2025 Volume 50 Issue 11 Pages 601-615
Neonatal growth and development are significantly influenced by maternal care and breastfeeding. Our previous research showed that maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dams reduced prolactin (PRL) levels, nursing behavior, and milk production during lactation. Intracerebroventricular infusion of PRL in TCDD-exposed dams partially reversed these defects in mothers and offspring. However, the mechanism by which maternal TCCD exposure causes reduced PRL levels and multigenerational effects remains unclear. This study aimed to investigate the multigenerational effects of maternal TCDD exposure and sought solutions to the developmental issues arising from low PRL levels due to TCDD exposure during gestation. Oral administration of TCDD (1 µg/kg) to pregnant rats on gestational day 15 (F0 dams) led to decreased PRL concentrations in female offspring (F1/F2) and impaired maternal licking behavior when F1 females had given birth, resulting in adverse effects on body weight and short-term memory in F2/F3 offspring. Aripiprazole (ARI), a partial dopamine D2 receptor (D2R) agonist, increases PRL levels by inhibiting the effect of dopamine during PRL synthesis and secretion. Importantly, administration of ARI to F0 dams not only restored PRL levels, nursing behavior, and milk volume in the treated mothers but also mitigated the developmental deficits observed in F2/F3 offspring. These findings highlight the critical role of PRL in maternal care and offspring development and suggest that ARI could be a potential therapeutic intervention to mitigate the effects of TCDD-induced multi-generational developmental disruptions.
Dioxins and dioxin-like compounds are a group of highly toxic and persistent organic pollutants that pervade the environment and accumulate in human tissues through biomagnification in the food chain. These compounds cause severe health problems, including cancer (Mead, 2008), reproductive and developmental disorder (Yu et al., 2020), immune system damage (Keller et al., 2004), nervous system dysfunction (Gileadi et al., 2021), and significant endocrine disruption (Liang et al., 2024). The toxicity of dioxins is primarily mediated by their interactions with the aryl hydrocarbon receptor (AHR). In their inactive form, dioxins bind to AHR in the cytoplasm, leading to its translocation into the nucleus, dimerizing with the AHR nuclear translocator. The binding of this dimer to dioxin-responsive elements in DNA alters the transcription of various genes (Pirkle et al., 1989). Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), characterized by chlorine atoms at the second, third, seventh, and eighth positions, is the most potent member of the group of polyhalogenated aromatic hydrocarbons. Owing to their high lipophilicity, dioxins are extremely persistent and difficult to eliminate from the body (Poland and Knutson, 1982). TCDD has an exceptionally prolonged half-life in humans, ranging between 7.1 and 11.3 years approximately (Denison and Nagy, 2003).
Maternal exposure to TCDD poses significant risks to developing fetuses and newborns because the compound can cross the placental barrier and be transferred through milk (Ishida et al., 2010). This exposure can lead to various developmental disorders in offspring (Peterson et al., 1993; Hojo et al., 2008), including reduced body weight and length, disturbances in sexual maturation (Yuan et al., 2023), learning and memory impairments (Zhang et al., 2018), and brain development and function (Gileadi et al., 2021).
Research indicated that gestational exposure to TCDD reduces pituitary prolactin (PRL) production in lactating dams (Takeda et al., 2020). PRL is a crucial polypeptide hormone known for its role in lactation. It is traditionally produced by lactotrophic cells in the anterior pituitary gland (Georgescu et al., 2021); however, this hormone is also synthesized in the central nervous and immune systems, uterus, and mammary glands. PRL is crucial in regulating mammary gland development, milk secretion, reproductive health, maternal behavior, immune responses, and cellular growth and differentiation (Ben-Jonathan et al., 1996).
TCDD-induced reduction in PRL level impairs maternal nursing behavior, disrupts mammary gland function, and affects milk protein expression (Takeda et al., 2020). This hormonal deficiency leads to atrophy of the mammary lobules and reduced milk ejection, further diminishing the dam’s ability to nurse effectively. These disruptions contribute to offspring growth retardation, including slow weight gain and impaired learning and memory. Overall, gestational exposure to TCDD negatively affects offspring development by compromising maternal PRL levels and nursing behaviors and by disrupting normal mammary gland development and milk secretion mechanisms. These further exacerbate developmental disorders in the offspring. However, the mechanism by which maternal TCCD exposure causes reduced PRL levels and multigenerational effects remains unclear.
