Background: The nervous system is a sensitive target for exposure to environmental endocrine-disrupting compounds (EDC). This vulnerability is particularly important during the critical windows of development and puberty and lasts even at later stages of life. Among these environmental EDC, phthalates have largely been described for their neurotoxic effects. These effects have been reported for a large majority of studies using high to very high doses of these substances, which are not relevant for environmental exposure. Summary: The aim of this review was to analyze specifically the male rodent studies using low doses of phthalates. This analysis focuses on reproductive and cognitive behaviors, given the described antiandrogenic effects of phthalates and the known regulation of these behaviors by sex steroids. We also analyze the other neural effects in the hypothalamus and hippocampus/cortex, the brain regions governing these behaviors. A particular focus is on the neurovascular unit, which is newly investigated in the field of endocrine disruption. Key Messages: Exposure to low doses of phthalates can induce modifications in reproductive and cognitive behaviors. Whether these changes are triggered by common initiating cellular and molecular mechanisms in the brain areas controlling these behaviors still needs to be extensively investigated. In this context, given the high sensitivity of the neurovascular unit to sex steroid regulation and its impairment by low doses of phthalates, it could represent a possible initiating trigger for behavioral alterations to assess for phthalate exposure.

Phthalates are widely detected chemicals in the environment due to their extensive use in the polyvinyl chloride plastic industry, cosmetics and personal care products, cleaning products, etc. Eight phthalates, including di(2-ethylhexyl) phthalate (DEHP), one of the most abundant phthalates in the environment, have been classified by the European Chemical Agency (ECHA) as substances of very high concern due to their toxicity for male reproduction and ability to reduce fetal testosterone production [1]. On the basis on this adverse effect, the use of DEHP has been restricted in Europe in some products such as toys for kids (authorization required for a mass >0.1% of the total product mass) or food contact material with a tolerable daily intake dose of 50 μg/kg/day [2, 3]. Despite these use restrictions, phthalates remain widespread in the environment. Indeed, data from the French Esteban study showed that in 80–99% of infants and adults, at least one phthalate metabolite was detected in urine samples, with high metabolite concentrations measured for monoethyl phthalate, mono-isobutyl phthalate, and the sum of DEHP metabolites [4].

In males, exposure to phthalates reduced the anogenital distance and impaired testicular and urogenital development through their antiandrogenic activity [2, 3]. The nervous system represents also a sensitive target for phthalate exposure. Previous publications have summarized the effects induced by exposure to phthalates on the nervous system, with a particular focus on the hippocampal and cognitive function [5, 6] and the underlying oxidative/inflammatory or neuroendocrine mechanisms [6, 7]. Here, we aimed to provide an updated overview of the male rodent studies addressing the effects of phthalate exposure on reproductive and cognitive behaviors given their known regulation by sex steroids and thus potential vulnerability to exposure to antiandrogenic compounds. The neural and behavioral effects of phthalate exposure in female rodents were reviewed in a recent article [8]. We also discuss newly described sensitive neural targets such as the blood-brain barrier (BBB). We focus on male rodent studies using relatively low doses of phthalates, which are below the non-observed adverse effect level of 4.8–5 mg/kg/day established for DEHP-induced effects on reproductive development and fetal testosterone production [2, 3, 9]. The choice of the DEHP reference dose is based on the use of this substance by the EFSA [3] as an index compound to establish a group-tolerable daily intake for other phthalates (dibutyl phthalate [DBP], butylbenzyl phthalate [BBP], di-isononyl phthalate [DINP], and di-isodecyl phthalate [DIDP]). Our goal is to question whether low doses of phthalates, which are closer to the estimated environmental exposure, can trigger in experimental models adverse effects and related endocrine(s) mode(s) of action that can be considered for risk assessment for human health and the environment.

We evaluated data from rodents (mice and rats) regarding the effects of exposure to phthalates on reproductive and cognitive behaviors and related neural mechanisms. We chose these endpoints based on the largely reported sensitivity of the brain regions underlying these behaviors to sex steroids in general and to androgens in particular. Indeed, androgens and their nuclear receptor play a key role in the functioning of the neural circuitries including the hypothalamus for reproduction and the hippocampus for cognition [10]. For example, neural deletion of the gene encoding the androgen receptor (AR) impairs sexual and temporal order memory in male mice [11, 12].

We identified publications up to November 2022 using PubMed, with the keywords “Phthalate and Nervous system”, “Phthalate and Brain” or “Phthalate and Behavior”. The first list of publications selected on the basis of the abstract is presented in Table S1 (for all online suppl. material, see https://doi.org/10.1159/000534836). Figure 1 provides general information on the percentage of studied species, periods of exposure, studied phthalates, and used doses. The second list of selected articles using doses in the range or lower than 5 mg/kg/day is reported in Table 1 for reproductive behaviors and Table 2 for learning and memory. When authors made neuroanatomical or neuroendocrine observations in the same studies, they are reported in Tables 1 and 2. Otherwise, this information can be found in Table 3. For the studies using both low and high doses, the data obtained for the high doses are also reported in Table 1-3 but not discussed in the following analysis. The prenatal/postnatal period of exposure corresponds to the whole prenatal and postnatal periods until the end of the third postnatal week. The age at analyses is also indicated in these tables.

Fig. 1.

Summary information on the experimental studies analyzed in this review. DEHP, di(2-ethylhexyl) phthalate; DBP, dibutyl phthalate; NOAEL, non-observed adverse effect level.

Fig. 1.

Summary information on the experimental studies analyzed in this review. DEHP, di(2-ethylhexyl) phthalate; DBP, dibutyl phthalate; NOAEL, non-observed adverse effect level.

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Table 1.

