Hari K. Bhat

Characterization of Phthalate Exposure among Pregnant Women Assessed by Repeat Air and Urine Samples

Background Although urinary concentrations of phthalate metabolites are frequently used as biomarkers in epidemiologic studies, variability during pregnancy has not been characterized. Methods We measured phthalate metabolite concentrations in spot urine samples collected from 246 pregnant Dominican and African-American women. Twenty-eight women had repeat urine samples collected over a 6-week period. We also analyzed 48-hr personal air samples (n = 96 women) and repeated indoor air samples (n = 32 homes) for five phthalate diesters. Mixed-effects models were fit to evaluate reproducibility via intraclass correlation coefficients (ICC). We evaluated the sensitivity and specificity of using a single specimen versus repeat samples to classify a woman’s exposure in the low or high category. Results Phthalates were detected in 85–100% of air and urine samples. ICCs for the unadjusted urinary metabolite concentrations ranged from 0.30 for mono-ethyl phthalate to 0.66 for monobenzyl phthalate. For indoor air, ICCs ranged from 0.48 [di-2-ethylhexyl phthalate (DEHP)] to 0.83 [butylbenzyl phthalate (BBzP)]. Air levels of phthalate diesters correlated with their respective urinary metabolite concentrations for BBzP (r = 0.71), di-isobutyl phthalate (r = 0.44), and diethyl phthalate (DEP; r = 0.39). In women sampled late in pregnancy, specific gravity appeared to be more effective than creatinine in adjusting for urine dilution. Conclusions Urinary concentrations of DEP and DEHP metabolites in pregnant women showed lower reproducibility than metabolites for di-n-butyl phthalate and BBzP. A single indoor air sample may be sufficient to characterize phthalate exposure in the home, whereas urinary phthalate biomarkers should be sampled longitudinally during pregnancy to minimize exposure misclassification.

Phytoestrogens and Breast Cancer Prevention: Possible Mechanisms of Action

OBJECTIVE Phytoestrogens display an array of pharmacologic properties, and in recent years investigation of their potential as anticancer agents has increased dramatically. In this article we review the published literature related to phytoestrogens and breast cancer as well as suggest the possible mechanisms that may underlie the relationship between phytoestrogens and breast cancer. DATA SOURCES Electronic searches on phytoestrogens and breast cancer were performed on MEDLINE and EMBASE in June 2007. No date restriction was placed on the electronic search. DATA EXTRACTION We focused on experimental data from published studies that examined the characteristics of phytoestrogens using in vivo or in vitro models. We also include human intervention studies in this review. DATA SYNTHESIS We evaluated evidence regarding the possible mechanisms of phytoestrogen action. Discussions of these mechanisms were organized into those activities related to the estrogen receptor, cell growth and proliferation, tumor development, signaling pathways, and estrogen-metabolizing enzymes. CONCLUSIONS We suggest that despite numerous investigations, the mechanisms of phytoestrogen action in breast cancer have yet to be elucidated. It remains uncertain whether these plant compounds are chemoprotective or whether they may produce adverse outcomes related to breast carcinogenesis.

Medicinal Plants and Cancer Chemoprevention

Cancer is the second leading cause of death worldwide. Although great advancements have been made in the treatment and control of cancer progression, significant deficiencies and room for improvement remain. A number of undesired side effects sometimes occur during chemotherapy. Natural therapies, such as the use of plant-derived products in cancer treatment, may reduce adverse side effects. Currently, a few plant products are being used to treat cancer. However, a myriad of many plant products exist that have shown very promising anti-cancer properties in vitro, but have yet to be evaluated in humans. Further study is required to determine the efficacy of these plant products in treating cancers in humans. This review will focus on the various plant-derived chemical compounds that have, in recent years, shown promise as anticancer agents and will outline their potential mechanism of action.