Dopamine, a neurotransmitter produced in the hypothalamus, inhibits PRL synthesis and secretion (Al-Kuraishy et al., 2022). Aripiprazole (ARI), a third-generation antipsychotic medication, is primarily used to treat schizophrenia and bipolar disorder (Correll et al., 2021). It has a slow dissolution rate and high affinity (Carboni et al., 2012) for dopamine D2 receptors (D2R). It acts as a dopamine system stabilizer, providing dual regulation of the dopamine system (Stelmach et al., 2023; Kikuchi et al., 2021). Although it remains to be established whether maternal exposure to TCDD promotes increases in dopamine levels in dams, a previous study (Russell et al., 1988) has reported that exposure to TCDD can contribute to an elevation in dopamine levels. This accordingly raises the possibility that maternal dopamine levels may also increase, potentially leading to a suppression of PRL expression and subsequent effects on maternal behavior. Given its mechanism of action, ARI is expected to counteract the inhibitory effects of TCDD on PRL secretion and restore normal PRL levels by modulating dopamine signaling pathways. This study aims to investigate whether developmental issues in newborns resulting from maternal TCDD exposure are inherited across generations. It also seeks to examine whether ARI supplementation can effectively restore low maternal PRL levels, thereby reversing growth retardation in affected newborns. Furthermore, we explored whether these recovery effects extend to subsequent generations and sought to elucidate the underlying mechanisms involved in these processes.
TCDD (purity > 99%, as determined by GC/MS) was purchased from AccuStandard, Inc. (New Haven, CT, USA). Standard chow (CE-2) was obtained from CLEA Japan Inc. (Tokyo, Japan). The oral administration tube (KN-349 for rat A) was purchased from Natsume Manufacturing Co., Ltd. ARI, used for preparing the mixed chow, was provided by FUJIFILM Wako Pure Chemical Corp. All other reagents were of the highest commercially available quality.
Animals and treatmentsAll animal experiments were approved by the Institutional Animal Care and Experimental Committee of the Kyushu University. Male Wistar (10 weeks old) and female Wistar rats (6−7 weeks old) were obtained from CLEA Japan Inc. The rats were housed under a 12-hr light/dark cycle and had access to food and tap water ad libitum. Female rats were paired with males, and pregnancy was confirmed by the presence of sperm, marking gestational day (GD) 0. On GD 15, the dams were administered TCDD at a dose of 1 µg/kg per oral, following our previous studies (Takeda et al., 2009; Takeda et al., 2014; Taura et al., 2014; Hattori et al., 2018). The control group received corn oil as a vehicle, which was sourced from Ajinomoto Co. Inc. (Tokyo, Japan). From GD20 to postpartum day (PD) 21, one of the TCDD and control groups was supplemented with 0.009% ARI mixed chow (CLEA Japan) as an F0 generation dam. Female offspring were allowed to grow, mate, and give birth naturally to produce F1 and F2 dams. Maternal behavior was assessed on PD 2, 4, 7, 14, and 21. Tissues from dams were collected on PD7 and PD21, and tissues from offspring were collected on post-neonatal day (PND) 21 and 28, with euthanasia performed using carbon dioxide. We measured the offspring body weights and lengths on PND 2, 4, 7, 14, and 21. Milk production ability and maternal serum PRL levels were evaluated. Additionally, the developmental status of the offspring was assessed based on their height, weight, anxiety, and activity levels, as measured using an open field experiment. There were no significant changes in litter size or sex ratio between these generations (Fig. S1 D, E).
Enzyme immunoassayBlood from euthanized rats was collected via decapitation, allowed to stand at room temperature for 1 to 2 hr, and then centrifuged at 12,000 rpm for 15 min at 4°C. We measured the serum PRL and transforming growth factor-β1 (TGF-β1) concentrations using specific enzyme immunoassay (EIA) kits. The Rat Prolactin EIA Kit (Bertin Pharma, Montigny le Bretonneux, France) was used for serum PRL, with serum samples diluted twice in the buffer. The TGF-β1 kit from R&D Systems was used for TGF-β1, with these samples diluted 90 times with the supplied buffer. The plates were washed five times in both assays using a shaker mode (diameter: 5 cm, speed: 120 to 300 rpm) with 300 µL of wash buffer per well for 1 min each. The antibody reaction for PRL was incubated at 25°C for 16−20 hr, with the color reaction performed at 25°C for 4 hr. For TGF-β1, the antibody reaction was incubated at room temperature for 2 hr, followed by a color reaction at room temperature for 30 min. Absorbance was measured at 405 nm for PRL and 450 nm for TGF-β1 using an automatic microplate reader (Immuno Mini NJ-2300; Nalge Nunc Corp.).
Maternal behaviorBehavioral tests were carried out to evaluate the maternal capacity of dams on PD 2, 4, 7, 14, and 21 following previous reports (Nephew and Bridges, 2011; Myers et al., 1989). Before conducting each test, dams were separated from their offspring for 30 min to enhance their motivation for maternal behaviors. Maternal behavior was observed immediately after returning the offspring to the maternal cage. Total licking time was measured as an index of maternal behavior.
