The global rise in the prevalence of neuropsychiatric disorders can be partially attributed to the widespread use of endocrine disrupting chemicals (EDCs) (Kajta and Wójtowicz, 2013; Mustieles et al., 2015). These environmental contaminants have been repeatedly associated with reproductive (Sifakis et al., 2017), metabolic (Janardhanan, 2018), and developmental (Mouritsen et al., 2010) abnormalities. Some of the most prevalent EDCs in the environment are the plasticizers bisphenol A (BPA) and diethylhexyl phthalate (DEHP), which are typically found in water bottles and food can linings (Chapin et al., 2008), medical devices (Erythropel et al., 2014), and personal care products (Rowdhwal and Chen, 2018). Humans are primarily exposed to these particular EDCs via ingestion of contaminated food and beverages containing BPA and/or DEHP that has leached from the plastics (Kang et al., 2006; Erythropel et al., 2014).

In pregnant females, these chemicals can readily cross the placental barrier and affect fetal brain development in utero (Singh et al., 1975; Balakrishnan et al., 2010). Additionally, there appears to be a sex bias in the prevalence of multiple neuropsychiatric disorders (Ramtekkar et al., 2010; Altemus et al., 2014; Baio et al., 2018). It is highly likely that sex differences in the risk for developing these disorders may stem from exposures to EDCs in utero, a sensitive critical period of fetal development (Mallozzi et al., 2016).

Exposure to BPA during the in utero and early postnatal stages exerts a variety of sex-specific effects on stress-related behaviors and hormones, including corticosterone (CORT). Low-dose prenatal BPA exposure (2–200 μg/kg/day) induces anxiogenic effects and reduces locomotor activity in the Open Field Test (OFT) in female mouse offspring; however, the reverse is observed in males (Kundakovic et al., 2013). These effects are compounded with increasing doses, suggesting a dose-dependent response. Furthermore, perinatal treatment with 5 μg/kg of BPA eliminates sex differences in rats in the Elevated Plus Maze (EPM), without affecting learning and memory in the Morris Water Maze (Jones and Watson, 2012). In addition, studies report increased basal and post-stress levels of CORT accompanied by increased anxiety-like behavior in female rat offspring with perinatal BPA exposure (40 μg/kg/day) (Poimenova et al., 2010). BPA-exposed male offspring, on the other hand, display an even higher post-stress CORT response than females. However, perinatal treatment with a lower BPA dose (2 μg/kg/day) can induce anxiolytic effects in female rat offspring without affecting CORT levels before or after stress exposure (Chen et al., 2014). Yet their BPA-exposed male counterparts have persistently heightened pre- and post-stress CORT concentrations. The variability observed in BPA effects across studies may be attributed to methodological differences, including window and route of BPA exposure, the species studied, and age of offspring at evaluation.

Perinatal DEHP appears to have a non-monotonic dose response on exploratory and locomotor activity. Low DEHP doses (5–400 μg/kg/day) are associated with increased home cage exploration in mouse offspring of both sexes (Quinnies et al., 2017), whereas higher doses (10–200 mg/kg/day) lead to decreased locomotor activity in the OFT in female mice (Xu et al., 2015). Anxiogenic effects in the OFT and EPM are additionally observed in both male and female mice following low-dose (5 and 40 μg/kg) (Quinnies et al., 2017), as well as high-dose (50–750 mg/kg) (Dai et al., 2015; Xu et al., 2015; Barakat et al., 2018) perinatal DEHP exposure. Moreover, male offspring appear to show increased dose-dependent deficits in learning and memory following prenatal DEHP exposure (Lin et al., 2015; Barakat et al., 2018). Although no changes were observed in CORT in male offspring with high-dose DEHP exposure (Martinez-Arguelles et al., 2011), prenatal treatment with 150 mg/kg of DEHP has been shown to transgenerationally reduce CORT concentrations in female mouse offspring instead (Quinnies et al., 2015). Thus, it appears that alterations in CORT levels in EDC-exposed offspring depend on the type of EDC and dose used.

An especially problematic aspect of these contaminants is that, in reality, they exist in combination with one another in the environment–as mixtures. Determining the mechanisms of action underlying EDC mixtures is a relatively novel area of research, but studies agree that EDC mixture effects are more elusive and vary from those of individual EDCs (Biemann et al., 2014; Suteau et al., 2020). EDC mixtures, including BPA and DEHP combinations, are associated with a variety of consequences on the metabolic (Naville et al., 2013; Labaronne et al., 2017), reproductive (Borman et al., 2017; Balci et al., 2020; Christiansen et al., 2020), and cardiovascular (Shang et al., 2019) systems in male and female rodents. Yet, there is an alarming lack of data on sex-specific outcomes on behavior following exposures to BPA and DEHP mixtures.

