Alteration of epigenetic methyl and acetyl marks by postnatal chromium(VI) exposure causes apoptotic changes in the ovary of the F1 offspring.

Hexavalent chromium, Cr(VI), is a heavy metal endocrine disruptor used widely in various industries worldwide and is considered a reproductive toxicant. Our previous studies demonstrated that lactational exposure to Cr(VI) caused follicular atresia, disrupted steroid hormone biosynthesis and signaling, and delayed puberty. However, the underlying mechanism was unknown. The current study investigated the effects of Cr(VI) exposure (25 ppm) during postnatal days 1-21 via dam's milk on epigenetic alterations in the ovary of F1 offspring. Data indicated that Cr(VI) disrupted follicle development and caused apoptosis by increasing DNMT3a /3b and histone methyl marks (H3K27me3 and H3K9me3) along with decreasing histone acetylation marks (H3K9ac and H3K27ac). Our study demonstrates that exposure to Cr(VI) causes changes in the epigenetic marks, partially contributing to the transcriptional repression of genes regulating ovarian development, cell proliferation (PCNA), cell survival (BCL-XL and BCL-2), and activation of genes regulating apoptosis (AIF and cleaved caspase-3), resulting in follicular atresia. The current study suggests a role for epigenetics in Cr(VI)-induced ovotoxicity and infertility.

Effects of selective inhibition of prostaglandin E2 receptors EP2 and EP4 on the miRNA profile in endometriosis.

Endometriosis is an estrogen-dependent, progesterone-resistant, chronic inflammatory gynecological disease of reproductive-age women. Two major clinical symptoms of endometriosis are chronic pelvic pain and infertility, which profoundly affect the quality of life in women. Current hormonal therapies to induce a hypoestrogenic state are unsuccessful because of undesirable side effects, reproductive health concerns, and failure to prevent disease recurrence. Prostaglandin E2 (PGE2) plays an important role in the survival and growth of endometriotic lesions. MicroRNAs (miRNAs) are small, noncoding RNAs that control gene expressions through multiple mechanisms and have important roles in the pathogenesis of endometriosis. The objective of the present study is to determine the effects of pharmacological inhibition of PGE2 receptors, EP2 and EP4, on miRNA profile in endometriosis. The novel results collectively indicate that inhibition of PGE2-EP2/EP4 signaling regulated several miRNA clusters associated with cell adhesion, migration, invasion, survival and growth in cell-specific and the chromosome-specific manner and reverses the epigenetic silencing of proapoptotic miRNAs 15a and 34c in the human endometriotic epithelial and stromal cells and experimental endometriotic lesions. Thus, selective inhibition of EP2/EP4 receptors could emerge as a potential nonsteroidal therapy for endometriosis.

Inhibition of Sirtuin-1 hyperacetylates p53 and abrogates Sirtuin-1-p53 interaction in Cr(VI)-induced apoptosis in the ovary.

Environmental contamination with hexavalent chromium, Cr(VI), has been increasing in the United States as well as in developing countries. Exposure to Cr(VI) predisposes the human population to various diseases, including cancer, infertility, and developmental problems in children. Previous findings from our laboratory reported that prenatal exposure to Cr(VI) caused premature ovarian failure through p53-mediated mechanisms. Sirtuin 1 (SIRT1) is an NAD+ -dependent histone deacetylase class III. SIRT1 deacetylates several histones and non-histone proteins such as p53 and NFkB. The current study determines a role for the SIRT1-p53 network in apoptosis induced by Cr(VI) in the ovary and establishes physical interaction between SIRT1 and p53. Adult pregnant dams were given regular drinking water or Cr(VI) (10 ppm potassium dichromate in drinking water, ad libitum), and treated with SIRT1 inhibitor, EX-527 (50 mg/kg body weight, i.p.,), during 9.5 - 14.5 days post-coitum. On postnatal day-1, ovaries from F1 offspring were collected for various analyses. Results indicated that Cr(VI) increased germ cell and somatic cell apoptosis, upregulated acetyl-p53, activated the apoptotic pathway, and inhibited cell survival pathways. Cr(VI) decreased acetyl-p53-SIRT1 co-localization in the ovary. In an immortalized rat granulosa cell line SIGC, Cr(VI) inhibited the physical interaction between SIRT1 and acetyl-p53 by altering the p53:SIRT1 ratio. EX-527 exacerbated Cr(VI)-induced mechanisms. The current study shows a novel mechanism for Cr(VI)-induced apoptosis in the ovary, mediated through the p53-SIRT1 network, suggesting that targeting the p53 pathway may be an ideal approach to rescue ovaries from Cr(VI)-induced apoptosis.

