Weight-of-the-evidence evaluation of 2,4-D potential for interactions with the estrogen, androgen and thyroid pathways and steroidogenesis.

A comprehensive weight-of-the-evidence evaluation of 2,4-dichlorophenoxyacetic acid (2,4-D) was conducted for potential interactions with the estrogen, androgen and thyroid pathways and with steroidogenesis. This assessment was based on an extensive database of high quality in vitro, in vivo ecotoxicological and in vivo mammalian toxicological studies. Epidemiological studies were also considered. Toxicokinetic data provided the basis for determining rational cutoffs above which exposures were considered irrelevant to humans based on exceeding thresholds for saturation of renal clearance (TSRC); extensive human exposure and biomonitoring data support that these boundaries far exceed human exposures and provide ample margins of exposure. 2,4-D showed no evidence of interacting with the estrogen or androgen pathways. 2,4-D interacts with the thyroid axis in rats through displacement of thyroxine from plasma binding sites only at high doses exceeding the TSRC in mammals. 2,4-D effects on steroidogenesis parameters are likely related to high-dose specific systemic toxicity at doses exceeding the TSRC and are not likely to be endocrine mediated. No studies, including high quality studies in the published literature, predict significant endocrine-related toxicity or functional decrements in any species at environmentally relevant concentrations, or, in mammals, at doses below the TSRC that are relevant for human hazard and risk assessment. Overall, there is no basis for concern regarding potential interactions of 2,4-D with endocrine pathways or axes (estrogen, androgen, steroidogenesis or thyroid), and thus 2,4-D is unlikely to pose a threat from endocrine disruption to wildlife or humans under conditions of real-world exposures.

Atrazine concentrations, gonadal gross morphology and histology in ranid frogs collected in Michigan agricultural areas.

The triazine herbicide atrazine has been suggested to be a potential disruptor of normal sexual development in male frogs. The goals of this study were to collect native ranid frogs from sites in agricultural and non-agricultural areas and determine whether hypothesised atrazine effects on the gonads could be observed at the gross morphological and histological levels. Juvenile and adult green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and leopard frogs (R. pipiens) were collected in the summers of 2002 and 2003. Atrazine concentrations were below the limit of quantification at non-agricultural sites, and concentrations did not exceed 2 microg/L at most agricultural sites. One concentration greater than 200 microg atrazine/L was measured once at one site in 2002. Hermaphroditic individuals with both male and female gonad tissue in either one or both gonads, were found at a low incidence at both non-agricultural and agricultural sites, and in both adults and juveniles. Testicular oocytes (TO) were found in male frogs at most of the sites, with the greatest incidence occurring in juvenile leopard frogs. TO incidence was not significantly different between agricultural and non-agricultural sites with the exception of juveniles collected in 2003. Atrazine concentrations were not significantly correlated with the incidence of hermaphroditism, but maximum atrazine concentrations were correlated with TO incidence in juvenile frogs in 2003. However, given the lack of a consistent relationship between atrazine concentrations and TO incidence, it is more likely the TOs observed in this study result from natural processes in development rather than atrazine exposure.

Plasma steroid hormone concentrations, aromatase activities and GSI in ranid frogs collected from agricultural and non-agricultural sites in Michigan (USA).

The triazine herbicide atrazine has been hypothesized to disrupt sexual development in frogs by up-regulating aromatase activity, resulting in greater estradiol (E2) concentrations and causing feminization in males. The goal of this study was to collect native ranid frogs from atrazine-exposed ponds and determine whether relationships exist between measured atrazine concentrations and the gonadosomatic index (GSI), plasma concentrations of testosterone (T), E2 or 11-ketotestosterone (KT), or with aromatase activity. In the summer of 2002 and 2003, adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana) and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south-central Michigan. Atrazine concentrations were below the limit of quantification at non-agricultural sites. Atrazine concentrations did not exceed 2 microg/L at most agricultural sites, but a concentration of 250 microg atrazine/L was measured in one sample from one site in 2002. Plasma steroid concentrations varied among locations. Aromatase activity was measurable in less than 11% of testes in adult males, and in less than 4% of testes in juvenile males. Median aromatase activities in ovaries of adult females ranged from 3 to 245 pmol/h/mg protein, and maximum activities were 2.5-fold greater in juveniles than in adults. Atrazine concentrations were not significantly correlated with any of the parameters measured in this study. These results indicate that atrazine does not up-regulate aromatase in green frogs in the wild, and does not appear to affect plasma steroid hormone concentrations.

Left ventricular shape analysis from three-dimensional echocardiograms.