Milk measurementOn PD 5, maternal rats were separated from their offspring for 2 hr to maintain constitutive milk production in the mammary glands. The dams were administered 2 μg/mL oxytocin intraperitoneally to induce milk secretion due to the relaxation of the mammary gland smooth muscles. They were anesthetized by the inhalation of 2% sevoflurane, and their mammary glands were suctioned for 30 min using a dry aspirator (ASONE Corp., Osaka, Japan) connected to a one-handed milker KN-591 (Natsume Seisakusho Co. Ltd., Tokyo, Japan). To avoid damage to the mammary glands by physical aspiration, the sucking duration per mammary gland was set at 5 min, with each interval lasting 10 sec.
Histopathological examination of Mammary glandMammary glands were collected from dams on PD 7 and fixed in 4% paraformaldehyde for 24 hr. After substituting with 10, 20, 30, and 30% sucrose for 4 days, the tissue was embedded in Tissue-Tek® OCT compound and frozen at −80°C. The frozen mammary gland was sliced into 25-µm-thick sections using a microtome (CM3050S; Leica Microsystems, Wetzlar, Germany), and each section was mounted on a glass slide. After washing with water and methanol, the sections were immersed in 0.1% Mayer’s hematoxylin solution and 1% Eosin Y solution (H&E) for 5 and 10 min, respectively. The sections were mounted in Entellan® New (Merck Millipore Corp., Billerica, MA, USA) and observed under an optical microscope (×10, ×40).
Protein determinationMilk samples were processed for protein determination analysis as follows: fresh milk was quickly frozen in liquid nitrogen and stored at -80°C until analysis. The protein concentration was determined using the Lowry method [28] with bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) as a standard.
Reverse transcription-polymerase chain reaction (RT-PCR)The mRNA expression of DOPA decarboxylase (Ddc), PRL, β-casein, leptin, TGF-β1, cytochrome P450 1A1 (CYP1A1), catechol-O-methyltransferase (COMT), D2R, and tyrosine hydroxylase (TH) was quantified using real-time RT-PCR. The total RNA was extracted from the hypothalamus using the RNeasy Kit (QIAGEN GmbH, Hilden, Germany), treated with genomic DNA (gDNA) Eraser (TaKaRa-bio, Shiga, Japan) to digest contaminating gDNA, and reverse-transcribed to cDNA. RNase-free water used to extract total RNA was treated with a gDNA eraser and reverse transcribed to prepare the negative control. Target mRNAs were amplified with Fast SYBR Green Master Mix (Thermo-Fisher Scientific, Waltham, MA, USA) using a StepOnePlus Real-Time PCR system (Applied Biosystems, Thermo-Fisher Scientific, Carlsbad, CA, USA). The primer design and PCR conditions are described in Table 1. The target mRNA level was normalized to that of β-actin mRNA.
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Intracerebroventricular PRL supplementation in dams was performed on the morning of PD 0, following our previous report (Takeda et al., 2014). Studies reported that administering 400 ng/day of PRL into the lateral ventricle of dams stimulated maternal behavior. Following anesthetization by an intraperitoneal administration of a mixture (10 mL saline/kg) consisting of medetomidine (0.15 mg/kg), midazolam (2 mg/kg), and butorphanol tartrate (2.5 mg/kg), a cannula (brain infusion kit 2: DURECT Corp., Cupertino, CA) was infused at the position of antero-posterior, −1.0 mm and lateral, 1.0 mm from the bregma, and depth of 4 mm. An osmotic mini pump (model 2002, DURECT) was filled with 200 μL PRL (50 ng/μL; Peptide Institute, Inc., Osaka, Japan), which was dissolved in sterilized saline. After surgery, the rats were administered atipamezole (0.3 mg/kg) intraperitoneally to ensure prompt post-anesthesia recovery. They were returned to the home cage with their 8 offspring (four males and females) randomly chosen to normalize the litter size. Control rats were similarly operated on and fitted with a cannula through which a vehicle not containing PRL was infused.
Short-term memory and alternation behaviorThe short-term memory of the 8–10-week-old offspring was evaluated using the Y-maze test. The test apparatus consisted of three plastic arms (60 cm × 12 cm) with walls (20 cm high). Each rat was placed in one of the arms and allowed to explore freely for 5 min. The behavior of the rats was monitored and recorded using video tracking software (Ethovision XT9, Noldus Information Technologies, Wageningen, The Netherlands). Spontaneous alternation behavior (SAB) was defined as the frequency of consecutive entries into three different arms, and the percentage of SAB entries relative to total arm entries was calculated as an index of short-term memory.