Critical role of oxidative stress in estrogen-induced carcinogenesis

Mechanisms of estrogen-induced tumorigenesis in the target organ are not well understood. It has been suggested that oxidative stress resulting from metabolic activation of carcinogenic estrogens plays a critical role in estrogen-induced carcinogenesis. We tested this hypothesis by using an estrogen-induced hamster renal tumor model, a well established animal model of hormonal carcinogenesis. Hamsters were implanted with 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethinylestradiol (αEE), menadione, a combination of αE2 and αEE, or a combination of αEE and menadione for 7 months. The group treated with βE2 developed target organ specific kidney tumors. The kidneys of hamsters treated with αE2, αEE, or menadione alone did not show any gross evidence of tumor. Kidneys of hamsters treated with a combination of αE2 and αEE showed early signs of proliferation in the interstitial cells. Kidneys of hamsters treated with a combination of menadione and αEE showed foci of tumor with congested tubules and atrophic glomeruli. βE2-treated tumor-bearing kidneys showed >2-fold increase in 8-iso-prostaglandin F2α (8-iso-PGF2α) levels compared with untreated controls. Kidneys of hamsters treated with a combination of menadione and αEE showed increased 8-iso-PGF2α levels compared with untreated controls, whereas no increase in 8-iso-PGF2α was detected in kidneys of αEE-treated group. A chemical known to produce oxidative stress or a potent estrogen with poor ability to produce oxidative stress, were nontumorigenic in hamsters, when given as single agents, but induced renal tumors, when given together. Thus, these data provide evidence that oxidant stress plays a crucial role in estrogen-induced carcinogenesis.

Skeletal Muscle Mitochondrial DNA Injury in Patients With Unilateral Peripheral Arterial Disease

BACKGROUND Patients with peripheral arterial disease (PAD) have exercise limitation due to claudication-limited pain and metabolic alterations in skeletal muscle. PAD is also associated with oxidative stress, which is a known cause of mitochondrial DNA (mtDNA) injury. The present study was designed to test the hypothesis that PAD is associated with mtDNA injury, as reflected by an increased frequency of a specific 4977-base pair (bp) mtDNA deletion mutation. METHODS AND RESULTS The deletion frequency was quantified in gastrocnemius muscle of 8 patients with unilateral PAD and 10 age-matched control subjects with the use of polymerase chain reaction methodologies. Muscle from the hemodynamically unaffected (less affected) PAD limb showed an 8-fold increased deletion frequency and the hemodynamically affected (worse affected) PAD limb had a 17-fold increased deletion frequency compared with muscle from control subjects. The frequency of the 4977-bp deletion in the worse-affected limb was positively correlated with the age of the patients but not the claudication-limited exercise performance of the patients. Total mtDNA content, citrate synthase activity, and cytochrome c oxidase activity were not different in the muscle from the 3 limb populations. However, the ratio of citrate synthase to cytochrome c oxidase was higher in the worse- versus less-affected limbs of PAD patients. CONCLUSIONS The present study demonstrates a large increase in the frequency of the mtDNA 4977-bp deletion in patients with PAD but in a distribution not limited to the hemodynamically affected limb.

Prenatal Di(2-ethylhexyl)Phthalate Exposure and Length of Gestation Among an Inner-City Cohort

OBJECTIVE: Our objective was to assess the relationship between di(2-ethylhexyl)phthalate (DEHP) exposure during pregnancy and gestational age at delivery among 311 African American or Dominican women from New York City. METHODS: Forty-eight-hour personal air and/or spot urine samples were collected during the third trimester. DEHP levels were measured in air samples and 4 DEHP metabolite levels were measured in urine. Specific gravity was used to adjust for urinary dilution. Gestational age was abstracted from newborn medical records (n = 289) or calculated from the expected date of delivery (n = 42). Multivariate linear regression models controlled for potential confounders. RESULTS: DEHP was detected in 100% of personal air samples (geometric mean: 0.20 μg/m3 [95% confidence interval [CI]: 0.18–0.21 μg/m3]); natural logarithms of air concentrations were inversely but not significantly associated with gestational age. Two or more of the DEHP metabolites were detected in 100% of urine samples (geometric mean: 4.8–38.9 ng/mL [95% CI: 4.1–44.3 ng/mL]). Controlling for potential confounders, gestational age was shorter by 1.1 days (95% CI: 0.2–1.8 days) for each 1-logarithmic unit increase in specific gravity-adjusted mono(2-ethylhexyl)phthalate concentrations (P = .01) and averaged 5.0 days (95% CI: 2.1–8.0 days) less among subjects with the highest versus lowest quartile concentrations (P = .001). Results were similar and statistically significant for the other DEHP metabolites. CONCLUSIONS: Prenatal DEHP exposure was associated with shorter gestation but, given inconsistencies with previous findings for other study populations, results should be interpreted with caution, and additional research is warranted.