Effects of exposure to phthalates on reproductive behaviors

Exposure periodSpeciesExposure durationExposure routePhthalate dosesAge at analysesBehavioral analysesNeuroanatomical and neuroendocrine analyses in the hypothalamus – other findingsReference
Prenatal/postnatal Wistar rats GD6-PND21 Oral (gavage) DEHP: 0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135, and 405 mg/kg bw/d PND110 No changes in mount, intromission or ejaculation latencies or frequency of intromission Serum levels of testosterone increased at DEHP-0.045, DEHP-0.405, and DEHP-405 Andrade et al. [13] (2006a) 
Reduced seminal vesicles weight at DEHP-405 
Reduced latency to first mount for DEHP-5 and DEHP-15 groups 
Incidence of cryptorchidism since the dose of 5 mg/kg/d and reduced sperm production since 15 mg/kg/d 
Prenatal/postnatal Wistar rats GD10-PND21 Oral (gavage) DiPeP: 1, 10, or 100 mg/kg/d PND82-140 Reduced partner preference for DiPeP-1 and DiPeP-10 groups Hypothalamic expression of estrogen receptor (ER) α was lower for DiPeP-10 group Neubert da Silva et al. [14] (2019) 
No effect of hypothalamic aromatase, androgen receptor (AR), and ERβ expression 
Increased latency to first mount and intromission for DiPeP-1 group 
Unchanged play behavior and anxiety-state level (EPM) for all treated groups 
Pubertal C57BL6/J mice PND30-PND60 Oral (diet) DEHP: 0.005 or 0.05 mg/kg/d PND120 Altered USV emission at the two doses, partner preference for DEHP-50 group Reduced AR protein amount in the preoptic area for DEHP-50 group Capela et al. [15] (2021) 
Increased latency to reach ejaculation and mating duration for DEHP-50 group 
Unchanged testosterone levels, weight of testis, Tyson gland, and seminal vesicles 
Unchanged olfactory preference and locomotor activity 
Increased anxiety-state level in the O-maze for DEHP-5 and DEHP-50 groups 
Adult C57BL6/J mice PND60-88, then during the whole period of behavioral analyses Oral (diet) DEHP: 0.005 or 0.05 mg/kg/d Since PND89 Reduced emission of ultrasonic vocalizations and partner preference for DEHP-5 and DEHP-50 groups Unchanged kisspeptin immunoreactivity in the hypothalamic medial preoptic area (MPOA) and arcuate nuclei, seminal vesicle and testis weights, and circulating levels of testosterone Dombret et al. [16] (2017) 
Increased latencies to first intromission and ejaculation for DEHP-5 and DEHP-50 groups 
Lower number of AR-ir cells in the medial POA, medial amygdala, and bed nucleus of stria terminalis; reduced AR mRNA levels in the MPOA. No effects on ERα-ir cells in the MPOA. 
Proteome analysis of the MPOA: identification of differentially expressed proteins between the vehicle and DEHP-5/DEHP-50 groups: increased levels of GFAP and NDRG2 
Unaltered olfactory preference 
Exposure periodSpeciesExposure durationExposure routePhthalate dosesAge at analysesBehavioral analysesNeuroanatomical and neuroendocrine analyses in the hypothalamus – other findingsReference
Prenatal/postnatal Wistar rats GD6-PND21 Oral (gavage) DEHP: 0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135, and 405 mg/kg bw/d PND110 No changes in mount, intromission or ejaculation latencies or frequency of intromission Serum levels of testosterone increased at DEHP-0.045, DEHP-0.405, and DEHP-405 Andrade et al. [13] (2006a) 
Reduced seminal vesicles weight at DEHP-405 
Reduced latency to first mount for DEHP-5 and DEHP-15 groups 
Incidence of cryptorchidism since the dose of 5 mg/kg/d and reduced sperm production since 15 mg/kg/d 
Prenatal/postnatal Wistar rats GD10-PND21 Oral (gavage) DiPeP: 1, 10, or 100 mg/kg/d PND82-140 Reduced partner preference for DiPeP-1 and DiPeP-10 groups Hypothalamic expression of estrogen receptor (ER) α was lower for DiPeP-10 group Neubert da Silva et al. [14] (2019) 
No effect of hypothalamic aromatase, androgen receptor (AR), and ERβ expression 
Increased latency to first mount and intromission for DiPeP-1 group 
Unchanged play behavior and anxiety-state level (EPM) for all treated groups 
Pubertal C57BL6/J mice PND30-PND60 Oral (diet) DEHP: 0.005 or 0.05 mg/kg/d PND120 Altered USV emission at the two doses, partner preference for DEHP-50 group Reduced AR protein amount in the preoptic area for DEHP-50 group Capela et al. [15] (2021) 
Increased latency to reach ejaculation and mating duration for DEHP-50 group 
Unchanged testosterone levels, weight of testis, Tyson gland, and seminal vesicles 
Unchanged olfactory preference and locomotor activity 
Increased anxiety-state level in the O-maze for DEHP-5 and DEHP-50 groups 
Adult C57BL6/J mice PND60-88, then during the whole period of behavioral analyses Oral (diet) DEHP: 0.005 or 0.05 mg/kg/d Since PND89 Reduced emission of ultrasonic vocalizations and partner preference for DEHP-5 and DEHP-50 groups Unchanged kisspeptin immunoreactivity in the hypothalamic medial preoptic area (MPOA) and arcuate nuclei, seminal vesicle and testis weights, and circulating levels of testosterone Dombret et al. [16] (2017) 
Increased latencies to first intromission and ejaculation for DEHP-5 and DEHP-50 groups 
Lower number of AR-ir cells in the medial POA, medial amygdala, and bed nucleus of stria terminalis; reduced AR mRNA levels in the MPOA. No effects on ERα-ir cells in the MPOA. 
Proteome analysis of the MPOA: identification of differentially expressed proteins between the vehicle and DEHP-5/DEHP-50 groups: increased levels of GFAP and NDRG2 
Unaltered olfactory preference 

AR, androgen receptor; bw, body weight; d, day; ER, estrogen receptor; GD, gestational day; GFAP, glial fibrillary acidic protein; ir, immunoreactivity; NDRG2, N-myc-downstream-regulated gene 2; PND, postnatal day.

Table 2.