Open field testIn this study, the open field apparatus was a white square plastic box (40×40×30 cm) with a defined central area (15×15 cm). Locomotor and anxiety-related behaviors were analyzed by calculating parameters from a 5-minute recording using video tracking software (Ethovision XT9, Noldus Information Technologies, Wageningen, The Netherlands). The locomotion parameters included total distance traveled, immobility duration, mean speed, and time spent in the inner area.
Statistical analysisAll data are expressed as mean ± S.E.M. Statistical differences were determined using either an unpaired two-tailed t-test for comparisons between two groups or a one-way analysis of variance (ANOVA) for comparisons involving more than two groups, with Tukey-Kramer post-hoc test. Statistical analyses were performed using GraphPad Prism 8.0 software. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were considered statistically significant.
Oral administration of 1 μg/kg TCDD on GD 15 considerably caused multigenerational effects and suppressed serum PRL concentrations in F1 and F2 female offspring at 4 weeks of age (Fig. 1A, B). Notably, when these females became mothers, their licking behavior was markedly reduced due to maternal TCDD exposure in F0 dams (Fig. 1C, D), adversely affecting the weight of F2 and F3 offspring (Fig. 1E, F). Moreover, we supplemented mothers with PRL from PD 0 to 14. PRL supplementation increased tendency for weight gain and improved short-term memory in F2 neonates (Fig. 1G, H). These results suggest that multigenerational developmental issues caused by maternal TCDD exposure are primarily due to insufficient PRL, highlighting PRL supplementation as a potential strategy to counteract TCDD-induced developmental toxicity.
Multigenerational effect of maternal exposure to TCDD (1 μg/kg at GD15) in F0 mothers. (A, B) PRL serum concentration in F1/F2 female rats at PND 28, separately. (C, D) Maternal behavior exhibited by F1 and F2 female rats when they became dams on PD 2, respectively. (E, F) Body weight at PND 21 of F2/F3 male offsprings, separately. (G, H) Intracerebroventricular supplementation of PRL to dams was performed in the morning from PD 0 to PD 14, and body weight on PND7 and short-term memory in 8–10-week-old F2 male offsprings were assessed. Bars represent the mean ± S.E.M. of four to six dams and four to six male and female rats. Statistical comparisons were performed using an unpaired two-tailed t-test for comparisons between two groups. *p<0.05. TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; PRL, prolactin.
In the present study, we used a 0.009% dietary concentration of ARI, which markedly restored the decreased serum PRL levels in F0 dams on PD 21. This recovery was multigenerational, as ARI supplementation also elevated PRL concentrations in the serum of F1 and F2 dams on PD 21 (Fig. 2A–C), although it did not increase PRL mRNA expression levels in these generations (Fig. S2A–C). TCDD exposure considerably reduced the total licking duration at all time points during the lactation period (Fig. 2D) and increased the latency to first licking on PD2 in F0 dams (Fig. S1E), while there were no changes in the latency to first sniffing or retrieving time between the groups of F0 dams (Fig. S1D, F). Based on these findings, we focused on licking behavior as a primary maternal measure in subsequent generations. Despite the gradual reduction in TCDD toxicity over time, a marked reduction in maternal behavior persisted in F1 and F2 dams (Fig. 2E, F). In contrast, ARI supplementation considerably improved licking behavior in F0, F1, and F2 dams on PD 7 (Fig. 2D–F). This study demonstrates that ARI supplementation effectively restores PRL secretion and maternal licking behavior in F0, F1, and F2 dams exposed to TCDD, highlighting its potential multigenerational benefits.
Effect of maternal exposure to TCDD (1 μg/kg on GD 15) and ARI supplementation on prolactin (PRL) concentration and licking behavior in F0/F1/F2 dams. (A–C) PRL serum concentrations in F0/F1/F2 dams on PD21. (D–F) Licking behavior in F0/F1/F2 dams on PD 2, PD 4, PD 7, PD 14, and PD 21. Bars represent the mean ± S.E.M. of four to six dams. Statistical comparisons were performed using a one-way analysis of variance for comparisons involving more than two groups, with Tukey–Kramer post-hoc test. *p<0.05, **p<0.01. ARI, aripiprazole.
Maternal exposure to TCDD considerably decreased COMT mRNA expression, leading to a marked increase in dopamine levels (Fig. 3F). ARI, acting as a partial dopamine D2 receptor agonist, may mitigate this effect by reducing dopamine transmission. Furthermore, ARI intervention markedly decreased the elevated TGF-β1 serum concentrations and TGF-β1 levels (Fig. 3D, E). This suggests that ARI may restore prolactin secretion affected by TCDD exposure by regulating dopamine signaling and TGF-β1 levels. However, the mRNA expression levels of Ddc, TH, and D2R did not significantly differ between the groups (Fig. 3A-C).
