PYRETHROID PESTICIDE EXPOSURE AND PLACENTAL EFFECTS

Abstract

Human exposures to pyrethroid pesticides have increased in recent years following the bans and sanctions placed on other families of pesticides. Although pyrethroids are currently widely used across the United States and throughout the world, and their overt neurological toxicity classified, the extent of their toxicity through low dose and chronic exposures on humans is less well characterized, particularly when it comes to prenatal exposures, their impacts on neurodevelopment, and any role for the placenta in those effects. In this review, we assess the state of research on pyrethroid pesticide exposure and placental effects. These studies presented hormone disrupting, genotoxic, neurodevelopmental and neurobehavioral effects, among others, following prenatal pyrethroid exposures, and highlights a need for future research to assess gaps relating to effects in the human placenta and mechanisms of toxicity as well as shortcomings in the reproducibility and standardization of the methodologies presented.

INTRODUCTION

Pyrethroid insecticides are a class of organic compounds that are synthetically derived from pyrethrins, naturally occurring insecticides found in certain chrysanthemum plants ((ATSDR), 2003; Burns & Pastoor, 2018). The synthesis of pyrethroids involves the addition of a wide array of acid and alcohol moieties, which allows for the creation of a range of pyrethroids with diverse chemical structures (Ujihara, 2019). Distinctions in these structures allow pyrethroids to be classified into two types: type I and type II. Type I pyrethroids lack an α-cyano moiety, while type II pyrethroids have this group (Nasuti et al., 2003). This difference in chemical structure imparts distinct chemical and physical properties to these two classes of pyrethroids, which then affects their mechanism of toxicity in biological systems (Soderlund et al., 2002). These two sub-classes of pyrethroid generally correspond to two poisoning syndromes, with type I pyrethroids causing tremor (T) syndrome and type II pyrethroids causing choreoathetosis with salivation (CS) syndrome in most cases, while a minority of both type I and type II pyrethroids cause symptoms characteristic of both syndromes (Nasuti et al., 2003; Soderlund, 2020). Some common pyrethroids are: allethrin, bifenthrin, cypermethrin, deltamethrin, fenvalerate, permethrin, and resmethrin, among others.

Pyrethroids are highly lipophilic, which allows them to cross the blood-brain barrier and deposit into the brain, where they can act in their key role as neurotoxicants (Carloni et al., 2013). Primarily, pyrethroids target Na⁺, Ca⁺, and Cl⁻ voltage-gated channels, GABA-gated chloride channels, acetylcholine nicotinic receptors, and intracellular gap junctions (Markus et al., 2018; Singh et al., 2016; Watkins et al., 2016). Cypermethrin, a type II pyrethroid that affects Na⁺ channels by altering their opening and closing, causes repetitive discharges and synaptic disturbances. It also interacts with Cl⁻ and K⁺ channels, causing hyperexcitability (Markus et al., 2018). Differential expression of isoforms of these channels in developing and newborn mammals as compared to adults also creates an increased risk of neurotoxicity from pyrethroids (Markus et al., 2018).

Beyond their canonical neurotoxicant properties, pyrethroids have also been shown to induce oxidative stress; in fact, exposure to different antioxidants has been shown to reduce the damaging oxidative effects seen with pyrethroid exposure (Imanishi et al., 2013). Another pyrethroid, fenvalerate, has been established as a reproductive and developmental toxicant, with recent evidence pointing toward its role as an environmental endocrine disruptor (Wang et al., 2017; Zhang et al., 2010).

Pyrethroid insecticides have many applications, with uses in agricultural, residential, and veterinary contexts in both indoor and outdoor environments (Saillenfait et al., 2015). However, due, in part, to changing regulations on pesticide use, human exposure to pyrethroids has increased over the years (Watkins et al., 2016). For urban populations, ingestion is the main route of exposure (Elser et al., 2022; Xu et al., 2020). Pyrethroid exposure may also occur more frequently in indoor environments, as a lack of exposure to sunlight and moisture allows them to persist for longer periods of time (Watkins et al., 2016). Beyond dietary ingestion, children are also exposed to pyrethroids through the ingestion of dust and through dermal absorption, if they are in an area where pyrethroid applications are frequent (Viel et al., 2015). Esterases in the human body metabolize pyrethroids and eliminate them with a half-life of about 6.5 hours. Thus, at low exposure levels, pyrethroids are seen as less harmful in terms of human toxicity as other pesticides due to their fast elimination (Ding et al., 2015).