Effects of exposure to phthalates on cognitive behaviors

Exposure periodSpeciesExposure durationExposure routePhthalate dosesAge at analysesBehavioral analysesNeuroanatomical and neuroendocrine analyses in the hippocampus/cortex – other findingsReference
Prenatal/postnatal Wistar rats GD0-PND21 Oral (gavage) DEHP: 0.005, 0.04, 0.4, and 300 mg/kg PND60 MWM: increased escape latency at days 2 and 3 for DEHP-0.005 group, increased escape latency at days 2–4 for DEHP-300 Nissl staining: higher dark cells in the CA1 and CA3 for DEHP-300 group Safarpour et al. [17] (2021) 
Increased hippocampal malondialdehyde (MDA) content for DEHP-0.4 and DEHP-300 groups. Reduced total antioxidant capacity content for DEHP-300 group 
Increased traveled distance at days 2–4 for DEHP-300 group, at day 3 for DEHP-0.005 group, and at days 2 and 4 for DEHP-0.4 group 
Higher GFAP-positive cells in the CA1 and CA3 for DEHP-300 group 
Reduced time spent in target quadrant for DEHP-300 group 
Reduced number of live puppies, reduced birth weight, and weight gain at DEHP-300. Reduced birth weight at PND10 and 25 for DEHP-0.4 
Y-maze alternation: reduced spontaneous alternation for DEHP-300 group 
Prenatal/postnatal Long-Evans rats GD2-PND10 Oral (food) Mixture (35% DEP, 21% DEHP, 15% DBP, 15% DiNP, 8% DiBP, and 5% BBP) at 0, 0.2, or 1 mg/kg bw PND92-103 Attentional set-shift at PND92: no effect of treatment on training Analyses at PND103 Kougias et al. [18] (2018) 
Reduced neurons in the medial prefrontal cortex (mPFC) but had no effect on glial number at both doses 
Reduced number of synaptophysin boutons in the mPFC for treated rats at both doses but no effect on the number of synaptophysin-labeled synapses per neuron 
Reduced total volume of the mPFC at both doses; unaffected volume of the white matter under the mPFC 
Rats exposed to the two doses of the mixture had an altered performance in extradimensional shift and showed more perseverative and omission errors than the vehicle group 
Prenatal/postnatal Sprague-Dawley rats GD2-GD21 Oral (gavage) DEHP: 1.25 mg/kg/d 21 months MWM: increased distance to reach platform and reduced time spent in target quadrant Reduced insulin gene expression in the hippocampus at 22 months Sun et al. [19] (2014) 
Reduced mRNA level of insulin growth factor (IGF) 1 and IGF 2, phospho-Akt, and phospho-GSK-3 beta. Increased expression level of Bad and Bim and phosphorylation level of Tau in the hippocampus 
Lower birth weight and at weaning 
Disrupted insulin homeostasis: impaired intra-peritoneal (ip) glucose tolerance tests, decreases ip insulin tolerance test at 19 months, delayed decreasing serum level after insulin injection and serum glucose increased faster. Higher fasting serum level of insulin and lower insulin level 
Prenatal/postnatal Long-Evans rats PND16-22 Ip injection DEHP: 1, 10, or 20 mg/kg/d PND55-78 Operant task to bar press for chocolate-flavored pellets Analyses at PND78 Holahan et al. [20] (2018) 
Reduced number of tyrosine hydroxylase (TH) positive cells (index of the biosynthetic activity of dopamine) in the substantia nigra and ventral tegmental area at DEHP-10 and DEHP-20 
Rats were trained from PND55 to PND63 
Higher bar pressing for food reward during acquisition and extinction at DEHP-10 
Unchanged mRNA expression for TH, increased expression of Pitx3 at DEHP-10, and unaffected expression genes related to apoptosis (Bad, Bax, Bcl-2, and caspase-3) 
Increased locomotor activity in press acquisition test for DEHP-20 group 
Prenatal/postnatal CD1 mice GD11-PND0 Oral (gavage) DEHP: 0.2, 500 and 750 mg/kg/d 16–22 months Y-maze spontaneous alternation: reduced number of arm entries for DEHP-0.2 group Nissl staining: reduced number of neurons in CA1 and CA2/3 at DEHP-0.2 and DEHP-750 Barakat et al. [21] (2018) 
Spatial light interference microscopy: reduced number of pyramidal neurons in CA1, CA2, and DG for DEHP-500 and DEHP-750 groups 
Increased expression of COX-2 in CA2/3 of the hippocampus for DEHP-0.2 and DEHP-750 groups 
Novel object recognition: reduced exploration time of novel object for DEHP-500 and DEHP-750 groups 
Qualitative increase in 8-hydroxyguanine (8-OHdG) in CA2, CA3, and DG and thymidine glycol in CA2 and DG (not quantified) 
Open field: reduced number of entries in central area for DEHP-0.2, DEHP-500, and DEHP-750 groups 
Reduced serum testosterone levels for DEHP-500 and DEHP-750 groups, a tendency to decrease at DEHP-0.2 
EPM: increased latency to enter in the open arms for DEHP-750 group 
Prenatal/postnatal C57BL6/J mice GD9.5-GD16.5 Oral (gavage) DEHP: 5, 50, 200 mg/kg/day PND89-210 MWM: increased latency to find the platform during learning at PND89 and PND-210 for DEHP-50 and DEHP-200 groups. In probe test, fewer platform crossing at PND89 and PND-210 for all groups Analyses in the fetal brain Lv et al. [22] (2022) 
Reduced free T3 levels for DEHP-5, DEHP-50, and DEHP-200 groups 
Reduced monocarboxylate transporter 8 and organic anion transporting polypeptides 1C1 protein amounts at DEHP-50 and DEHP-200 and mRNA levels at all doses 
Open field: reduced total distance and central area distance for DEHP-5, DEHP-50, and DEHP-200 groups at PND89. Increased latency to enter in the central zone for DEHP-200 group 
Protein amount reduced for deiodinase 2 and increased for deiodinase 3 at all doses, mRNA levels reduced for deiodinase 2 at DEHP-200 and increased for deiodinase 3 at DEHP-50 and DEHP-200 
Forced swim test: reduced climbing number for all groups, reduced time spent in swimming and higher time spent in immobility for DEHP-50 and DEHP-200 groups at PND89 
Reduced TR alpha and beta and BDNF proteins at all DEHP doses and reduced mRNA levels at DEHP-200 
Prepubertal/pubertal ICR mice PND21-42 Oral (gavage) DEHP: 0.18, 1.8, 18, and 180 mg/kg/bw/d MWM: decreased swimming speed in time spent in target quadrant at all doses, increased latency to reach platform for all doses, and decreased time in platform spot for DEHP-1.8, DEHP-18, and DEHP-180 groups Reduced acetylcholine esterase activity for all doses and GSH peroxidase and superoxide dismutase for DEHP-1.8, DEHP-18, and DEHP-180 in the brain. mRNA levels reduced for Slc6a4, Tph2, Gabrr1, and Pax8 and higher for Fgf17 and Avp at all DEHP doses Feng et al. [23] (2020) 
Reduced 5-HT and GABA levels together with reduced cAMP content and higher Ca2+ levels and increase of CaM and p-CaMKII at all DEHP doses 
Decreased of p-PKA/PKA, p-ERK1/2/ERK1/2, CREB, and p-CREB/CREB at all doses 
Open field: decreased distance for DEHP-18 and DEHP-180 groups, decreased clock-wise rotation count, and increased time in the central area for DEHP-1.8, DEHP-18, and DEHP-180 
