Morphological and histological abnormalities of the neotropical toad, Rhinella arenarum (Anura: Bufonidae) larvae exposed to dexamethasone.

Dexamethasone (DEX) is a glucocorticoid highly effective as an anti-inflammatory, immunosuppressant and decongestant drug. In the present study, a preliminary acute toxicity test was assayed in order to determinate DEX median-lethal, lowest-observed-effect and the no-observed-effect concentrations (LC50, LOEC and NOEC, respectively) on the common toad embryos (Rhinella arenarum). Also, morphological and histological abnormalities from five body larval regions, liver melanomacrophages (MM) and glutathione S-transferase (GST) activity were evaluated in the toad larvae to characterize the chronic sublethal effects of DEX (1-1,000 µg L-L). Results of the acute test showed that the LC50 of DEX at 96 h of exposure for the toad embryos (GS 18-20) was 10.720 mg L-g, and the LOEC was 1 µg L-g. In the chronic assay, the larval development and body length were significantly affected. DEX exposition also induced teratogenic effects. Most frequent external abnormalities observed in DEX-treated larvae included abdominal edema and swollen body, abnormal gut coiling and visceral congestion. Intestinal dysplasia was recurrent in cross-section of all DEX-treated larvae. Neural, conjunctive and renal epithelial cells were also affected. Significant increase in liver MM number and size, and GST activity levels were also registered in DEX treatments with respect to controls. The evaluation of a variety of biomarkers provided clear evidence of toad larvae sensitivity to DEX, and the ecotoxicological risk of these pharmaceuticals, commonly found in different water bodies worldwide on aquatic animals.

Developmental Toxicity of a Neonicotinoid Insecticide, Acetamiprid to Zebrafish Embryos.

Agricultural use of neonicotinoid insecticides is increasing worldwide, posing a risk to nontarget organisms. The present study investigated developmental toxicity of a widely used neonicotinoid, acetamiprid, to zebrafish embryos. Sublethal (malformations, hatchability, heart rate, body length, alteration of spontaneous movement and touch responses) and lethal effects were monitored during exposure period from 6 h post fertilization (hpf) to 120 hpf. Zebrafish embryos exhibited significant mortality and teratogenic effects at acetamiprid concentration greater than 263 mg/L, with bent spine being the main malformation. Toxicity spectra were constructed to rank the sensitivity of individual end points to acetamiprid exposure and impaired spontaneous movement was the most sensitive end point of those tested. The present study provides the basis for understanding developmental toxicity of acetamiprid exposure to zebrafish embryos. This information is critical for future studies evaluating aquatic risk from neonicotinoids as little is known regarding adverse effects of neonicotinoids to aquatic vertebrate species.

The effects of Roundup on gametes and early development of common carp (Cyprinus carpio L).

To determine the effects of Roundup, a commercial formulation of glyphosate, gametes, and embryos of common carp (Cyprinus carpio L) was exposed to wide range of herbicide concentrations (0.0, 0.1, 0.5, 2.0, 5.0, 10.0, 20.0, and 50.0 mg/l). The obtained results showed different effects of Roundup on common carp gametes. Herbicide reduced swelling of eggs (but the effect was not concentration-related), while sperm showed low sensitivity to Roundup (time of spermatozoa motility was reduced in a significant way only at 20 mg/l, and at remaining concentrations, only a slight tendency was observed). During the embryonic development, Roundup caused a decrease of common carp embryonic survival (and the effect was concentration-related); however, it had no effect on development rate. During the embryogenesis, three types of embryo body malformation were observed: yolk sac edema, spine curvature, and shortening of body, but their frequencies were not associated with the presence or concentration of herbicide. However, Roundup affected quality of newly hatched larvae of common carp by increasing their mortality. No effect of herbicide on percentage of deformed larvae was observed but larvae hatched in water with Roundup tended to show more complex anomalies compared to those from the control. Obtained data showed that even low concentrations of this herbicide in waters can significantly reduce egg swelling, survival of embryos, and quality of fish larvae.

Multiple midline defects identified in a litter of golden retrievers following gestational administration of prednisone and doxycycline: a case series.

