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In fish, exposure to estrogen or estrogen-mimicking chemicals (xenoestrogens) during a critical period of development can irreversibly invert sex differentiation. In medaka, a male-to-female reversal upon exposure to a xenoestrogen is accompanied by an increase in brain aromatase expression and activity. However, whether this increase is the direct cause of sex reversal is unknown. In this study we further examined the role brain aromatase plays in genesis of developmental abnormalities in response to endocrine-disrupting chemicals (EDCs). Further, the effects of a mixture of apparent antagonistic environmentally relevant EDCs on development were examined to determine if their combined actions could lessen each other's impacts. To this end, hatchling medaka were subjected in a 2-week flow-through immersion exposure to an estrogen mimic [dichlorodiphenyltrichloroethane (o,p -DDT)] and to pharmaceutical [fadrozole (FAD)] and environmental aromatase inhibitors [tributyltin (TBT)] alone and in combination. Brain aromatase expression and enzyme activity were measured on exposure days 5, 9, and 14 by real-time reverse-transcriptase polymerase chain reaction and tritiated water release assay, respectively. We recorded sex reversals at sexual maturity by examining the phenotypic and genotypic sex of d-rR-strain medaka. Results indicate that FAD and TBT inhibit aromatase activity in o,p -DDT-treated fish but do not prevent feminization, indicating that increased brain aromatase activity is not critical to EDC-induced male-to-female sex inversion. The observation that estradiol biosynthesis inhibitors do not block the effect of the xenoestrogen suggests that in the environment, exposure to seemingly antagonistic EDCs does not necessarily lessen the harmful impacts of these compounds. [ABSTRACT FROM AUTHOR]

In order to simulate an offshore oil spill event, we assessed the acute toxicity of the non-dispersed and the chemically dispersed water-accommodated fraction (WAF) of crude oil using Louisiana sweet crude and Corexit(®) 9500A with juvenile Harris mud crabs (Rhithropanopeus harrisii), an important Gulf of Mexico benthic crustacean. The chemical dispersion of crude oil significantly increased acute toxicity of the WAF in juvenile mud crabs compared to naturally dispersed oil. The majority of the mortality in the chemically dispersed treatments occurred within 24 h. While higher concentrations of chemically dispersed WAF had no survivors, at lower concentrations surviving juvenile crabs displayed no long-term effects. These results suggest that if the juvenile crabs survive initial exposure, acute exposure to dispersed or non-dispersed crude oil may not induce long-term effects.

Chemical dispersants are an important technology in the remediation of oil spills in the aquatic environment, facilitating degradation of crude oil and salinity is an important factor in dispersant effectiveness. The aim of the present study was to explore the role of salinity on the degradation chemistry of crude oil polycyclic aromatic hydrocarbons (PAHs) and acute toxicity of the water accommodated fraction (WAF) of the dispersant COREXIT 9500A and chemically dispersed crude oil on a common estuarine fish. Laboratory microcosms were designed at salinities of 4 parts per thousand (ppt), 12 ppt, or 18 ppt and spiked with crude oil, COREXIT 9500A, or a combined exposure to crude oil and COREXIT and allowed to biodegrade for 1 wk, 4 wk, and 16 wk. The WAF was harvested for analytical PAH analysis and acute toxicity testing in juvenile Fundulus grandis. Compared with undispersed oil, COREXIT exponentially increased the PAH concentrations in the WAF for up to 16 wk; hopane-normalized concentrations indicated that biodegradation was slowed for the first 4 wk. Dispersed crude oil and COREXIT were acutely toxic following 1 wk of biodegradation with no correlation between PAH concentrations and crude oil WAF mortality. Both dispersant and dispersant oil mixtures remained toxic for at least 4 wk at the lowest salinity tested, suggesting increased sensitivity or reduced biodegradation of toxic components in low-saline environments. At the lowest salinity, oil dispersed with COREXIT was more toxic than either the COREXIT alone or oil alone, even after 16 wk of biodegradation.