Exposure to pyrethroids early in the life course, particularly during gestation where critical periods of neurodevelopment occur, presents an exposure window of interest, given the unique vulnerabilities present to both the mother and the fetus during this time. Despite the vulnerabilities developing fetuses may have toward pyrethroids, few studies on pyrethroid exposure during gestation are currently present in the literature. Furthermore, these studies also present conflicting results (Ding et al., 2015), and there has been limited discussion about potential modes of action, beyond their neurotoxic mechanisms, that could be involved in the multiple outcomes that have been associated with pyrethroid exposures. One of these additional modes of action could be through direct impacts on the function of the placenta, the master regulator of fetal development and developmental programming, owed to a variety of critical functions in nutrient, water, gas and waste transport, metabolism, and barrier functions. In this review, we will provide an overview of the existing literature with a particular focus on impacts to the placenta, given the critical role of the placenta in pregnancy, in utero development, and lifelong health programming.

METHODS

This review focuses on research relating to the effects of exposure to pyrethroid pesticides on the human placenta. We used both PubMed and Google Scholar to search for toxicological publications that evaluated placental-mediated mechanisms of pyrethroid toxicity and any associated health outcomes. This review encompasses research published from 2010 to 2023 in an effort to reflect the current body of research relating to the effects of pyrethroid exposure on the placenta. The list of terms used to search for the articles included in this review is present in the Supplementary Material. Papers included in this review are peer-reviewed articles originally published in English, selected with a focus on pyrethroid effects on the placenta but also including broader search terms such as pyrethroid and fetal to be more inclusive. A full listing of the search terms is provided in the supplementary materials. Initial screening of the databases identified 62 possible studies of interest. Further screening was performed by reading paper abstracts and then the full articles to ultimately include in this review those which mentioned any effects of pyrethroids on the placenta or discussed any findings in relation to the placenta.

Toxicology studies on model animal systems were also included in this review as a supplement to the epidemiologic studies presented here. These studies offer a unique perspective on the toxicological effects of mammalian pyrethroid exposure at a wide range of dosage levels, which can mimic human exposure levels and inform human health outcomes. Additionally, in vitro studies included in this review inform assessments of pyrethroid toxicities in various model systems. They then help uncover biologically plausible mechanisms behind pyrethroid toxicity.

RESULTS & DISCUSSION

Following the inclusion criteria listed above, this review includes 15 studies (Table 1), consisting of eight studies based on animal models, four epidemiological studies, and three in vitro studies. While some of these studies were unspecific in the compounds they investigated and reported looking at pyrethroids as a whole or common metabolites of different pyrethroid compounds, others focused on one or two compounds in the pyrethroid family. Specifically, these studies looked at bifenthrin, cypermethrin, deltamethrin, fenvalerate, and permethrin. It is worth noting that two of the studies on cypermethrin focused solely on α-cypermethrin while the other three did not specify beyond cypermethrin.

Table 1.

Studies included in review of pyrethroid effects to the placenta.