Effect of maternal exposure to TCDD (1 μg/kg on GD 15) and ARI supplementation on dopamine pathway in F0 dams. Hypothalamic mRNA levels of (A) Ddc, (B) TH, (D) COMT and (E) TGF-β1 in F0 dams on PD7. (C) D2R mRNA levels in the pituitary on PD 7 and (F) Serum TGF-β1 concentration on PD 21 in F0 dams. Bars represent the mean ± S.E.M. of four to six dams. Statistical comparisons were performed using a one-way analysis of variance for comparisons involving more than two groups, with Tukey–Kramer post-hoc test. *p<0.05, **p<0.01. TGF, transforming growth factor; COMT, catechol-O-methyltransferase; TH, tyrosine hydroxylase; Ddc, DOPA decarboxylase; D2R: dopamine D2 receptor.
In the present study, TCDD exposure in F0 dams markedly reduced milk production across generations. Notably, ARI supplementation in F0 dams effectively restored the milk volume across generations (Fig. 4A–C). To understand the mechanisms underlying this restoration, we examined the mammary gland development in F0 dams during lactation. The results showed that ARI supplementation reversed mammary duct stenosis compared to the TCDD-exposed group (Fig. 4D). Additionally, mRNA analysis revealed that ARI intervention considerably suppressed the elevated leptin expression induced by TCDD exposure (Fig. 4E). Moreover, our results showed that TCDD exposure decreased the expression of key nutrients such as β-casein in the mammary glands on PD 7 and overall milk protein content on PD 5 (Fig. 4F, G). In contrast, ARI supplementation restored these levels in F0 dams (Fig. 4F, G). In summary, maternal TCDD exposure severely impaired milk production and nutrient content across generations; however, ARI supplementation effectively restored these deficits. This highlights the potential of ARI as a therapeutic intervention to enhance milk production and support mammary gland development by improving PRL levels.
Effect of maternal exposure to TCDD (1 μg/kg at GD 15) and ARI supplementation on milk production, mammary gland development, milk protein concentration, and β-casein in the mammary glands of F0 dams. (A–C) Average milk secretion ability on PD 5 in F0, F1, and F2 dams, respectively. (D) Hematoxylin and eosin staining (0.1% hematoxylin and 1% eosin) of the mammary glands in F0 dams was performed on PD 7 to assess mammary gland development. Arrows indicate mammary ducts, which are part of the ductal structure of the mammary gland. (E, F) mRNA expression levels of genes related to mammary gland development and milk nutritional factor in the mammary glands of F0 dams on PD 7. (G) Total protein concentration was determined on PD 5 from F0 dams. Bars represent the mean ± S.E.M. of four to six dams. Statistical comparisons were performed using a one-way analysis of variance for comparisons involving more than two groups, with Tukey–Kramer post-hoc test. *p<0.05, **p<0.01.
The effect of maternal TCDD exposure on the spontaneous locomotion of female offspring was assessed using an open-field test. We observed that the trajectories were denser in the 1 μg/kg TCDD group compared to the control group (Fig. 5A). Consistent with this higher trajectory density, there was a notable increase in total distance traveled and mean speed in the 1 μg/kg TCDD group compared to those in the control group (Fig. 5B, C). In line with the increased locomotor activity, immobility was markedly reduced in the 1 μg/kg TCDD group compared with that in the control (Fig. 5D). Notably, all hyperactivity-like behaviors induced by TCDD exposure in dams were reversed by ARI (Fig. 5A–D). The inner time was not significantly different between the groups (Fig. 5E). These results suggest that maternal TCDD exposure enhances locomotion in female offspring and that ARI can reverse these effects.
Effect of maternal exposure to TCDD (1 μg/kg at GD 15) and ARI supplementation in F0 dams on hyperactivity-like behaviors in 7–8-week-old F1 female rats, as assessed via the open field test. (A) Representative trajectories of the offspring in an open field test, (B) total distance, (C) mean speed, (D) immobility, and (E) inner time of the movement were calculated based on the collected data. Bars represent the mean ± S.E.M. of five female rats. Statistical comparisons were performed using a one-way analysis of variance for comparisons involving more than two groups, with Tukey–Kramer post-hoc test. *p<0.05.
