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Markus Hecker

Publications and source records attributed to Markus Hecker.

At least 19 recordsLinked to original sources

The H295R system for evaluation of endocrine-disrupting effects.

The present studies were undertaken to evaluate the utility of the H295R system as an in vitro assay to assess the potential of chemicals to modulate steroidogenesis. The effects of four model chemicals on the expression of ten steroidogenic genes and on the production of three steroid hormones were examined. Exposures with individual model chemicals as well as binary mixtures were conducted. Although the responses reflect the known mode of action of the various compounds, the results show that designating a chemical as "specific inducer or inhibitor" is unwise. Not all changes in the mixture exposures could be predicted based on results from individual chemical exposures. Hormone production was not always directly related to gene expression. The H295R system integrates the effects of direct-acting hormone agonists and antagonists as well as chemicals affecting signal transduction pathways for steroid production and provides data on both gene expression and hormone secretion which makes this cell line a valuable tool to examine effects of chemicals on steroidogenesis.

Aminoglutethimide↗

Human adrenocarcinoma (H295R) cells for rapid in vitro determination of effects on steroidogenesis: hormone production.

To identify and prioritize chemicals that may alter steroidogenesis, an in vitro screening assay based on measuring alterations in hormone production was developed using the H295R human adrenocortical carcinoma cell line. Previous studies indicated that this cell line was useful to screen for effects on gene expression of steroidogenic enzymes. This study extended that work to measure the integrated response on production of testosterone (T), estradiol (E2), and progesterone/pregnenolone (P) using an ELISA. Under optimized culture and experimental conditions, the basal release of P, T and E2 into the medium was 7.0+/-1.2 ng/ml, 1.6+/-0.4 ng/ml, and 0.51+/-0.13 ng/ml, respectively. Model chemicals with different modes of action on steroidogenic systems were tested. Exposure to forskolin resulted in dose-dependent increases in all three hormones with the greatest relative increase being observed for E2. This differed from cells exposed to prochloraz or ketoconazole where P concentrations increased while T and E2 concentrations decreased in a dose-dependent manner. In cells exposed to fadrozole, E2 decreased in a dose-dependent manner while T and P only decreased at the greatest dose tested. Aminoglutethimide decreased P and E2 concentrations but increased T concentrations. Vinclozolin reduced both P and T but resulted in a slight increase in E2. The alteration in the patterns of hormone production in the H295R assay was consistent with the modes of action of the chemicals and was also consistent with observed effects of these chemicals in animal models. Based on these results, the H295R in vitro system has potential for high throughput screening to not only characterize the effects of chemicals on endocrine systems but also to prioritize chemicals for additional testing.

Adrenal Cortex↗

Suppression of aromatase activity in populations of bream (Abramis brama) from the river Elbe, Germany.

Aromatase activity was determined in brain and gonads of wild bream collected along the river Elbe, Germany, and correlated with other endocrine and reproductive endpoints such as plasma sex steroid concentrations, secondary sex characteristics (STI), plasma vitellogenin, gonad size (GSI), and maturation stages of germ cells (MS) that were reported for the same fish in a previous study. Furthermore, regional patterns of aromatase activity were correlated to a number of environmental factors such as exposure to environmental contaminants and parasitism. While aromatase activity was not detectable in the gonads of male and female fish with the assay used, fish of both genders revealed relatively great brain enzyme activities. As for most of the endocrine and reproductive parameters, with the exception of plasma testosterone (T), aromatase activities were significantly less in fish from a river stretch characterized by elevated exposures to organic contaminants and metals. Brain aromatase activity was positively and significantly correlated with plasma estradiol (E2) and MS in females, and showed a similar trend with plasma 11-ketotestosterone (11KT) and STI in males. No comparable trend occurred for T. This decrease of the reproductively relevant hormones 11KT and E2 may be indicative of a disruption of the last step in sex hormone synthesis, a hypothesis that was supported for E2 by the strong (R2=0.78, p<0.05) linear regression between aromatase activity and E2 in female bream. It is also hypothesized that the effects on brain aromatase activity were likely to be related to the disruption of other reproductive parameters including sexual maturity and expression of secondary sex characteristics. Although a number of factors such as exposure to pollutants and prevalence of the tapeworm Ligula intestinalis correlated with the suppression of aromatase activity, the exact causes for the regional decrease in brain aromatase activity remain unclear due to inconsistencies of these correlations between sampling events or gender.

