Steffen Schneider

MicroRNAs as potential biomarkers in diseases and toxicology

MiRNAs (microRNAs) are single-stranded non-coding RNAs of approximately 21-23 nucleotides in length whose main function is to inhibit gene expression by interfering with mRNA processes. MicroRNAs suppress gene expression by affecting mRNA (messenger RNAs) stability, targeting the mRNA for degradation, or both. In this review, we have examined how microRNA expression could be altered following exposure to chemicals and how they could represent appropriate tissue and more interestingly circulating biomarkers. Among the key questions before using the microRNA for evaluation of risk toxicity, it remains still to clarify how they could be causally involved in the adverse effects and how stable their changes are.

Vinclozolin--no transgenerational inheritance of anti-androgenic effects after maternal exposure during organogenesis via the intraperitoneal route.

The goal of this study was to examine the potential transgenerational inheritance of anti-androgenic effects induced by Vinclozolin administered intraperitoneally to pregnant Wistar rats (Crl:WI[Han]). Dams were dosed with Vinclozolin at 0, 4 or 100mg/kg bw/d on gestation days 6-15. Male offspring of F1-F3 generations were bred with untreated females to yield F2-F4 offspring. No evident anti-androgenic effects were observed at 4mg/kg bw/d, but a case of hypospadias as well as delayed sexual maturation in F1 male offspring was observed as a sign of anti-androgenicity at 100mg/kg bw/d. However, F1-F3 males developed normally to sexual maturity and were able to mate and to generate healthy progeny. Sperm count, morphology and motility were not affected in F1-F4 generation male offspring. In conclusion, transgenerational inheritance of Vinclozolins anti-androgenic effects was not evident in outbred Wistar rats.

Extended evaluation on the ES‑D3 cell differentiation assay combined with the BeWo transport model, to predict relative developmental toxicity of triazole compounds

The mouse embryonic stem D3 (ES-D3) cell differentiation assay is based on the morphometric measurement of cardiomyocyte differentiation and is a promising tool to detect developmental toxicity of compounds. The BeWo transport model, consisting of BeWo b30 cells grown on transwell inserts and mimicking the placental barrier, is useful to determine relative placental transport velocities of compounds. We have previously demonstrated the usefulness of the ES-D3 cell differentiation assay in combination with the in vitro BeWo transport model to predict the relative in vivo developmental toxicity potencies of a set of reference azole compounds. To further evaluate this combined in vitro toxicokinetic and toxicodynamic approach, we combined ES-D3 cell differentiation data of six novel triazoles with relative transport rates obtained from the BeWo model and compared the obtained ranking to the developmental toxicity ranking as derived from in vivo data. The data show that the combined in vitro approach provided a correct prediction for in vivo developmental toxicity, whereas the ES-D3 cell differentiation assay as stand-alone did not. In conclusion, we have validated the combined in vitro approach for developmental toxicity, which we have previously developed with a set of reference azoles, for a set of six novel triazoles. We suggest that this combined model, which takes both toxicodynamic and toxicokinetic aspects into account, should be further validated for other chemical classes of developmental toxicants.

Epoxiconazole‐Induced Degeneration in Rat Placenta and the Effects of Estradiol Supplementation

Epoxiconazole (CAS-No. 133855-98-8) was recently shown to cause both a marked depletion of maternal estradiol blood levels and a significantly increased incidence of late fetal mortality when administered to pregnant rats throughout gestation (GD 7-18 or 21); estradiol supplementation prevented this epoxiconazole effect in rats (Stinchcombe et al., 2013), indicating that epoxiconazole-mediated estradiol depletion is a critical key event for induction of late fetal resorptions in rats. For further elucidation of the mode of action, the placentas from these modified prenatal developmental toxicity experiments with 23 and 50 mg/kg bw/d epoxiconazole were subjected to a detailed histopathological examination. This revealed dose-dependent placental degeneration characterized by cystic dilation of maternal sinuses in the labyrinth, leading to rupture of the interhemal membrane. Concomitant degeneration occurred in the trophospongium. Both placentas supporting live fetuses and late fetal resorptions were affected; the highest degree of severity was observed in placentas with late resorptions. Placental degeneration correlated with a severe decline in maternal serum estradiol concentration. Supplementation with 0.5 and 1.0 μg of the synthetic estrogen estradiol cyclopentylpropionate per day reduced the severity of the degeneration in placentas with live fetuses. The present study demonstrates that both the placental degeneration and the increased incidence of late fetal resorptions are due to decreased levels of estrogen, since estrogen supplementation ameliorates the former and abolishes the latter.

Anti-androgenicity can only be evaluated using a weight of evidence approach.

Preputial separation (PPS) is a commonly used external marker for the onset of male puberty in experimental animal studies. While treatment-related delays in PPS may be indicative of specific anti-androgenic activity, impaired general growth also alters the onset of puberty. To differentiate between specific and non-specific effects on the age at PPS--and thereby evaluate the validity of the endpoint PPS-two-generation toxicity studies of 23 substances were evaluated. The 23 substances were assessed regarding anti-androgenicity using all available data and external assessments in a weight-of-evidence evaluation (WoE). Correlation of individual pup body weight with age at PPS revealed that delays in pubertal development coincided with reduced pup body weight. After comparison with the WoE assessment, we concluded that inclusion of body weight analysis into the PPS evaluation of each study was able to correctly identify three compounds which specifically induced delayed PPS and 16 which only showed unspecific changes. A further two compounds which might be categorized as anti-androgens based on delayed PPS, were correctly regrouped using our refined methodology. Based on this analysis and in comparison to the WoE evaluation, it was found, that caution should be exercised when using the endpoint PPS in hazard assessment.

