mice and their developing embryo-placental units, with a specific focus on the placenta as a hypothesized target.

Methods: Pregnant CD-1 mice were exposed daily to PFOA (0, 1 or 5 mg/kg) or GenX (0, 2 or 10 mg/kg) via oral gavage from embryonic day (E) 1.5 to 11.5 or 17.5 to evaluate exposure effects on the dam and embryo-placental unit. Gestational weight gain (GWG), maternal clinical chemistry, maternal liver histopathology, placental histopathology, embryo weight, placental weight, internal chemical dosimetry, and placental thyroid hormone levels were determined.

Results: Exposure to GenX or PFOA resulted in increased GWG, with increase in weight most prominent and of shortest latency with 10 mg/kg/d GenX exposure. Embryo weight was significantly lower after exposure to 5 mg/kg/d PFOA (9.4% decrease relative to controls). Effect sizes were similar for higher doses (5 mg/kg/d PFOA and 10 mg/kg/d GenX) and lower doses (1 mg/kg/d PFOA and 2 mg/kg/day GenX), including higher maternal liver weights, changes in liver histopathology, higher placental weights and embryo-placental weight ratios, and greater incidence of placental abnormalities relative to controls. Histopathological features in placentas suggested that PFOA and GenX may exhibit divergent mechanisms of toxicity in the embryo:placental unit, whereas PFOA- and GenX-exposed livers shared a similar constellation of adverse pathological features.

Conclusions: Gestational exposure to GenX recapitulated many documented effects of PFOA in CD-1 mice, regardless of its much shorter reported half-life; however adverse effects towards the placenta appear to have compound-specific signatures.

Figures

Figure 1. Internal dosimetry of PFOA and GenX (also HFPO-DA) in maternal serum and liver at embryonic day (E) 11.5 and E17.5

(A) Maternal serum concentration (µg PFOA/ mL serum) at E11.5 and E17.5, (B) maternal serum concentration (µg GenX/ mL serum) at E11.5 and E17.5, (C) maternal liver concentration (µg PFOA/ g liver) at E11.5 and E17.5, and (D) maternal liver concentration (µg GenX/ g liver) at E11.5 and E17.5 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 grey circles (N = 6-8). Vehicle control (VC) samples were quantified for PFOA and GenX; all VC means were below the LOD of 10 ng/mL for both PFOA and GenX except for maternal serum (0.211 ± 0.55 µg/mL). Statistical comparisons of internal dosimetry across all treatment groups are shown in Tables S2 and S3.

Figure 2. Internal dosimetry of PFOA and GenX (also HFPO-DA) in amniotic fluid and whole embryos

(A) Amniotic fluid concentration (µg PFOA/ mL amniotic fluid) at embryonic day (E) 11.5, (B) whole embryo concentration (µg PFOA/ g embryo) at E11.5 and E17.5, (C) amniotic fluid concentration (µg GenX/ mL amniotic fluid) at E11.5, and (D) whole embryo concentration (µg GenX/ g embryo) at E11.5 and E17.5 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 grey squares, circles, or triangles (N = 6-8). Triangles = E17.5 male embryos, circles = E17.5 female embryos, and squares = pooled E11.5 embryos (B and D). Vehicle control (VC) samples were quantified for PFOA and GenX; all VC means were below the LOD of 10 ng/mL for both PFOA and GenX. Statistical comparisons of internal dosimetry across all treatment groups are shown in Tables S2 and S3.

Figure 3. Gestational weight gain (GWG) repeated measure mixed effect model estimates for pregnant dams exposed to PFOA or GenX (also HFPO-DA)

Effect estimates for pregnant dams exposed through embryonic day 11.5 (A) or 17.5 (B) are centered around the vehicle control group (y = 0) and show the point estimate of the relative change in dam weight (% change from E0.5) with 95% confidence intervals (CI). (C) Boxplots of relative weight gain over time, with the upper and lower hinges corresponding to the first and third quartiles (25th and 75th percentiles), the middle hinge corresponding to the median, and the upper whisker extending to the highest value that is within 1.5 times the distance between the first and third quartiles (inter-quartile range, IQR) of the hinge and the lower whisker extending to the lowest value within 1.5 times the IQR of the hinge. N = 11-13 dams per treatment group. *P < 0.05, **P < 0.01, ***P <0.001; Beta estimate 95% confidence intervals do not overlap zero (Repeated measures mixed effect model adjusting a priori for litter size and gestational (embryonic) day as fixed effects and the dam as a random effect, vehicle control as reference group)

Figure 4. Light and transmission electron microscopy (TEM) of liver from vehicle control (VC) and PFOA-exposed pregnant dams at embryonic day (E) 17.5