Prepubertal/pubertal Kunming mice PND28-49 Oral (gavage) DIDP: 0.15, 1.5, 15, and 150 mg/kg/d PND41-49 MWM: in spatial probe test, swimming trajectories were scattered and disordered for DIDP-15 and DIDP-150 groups Hematoxylin/eosin staining in CA1: more disordered and swelling pyramidal neurons when exposed to the higher DIDP doses (not quantified) Ge et al. [24] (2019) 
Nissl staining: loss of Nissl substance in the CA1 and swelling deformation and some were deeply stained and shrunken at the higher doses (not quantified) 
Reduced brain levels of testosterone and estradiol for DIDP-15 and DIDP-150 groups 
Increased level of reactive oxygen species (ROS), MDA and 8-OHdG and glutathione (GSH) decreased at DIDP-15 and DIDP-150 
Increased level of NF-kappaB and Casp-3 in the brain at DIDP-15 and DIDP-150 
Decreased levels of BDNF at DIDP-15 and DIDP-150 and 
p-CREB at DIDP-150 
Reduced testosterone levels in serum and increased estradiol at DIDP-15 and DIDP-150 
DIDP-15 and DIDP-150 groups spent less time in the target quarter than the control group 
Adult NMRI mice Around 7 weeks of age: 14 days of exposure Oral (gavage) DBP: 6.25, 12.5, 25, 50, 100, and 200 mg/kg Since day 15 after the end of exposure Passive avoidance test: decreased avoidance latency for DBP-25, DBP-50, DBP-100, and DBP-200 groups Hematoxylin/eosin staining: decreased nuclei area in dentate gyrus for DBP-25 and DBP-100 groups Farzanehfar et al. [25] (2016) 
Open field: decreased total distance movement and time spent in center area at DBP-12.5 to DBP-200 
EPM: decreased time in open arm for DBP-25, DBP-50, DBP-100, and DBP-200 groups 
Spontaneous alternation Y-maze, rotarod and grip strength: no changes 
Adult Kunming mice 6 weeks of age: 14 days of exposure Oral (gavage) DINP: 0.2, 2, 20, 200 mg/kg/day MWM: days 6 to 12 OFT: day 14 MWM: increased escape latency increased during learning phase and increased escape latency at day 7 at DINP-200. During the test phase, decreased time spent and frequency to enter in the target quadrant at DINP-200 Hematoxylin/eosin staining: disorganized and loose pyramidal cells with swelling deformation in CA1 for DINP-20 and DINP-200 groups (no quantification) Ma et al. [26] (2015) 
Nissl staining showed loss of substance in cells with increasing exposure concentration 
Upregulation of caspase-3 and GFAP in the CA1 and cortex at DINP-20 and DINP-200 
Open field test (OFT): decreased central area entries for DINP-200 
Increased brain ROS levels for DINP-20 and DINP-200 groups, decreased GSH content at DINP-200, and SOD at DINP-20 and DINP-200. Increased level of DNA-protein crosslinks (DINP-200) and 8-OH-dG (DINP-20 and DINP-200) 
Increased expression levels of TNF-α and IL-1β at DINP-20 and DINP-200 
Adult Kunming mice 7 weeks of age: 9 days of exposure Oral (gavage) DINP: 1.5, 15, and 150 mg/kg/day During the 9 days of treatment MWM: increased latency to reach platform, reduced time spent, and number of entries in target quadrant for DINP-150 group Hematoxylin/eosin staining: CA1 pyramidal cells were loose and disordered, cell swelled, apical dendrites shortened, or disappeared at DINP-150 (not quantified) Peng et al. [27] (2015) 
Nissl staining: increased staining at DINP-15 and DINP-150 
Increased ROS, MDA, SOD, TNF-α, IL-1, caspase-3 activity in the brain at DINP-150 and decreased GSH. 
Lower gain weight in the DINP-150 group 
Exposure periodSpeciesExposure durationExposure routePhthalate dosesAge at analysesBehavioral analysesNeuroanatomical and neuroendocrine analyses in the hippocampus/cortex – other findingsReference
Prenatal/postnatal Wistar rats GD0-PND21 Oral (gavage) DEHP: 0.005, 0.04, 0.4, and 300 mg/kg PND60 MWM: increased escape latency at days 2 and 3 for DEHP-0.005 group, increased escape latency at days 2–4 for DEHP-300 Nissl staining: higher dark cells in the CA1 and CA3 for DEHP-300 group Safarpour et al. [17] (2021) 
Increased hippocampal malondialdehyde (MDA) content for DEHP-0.4 and DEHP-300 groups. Reduced total antioxidant capacity content for DEHP-300 group 
Increased traveled distance at days 2–4 for DEHP-300 group, at day 3 for DEHP-0.005 group, and at days 2 and 4 for DEHP-0.4 group 
Higher GFAP-positive cells in the CA1 and CA3 for DEHP-300 group 
Reduced time spent in target quadrant for DEHP-300 group 
Reduced number of live puppies, reduced birth weight, and weight gain at DEHP-300. Reduced birth weight at PND10 and 25 for DEHP-0.4 
Y-maze alternation: reduced spontaneous alternation for DEHP-300 group 
Prenatal/postnatal Long-Evans rats GD2-PND10 Oral (food) Mixture (35% DEP, 21% DEHP, 15% DBP, 15% DiNP, 8% DiBP, and 5% BBP) at 0, 0.2, or 1 mg/kg bw PND92-103 Attentional set-shift at PND92: no effect of treatment on training Analyses at PND103 Kougias et al. [18] (2018) 
Reduced neurons in the medial prefrontal cortex (mPFC) but had no effect on glial number at both doses 
Reduced number of synaptophysin boutons in the mPFC for treated rats at both doses but no effect on the number of synaptophysin-labeled synapses per neuron 
Reduced total volume of the mPFC at both doses; unaffected volume of the white matter under the mPFC 
Rats exposed to the two doses of the mixture had an altered performance in extradimensional shift and showed more perseverative and omission errors than the vehicle group 
Prenatal/postnatal Sprague-Dawley rats GD2-GD21 Oral (gavage) DEHP: 1.25 mg/kg/d 21 months MWM: increased distance to reach platform and reduced time spent in target quadrant Reduced insulin gene expression in the hippocampus at 22 months Sun et al. [19] (2014) 
Reduced mRNA level of insulin growth factor (IGF) 1 and IGF 2, phospho-Akt, and phospho-GSK-3 beta. Increased expression level of Bad and Bim and phosphorylation level of Tau in the hippocampus 
Lower birth weight and at weaning 
Disrupted insulin homeostasis: impaired intra-peritoneal (ip) glucose tolerance tests, decreases ip insulin tolerance test at 19 months, delayed decreasing serum level after insulin injection and serum glucose increased faster. Higher fasting serum level of insulin and lower insulin level 
Prenatal/postnatal Long-Evans rats PND16-22 Ip injection DEHP: 1, 10, or 20 mg/kg/d PND55-78 Operant task to bar press for chocolate-flavored pellets Analyses at PND78 Holahan et al. [20] (2018) 
Reduced number of tyrosine hydroxylase (TH) positive cells (index of the biosynthetic activity of dopamine) in the substantia nigra and ventral tegmental area at DEHP-10 and DEHP-20 
Rats were trained from PND55 to PND63 
Higher bar pressing for food reward during acquisition and extinction at DEHP-10 
Unchanged mRNA expression for TH, increased expression of Pitx3 at DEHP-10, and unaffected expression genes related to apoptosis (Bad, Bax, Bcl-2, and caspase-3) 
