Review of the state of science and evaluation of currently available in silico prediction models for reproductive and developmental toxicity: A case study on pesticides.

BACKGROUND: In silico methods for toxicity prediction have increased significantly in recent years due to the 3Rs principle. This also applies to predicting reproductive toxicology, which is one of the most critical factors in pesticide approval. The widely used quantitative structure-activity relationship (QSAR) models use experimental toxicity data to create a model that relates experimentally observed toxicity to molecular structures to predict toxicity. Aim of the study was to evaluate the available prediction models for developmental and reproductive toxicity regarding their strengths and weaknesses in a pesticide database. METHODS: The reproductive toxicity of 315 pesticides, which have a GHS classification by ECHA, was compared with the prediction of different in silico models: VEGA, OECD (Q)SAR Toolbox, Leadscope Model Applier, and CASE Ultra by MultiCASE. RESULTS: In all models, a large proportion (up to 77%) of all pesticides were outside the chemical space of the model. Analysis of the prediction of remaining pesticides revealed a balanced accuracy of the models between 0.48 and 0.66. CONCLUSION: Overall, predictions were only meaningful in rare cases and therefore always require evaluation by an expert. The critical factors were the underlying data and determination of molecular similarity, which offer great potential for improvement.

Food grade safflower concentrate: No evidence for reproduction and early developmental toxicity.

Safflower (Carthamus tinctorius) petals have been used for centuries as a spice, in tea blends and in traditional Asian medicine. Aqueous extracts of Safflower petals have been used as a colouring food over the last 30 years due to their bright colour. Publications in the past raised concerns about fertility impairing, maternal toxicity, fetotoxic and teratogenic properties in rodents. As the tested extracts were poorly characterized and the studies were not performed according to guidelines, a need for further evaluation was seen. In silico predictions for the main pigments provided negative results for bacterial mutagenicity. Further, in vitro genotoxicity and in vivo reproductive toxicity studies of a well-characterized aqueous safflower concentrate generated more relevant data for risk assessment of its use in food. In vitro AMES tests and a mouse lymphoma cell assay were negative. An OECD guideline 421 screening study was performed in rats with oral daily doses of up to 1000 mg/kg bodyweight, applied via gavage to simulate a bolus effect. The highest dose reflected a toxicological limit test. The study did not give indications of general toxicity, did not show any effect on fertility and reproduction nor any effect on prenatal development and, also in contrast to previous results, treatment did not affect estradiol and FSH levels. Furthermore, pups raised until PND 14-16, developed normally with no adverse effects observed. With the established NOAEL of at least 1000 mg/kg/d, a considerable margin of exposure is achieved when compared with human intake estimates.

Critical role of transcription factor cyclic AMP response element modulator in beta1-adrenoceptor-mediated cardiac dysfunction.

BACKGROUND: Chronic stimulation of the beta(1)-adrenoceptor (beta(1)AR) plays a crucial role in the pathogenesis of heart failure; however, underlying mechanisms remain to be elucidated. The regulation by transcription factors cAMP response element-binding protein (CREB) and cyclic AMP response element modulator (CREM) represents a fundamental mechanism of cyclic AMP-dependent gene control possibly implicated in beta(1)AR-mediated cardiac deterioration. METHODS AND RESULTS: We studied the role of CREM in beta(1)AR-mediated cardiac effects, comparing transgenic mice with heart-directed expression of beta(1)AR in the absence and presence of functional CREM. CREM inactivation protected from cardiomyocyte hypertrophy, fibrosis, and left ventricular dysfunction in beta(1)AR-overexpressing mice. Transcriptome and proteome analysis revealed a set of predicted CREB/CREM target genes including the cardiac ryanodine receptor, tropomyosin 1alpha, and cardiac alpha-actin as altered on the mRNA or protein level along with the improved phenotype in CREM-deficient beta(1)AR-transgenic hearts. CONCLUSIONS: The results imply the regulation of genes by CREM as an important mechanism of beta(1)AR-induced cardiac damage in mice.

Cardiomyocyte-specific inactivation of transcription factor CREB in mice.

