Rao L. Divi

Original ArticlesAnti-Thyroid Isoflavones from Soybean: Isolation, Characterization, and Mechanisms of Action

The soybean has been implicated in diet-induced goiter by many studies. The extensive consumption of soy products in infant formulas and in vegetarian diets makes it essential to define the goitrogenic potential. In this report, it was observed that an acidic methanolic extract of soybeans contains compounds that inhibit thyroid peroxidase- (TPO) catalyzed reactions essential to thyroid hormone synthesis. Analysis of the soybean extract using HPLC, UV-VIS spectrophotometry, and LC-MS led to identification of the isoflavones genistein and daidzein as major components by direct comparison with authentic standard reference isoflavones. HPLC fractionation and enzymatic assay of the soybean extract showed that the components responsible for inhibition of TPO-catalyzed reactions coeluted with daidzein and genistein. In the presence of iodide ion, genistein and daidzein blocked TPO-catalyzed tyrosine iodination by acting as alternate substrates, yielding mono-, di-, and triiodoisoflavones. Genistein also inhibited thyroxine synthesis using iodinated casein or human goiter thyroglobulin as substrates for the coupling reaction. Incubation of either isoflavone with TPO in the presence of H2O2 caused irreversible inactivation of the enzyme; however, the presence of iodide ion in the incubations completely abolished the inactivation. The IC50 values for inhibition of TPO-catalyzed reactions by genistein and daidzein were ca. 1-10 microM, concentrations that approach the total isoflavone levels (ca. 1 microM) previously measured in plasma from humans consuming soy products. Because inhibition of thyroid hormone synthesis can induce goiter and thyroid neoplasia in rodents, delineation of anti-thyroid mechanisms for soy isoflavones may be important for extrapolating goitrogenic hazards identified in chronic rodent bioassays to humans consuming soy products.

Mitochondrial damage and DNA depletion in cord blood and umbilical cord from infants exposed in utero to Combivir.

Objective: Although most uninfected infants born to women infected with HIV-1 show no clinical evidence of mitochondrial compromise, mitochondrial dysfunction has been reported in children born to women receiving zidovudine and/or lamivudine during pregnancy. In this pilot study we examined mitochondrial integrity in HIV-1-uninfected infants born to HIV-1-infected women receiving Combivir during pregnancy. Design: Samples of umbilical cord and cord blood were obtained from HIV-1-uninfected infants born to either HIV-1-infected women receiving Combivir therapy during pregnancy (n = 10) or HIV-1-uninfected women (n = 9). Methods: Mitochondrial morphological integrity was examined in umbilical cords (n = 16) by electron microscopy and mtDNA quantity was determined in DNA from cord blood (n = 18) and umbilical cord (n = 18) by PCR-chemiluminescence immunoassay detection. Results: In umbilical cords from six of nine infants born to HIV-1-infected mothers taking Combivir moderate to severe mitochondrial morphological damage was observed (P = 0.011), while none of seven unexposed infants showed similar damage. Compared to unexposed infants, statistically significant mtDNA depletion was observed in umbilical cord (P = 0.006) and cord blood (P = 0.003) from drug-exposed infants. Conclusions: A cohort of HIV-1-uninfected Combivir-exposed infants with no clinical symptoms showed morphological and molecular evidence of mitochondrial damage.

Mitochondrial Toxicity in Fetal Erythrocebus patas Monkeys Exposed Transplacentally to Zidovudine Plus Lamivudine

This study was designed to investigate fetal mitochondrial toxicity in Erythrocebus patas monkeys exposed in utero to zidovudine (AZT) and lamivudine (3TC), and taken at term. Pregnant patas monkeys were given a daily dose of 40 mg AZT (86% of the human daily dose, based on body weight), for the last 10 weeks (50%) of gestation, and a daily dose of 24 mg 3TC (84% of the human daily dose, based on body weight) for the last 4 weeks of gestation. At term, AZT was found to be incorporated into fetal mitochondrial DNA from skeletal muscle, liver, kidney, and placenta. By transmission electron microscopy (EM) drug-exposed fetal cardiac and skeletal muscle cells showed mitochondrial membrane compromise, mitochondrial proliferation, and damaged sarcomeres, while mitochondria in brain cerebrum and cerebellum were morphologically normal. Substantial depletion of oxidative phosphorylation (OXPHOS) Complex I specific activities was observed in heart (87% reduction in mean, p = 0.02) and skeletal muscle (98% reduction in mean, p = 0.002) from drug-exposed fetuses, compared to unexposed fetuses. In addition Complex IV activity was highly depleted (85% reduction in mean, p = 0.004) in skeletal muscle from the drug-exposed fetuses (p = 0.004). Brain cerebrum and cerebellum showed no statistically significant OXPHOS changes with drug exposure. Mitochondrial DNA quantity was substantially depleted (>50%) in heart, skeletal muscle, cerebellum, and cerebrum from drug-exposed fetuses compared to unexposed controls. Overall, the data indicate that significant mitochondrial damage was observed at birth in monkey fetuses exposed in utero to AZT plus 3TC in a human-equivalent dosing protocol.