The objective of the present study was to assess sex differences in stress-related behaviors, cognition, and the hypothalamic-pituitary-adrenal (HPA) axis following prenatal BPA or DEHP exposure individually or in combination at low and high doses. Our hypothesis was that prenatal EDC exposure, particularly in combination, would induce sex-specific and dose-dependent alterations in behavioral responses to stress that are mediated by the HPA axis.

In this study, we aimed to determine sex differences in the adverse behavioral and cognitive effects resulting from prenatal EDC exposure. The results of our study revealed a myriad of effects on stress-related behaviors, CORT, and adrenal gland weights. Firstly, BPA-treated female offspring displayed a near-significant increase in time spent in the center zone of the OFT, indicative of decreased anxiety-like behavior. Each of the behavioral paradigms incorporated in our study evaluates distinct aspects of stress-related behaviors, with the OFT assessing exploration of a novel environment (Choleris et al., 2001; Ramos, 2008). Hence, our BPA female offspring showed anxiolytic effects in response to novelty. BPA males, on the other hand, spent less time burying in the SPDB. Burying is an adaptive coping style since it embodies an active coping behavioral response to stress (Hori et al., 2004; Bondi et al., 2007). A decrease in burying represents a maladaptive reduction in active coping mechanisms (Fucich and Morilak, 2018). The BPA females in our study also exhibited altered defensive behaviors, but in the form of reduced shock reactivity in the SPDB. These effects taken together suggest that prenatal exposure to BPA at a low dose of 5 µg may lead to aberrant effects on sex-specific defensive behaviors and fear responses.

Prenatal exposure to DEHP at low and high doses yielded varying effects in each of the behavioral tests. In the OFT, both LD and HD DEHP-treated female offspring had fewer entries into the perimeter zone, but showed no changes in perimeter zone time. While a reduction of entries into the perimeter zone of the OFT may represent an anxiolytic-like effect due to reduced activity in the periphery, changes in exploration of the center zone in this test are typically used to measure anxiety-like behavior (Seibenhener and Wooten, 2015; Scholl et al., 2019). DEHP females were unaffected in the center zone parameters, rendering unclear anxiety-related effects.

DEHP (HD) males exhibited decreased anxiety-like behavior in the EPM, which represents a riskier environment and evaluates unconditioned anxiety (Lapiz-Bluhm et al., 2008; Ramos, 2008). This contrasts with studies that have observed anxiogenic effects in the EPM in males with perinatal low-dose (5–40 µg) and high-dose (10–50 mg) DEHP exposure (Xu et al., 2015; Quinnies et al., 2017). These inconsistencies could be attributed to differences in DEHP exposure period, species tested, and age at evaluation. In the aforementioned studies, DEHP was administered to mice throughout the gestational and early postnatal stages, and increased EPM anxiety levels were observed in pubertal male offspring. The anxiolytic effect in our DEHP (HD) males additionally resembled the behavior of control females. The sex difference between HD males and females in the proportion of time spent in the open arms was also reversed compared to controls, and this particular effect was not noted in any other EDC group. This suggests a potential feminization of HD males in anxiety-like behavior. This is consistent with the anti-androgenic effects of DEHP commonly discerned in exposed males (Moore et al., 2001; Vo et al., 2009a; Vo et al., 2009b; Christiansen et al., 2010; Carbone et al., 2013; Wang et al., 2016). We can further confirm based on our results that this particular effect is not present at a lower DEHP dose of 5 µg.

In the SPDB, we observed robust decreases in burying in LD and HD males in the DEHP-exposed groups. LD males and females also engaged in increased immobility, which was not reflected in HD offspring. In addition, we observed significantly reduced shock reactivity in LD, but not HD, females. These findings imply aberrant changes in defensive behaviors–reduced active coping in DEHP males, regardless of dose, and a definite shift to passive coping in DEHP (LD) male and female offspring. An increase in immobility reflects a preference for passive reactive coping mechanisms (Koolhaas et al., 1999; Fucich and Morilak, 2018), a maladaptive stress response that is often observed in individuals with affective disorders, including depression (Koolhaas et al., 1999; Olff et al., 2005; Southwick et al., 2005; Mahmoud et al., 2012). Immobility can also be interpreted as a measure of anxiety-like behavior (Nijssen and Schelvis, 1987).

Heightened immobility responses in the Forced Swim Test, an indicator of depressive-like behavior, have previously been reported in both male and female offspring perinatally treated with DEHP at high doses (10–200 mg/kg) (Xu et al., 2015). Our study is the first to demonstrate an acute stress-induced passive behavioral phenotype in the SPDB in male and female offspring exposed to low-dose DEHP in utero. Furthermore, our DEHP (LD) males also showed a reduction in adrenal gland relative weights. This is intriguing because adrenal hormones have been implicated in freezing behavior, with adrenalectomy impairing the duration of fear-induced freezing (Bohus, 1987). Hence, the lower adrenal weights in our DEHP (LD) males may have contributed to their behavioral outcomes. Additional experiments should examine mineralocorticoid and glucocorticoid receptor alterations for further insight.