Authors	Year	Country			Pesticide(s)
Epidemiological Studies
			Sample Size (N)
Chevrier et al.	2019	South Africa	751		Metabolites (3-PBA, 4-F-3-PBA, cis-DCCA and trans-DCCA, cis-DBCA)
Matsuki et al.	2020	Japan	93,718		Pyrethroids (unspecified)
Schmidt et al.	2016	USA	47		Pyrethroids (unspecified)
Zhang et al.	2014	Japan	147		Metabolites (3-PBA)
in vitro Studies
			Model	Tissue
Markus et al.	2018	Turkey	hpGSTP1–1 enzyme	Placenta	Deltamethrin
Mathiesen et al.	2020	Denmark	BeWo b30 cell line	Placenta	Cypermethrin
Zhao et al.	2014	China	JEG-3 cell line	Placenta	Bifenthrin
Animal Studies
			Model	Tissue(s)
Elser et al.	2020	USA	CD1 mice	Amniotic fluid, placenta Fetal: brain, body Maternal: liver, serum	α-Cypermethrin
Elser et al.	2022	USA	CD-1-IGS mice	Amniotic fluid, fetal body, placenta Maternal: serum, liver, ovaries, brain	α-Cypermethrin and permethrin
Guo et al.	2019	China	ICR mice	Placenta	Fenvalerate
Irani et al.	2022	India	Holtzman strain rats	F1 offspring: testes, ovaries, spermatozoa	Cypermethrin
Lesseur et al.	2023	USA	C57BL/6 J mice	Fetal: brain, placenta	Deltamethrin
Murkunde et al.	2012	India	Crl: Wistar rats	Cord blood, Fetal: body, liver	Cypermethrin
Vester et al.	2020	USA	C57BL/6NCrl wild-type mice	Fetal: midbrain	Deltamethrin
Wang et al.	2017	China	ICR mice	Placenta Fetal: serum Maternal: serum	Fenvalerate

EXPERIMENTAL STUDIES

Exposure to pyrethroids during prenatal development can affect the placenta in multiple ways. Mouse models suggest that fenvalerate exposures in the late stage of gestation can affect the morphology of the placenta by decreasing the sinusoid area of the placental labyrinth layer, resulting in placental insufficiency (Guo et al., 2019; Wang et al., 2017). Reduction in the vasculature in the placenta can affect the transport capacity of the placenta, which, in turn, may contribute to intrauterine growth restriction (IUGR) seen in offspring as the placenta has suppressed expression of nutrient transporters. Mechanistic possibilities for fenvalerate-induced IUGR include oxidative stress, and the consequent activation of the placental antioxidant system, and the disruption of placental thyroid hormone receptor signaling (Guo et al., 2019; Wang et al., 2017).

Similarly, cypermethrin exposures may result in genotoxicity. Murkunde et al. (2012) found that exposure to cypermethrin across the placenta resulted in low-level DNA damage in rat fetuses, although these results were not significant (Murkunde et al., 2012). Cypermethrin may induce DNA damage mechanistically through certain chemical properties, namely its size and lipophilicity, that allow it to enter the nucleus through the cell membrane where it can then bind with the DNA, destabilizing it and causing it to unwind. Alternatively, cypermethrin has been shown to induce oxidative stress in rats through the generation of free radicals, inducing DNA damage. However, the potential teratogenic capabilities of cypermethrin presented by Murkunde et al. (2012) warrant further investigation. In another study, cypermethrin was shown to transfer across the human placenta from maternal to fetal tissue. Furthermore, the increasing presence of 3-phenoxybenzoic acid (3-PBA), a metabolite of cypermethrin and several other synthetic pyrethroids, over time in maternal and fetal circulation indicates the metabolism of cypermethrin by the placenta itself (Mathiesen et al., 2020).

This then begs the question as to whether cypermethrin-induced fetal toxicity occurs via a direct or indirect mechanism. A model study found minimal transfer of α-cypermethrin across the placenta from the mother to the fetus by measuring α-cypermethrin levels below the limit of quantification in amniotic fluid despite considerable levels of the pesticide in maternal tissue (Elser et al., 2020). This finding, coupled with the fact that researchers also did not find measurable levels of metabolites in fetal samples, leads to considerations of indirect or placenta effects on the fetus. In the same study, exposure to α-cypermethrin resulted in an increased expression of multiple genes in placental tissues important in hypoxia and oxidative stress responses as well as in the upregulation of the expression of nutrient transporters in the placenta, providing evidence implicating the placenta in the toxicant’s mode of action. In another study involving α-cypermethrin and permethrin, pyrethroid levels in both maternal and placenta tissues were significantly higher than levels measured in fetal tissues as well as in amniotic fluid, providing more evidence supporting a low transfer rate of these compounds across the placenta to the fetus following oral administration (Elser et al., 2022). The authors also note that the aqueous nature of amniotic fluid would make it a poor reservoir for lipophilic compounds such as cypermethrin, accounting for the low levels reported in the study.