BACKGROUND: The teratogenic effects of immunomodulatory and certain antimicrobial therapies are described in small rodents and humans. While the described teratogenic effects in small rodents have been extrapolated to make conclusions about its use in the pregnant dam, teratogenic effects of prednisone and doxycycline have not yet been reported in the dog. Here we report and describe midline defects observed in a litter of golden retriever puppies exposed to mid-gestational immunosuppressive and antimicrobial therapy. CASE PRESENTATION: Twenty-one days into gestation, the dam of a litter of eight golden retriever puppies was administered prednisone, doxycycline, and tramadol as treatment for immune-mediated polyarthritis. The individuals in the litter were subsequently diagnosed with a variety of midline defects and congenital cardiac defects. This case series describes the variety of identified defects and presents a descriptive account of complex congenital abnormalities that are likely secondary to teratogenic effects of one or more drugs administered during gestation. The available puppies, dam, and grand dam underwent thorough physical examination, complete echocardiogram, and where indicated, advanced imaging with various surgical corrections when possible. Numerous midline congenital defects and congenital heart disease were identified in the puppies evaluated. Ultimately 5 of 8 puppies born to the dam were presented for thorough evaluation. The midline defects include: gastroschisis (1), peritoneopericardial diaphragmatic hernias (4, PPDH), umbilical hernia (4), unilateral cryptorchidism (1 of 4 males), cleft palate (1), renal agenesis (1), renal abnormalities (1), sternal and vertebral abnormalities (3), remnant liver lobe (1) and malformations consistent with ductal plate malformations with congenital hepatic fibrosis (1). The congenital cardiac defects include: ventricular septal defect (4, VSD) and subaortic stenosis (4, SAS). The presence of greater than one congenital defect was noted in all 5 of the dogs evaluated. Surgical correction was necessary for PPDH in 4 puppies. Medical intervention was recommended for congenital cardiac disease in 1 puppy. CONCLUSION: This case report is the first to describe midline defects in dogs that have been exposed to immunomodulatory therapy during gestation. A causative relationship between mid-gestational immunomodulatory exposure and midline defects cannot be proven, however, this case supports a clear association and provides case-based evidence to support its avoidance when possible.

Categorization of fetal external findings in developmental toxicology studies by the Terminology Committee of the Japanese Teratology Society.

Categorization of fetal external findings in common laboratory animals, intended to make the agreement at Berlin Workshop in 2014 more practical, was proposed by the Terminology Committee of the Japanese Teratology Society at the Workshop in the 55th Japanese Teratology Society Annual Meeting in 2015. In the Workshop, 73 external findings, which had been categorized as "Gray zone" anomalies but not as "Malformation" or "Variation" in the 2014 Berlin Workshop, were discussed and classified as Malformation, "Non-structural abnormality," Variation, and "Not applicable." The proposal was based on the results of a survey conducted in 2014, where 20 facilities (including pharmaceutical, chemical, and pesticide companies and contract laboratories) and 2 selected expert teratologists in Japan were asked for their opinions on the categorization of these findings. Based on the discussion, Japanese Teratology Society members have agreed that 42 out of the 73 findings can be classified as Malformations (38), Non-structural abnormalities (3), Malformations/Non-structural abnormalities (1), and Variations (0), while the remaining 31 findings were recommended to be categorized as Not applicable for fetuses. The details of the classification are shown on the website of the Japanese Teratology Society (http://www.umin.ac.jp/cadb/External.pdf).

Larval Red Drum (Sciaenops ocellatus) Sublethal Exposure to Weathered Deepwater Horizon Crude Oil: Developmental and Transcriptomic Consequences.

