Out of the Frying Pan and into Fetal Development: Who’s Really Burnt by Unchecked Per- and Polyfluoroalkyl Substances

Introduction

Per- and Polyfluoroalkyl Substances (PFAS) are a class of chemicals that are commonly used in consumer products such as non-stick cookware, water repellent clothing, and cosmetic products including waterproof mascara and foundations due to their water and grease repellent properties (NIEHS, 2023). PFAS contain an alkyl chain with at least one fully fluorinated carbon atom (Figure 1A) and they are known as “forever chemicals” as the strong carbon-fluorine bonds give these chemicals resistance within the environment (NIEHS, 2023). As a result, these chemicals are known to bioaccumulate in humans and animals (Pelch, et al, 2019). In Canada, Perfluorooctane Sulfonate (PFOS), Perfluorooctanoic Acid (PFOA), and Long-Chain Perfluorocarboxylic Acids (LC-PFCAs) are prohibited for manufacture, sale, and use (Government of Canada, 2023). This regulation was determined in 2016 following research which characterized the adverse human and environmental effects these PFAS have. Specifically, attention was brought to PFOA because of its use in Teflon for non-stick cookware by the company DuPont (Soechtig, 2018). In the early 2000’s, Parkersburg, West Virginia, the town in which Teflon was produced, observed abundant cases of human cancers and developmental defects upon birth (Soechtig, 2018). This brought more attention to the adverse effects of PFOA. 

Human cytochrome P450s (CYP450s) are a family of heme-thiolate monooxygenases that metabolize both endogenous and exogenous substrates (Li and Lampe, 2019). The CYP3A is a subfamily of CYP450s that play a significant role in the metabolism of exogenous drugs. CYP3A7 is an isoform that is predominantly present within fetal liver tissue but also found in other fetal tissues such as the adrenal gland, kidney, and lung (Li and Lampe, 2019). This enzyme plays an important role in fetal development as it metabolizes both endogenous and exogenous compounds in the developing liver (Hvizdak, et al, 2023). Some of these endogenous compounds include dehydroepiandrosterone 3-sulfate (DHEA-S) which is essential to estradiol synthesis during pregnancy and the morphogen, all-trans-retinoic acid (atRA) (Figure 1B) (Hvizdak, et al, 2023). Both of these compounds are essential to the development of the embryo. Like fatty acids, PFAS have long alkyl chains. Some PFAS also contain particular functional groups such as the sulfonic acid head group as seen in DHEA-S or the carboxylic head group as seen in atRA (Hvizdak, et al, 2023). Due to these structure similarities, the effect that PFAS can have on the CYP3A7 enzyme is a concern that will be analyzed in this paper to explore the risk that these chemicals may have on human fetal development. 

Figure 1. Structure of PFAS. (A) Chemical structures of PFAS including Perfluorooctanoic Acid (PFOA), Perfluorooctane Sulfonate (PFOS), the ammonium salt of hexafluoropropylene oxide dimer acid (GenX), Perfluorononanoic Acid (PFNA), Perfluorononanesulfonic acid (PFNS), and Perfluorohexanesulphonic acid (PFHxS). (B) The chemical structures of the endogenous compounds of CYP3A7, dehydroepiandrosterone 3-sulfate (DHEA-S) and all-trans-retinoic acid (atRA). Modified from Hvizdak, et al, (2023).

PFAS Inhibit the Activity of the CYP3A7 Enzyme Within in vitro Conditions

Given the structure similarity of PFAS to the endogenous compounds of CYP3A7 and the association of exposure to PFAS with developmental defects, the ability of these PFAS to bind and alter the activity of the CYP3A7 fetal enzyme was questioned. Hvizdak, et al (2023) performed in vitro binding assays of purified CYP3A7 and six different PFAS including, PFOA, PFOS, GenX, PFNA, PFNS, and PFHxS. The binding of the PFAS was measured through UV-visible difference spectroscopy from which the dissociation constant (Kd) could be calculated from. PFOA, PFOS, PFNA, and PFHxS all resulted in a unique UV-vis spectroscopy spectra showing characteristics negative cooperativity with initial Type I binding of the CYP37A heme group followed by reverse Type I binding upon greater concentrations of the added PFAS (Hvizdak, et al, 2023). Type I and reverse Type I binding involves the displacement of the water molecule within the heme group of the enzyme by the substrate. However, reverse Type I binding further involves an interaction of a heteroatom on the substrate with the heme iron (Hvizdak, et al, 2023). Both of these binding activities produce a unique binding spectra. The reverse Type I binding activity demonstrated a stronger interaction with the active site of CYP3A7 as it produced lower Kd values in the case of all the PFAS (Figure 2A). Additionally, longer chained PFAS that contain a sulfonic acid head showed to bind with greater affinity. This was the case with PFOS which bound with the lowest Kd values (Figure 2A). Notably, GenX and PFNS did not produce a UV-vis spectroscopy spectra and thus, are assumed to not bind CYP3A7 in vitro. From these results, in-silico binding configurations were produced (Figure 2B), from which the higher affinity reverse Type I binding shows the hydrophilic group on PFAS interacts with the heme iron.