Increased locomotor activity in press acquisition test for DEHP-20 group 
Prenatal/postnatal CD1 mice GD11-PND0 Oral (gavage) DEHP: 0.2, 500 and 750 mg/kg/d 16–22 months Y-maze spontaneous alternation: reduced number of arm entries for DEHP-0.2 group Nissl staining: reduced number of neurons in CA1 and CA2/3 at DEHP-0.2 and DEHP-750 Barakat et al. [21] (2018) 
Spatial light interference microscopy: reduced number of pyramidal neurons in CA1, CA2, and DG for DEHP-500 and DEHP-750 groups 
Increased expression of COX-2 in CA2/3 of the hippocampus for DEHP-0.2 and DEHP-750 groups 
Novel object recognition: reduced exploration time of novel object for DEHP-500 and DEHP-750 groups 
Qualitative increase in 8-hydroxyguanine (8-OHdG) in CA2, CA3, and DG and thymidine glycol in CA2 and DG (not quantified) 
Open field: reduced number of entries in central area for DEHP-0.2, DEHP-500, and DEHP-750 groups 
Reduced serum testosterone levels for DEHP-500 and DEHP-750 groups, a tendency to decrease at DEHP-0.2 
EPM: increased latency to enter in the open arms for DEHP-750 group 
Prenatal/postnatal C57BL6/J mice GD9.5-GD16.5 Oral (gavage) DEHP: 5, 50, 200 mg/kg/day PND89-210 MWM: increased latency to find the platform during learning at PND89 and PND-210 for DEHP-50 and DEHP-200 groups. In probe test, fewer platform crossing at PND89 and PND-210 for all groups Analyses in the fetal brain Lv et al. [22] (2022) 
Reduced free T3 levels for DEHP-5, DEHP-50, and DEHP-200 groups 
Reduced monocarboxylate transporter 8 and organic anion transporting polypeptides 1C1 protein amounts at DEHP-50 and DEHP-200 and mRNA levels at all doses 
Open field: reduced total distance and central area distance for DEHP-5, DEHP-50, and DEHP-200 groups at PND89. Increased latency to enter in the central zone for DEHP-200 group 
Protein amount reduced for deiodinase 2 and increased for deiodinase 3 at all doses, mRNA levels reduced for deiodinase 2 at DEHP-200 and increased for deiodinase 3 at DEHP-50 and DEHP-200 
Forced swim test: reduced climbing number for all groups, reduced time spent in swimming and higher time spent in immobility for DEHP-50 and DEHP-200 groups at PND89 
Reduced TR alpha and beta and BDNF proteins at all DEHP doses and reduced mRNA levels at DEHP-200 
Prepubertal/pubertal ICR mice PND21-42 Oral (gavage) DEHP: 0.18, 1.8, 18, and 180 mg/kg/bw/d MWM: decreased swimming speed in time spent in target quadrant at all doses, increased latency to reach platform for all doses, and decreased time in platform spot for DEHP-1.8, DEHP-18, and DEHP-180 groups Reduced acetylcholine esterase activity for all doses and GSH peroxidase and superoxide dismutase for DEHP-1.8, DEHP-18, and DEHP-180 in the brain. mRNA levels reduced for Slc6a4, Tph2, Gabrr1, and Pax8 and higher for Fgf17 and Avp at all DEHP doses Feng et al. [23] (2020) 
Reduced 5-HT and GABA levels together with reduced cAMP content and higher Ca2+ levels and increase of CaM and p-CaMKII at all DEHP doses 
Decreased of p-PKA/PKA, p-ERK1/2/ERK1/2, CREB, and p-CREB/CREB at all doses 
Open field: decreased distance for DEHP-18 and DEHP-180 groups, decreased clock-wise rotation count, and increased time in the central area for DEHP-1.8, DEHP-18, and DEHP-180 
Prepubertal/pubertal Kunming mice PND28-49 Oral (gavage) DIDP: 0.15, 1.5, 15, and 150 mg/kg/d PND41-49 MWM: in spatial probe test, swimming trajectories were scattered and disordered for DIDP-15 and DIDP-150 groups Hematoxylin/eosin staining in CA1: more disordered and swelling pyramidal neurons when exposed to the higher DIDP doses (not quantified) Ge et al. [24] (2019) 
Nissl staining: loss of Nissl substance in the CA1 and swelling deformation and some were deeply stained and shrunken at the higher doses (not quantified) 
Reduced brain levels of testosterone and estradiol for DIDP-15 and DIDP-150 groups 
Increased level of reactive oxygen species (ROS), MDA and 8-OHdG and glutathione (GSH) decreased at DIDP-15 and DIDP-150 
Increased level of NF-kappaB and Casp-3 in the brain at DIDP-15 and DIDP-150 
Decreased levels of BDNF at DIDP-15 and DIDP-150 and 
p-CREB at DIDP-150 
Reduced testosterone levels in serum and increased estradiol at DIDP-15 and DIDP-150 
DIDP-15 and DIDP-150 groups spent less time in the target quarter than the control group 
Adult NMRI mice Around 7 weeks of age: 14 days of exposure Oral (gavage) DBP: 6.25, 12.5, 25, 50, 100, and 200 mg/kg Since day 15 after the end of exposure Passive avoidance test: decreased avoidance latency for DBP-25, DBP-50, DBP-100, and DBP-200 groups Hematoxylin/eosin staining: decreased nuclei area in dentate gyrus for DBP-25 and DBP-100 groups Farzanehfar et al. [25] (2016) 
Open field: decreased total distance movement and time spent in center area at DBP-12.5 to DBP-200 
EPM: decreased time in open arm for DBP-25, DBP-50, DBP-100, and DBP-200 groups 
Spontaneous alternation Y-maze, rotarod and grip strength: no changes 
Adult Kunming mice 6 weeks of age: 14 days of exposure Oral (gavage) DINP: 0.2, 2, 20, 200 mg/kg/day MWM: days 6 to 12 OFT: day 14 MWM: increased escape latency increased during learning phase and increased escape latency at day 7 at DINP-200. During the test phase, decreased time spent and frequency to enter in the target quadrant at DINP-200 Hematoxylin/eosin staining: disorganized and loose pyramidal cells with swelling deformation in CA1 for DINP-20 and DINP-200 groups (no quantification) Ma et al. [26] (2015) 
Nissl staining showed loss of substance in cells with increasing exposure concentration 
Upregulation of caspase-3 and GFAP in the CA1 and cortex at DINP-20 and DINP-200 
Open field test (OFT): decreased central area entries for DINP-200 
Increased brain ROS levels for DINP-20 and DINP-200 groups, decreased GSH content at DINP-200, and SOD at DINP-20 and DINP-200. Increased level of DNA-protein crosslinks (DINP-200) and 8-OH-dG (DINP-20 and DINP-200) 
Increased expression levels of TNF-α and IL-1β at DINP-20 and DINP-200 
Adult Kunming mice 7 weeks of age: 9 days of exposure Oral (gavage) DINP: 1.5, 15, and 150 mg/kg/day During the 9 days of treatment MWM: increased latency to reach platform, reduced time spent, and number of entries in target quadrant for DINP-150 group Hematoxylin/eosin staining: CA1 pyramidal cells were loose and disordered, cell swelled, apical dendrites shortened, or disappeared at DINP-150 (not quantified) Peng et al. [27] (2015) 
Nissl staining: increased staining at DINP-15 and DINP-150 
Increased ROS, MDA, SOD, TNF-α, IL-1, caspase-3 activity in the brain at DINP-150 and decreased GSH. 
Lower gain weight in the DINP-150 group 