The objective of this study was to develop and validate a three-dimensional technique of left ventricular shape analysis. Geometric phantoms and left ventricles of excised calf hearts, normal human subjects, and one subject each with aortic stenosis and dilated cardiomyopathy were reconstructed from three-dimensional echocardiograms. The fit between the reconstructions and true surfaces of the geometric phantoms and excised ventricles was determined. To evaluate in vivo left ventricular shape, a center axis was constructed from the centroid of the mitral annulus to the furthest endocardial point. Regional shape was evaluated as the relative distances of 16 separate myocardial segments from the center axis compared with a population-derived mean value. Global shape was evaluated as the average standard deviation from the normal value over the 16 segments. The system precisely reproduced the shapes of the phantoms and excised left ventricles (root-mean-square error between true and reconstructed surface 1.0 0.2 mm and 1.2 0.8 mm, respectively). The in vivo shape analysis differentiated the pathological from normal left ventricles.

Organochlorine insecticides, polychlorinated biphenyls, and metals in water, sediment, and green frogs from southwestern Michigan.

In an attempt to explain the etiology of frog deformities and population declines, many possible causative factors have been examined, including the input of synthetic chemicals into aquatic systems, where frogs spend much of their lives, including their entire developmental stages. Deformities in populations of green frogs in wetlands of southwestern Michigan that are influenced by agricultural, urban, or industrial inputs were assessed in this study. Of the 1445 green frogs (Rana clamitans) examined, only four (0.3%) exhibited morphological deformities. This deformity rate is less than the recognized background level of deformities for this species, which is approximately 1%. Concentrations of organochlorine insecticides, polychlorinated biphenyls (PCBs), and metals were determined in water, sediment, frog eggs, tadpoles, and adult green frog tissues. Concentrations of all individual organochlorine insecticides in tissue were less than 6 ng/g, wet wt. Concentrations of sigmaPCBs in tissue did not exceed 100 ng/g, wet wt. Concentrations of toxic metals were less than the limits of detection. Because no significant numbers of green frog deformities were observed in this region, it can be assumed that at these low concentrations, physical malformations in green frogs should not be observed. Significance of study. This study provides information on the incidence of deformities in green frog populations in southwestern Michigan and offers background data on chemical residues in green frogs and their environment.

2,3,7,8-Tetrachlorodibenzo-p-dioxin equivalents in tissue samples from three species in the Denver, Colorado, USA, metropolitan area.

The Rocky Mountain Arsenal (RMA), a Superfund site near Denver, Colorado, USA, has a history of various industrial processes that may have led to the release of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs). The PCDDs, PCDFs, and non-ortho- and mono-ortho-substituted PCBs cause a common set of toxic effects that are mediated through the aryl hydrocarbon receptor (AhR). The total AhR-mediated activity of complex mixtures in biota samples from the RMA and surrounding reference areas was determined by both instrumental and bioanalytical techniques. Mean concentrations of bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TCDD-EQ) in carp eggs, owl livers, and kestrel eggs ranged from 0 to 8.5, 17 to 130, and 2.4 to 18 pg/g, respectively. For most samples analyzed, concentrations of TCDD-EQ and instrumentally derived TCDD equivalents (TEQs) were not significantly different. In a few tissue samples, concentrations of TEQs and TCDD-EQs were not equivalent. This can indicate the presence of AhR-active compounds that were not identified or quantified by instrumental techniques or the existence of nonadditive interactions among congeners when samples are analyzed by the bioassay. Overall, mean concentrations of TCDD-EQs in extracts of carp and kestrel eggs were not significantly different between groups of samples collected on and off the RMA, whereas concentrations of TCDD-EQs in several owl livers collected on the RMA were significantly greater than concentrations in owl livers from off the RMA.

The diagnosis and treatment of pyoderma gangraenosum.

Pyoderma gangraenosum is a rare condition and its symptoms are often misdiagnosed. This article presents of an overview of this form of ulceration, and includes a case study showing how it was diagnosed and treated in a patient following an arthroscopy to the left ankle.

Quantification of total thyroxine in plasma from Xenopus laevis.

There is evidence that the endocrine systems of certain fish and wildlife can be affected by chemical contaminants, possibly resulting in developmental and reproductive problems. Perturbations in the hypothalamus-pituitary-thyroid (HPT) axis, in particular, can be detrimental during early development. Because the rate of amphibian metamorphosis is controlled by circulating thyroid hormones, tadpoles undergoing metamorphosis have been selected as relevant test organisms for evaluating the potential effects of a substance on the HPT axis in vertebrates. An indicative measure of HPT functioning in these assays is the concentration of the thyroid hormone, thyroxine (T4), in frog plasma. Therefore, there is a need for a validated method to measure T4 in plasma during amphibian metamorphosis. This study describes a method involving mixed-mode strong cation exchange solid-phase extraction (SPE) with ultrahigh-performance liquid chromatography and isotope dilution tandem mass spectrometry (UPLC-ID-MS-MS) to quantify total T4 in a small volume (10 µL) of plasma from Xenopus laevis (African clawed frog). The SPE procedure, together with MS detection, produced the required selectivity for the analysis of both T4 and the T4 internal standard. The limit of detection of the method was determined to be 0.2 ng/mL, whereas the lower limit of quantification was 0.5 ng/mL. The intra-day and inter-day precision values were less than ± 5 and ±10%, respectively. Concentrations of total T4 in the plasma of X. laevis tadpoles at metamorphic peak were calculated to be 10.7 ± 0.8 ng/mL, which is comparable to the results from radioimmunoassay. This validated UPLC-ID-MS-MS method for the determination of total T4 in plasma has demonstrated good accuracy and precision, with low susceptibility to interferences with the utilization of multiple reaction monitoring and ID.