Light microscopic image (A) at 40X magnification of liver from a VC pregnant dam (control) showing centrilobular hepatocellular hypertrophy with karyomegaly, increased basophilic granular cytoplasm and decreased glycogen. Corresponding (B) TEM magnification shows prominent rough endoplasmic reticulum (arrows) with abundant ribosomes and evenly dispersed, abundant glycogen (asterisk) (see Figure S2A). Light microscopic image (C) at 40X magnification of liver from a pregnant dam at E17.5 and treated with 1 mg/kg/day PFOA. Although this liver appears to be within normal limits when viewed with light microscopy, TEM (D) reveals an increase in scattered vacuoles (see Figure S2B), decreased, evenly dispersed glycogen (asterisks), as well as abundant mitochondria (arrows) and peroxisomes (arrowheads). Light microscopic image (E) at 40X magnification of liver from a pregnant dam at E17.5 and treated with 5 mg/kg/day PFOA. Increased cytoplasmic vacuoles are evident at this light microscopic level. TEM (F) reveals abundant cytoplasmic organelles consistent with mitochondria (M) and peroxisomes (P), extensive vacuoles (V), less prominent rough endoplasmic reticulum (arrows) with fewer ribosomes and less abundant glycogen (see S2C and S2D) TEM = transmission electron microscopy, NU = nucleolus, N = nucleus.

Figure 5. Light and transmission electron microscopy (TEM) of liver from vehicle control (VC) and Gen-X -exposed pregnant dams at embryonic day (E) 17.5

Light microscopic image (A) at 40X magnification of liver from a VC pregnant dam showing centrilobular hepatocellular hypertrophy with karyomegaly, increased basophilic granular cytoplasm and decreased glycogen. Corresponding medium (B) TEM magnification shows prominent rough endoplasmic reticulum (arrows) with abundant ribosomes and evenly dispersed, abundant glycogen (asterisk) (see Figure S2A). Light microscopy at 40X magnification (C) and transmission electron microscopy (D) of liver from a pregnant dam at E17.5 treated with 2 mg/kg/day GenX (also HFPO-DA) or 10 mg/kg/day GenX (E and F). Marked cytoplasmic alteration is evident in (C) and (E). TEM (D and F, see Figure S2E and S2F, respectively) reveals an abundance of cytoplasmic organelles, consistent with mitochondria (M) and peroxisomes (P), that increase with increasing dose (D compared to F). Note also the decreased glycogen (asterisks) as well as the vacuole (V) and rough endoplasmic reticulum (arrows) and N = nucleus.

Figure 6. Mixed effect model estimates for (A) embryo weight (mg), (B) placental weight (mg), & (C) embryo:placental weight ratios (mg:mg) after exposure in utero to PFOA or GenX at E17.5

Effect estimates are centered around the vehicle control group (y = 0) and show the point estimate of the relative change in weight (mg; A & B) or weight ratio (mg:mg; C) with 95% confidence intervals (CI). *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% CI are shown in Table S8.

Figure 7. Representative examples of histopathological placenta findings observed in dams at embryonic day (E) 11.5 and E17.5, treated with PFOA or GenX (also HFPO-DA)

A) Normal labyrinth from a vehicle control dam at E17.5. (B) Labyrinth congestion in a dam at E17.5 that was treated with 10 mg/kg/day GenX (C) Moderate labyrinth atrophy of the trilaminar trophoblast layer at E17.5 in a dam treated with 10 mg/kg/day GenX. (D) Labryrinth necrosis (arrows) in a E17.5 dam that was treated with 10 mg/kg/day GenX. All images at 20X magnification.

Figure 8. Incidence of placenta lesions across treatment groups at embryonic day 17.5

N = 5-6 litters with 31-41 placentas evaluated per treatment group (an average of 6-8 placentas per litter). Incidence values <4% are not numerically indicated, but all values and statistical comparisons of placenta lesion incidences across treatment groups at E17.5 are shown in Table S10.

Tables

Table 1. Maternal indices at embryonic day 11.5 and 17.5 (Mean ± SD, N = 11-13)

Table 2. Clinical chemistry panel of dam serum at embryonic day 11.5

Table 3. Clinical chemistry panel of dam serum at embryonic day 17.5

Table 4. Placental thyroid hormone measurements at embryonic day 17.5

Supplemental: Tables

Table S1. Number of total observations and litters represented in mixed effect models (observations, litters)

Table S2: Internal dosimetry of tissues at embryonic day 11.5 including maternal serum, maternal serum, maternal liver, amniotic fluid and whole embryo (Mean ± SD, N=6-8)

Table S3. Internal dosimetry of tissues at embryonic day 17.5 including maternal serum, maternal liver, male whole embryo and female whole embryo (Mean ± SD, N=6-8)

Table S4. Litter parameters in mice gestationally exposed to PFOA or GenX from embryonic day 0.5 to 11.5 or 17.5 (Mean ± SD, N=11-13)

Table S5. Relative gestational weight gain (% gain from embryonic day 0.5) at necropsy adjusting for litter size (adjusted model estimate and 95% confidence intervals; N=11-13)