The Deepwater Horizon (DWH) incident resulted in extensive oiling of the pelagic zone and shoreline habitats of many commercially important fish species. Exposure to the water-accommodated fraction (WAF) of oil from the spill causes developmental toxicity through cardiac defects in pelagic fish species. However, few studies have evaluated the effects of the oil on near-shore estuarine fish species such as red drum (Sciaenops ocellatus). Following exposure to a certified weathered slick oil (4.74 µg/L ∑PAH50) from the DWH event, significant sublethal impacts were observed ranging from impaired nervous system development [average 17 and 22% reductions in brain and eye area at 48 h postfertilization (hpf), respectively] to abnormal cardiac morphology (100% incidence at 24, 48, and 72 hpf) in red drum larvae. Consistent with the phenotypic responses, significantly differentially expressed transcripts, enriched gene ontology, and altered functions and canonical pathways predicted adverse outcomes in nervous and cardiovascular systems, with more pronounced changes at later larval stages. Our study demonstrated that the WAF of weathered slick oil of DWH caused morphological abnormalities predicted by a suite of advanced bioinformatic tools in early developing red drum and also provided the basis for a better understanding of molecular mechanisms of crude oil toxicity in fish.

Pollutants Induced Oxidative Stress, DNA Damage and Cellular Deformities in Clarias gariepinus (Burchell), from River Yamuna in Delhi Region, India.

The purpose of the present study was to evaluate the severity of pollution impact at two extreme sites of river Yamuna in Delhi region utilizing oxidative stress, genotoxic and histopathological biomarkers in gonad (ovary) of Clarias gariepinus. To evaluate oxidative stress, TBAR's and ferric reducing antioxidant power (FRAP) assay were employed while comet assay and classic histology were used to estimate genotoxicity and cellular damage respectively. The results indicated significant increase (p < 0.001) in TBARs level (µmol/g wet tissue); significant decrease (p < 0.001) in FRAP value (U/mg tissue), significant increase (p < 0.001) in DNA damage and extensive abnormal histoarchitecture in ovarian samples procured from Okhla as compared to Wazirabad barrage. Thus, exposure to the increasing toxicity downstream the river is altering the activity of cellular total antioxidant capacity persuading oxidative stress and cellular damage, eventually distressing the heath of fish fauna directly and humans indirectly.

Congenital malformations and other reproductive losses in goats due to poisoning by Poincianella pyramidalis (Tul.) L.P. Queiroz (=Caesalpinia pyramidalis Tul.).

In the semiarid region of Brazil, in areas with vegetation composed mainly of Poincianella pyramidalis, several cases of congenital malformation and reproductive losses were observed in goats and sheep from 2012 to 2014. To determine the teratogenic effect of P. pyramidalis, two groups of eight goats each were used. Goats from Group 1 received fresh P. pyramidalis, harvested daily, as the only roughage during the whole breeding and pregnancy period. Goats in Group 2 (control) received Cynodon dactylon (tifton) hay free choice. Ultrasound examination for pregnancy diagnosis was performed every 28 days. Four goats from Group 1 were pregnant on day 28 but not on day 56, suggesting embryonic death or abortion. Another goat from Group 1 died at day 70 of pregnancy, and the fetuses exhibited micrognathia. The other three goats bore six kids, three of which showed bone malformations in the limbs, spine, ribs, sternum, and head, including arthrogryposis, scoliosis and micrognathia. One kid also showed hypoplasia of the left pulmonary lobes. In the control group, all goats bore a total of 13 kids and none of them exhibited malformations. These results demonstrated that P. pyramidalis causes congenital malformations and other reproductive losses in goats.

Cerebellar abnormalities typical of methylmercury poisoning in a fledged saltmarsh sparrow, Ammodramus caudacutus.

A fledged, 12-15 day-old saltmarsh sparrow, Ammodramus caudacutus, was collected from an accidental kill on Cinder Island, Long Island, NY, USA. The sparrow was assessed for feather mercury levels and the brain analyzed for cerebellar abnormalities by microscopic examination. In humans, fetal Minamata disease is caused by maternal ingestion of mercury. It is characterized by disrupted and disordered cerebellar neuronal migration in the fetus or infant. Results from this sparrow show cerebellar abnormalities typical of Minamata disease. It is the first known avian or mammalian specimen taken from the wild to show the abnormalities typical of the human fetal syndrome.

Possible chemical causes of skeletal deformities in natural populations of Aphanius fasciatus collected from the Tunisian coast.