Controlling the Colonization of Clostridium perfringens in Broiler Chickens by an Electron-Beam-Killed Vaccine.

Clostridium perfringens (Cp) is a Gram-positive anaerobe that is one of the causative agents of necrotic enteritis (NE) in chickens, which leads to high mortality. Owing to the ban of administering antibiotics in feed to chickens, there has been an increase in the number of NE outbreaks all over the world, and the estimated loss is approximately 6 billion U.S. dollars. The best alternative method to control NE without antibiotics could be vaccination. In this study, we exposed three different strains of Cp to electron beam (eBeam) irradiation to inactivate them and then used them as a killed vaccine to control the colonization of Cp in broiler chickens. The vaccine was delivered to 18-day old embryos in ovo and the chickens were challenged with the respective vaccine strain at two different time points (early and late) to test the protective efficacy of the vaccine. The results indicate that an effective eBeam dose of 10 kGy inactivated all three strains of Cp, did not affect the cell membrane or epitopes, induced significant levels of IgY in the vaccinated birds, and further reduced the colonization of Cp strains significantly (p < 0.0001) in late challenge (JGS4064: 4 out of 10; JGS1473: 0 out of 10; JGS4104: 3 out of 10). Further studies are necessary to enhance the efficacy of the vaccine and to understand the mechanism of vaccine protection.

Sexually Dimorphic Impact of Chromium Accumulation on Human Placental Oxidative Stress and Apoptosis.

Environmental contamination with hexavalent chromium (CrVI) is a growing problem both in the United States and developing countries. Hexavalent chromium is widely used in numerous industries. Environmental exposure to CrVI adversely affects pregnancy outcomes and subsequent health of 2 generations, resulting in higher pregnancy loss, spontaneous abortion and low birth rate. Pregnant women exposed to CrVI through occupational settings experience increased risk of spontaneous abortion, stillbirth, preterm birth, and neonatal death. Children of the CrVI exposed women experience respiratory problems, perinatal jaundice, and increased birth defects. Because placental dysfunction may have a role in such adverse pregnancy outcome, we tested the hypothesis that environmental Cr exposure in pregnant women results in Cr accumulation in the human placenta, which could increase placental oxidative stress by disrupting antioxidant machinery and inducing apoptosis. Studies using frozen, deidentified human term placenta samples indicated that: (1) Cr accumulates in human term placenta tissues and (2) increase in Cr accumulation is positively correlated with oxidative stress and apoptotic markers, and altered antioxidants levels. Interestingly, there was a sexual dimorphism in the correlation between Cr accumulation and oxidative stress, and expression of apoptotic and antioxidant markers. Mechanistic in vitro studies using human trophoblast cells BeWo confirmed the detrimental effects of Cr in altering antioxidant genes. For the first time, this study provides evidence in support of a positive correlation between Cr accumulation in the human placenta and accelerated oxidative stress, with a gender bias toward the male sex.

Editor's Highlight: Exposure to CrVI during Early Pregnancy Increases Oxidative Stress and Disrupts the Expression of Antioxidant Proteins in Placental Compartments.