Low PRL levels due to maternal TCDD exposure may cause developmental disorders in offspring (Fig. 1E–H). Therefore, we verified whether ARI supplementation could reduce the next-generation toxicity of TCDD. Consistent with the results shown in Fig. 1, maternal exposure to TCDD markedly reduced the body weight of male offspring in the F1, F2, and F3 generations. Moreover, 0.009% ARI supplementation, although beneficial only for F0 mothers, led to a notable recovery in the height and body length of both male and female offspring in the F1, F2, and F3 generations on PND 2 (Fig. 6A, B, D, E, G, H and Fig. S2A–C). Notably, ARI intervention in F0 dams completely reversed developmental issues in F3 males and females (Fig. 6G, H and Fig. S2C). There were no significant changes in litter size or sex ratio between these generations (Fig. S2D, E).
Effect of maternal exposure to TCDD (1 μg/kg at GD 15) and co-treatment with ARI in F0 mothers on body weight and body length of F1/F2/F3 male offspring at PND 2. (A, D, G) Body weight of F1/F2/F3 male offspring at PND 2. (B, E, H) Body length of F1/F2/F3 male offspring at PND 2. (C, F, I) Hepatic mRNA expression levels of CYP1A1 at PND 21 of F1/F2/F3 male offspring, respectively. Bars represent the mean ± S.E.M. of offspring from four to six dams and four to six male offsprings. Statistical comparisons were performed using a one-way analysis of variance for comparisons involving more than two groups, with Tukey–Kramer post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
In addition, CYP1A1 expression levels in the livers of F0 dam rats and F1 male offspring were comparable between the TCDD-exposed and TCDD+ARI groups, indicating that both groups were exposed to similar amounts of TCDD from their dams. Gradually, the levels and toxicity of TCDD in F2 offspring decreased and were completely reversed in F3 due to ARI supplementation in F0 dams (Fig. 6C, F, I). These results suggest that maternal ARI supplementation mitigates growth and developmental toxicity in the offspring by restoring maternal PRL levels. This, in turn, triggers positive parenting behaviors and increases milk production, thereby addressing multigenerational developmental issues in the offspring.
In this study, we primarily focus on the growth and developmental health of offspring, while previous research mostly targeted the reproductive system. We further place greater emphasis on the condition of the dams rather than the offspring. Moreover, we do not only address the multigenerational effects of TCDD exposure but, more importantly, explores feasible interventions to improve reduced prolactin levels. Our results indicate that restoration of maternal prolactin levels can improve maternal behavior and milk production, thereby alleviating growth and developmental impairments in offspring.
This study investigated the multigenerational effects of gestational exposure to TCDD and ARI intervention on PRL levels, milk production, and maternal behavior in female rats. We found that TCDD exposure in F0 dams significantly reduced serum PRL concentrations in successive females, leading to impaired maternal nursing behavior and subsequent growth retardation of their offspring. PRL is crucial in mammary gland development and milk production. Therefore, it is reasonable to suppose that PRL suppression is linked to increased dopamine levels induced by TCDD, affecting lactotroph proliferation. ARI partially restored PRL levels and ameliorated negative effects on body weight and cognitive function in offspring by reducing dopamine signaling. Additionally, maternal TCDD exposure diminishes total milk production and alters protein composition, particularly affecting the key proteins involved in milk synthesis. Notably, whereas histopathological analysis revealed impaired mammary gland development in the TCDD-exposed dams, ARI treatment in F0 dams was found to effectively reverse these adverse effects in both F1 and F2 dams. Overall, these findings emphasize the significant impact of TCDD on maternal and offspring health across generations, highlighting the potential of pharmacological interventions to mitigate these effects.
PRL is a hormone primarily produced by the anterior pituitary gland and encoded by the PRL gene. PRL secretion is regulated by endocrine neurons in the hypothalamus, particularly the tuberoinfundibulum neurons of the arcuate nucleus, which release dopamine to inhibit PRL secretion via D2R in lactotrophs (Ben-Jonathan, 1985). Other hormones, such as prolactin-releasing hormone, thyrotropin-releasing hormone, and kisspeptin can also influence PRL levels (Freeman et al., 2000; Taylor and Samson, 2001; Szawka et al., 2010); however, their effects are less affected by TCDD (Yuan et al., unpublished data, 2023). Due to the study design, we did not determine the dopamine levels in dams after maternal exposure to TCDD. However, considering the results of ARI treatment, our results strongly suggest that the decrease in PRL caused by TCDD is primarily due to abnormally high levels of dopamine, enhancing its inhibitory effects. Given that dopamine can inhibit PRL at low concentrations, we used ARI to investigate how TCDD affects PRL secretion.
ARI stimulates PRL release due to its slow dissociation from D2R, and repeated dosing can enhance its effects. Studies have shown that a dose of 10 mg/kg (i.p.) is the upper limit of (PRL) release (Carboni et al., 2012). Furthermore, ARI significantly reduced dopamine release in the nucleus accumbens and medial prefrontal cortex at the same dose (Li et al., 2004). Thus, we selected a feed containing 0.009% ARI as a non-invasive supplementation method from GD 20 to PD 21. This feed resulted in an average daily intake of approximately 15 mg/kg ARI by F0 dams, which significantly increased PRL serum concentrations (Fig. S3), and licking time in successive dams compared to TCDD-exposed dams.
