Two Hits of EDCs Three Generations Apart: Effects on Social Behaviors in Rats, and Analysis by Machine Learning

All individuals are directly exposed to extant environmental endocrine-disrupting chemicals (EDCs), and indirectly exposed through transgenerational inheritance from our ancestors. Although direct and ancestral exposures can each lead to deficits in behaviors, their interactions are not known. Here we focused on social behaviors based on evidence of their vulnerability to direct or ancestral exposures, together with their importance in reproduction and survival of a species. Using a novel “two hits, three generations apart” experimental rat model, we investigated interactions of two classes of EDCs across six generations. PCBs (a weakly estrogenic mixture Aroclor 1221, 1 mg/kg), Vinclozolin (antiandrogenic, 1 mg/kg) or vehicle (6% DMSO in sesame oil) were administered to pregnant rat dams (F0) to directly expose the F1 generation, with subsequent breeding through paternal or maternal lines. A second EDC hit was given to F3 dams, thereby exposing the F4 generation, with breeding through the F6 generation. Approximately 1200 male and female rats from F1, F3, F4 and F6 generations were run through tests of sociability and social novelty as indices of social preference. We leveraged machine learning using DeepLabCut to analyze nuanced social behaviors such as nose touching with accuracy similar to a human scorer. Surprisingly, social behaviors were affected in ancestrally exposed but not directly exposed individuals, particularly females from a paternally exposed breeding lineage. Effects varied by EDC: Vinclozolin affected aspects of behavior in the F3 generation while PCBs affected both the F3 and F6 generations. Taken together, our data suggest that specific aspects of behavior are particularly vulnerable to heritable ancestral exposure of EDC contamination, that there are sex differences, and that lineage is a key factor in transgenerational outcomes.

La sobre-expresión de PtCSP4 del álamo promueve un mayor potencial para la fitorremediación de PCB

La fitorremediación es un método biotecnológico ampliamente aceptado que reduce, absorbe y degrada los contaminantes tóxicos. Los bifenilos policlorados (PCB) son contaminantes orgánicos persistentes que causan efectos nocivos para el medio ambiente. Se reconoce que el álamo (Populus trichocarpa) tiene un crecimiento rápido, alta transpiración y se tiene una secuencia completa de su genoma; por lo que se considera un modelo perfecto en fitorremediación. Las plantas genéticamente modificadas pueden ser más eficientes en la remediación de sitios, cuyos suelo y agua están contaminados con bifenilos policlorados (PCB). Yb-1 es una proteína multifuncional involucrada en la regulación de la transcripción, traducción, empalme de ARNm y reparación de ADN. El factor de transcripción Yb1 se conoce en humanos y mamíferos, pero muy poco en plantas. Yb-1 en plantas es más conocido como CSP (proteínas de choque frío). En este trabajo, el gen PtCSP4 (Potri.004G172600) se aisló y amplificó a partir del álamo. El sistema de recombinación de puerta de enlace se usó para clonar PtCSP4 en un vector binario con un promotor constitutivo. PtCSP4 se introdujo en Arabidopsis thaliana y se probó su tolerancia en presencia de PCB (Aroclor 1221). Ocho líneas transgenicas de Arabidopsis cultivadas in vitro fueron expuestas a PCB. Todos los resultados sugieren que PtCSP4 es un buen candidato para la fitorremediación de PCB.

Transgenerational effects of polychlorinated biphenyls: 2. Hypothalamic gene expression in rats