Animals↗

STAT-1 and AP-1 decoy oligonucleotide therapy delays acute rejection and prolongs cardiac allograft survival.

OBJECTIVE: Acute myocardial rejection is a cell-mediated process characterized by increased leukocyte recruitment into the graft myocardial tissue. Transcription factors like STAT-1 and AP-1 are critically involved in this process by regulating vascular adhesion molecule expression. The aim of our study was to investigate the effect of decoy oligodeoxynucleotide (dODN) treatment targeting transcription factors AP-1 and STAT-1 on acute cardiac allograft rejection in a rat transplant model. METHODS: Wistar-Furth (WF) cardiac allografts were transplanted into Lewis (LEW) rats after perfusion with STAT-1 or AP-1 dODN solution (5 micromol/l), buffer or the corresponding mutant control ODNs. Grafts were harvested and processed for histologic and immunohistochemical evaluation. RESULTS: As demonstrated by fluorescence dye-labelled dODN, exposure of the grafts to the dODNs during 45 min of warm ischemia resulted in a dominant uptake of naked DNA by the graft endothelium. Treatment with AP-1 and STAT-1 dODNs, but not with vehicle or the control dODNs, significantly prolonged cardiac allograft survival by approximately 40% from 5.6+/-0.5 days to 7.8+/-1.3 days and 7.4+/-0.5 days, respectively (mean+/-S.D., p<0.01, n=5 in each group). Immunohistochemical examination on days 1, 3 and 6 revealed a marked reduction of infiltrating leukocytes (AP-1 dODN: 85%, STAT-1 dODN: 50%), namely T-cells, in the dODN-perfused grafts at day 3 post transplantation. In addition, as demonstrated by immunohistochemical analysis, endothelial expression of ICAM-1 and VCAM-1 was found to be markedly reduced in dODN-treated grafts. CONCLUSION: Both AP-1 and STAT-1 dODN treatments suppress graft endothelial adhesion molecule expression, reduce graft infiltration and in turn significantly delay acute rejection. The utilization of dODNs in the cardioplegic solution might be a novel strategy to protect transplanted organs from early damage during transplantation, to preserve organ function and bridge the critical phase after transplantation when standard immunosuppression is not yet completely effective.

Acute Disease↗

Arginine challenge unravels persistent disturbances of urea cycle and gluconeogenesis in abstinent alcoholics.

AIMS: Data on recovery from hormonal and metabolic sequelae of alcoholism in strictly controlled alcohol abstinence are mainly restricted to short-term abstention. Our previous findings of persistently decreased plasma and urinary urea concentrations in long-term abstinent alcoholics prompted us to further elucidate this unexplained phenomenon. METHODS: The response of circulating urea cycle metabolites and glucose-regulating hormones to an intravenous load (30 g) of arginine hydrochloride was investigated in abstinent male alcoholics (n = 14) after complete recovery of all routine liver parameters and compared with that in healthy male controls (n = 15). RESULTS: The arginine challenge provoked (i) higher peak concentrations of arginine and increased arginine/ornithine and ornithine/citrulline ratios in the plasma of abstinent alcoholics; (ii) augmented plasma glutamine concentrations in alcoholics in the presence of comparable levels in both experimental groups of plasma glutamate, ammonia, and nitrate/nitrite; (iii) parallel increases in plasma urea concentrations over the respective baseline levels but distinctly higher urinary urea excretion in controls; (iv) a blunted blood glucose response to arginine in alcoholics together with a reduced insulin and glucagon surge; and (v) an elevated growth hormone peak as compared with controls. CONCLUSIONS: Application of an intravenous arginine challenge reveals profound and lasting metabolic and hormonal disturbances in abstinent alcoholics, affecting urea cycle and gluconeogenesis. The common denominator of many of these changes may be an acquired irreversible deficiency in cellular energy regulation.

Adult↗

Alteration of steroidogenesis in H295R cells by organic sediment contaminants and relationships to other endocrine disrupting effects.