Bad, Bcl-2-associated death protein; Bax, Bcl-2–associated X protein; Bcl-2, B-cell lymphoma 2; BDNF, brain-derived neurotrophic factor; Bim, Bcl-2-interacting mediator of cell death; bw, body weight; COX-2, cyclooxygenase 2; CREB, cAMP response element-binding protein; d, day; DG, dentate gyrus; EPM, elevated plus maze; GD, gestational day; GSH, glutathione; IL, interleukin; MWM, Morris water maze; PND, postnatal day; T3, tri-iodothyronine; Tau, tubulin-associated unit; TNF, tumor necrosis factor; TR, thyroid hormone receptor.

Table 3.

Neuroanatomical and neuroendocrine effects of exposure to phthalates

Exposure periodSpeciesExposure durationExposure routePhthalate dosesAge at analysesNeuroanatomical and neuroendocrine analyses in the hypothalamus/hippocampus/cortex – other findingsReference
Prenatal/postnatal Wistar rats GD6 to PND21 Oral (gavage) DEHP: 0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135, and 405 mg/kg bw/d PND1 PND1: reduced aromatase activity for DEHP-0.135 and DEHP-0.405 groups Andrade et al. [28] (2006b) 
PND22 Increased aromatase activity for DEHP-15, DEHP-45 and DEHP-405 groups 
PND22: increased aromatase activity for DEHP-0.405 group 
Prenatal/postnatal Wistar rats GD0 to PND21 Oral (water) DEHP: 3 and 30 mg/kg bw/d PND30 Reduced serum level of follicle-stimulating hormone for DEHP-30. Decreased hypothalamic content of aspartate and increased level of GABA at DEHP-30 Carbone et al. [29] (2010) 
Reduced absolute testis weight in the DEHP-30 
Prenatal/postnatal Wistar rats GD0 to PND15 Oral (water) DEHP: 3 and 30 mg/kg bw/d PND15 Increased serum level of luteinizing hormone and follicle-stimulating hormone and hypothalamic aspartate concentration at DEHP-30. Decreased GABA content at DEHP-30 Carbone et al. [30] (2012) 
Reduced absolute testis weight at DEHP-30 
Prenatal/postnatal Sprague-Dawley rats GD14 to GD19 Oral (gavage) DEHP: 2, 10, or 50 mg/kg PND1-70 PND1: downregulation of hypothalamic ERβ, Clock, Dbp at DEHP-2 and DEHP-10, and Cyp19a1, Grin2a, Avpr1a, Kiss1r, Tac3r, Mtnr1a, Per2 at DEHP-10, and Arntl and Mtnr1a at DEHP-10 and DEHP-50 Gao et al. [31] (2018) 
PND70: altered Crhr1, Drd2 in the AVPV at DEHP-2, Avp and Tac3r at DEHP-10 and DEHP-50 and Hcrtr2 in the MPOA at DEHP-50. Altered Avp, ERα, ERβ, Ghrh, Kiss1, Tac2 at DEHP-50 and Npy, Pomc, and Trh at DEHP-10 and DEHP-50 in the arcuate nucleus (ARC) 
Reduced ERα immunofluorescence at DEHP-50 in the ARC; no alteration in anteroventral periventricular nucleus and MPOA 
Birth weight lower at DEHP-10 and DEHP-50; anogenital distance increased at DEHP-2. Reduced body weight before 8 weeks of age. Increased organ coefficient of prostate for DEHP-2 group with no changes for testis, epididymis, seminal vesicle, preputial glands, and levator ani 
Prenatal/postnatal Long-Evans rats GD2 to PND0 GD2 to PND10 Oral (diet) 35% DEP, 21% DEHP, 15% DBP, 15% DiNP, 8% DiBP, and 5% BBP at 0, 1, or 5 mg/kg ED18, PND5, PND10 Prenatal exposure Sellinger et al. [32] (2021) 
Increased apoptosis (Tunel cell density) on ED18 in the mPFC of the group exposed to 1 mg/kg 
Prenatal/postnatal exposure 
Increased apoptosis on PND10 in the medial prefrontal cortex for the dose of 5 mg/kg 
Prenatal/postnatal Long-Evans rats PND16 to PND22 Ip injection DEHP: 1, 10, or 20 mg/kg/d PND78 Reduced 21, 25, and 44 microRNAs in the hippocampus at DEHP-1, DEHP-10, or DEHP-20 and upregulated rno-let7e-p5 at DEHP-20 Luu et al. [33] (2017) 
Reduced rno-miR-191a-5p (involved in synaptic plasticity, associated with increased spine area, and decreased spine elimination) for DEHP-20 group 
Adult C57BL6/J mice PND60: 6 weeks of exposure Oral (diet) DEHP: 0.005 or 0.05 mg/kg/d. Mixture (mg/kg/d): DEHP 0.005, DBP 0.0005, BBP 0.0005, DiBP 0.0005, DEP 0.00025 End of treatment Medial preoptic area (mPOA) Ahmadpour et al. [34] (2021) 
Increased blood-brain barrier (BBB) leakage for DEHP-5, 50 and mixture groups 
Tight junction components: reduced protein levels of ZO-1 for DEHP-5, 50 and mixture and Cav-1 for DEHP-50 and mixture 
Increased NDRG2 and GFAP immunofluorescence in the parenchyma for DEHP-50 and mixture groups; increased iNOS immunoreactivity colocalized with GFAP surrounding capillaries for DEHP-5, 50, and mixture groups, and Iba-1 levels in mPOA parenchyma at DEHP-50 
Hippocampus 
Increased BBB leakage in CA1 and CA3 but not in the dentate gyrus, at DEHP-5 and 50 
Increased protein amount of ZO-1 for DEHP-50, and reduced Cav-1 in the CA1 and CA3 capillary walls for DEHP-50 and mixture groups 
Unchanged GFAP and NDRG2 and S100 beta immunolabeling in CA1, CA3, and DG; increased iNOS and Iba-1 immunoreactivity in the DG at DEHP-5 
Adult C57BL6/J mice PND60: 6 weeks of exposure Oral (diet) DEHP: 0.005 or 0.05 mg/kg/d. Mixture (mg/kg/d): DEHP 0.005, DBP 0.0005, BBP 0.0005, DiBP 0.0005, DEP 0.00025 End of treatment Medial preoptic area/hypothalamus Ahmadpour et al. [35] (2022) 
Reduced AR protein levels in microvessel-enriched fractions for DEHP-50 and mixture groups 
Increased gelatinase-immunoreactivity and reduced laminin α-1 immunoreactivity for DEHP-5, 50, and mixture groups 
Reduced type IV collagen (COL-IVα-1) amount in hypothalamic microvessel-enriched fraction for DEHP-5, 50, and mixture groups, reduced beta-dystroglycan-immunofluorescence for DEHP-50 in the mPOA. 
Increased Fluoro-Jade C labeling in DEHP-5, 50, and mixture groups 
Exposure periodSpeciesExposure durationExposure routePhthalate dosesAge at analysesNeuroanatomical and neuroendocrine analyses in the hypothalamus/hippocampus/cortex – other findingsReference
Prenatal/postnatal Wistar rats GD6 to PND21 Oral (gavage) DEHP: 0.015, 0.045, 0.135, 0.405, 1.215, 5, 15, 45, 135, and 405 mg/kg bw/d PND1 PND1: reduced aromatase activity for DEHP-0.135 and DEHP-0.405 groups Andrade et al. [28] (2006b) 
PND22 Increased aromatase activity for DEHP-15, DEHP-45 and DEHP-405 groups 
PND22: increased aromatase activity for DEHP-0.405 group 
Prenatal/postnatal Wistar rats GD0 to PND21 Oral (water) DEHP: 3 and 30 mg/kg bw/d PND30 Reduced serum level of follicle-stimulating hormone for DEHP-30. Decreased hypothalamic content of aspartate and increased level of GABA at DEHP-30 Carbone et al. [29] (2010) 
Reduced absolute testis weight in the DEHP-30 
Prenatal/postnatal Wistar rats GD0 to PND15 Oral (water) DEHP: 3 and 30 mg/kg bw/d PND15 Increased serum level of luteinizing hormone and follicle-stimulating hormone and hypothalamic aspartate concentration at DEHP-30. Decreased GABA content at DEHP-30 Carbone et al. [30] (2012) 
Reduced absolute testis weight at DEHP-30 
Prenatal/postnatal Sprague-Dawley rats GD14 to GD19 Oral (gavage) DEHP: 2, 10, or 50 mg/kg PND1-70 PND1: downregulation of hypothalamic ERβ, Clock, Dbp at DEHP-2 and DEHP-10, and Cyp19a1, Grin2a, Avpr1a, Kiss1r, Tac3r, Mtnr1a, Per2 at DEHP-10, and Arntl and Mtnr1a at DEHP-10 and DEHP-50 Gao et al. [31] (2018) 
PND70: altered Crhr1, Drd2 in the AVPV at DEHP-2, Avp and Tac3r at DEHP-10 and DEHP-50 and Hcrtr2 in the MPOA at DEHP-50. Altered Avp, ERα, ERβ, Ghrh, Kiss1, Tac2 at DEHP-50 and Npy, Pomc, and Trh at DEHP-10 and DEHP-50 in the arcuate nucleus (ARC) 
Reduced ERα immunofluorescence at DEHP-50 in the ARC; no alteration in anteroventral periventricular nucleus and MPOA 
Birth weight lower at DEHP-10 and DEHP-50; anogenital distance increased at DEHP-2. Reduced body weight before 8 weeks of age. Increased organ coefficient of prostate for DEHP-2 group with no changes for testis, epididymis, seminal vesicle, preputial glands, and levator ani 
Prenatal/postnatal Long-Evans rats GD2 to PND0 GD2 to PND10 Oral (diet) 35% DEP, 21% DEHP, 15% DBP, 15% DiNP, 8% DiBP, and 5% BBP at 0, 1, or 5 mg/kg ED18, PND5, PND10 Prenatal exposure Sellinger et al. [32] (2021) 
Increased apoptosis (Tunel cell density) on ED18 in the mPFC of the group exposed to 1 mg/kg 
Prenatal/postnatal exposure 
Increased apoptosis on PND10 in the medial prefrontal cortex for the dose of 5 mg/kg 
Prenatal/postnatal Long-Evans rats PND16 to PND22 Ip injection DEHP: 1, 10, or 20 mg/kg/d PND78 Reduced 21, 25, and 44 microRNAs in the hippocampus at DEHP-1, DEHP-10, or DEHP-20 and upregulated rno-let7e-p5 at DEHP-20 Luu et al. [33] (2017) 
Reduced rno-miR-191a-5p (involved in synaptic plasticity, associated with increased spine area, and decreased spine elimination) for DEHP-20 group 
Adult C57BL6/J mice PND60: 6 weeks of exposure Oral (diet) DEHP: 0.005 or 0.05 mg/kg/d. Mixture (mg/kg/d): DEHP 0.005, DBP 0.0005, BBP 0.0005, DiBP 0.0005, DEP 0.00025 End of treatment Medial preoptic area (mPOA) Ahmadpour et al. [34] (2021) 
Increased blood-brain barrier (BBB) leakage for DEHP-5, 50 and mixture groups 
Tight junction components: reduced protein levels of ZO-1 for DEHP-5, 50 and mixture and Cav-1 for DEHP-50 and mixture 
Increased NDRG2 and GFAP immunofluorescence in the parenchyma for DEHP-50 and mixture groups; increased iNOS immunoreactivity colocalized with GFAP surrounding capillaries for DEHP-5, 50, and mixture groups, and Iba-1 levels in mPOA parenchyma at DEHP-50 
Hippocampus 
Increased BBB leakage in CA1 and CA3 but not in the dentate gyrus, at DEHP-5 and 50 
Increased protein amount of ZO-1 for DEHP-50, and reduced Cav-1 in the CA1 and CA3 capillary walls for DEHP-50 and mixture groups 
Unchanged GFAP and NDRG2 and S100 beta immunolabeling in CA1, CA3, and DG; increased iNOS and Iba-1 immunoreactivity in the DG at DEHP-5 
Adult C57BL6/J mice PND60: 6 weeks of exposure Oral (diet) DEHP: 0.005 or 0.05 mg/kg/d. Mixture (mg/kg/d): DEHP 0.005, DBP 0.0005, BBP 0.0005, DiBP 0.0005, DEP 0.00025 End of treatment Medial preoptic area/hypothalamus Ahmadpour et al. [35] (2022) 
Reduced AR protein levels in microvessel-enriched fractions for DEHP-50 and mixture groups 
Increased gelatinase-immunoreactivity and reduced laminin α-1 immunoreactivity for DEHP-5, 50, and mixture groups 
Reduced type IV collagen (COL-IVα-1) amount in hypothalamic microvessel-enriched fraction for DEHP-5, 50, and mixture groups, reduced beta-dystroglycan-immunofluorescence for DEHP-50 in the mPOA. 
Increased Fluoro-Jade C labeling in DEHP-5, 50, and mixture groups 