Many known inhibitors of CYP450 enzymes have shown to bind through reverse type I geometry (Shimada, et al, 2009). Therefore, it is necessary to determine if the binding of PFAS inhibits the activity of the CYP3A7 enzyme. Hvizdak, et al (2023) demonstrated that all four of the PFAS that bind to CYP3A7, inhibit its oxidative activity (Figure 2C). This was shown using a known fluorescent substrate of CYP450 enzymes, Dibenzylfluorescein (DBF) and measuring its oxidation upon the addition of PFAS. PFOS and PFNA showed the greatest inhibition of the CYP3A7 enzyme (Figure 2C) with PFOS having the lowest half maximal inhibitory concentration (IC50) of 20.39uM (Hvizdak, et al, 2023). Additionally, PFOS showed to inhibit the hydroxylation activity of the endogenous substrate DHEA-S in vitro in a similar manner, obtaining an IC50 value of 16.75uM (Hvizdak, et al, 2023). This data provides evidence that PFAS can bind to and inhibit the activity of the CYP3A7 fetal enzyme in vitro. However, it remains to be determined if this is representative of an in vivo system and if these concentrations are biologically relevant within humans.  

Figure 2. Binding of PFAS to CYP3A7. (A) The dissociation constants (Kd) in uM for PFOA, PFOS, PFNA, and PFHxS upon binding to CYP3A7 with reverse Type I and Type I binding. The 95% confidence intervals are shown. (B) The in-silico binding configurations of (A&B) PFOA, (C&D) PFOS, (E&F) PFNA, and (G&H) PFHxS showing reverse Type I binding on the left (A,C,E,G) and Type I binding on the right (B,D,F,H). (C) Percent inhibition of the CYP37A enzyme oxidation of fluorescent Dibenzylfluorescein (DBF) substrate upon exposure to PFOA, PFOS, PFNA, and PFHxS at 10uM and 100uM. Modified from Hvizdak, et al, (2023).

The Result of Exposure to PFAS in Rats and Mice Could Point Toward Issues in Fetal Development upon Human Exposure

To determine if PFAS pose a risk to human fetal development it is necessary to analyze in vivo data. Through prenatal exposure of PFAS to pregnant rats and mice, a more comprehensive measure as to whether or not PFAS cause adverse in vivo effects can be obtained. Lau, et al, (2003) exposed pregnant mice and rats to different concentrations of PFOS and observed the survival, growth, and development of the newborn rodents. The pregnant rats were exposed to different concentrations of PFOS ranging from 1-10 mg/kg and the pregnant mice were exposed to concentrations of PFOS ranging from 1-20 mg/kg. PFOS was administered daily through ingestion for about 20 days for each group and upon birth, the newborns were observed for survival and their growth and development was monitored over time. The survival of the rat pups was significantly impacted at concentrations of PFOS as low as 2 mg/kg with survival decreasing upon greater concentrations of PFOS (Figure 3A) (Lau, et al, 2003). More than 95% of the rat pups failed to survive for 24 hours following birth. A similar result was observed in newborn mice where greater concentrations of PFOS resulted in decreased survival of the newborns (Figure 3B). A significant effect on newborn survival was observed for mice treated with 15 mg/kg and 20 mg/kg (Lau, et al, 2003). For the newborn rat pups and mice that survived for longer than a few days, the body weight gain was much lower than the controls and the development measured by eye opening was delayed by about a day for the rat pups treated with 2 mg/kg or more of PFOS (Lau, et al, 2003). These results indicate that prenatal exposure to PFAS, specifically PFOS has adverse effects on the fetal survival, growth, and development of rats and mice. The difference in tolerance levels between the rats and mice is interesting and more research is needed to determine the tolerance level in pregnant humans. Observational studies with pregnant humans have shown decreased birth weight and delayed development of newborns upon prenatal exposure to PFAS (Szilagyi, et al, 2020). Thus, the results of exposure to PFAS in rats and mice combined with the results from observational studies could point toward issues in fetal development upon human exposure to PFAS. 