Guidance on the selection of cohorts for the extended one-generation reproduction toxicity study (OECD test guideline 443).

The extended one-generation reproduction toxicity study (EOGRTS; OECD test guideline 433) is a new and technically complex design to evaluate the putative effects of chemicals on fertility and development, including effects upon the developing nervous and immune systems. In addition to offering a more comprehensive assessment of developmental toxicity, the EOGRTS offers important improvements in animal welfare through reduction and refinement in a modular study design. The challenge to the practitioner is to know how the modular aspects of the study should be triggered on the basis of prior knowledge of a particular chemical, or on earlier findings in the EOGRTS itself, requirements of specific regulatory frameworks notwithstanding. The purpose of this document is to offer guidance on science-based triggers for these extended evaluations.

Species differences in developmental toxicity of epoxiconazole and its relevance to humans.

Epoxiconazole, a triazole-based fungicide, was tested in toxicokinetic, prenatal and pre-postnatal toxicity studies in guinea pigs, following oral (gavage) administration at several dose levels (high dose: 90 mg/kg body weight per day). Maternal toxicity was evidenced by slightly increased abortion rates and by histopathological changes in adrenal glands, suggesting maternal stress. No compound-related increase in the incidence of malformations or variations was observed in the prenatal study. In the pre-postnatal study, epoxiconazole did not adversely affect gestation length, parturition, or postnatal growth and development. Administration of epoxiconazole did not alter circulating estradiol levels. Histopathological examination of the placentas did not reveal compound-related effects. The results in guinea pigs are strikingly different to those observed in pregnant rats, in which maternal estrogen depletion, pathological alteration of placentas, increased gestation length, late fetal death, and dystocia were observed after administration of epoxiconazole. In the studies reported here, analysis of maternal plasma concentrations and metabolism after administration of radiolabeled epoxiconazole demonstrated that the different results in rats and guinea pigs were not due to different exposures of the animals. A comprehensive comparison of hormonal regulation of pregnancy and birth in murid rodents and primates indicates that the effects on pregnancy and parturition observed in rats are not applicable to humans. In contrast, the pregnant guinea pig shares many similarities to pregnant humans regarding hormonal regulation and is therefore considered to be a suitable species for extrapolation of related effects to humans.

Developmental toxicity of polyethylene glycol-g-polyvinyl alcohol grafted copolymer in rats and rabbits

Polyethylene glycol-g-polyvinyl alcohol (PEG-PVA) grafted copolymer was evaluated in developmental toxicity studies with Wistar rats and Himalayan rabbits. Pregnant Wistar rats were gavaged with 0 (vehicle control), 100, 300, or 1000 mg PEG-PVA grafted copolymer/kg bw/day from gestation day (GD) 6-15. Pregnant Himalayan rabbits received the same treatment from GD 6 to 19. On GD 20 and 29 for rats and rabbits, respectively, the animals were euthanized and were examined grossly. For each dam, corpora lutea were counted and number and distribution of implantation sites were determined. The fetuses were removed, sexed, weighed, and evaluated for any external, soft tissue, and skeletal findings. No significant findings were found that could be attributed to administration of PEG-PVA grafted copolymer. Under the conditions of these studies, the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity in both species was the highest dose tested of 1000 mg/kg bw/day.

Investigations on the dose–response relationship of combined exposure to low doses of three anti-androgens in Wistar rats

The current investigation examines whether combined exposure to three anti-androgens (flutamide, prochloraz, vinclozolin) result in interference with endocrine homeostasis when applied at very low dose levels, and whether the results of combined exposure are more pronounced than to the individual compounds. A pre–post-natal in vivo study design was chosen with more parameters than regulatory testing protocols require (additional endpoints addressing hormone levels, morphology and histopathological examinations). Dose levels were chosen to represent the lowest observed adverse effect level (LOAEL), the no observed adverse effect level (NOAEL), and the acceptable daily intake for each individual substance. Anti-androgenic changes were observable at the effect level (LOAEL) but not at lower exposures. Nipple/areola counts appeared to be a sensitive measure of effect, in addition to male sex organ weights at sexual maturation, and finally gross findings. The results indicate the absence of evidence for effects at low or very low dose levels. No (adverse) effects were seen at the NOAEL dose. A non-monotonic dose–response relationship was not evident. Combined exposure at LOAEL level resulted in enhanced responses for anogenital index, number of areolas/nipples, delayed preputial separation and reduced ventral prostate weight in comparison to the individual compounds.

Postnatal fate of prenatal-induced fetal alterations in laboratory animals

Currently it is common practice to evaluate the developmental toxicity hazard of chemicals or pharmaceuticals by evaluation of fetuses after administration of the compound to pregnant animals. These studies are designed to provide possible compound-related fetal changes near term, which are usually classified into malformations or variations. Malformations, but not variations are expected to adversely affect the survival or health. Therefore, classification has striking different regulatory consequences. For categorization as variation reversibility is an important criterion, but it is usually not examined in a standard guideline study. Although this issue has already been recognized long time ago, data dealing with the postnatal reversibility of fetal alterations are still rare. In the current review, literature data, regulatory documents as well as in-house data were compiled. Beside skeletal alterations of skull, vertebral column, ribs, shoulder and pelvic girdle, and extremities, kidney and heart defects are discussed and assessed.