Angela M. Betancourt

Altered Carcinogenesis and Proteome in Mammary Glands of Rats after Prepubertal Exposures to the Hormonally Active Chemicals Bisphenol A and Genistein

Through our diet, we are exposed to numerous natural and man-made chemicals, including polyphenols with hormone-like properties. The most abundant hormonally active polyphenols are characterized as weak estrogens. These chemicals are hypothesized to interfere with signaling pathways involved in important diseases such as breast cancer, which in most cases is initially estrogen dependent. Two such chemicals are bisphenol A (BPA), a plasticizer, and genistein, a component of soy. In spite of both possessing estrogenic properties, BPA and genistein yield different health outcomes. The exposure of rats during the prepubertal period to BPA increases the susceptibility of adult animals for mammary cancer development, whereas genistein decreases this susceptibility in a chemically induced model. Because both BPA and genistein possess estrogenic properties, it is certainly plausible that additional mechanisms are affected by these chemicals. Hence, it was our goal to investigate at the protein level how exposure to these 2 chemicals can contribute to mammary cancer causation as opposed to cancer chemoprevention. Using 2-dimensional gel electrophoresis followed by MS analysis, we identified differentially regulated proteins from the mammary glands of rats prepubertally exposed to BPA and genistein. Following protein identification, we used immunoblotting techniques to validate the identity and regulation of these proteins and to identify downstream signaling proteins. Our studies highlight the importance of proteomics technology in elucidating signaling pathways altered by exposure to hormonally active chemicals and its potential value in identifying biomarkers for mammary cancer.

In Utero Exposure to Bisphenol A Shifts the Window of Susceptibility for Mammary Carcinogenesis in the Rat

Background Bisphenol A (BPA) is a ubiquitous environmental chemical with reported endocrine-disrupting properties. Objective Our goal in this study was to determine whether prenatal exposure to BPA predisposes the adult rat mammary gland to carcinogenesis. Methods Pregnant rats were treated orally with 0, 25, or 250 μg BPA/kg body weight (BW) from gestation day (GD) 10 to GD21. For tumorigenesis experiments, prenatally exposed female offspring received a single gavage of 7,12-dimethylbenz(a)anthracene (DMBA; 30 mg/kg BW) on postnatal day (PND) 50, or PND100. Results Prenatal exposure of the dam to 250 μg BPA/kg BW combined with a single exposure of female offspring to DMBA on PND100, but not on PND50, significantly increased tumor incidence while decreasing tumor latency compared with the control group. Prenatal exposure of the dam to 250 μg BPA/kg BW, in the absence of DMBA to the female offspring, increased cell proliferation and elicited differential effects at the protein level at PND100 compared with PND50. Differentially regulated proteins in the mammary gland included estrogen receptor-α, progesterone receptor-A, Bcl-2, steroid receptor coactivators, epidermal growth factor receptor, phospho-insulin-like growth factor 1 receptor, and phospho-Raf. Conclusions Our study demonstrates that oral prenatal exposure to BPA increases mammary cancer susceptibility in offspring and shifts the window of susceptibility for DMBA-induced tumorigenesis in the rat mammary gland from PND50 to PND100. These changes are accompanied by differential effects of prenatal BPA exposure on the expression of key proteins involved in cell proliferation.

Chronic Exposure to a High-Fat Diet Induces Hepatic Steatosis, Impairs Nitric Oxide Bioavailability, and Modifies the Mitochondrial Proteome in Mice