Figure 3. Postnatal survival of rats and mice upon exposure to PFOS. (A) The percent of rat pups that survived after prenatal exposure to PFOS at concentrations ranging from 1 mg/kg to 10 mg/kg measured over the days after birth. (B) The percent of mice that survived after prenatal exposure to PFOS at concentrations ranging from 1 mg/kg to 20 mg/kg measured over the days after birth. Modified from Lau, et al, (2003).

There Should be Stronger Regulations of PFAS and more Research Surrounding the Safety of Newer PFAS being used as Replacements

The toxicity effects of PFAS didn’t come to the attention of policy makers and governments until the early 2000’s. PFOS was being detected in aquatic ecosystems and food webs and thus, it was determined that human exposure was mainly occurring through seafood and drinking water (Sunderland, 2019). The U.S. Centers For Disease Control and Prevention (CDC) determined that PFOS, PFOA, PFHxS, and PFNA was present in the tissues of 98% of Americans in 2003 to 2004 (Calafat, et al, 2007). In 2009, PFOS was added to the Stockholm’s Convention list of globally restricted Persistent Organic Pollutants (POPs) and as of 2016, the manufacture, use, and sale of PFOA, and LC-PFCAs have been prohibited in Canada with some exceptions. For instance, they can still be used in some aqueous film forming foams (AFFF) that are used as fire suppressants (Government of Canada, 2023). While these regulations have been successful in decreasing human exposures to PFOA, PFOS, and LC-PFCAs, companies have since transitioned to using different PFAS which serve as replacements within consumer products (Sunderland, 2019). Some of these replacement PFAS include hexafluoropropylene oxide dimer acid (GenX), 6:2 chlorinated polyfluorinated ether sulphonate (6:2 Cl-PFAES), and perfluoroethylcyclohexane sulphonate (PFECHS) (Mahoney, 2022). There is very little research surrounding the toxicity effects of these replacement PFAS in human health and the environment regardless of their use in common consumer products such as non-stick cookware, food wrappers, and cosmetics (Sunderland, 2019). The detection and quantification of PFAS in consumer products is a difficult task due to a lack of analytical methods to achieve this. In addition, the presence of many PFAS in consumer products may be unintentional due to contamination during the manufacturing process of these products (Dewapriya, et al, 2023). While novel PFAS such as GenX has not yet been detected in the tissues of humans at significant concentrations (Mahoney, 2022), with persistent exposure, bioaccumulation is a concern. Especially as these PFAS are present within consumer products that humans are exposed to everyday, the risk of exposure to pregnant humans is unavoidable. As the research surrounding these replacement PFAS is still limited, it is not known if they pose the same risk to fetal development as PFAS such as PFOA and PFOS. However, given our history with these well-studied PFAS, there should be stronger regulations on the use of replacement PFAS in common consumer products and further research into the health and environmental toxicity of these compounds.

Conclusions

Per- and Polyfluoroalkyl Substances (PFAS) are a class of chemicals that are highly resistant within the environment due to their strong carbon to fluorine bonds (NIEHS, 2023). Through observational studies, it has been shown that these chemicals may be of risk to human fetal development (Szilagyi, et al, 2020). However, there is no current research that shows a direct cause of abnormal human fetal development to prenatal exposure of PFAS. The in vitro binding assays of PFAS with the fetal CYP3A7 enzyme demonstrate the ability of particular PFAS to bind to the heme active site of the enzyme and inhibit its oxidative activity (Hvizdak, et al, 2023). If these results are representative of an in vivo system, then this would provide a direct link to PFAS and fetal development as CYP3A7 oxidizes endogenous compounds such as DHEA-S and atRA which play essential roles in development (Hvizdak, et al, 2023). The results of prenatal PFAS exposure to rats and mice provide in vivo evidence that these chemicals have a negative effect on fetal survival, growth and development (Lau, et al, 2003). The discrepancy of tolerance to PFAS between rats and mice provides reason that further research needs to focus on the tolerance of PFAS within humans and other species. Additionally, further research should investigate if this abnormal fetal development is partly caused by the inhibition of CYP3A7 by the PFAS. The manufacture, use, and sale of PFOA, PFOS, and LC-PFCAs have been prohibited in Canada however, companies have resorted to using replacement PFAS such as GenX, 6:2 Cl-PFAES, and PFECHS in consumer products such as water repellent clothing, non-stick cookware, and cosmetic products (Sunderland, 2019). As there is little research about the toxicity of these replacement PFAS within humans, there needs to be stronger regulations surrounding the use of these chemicals given how likely it is that pregnant humans are exposed through the use of everyday consumer products. Additionally, future research should investigate the safety of these replacement PFAS in the context of their effects on fetal development. 

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