Extracellular vesicles: potential applications in cancer diagnosis, prognosis, and epidemiology.

Both normal and diseased cells continuously shed extracellular vesicles (EVs) into extracellular space, and the EVs carry molecular signatures and effectors of both health and disease. EVs reflect dynamic changes that are occurring in cells and tissue microenvironment in health and at a different stage of a disease. EVs are capable of altering the function of the recipient cells. Trafficking and reciprocal exchange of molecular information by EVs among different organs and cell types have been shown to contribute to horizontal cellular transformation, cellular reprogramming, functional alterations, and metastasis. EV contents may include tumor suppressors, phosphoproteins, proteases, growth factors, bioactive lipids, mutant oncoproteins, oncogenic transcripts, microRNAs, and DNA sequences. Therefore, the EVs present in biofluids offer unprecedented, remote, and non-invasive access to crucial molecular information about the health status of cells, including their driver mutations, classifiers, molecular subtypes, therapeutic targets, and biomarkers of drug resistance. In addition, EVs may offer a non-invasive means to assess cancer initiation, progression, risk, survival, and treatment outcomes. The goal of this review is to highlight the current status of information on the role of EVs in cancer, and to explore the utility of EVs for cancer diagnosis, prognosis, and epidemiology.

Next generation analytic tools for large scale genetic epidemiology studies of complex diseases.

Over the past several years, genome‐wide association studies (GWAS) have succeeded in identifying hundreds of genetic markers associated with common diseases. However, most of these markers confer relatively small increments of risk and explain only a small proportion of familial clustering. To identify obstacles to future progress in genetic epidemiology research and provide recommendations to NIH for overcoming these barriers, the National Cancer Institute sponsored a workshop entitled “Next Generation Analytic Tools for Large‐Scale Genetic Epidemiology Studies of Complex Diseases” on September 15–16, 2010. The goal of the workshop was to facilitate discussions on (1) statistical strategies and methods to efficiently identify genetic and environmental factors contributing to the risk of complex disease; and (2) how to develop, apply, and evaluate these strategies for the design, analysis, and interpretation of large‐scale complex disease association studies in order to guide NIH in setting the future agenda in this area of research. The workshop was organized as a series of short presentations covering scientific (gene‐gene and gene‐environment interaction, complex phenotypes, and rare variants and next generation sequencing) and methodological (simulation modeling and computational resources and data management) topic areas. Specific needs to advance the field were identified during each session and are summarized. Genet. Epidemiol. 36 : 22–35, 2012.

Cardiac mitochondrial compromise in 1-yr-old Erythrocebus patas monkeys perinatally-exposed to nucleoside reverse transcriptase inhibitors.

Hearts from 1-yr-old Erythrocebus patas monkeys were examined after in utero and 6-wk-postbirth exposure to antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs). Protocols were modeled on those given to human immunodeficiency virus (HIV)-1-infected pregnant women. NRTIs were administered daily to the dams for the last 20% or 50% of gestation, and to the infants for 6 wk after birth. Exposures included: no drug (n=4); Zidovudine, 3′-azido-3′-deoxythymidine (AZT; n=4); AZT/Lamivudine, (−)-β-l-2′, 3′-Dideoxy-3′-thiacytidine (Epivir, 3TC) (n=4); AZT/Didanosine (Videx, ddl) (n=4); and Stavudine (Zerit, d4T)/3TC (n=4). Echocardiograms and clinical chemistry showed no drug-related changes, but the d4T/3TC-exposed fetuses at 6 and 12 mo had increased white cell counts (p<0.05). At 1 yr of age, oxidative phosphorylation (OXPHOS) enzyme activities were similar in heart mitochondria from all groups. Mitochondrial pathology, that included clones of damaged mitochondria (p<0.05), was found in hearts of all 1-yr drug-exposed infants. Levels of mtDNA were elevated (p<0.05) in hearts of all NRTI-exposed monkeys in the following order: control < d4T/3TC<AZT<AZT/3TC<AZT/ddl. The clinical status of NRTI-exposed infants, as evidenced by behavior, clinical chemistry, OXPHOS activity and echocardiogram, was normal. However, extensive mitochondrial damage with clusters of similar-appearing damaged heart mitochondria observed by electron microscopy, and an increase in mtDNA quantity, that persisted at 1 yr of age, suggest the potential for cardiotoxicity later in life.