Interestingly, Elser et al. (2022) also discussed isomeric differences in transfer rates for permethrin, with higher levels of cis-permethrin found in the fetal compartment. They note this could be due to a higher permeability of the cis isomer in placental tissues, and, separately, they also highlight the fact that previous studies show rats have a higher rate of metabolism for trans isomers of pyrethroids over their corresponding cis isomers. Other studies have also shown the enantioselectivity of pyrethroids and the differing effects seen in chiral pesticides on the endocrine system. For instance, S-bifenthrin was shown to have greater hormone disrupting effects on estrogen receptors (ERs) in vitro than R-bifenthrin, which was due, in part, to the stronger binding affinity the S enantiomer has for ERs over the R enantiomer (Zhao et al., 2014). These effects seen in trophoblast cells at the maternal-fetal interface pose a unique risk for the developing fetus as these cells separate the two compartments, providing protection for the developing embryo.

Deltamethrin, another type 2 pyrethroid, can act through the noncompetitive inhibition of human placental glutathione transferase P1-1 (hpGSTP1-1). The H-site of hpGSTP1-1 is not well conserved, giving it a broader specificity for binding a range of both endogenous and exogenous substances. It is hypothesized that deltamethrin binding at this site promotes allosteric inhibition of hpGSTP1-1 through a conformational change that prevents the binding of substrate in the active site of the enzyme (Markus et al., 2018). This then inhibits the enzyme from performing its detoxification, and other, functions. For developing fetuses, inhibition of hpGSTP1-1 can pose a greater risk as immune and organ systems that are not fully developed and thus more susceptible to harmful interactions and any resultant dysfunction. Deltamethrin can also have transcriptional-level effects in mice, causing changes on a larger scale and across multiple organs. Affecting placental processes related to spliceosome, lysosome, cell cycle and extra cellular matrix processes by altering specific genes, early-life, gestational deltamethrin exposure can lead to various adverse neurobehavioral and somatic outcomes (Lesseur et al., 2023).

Additionally, exposure to deltamethrin and other environmental factors, like stress, can have synergistic effects. In male mice, exposure to deltamethrin and corticosterone, to mimic stress response, has been associated with hypermethylation of Nr3c1 in the midbrain. As Nr3c1 encodes the glucocorticoid receptor and is critical in stress response signaling, alterations in DNA methylation patterns around and on this gene may result in neurodevelopmental and neurobehavioral changes. Furthermore, methylation of an NR3C1 promoter in human placenta has shown a significant and positive association with attention and movement (Vester et al., 2020). While generating developmental and physiological effects, pyrethroid interactions with the placenta can also affect neurobehavioral outcomes.

Pyrethroid exposure can also have intergenerational effects. In mice, perinatal exposure to cypermethrin resulted in aberrations in the methylation patterns of certain genes during testicular steroidogenesis in offspring males. Alterations in the methylation patterns of DNA around genes insulin-like growth factor 2 (Igf2) and H19 can affect the regulation of fetal growth and development in the offspring of perinatally exposed male mice (Irani et al., 2022). Following exposure to fenvalerate in mice, placental Igf2 has also been downregulated, a target for the disruption of placental thyroid hormone receptor signaling (Wang et al., 2017). These findings bring light to the lasting, multigenerational effects of pyrethroid exposures.

OBSERVATIONAL STUDIES

Although data collected from in vitro studies and animal models more readily support hypotheses of placenta-mediated pyrethroid toxicity and pyrethroid effects on the placenta specifically, human epidemiologic studies which involve or discuss potential placental impacts appear to have more contentious results. A large, representative cohort study in Japan, found that those reporting exposure to insecticidal fumigants and increased frequency of exposure to mosquito coils, both containing pyrethroids, was associated with small reductions in birth weight and reduced neonatal length, respectively (Matsuki et al., 2020). These researchers found that placental weight was not associated with pesticide exposure and infant body weight, which allowed them to hypothesize that the development of the placenta was not involved in the mechanism of action (Matsuki et al., 2020). On the other hand in studies using biomarkers of exposure, one found that maternal urinary 3-PBA metabolites were associated with a positive increase in birth size (Zhang et al., 2014), and another observed associations between metabolites of dichlorodiphenyltrichloroethane (DDT) with elevated birth weight, birth length, and head circumference in female infants, but no associations with between birth characteristics and pyrethroid metabolites (Chevrier et al., 2019).