MicroRNA-93 regulates NRF2 expression and is associated with breast carcinogenesis

MicroRNAs (miRNA) are small non-coding RNAs that regulate the expression of approximately 60% of all human genes and play important roles in disease processes. Recent studies have demonstrated a link between dysregulated expression of miRNAs and breast carcinogenesis. Long-term estrogen exposure is implicated in development of human breast cancers, yet underlying mechanisms remain elusive. We have recently demonstrated that antioxidant vitamin C (vit C) prevents estrogen-induced breast tumor development. In this study, we investigated the role of vit C in the regulation of microRNA-93 (miR-93) and its target gene(s) in a rat model of mammary carcinogenesis. Female August Copenhagen Irish (ACI) rats were treated with vit C in the presence or absence of 17β-estradiol (E2) for 8 months. We demonstrate an increased expression of the miR-93 in E2-treated mammary tissues and in human breast cell lines and vit C treatment reverted E2-mediated increase in miR-93 levels. MiRNA target prediction programs suggest one of the target genes of miR-93 to be nuclear factor erythroid 2-related factor 2 (NRF2). In contrast with miR-93 expression, NRF2 protein expression was significantly decreased in E2-treated mammary tissues, mammary tumors, and in breast cancer cell lines, and its expression was significantly increased after vit C treatment. Ectopic expression of miR-93 decreased protein expression of NRF2 and NRF2-regulated genes. Furthermore, miR-93 decreased apoptosis, increased colony formation, mammosphere formation, cell migration and DNA damage in breast epithelial cells, whereas silencing of miR-93 in these cells inhibited these carcinogenic processes. Taken together, our findings suggest an oncogenic potential of miR-93 during E2-induced breast carcinogenesis.

Resveratrol inhibits estrogen-induced breast carcinogenesis through induction of NRF2-mediated protective pathways

The importance of estrogens in the etiology of breast cancer is widely recognized. Estrogen-induced oxidative stress has been implicated in this carcinogenic process. Resveratrol (Res), a natural antioxidant phytoestrogen has chemopreventive effects against a variety of illnesses including cancer. The objective of the present study was to characterize the mechanism(s) of Res-mediated protection against estrogen-induced breast carcinogenesis. Female August Copenhagen Irish rats were treated with 17β-estradiol (E2), Res and Res + E2 for 8 months. Cotreatment of rats with Res and E2 inhibited E2-mediated proliferative changes in mammary tissues and significantly increased tumor latency and reduced E2-induced breast tumor development. Resveratrol treatment alone or in combination with E2 significantly upregulated expression of nuclear factor erythroid 2-related factor 2 (NRF2) in mammary tissues. Expression of NRF2-regulated antioxidant genes NQO1, SOD3 and OGG1 that are involved in protection against oxidative DNA damage was increased in Res- and Res + E2-treated mammary tissues. Resveratrol also prevented E2-mediated inhibition of detoxification genes AOX1 and FMO1. Inhibition of E2-mediated alterations in NRF2 promoter methylation and expression of NRF2 targeting miR-93 after Res treatment indicated Res-mediated epigenetic regulation of NRF2 during E2-induced breast carcinogenesis. Resveratrol treatment also induced apoptosis and inhibited E2-mediated increase in DNA damage in mammary tissues. Increased apoptosis and decreased DNA damage, cell migration, colony and mammosphere formation in Res- and Res + E2-treated MCF-10A cells suggested a protective role of Res against E2-induced mammary carcinogenesis. Small-interfering RNA-mediated silencing of NRF2 inhibited Res-mediated preventive effects on the colony and mammosphere formation. Taken together, these results suggest that Res inhibits E2-induced breast carcinogenesis via induction of NRF2-mediated protective pathways.