A novel bioassay with the human adrenocortical carcinoma cell line H295R can be used to screen for endocrine disrupting chemicals that affect the expression of genes important in steroidogenesis. This assay was employed to study the effects of organic contaminants associated with the freshwater pond sediments collected in the Ostrava-Karvina region, Czech Republic. The modulation of ten major genes involved in the synthesis of steroid hormones (CYP11A, CYP11B2, CYP17, CYP19, 17betaHSD1, 17betaHSD4, CYP21, 3betaHSD2, HMGR, StAR) after exposure of H295R cells to sediment extracts was investigated using quantitative real-time polymerase chain reaction (PCR). Crude sediment extracts, containing high concentrations of polycyclic aromatic hydrocarbons (PAHs) and moderate amounts of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) significantly stimulated expression of the CYP11B2 gene (up to 10-fold induction), and suppressed expression of 3betaHSD2 and CYP21 genes. A similar pattern was observed with the extracts after treatment with concentrated sulfuric acid to remove labile chemicals (including PAHs) leaving only persistent PCBs, OCPs and potentially PCDD/Fs. Comparison of the results with other mechanistically based bioassays (arylhydrocarbon receptor, AhR, mediated responses in H4IIE-luc cells, and estrogen receptor mediated effects in MVLN cells) revealed significant endocrine disrupting potencies of organic contaminants present in the sediments (most likely antiestrogenicity). Pronounced effects were observed particularly in sediment extracts from the Pilnok Pond which harbors an unusual intersexual population of the narrow-cawed crayfish Pontastacus leptodactylus (Decapoda, Crustacea). This pilot study provided the first experimental evidence of the wider application of the H295R bioassay for screening complex environmental samples, and the results support the hypothesis of chemical-induced endocrine disruption in intersexual crayfish.

Cell Line, Tumor↗

Development and optimization of a Q-RT PCR method to quantify CYP19 mRNA expression in testis of male adult Xenopus laevis: comparisons with aromatase enzyme activity.

Due to limitations of the currently used enzymatic assays, it is difficult to determine aromatase activity in testicular tissue of amphibians. Quantitative reverse transcription polymerase chain reaction (Q-RT PCR) is a sensitive and reliable technique to detect low amounts of mRNA for specific genes. This study was designed to develop and optimize a SYBR Green I-based Q-RT PCR method to quantify CYP19 mRNA in testicular tissue from male Xenopus laevis. Four quantification methods for measuring CYP19 mRNA expression were compared. The established test system proved to be highly sensitive (detectable mRNA copies < 10), reproducible (interassay CV < 5.4%, intraassay CV < 0.9%), precise and specific for the CYP19 gene. To confirm the validity of the applied test system, an ex vivo testicular and ovarian explant study with a known inducer of aromatase, forskolin, was conducted. Forskolin induced CYP19 gene expression in both ovarian (3.7-fold) and testicular (2.6-fold) explants. Of the four quantification methods, the absolute standard curve and the comparative CT method appear to be optimal as indicated by their highly significant correlation (r2 = 0.998, p < 0.001). In conclusion, we recommend the comparative CT method over the standard curve method because it is more economical in terms of both cost and labor. Although both aromatase activity and CYP19 mRNA were clearly detectable in testes of X. laevis, both aromatase enzyme activity and CYP19 gene expression were very low. Also, no significant relationships were found between aromatase enzyme activity and gene expression. This is likely due the fact that the aromatase enzyme may have been dormant at the developmental stage the frogs were in during the experiment.

Animals↗

The -786C/T single-nucleotide polymorphism in the promoter of the gene for endothelial nitric oxide synthase: insensitivity to physiologic stimuli as a risk factor for rheumatoid arthritis.

