Catherine A. Richter

Conformational flexibility of bacterial RNA polymerase.

The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 Å. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the high-resolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA/RNA channel by nearly 25 Å, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel.

Atrazine reduces reproduction in fathead minnow (Pimephales promelas)

Atrazine, the widely used herbicide, has shown to affect the hypothalamus-pituitary-gonad axis in certain vertebrate species, but few studies have examined reproductive effects of this chemical on fish. Our study was designed to evaluate a population endpoint (egg production) in conjunction with histological (e.g., gonad development) and biochemical (e.g., hormone production) phenotypes associated with atrazine exposure in fathead minnows. Adult virgin breeding groups of 1 male and 2 females were exposed to nominal concentrations of 0, 0.5, 5.0, and 50 microg/L of atrazine in a flow-through diluter for 14 or 30 days. Total egg production was lower (19-39%) in all atrazine-exposed groups as compared to the controls. The decreases in cumulative egg production of atrazine treated fish were significant by 17-20 days of exposure. Reductions in egg production in atrazine treatment groups were most attributable to reduced numbers of spawning events with increased atrazine exposure concentrations. Gonad abnormalities were observed in both male and female fish of atrazine-exposed fish. Our results also indicate that atrazine reduces egg production through alteration of final maturation of oocytes. The reproductive effects observed in this study warrant further investigation and evaluation of the potential risks posed by atrazine, particularly feral populations of fish from streams in agricultural areas with high use of this herbicide.

Estradiol and Bisphenol A Stimulate Androgen Receptor and Estrogen Receptor Gene Expression in Fetal Mouse Prostate Mesenchyme Cells

Background Hormonal alterations during development have lifelong effects on the prostate gland. Endogenous estrogens, including 17β-estradiol (E2), and synthetic estrogenic endocrine disruptors, such as bisphenol A (BPA), have similar effects on prostate development. Increasing exposure to estrogens within the low-dose, physiologic range results in permanent increases in the size and androgen responsiveness of the prostate, whereas exposure within the high-dose, pharmacologic range has the opposite effects. Objectives We tested the hypothesis that the low-dose effects of estrogens on the developing prostate are associated with increased expression of androgen receptor (Ar) and estrogen receptor 1 (α) (Esr1) genes in mesenchyme cells. Methods Ar and Esr1 mRNA levels were quantified in primary cultures of fetal mouse prostate mesenchyme cells treated with E2 and BPA. Discussion Ar and Esr1 mRNA expression increased in response to E2, with thresholds of 0.001 and 0.037 nM, respectively; and in response to BPA, with a threshold of 1 nM for both mRNAs. We did not observe the expected inhibition of Ar mRNA expression by pharmacologic levels of E2 relative to unexposed cells. Conclusions The observed induction of gene expression occurred at concentrations within the range of free E2 previously shown to permanently increase prostate size, thus supporting the involvement of direct effects of estrogens on gene expression in prostate mesenchyme. The effects of BPA occurred within the range of concentrations currently measured in human serum, demonstrating the vulnerability of developing tissues to xenoestrogens.

Estrogenic Environmental Chemicals and Drugs: Mechanisms for Effects on the Developing Male Urogenital System

Development and differentiation of the prostate from the fetal urogenital sinus (UGS) is dependent on androgen action via androgen receptors (AR) in the UGS mesenchyme. Estrogens are not required for prostate differentiation but do act to modulate androgen action. In mice exposure to exogenous estrogen during development results in permanent effects on adult prostate size and function, which is mediated through mesenchymal estrogen receptor (ER) alpha. For many years estrogens were thought to inhibit prostate growth because estrogenic drugs studied were administered at very high concentrations that interfered with normal prostate development. There is now extensive evidence that exposure to estrogen at very low concentrations during the early stages of prostate differentiation can stimulate fetal/neonatal prostate growth and lead to prostate disease in adulthood. Bisphenol A (BPA) is an environmental endocrine disrupting chemical that binds to both ER receptor subtypes as well as to AR. Interest in BPA has increased because of its prevalence in the environment and its detection in over 90% of people in the USA. In tissue culture of fetal mouse UGS mesenchymal cells, BPA and estradiol stimulated changes in the expression of several genes. We discuss here the potential involvement of estrogen in regulating signaling pathways affecting cellular functions relevant to steroid hormone signaling and metabolism and to inter- and intra-cellular communications that promote cell growth. The findings presented here provide additional evidence that BPA and the estrogenic drug ethinylestradiol disrupt prostate development in male mice at administered doses relevant to human exposures.

The genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute exposure to the androgen, 17β-trenbolone

We investigated the genomic transcriptional response of female fathead minnows (Pimephales promelas) to an acute (4 days) exposure to 0.1 or 1.0microg/L of 17beta-trenbolone (TB), the active metabolite of an anabolic androgenic steroid used as a growth promoter in cattle and a contaminant of concern in aquatic systems. Our objectives were to investigate the gene expression profile induced by TB, define biomarkers of exposure to TB, and increase our understanding of the mechanisms of adverse effects of TB on fish reproduction. In female gonad tissue, microarray analysis using a 22K oligonucleotide microarray (EcoArray Inc., Gainesville, FL) showed 99 significantly upregulated genes and 741 significantly downregulated genes in response to 1microg TB/L. In particular, hydroxysteroid (17beta) dehydrogenase 12a (hsd17b12a), zona pellucida glycoprotein 2.2 (zp2.2), and protein inhibitor of activated STAT, 2 (pias2) were all downregulated in gonad. Q-PCR measurements in a larger sample set were consistent with the microarray results in the direction and magnitude of these changes in gene expression. However, several novel potential biomarkers were verified by Q-PCR in the same samples, but could not be validated in independent samples. In liver, Q-PCR measurements showed a significant decrease in vitellogenin 1 (vtg1) mRNA expression. In brain, cytochrome P450, family 19, subfamily A, polypeptide 1b (cyp19a1b, previously known as aromatase B) transcript levels were significantly reduced following TB exposure. Our study provides a candidate gene involved in mediating the action of TB, hsd17b12a, and two potential biomarkers sensitive to acute TB exposure, hepatic vtg1 and brain cyp19a1b.

GENE EXPRESSION CHANGES IN FEMALE ZEBRAFISH (DANIO RERIO) BRAIN IN RESPONSE TO ACUTE EXPOSURE TO METHYLMERCURY

Methylmercury (MeHg) is a potent neurotoxicant and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. The gene expression profile in adult female zebrafish whole brain induced by acute (96 h) MeHg exposure was investigated. Fish were exposed by injection to 0 or 0.5 µg MeHg/g. Gene expression changes in the brain were examined using a 22,000-feature zebrafish microarray. At a significance level of p < 0.01, 79 genes were up-regulated and 76 genes were down-regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxicants and will investigate responsive genes as potential biomarkers of MeHg exposure.

Atrazine reduces reproduction in Japanese medaka (Oryzias latipes)

Atrazine is an effective broadleaf herbicide and the second most heavily used herbicide in the United States. Effects along the hypothalamus-pituitary-gonad axis in a number of vertebrate taxa have been demonstrated. Seasonally elevated concentrations of atrazine in surface waters may adversely affect fishes, but only a few studies have examined reproductive effects of this chemical. The present study was designed to evaluate a population endpoint (egg production) in conjunction with histological (reproductive stage, gonad pathology) and biochemical (aromatase activity, sex hormone production) phenotypes associated with atrazine exposure in Japanese medaka. Adult virgin breeding groups of one male and four females were exposed to nominal concentrations of 0, 0.5, 5.0, and 50 μg/L (0, 2.3, 23.2, 231 nM) of atrazine in a flow-through diluter for 14 or 38 days. Total egg production was lower (36-42%) in all atrazine-exposed groups as compared to the controls. The decreases in cumulative egg production of atrazine-treated fish were significant by exposure day 24. Reductions in total egg production in atrazine treatment groups were most attributable to a reduced number of eggs ovulated by females in atrazine-treated tanks. Additionally, males exposed to atrazine had a greater number of abnormal germ cells. There was no effect of atrazine on gonadosomatic index, aromatase protein, or whole body 17 β-estradiol or testosterone. Our results suggest that atrazine reduces egg production through alteration of final maturation of oocytes. The reduced egg production observed in this study was very similar to our previously reported results for fathead minnow. This study provides further information with which to evaluate atrazines risk to fish populations.