This study attempt to quantify and identify skeletal deformities in natural populations of Aphanius fasciatus collected from the Tunisian coast and tends to found a possible relationship between these anomalies and several types of pollutants presents in the environment. Fish were collected from one reference area (S1: coast of Luza) and three polluted areas (S2: industrialized coast of Sfax, S3: coast of Khniss and S4: Hamdoun'Oued). Various patterns of skeletal deformities were diagnosed using double staining technics, and the levels of heavy metals (Cd, Cu and Zn), various polycyclic aromatic hydrocarbons (PAHs) and estrogenic compounds were determined in water and sediment from different sites. Spatio-temporal variation of the spinal deformities frequencies in A. fasciatus show that high incidence of spinal anomalies has been recorded in population collected from S2 in comparison to three other ones. Morphological results indicated that skeletal deformities of adult A. fasciatus were grouped into 13 categories that described abnormalities on spines, vertebrae, arcs and mandibles. A total of 1025 abnormalities were quantified. The results of chemical analysis showed that the levels of heavy metals and PAHs were significantly higher in S2 than in S1, S3 and S4. High level of estrogenic activity was observed only in S4. A possible correlation between environmental exposures to a mixture of pollutants in coastal waters in S2 and spinal deformities in A. fasciatus was suggested.

Structural and molecular analysis of pollution-linked deformities in a natural Aphanius fasciatus (Valenciennes, 1821) population from the Tunisian coast.

The Eastern coast of Tunisia hosts a mosaic of populations of the Mediterranean killifish A. fasciatus, an excellent monitoring species for the analysis of environmental impact in coastal Mediterranean areas. Among these populations, fish from the Sfax area, in the Gulf of Gabès, are known for presenting a high prevalence of different physiological alterations, including spinal deformation. Compared to a reference clean area, Luza, Sfax water and sediments are polluted with heavy metals (20 to 40 times more Cd, for example) and other compounds like PAHs (10 times higher levels). Histological analyses of deformed fish from this area showed an abnormal abundance of collagen fibers infiltrated in the spinal area, either as a physiological mechanism to alleviate the consequences of the deformation of the bones or as a secondary effect of the same pathological/toxicity mechanism inducing bone deformation. Deformed fish from Sfax showed elevated (4-5 fold) levels of transcripts from the collagen COL1A2 gene in bone tissue, compared to either non-deformed animals from the same location or normal animals from the reference Luza site. These results are consistent with an overexpression of collagen in the bone tissue, in agreement with the histological results. We propose that the deformation observed in the Sfax Aphanius population is a degenerative process occurring in adult animals and probably terminal, as it results in severe limitations on the fish capacity to swim, to catch food, and ultimately to survive.

Absorption levels and morphological features of fetal organs in wistar rats treated with lead acetate / Niveles de absorción y características morfológicas de los órganos fetales en ratas wistar tratadas con acetato de plomo

Lead is one of the heavy metals most used in industry. Poisoning due to long-term lead exposure is known as saturnism, and is an occupational illness that has been known for many years. Lead is highly toxic and can compromise the structural and functional patterns of organs and systems. The aim of this study was to examine the lungs and kidneys of fetuses from female Wistar rats exposed to lead acetate. In this study, the lungs and kidneys of 20 fetuses from female rats that had previously been treated with lead acetate were dissected, fixed, embedded in paraffin and stained with hematoxylin and eosin. Macroscopic changes to the shape, color and consistency of organs from fetuses treated with this heavy metal were observed, in comparison with organs from control fetuses. Microscopic lesions characterized by vascular sclerosis, cell atrophy or hyperplasia, progressive interstitial fibrosis, inclusion bodies containing lead acetate and glomerular sclerosis were found in the kidneys. The lesions found in the lungs consisted of destructuring of the parenchyma, impregnation with lead acetate, formation of fibrosis, extravasation of vascular fluids, reduction of the alveolar spaces and formation of alveolar edema. These changes were correlated with the level of lead acetate absorption, as determined using atomic spectrophotometry.