OBJECTIVE: Shear stress is the main physiologic stimulus for the expression of NOS3, the gene for human endothelial nitric oxide synthase. Interestingly, a promoter variant of the NOS3 gene, the -786C variant, is insensitive to shear stress, and individuals homozygous for this single-nucleotide polymorphism (SNP) have an increased risk of developing coronary artery disease. The cytokine interleukin-10 (IL-10) is also capable of up-regulating endothelial NOS3 expression through binding of the transcription factor STAT-3 to a nearby promoter sequence. The aim of this study was to explore the possibility that the -786C variant of the NOS3 gene is also insensitive to IL-10 and that individuals with the -786C/C genotype are more prone to developing rheumatoid arthritis (RA). METHODS: Endothelial cells were isolated from human umbilical cord veins, clonally expanded, and analyzed for NOS3 and IL-12 expression by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Umbilical cord arteries and blood samples from RA patients were genotyped for the -786C/T SNP of the NOS3gene. RESULTS: In contrast to cells of other genotypes, endothelial cells of the -786C/C genotype did not reveal an increase in NOS3 expression upon exposure to IL-10, and the cytokine failed to suppress IL-12 expression upon stimulation of CD40. Preincubation of these cells with a 16-mer C-type decoy oligonucleotide fully reconstituted the defective IL-10-induced suppression of IL-12 synthesis. The frequency of the -786C/C genotype was significantly higher in the 596 RA patients than in the general population (19.1% versus 12.1%; P < 0.0001). CONCLUSION: Individuals with the -786C/C genotype have an increased risk of developing RA. This may be explained by the IL-10 insensitivity of the C-type NOS3 gene promoter and the resulting failure to subdue CD40-mediated proinflammatory gene expression.

Adult↗

Attenuation of murine antigen-induced arthritis by treatment with a decoy oligodeoxynucleotide inhibiting signal transducer and activator of transcription-1 (STAT-1).

The transcription factor STAT-1 (signal transducer and activator of transcription-1) plays a pivotal role in the expression of inflammatory gene products involved in the pathogenesis of arthritis such as various cytokines and the CD40/CD40 ligand (CD40/CD40L) receptor-ligand dyad. The therapeutic efficacy of a synthetic decoy oligodeoxynucleotide (ODN) binding and neutralizing STAT-1 was tested in murine antigen-induced arthritis (AIA) as a model for human rheumatoid arthritis (RA). The STAT-1 decoy ODN was injected intra-articularly in methylated bovine serum albumin (mBSA)-immunized mice 4 h before arthritis induction. Arthritis was evaluated by joint swelling measurement and histological evaluation and compared to treatment with mutant control ODN. Serum levels of pro-inflammatory cytokines, mBSA-specific antibodies and auto-antibodies against matrix constituents were assessed by enzyme-linked immunosorbent assay (ELISA). The transcription factor neutralizing efficacy of the STAT-1 decoy ODN was verified in vitro in cultured synoviocytes and macrophages. Single administration of STAT-1 decoy ODN dose-dependently suppressed joint swelling and histological signs of acute and chronic arthritis. Delayed-type hypersensitivity (DTH) reaction, serum levels of interleukin-6 (IL-6) and anti-proteoglycan IgG titres were significantly reduced in STAT-1 decoy ODN-treated mice, whereas mBSA, collagen type I and type II specific immunoglobulins were not significantly affected. Intra-articular administration of an anti-CD40L (anti-CD154) antibody was similarly effective. Electrophoretic mobility shift analysis (EMSA) of nuclear extracts from synoviocytes incubated with the STAT-1 decoy ODN in vitro revealed an inhibitory effect on STAT-1. Furthermore, the STAT-1 decoy ODN inhibited the expression of CD40 mRNA in stimulated macrophages. The beneficial effects of the STAT-1 decoy ODN in experimental arthritis presumably mediated in part by affecting CD40 signalling in macrophages may provide the basis for a novel treatment of human RA.

Animals↗

Induction of cytochrome P4501A in African brown house snake (Lamprophis fuliginosus) primary hepatocytes.

Although there have been numerous studies involving fish, birds, and mammals, little is known about the response of the cytochrome P4501A system of snakes to halogenated aromatic hydrocarbons (HAHs). The present study describes the induction of ethoxyresorufin-O-deethylase (EROD) in primary hepatocytes of the African brown house snake (Lamprophis fuliginosus). Hepatocytes were exposed in multiwell plates to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and four different non-ortho-substituted coplanar polychlorinated biphenyls (PCBs 77, 81, 126, and 169). Exposure to TCDD and PCB 126 resulted in a dose-dependent increase in EROD activity, with maximum inducible EROD activities of 177 +/- 56 (mean +/- SEM) and 101.1 +/- 55 pmol/min/mg protein for TCDD and PCB 126, respectively. None of the other PCBs caused a measurable induction of EROD, which suggests reduced inducibility of snake hepatocytes compared to some vertebrate taxa. Median effective concentrations (EC50s) were 0.16 +/- 0.03 nM for TCDD and 8.25 +/- 4.14 nM for PCB 126. The relative potency (REP20-80) range for PCB 126 was 0.044 to 0.046. Compared to results from in vitro systems using other vertebrate species, both the maximum inducibility and the REPs estimated for L. fuliginosus were within the same range as those reported for mammals and the more sensitive bird species but were greater than the values reported for most fish species. In conclusion, induction of EROD activity in primary hepatocytes appears to be a useful approach for evaluating the dioxin-like potencies of aryl hydrocarbon-receptor agonists in snakes. The test system offers a method for rapid screening of reptilian responsiveness to these compounds using smaller numbers of organisms than with in vivo studies, an important consideration for many declining reptile species.