Research has shown that TCDD exposure significantly elevates TGF-β1 levels (Takeda et al., 2020). Dopamine binds to D2R, reducing lactotroph proliferation, upregulating TGF-β1 expression and secretion, and inhibiting lactotroph proliferation (Ben-Jonathan, 2005; Sarkar et al., 1992; Sarkar et al., 2005). Tyrosine hydroxylase and DOPA decarboxylase are the crucial enzymes involved in dopamine synthesis. However, TCDD exposure and ARI treatment had minimal effects on the mRNA abundances of these enzymes and D2R (Fig. 3A-C). We investigated the enzymes involved in dopamine regulation, and our results demonstrated that TCDD suppressed the expression of COMT. COMT catalyzes the methylation of dopamine, converting it to its inactive form. TCDD-induced suppression of COMT leads to decreased methylation of dopamine, indicating that less dopamine is converted into its inactive form, which can result in the accumulation of dopamine. To our knowledge, this is the first study to demonstrate that TCDD can elevate dopamine levels by inhibiting its degradation, leading to dopamine accumulation in the hypothalamus, and subsequently altering prolactin expression. As a partial agonist, ARI can contribute to the suppression of excessively high dopamine levels, thereby alleviating the dopamine-mediated inhibition of prolactin secretion.
Specifically, we determined that gestational TCDD exposure in F0 dams attenuated the pituitary PRL serum concentration in successive generations (F1/F2) of females. This suppressed their maternal nursing behavior to care for subsequent generations. Maternal licking is one of the most representative maternal behaviors which is essential for offspring development by stimulating the physiological functions of the offspring. It promotes growth and development and encourages nursing to ensure adequate nutrition and reduce fear under conditions of novelty. Environmental manipulation that alters maternal behavior during early development can affect the transmission patterns of maternal behavior in subsequent generations (Francis et al., 1999; Stürtz et al., 2008).
For instance, perinatal dioxin exposure affects neurodevelopment and impairs various brain functions in offspring, including cognition, language, learning, and emotion. Reportedly, maternal exposure to TCDD, particularly at low doses, may enhance movement ability, novelty exploration, and anxiety-related behaviors in offspring (Sha et al., 2021). These findings are in line with a report that mothers of predominantly hyperactive-impulsive-type children exhibited significantly greater parenting stress and engaged in more negative parenting behaviors. Additionally, when maternal inhibitory control was low, hyperactive-impulsive symptoms were particularly associated with negative parenting behaviors (Zaidman-Zait and Shilo, 2021).
In this study, gestational exposure to TCDD decreased PRL levels and induced hyperactivity-like behaviors in female offspring. When these females became mothers, they exhibited negative parenting patterns, contributing to the developmental issues in subsequent generations. ARI, known for its dopaminergic and serotonergic antagonistic effects, is considered a first-line intervention for addressing hyperactivity, impulsivity, and aggression (Hirota and King, 2023; Persico et al., 2021). Treatment with ARI in F0 dams successfully mitigated hyperactivity-like behaviors and restored maternal behaviors in these offspring, reversing TCDD-induced developmental and behavioral abnormalities.
In mammals, the main functions of PRL include mammary gland development, milk production, and breastfeeding during lactation (Berryhill et al., 2016; Freeman et al., 2000). Our results indicate that the maternal exposure of F0 dams to TCDD significantly reduces the total amount of milk secreted by dams. Notably, maternal intervention with ARI completely reversed the reduced milk production, which is consistent with previous reports on the recovery effects of PRL supplementation. The allometric growth periods of the mammary glands, which occur at the onset of puberty and pregnancy, are critical for reproductive development. H&E staining was used for the histopathological analysis of mammary glands to explore the effect of F0 dam exposure to TCDD on mammary gland development during lactation. TCDD exposure resulted in mammary duct stenosis compared to that in the TCDD + ARI group. To further explore the mechanism behind the recovery effect of ARI in the mammary gland, we measured mRNA expression levels. We observed a significant increase in leptin expression following TCDD exposure. Maternal supplementation with ARI restored leptin levels to baseline. High levels of leptin during lactation inhibit mammary cell proliferation and differentiation (Kamikawa et al., 2009). This reduces energy and nutrient availability for milk synthesis and secretion. Analysis of milk protein content in F0 dams exposed to TCDD revealed a significant reduction in total protein concentration during early lactation. mRNA levels of β-casein in the mammary gland at PD 7 showed significant decreases, thus reducing the milk nutrient content. These findings suggest that maternal ARI intervention has a restorative effect on F0 rat dams by increasing PRL levels, thereby promoting mammary gland development and enhancing milk production and nutrient availability in the female offspring during lactation.