El plomo es un metal pesado utilizado en la industria. El envenenamiento debido a la exposición prolongada por plomo es una enfermedad profesional conocida por muchos años. La toxicidad del plomo es muy expresiva y puede poner en peligro el modelo estructural y funcional de los órganos y sistemas. El objetivo de este estudio fue examinar los pulmones y riñones de fetos de ratas Wistar expuestos al acetato de plomo. En este estudio, 20 fetos de ratas Wistar previamente tratados con acetato de plomo durante la gestación, tuvieron sus órganos disecados, fijados, incluidos en parafina y teñidos con hematoxilina y eosina. Macroscópicamente, los órganos fetales tratados por este metal fueron comparados con los órganos de fetos controles en relación a forma, color y consistencia. Microscópicamente, se encontraron lesiones en el riñón que se caracterizaron por esclerosis vascular, atrofia o hiperplasia de células, fibrosis intersticial progresiva, presencia de cuerpos de inclusión que contenían acetato de plomo y esclerosis glomerular. En el pulmón se observó desorganización del parénquima impregnado con acetato de plomo, formación de fibrosis, líquido intersticial, reducción de los espacios alveolares y edema alveolar. Estos cambios se correlacionaron con el nivel de absorción de acetato de plomo, determinado por espectrometría atómica.

A joint modeling approach to data with informative cluster size: robustness to the cluster size model.

In many biomedical and epidemiological studies, data are often clustered due to longitudinal follow up or repeated sampling. While in some clustered data the cluster size is pre-determined, in others it may be correlated with the outcome of subunits, resulting in informative cluster size. When the cluster size is informative, standard statistical procedures that ignore cluster size may produce biased estimates. One attractive framework for modeling data with informative cluster size is the joint modeling approach in which a common set of random effects are shared by both the outcome and cluster size models. In addition to making distributional assumptions on the shared random effects, the joint modeling approach needs to specify the cluster size model. Questions arise as to whether the joint modeling approach is robust to misspecification of the cluster size model. In this paper, we studied both asymptotic and finite-sample characteristics of the maximum likelihood estimators in joint models when the cluster size model is misspecified. We found that using an incorrect distribution for the cluster size may induce small to moderate biases, while using a misspecified functional form for the shared random parameter in the cluster size model results in nearly unbiased estimation of outcome model parameters. We also found that there is little efficiency loss under this model misspecification. A developmental toxicity study was used to motivate the research and to demonstrate the findings.

Congenital neuroglial heterotopia in a neonatal harbor seal (Phoca vitulina richardsi ) with evidence of recent exposure to polycyclic aromatic hydrocarbons.

A male neonatal Pacific harbor seal (Phoca vitulina richardsi) stranded off the coast of California, USA, was presented for rehabilitation with numerous partially haired, soft tissue masses around the mouth and in the oropharynx. Because of the extent of the lesions, the seal was humanely euthanized. Histologically, the masses consisted of subepithelial connective tissue and subcutis expanded by a proliferation of streams and bundles of spindle to stellate cells. Morphology of these cells suggested a neural origin, which was confirmed by positive immunohistochemistry for two neural markers, S-100 protein and glial fibrillary acidic protein, so the masses were diagnosed as neuroglial heterotopia. Heterotopic neuroglial tissue is a rare lesion comprised of benign mature neural tissue in an ectopic location with no connection to the central nervous system. Results of polycyclic aromatic hydrocarbon (PAH) metabolite analysis of bile indicated recent exposure to a petroleum source. Although fetal exposure to PAHs in utero can cause neurotoxicity and affect normal embryonic development, it is unknown whether gestational exposure occurred in this case.

Phenological changes in the concentration of alkaloids of Carex brevicollis in an Alpine rangeland.

Carex brevicollis (Cyperaceae) is a plant of mesic grasslands in calcareous mountains of southern Europe. It contains two different ß-carboline alkaloids, brevicolline and brevicarine, the first of which is thought to produce abortions in mammals. In the rangeland of Aliva, within the Picos de Europa massif in northern Spain, the abundance of Carex brevicollis has been linked with the occurrence of teratogenesis in early gestating cows grazing in early summer. The concentration of alkaloids was measured in the summers of 2007 and 2008, at intervals of 2 weeks, at different altitudes within the rangeland (1,350, 1,600, and 1,850 m) and from different parts of the sedge (leaves, reproductive stems, and inflorescences). Estimated growing degree days were related to the flowering phenology of Carex brevicollis and were used to analyse its relation with the concentration of alkaloids. Brevicarine concentration was higher in inflorescences and brevicolline in leaves. Although it also depended on the zone and year, the concentrations of both alkaloids were related one to another in leaves and inflorescences but not in stems. Both alkaloids decreased with growing degree days in the inflorescences and showed no response in leaves. Our findings suggest that brevicarine, not brevicolline, could be the teratogen in pregnant cattle in this region. This hypothesis is supported by the observed frequent consumption of inflorescences and scarce consumption of leaves of Carex brevicollis by grazing livestock, and also by the coincidence of the toxicity in early pregnant cows with the flowering time of the sedge.