There could be a number of reasons for these discrepancies, and these studies described how the placenta could be implicated in some of these differences. Through oral exposure, the most common method of exposure in urban populations, pyrethroids may be partially or fully metabolized by the time they reach the placenta. Thus, the developing fetus may be exposed to metabolites such as 3-PBA alongside the parent pyrethroid compounds. However, even if pyrethroids or their metabolites have an effect on placental or fetal hormone receptors, these effects may not be reflected in the circulating hormone levels in the fetus, making determination of the disrupting effects of pyrethroids on the fetus more challenging (Zhang et al., 2014). Zhang et al. (2014) also note the magnitude of difference between the levels of pyrethroids administered in animal models and the environmental exposures of humans in epidemiologic studies are enough that exposures in humans can at times be considered below the no-observed-adverse-effect level of rats, as mentioned in their study. Determination of pyrethroid exposures through common metabolites and nonspecific groupings, such as type 1 and type 2 designations, can also introduce problems with determination of their effects, as different compounds will have differing structural elements that influence their mechanism of toxicity within biologic systems.

One study looked more directly at the impacts of reported exposures to pyrethroids and placental molecular effects, through an examination of placental DNA methylation, using Methyl-C sequencing. Professionally applied pesticides in the form of foggers and sprays were found to be highly correlated with a reduction of the proportion of placental DNA in partially methylated domains (PMDs) as well as with a higher average measure of methylation within the PMDs (Schmidt et al., 2016). Alterations in methylation patterns across the placental genome in response to pyrethroid exposures can result in alterations of gene expression, ultimately affecting functionality and development.

LIMITATIONS OF PRIOR RESEARCH

While interest in placenta-mediated mechanisms of pyrethroid toxicity and pyrethroid effects on the placenta has risen in recent years, research in this vein is still scarce and faces a variety of limitations. Many studies attempting to uncover mechanisms of pyrethroid toxicity involving the placenta are based on animal models, as reflected in this review. Animal models can provide invaluable information, but they do not act as a perfect simulation for human systems. Human placentation involves human-specific factors not found in other animal or mammalian cells (Schmidt et al., 2015). Applications of model systems can then fall short as they do not fully represent the developmental and gestational processes present in humans. The cell lines used in the described studies, BEWO and JEG-3, are derived from choriocarcinomas, and while they may express placental specific markers and undergo functions including syncytialization, they may suffer from limitations that exist for all tumor-based cell line models, including aneuploidy and difficulty in fully recapitulating placenta function. Furthermore, discussion circulates regarding issues relating to the translatability of experimental research to clinical research settings in terms of determining human toxicity and drug safety (Van Norman, 2019; Bailey et al., 2014). As such, the research surrounding the extent of transfer of pyrethroids across the human placenta is rather limited (Elser et al., 2022). For instance, multiple studies have reported a dose-dependent relationship with pyrethroid exposures and adverse health outcomes (Matsuki et al., 2020; Murkunde et al., 2012; Singh et al., 2016). However, at low-level doses in humans, the health benefits of consuming fruits treated with these pesticides may outweigh the potential adverse health effects, resulting in some observed positive associations (Xu et al., 2020; Zhang et al., 2014). This potential association, like many other aspects surrounding the placenta and pyrethroid exposure, warrants further study.