Progressive Mitochondrial Compromise in Brains and Livers of Primates Exposed In Utero to Nucleoside Reverse Transcriptase Inhibitors (NRTIs)

Mitochondrial compromise has been documented in infants born to women infected with the human immunodeficiency virus (HIV-1) who received nucleoside reverse transcriptase inhibitor (NRTI) therapy during pregnancy. To model these human exposures, we examined mitochondrial integrity at birth and 1 year in brain cortex and liver from offspring of retroviral-free Erythrocebus patas dams-administered human-equivalent NRTI doses for the last half (10 weeks) of gestation. Additional infants, followed for 1 year, were given the same drugs as their mothers for the first 6 weeks of life. Exposures included: no drug, Zidovudine (AZT), Lamivudine (3TC), AZT/3TC, AZT/Didanosine (ddI), and Stavudine (d4T)/3TC. In brain and liver, oxidative phosphorylation (OXPHOS) enzyme activities (complexes I, II, and IV) showed minimal differences between unexposed and NRTI-exposed offspring at both times. Brain and liver mitochondria from most NRTI-exposed patas, both at birth and 1 year of age, contained significant (p < 0.05) morphological damage observed by electron microscopy (EM), based on scoring of coded photomicrographs. Brain and liver mitochondrial DNA (mtDNA) levels in NRTI-exposed patas were depleted significantly in the 3TC and d4T/3TC groups at birth and were depleted significantly (p < 0.05) at 1 year in all NRTI-exposed groups. In 1-year-old infants exposed in utero to NRTIs, mtDNA depletion was 28.8-51.8% in brain and 37.4-56.5% in liver. These investigations suggest that some NRTI-exposed human infants may sustain similar mitochondrial compromise in brain and liver and should be followed long term for cognitive integrity and liver function.

Persistence of mitochondrial toxicity in hearts of female B6C3F1 mice exposed in utero to 3′-azido-3′-deoxythymidine

Cardiac toxicity has been associated with HIV infection and exposure to nucleoside reverse transcriptase inhibitors (NRTIs), but the role of the latter in the development of cardiac disease of HIV-infected patients is uncertain. To investigate the cardiotoxicity of transplacentally administered zidovudine (AZT) or AZT plus lamivudine (3TC) in the absence of HIV infection, we evaluated several biomarkers of cardiac mitochondrial structure and cardiac structure and function in a B6C3F1 mouse model. In utero exposure to AZT-3TC resulted in ultrastructural pathology, loss of mitochondria, and altered echocardiographic measurements in newborn mice. Cardiac pathology and dysfunction persisted into the adult life of female mice exposed in utero to AZT, as evidenced by significant dose-dependent heart enlargement, clusters of atypical mitochondria and myofibril alterations, significantly increased cytochrome c oxidase activity, and significantly higher numbers of mutations in mitochondrial tRNA genes compared with unexposed controls at 18 to 24 mo of age. These data led to the hypothesis that the long-term pathology of perinatal exposure to these NRTIs is related to persistent mitochondrial DNA mutations in cardiac tissue; that is, the primary damage during drug treatment is mutational (as opposed to affecting polymerase γ and/or other mitochondrial elements) and leads over time to delayed, progressive cardiotoxicity.

Perinatal Exposure of Patas Monkeys to Antiretroviral Nucleoside Reverse-Transcriptase Inhibitors Induces Genotoxicity Persistent for up to 3 Years of Age

BACKGROUND Erythrocebus patas (patas) monkeys were used to model antiretroviral (ARV) drug in human immunodeficiency virus type 1-infected pregnant women. METHODS Pregnant patas dams were given human-equivalent doses of ARVs daily during 50% of gestation. Mesenchymal cells, cultured from bone marrow of patas offspring obtained at birth and at 1 and 3 years of age, were examined for genotoxicity, including centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes. RESULTS Compared with controls, statistically significant increases (P < .05) in centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes were found in mesenchymal cells from most groups of offspring at the 3 time points. CONCLUSIONS Transplacental nucleoside reverse-transcriptase inhibitor exposures induced fetal genotoxicity that was persistent for 3 years.

Epigenetic Research in Cancer Epidemiology: Trends, Opportunities, and Challenges

Epigenetics is emerging as an important field in cancer epidemiology that promises to provide insights into gene regulation and facilitate cancer control throughout the cancer care continuum. Increasingly, investigators are incorporating epigenetic analysis into the studies of etiology and outcomes. To understand current progress and trends in the inclusion of epigenetics in cancer epidemiology, we evaluated the published literature and the National Cancer Institute (NCI)–supported research grant awards in this field to identify trends in epigenetics research. We present a summary of the epidemiologic studies in NCIs grant portfolio (from January 2005 through December 2012) and in the scientific literature published during the same period, irrespective of support from the NCI. Blood cells and tumor tissue were the most commonly used biospecimens in these studies, although buccal cells, cervical cells, sputum, and stool samples were also used. DNA methylation profiling was the focus of the majority of studies, but several studies also measured microRNA profiles. We illustrate here the current status of epidemiologic studies that are evaluating epigenetic changes in large populations. The incorporation of epigenomic assessments in cancer epidemiology studies has and is likely to continue to provide important insights into the field of cancer research. Cancer Epidemiol Biomarkers Prev; 23(2); 223–33. ©2013 AACR.