Toxicity of short-term copper exposure to early life stages of red sea bream, Pagrus major.

Acute (0, 0.1, 0.2, 0.4, 0.8, 1.6 mg Cu/L) and chronic (0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12 mg Cu/L) toxicity tests of Cu with embryonic and larval red sea bream, Pagrus major, were carried out to investigate their biological responses to Cu exposure in static water at 18 +/- 1 degrees C (dissolved organic carbon, 1.8 +/- 0.65 mg C/L; hardness, 6,183 +/- 360 mg CaCO3/L; salinity, 33 +/- 1 per thousand). The 24- and 48-h LC50 (median lethal concentration) values of Cu for embryos were 0.23 and 0.15 mg/L, whereas the 48-, 72-, and 96-h LC50 values for larvae were 0.52, 0.19, and 0.13 mg/L, respectively, suggesting that embryos were more sensitive to Cu toxicity than larvae. Copper exposures at > or =0.06 mg concentrations caused low hatching success, a delay in the time to hatching of embryos, and reductions in the growth and yolk absorption of the larvae, whereas high mortality and morphological malformations occurred in the embryos and larvae at > or =0.08 mg/L concentrations. Copper concentration did not significantly affect the heart rate of the embryos, but it significantly decreased the heart rate of the newly hatched larvae when the Cu concentration was > or =0.08 mg/L, suggesting that Cu at high concentrations could induce heartbeat disturbances in red sea bream more easily at the larval stage than at the embryonic stage. Hatching success, time to hatching, growth rate, morphological abnormality, yolk absorption, and heart rate were Cu concentration-dependent and could be effective endpoints for evaluating Cu toxicity to the early life stages of red sea bream in nature.

Study of the toxic effects of an insecticide and copper sulphate on chicken embryos.

The toxic effects of the BI 58 EC insecticide (38% dimethoate) applied alone or in combination with copper sulphate were studied on chicken embryo in the early phase of development. The test materials were injected in 0.1-0.1 ml volume into the air chamber of eggs on the first day of incubation. Subsequently, on days 2 and 3 of incubation permanent preparations were made from the embryo in order to study the early developmental stage. Embryos fixed on slides and stained with osmium tetroxide solution were studied under light microscope. According to the result of the statistical evaluation, to sum up, we can say that the simultaneous administration of the test materials did not result in a significant increase in the embryo mortality, but after the combined administration the rate of embryonic mortality markedly increased. As a result of combined administrations the developmental anomalies included the apperance of a blood ring, poor development or absence of somites, the retarted development of the vascular system, the head and the body, irregular differentiation of the brain vesicles. Summarising the findings, it can be established that the insecticide treatment combined with heavy metal resulted in enhanced embryotoxicity in the case of both combinations, which was primarily manifested in an increased embryonic mortality rate.

The alkaloid profiles of Lupinus sulphureus.

Lupines are common plants on the rangelands in the western United States. Lupines contain alkaloids that can be toxic and teratogenic causing congenital birth defects (crooked calf disease). One such lupine, Lupinus sulphureus, occurs in parts of Oregon, Washington, and British Columbia. Specimens of L. sulphureus from field collections and herbaria were evaluated taxonomically and by chemical means. A total of seven distinct alkaloid profiles and the individual alkaloids associated with each profile were identified. Each alkaloid profile was unique in its geographical distribution and its potential risk to livestock. In conclusion, taxonomic classification is not sufficient to determine risk, as chemical characterization of the alkaloids must also be performed.