Sediment TCDD-EQs and EROD and MROD activities in Ranid frogs from agricultural and nonagricultural sites in Michigan (USA).

In vitro studies have demonstrated atrazine-mediated induction of 7-ethoxyresorufin O-deethylase (EROD) activity. EROD is an enzyme active in the metabolism of many compounds, including many xenobiotics. These studies have suggested that atrazine may affect reproductive function by altering steroid metabolism. The goal of this study was to determine whether relationships could be detected between measured atrazine concentrations in surface waters and the liver-somatic index (LSI) and EROD and 7-methoxyresorufin O-deethylase (MROD) activities in the livers of ranid frogs. In addition, sediment dioxin toxic equivalents (TCDD-EQs) were determined using the H4IIE-luc cell bioassay. Adult and juvenile green frogs (Rana clamitans), bullfrogs (R. catesbeiana), and Northern leopard frogs (R. pipiens) were collected from areas with extensive corn cultivation and areas where there was little agricultural activity in south central Michigan in the summer of 2003. Atrazine concentrations at nonagricultural sites ranged from less than the limit of quantification (0.17 microg atrazine/L) to 0.23 microg atrazine/L and did not exceed 1.2 microg atrazine/L at agricultural sites. Sediment TCDD-EQs were measurable only at one agricultural site. Of the measured parameters, only LSI values in adult male frogs differed significantly between agricultural and nonagricultural sites, with greater values observed at agricultural sites. In green frogs, EROD and MROD activities were measurable in both adult and juvenile frogs and were similar among sites. Median EROD activities ranged from 13 to 21 pmol/min/mg protein in adult male green frogs and from 5 to 13 pmol/min/mg protein in adult female green frogs. Juvenile frogs had greater EROD and MROD activities than adult frogs. Bullfrogs and leopard frogs had greater activities than did green frogs. Atrazine concentrations were significantly and negatively correlated with MROD activity in adult male green frogs (Spearman R = -0.800). LSI and EROD and MROD activities of adult female or juvenile green frogs were not significantly correlated with atrazine concentrations. These results suggest that atrazine does not appear to have a consistent association with EROD or MROD activities in wild-caught green frogs.

Uric acid synthesis by avian exocrine pancreas.

1. Chicken pancreas has been shown to synthesize and secrete uric acid. Uric acid synthesis from xanthine in vitro by isolated pancreatic acinii is saturable and dependent on the activity of xanthine dehydrogenase. 2. Chicken pancreas is unable to synthesize uric acid de novo but the variety of substrates which support urate synthesis suggests that it occurs by the purine degradation pathway.

Measurement of right ventricular mass and volume by three-dimensional echocardiography by freehand scanning.

OBJECTIVE: Three-dimensional echocardiography (3D echo) by freehand scanning provides highly accurate measurements of left mass & volume using the piecewise smooth subdivision surface reconstruction method. The complexity of right ventricular (RV) geometry presents a challenge in accurate 3D assessments of its physical parameters. The authors measured the accuracy of their 3D echo technology in measuring RV mass and volume. METHODS: From 10 bovine hearts, the freewall mass and endocardial volume were measured. The hearts were imaged by 3D and the RV contours were manually traced. The endo- and epicardial surfaces were reconstructed. RV volume and mass from the reconstructions were compared to true measurements. RESULTS: 3D echo RV volume overestimated true volume (y=1.2x, r=0.998, SEE=3.2 ml, p<0.001), over a range of volumes (14-62 ml). The mean overestimation of RV volume by 3D echo was 6.2 ml, or 15.8% of the mean true volume. 3D echo mass agreed well with RV mass (y=1.0x, r=0.996, SEE=4.5 g, p=0.031) over a range of 47 to 79 g. Mean difference between 3D and true mass was 3.4 g, or 5.4% of the mean true mass. CONCLUSION: RV mass and volume can be measured accurately from 3D echocardiograms acquired using freehand scanning and reconstruction by the piecewise smooth subdivision method.