The transcription factor cAMP response element (CRE)-binding protein (CREB, Creb1) plays a critical role in regulating gene expression in response to activation of the cAMP-dependent signaling pathway, which is implicated in the pathophysiology of heart failure. Using the Cre-loxP system, we generated mice with a cardiomyocyte-specific inactivation of CREB and studied in this model whether CREB is critical for cardiac function. CREB-deficient mice were viable and displayed neither changes in cardiac morphology nor alterations of basal or isoproterenol-stimulated left ventricular function in vivo or of important cardiac regulatory proteins. Since CREB was proposed as a negative regulator of cardiomyocyte apoptosis by enhancing the expression of the antiapoptotic protein Bcl-2, we analyzed the fragmentation of DNA, the activity of caspases 3/7 and the expression of Bcl-2 and did not observe any differences between CREB-deficient and CREB-normal hearts. Our results suggest that the presence of CREB is not critical for normal cardiac function in mice.

Pre- and Postnatal Lung Development: An Updated Species Comparison.

The purpose of this review is to give an outline of respiratory tract morphological and functional development with an emphasis on perinatal and postnatal maturational processes. In view of the rising need for qualitative and quantitative data for the development of pediatric pharmaceuticals, a comparison of the human situation to experimental animal models is made, and functional data as well as suitable models for human airway diseases and functional testing are presented. Birth Defects Research 109:1519-1539, 2017. © 2017 Wiley Periodicals, Inc.

Screening for potential endocrine disruptors in fish: evidence from structural alerts and in vitro and in vivo toxicological assays.

BACKGROUND: The European chemicals' legislation REACH aims to protect man and the environment from substances of very high concern (SVHC). Chemicals like endocrine disruptors (EDs) may be subject to authorization. Identification of (potential) EDs with regard to the environment is limited because specific experimental assessments are not standard requirements under REACH. Evidence is based on a combination of in vitro and in vivo experiments (if available), expert judgement, and structural analogy with known EDs. OBJECTIVES: The objectives of this study are to review and refine structural alerts for the indication of potential estrogenic and androgenic endocrine activities based on in vitro studies; to analyze in vivo mammalian long-term reproduction studies with regard to estrogen- and androgen-sensitive endpoints in order to identify potential indicators for endocrine activity with regard to the environment; to assess the consistency of potential estrogenic and androgenic endocrine activities based on in vitro assays and in vivo mammalian long-term reproduction studies and fish life-cycle tests; and to evaluate structural alerts, in vitro assays, and in vivo mammalian long-term reproduction studies for the indication of potential estrogenic and androgenic endocrine disruptors in fish. RESULTS: Screening for potential endocrine activities in fish via estrogenic and androgenic modes of action based on structural alerts provides similar information as in vitro receptor-mediated assays. Additional evidence can be obtained from in vivo mammalian long-term reproduction studies. Conclusive confirmation is possible with fish life-cycle tests. Application of structural alerts to the more than 33,000 discrete organic compounds of the EINECS inventory indicated 3585 chemicals (approx. 11%) as potential candidates for estrogenic and androgenic effects that should be further investigated. Endocrine activities of the remaining substances cannot be excluded; however, because the structural alerts perform much better for substances with (very) high estrogenic and androgenic activities, there is reasonable probability that the most hazardous candidates have been identified. CONCLUSIONS: The combination of structural alerts, in vitro receptor-based assays, and in vivo mammalian studies may support the priority setting for further assessments of chemicals with potential environmental hazards due to estrogenic and androgenic activities.

Impaired cardiac contraction and relaxation and decreased expression of sarcoplasmic Ca2+-ATPase in mice lacking the CREM gene.

Congestive heart failure is the common endpoint of various cardiac diseases representing a leading cause of cardiovascular mortality in Western countries. Characteristic functional alterations of the failing heart are explained by expressional changes of myocardial regulatory proteins; however, little is known about underlying mechanisms regulating cardiac gene expression in the failing heart. Here, we address the specific role of transcription factor CREM for cardiac function in CREM mutant mice with complete inactivation of the CREM gene. We show that CREM mutant mice display distinct alterations of cardiac function resembling characteristic functional defects of the failing heart. Left ventricular hemodynamic assessment of CREM mutant mice revealed impairment of both cardiac contraction and relaxation in basal state, as well as a decreased responsiveness to beta-adrenergic stimulation. The diminished cardiac contractile performance was associated with a selective down-regulation of beta1-adrenergic receptors and a decreased ventricular expression of SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum. The cardiac phenotype of CREM mutant mice provides the first evidence that CREM represents an important key regulator of cardiac gene expression, which is essential for normal left ventricular contractile performance and response to beta-adrenoreceptor stimulation.