Epidemiologic studies document relationships between chromium VI (CrVI) exposure and increased risk of spontaneous abortion, stillbirth, preterm birth, and neonatal death in pregnant women. Environmental contamination with CrVI is a growing problem both in the United States and developing countries. CrVI is widely used in numerous industries. This study was designed to understand the mechanism of CrVI toxicity on placental oxidative stress and antioxidant (AOX) machinery. Pregnant mother rats were treated with or without CrVI (50 ppm K2Cr2O7) through drinking water from gestational day (GD) 9.5-14.5, and placentas were analyzed on GD 18.5. Results indicated that CrVI reduced the trophoblast cell population. CrVI increased reactive oxygen species (ROS) and decreased the expression of AOX proteins. CrVI disrupts the trophoblast proliferation of the placenta. This study provides insight into the critical role of AOXs in placental function.

Chromium VI - Induced developmental toxicity of placenta is mediated through spatiotemporal dysregulation of cell survival and apoptotic proteins.

Environmental contamination with hexavalent chromium (CrVI) is a growing problem both in the U.S and developing countries. CrVI is a heavy-metal endocrine disruptor; women working in Cr industries exhibit an increased incidence of premature abortion and infertility. The current study was designed to understand the mechanism of CrVI toxicity on placental cell survival/death pathways. Pregnant mothers were treated with or without CrVI (50ppmK2Cr2O7) through drinking water from gestational day (GD) 9.5-14.5, and placentas were analyzed on GD 18.5. Results indicated that CrVI increased apoptosis of trophoblasts, vascular endothelium of the metrial glands and yolk sac epithelium through caspase-3 and p53-dependent pathways. CrVI increased apoptosis in labyrinth and basal zones in a caspase-3-independent manner via AIF, and through an ATM-p53-NOXA-PUMA-p27 network. CrVI downregulated cell survival proteins Bcl-2, Bcl-XL and XIAP in the placenta. CrVI disrupts placental histoarchitecture and increases cell death by spatiotemporal modulation of apoptotic signaling.

Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol.

Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) have not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, polycystic ovary syndrome, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in >50 industries. Environmental contamination with CrVI in the US is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. Our lab has been involved in determining the molecular mechanism of CrVI-induced female infertility and intervention strategies to mitigate CrVI effects. Lactating mother rats were exposed to CrVI (50ppm potassium dichromate) from postpartum days 1-21 through drinking water with or without RVT (10mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother's milk. On postnatal day (PND) 25, blood and the ovary, kidney and liver were collected from the F1 females for analyses. CrVI increased atresia of follicles by increasing cytochrome C and cleaved caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E2) biosynthesis and enhancing metabolic clearance of E2, increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by upregulating cell survival proteins and AOXs; and restored E2 levels by inhibiting hydroxylation, glucuronidation and sulphation of E2. This is the first study to report the protective effects of RVT against any toxicant in the ovary.

Identifying a novel role for X-prolyl aminopeptidase (Xpnpep) 2 in CrVI-induced adverse effects on germ cell nest breakdown and follicle development in rats.

Environmental exposure to endocrine-disrupting chemicals (EDCs) is one cause of premature ovarian failure (POF). Hexavalent chromium (CrVI) is a heavy metal EDC widely used in more than 50 industries, including chrome plating, welding, wood processing, and tanneries. Recent data from U.S. Environmental Protection Agency indicate increased levels of Cr in drinking water from several American cities, which potentially predispose residents to various health problems. Recently, we demonstrated that gestational exposure to CrVI caused POF in F1 offspring. The current study was performed to identify the molecular mechanism behind CrVI-induced POF. Pregnant rats were treated with 25 ppm of potassium dichromate from Gestational Day (GD) 9.5 to GD 14.5 through drinking water, and the fetuses were exposed to CrVI through transplacental transfer. Ovaries were removed from the fetuses or pups on Embryonic Day (ED) 15.5, ED 17.5, Postnatal Day (PND) 1, PND 4, or PND 25, and various analyses were performed. Results showed that gestational exposure to CrVI: 1) increased germ cell/oocyte apoptosis and advanced germ cell nest (GCN) breakdown; 2) increased X-prolyl aminopeptidase (Xpnpep) 2, a POF marker in humans, during GCN breakdown; 3) decreased Xpnpep2 during postnatal follicle development; and 4) increased colocalization of Xpnpep2 with Col3 and Col4. We also found that Xpnpep2 inversely regulated the expression of Col1, Col3, and Col4 in all the developmental stages studied. Thus, CrVI advanced GCN breakdown and increased follicle atresia in F1 female progeny by targeting Xpnpep2.