Estrogen-induced breast cancer: Alterations in breast morphology and oxidative stress as a function of estrogen exposure

Epidemiological evidence indicates that prolonged lifetime exposure to estrogen is associated with elevated breast cancer risk in women. Oxidative stress and estrogen receptor-associated proliferative changes are suggested to play important roles in estrogen-induced breast carcinogenesis. In the present study, we investigated changes in breast morphology and oxidative stress following estrogen exposure. Female ACI rats were treated with 17beta-estradiol (E(2), 3 mg, s.c.) for either 7, 15, 120 or 240 days. Animals were euthanized, tissues were excised, and portions of the tissues were either fixed in 10% buffered formalin or snap-frozen in liquid nitrogen. Paraffin-embedded tissues were examined for histopathologic changes. Proliferative changes appeared in the breast after 7 days of E(2) exposure. Atypical ductal proliferation and significant reduction in stromal fat were observed following 120 days of E(2) exposure. Both in situ and invasive carcinomas were observed in the majority of the mammary glands from rats treated with E(2) for 240 days. Palpable breast tumors were observed in 82% of E(2)-treated rats after 228 days, with the first palpable tumor appearing after 128 days. No morphological changes were observed in the livers, kidneys, lungs or brains of rats treated with E(2) for 240 days compared to controls. Furthermore, 8-isoprostane (8-isoPGF(2alpha)) levels as well as the activities of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase and catalase, were quantified in the breast tissues of rats treated with E(2) for 7, 15, 120 and 240 days and compared to activity levels in age-matched controls. 8-isoPGF(2alpha) levels displayed time-dependent increases upon E(2) treatment and were significantly higher than control levels at the 15, 120 and 240 day time-points. 8-isoPGF(2alpha) observed in E(2)-induced mammary tumors were significantly higher than levels found in control mammary tissue from age-matched animals. Similarly, alterations in glutathione peroxidase and superoxide dismutase activities were detected in both mammary and tumor tissue from E(2)-treated rats. Taken together, our data reveal that proliferative changes in the breast tissue of ACI rats are associated with increases in 8-isoPGF(2alpha) formation as well as changes in the activities of antioxidant enzymes. These oxidative changes appear to be a function of E(2) exposure and occur prior to tumor development.

Superoxide dismutase 3 is induced by antioxidants, inhibits oxidative DNA damage and is associated with inhibition of estrogen-induced breast cancer.

Epidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Oxidative stress has been implicated in this carcinogenic process. We have recently demonstrated that antioxidants vitamin C or butylated hydroxyanisole (BHA) severely inhibit 17β-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of this study was to characterize the mechanism of antioxidant-mediated prevention of breast cancer. Female August Copenhagen Irish (ACI) rats were treated with E2, vitamin C, vitamin C + E2, BHA and BHA + E2 for up to 8 months. Superoxide dismutase 3 (SOD3) was suppressed in E2-exposed mammary tissues and in mammary tumors of rats treated with E2. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. 8-Hydroxydeoxyguanosine (8-OHdG) levels determined as a marker of oxidative DNA damage were higher in E2-exposed mammary tissues and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissues and in MCF-10A cells. Increased DNA damage, colony and mammosphere formation, and migration in SOD3 knocked down MCF-10A cells, and nuclear translocation of SOD3 in vitamin C-treated mammary tissues and in MCF-10A cells suggest protective role of SOD3 against DNA damage and mammary carcinogenesis. Our studies further demonstrate that SOD3, but not SOD2 and SOD1, is induced by antioxidants and is regulated through NRF2. SOD3 may thus be an important gene in defense against oxidative stress and in the prevention of estrogen-mediated breast cancer.