Polychlorinated biphenyls (PCBs) are endocrine-disrupting chemicals (EDCs) with well-established effects on reproduction and behavior in developmentally-exposed (F1) individuals. Because of evidence for transgenerational effects of EDCs on the neuroendocrine control of reproductive physiology, we tested the hypothesis that prenatal PCB exposure leads to unique hypothalamic gene expression profiles in three generations. Pregnant Sprague–Dawley rats were treated on gestational days 16 and 18 with the PCB mixture Aroclor 1221 (A1221), vehicle (3% DMSO in sesame oil), or estradiol benzoate (EB, 50 μg/kg), the latter a positive control for estrogenic effects of A1221. Maternal- and paternal-lineage F2 and F3 generations were bred using untreated partners. The anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), involved in the hypothalamic control of reproduction, were dissected from F1-F3 females and males, RNA extracted, and gene expression measured in a qPCR array. We detected unique gene expression profiles in each generation, that were sex- and lineage-specific. In the AVPV, treatment significantly changed 10, 25, and 11 transcripts in F1, F2, and F3 generations, whereas 10, 1, and 12 transcripts were changed in these generations in the ARC. In the F1 AVPV and ARC, most affected transcripts were decreased by A1221. In the F2 AVPV, most effects of A1221 were observed in females of the maternal lineage, whereas only Pomc expression changed in the F2 ARC (by EB). The F3 AVPV and ARC were mainly affected by EB. It is notable that results in one generation do not predict results in another, and that lineage was a major determinant in results. Thus, transient prenatal exposure of F1 rats to A1221 or EB can alter hypothalamic gene expression across 3 generations in a sex- and lineage-dependent manner, leading to the conclusion that the legacy of PCBs continues for generations.

Prenatal polychlorinated biphenyl exposure promotes invasion of progeny ectopic endometrial stromal cells via epigenetic modification of EZH2

Polychlorinated biphenyls(PCBs) are persistent environmental endocrine disruptor. This study aim to investigate the changes of the ectopic endometrium invasive ability and the possible mechanism after exposure to PCBs during pregnancy. In total, 12 female Sprague Dawley rats were intraperitoneally injected with Aroclor 1221 (1 mg/kg) or dimethyl sulfoxide (1 mg/kg) at 16 and 18 days of gestation. The endometriosis model was established by autogenous uterine abdominal wall implantation 2 months after birth. The degree of adhesion between the endometriosis and the greater omentum, as well as cell morphology and Transwell invasion patterns were used to evaluate the invasive ability of progeny ectopic endometrial stromal cells (ESCs). Moreover, the effect of the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)/ trimethylation of Histone 3 lysine 27 (H3K27me3) axis was examined using a highly selective EZH2 inhibitor, GSK126. After gestational PCBs exposure, the adhesion between the endometrium and the greater omentum was enhanced (P<0.05), and the length and number of microvilli were significantly increased (P<0.01). Exposure to PCBs during pregnancy increased the invasive ability of the ectopic ESCs (P<0.01), with upregulated expression levels of EZH2 and H3k27me3, which were abrogated by the EZH2 inhibitor GSK126. In conclusion, exposure to PCBs during pregnancy increased the invasive ability of the ectopic endometrium, which may be mediated by EZH2.

Prenatal EDCs Impair Mate and Odor Preference and Activation of the VMN in Male and Female Rats

Environmental endocrine-disrupting chemicals (EDCs) disrupt hormone-dependent biological processes. We examined how prenatal exposure to EDCs act in a sex-specific manner to disrupt social and olfactory behaviors in adulthood and underlying neurobiological mechanisms. Pregnant rat dams were injected daily from embryonic day 8 to 18 with 1 mg/kg Aroclor 1221 (A1221), 1 mg/kg vinclozolin, or the vehicle (6% DMSO in sesame oil). A1221 is a mixture of polychlorinated biphenyls (weakly estrogenic) while vinclozolin is a fungicide (anti-androgenic). Adult male offspring exposed to A1221 or vinclozolin, and females exposed to A1221, had impaired mate preference behavior when given a choice between 2 opposite-sex rats that differed by hormone status. A similar pattern of impairment was observed in an odor preference test for urine-soaked filter paper from the same rat groups. A habituation/dishabituation test revealed that all rats had normal odor discrimination ability. Because of the importance of the ventrolateral portion of the ventromedial nucleus (VMNvl) in mate choice, expression of the immediate early gene product Fos was measured, along with its co-expression in estrogen receptor alpha (ERα) cells. A1221 females with impaired mate and odor preference behavior also had increased neuronal activation in the VMNvl, although not specific to ERα-expressing neurons. Interestingly, males exposed to EDCs had normal Fos expression in this region, suggesting that other neurons and/or brain regions mediate these effects. The high conservation of hormonal, olfactory, and behavioral traits necessary for reproductive success means that EDC contamination and its ability to alter these traits has widespread effects on wildlife and humans.