Edaravone mitigates hexavalent chromium-induced oxidative stress and depletion of antioxidant enzymes while estrogen restores antioxidant enzymes in the rat ovary in F1 offspring.

Environmental contamination of drinking water with chromium (Cr) has been increasing in more than 30 cities in the United States. Previous studies from our group have shown that Cr affects reproductive functions in female Sprague Dawley rats. Although it is impossible to completely remove Cr from the drinking water, it is imperative to develop effective intervention strategies to inhibit Cr-induced deleterious health effects. Edaravone (EDA), a potential inhibitor of free radicals, has been clinically used to treat cancer and cardiac ischemia. This study evaluated the efficacy of EDA against Cr-induced ovarian toxicity. Results showed that maternal exposure to CrVI in rats increased follicular atresia, decreased steroidogenesis, and delayed puberty in F1 offspring. CrVI increased oxidative stress and decreased antioxidant (AOX) enzyme levels in the ovary. CrVI increased follicle atresia by increased expression of cleaved caspase 3, and decreased expression of Bcl2 and Bcl2l1 in the ovary. EDA mitigated or inhibited the effects of CrVI on follicle atresia, pubertal onset, steroid hormone levels, and AOX enzyme activity, as well as the expression of Bcl2 and Bcl2l1 in the ovary. In a second study, CrVI treatment was withdrawn, and F1 rats were injected with estradiol (E2) (10 µg in PBS/ethanol per 100 g body weight) for a period of 2 wk to evaluate whether E2 treatment will restore Cr-induced depletion of AOX enzymes. E2 restored CrVI-induced depletion of glutathione peroxidase 1, catalase, thioredoxin 2, and peroxiredoxin 3 in the ovary. This is the first study to demonstrate the protective effects of EDA against any toxicant in the ovary.

Prenatal exposure to chromium induces early reproductive senescence by increasing germ cell apoptosis and advancing germ cell cyst breakdown in the F1 offspring.

Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries such as chrome plating, welding, wood processing and tanneries. As one of the world's leading producers of chromium compounds, the U.S. is facing growing challenges in protecting human health against multiple adverse effects of CrVI. CrVI is rapidly converted to CrIII intracellularly, and can induce apoptosis through different mechanisms. Our previous studies demonstrated postnatal exposure to CrVI results in a delay or arrest in follicle development and puberty. Pregnant rats were treated with 25 ppm potassium dichromate (CrVI) from gestational day (GD) 9.5 to 14.5 through drinking water, placentae were removed on GD 20, and total Cr was estimated in the placentae; ovaries were removed from the F1 offspring on postnatal day (PND)-1 and various analyses were performed. Our results show that gestational exposure to CrVI resulted in (i) increased Cr concentration in the placenta, (ii) increased germ cell apoptosis by up-regulating p53/p27-Bax-caspase-3 proteins and by increasing p53-SOD-2 co-localization; (iii) accelerated germ cell cyst (GCC) breakdown; (iv) advanced primordial follicle assembly and primary follicle transition and (v) down regulation of p-AKT, p-ERK and XIAP. As a result of the above events, CrVI induced early reproductive senescence and decrease in litter size in F1 female progeny.