Obesity-related pathologies, such as nonalcoholic fatty liver disease, are linked to mitochondrial dysfunction and nitric oxide (NO) deficiency. Herein, we tested the hypothesis that a high-fat diet (HFD) modifies the liver mitochondrial proteome and alters proteins involved in NO metabolism, namely arginase 1 and endothelial NO synthase. Male C57BL/6 mice were fed a control or HFD and liver mitochondria were isolated for proteomics and reactive oxygen species measurements. Steatosis and hepatocyte ballooning were present in livers of HFD mice, with no pathology observed in the controls. HFD mice had increased serum glucose and decreased adiponectin. Mitochondrial reactive oxygen species was increased after 8 weeks in the HFD mice, but decreased at 16 weeks compared with the control, which was accompanied by increased uncoupling protein 2. Using proteomics, 22 proteins were altered as a consequence of the HFD. This cohort consists of oxidative phosphorylation, lipid metabolism, sulfur amino acid metabolism, and chaperone proteins. We observed a HFD-dependent increase in arginase 1 and decrease in activated endothelial NO synthase. Serum and liver nitrate + nitrite were decreased by HFD. In summary, these data demonstrate that a HFD causes steatosis, alters NO metabolism, and modifies the liver mitochondrial proteome; thus, NO may play an important role in the processes responsible for nonalcoholic fatty liver disease.

Exposure to the Endocrine Disruptor Bisphenol A Alters Susceptibility for Mammary Cancer

Abstract Bisphenol A (BPA) is a synthetically made chemical used in the production of polycarbonate plastics and epoxy resins. Recent studies have shown that >90% of humans investigated have detectable BPA concentrations. Yet, the biggest concern for BPA is exposure during early development because BPA has been shown to bind to the estrogen receptors (ERs) and cause developmental and reproductive toxicity. We have investigated the potential of perinatal BPA to alter susceptibility for chemically induced mammary cancer in rats. We demonstrate that prepubertal exposure to low concentrations of orally administered BPA given to lactating dams resulted in a significantly decreased tumor latency and increased tumor multiplicity in the dimethylbenz[a]anthra-cene model of rodent mammary carcinogenesis. Our data suggested that the mechanism of action behind this carcinogenic response was mediated through increased cell proliferation, decreased apoptosis, and centered on an upregulation of steroid receptor coactivators (SRCs) 1–3, erbB3, and increased Akt signaling in the mammary gland. Also, we demonstrate that prenatal exposure to BPA shifts the time of susceptibility from 50 days to 100 days for chemically induced mammary carcinogenesis. Proteomic data suggest that prenatal BPA exposure alters the expression of several proteins involved in regulating protein metabolism, signal transduction, developmental processes, and cell cycle and proliferation. Increases in ER-α, SRCs 1–3, Bcl-2, epidermal growth factor-receptor, phospho-IGF-1R, phospho-c-Raf, phospho-ERKs 1/2, phospho-ErbB2, and phospho-Akt are accompanied by increases in cell proliferation. We conclude that exposure to low concentrations of BPA during the prenatal and early postnatal periods of life can predispose for chemically induced mammary cancer.

Proteomic analysis in mammary glands of rat offspring exposed in utero to bisphenol A.

Bisphenol A (BPA) is a ubiquitous environmental contaminant with established endocrine disruptor properties. The objective of our study was to determine the effects of prenatal exposure to BPA on the rat mammary gland proteome in postnatal rats as a first step toward the investigation of translational biomarkers of susceptibility in the human population. Pregnant rats were treated orally with 0, 25 or 250 microg BPA/kg body weight from days 10 to 21 post-conception. Female offspring were euthanized at 21 and 50 days, and mammary glands were collected. Proteomic analysis was conducted using 2-DE, followed by a combination of MALDI-TOF-TOF and LC-MS/MS, which led to the identification of 21 differentially abundant proteins including vimentin, SPARC and 14-3-3. Western blot analysis of key downstream signaling proteins demonstrated increased phospho-AKT, c-Raf, phospho-ERKs-1 and 2, but decreased TGF-beta in mammary glands of 50 day old rats exposed prenatally to BPA. Our studies indicate for the first time that key proteins involved in signaling pathways such as cellular proliferation are regulated at the protein level by BPA. This data is expected to aid in the understanding of how BPA may be influencing the susceptibility of the mammary gland to cancer transformation.

Nitric oxide and hypoxia exacerbate alcohol-induced mitochondrial dysfunction in hepatocytes.