Table S6. Incidence of liver histopathology in maternal livers at embryonic day 11.5

Table S7. Incidence of liver histopathology in maternal livers at embryonic day 17.5

Table S8. Embryo and placental mixed effect model adjusted estimates and 95% confidence intervals (N=11-13 dams with 62-80 observations per group)

Table S9. Placental lesion index at embryonic day 11.5

Table S10. Placental lesion incidence at embryonic day 17.5

Table S11. Sex stratified placental thyroid hormone measurements at embryonic day 17.5 (Mean ± SD, N=1-3)

Supplemental: Figures

Figure S1. Representative examples of liver histology in pregnant dams at gestation days 11.5 & 17.5 exposed to either vehicle control (A, B, C, D) or treated with GenX (E, F) or PFOA (G, H)

(A) Liver from a pregnant dam at 11.5 days of gestation, exposed to vehicle control (4X). (B) Higher magnification of (A) illustrating the normal uniform hepatocellular size and cytoplasmic glycogen accumulation (20X). (C) Example of a liver from a pregnant dam at 17.5 days of gestation, exposed to vehicle control. The features of centrilobular hepatocellular hypertrophy (arrows), karyomegaly, increased mitotic figures, decreased glycogen, and increased basophilic granular cytoplasm are normal features for dam livers at this stage of pregnancy (4X). (D) Higher magnification of (C) illustrating the increased mitotic figures (arrow) decreased glycogen, and increased basophilic granular cytoplasm in the areas of centrilobular hepatocellular hypertrophy (20X). (E) Example of a liver from a pregnant dam at 11.5 days of gestation, exposed to 10 mg/kg/day of GenX. There is diffuse moderate cytoplasmic alteration in this liver affecting the centrilobular, midzonal and periportal regions (4X). (F) Higher magnification of (E) illustrating the hepatocellular hypertrophy with decreased glycogen and eosinophilic granular cytoplasm. The arrows show examples of early hepatocellular apoptosis with condensed cytoplasm and condensed dark basophilic nuclear chromatin (20X). (G) Example of a liver from a pregnant dam at gestation day 17.5, exposed to 5 mg/kg/day PFOA with diffuse cytoplasmic alteration (4X). (H) Higher magnification of the boxed region in (G) showing cytoplasmic alteration with apoptosis (arrowheads) as well as accumulation of hepatocellular cytoplasmic small vacuoles with distinct borders (arrow; 20X).

Figure S2. Transmission electron microscopy (TEM) of normal liver and livers exposed to PFOA or GenX (also HFPO-DA)

A) TEM of normal liver from a vehicle control pregnant dam at gestation day 17.5 showing prominent rough endoplasmic reticulum with abundant ribosomes and evenly dispersed, abundant glycogen (see Figures 4A or 5A H&E and 4B or 5B TEM). (B) TEM of liver from a pregnant dam at gestation day 17.5 and treated with 1 mg/kg/day PFOA. Although at 40X magnification light microscopy this liver appeared to be within normal limits (see Figures 4C H&E and D TEM), TEM reveals increased vacuolation (V), evenly dispersed glycogen, as well as abundant mitochondria and peroxisomes. (C and D) TEM of liver from a pregnant dam at gestation day 17.5 and treated with 5 mg/kg/day PFOA (see figures 4E H&E and 4F TEM). Note the abundant cytoplasmic organelles consistent with mitochondria (M) and peroxisomes (P), extensive vacuoles (V), less prominent rough endoplasmic reticulum (arrow) with fewer ribosomes and less abundant glycogen (asterisk). (E and F) Transmission electron microscopy of liver from a pregnant dam at gestation day 17.5 treated with 2 mg/kg/day GenX (E; see Figures 5C H&E and 5D TEM) or 10 mg/kg/day GenX (F; see Figures 5E H&E and 5F TEM). Note the abundance of cytoplasmic organelles consistent with mitochondria (M) and peroxisomes (P). K = Kupffer cell, N = nucleus, NU = nucleolus.

Figure S3. Representative examples of occasional histopathological placenta findings observed in dams at gestation day 17.5

(A) Early clot formation in a maternal artery in the decidua region of the placenta (20X). This dam was at gestation day 17.5 and treated with 10 mg/kg/day GenX (also HFPO-DA). Note the fibrin formation with trapped cells. (B) Nodule (arrow) of tissue from the junction zone of the placenta from a dam at gestational day 17.5 that was treated with 2 mg/kg/day GenX (2X).

Figure S4. Comparison of maternal serum or plasma levels (mean ± SD) in CD-1 mice gestationally exposed to GenX at varying dose levels in a study conducted by DuPont & the present study

Maternal serum or plasma was collected less than 6 hours after oral gavage in both studies. Administered GenX dose and maternal serum or plasma concentration was linearly correlated across data from both studies (R2 = 0.959, P < 0.05 for non-zero slope).