Oxidative stress in liver and brain of the hatchling chicken (Gallus domesticus) following in ovo injection with TCDD.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was injected into chicken eggs prior to incubation to study possible mechanisms of toxicity and teratogenicity. One of the suggested mechanisms of teratogenicity is oxidative stress. Eggs were injected simultaneously with TCDD and cotreatment compounds in an attempt to prevent oxidative stress or to block cytochrome P450 activity. Indicators of oxidative stress were assessed in livers and brains of hatchling chicks. In ovo, exposure to TCDD caused significant effects on indicators of oxidative stress in liver, but not in the brain of the hatchling chicks. TCDD did not significantly affect superoxide production. In liver, TCDD treatment caused a decrease in glutathione content and glutathione peroxidase activity and an increase in the ratio of oxidized to reduced glutathione. TCDD increased the susceptibility to lipid peroxidation and oxidative DNA damage in liver. Administration of the antioxidants vitamin E and vitamin A provided partial protection against TCDD-induced oxidative stress in liver. The lack of effect of TCDD in chicken brain could be due to the low cytochrome P4501A activity in this tissue and little accumulation of TCDD in brain compared to liver. Phenytoin, a known inducer of oxidative stress, caused a decrease in glutathione content and an increase in susceptibility to lipid peroxidation in both liver and brain and increased oxidative DNA damage in brain. Responsiveness varied among individual animals, but measures of the oxidative stress were correlated.

Mechanisms of TCDD-induced abnormalities and embryo lethality in white leghorn chickens.

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in birds has been well-established in laboratory and field studies. Observed effects of TCDD and related chemicals in birds include developmental deformities, reproductive failure, liver damage, wasting syndrome and death. The mechanism of action of TCDD at the cellular level is primarily mediated through the aryl hydrocarbon receptor (AhR). However, the mechanism of toxic action at the organism level is poorly understood. In this study, the role of radical oxygen species and mixed function oxidize (MFO; cytochrome P4501A) in the mechanism of TCDD-induced abnormalities and lethality were examined by co-injecting radical scavengers and an MFO inhibitor (piperonyl butoxide). Egg injection studies were conducted to determine if in ovo TCDD exposure can cause oxidative stress in white leghorn chicken eggs. Test agents were injected into the yolk prior to incubation. Treatments included TCDD (150 ng/kg), triolein (vehicle control), and various co-treatments including MnTBAP (a mimetic of superoxide dismutase), piperonyl butoxide, piroxicam, vitamin A acetate, and vitamin E succinate. Phenytoin, which is known to cause teratogenesis through oxidative stress was used as a positive control. Eggs were incubated until hatch and then the following parameters were assessed: mortality, hatching success, abnormalities, weights for whole body, liver, heart and brain, and biochemical endpoints for oxidative stress. As a measure of exposure, concentrations of TCDD and ethoxyresorufin-O-deethylase (EROD) activities were measured in tissues of hatchlings. While greater mortality and abnormalities were observed in the TCDD treatment groups, the number of the replicates were not great enough to detect statistically significant differences in abnormality rates for the co-treatments. Some of the observed developmental abnormalities included edema, liver necrosis and bill, eye and limb deformities with TCDD treatments, bill and brain deformities with phenytoin treatments, eye abnormalities with Vitamin E treatments, and abnormal feather pigmentation with piperonyl butoxide treatments.

Oxidative stress in laboratory-incubated double-crested cormorant eggs collected from the Great Lakes.

Double-crested cormorant (Phalacrocorax auritus) eggs were collected in 1998 from three sites on Lakes Huron and Superior and either analyzed for 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-like residues or artifically incubated. Some of the incubated eggs were injected with vitamin E (antioxidant) or piperonyl butoxide (CYPIA blocker) to examine the role of CYPIA and oxidative stress in normal bird development. Embryos (day 23) were analyzed for hepatic ethoxyresorufin O-deethylase (EROD) activity and different measures of oxidative stress. Glutathione-related parameters were also measured in brain. In contrast to the historical data, there were no statistically significant differences in concentrations of chlorinated dioxins, furans, dioxin-like PCBs, or total TCDD-equivalents (TEQs) in eggs among sites. Survival and incidence of abnormalities were comparable at all study sites. Slight differences in liver, heart, and egg weight were observed among sites. A greater incidence of eye deformities was observed in embryos treated with vitamin E. Treatment with the CYPIA blocker, piperonyl butoxide, decreased the body weights of embryos. EROD activities were similar at all locations, but measures of oxidative stress varied among locations. There were greater levels of oxidized glutathione and oxidative DNA damage at Little Charity Island in Saginaw Bay. There was relatively great interindividual variation in biochemical responses and significant interrelation of the parameters of oxidative stress. While exposure to PCDD/DF and PCBs does not seem to explain the observed oxidative stress, the potential of these compounds to cause the observed effects can not be completely excluded.