Owing to the restoration of maternal PRL levels, improved maternal behavior, and increased quantity of milk and nutrients, the height and weight of the offspring were restored. These recovery effects showed significant improvement across generations, completely reversing the adverse effects of maternal TCDD exposure in the F3 generation. CYP1A1 expression levels in the F1 generation were consistent, regardless of maternal TCDD exposure or ARI supplementation, indicating that the F1 generation received similar levels of TCDD from the mother via the placenta or milk. Although ARI supplementation did not alleviate TCDD toxicity through AHR inhibition over time or across generations, CYP1A1 expression levels in the F3 generation were restored. This was consistent with the complete reversal of height and weight in the F3 generation compared with F0 maternal exposure. These results suggest that maternal PRL restoration plays a positive role in eliminating TCDD toxicity during multigenerational inheritance.
The significant induction of hepatic CYP1A1 expression in the F3 generation of the TCDD line, despite no direct TCDD exposure, indeed suggests a potential multigenerational epigenetic inheritance. Environmental toxicants such as TCDD are known to induce heritable epigenetic modifications, including changes in DNA methylation, histone modifications, and non-coding RNA expression, which may alter gene regulation across generations.
The mechanism underlying improvements in the F1 and subsequent generations may be related to normalization of PRL levels in the F0 generation, which alleviated deficits in maternal care and led to an imprinting effect that contributed to improvements in the F1 and later generations.
One limitation of this study is that dopamine levels in the maternal brain were not directly measured, which limits our understanding of the neuroendocrine mechanisms underlying the observed changes in maternal behavior. Additionally, while multigenerational effects were observed in the F3 generation, we did not directly assess epigenetic modifications in germ cells or target tissues in offspring. Future studies involving molecular analysis of epigenetic marks, such as DNA methylation or histone modifications, would be valuable to confirm the potential mechanisms of inheritance.
This study revealed the positive effects of ARI administered to F0 dams in restoring maternal PRL levels. ARI mitigated the effects of TCDD toxicity on offspring growth and health with improved multigenerational (F1/F2/F3) effects. The recovery mechanism is linked to the dopamine pathway, and our experiments strongly suggest that TCDD exposure increases dopamine secretion by reducing COMT levels, thereby inhibiting PRL expression. As a partial agonist, in the presence of excess dopamine, ARI can reduce dopamine signaling via D2 receptors, thereby restoring prolactin expression suppressed by the elevated dopamine levels. ARI is a psychotropic medication and should be cautiously administered during pregnancy and lactation. Our future studies will focus on identifying effective medications against environmental pollutants that can restore low PRL levels with minimal adverse effects by targeting the dopamine pathways during lactation.
FootnotesThis study was presented in part at the meetings of Zou et al., Forum 2024: Pharmaceutical Health Science-Environmental Toxicology, Pharmaceutical Society of Japan, Sendai (September 2024) and Ishii et al., International ISSX meeting, Chicago (September 2025).
FundingThis work was supported by the Japan Society for the Promotion of Science (JSPS) [Scientific Research (A) JSPS KAKENHI JP17H00788, JSPS KAKENHI JP21H04928, Recipient Y.I], the Ministry of Health, Labor and Welfare, Japan [Research on Food Safety (H30-Designated Research-005, R3-Designated Research JP21KA2003, and R6-Designated Research JP24KA2001, Recipient Y.I)].
Conflict of interestThe authors declare that there is no conflict of interest.
Data availabilityAll data generated or analyzed during this study are included in this manuscript. Data are provided upon request.
Author contributionsConceptualization: Xing Zou, Ming Yuan, Tomoki Takeda, Yuji Ishii;
Methodology: Xing Zou, Ming Yuan, Tomoki Takeda, Yuji Ishii;
Formal analysis and investigation: Xing Zou, Ming Yuan, Tomoki Takeda, Yuji Ishii;
Writing - original draft preparation: Xing Zou, Ming Yuan, Yuji Ishii;
Writing - review and editing: Xing Zou, Ming Yuan, Tomoki Takeda, Takayuki Koga, Yoshitaka Tanaka, Yuji Ishii;
Funding acquisition: Yuji Ishii;
Resources: Yuji Ishii;
Supervision: Yuji Ishii.
Ethical approval and consent to participateAll animal experiments were conducted in accordance with a protocol approved by the Institutional Animal Ethics Committee of Kyushu University, Fukuoka, Japan (project numbers A22-060 and A24-116).
Patient consent for publicationNot applicable.