Treatment with BPA and DEHP in combination altered the behavior of female offspring more than male offspring. B + D (LD) females exhibited anxiolytic effects in the OFT, but passive coping responses in the SPDB. B + D (HD) females showed unclear and slightly conflicting anxiety-like behavioral responses as well. In the OFT, they displayed significantly fewer entries into the perimeter zone, but they also spent the least amount of time in the center zone compared to other female offspring. However, this was not significantly different from control females. Moreover, B + D (HD) females had elevated CORT concentrations relative to their control counterparts. CORT levels were not significantly altered in any of the other treatment groups, regardless of sex. Studies have identified higher CORT levels in female offspring with perinatal BPA exposure at 40 μg/kg (Poimenova et al., 2010; Panagiotidou et al., 2014), but blunted CORT levels in female offspring three generations following prenatal high-dose DEHP exposure (150 mg/kg) (Quinnies et al., 2015). To our knowledge, ours is the first study to report a sex-specific increase in CORT following treatment with a mixture of BPA + DEHP. Interestingly, this outcome was not coupled with any robust behavioral changes.

Similarly, B + D (LD) males were not affected in any of the behavioral measures, but instead had lower adrenal gland relative weights, comparable to DEHP (LD) males. This result is particularly striking because it suggests that B + D (LD) male offspring may be more impacted at the organ systems level. Ours is the first study to our knowledge to determine associations between B + D exposure and reductions in these organ weights in male offspring. B + D (HD) males, on the other hand, did show behavioral alterations. They demonstrated significant reductions in active coping in the SPDB, with no accompanying alterations in CORT or adrenal weights. Therefore, the dose of B + D at which male offspring are exposed to determines the nature of the outcomes they will exhibit.

A major limitation of our study was the fact that the pregnant dams were group housed during the EDC treatment period. As a result, any potential contamination of the animals with bisphenol metabolites released through urine or feces in the cages was not controlled for. However, there were no variations in the types of cages used and materials provided within the cages, and all animals were provided water in glass bottles. Therefore, any additional bisphenol exposure from the environment would have been similar across treatments. Measuring the bisphenol levels in the animals and identifying any differences between EDC-exposed and control animals would help determine if there was bisphenol contamination. Moreover, larger sample sizes in all of our groups may have potentially exposed further significant effects, especially in those parameters that were near-significant.

In addition, all of the female offspring in our study were tested while in the estrus stage of the estrous cycle. This stage is characterized by reduced levels of estradiol (E2) and progesterone P), and is generally correlated with lower stress hormone levels relative to the proestrus stage, during which E2 and P levels are higher (Viau and Meaney, 1991). Anxiety-like behavioral responses are therefore influenced by ovarian steroids such as E2 in females (MohanKumar et al., 2018), but the effects vary based on a multitude of factors, including age. A prior study from our lab reported that chronic exposure to low-dose E2 (20 ng/day for 90 days) increases anxiety-like behavior in female rats (Balasubramanian et al., 2014). Studies have also demonstrated that certain anxiety-related behaviors are independent of estrous cycle stage in female rodents (Scholl et al., 2019). Hence, the fact that only estrus females were used in our study is not a major limitation.

To summarize, offspring with prenatal EDC exposures were dose- and sex-specifically affected in each of the behavioral measures assessed. BPA offspring were impacted in anxiety-like and defensive behaviors. DEHP (LD) offspring were more severely affected in terms of defensive behaviors, whereas DEHP (HD) offspring displayed aberrant anxiety-like behavior. Low dose B + D exposure modified anxiety-like and defensive behaviors, but high-dose exposure affected defensive behaviors and CORT. DEHP and B + D males showed a reduction in adrenal gland weights following low-dose exposure. These findings raise significant health concerns, especially considering the detectable levels of these chemicals in pregnant women’s urine (Arbuckle et al., 2014; Jensen et al., 2016; Muerkoster et al., 2020). Exposure pathways include dermal contact via clothing (Kaylor et al., 2023), food packaging (Pacyga et al., 2019) and the use of personal care products (Fisher et al., 2019). The potential long-term repercussions of these low-dose exposures on offspring behavior warrant attention, emphasizing the need for future rodent studies to delve into molecular mechanisms that influence these behavioral changes. Exploring neurotransmitter systems and hormone receptor dysregulation in the brain could offer valuable insights. Additionally, conducting extensive long-term studies in humans can further elucidate potential behavioral risks.