Arntl, aryl hydrocarbon receptor nuclear translocator-like protein 1; Avp, arginine vasopressin; Avpr1a, arginine vasopressin receptor type 1a; AVPV, anteroventral periventricular nucleus; Cav-1, caveolin-1; Crhr1, corticotropin-releasing hormone receptor 1; Cyp19a1, cytochrome P450 family 19 subfamily A member 1; Drd2, dopamine receptor D2; ED, embryonic day; ER, estrogen receptor; GD, gestational day; GFAP, glial fibrillary acidic protein; Ghrh, growth hormone releasing hormone; Grin2a, glutamate ionotropic receptor NMDA type subunit 2A; Hcrtr2, hypocretin receptor 2; Iba-1, ionized calcium-binding adapter molecule 1; iNOS, inducible nitric oxide synthase; Kiss1r, kisspeptin 1 receptor; Mtnr1a, melatonin receptor 1A; NDRG2, N-myc downstream-regulated gene 2; Npy, neuropeptide y; Pomc, Pro-opiomelanocortin; Per2, period circadian regulator 2; PND, postnatal day; Tac2, tachykinin 2; Tac3r, tachykinin receptor 3; Trh, thyrotropin-releasing hormone; ZO-1, zonula occludens-1.

Reproductive Behaviors and Related Neural Processes

Prenatal/Postnatal Exposure

Andrade et al. [13] showed that rats exposed prenatally and postnatally to 5 mg/kg/day DEHP exhibited a decreased latency to the first mount, without changes in the other behavioral components at any of the tested doses. Under comparable experimental conditions, the hypothalamic aromatase activity was reduced by the lowest DEHP doses and increased by the highest ones [28]. In another rat study [14], prenatal and postnatal exposure of rats to 1 mg/kg/day of di-isopentyl phthalate (DiPeP) resulted in an increased latency to the first mount and intromission and reduced partner preference (Table 1). The expression of male sexual behavior relies on the activation by gonadal testosterone of the neural circuitry underlying this behavior. Testosterone activates directly the AR but can also be metabolized into neural estradiol, which stimulates the estrogen receptors (ER). In the study of Neubert da Silva et al. [14], no modifications in the hypothalamic expression of genes encoding ERα (esr1), ERβ (esr2), or AR were associated with the observed behavioral changes.

In some studies, the authors did not investigate reproductive behavior but did explore the effects of perinatal exposure to phthalates on the hypothalamus (Table 3). Gao et al. [31] showed that prenatal exposure of rats to DEHP at 2 mg/kg/day altered the expression of genes encoding the corticotropin-releasing hormone receptor 1 (Crhr1) and the D2 dopamine receptor, known to be involved in GnRH secretion inhibition [36, 37], in the anteroventral periventricular nucleus of adult animals. The dose of 2 mg/kg/day altered the anogenital distance, an androgen-sensitive endpoint, suggesting that the observed gene expression modifications were maybe induced by early hormonal changes. Whether these hypothalamic modifications impaired some of the functions and behaviors controlled by the hypothalamus requires further investigation. In two rat studies using DEHP at 3 mg/kg/day, the researchers found no effect on hypothalamic aspartate and GABA content and serum luteinizing hormone and follicle-stimulating hormone levels [29, 30].

Pubertal Exposure

Capela and Mhaouty-Kodja [15] showed that pubertal exposure of mice to DEHP at 5 and 50 μg/kg/day decreased the duration and number of ultrasonic vocalizations emitted by adult males in the presence of receptive females (Table 1). Such exposure also altered mate preference as receptive females spent more time with control than phthalate-exposed males and increased the latency to ejaculation as well as the number of pelvic thrusts [15]. These behavioral alterations were associated with unchanged testosterone levels and weight of androgen-sensitive tissues (seminal vesicles, testis, and the Tyson gland). There were reduced AR protein amounts in the medial preoptic area, the key hypothalamic area underlying the expression of male sexual behavior. Interestingly, these effects were observed 2 months after the exposure arrest, indicating that pubertal exposure induced long-term changes at the neural level [15].

Adult Exposure

Exposure of adult male mice to DEHP at 5–50 μg/kg/day reduced their emission of ultrasonic vocalizations and their ability to attract females and increased the latency to initiate mating and reach ejaculation [16]. At the neural level, DEHP did not affect the gonadotropic axis as evidenced by unchanged kisspeptin immunoreactivity, Kiss1 gene expression in the RP3V or arcuate nucleus, and circulating testosterone levels. Proteomic analysis of the medial preoptic area showed increased levels of astrocytic proteins such as glial fibrillary acidic protein (GFAP) and N-myc downstream-regulated gene 2 (NDRG2). These behavioral and protein level alterations were associated with fewer AR-immunoreactive cells in the medial preoptic area and other chemosensory regions involved in male sexual behavior (medial amygdala and the bed nucleus of the stria terminalis). These findings led us to suggest that low doses of DEHP possibly act directly at the neural level to induce antiandrogenic effects through AR downregulation, without any changes in the number of estrogen receptor alpha (ERα)-immunoreactive neurons.

Learning and Memory and Related Neural Processes

The majority of studies investigating the impact of phthalate exposure on learning and memory have performed spatial memory tests using the Morris water maze (MWM). The most studied brain region was the hippocampus with a few studies focusing also on the medial prefrontal cortex (Tables 2, 3).

Prenatal/Postnatal Exposure

Two studies assessing spatial memory in the MWM showed an increased latency or traveled distance to reach the platform and reduced time spent in the target quadrant in rats exposed to DEHP at 5 μg/kg/day [17] or 1.25 mg/kg/day [19]. These behavioral changes were associated in the adult hippocampus with reduced gene expression of insulin and its signaling pathway involving phospho-Akt and phospho-glycogen synthase kinase 3 beta (GSK3β) [19]. The impairment of insulin signaling pathway is suggested to contribute to neurodegeneration and cognitive dysfunction in Alzheimer disease [38]. There was also increased expression of Bcl-2-associated death protein (Bad) and Bcl-2-interacting mediator of cell death (Bim) and tubulin-associated unit (Tau) phosphorylation; Tau is an axonal marker associated with altered structural plasticity. In contrast, there were no effects on spatial memory using the same test in mice prenatally exposed to 5 mg/kg/day of DEHP [22]. In this study, there were altered free tri-iodothyronine (T3) levels and protein expression of deiodinases 2 and 3 as well as thyroid hormone receptors α and β and brain-derived neurotrophic factor (BDNF) in the fetal brain.

In the attentional shift test, while there was no effect of the phthalate mixture exposure during the training phase, rats exposed to the two-mixture doses displayed altered performance in extradimensional shift and showed more perseverative and omission errors than the control group (Table 2 and [18]). This was associated with fewer neurons and synaptophysin-positive boutons in the medial prefrontal cortex [18]. Using a comparable phthalate mixture, Sellinger et al. [32] reported a decreased bromodeoxyuridine-labeled cell density and more terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the medial prefrontal cortex at embryonic day 18 or postnatal day 10.

In the Y-maze spontaneous alternation test, mice exposed during the prenatal period to 0.2 mg/kg/day of DEHP displayed fewer arm entries [21]. Whether this effect was related to impaired memory and/or increased anxiety-related behavior is not clear because opposite data on the anxiety-state level were obtained using the elevated plus maze and the open field tests. In the same study, the authors showed that prenatal exposure to DEHP at 0.2 m/kg/day increased the expression of cyclooxygenase 2 (COX-2), a neuroinflammatory marker, in pyramidal neurons of the hippocampal CA2/3 region in adult male mice [21]. Holahan et al. [20] used an operant task to bar press for chocolate-flavored pellets and found no behavioral effect in rats postnatally exposed to 1 mg/kg of DEHP. Postnatal exposure to an equivalent dose of DEHP induced changes in the expression of several microRNAs in the hippocampus [33], as shown in Table 3.