Postnatal exposure to chromium through mother's milk accelerates follicular atresia in F1 offspring through increased oxidative stress and depletion of antioxidant enzymes.

Hexavalent chromium, CrVI, is a heavy metal endocrine disruptor, known as a mutagen, teratogen, and a group A carcinogen. Environmental contamination with CrVI, including drinking water, has been increasing in more than 30 cities in the United States. CrVI is rapidly converted to CrIII intracellularly, and CrIII can cause DNA strand breaks and cancer or apoptosis through different mechanisms. Our previous study demonstrated that lactational exposure to chromium results in a delay or arrest in follicle development and a decrease in steroid hormone levels in F1 female rats, both of which are mitigated (partial inhibition) by vitamin C. The current study tested the hypothesis that lactational exposure to CrIII accelerates follicle atresia in F1 offspring by increasing reactive oxygen species (ROS) and decreasing cellular antioxidants. Results showed that lactational exposure to CrIII dose-dependently increased follicular atresia and decreased steroidogenesis in postnatal day 25, 45, and 65 rats. Vitamin C mitigated or inhibited the effects of CrIII at all doses. CrIII increased hydrogen peroxide and lipid hydroperoxide in plasma and ovary; decreased the antioxidant enzymes (AOXs) GPx1, GR, SOD, and catalase; and increased glutathione S-transferase in plasma and ovary. To understand the effects of CrVI on ROS and AOXs in granulosa (GC) and theca (TC) cell compartments in the ovary, ROS levels and mRNA expression of cytosolic and mitochondrial AOXs, such as SOD1, SOD2, catalase, GLRX1, GSTM1, GSTM2, GSTA4, GR, TXN1, TXN2, TXNRD2, and PRDX3, were studied in GCs and TCs and in a spontaneously immortalized granulosa cell line (SIGC). Overall, CrVI downregulated each of the AOXs; and vitamin C mitigated the effects of CrVI on these enzymes in GCs and SIGCs, but failed to mitigate CrVI effects on GSTM1, GSTM2, TXN1, and TXN2 in TCs. Thus, these data for the first time reveal that lactational exposure to CrIII accelerated follicular atresia and decreased steroidogenesis in F1 female offspring by altering the ratio of ROS and AOXs in the ovary. Vitamin C is able to protect the ovary from CrIII-induced oxidative stress and follicle atresia through protective effects on GCs rather than TCs.

Detection of autoinducer (AI-2)-like activity in food samples.

The contamination, survival, and possible foodborne disease outbreaks are major issues confronting the food industry. However, from a microbial perspective, any food whether natural or processed is just another environmental niche that is available for colonization. Quorum sensing or cell-cell communication is a process by which microorganisms are thought to communicate with each other using a variety of small molecules termed autoinducers. The autoinducer AI-2 is thought to be a universal signaling molecule due to its ability to modulate the gene expression of a number of different bacterial species and genera. Pathogens such as Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio anguillarum, Streptococcus sp., and Burkholderia cepacia form biofilms on a variety of man-made and natural surfaces using cell-cell mechanisms. It is important to detect and study autoinducers and their activities in foods, since a better understanding of these molecules in food and food ingredients may help in designing new approaches to thwart microbial persistence and biofilm formation. The autoinducer AI-2 is thought to be involved in microbial attachment and biofilm formation leading to food spoilage. To better understand microbial cell-cell signaling in foods especially as it relates to pathogen persistence, biofilm formation, and food spoilage, methods to process, extract, and purify autoinducer molecules need to be developed. This chapter details methods to process food samples to obtain cell-free supernatants (CFS), which could subsequently be tested for the presence of AI-2 or "AI-2-like activity" in the extracted CFS using autoinducer bioassays. Additionally, the method of synthesizing AI-2 in the laboratory is also provided. The methods that are presented in this chapter are based on previously published research articles from the authors' laboratory.