Chronic alcohol consumption results in hepatotoxicity, steatosis, hypoxia, increased expression of inducible nitric oxide synthase (iNOS) and decreased activities of mitochondrial respiratory enzymes. The impact of these changes on cellular respiration and their interaction in a cellular setting is not well understood. In the present study we tested the hypothesis that nitric oxide (NO)-dependent modulation of cellular respiration and the sensitivity to hypoxic stress is increased following chronic alcohol consumption. This is important since NO has been shown to regulate mitochondrial function through its interaction with cytochrome c oxidase, although at higher concentrations, and in combination with reactive oxygen species, can result in mitochondrial dysfunction. We found that hepatocytes isolated from alcohol-fed rats had decreased mitochondrial bioenergetic reserve capacity and were more sensitive to NO-dependent inhibition of respiration under room air and hypoxic conditions. We reasoned that this would result in greater hypoxic stress in vivo, and to test this, wild-type and iNOS(-/-) mice were administered alcohol-containing diets. Chronic alcohol consumption resulted in liver hypoxia in the wild-type mice and increased levels of hypoxia-inducible factor 1 α in the peri-venular region of the liver lobule. These effects were attenuated in the alcohol-fed iNOS(-/-) mice suggesting that increased mitochondrial sensitivity to NO and reactive nitrogen species in hepatocytes and iNOS plays a critical role in determining the response to hypoxic stress in vivo. These data support the concept that the combined effects of NO and ethanol contribute to an increased susceptibility to hypoxia and the deleterious effects of alcohol consumption on liver.

Mitochondrial-nuclear genome interactions in non-alcoholic fatty liver disease in mice.

NAFLD (non-alcoholic fatty liver disease) involves significant changes in liver metabolism characterized by oxidative stress, lipid accumulation and fibrogenesis. Mitochondrial dysfunction and bioenergetic defects also contribute to NAFLD. In the present study, we examined whether differences in mtDNA influence NAFLD. To determine the role of mitochondrial and nuclear genomes in NAFLD, MNX (mitochondrial-nuclear exchange) mice were fed an atherogenic diet. MNX mice have mtDNA from C57BL/6J mice on a C3H/HeN nuclear background and vice versa. Results from MNX mice were compared with wild-type C57BL/6J and C3H/HeN mice fed a control or atherogenic diet. Mice with the C57BL/6J nuclear genome developed more macrosteatosis, inflammation and fibrosis compared with mice containing the C3H/HeN nuclear genome when fed the atherogenic diet. These changes were associated with parallel alterations in inflammation and fibrosis gene expression in wild-type mice, with intermediate responses in MNX mice. Mice with the C57BL/6J nuclear genome had increased State 4 respiration, whereas MNX mice had decreased State 3 respiration and RCR (respiratory control ratio) when fed the atherogenic diet. Complex IV activity and most mitochondrial biogenesis genes were increased in mice with the C57BL/6J nuclear or mitochondrial genome, or both fed the atherogenic diet. These results reveal new interactions between mitochondrial and nuclear genomes and support the concept that mtDNA influences mitochondrial function and metabolic pathways implicated in NAFLD.

Involvement of the mitochondrial permeability transition pore in chronic ethanol-mediated liver injury in mice