A common limitation mentioned in studies pertaining to the effects of prenatal pyrethroid exposure often deals with the quantification of pesticide levels and limits of measurement tools. Current tools and methods of measurement pose limitations in recognizing and quantifying low-level exposures that are often reported in fetal exposures studies (Elser et al., 2022; Watkins et al., 2016). Similarly, there are low detection frequencies and concentrations in both pregnant and nonpregnant adult populations (Huang et al., 2018). Difficulties assessing the presence of a compound within a sample make determination between its presence in quantities smaller than those that can be detected by the measurement tool and its presence at all in the sample difficult, which then affects possible outcomes and conclusions of the study. However, epidemiological studies included in this review that determined detection rates of pesticides reported extremely high detection rates, contradicting reported detection difficulties seen in the literature (Table 2). Similarly, as is the case with many epidemiologic studies relating to pyrethroid exposures, there are difficulties in measuring chronic exposures of pyrethroids due to their quick metabolism and the use of spot sampling. Many studies rely on spot urine samples to measure short-term pyrethroid exposures due to their rapid metabolism and excretion through urine; however, these samples may not be representative of exposures over longer periods of time (Chevrier et al., 2019; Viel et al., 2015; Xu et al., 2020). Thus, depending on when samples are taken in these studies, exposures can be misattributed if this temporal relationship is not accounted for. Furthermore, as is the case with studying any environmental exposure, drawing associations between exposures and outcomes is difficult due to the complexities of real-world exposures, although this limitation can be addressed with thorough inclusion of confounders, covariates, and mediating variables. Reliance on the self-reporting of exposures as opposed to utilizing biologic samples presents another limitation, although time, ethics, and economic considerations are restrictive in this regard. Lastly, comparison between epidemiologic studies can be limited due to variances between study populations and exposure assessments and methodologies among different studies (Watkins et al., 2016). While some epidemiological studies included in this review utilized direct detection methods through biological sampling and multiple analytical techniques, others relied on self-reported exposures from self-administered questionnaires (Table 2). This then precludes the reproducibility of study findings and further establishing observed associations and mechanisms.

Table 2.

Pyrethroid detection methods and analysis in observational studies.

Study	Pesticide(s)	Detection method	Detection or Use rate* (%)		Variable analysis (presence/absence, continuous)	Mixtures analysis ?
Chevrier et al. (2019)	Metabolites (3-PBA, 4-F-3-PBA, cis-DCCA and trans-DCCA, cis-DBCA)	Gas chromatography –mass spectrometry	3-PBA	100	Continuous	No
			4-F-3-PBA	-
			cis-DCCA	100
			trans-DCCA	100
			cis-DBCA	100
Matsuki et al. (2020)	Pyrethroids (unspecified)	Self-reported exposures	Any use	18.65	-	-
Schmid t et al. (2016)	Pyrethroids (unspecified)	Self-reported exposures	Lawn/garden use	27.3	-	-
			Pet use	34.1
Zhang et al. (2014)	Metabolites (3-PBA)	High performance liquid chromatography tandem mass spectrometry	3-PBA	98.7	Continuous	No

^*Detection rates are provided for studies examining metabolite biomarkers, and use rates for studys reporting self-reported exposures.

CONCLUSION AND FUTURE DIRECTIONS

This review examined the state of the current literature relating to pyrethroid effects on the placenta. Overall, the literature reports some conflicting results, especially between the utilized study designs and the specific compounds of interest across these studies. Despite this, associations between prenatal pyrethroid exposures and adverse placental development and function have been observed and established. Animal model studies point toward mechanistic pathways involving the induction of oxidative stress as well as hormone disrupting pathways, among others, while in vitro studies shed light on hormonal and genetic effects with epidemiologic studies reporting on neurobehavioral and developmental outcomes as well.

There are a number of potential directions needed in future research. Given the limitations regarding the comparability of murine and human placenta, in vitro research utilizing stem cell derived placenta cell lines or human explants and organoids could offer an opportunity to perform experimental toxicologic research, and would not suffer from the limitations seen in tumor-cell lines. These could be in parallel with more extensive molecular epidemiologic research which should focus on the placenta as a target tissues. Both the experimental and observational approaches could utilize state-of-the-art techniques, including single-cell sequencing, and assessments of a broad array of molecular features, such as transcriptomics, metabolomics, proteomics, and epigenomics. In addition, inclusion of placenta research into exposomic studies could allow for novel insights to how pyrethroid pesticides can be acting in combination with other chemical classes as well as social and behavioral elements to impact health. By using a core functional tissue, such as placenta, key alterations could be identified which can allow for the development of new intervention approaches based on the holistic, exposome framework.