Animals↗

Terminology of gonadal anomalies in fish and amphibians resulting from chemical exposures.

Given the recent increase in the number of studies describing the ability of chemicals to exert endocrine-disrupting effects, not only in fish but in a variety of other oviparous groups such as amphibians and reptiles, there is an urgent need to harmonize the terminology currently used in describing pathological changes of the gonads. In addition to difficulties in comparing results from different studies, there is also the risk of miscommunication by using terms that imply a certain clinical relevance which may not be true for the species examined. Especially in the case of the recent and controversial issue about potential effects of the triazine herbicide atrazine on amphibians, clinical terminology has been utilized beyond its true meaning by using terms such as "chemical castration" to describe occurrence of TOs or ovarian tissue in the testis of male frogs exposed to environmental chemicals (Hayes 2004). In clinical terminology, castration is defined as the removal of the gonads or their destruction by an external influence, resulting in a nonfertile organism. However, Hayes (2004) did not investigate any possible effects on the fertility of the test animals and thus did not know if these animals were truly castrated. Similarly, terms such as intersex, hermaphrodite, and sex reversal have been used in ways that appear inappropriate with regard to their clinical meaning in a series of different studies with fish or frogs (see previous sections for a detailed discussion). To ensure the appropriate use of certain terminology in a field as controversial and complex as the study of endocrine disruption, we have attempted, in this chapter, to harmonize the terminology used to describe changes in gonadal development of vertebrates such as fish and amphibians, especially frogs (see Table 3). Where appropriate, the terminology suggested was adopted directly from the clinical terminology. However, as outlined here there are substantial differences between the developmental biology of oviparous vertebrates and mammals, and especially humans, that necessitate modification of the definitions of some of the clinical terms. Where appropriate, therefore, the terminology proposed in this manuscript was redefined based on the biological meanings of the terms used in clinical diagnosis. Considering the large increase in research in the area of reproductive endocrine disruption over the past decades, the authors see an increasing need for a harmonization of terms to be used to describe effects observed in the investigated species. Agreement on a common terminology will allow scientists to better communicate and compare their work, and will enable risk assessors to conduct large-scale evaluations of environmental endocrine disruption by fitting the information from individual studies into a synthesis of normal and abnormal conditions of gonadal tissues.

Abnormalities, Drug-Induced↗

Effects of atrazine on CYP19 gene expression and aromatase activity in testes and on plasma sex steroid concentrations of male African clawed frogs (Xenopus laevis).

Some investigators have suggested that the triazine herbicide atrazine can cause demasculinization of male amphibians via upregulation of the enzyme aromatase. Male adult African clawed frogs (Xenopus laevis) were exposed to three nominal concentrations of atrazine (1, 25, or 250 microg atrazine/l) for 36 days, and testicular aromatase activity and CYP19 gene expression, as well as concentrations of the plasma sex steroids testosterone (T) and 17beta-estradiol (E2), and gonad size (GSI) were measured. There were no effects on any of the parameters measured, with the exception of plasma T concentrations. Plasma T concentrations in X. laevis exposed to the greatest concentration of atrazine were significantly less (p = 0.034) than those in untreated frogs. Both CYP19 gene expression and aromatase activities were low regardless of treatment, and neither parameter correlated with the other. We conclude that aromatase enzyme activity and gene expression were at basal levels in X. laevis from all treatments, and that the tested concentrations of atrazine did not interfere with steroidogenesis through an aromatase-mediated mechanism of action.

Animals↗

Plasma concentrations of estradiol and testosterone, gonadal aromatase activity and ultrastructure of the testis in Xenopus laevis exposed to estradiol or atrazine.