Prepubertal/Pubertal Exposure

In mice exposed to DEHP (0.18 and 1.8 mg/kg/day), Feng et al. [23] reported increased latency to reach the platform and decreased time spent in the target quadrant and platform area of the MWM. In this study, the authors found decreased activity of acetylcholine esterase and increased oxidative stress with reduced levels of glutathione and superoxide dismutase (SOD) in the whole brain. There were altered messenger RNA (mRNA) levels of several genes involved in neurotransmitter synthesis (tryptophan hydroxylase 2 [Tph2]), neurotransmitter and neuropeptide levels (serotonin, GABA, arginine vasopressin [Avp]), and neurotransmission and neuroplasticity (cyclic adenosine monophosphate [cAMP] and Ca2+ contents, phosphorylated protein kinase A [PKA], phosphorylated extracellular signal-regulated kinase [ERK], cAMP response element-binding protein [CREB], and calcium/calmodulin-dependent protein kinase [CamK]). Ge et al. [24] found that exposure to 0.15 or 1.5 mg/kg/day of DIDP did not affect spatial memory, hippocampal structure, or BDNF and CREB levels.

Adult Exposure

Three studies have addressed adult exposure to 6.25 mg/kg/day of DBP [25], or 0.2, 1.5, or 2 mg/kg/day of DINP [26, 27]. There were no behavioral alterations based on the MWM or passive avoidance test and no neural effects (Table 2).

The BBB: A New Relevant Neural Target for Exposure to Endocrine-Disrupting Compounds?

Behavioral and brain functions rely on neuronal and glial activities, which are ensured by cerebral homeostasis maintained by the BBB. This neurovascular unit comprises endothelial cells of the capillary wall and their surrounding parenchyma. Cerebral endothelial cells limit the passage across the BBB through inter-endothelial tight junctions, selective transporters located in the luminal/abluminal side of endothelial cells, and caveolae-mediated transcellular vesicular transport [39].

Interestingly, cerebral vessels are also sensitive to androgens, which influence BBB properties and modulate neuroinflammatory responses in males (for a review, see [40]). In particular, we have shown that chronic androgen depletion of adult male mice by castration-induced neural AR downregulation increased BBB permeability and activation of astrocytes and microglia and upregulated inflammatory molecules in the medial preoptic area [41]. These findings suggest that exposure to chemical substances with antiandrogenic activity such as phthalates could also interfere with BBB integrity.

Effects of Adult Exposure to Phthalates on the Hypothalamic Neurovascular Unit

We have recently shown that adult exposure to DEHP alone at 5 or 50 µg/kg/day, or to an environmentally relevant mixture of phthalates, led to BBB leakage in the medial preoptic area [34]. This was evidenced by reduced levels of the endothelial tight junction accessory proteins zonula occludens-1 (ZO-1) and caveolin-1 isoform protein (Cav-1), the main caveolar transmembrane protein. These changes were associated with activation of capillary-associated microglia and astrocytes. Interestingly, there was degradation of the basement membrane surrounding capillary walls, which play a pivotal role in vascular integrity including BBB maintain, with a significant increase in the gelatinase activity of matrix metalloproteinase (MMP) 2 and 9 [35] and an increase of inducible nitric oxide synthase (NOS) protein [34]. Given the interaction between Cav-1, nitric oxide, and MMP signaling pathways and their reported involvement to ensure vascular protection [42], it is possible that elevated MMP activity results, at least partly, from the reduced Cav-1 protein levels leading to a disinhibition of NOS and an increase in NO production.

On the other hand, adult exposure to DEHP alone or in a phthalate mixture reduced the protein levels of AR but not of ERα in the capillaries of the hypothalamus in male mice [35]. Reduced AR and/or Cav-1 protein expression in brain capillaries could lead to a low interaction between Cav-1 and AR [43], and thus to lower AR transactivation. This lower AR/Cav-1 interaction and the resulting alteration of AR-mediated cellular pathway can participate also to the disruption of the glio-neurovascular in the medial preoptic area and consequently to the impaired male sexual behavior [16].

Effects of Adult Exposure to Phthalates on the Hippocampal Neurovascular Unit

Exposure to low doses of DEHP alone at 5 or 50 µg/kg/day or DEHP at 5 µg/kg/day in a phthalate mixture significantly induced microglial activation and increased BBB permeability in the hippocampal CA1 and CA3 regions [34]. This BBB leakage was associated with increased levels of the accessory junctional protein ZO-1 in hippocampal microvessels following exposure to DEHP at 50 µg/kg/day, with no effect on the levels of the major tight junction transmembrane proteins, claudin-5 and occludin, regardless of the dose. These findings suggest a possible impairment in the organization of endothelial tight junctions [34]. In addition, adult exposure to DEHP at 50 µg/kg/day or to DEHP at 5 µg/kg/day in a phthalate mixture led to a significant decrease in the hippocampal CA1 and CA3 regions of Cav-1, the main transendothelial vesicular transport transmembrane protein. By contrast, in the hippocampal dentate gyrus, none of these modifications induced by phthalate exposure were observed, indicating that BBB integrity was preserved in this hippocampal vascular network [34]. These data suggest that, within the same brain structure, the BBB may exhibit differential sensitivity to exposure to DEHP alone or in a phthalate mixture. A possible explanation for these data is that the hippocampal CA1 and CA3 regions present higher AR expression than the dentate gyrus in mice [11].

Based on our review analyzing studies using low doses of phthalates (around 41% of the total studies; Fig. 1), behavioral modifications can be induced when the exposure occurs during critical periods of development, including the prenatal/postnatal and prepubertal/pubertal periods [14, 15, 17‒19, 21, 23]. The associated neural modifications involve neuropeptides or receptors related to sex steroids, insulin, and insulin growth factor (IGF) 1/IGF 2, or Avp systems [15, 19, 23, 28], as well as neurotransmitters, pre- and postsynaptic proteins, and mediators [18, 23], or proinflammatory proteins such as COX-2 [21]. It is not yet clear whether these changes can occur in the whole brain or are rather specific to some brain areas, and if a (and which) common initiating molecular mechanism can trigger these modifications. For adult exposure, more studies addressing chronic exposure to phthalates are needed, given the vulnerability of the nervous system also at late stages. In particular, assessing the impact of chronic low-dose exposure on learning and memory still needs to be documented because only three studies using acute exposure to phthalates are available [25‒27].

An interesting result regarding the effects of adult exposure to phthalates at low doses in the hypothalamus and hippocampus is disruption of the BBB integrity associated with increased glial activation and/or neuroinflammatory processes [34, 35]. In the field of environmental toxicology in general and endocrine disruption in particular, physiological barriers including the BBB are considered mainly for the passage of substances and less for the impact of chemical substances on their integrity. The BBB has endocrine properties through response to endocrine systems and secretion of endocrine molecules [44], and it is also considered a key component and perhaps an initiating trigger of neurological and psychiatric disorders [45]. This suggests that BBB integrity should be one of the key targets to assess for the impact of adult exposure to phthalates or other endocrine-disrupting compounds on the nervous system [46]. Much remains to be done concerning evaluation of exposure to phthalates on the developing BBB, which is not only present in the developing brain but must play a key protecting role in the developmental exposure of the brain to chemical substances [47, 48].

The authors declare that they have no known financial interest or personal relationships that could have appeared to influence the work reported in this paper.

This work was supported by the Agence Nationale de la Recherche (Phtailure, 2018) in France.

S.D. and S.M.K. performed the literature review; S.M.K. and V.G.M. wrote the manuscript. All the authors read and approved the final version of the manuscript.

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