Development of hematological and immunological characteristics in neonatal rats.

As major immunological and hematological parameters evolve during the early period of life, laboratory data must be interpreted in relation to developmental changes. Wistar (WU) rats were sacrificed on PND2, 4, 7, 10, 14, 17 and 21. Peripheral blood, bone marrow, thymus samples and spleen cells were collected and a bronchoalveolar lavage (BAL) performed. Parameters of blood counts changed considerably between time points. IgM and IgG levels steadily increased. Spontaneous spleen cell proliferation was low before PND21, although mitogens had stimulatory effects above baseline. In the spleen, T-lymphocyte counts tripled by PND17 (mainly attributed to CD8(+) cytotoxic T-cells and CD4(+) T-helper cells). In peripheral blood an increase in B-lymphocytes to about 60% of the cell number was observed. In BAL fluid, macrophages represented 95-98% of the cells. In thymus architecture, lymphoblast migration was seen and epithelial structures appeared. The data presented will help to distinguish between maturational changes and treatment-related effects.

Structural features of endocrine active chemicals--A comparison of in vivo and in vitro data.

Studies on reproductive toxicity need high numbers of test animals. Therefore, we investigated whether chemical structural features (SF) in combination with in vitro data on specific adverse outcome pathways (AOPs) may be used for predicting reproductive toxicity of untested chemicals. Using the OECD Toolbox and expert judgment, we identified 89 structure groups for 275 chemicals for which the results of prenatal developmental toxicity or multigeneration studies were present in the Fraunhofer database on Fertility and Developmental Toxicity in experimental animals (FeDTex) database. Likewise, we evaluated 220 chemicals which had been tested in reporter gene assays on endocrine ((anti)estrogenic and (anti)androgenic) properties in the CALUX(®) test battery. There was a large spread of effect levels for substances within the chemical structure groups for both, in vivo and in vitro results. The groups of highest concern (diphenyl derivatives, planar conjugated systems with fused rings, phenols and organophosphates) correlated quite well, however, between the in vivo and in vitro data on estrogenic activity. For the 56 chemicals represented in both databases, lowest effect doses in vivo correlated well with the estrogenic activity in vitro. These results suggest that a panel of assays covering relevant AOPs and data on metabolism and toxicokinetics may allow prediction of relative reproductive or development toxicity potency within the identified chemical structure groups.

Evaluation of an alternative in vitro test battery for detecting reproductive toxicants in a grouping context.

Previously we showed a battery consisting of CALUX transcriptional activation assays, the ReProGlo assay, and the embryonic stem cell test, and zebrafish embryotoxicity assay as 'apical' tests to correctly predict developmental toxicity for 11 out of 12 compounds, and to explain the one false negative [7]. Here we report on applying this battery within the context of grouping and read across, put forward as a potential tool to fill data gaps and avoid animal testing, to distinguish in vivo non- or weak developmental toxicants from potent developmental toxicants within groups of structural analogs. The battery correctly distinguished 2-methylhexanoic acid, monomethyl phthalate, and monobutyltin trichloride as non- or weak developmental toxicants from structurally related developmental toxicants valproic acid, mono-ethylhexyl phthalate, and tributyltin chloride, respectively, and, therefore, holds promise as a biological verification model in grouping and read across approaches. The relevance of toxicokinetic information is indicated.

A high throughput screening system for predicting chemically-induced reproductive organ deformities.

There is a great need for alternative testing methods for reproductive toxicants that are practical, fast, cost-effective and easy to interpret. Previously we followed a pragmatic approach using readily available tests, which was successful in predicting reproductive toxicity of chemicals [13]. This initial battery still contained apical tests and is fairly complex and low in its throughput. The current study aimed to simplify this screening battery using a mechanistic approach and a panel of high throughput CALUX reporter gene assays. A mechanistic approach was taken to validate this high throughput test battery. To this end it was challenged with two preselected sets of chemicals addressing two major apical effect classes relevant in reproductive toxicity. We found selectivity in this battery in that 82% of the compounds inducing reproductive organ deformities were predicted correctly, while for compounds inducing neural tube defects this was the case in 47% only. This is consistent with the mechanisms of toxicity covered in the battery. The most informative assays in the battery were ERalpha CALUX to measure estrogenicity and the AR-anti CALUX assay to measure androgen receptor antagonism.