The ultrastructure of testicular cells of adult male African clawed frogs (Xenopus laevis) exposed to either estradiol (0.1 microg/L) or 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine (atrazine; 10 or 100 microg/L) was examined by electron microscopy and compared to plasma concentrations of the steroid hormones, testosterone (T) and estradiol (E2), testicular aromatase activity and gonad growth expressed as the gonado-somatic index (GSI). Exposure to E2 caused significant changes both at the sub-cellular and biochemical levels. Exposure to E2 resulted in significantly fewer sperm cells, inhibition of meiotic division of germ cells, more lipid droplets that are storage compartments for the sex steroid hormone precursor cholesterol, and lesser plasma T concentrations. Although not statistically significant, frogs exposed to E2 had slightly smaller GSI values. These results may be indicative of an inhibition of gonad growth and disrupted germ cell development by E2. Concentrations of E2 in plasma were greater in frogs exposed to E2 in water. Exposure to neither concentration of atrazine caused effects on germ cell development, testicular aromatase activity or plasma hormone concentrations. These results suggest that atrazine does not affect testicular function. In contrast, exposure of male X. laevis to E2 led to sub-cellular events that are indicative of disruption of testicular development, and demasculinization processes (decrease of androgen hormone titers). These results indicate that atrazine does not cause responses that are similar to those caused by exposure to E2.

Analysis of Variance↗

Parasitism in fish--an endocrine modulator of ecological relevance?

Wild bream (Abramis brama) were collected from the river Elbe, and the influences of parasitic infection by the tapeworm Ligula intestinalis on endocrine and related functions (vitellogenin [VTG]; plasma sex steroids: 17beta-estradiol [E2], 11-ketotestosterone [11-KT] and testosterone [T]; relative gonad [GSI] and liver [HSI] growth; maturation stages of germ cells [MS]; prominence of spawning tubercles [STI]) were investigated. Distinct regional differences in infection rates of bream with L. intestinalis were observed along the Elbe with the highest prevalences at the Czech border (up to 45%) and Magdeburg (up to 65%), areas that are heavily contaminated with complex mixtures of organic chemicals and metals. Parasitized fish of both sexes had significantly lower GSIs and poorly developed gonads (low MS). In males, a significant reduction in the prominence of spawning tubercles occurred. Infected females had significantly lower plasma VTG concentrations. A selective suppression of the sex steroids 11-KT and E2 was observed in male and female bream, respectively. Testosterone was not affected in the same manner in fish of both sexes. At sites with an elevated prevalence of L. intestinalis, the extent of the infection of an individual was significantly correlated with the suppression of the measured biomarkers. However, when applying a linear model to compare regional differences in infection prevalence with biological parameters, not all of the observed differences could be explained by parasitization by L. intestinalis. This indicates that other factors such as pollution may have contributed to the effects on reproductive and endocrine processes that occurred along the river. Given that sites with high prevalences of L. intestinalis were also characterized by elevated pollution, it is possible that there exists a combinatory effect of both pollution and parasitization that can have a serious impact on the reproductive capacity of a population, such as was observed at the sampling site at Magdeburg.

Animals↗

Quantitative RT-PCR methods for evaluating toxicant-induced effects on steroidogenesis using the H295R cell line.

Gene expression profiles show considerable promise for the evaluation of the toxic potential of environmental contaminants. For example, any alterations in the pathways of steroid synthesis or breakdown have the potential to Cause endocrine disruption. Therefore monitoring these pathways can provide information relative to a chemical's ability to impact endocrine function. One approach to monitoring these pathways has been to use a human adrenocortical carcinoma cell line (H295R) that expresses all the key enzymes necessary for steroidogenesis. In this study we have further developed these methods using accurate and specific quantification methods utilizing molecular beacon-based quantitative RT-PCR (Q-RT-PCR). The assay system was used to analyze the expression patterns of 11 steroidogenic genes in H295R cells. The expression of gene transcripts was measured using a real-time PCR system and quantified based on both a standard curve method using a dilution series of RNA standards and a comparative Ct method. To validate the optimized method, cells were exposed to specific and nonspecific model compounds (inducers and inhibitors of various steroidogenic enzymes) for gene expression profiling. Similar gene expression profiles were exhibited by cells treated with chemicals acting through common mechanisms of action. Overall, our findings demonstrated that the present assay can facilitate the development of compound-specific response profiles, and will provide a sensitive and integrative screen for the effects of chemicals on steroidogenesis.