Chronic ethanol consumption increases sensitivity of the mitochondrial permeability transition (MPT) pore induction in liver. Ca(2+) promotes MPT pore opening, and genetic ablation of cyclophilin D (CypD) increases the Ca(2+) threshold for the MPT. We used wild-type (WT) and CypD-null (CypD(-/-)) mice fed a control or an ethanol-containing diet to investigate the role of the MPT in ethanol-mediated liver injury. Ca(2+)-mediated induction of the MPT and mitochondrial respiration were measured in isolated liver mitochondria. Steatosis was present in WT and CypD(-/-) mice fed ethanol and accompanied by increased terminal deoxynucleotidyl transferase dUTP-mediated nick-end label-positive nuclei. Autophagy was increased in ethanol-fed WT mice compared with ethanol-fed CypD(-/-) mice, as reflected by an increase in the ratio of microtubule protein 1 light chain 3B II to microtubule protein 1 light chain 3B I. Higher levels of p62 were measured in CypD(-/-) than WT mice. Ethanol decreased mitochondrial respiratory control ratios and select complex activities in WT and CypD(-/-) mice. Ethanol also increased CypD protein in liver of WT mice. Mitochondria from control- and ethanol-fed WT mice were more sensitive to Ca(2+)-mediated MPT pore induction than mitochondria from their CypD(-/-) counterparts. Mitochondria from ethanol-fed CypD(-/-) mice were also more sensitive to Ca(2+)-induced swelling than mitochondria from control-fed CypD(-/-) mice but were less sensitive than mitochondria from ethanol-fed WT mice. In summary, CypD deficiency was associated with impaired autophagy and did not prevent ethanol-mediated steatosis. Furthermore, increased MPT sensitivity was observed in mitochondria from ethanol-fed WT and CypD(-/-) mice. We conclude that chronic ethanol consumption likely lowers the threshold for CypD-regulated and -independent characteristics of the ethanol-mediated MPT pore in liver mitochondria.

Proteomic discovery of genistein action in the rat mammary gland.

Genistein, the primary isoflavone component of soy, consumed in diet during the prepubertal period suppresses chemically induced mammary cancer in rats. The current study used two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology to identify proteins responsible for genistein breast cancer protection In Vivo. Female offspring were exposed via lactating dams treated with 250 mg genistein/kg AIN-76A diet from days 1 to 21 postpartum (prepubertal period). Mammary glands were collected at 21 and 50 day of age and subjected to 2-DE/MS and immuno-blot analyses. Twenty-three proteins were determined to be differentially regulated (p < 0.05) and identified using 2-DE, followed by MALDI-TOF/TOF or LC-ESI-MS/MS. Five of these proteins were validated by immuno-blots. Annexin A2 was significantly increased at 21 days yet found to be decreased at 50 days. Fetuin B was found to be unchanged at day 21 but increased at day 50. Phosphoglycerate kinase 1 (PGK1) was unchanged at day 21 but decreased at day 50. Gelsolin was increased at day 21 but not at day 50. Protein disulfide-isomerase A3 (PDIA3) was decreased at day 21 and unchanged at day 50. Also, we found that vascular endothelial growth factor receptor 2 (VEGF-R2) and epidermal growth factor receptor (EGF-R) were decreased in mammary glands of 50-day-old rats treated prepubertally with genistein. This study demonstrates the usefulness of proteomics for the discovery of key proteins involved in signaling pathways to understand genistein mechanisms of action in breast cancer prevention.

Alterations in the rat serum proteome induced by prepubertal exposure to bisphenol a and genistein.

Humans are exposed to an array of chemicals via the food, drink and air, including a significant number that can mimic endogenous hormones. One such chemical is Bisphenol A (BPA), a synthetic chemical that has been shown to cause developmental alterations and to predispose for mammary cancer in rodent models. In contrast, the phytochemical genistein has been reported to suppress chemically induced mammary cancer in rodents, and Asians ingesting a diet high in soy containing genistein have lower incidence of breast and prostate cancers. In this study, we sought to: (1) identify protein biomarkers of susceptibility from blood sera of rats exposed prepubertally to BPA or genistein using Isobaric Tandem Mass Tags quantitative mass spectrometry (TMT-MS) combined with MudPIT technology and, (2) explore the relevance of these proteins to carcinogenesis. Prepubertal exposures to BPA and genistein resulted in altered expression of 63 and 28 proteins in rat sera at postnatal day (PND) 21, and of 9 and 18 proteins in sera at PND35, respectively. This study demonstrates the value of using quantitative proteomic techniques to explore the effect of chemical exposure on the rat serum proteome and its potential for unraveling cellular targets altered by BPA and genistein involved in carcinogenesis.