Identifying placental epigenetic alterations in an intrauterine growth restriction (IUGR) rat model induced by gestational protein deficiency.

Poor maternal nutrition during gestation can lead to intrauterine growth retardation (IUGR), a main cause of low birth weight associated with high neonatal morbidity and mortality. Such early uterine environmental exposures can impact the neonatal epigenome to render later-in-life disease susceptibility. We established in Wistar Han rats a mild IUGR model induced by gestational protein deficiency (i.e. 9% crude protein in low protein diet vs. 21% in control, from GD 0 to 21) to identify alterations in gene expression and methylation patterns in certain genes implicated in human IUGR or in placental development. We found differential gene expression of Wnt2 and Dlk1 between IUGR and control. Notably, Wnt2 exhibited significant decrease while Dlk1 increase in IUGR placentas, correlating to decrease in fetal and placental weight. Methylation patterns encompassing 30 CpGs in the Wnt2 promoter region revealed variability in both IUGR and control placentas, but a site-specific hypomethylation was evident in IUGR placentas. Our present findings further support a key role of maternal gestational nutrition in defining the neonatal epigenome.

Assessment of pulmonary function and serum substance levels in newborn and juvenile rats.

In the context of pharmaceutical development today, studies for pediatric drug approval are requested more and more often by the regulatory authorities. The developing lung represents a potential target in juvenile toxicity studies. Due to physiological differences in prenatal and postnatal development between humans and standard animal models, experimental methods have to be modified to assess pulmonary function, and basic data on respiratory parameters need to be provided. Daily nose-only inhalation exposure from postnatal days 4 to 21 using a model substance (verapamil HCl) and plethysmographic measurements between postnatal days 2 and 50 were performed noninvasively in conscious juvenile Wistar (WU) rats. The methods proved to be feasible and did not interfere with normal growth and development of the animals. Both techniques therefore permit new insights to support human neonatal risk assessment and therefore these animal models are suitable for regulatory studies.

Nitric oxide synthase in muscular dystrophies: a re-evaluation.

Duchenne and Becker muscular dystrophies (DMD and BMD) are associated with decreased total nitric oxide (NO). However, mechanisms leading to NO deficiency with consequent muscle-cell degeneration remain unknown. To address this issue, we examined skeletal muscles of DMD and BMD patients for co-expression of NO synthase (NOS) with nitrotyrosine and transcription factor CREB, as well as with enzymes engaged in NO signaling. Employing immunocytochemical labeling, Western blotting and RT-PCR, we found that, in contrast to the most commonly accepted view, neuronal NOS was not restricted to the sarcolemma and that muscles of DMD and BMD patients retained all three NOS isoforms with an up-regulation of the inducible NOS isoform, CREB and nitrotyrosine. We suggest that enhanced nitrotyrosine immunostaining in muscle fibers as well as in the vasculature of DMD and BMD specimens reflects massive oxidative stress, resulting in withdrawal of NO from its regular physiological course via the scavenging actions of superoxides.

Heart-directed expression of a human cardiac isoform of cAMP-response element modulator in transgenic mice.

The transcriptional activation mediated by cAMP-response element (CRE) and transcription factors of the CRE-binding protein (CREB)/CRE modulator (CREM) family represents an important mechanism of cAMP-dependent gene regulation possibly implicated in detrimental effects of chronic beta-adrenergic stimulation in end-stage heart failure. We studied the cardiac role of CREM in transgenic mice with heart-directed expression of CREM-IbDeltaC-X, a human cardiac CREM isoform. Transgenic mice displayed atrial enlargement with atrial and ventricular hypertrophy, developed atrial fibrillation, and died prematurely. In vivo hemodynamic assessment revealed increased contractility of transgenic left ventricles probably due to a selective up-regulation of SERCA2, the cardiac Ca(2+)-ATPase of the sarcoplasmic reticulum. In transgenic ventricles, reduced phosphorylation of phospholamban and of the CREB was associated with increased activity of serine-threonine protein phosphatase 1. The density of beta(1)-adrenoreceptor was increased, and messenger RNAs encoding transcription factor dHAND and small G-protein RhoB were decreased in transgenic hearts as compared with wild-type controls. Our results indicate that heart-directed expression of CREM-IbDeltaC-X leads to complex cardiac alterations, suggesting CREM as a central regulator of cardiac morphology, function, and gene expression.