Dynamic Postnatal Developmental and Sex-Specific Neuroendocrine Effects of Prenatal Polychlorinated Biphenyls in rats

Gestational exposures to estrogenic compounds, both endogenous hormones and exogenous endocrine-disrupting chemicals (EDCs), have long-term effects on reproductive physiology and behavior. We tested the hypothesis that prenatal treatment of rats with low doses of Aroclor 1221 (A1221), a weakly estrogenic polychlorinated biphenyl mix previously used in industry, or estradiol benzoate (EB), alters development of the hypothalamus in a sexually dimorphic manner and subsequently perturbs reproductive function. Pregnant Sprague-Dawley rats were injected on embryonic days 16 and 18 with vehicle (dimethylsulfoxide), A1221 (1 mg/kg), or EB (50 μg/kg). Developmental milestones were monitored, and on postnatal days 15, 30, 45, and 90, 1 male and 1 female per litter were euthanized. Because of their key roles in the mediation of steroid actions on reproductive function, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) were punched for a low-density quantitative PCR array of 48 neuroendocrine genes and analysis of DNA methylation of a subset of genes. Gestational exposure to A1221 or EB delayed the timing of puberty in males and disrupted estrous cyclicity in females. In the AVPV, 28 genes were affected by treatment in a developmental stage–specific manner, mostly in females, which exhibited a masculinized expression profile. This included 2 clock genes, Per2 and Arntl, implicating circadian circuits as being vulnerable to endocrine disruption. DNA methylation analysis of 2 genes, Per2 and Ar, showed no effect of EDCs and suggested alternative mechanisms for the altered mRNA levels. In the ARC, 12 genes were affected by treatment, mostly in males, again with dynamic developmental changes. Bionetwork analysis of relationships among genes, hormones, and physiological markers showed sexually dimorphic effects of estrogenic EDC exposures, with the female AVPV and the male ARC being most vulnerable, and provided novel relationships among hypothalamic genes and postnatal reproductive maturation.

Disruption of Reproductive Aging in Female and Male Rats by Gestational Exposure to Estrogenic Endocrine Disruptors

Polychlorinated biphenyls (PCBs) are industrial contaminants and known endocrine-disrupting chemicals. Previous work has shown that gestational exposure to PCBs cause changes in reproductive neuroendocrine processes. Here we extended work farther down the life spectrum and tested the hypothesis that early life exposure to Aroclor 1221 (A1221), a mixture of primarily estrogenic PCBs, results in sexually dimorphic aging-associated alterations to reproductive parameters in rats, and gene expression changes in hypothalamic nuclei that regulate reproductive function. Pregnant Sprague Dawley rats were injected on gestational days 16 and 18 with vehicle (dimethylsulfoxide), A1221 (1 mg/kg), or estradiol benzoate (50 μg/kg). Developmental parameters, estrous cyclicity (females), and timing of reproductive senescence were monitored in the offspring through 9 months of age. Expression of 48 genes was measured in 3 hypothalamic nuclei: the anteroventral periventricular nucleus (AVPV), arcuate nucleus (ARC), and median eminence (females only) by real-time RT-PCR. Serum LH, testosterone, and estradiol were assayed in the same animals. In males, A1221 had no effects; however, prenatal estradiol benzoate increased serum estradiol, gene expression in the AVPV (1 gene), and ARC (2 genes) compared with controls. In females, estrous cycles were longer in the A1221-exposed females throughout the life cycle. Gene expression was not affected in the AVPV, but significant changes were caused by A1221 in the ARC and median eminence as a function of cycling status. Bionetwork analysis demonstrated fundamental differences in physiology and gene expression between cycling and acyclic females independent of treatment. Thus, gestational exposure to biologically relevant levels of estrogenic endocrine-disrupting chemicals has sexually dimorphic effects, with an altered transition to reproductive aging in female rats but relatively little effect in males.