Cell Line, Tumor↗

Effects of atrazine on metamorphosis, growth, laryngeal and gonadal development, aromatase activity, and sex steroid concentrations in Xenopus laevis.

African clawed frogs (Xenopus laevis) were exposed to one of eight nominal waterborne concentrations including 0, 0.1, 1.0, 10, or 25 microg/L atrazine, 0.005% ethanol (EtOH), or 0.1mg/L estradiol (E2) or dihydrotestosterone (DHT) containing 0.005% EtOH. Frogs were exposed from 72 h posthatch until 2--3 months postmetamorphosis via a 3-day static renewal exposure regimen. Atrazine at concentrations between 0.1 and 25 microg/L did not significantly affect mortality, growth, gonad development, laryngeal muscle size, or aromatase activity in juvenile X. laevis. Male frogs exposed to 1.0 microg/L atrazine had lower E2 levels compared to controls, but this response was not consistent among other concentrations of atrazine. Male and female frogs exposed to DHT had larger laryngeal dilator muscle areas compared to controls. E2-exposed female frogs had decreased gonadal aromatase activity, and E2-exposed male frogs had statistically greater plasma concentrations of E2 compared to controls.

Animals↗

Shear stress insensitivity of endothelial nitric oxide synthase expression as a genetic risk factor for coronary heart disease.

Coronary heart disease (CHD) is based on the development of atherosclerosis in coronary arteries. Shear stress-induced endothelial nitric oxide (NO) release not only contributes to local blood pressure control but also effectively helps to retard atherosclerosis. Therefore, functionally relevant polymorphisms in the endothelial NO synthase (NOS-3) gene may contribute to the development of CHD. NOS-3 expression was analyzed in endothelial cells isolated from umbilical cords genotyped for the -786C/T single nucleotide polymorphism (SNP) of the human nos-3 gene. Moreover, NO-dependent relaxation was examined in segments of saphenous vein isolated from genotyped patients undergoing aortocoronary bypass surgery, and patients subjected to quantitative coronary angiography were genotyped to verify an association between this SNP and CHD. Shear stress-induced NOS-3 mRNA and protein expression was present in TT and CT genotype cells but absent in cells with CC genotype. Pretreatment of these cells with a decoy oligonucleotide comprising position -800 to -779 of the C-type nos-3 promoter reconstituted shear stress-induced NOS-3 expression. These results were confirmed by reporter gene analysis with the corresponding nos-3 promoter luciferase constructs. In addition, the NO-mediated relaxant response of vein grafts from CC genotype patients was significantly attenuated as compared with the CT or TT genotype, and in CHD-positive patients, the CC genotype was significantly more frequent (19.0%) than in CHD-negative patients (4.4%). The -786C/T SNP of the nos-3 gene thus constitutes a genetic risk factor for CHD, presumably due to binding of an inhibitory transcription factor to the C-type promoter blocking shear stress-dependent maintenance of NOS-3 expression.

Adult↗

Effects of atrazine on metamorphosis, growth, and gonadal development in the green frog (Rana clamitans).

Embryos of the green frog (Rana clamitans) were collected from the field and exposed to 1 of 6 water-borne treatments for 273 d (mid July 2001 to mid April 2002). The treatments were 0, 10, or 25 microg/L atrazine, 0.005% ethanol (EtOH), or 0.1 mg/L estradiol or dihydrotestosterone carried in 0.005% EtOH. Treatments were applied in a static renewal system with a 50% test solution replacement approximately every 3 d. Following the exposure period, tadpoles were reared in freshwater until metamorphosis or until study termination (at d 506). Time to initiate and complete metamorphosis, stage-specific mortality, length and weight at metamorphosis, and gross morphology and histology of the gonads were examined. At environmentally relevant concentrations, atrazine did not consistently affect growth or metamorphosis. Compared to controls, the length of the larval period was greater in tadpoles exposed to 10 microg/L atrazine. However, the length of the larval period was not markedly different between tadpoles in the control and 25 microg/L atrazine treatments. Neither gross gonadal morphology nor histopathology of the gonads in postmetamorphic frogs was significantly altered in response to atrazine exposure. This study provides evidence that environmentally relevant concentrations of atrazine do not adversely affect the growth or reproductive development of R. clamitans.

Animals↗