Prolonged in utero exposure of fetal male rats to dibutyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples, among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes and, therefore, reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semiquantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including steroidogenic acute regulatory, scavenger receptor B-1, cytochrome P450 side chain cleavage, and cytochrome P450 17A1, that are down-regulated after an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by EMSA, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein beta (c/ebp beta) and steroidogenic factor-1 (SF-1). Chromatin immunoprecipitation confirmed declines in DNA-protein interactions of c/ebp beta in DBP treated animals, whereas SF-1 was reduced in both diethyl phthalate (nontoxic) and DBP (toxic) treatments. These results suggest that inhibition of c/ebp beta, and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. In addition, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure.

Exposure to environmental chemicals often induces changes in gene expression leading to a variety of developmental and physiological problems. Understanding the underlying mechanism of these changes will aid in assessing human risk to these chemicals. Traditional methods for analyzing protein-DNA interactions include in vivo footprinting and chromatin immunoprecipitation (ChIP). However, ChIP does not provide binding location, and conventional footprinting is too subjective and time consuming for comparing protein binding in toxicological studies. Here, in vivo DNase I footprinting is adapted for use with the automated DNA sequencer to provide a semiquantitative map of changes in DNA-protein interactions in the promoter of steroidogenic acute regulatory (StAR) protein. StAR is the rate-limiting step in testosterone biosynthesis and is downregulated following in utero di-butyl phthalate (DBP) treatment in rats through an unknown mechanism. In vivo footprinting identified three regions of altered DNase digestibility following DBP treatment, and EMSA identified the corresponding transcription factors as SF-1, c/ebp beta, and GATA4. ChIP assays confirmed changes in protein-binding activity of SF-1 and c/ebp beta, but only c/ebp beta gesponds to only DBP. This suggests that c/ebp beta ginding is involved in DBP-induced transcriptional changes. By tailoring in vivo footprinting for toxicological studies, it can provide a detailed and accurate map of protein-DNA interactions and is an excellent first step in determining the changes in the structure of transcriptional machinery following an exogenous chemical treatment., ((c) 2007 Wiley Periodicals, Inc.)

In female fish estrogen is required for the development of primary and secondary sex characteristics and is derived from the aromatization of androgens by aromatase. There are two isoforms of aromatase in several teleost species, brain and ovarian. The objective of this study was two-fold: clone and sequence the coding and promoter region of brain aromatase in medaka, and determine the effects of exposure to an environmental estrogen (o,p-DDT) on sex determination and brain aromatase transcription and activity. The brain aromatase coding sequence was obtained by reverse transcription polymerase chain reaction (RT-PCR) and PCR-based genomic DNA walking was used to clone the promoter of the brain aromatase gene. The promoter sequence revealed potential binding sites for the estrogen receptor and for transcription factors involved in primary neurogenesis and sex determination. Medaka fry were exposed to increasing o,p-DDT concentrations (0-5.5 microg/L) from days 1 to 15 after hatch and brain aromatase expression and activity were measured on days 5, 9, and 14. A complete male-to-female sex reversal occurred at 5.5 microg/L o,p-DDT and aromatase activity and expression data showed a significant five-fold increase at this concentration at day 14. This information suggests that brain aromatase is involved in the abnormal sexual differentiation of fish treated with xenoestrogens.

The electrophysiologic effects of sotalol were studied in 11 patients with Wolff-Parkinson-White syndrome and 9 patients with AV nodal reentrant tachycardia. Electrophysiologic studies were performed before and after intravenous infusion of 80 mg sotalol over a period of 5 minutes. Sotalol prolonged the effective refractory period of the right atrium and the right ventricle. Both AV node and accessory pathway conduction were depressed by sotalol in antegrade and retrograde directions. Induction of reentrant tachycardia was prevented in 6 of 18 patients. The rate of reentrant tachycardia decreased from 182 +/- 29/min to 153 +/- 14/min (p less than 0.01) and the ventricular rate during atrial fibrillation from 148 +/- 14/min to 112 +/- 12/min (p less than 0.05). Sotalol exhibited a depressant effect on all parts of the reentrant circuit: atrium, ventricle, AV node, and accessory pathway. Thus, sotalol is effective in the therapy of patients with recurrent supraventricular tachycardias.