Dreissenid mussels from the Great Lakes contain elevated thiaminase activity

ABSTRACT We examined thiaminase activity in dreissenid mussels collected at different depths and seasons, and from various locations in Lakes Michigan, Ontario, and Huron. Here we present evidence that two dreissenid mussel species (Dreissena bugensis and D. polymorpha) contain thiaminase activity that is 5–100 fold greater than observed in Great Lakes fishes. Thiaminase activity in zebra mussels ranged from 10,600 to 47,900 pmol g-1·min-1 and activities in quagga mussels ranged from 19,500 to 223,800 pmol g-1 ·min-1. Activity in the mussels was greatest in spring, less in summer, and least in fall. Additionally, we observed greater thiaminase activity in dreissenid mussels collected at shallow depths compared to mussels collected at deeper depths. Dreissenids constitute a significant and previously unknown pool of thiaminase in the Great Lakes food web compared to other known sources of this thiamine (vitamin B1)-degrading enzyme. Thiaminase in forage fish of the Great Lakes has been causally linked to thiamine deficiency in salmonines. We currently do not know whether linkages exist between thiaminase activities observed in dreissenids and the thiaminase activities in higher trophic levels of the Great Lakes food web. However, the extreme thiaminase activities observed in dreissenids from the Great Lakes may represent a serious unanticipated negative effect of these exotic species on Great Lakes ecosystems.

Methylmercury-induced changes in gene transcription associated with neuroendocrine disruption in largemouth bass (Micropterus salmoides)

Methyl-mercury (MeHg) is a potent neuroendocrine disruptor that impairs reproductive processes in fish. The objectives of this study were to (1) characterize transcriptomic changes induced by MeHg exposure in the female largemouth bass (LMB) hypothalamus under controlled laboratory conditions, (2) investigate the health and reproductive impacts of MeHg exposure on male and female largemouth bass (LMB) in the natural environment, and (3) identify MeHg-associated gene expression patterns in whole brain of female LMB from MeHg-contaminated habitats. The laboratory experiment was a single injection of 2.5 μg MeHg/g body weight for 96 h exposure. The field survey compared river systems in Florida, USA with comparably lower concentrations of MeHg (Wekiva, Santa Fe, and St. Johns Rivers) in fish and one river system with LMB that contained elevated concentrations of MeHg (St. Marys River). Microarray analysis was used to quantify transcriptomic responses to MeHg exposure. Although fish at the high-MeHg site did not show overt health or reproductive impairment, there were MeHg-responsive genes and pathways identified in the laboratory study that were also altered in fish from the high-MeHg site relative to fish at the low-MeHg sites. Gene network analysis suggested that MeHg regulated the expression targets of neuropeptide receptor and steroid signaling, as well as structural components of the cell. Disease-associated gene networks related to MeHg exposure, based upon expression data, included cerebellum ataxia, movement disorders, and hypercalcemia. Gene responses in the CNS are consistent with the documented neurotoxicological and neuroendocrine disrupting effects of MeHg in vertebrates.

Immunoassay monitoring of polychlorinated biphenyls (PCBs) in the Great Lakes

Although polychlorinated biphenyls (PCBs) are no longer manufactured, they are still entering the environment. In some compartments of the environment, PCB concentrations are a serious concern. This is especially true in compartments which accumulate PCBs, and in food items consumed by humans and wildlife. Also, there are situations in which management decisions require rapid, sensitive, accurate measurements, which can be made in real time under field conditions. Methods to use an enzyme-linked immunosorbent assay (ELISA) for PCBs were developed and applied to sediments and fish muscle homogenates collected from the Great Lakes. The extraction methods developed can be applied in the field with non-hazardous solvents, in the absence of sophisticated laboratory equipment. The method detection limit for PCBs in dimethyl sulfoxide (DMSO) extracts of sediment was 0.9 mg/kg. For PCBs in isopropanol extracts of fish tissue, the method detection limit was 0.6 mg/kg. The resolution of the ELISA was 0.83 mg/kg at 1.1 mg/kg and 1.6 mg/kg at 1.7 mg/kg, for sediment and fish tissue, respectively.