In Vivo Assessment of Drug-Induced Hepatotoxicity Using Xenopus Embryos.

Failure to predict drug-induced toxicity reactions is a major problem contributing to a high attrition rate and tremendous cost in drug development. Drug screening in X. laevis embryos is high-throughput relative to screening in rodents, potentially making them ideal for this use. Xenopus embryos have been used as a toxicity model in the frog embryo teratogenesis assay on Xenopus (FETAX) for the early stages of drug safety evaluation. We previously developed compound-screening methods using Xenopus embryos and believe they could be used for in vitro drug-induced toxicity safety assessment before expensive preclinical trials in mammals. Specifically, Xenopus embryos could help predict drug-induced hepatotoxicity and consequently aid lead candidate prioritization. Here we present methods, which we have modified for use on Xenopus embryos, to help measure the potential for a drug to induce liver toxicity. One such method examines the release of the liver-specific microRNA (miRNA) miR-122 from the liver into the vasculature as a result of hepatocellular damage, which could be due to drug-induced acute liver injury. Paracetamol, a known hepatotoxin at high doses, can be used as a positive control. We previously showed that some of the phenotypes of mammalian paracetamol overdose are reflected in Xenopus embryos. Consequently, we have also included here a method that measures the concentration of free glutathione (GSH), which is an indicator of paracetamol-induced liver injury. These methods can be used as part of a panel of protocols to help predict the hepatoxicity of a drug at an early stage in drug development.

Assessment of Gross Fetal Malformations: The Modernized Wilson Technique and Skeletal Staining.

Teratology is the study of anatomical and physiological abnormalities, commonly known as birth defects. If an embryo is exposed to a harmful substance, or teratogen, during the critical period of development, an ensuing malformation may occur. These malformations and their associated mechanisms are studied and analyzed in laboratory animals in order to prevent them from occurring in humans. Rodents such as rats and mice have commonly been used in such studies because of their similarity to humans. In 1959, James G. Wilson designed, developed, and tested a protocol on how to observe and analyze structural malformations in rodent fetuses, which included: external examination, skeletal evaluation, soft tissue analysis, and data collection/analysis. For standardization purposes, i.e., to normalize findings from one lab to another, it is important that this protocol be followed with precision. Although many years have passed since Wilson initially created this protocol, it is still widely used to this day, and only minor changes have been made to his instructions such as the chemical reagents used in the experiments and methods of analysis of the experimental data. Such testing has resulted in major advances in the dissemination of teratology information, including the identification of an increasing number of teratogens and the understanding of the pathogenesis of birth defects. While mechanistically birth defect prevention will include the understanding of individual genomes and pharmacogenomics, overall, morphological assessment will still be required as an integral part of birth defects research. As the interaction between teratogenic and genetic factors is better understood, it is anticipated that the incidence of most types of defects will substantially be reduced.

Fetal alcohol spectrum disorders: an overview for pediatric and adolescent care providers.

Fetal alcohol spectrum disorder (FASD) is a term used to describe the spectrum of conditions associated with prenatal alcohol exposure. These are characterized by facial dysmorphia, growth deficits and central nervous system abnormalities. FASDs are the most common preventable cause of intellectual disability in the United States and have high financial costs. Therefore, efforts at prevention are paramount. When an individual with an FASD goes undiagnosed and when appropriate interventions are not instituted, secondary disabilities such as substance abuse, school dropout, and criminal involvement are common with corresponding suffering endured by both the affected individual and the family. The diagnostic process opens up access to existing tools and resources, including the new American Academy of Pediatrics (AAP) FASD algorithm for the evaluation of FASDs, the new AAP FASD toolkit and evidence-based interventions specific to FASDs. Pediatric and adolescent clinicians are challenged to participate in the continuum of care from FASD prevention to identification, diagnosis, and management, including provision of supportive services for families in order for clinicians to make a difference in this 100% preventable disorder.

Angiotensin-II receptor 1 antagonist fetopathy--risk assessment, critical time period and vena cava thrombosis as a possible new feature.

AIMS: Angiotensin-II receptor 1 antagonists (AT1-antagonists) may cause severe and even lethal fetopathy in late pregnancy. However, exposure still occurs in spite of warnings in package leaflets. This study aimed to assess the risk of fetopathy, the sensitive time window, and possible new symptoms in prospective as well as retrospective cases with AT1-antagonist treatment during the second or third trimester of pregnancy. METHODS: Patients were enrolled by the Berlin Institute for Clinical Teratology and Drug Risk Assessment in Pregnancy between 1999 and 2011 through risk consultation. Symptoms defined as indicative of AT1-antagonist fetopathy were: oligo-/anhydramnios, renal insufficiency, lung hypoplasia, joint contractures, skull hypoplasia and fetal/neonatal death. RESULTS: In 5/29 (17%) prospectively enrolled cases with AT1-antagonist exposure beyond the first trimester oligo-/anhydramnios was diagnosed. Two infants showed additional symptoms of fetopathy. The risk is more than 30% if treatment continues beyond the 20th week of pregnancy. Oligo-/anhydramnios was reversible after AT1-antagonist withdrawal. Among 16 retrospective case reports, three infants presented with a thrombosis of the inferior vena cava in the vicinity of the renal veins. Four out of 13 live births did not survive. CONCLUSIONS: Our survey suggests that the risk increases with duration of AT1-antagonist treatment into late pregnancy and oligo-/anhydramnios may be reversible after AT1-antagonist discontinuation. Thrombosis of inferior vena cava may be a new feature of AT1-antagonist fetopathy. AT1-antagonist medication during pregnancy constitutes a considerable risk and must be discontinued immediately. In case of indicative diagnostic findings in either the fetus or newborn, previous maternal AT1-antagonist exposure should be considered.

Assessment of the developmental and reproductive toxicity of diiodomethyl-p-tolylsulfone in rats and rabbits.

Diiodomethyl-p-tolylsulfone (DIMPTS) was tested in developmental toxicity (DT) and reproductive toxicity studies. In the rat DT study, DIMPTS was administered at 0, 100, 300 or 1000 mg/kg/day. Maternal toxicity as evidenced by reductions in body weight gain or feed consumption at 1000 and, to a lesser extent, 300 mg/kg/day. The only developmental effect was umbilical hernia at 1000 mg/kg/day; therefore, NOELs for maternal and developmental toxicity were 100 and 300 mg/kg/day, respectively. In the rabbit DT study, NZW rabbits were gavaged with 0, 0.05, 0.5 and 2mg/kg/day DIMPTS. The NOEL for maternal toxicity was 0.5mg/kg/day, based on thyroid weight increase with histopathology. There were no observed developmental effects. In the two-generation study, CD rats were fed 0, 2.5, 10 or 40 mg/kg/day DIMPTS. Increased thyroid weight and histopathology were observed at all doses with associated pituitary findings in males. Reproductive toxicity at 40 mg/kg/day consisted of increased postimplantation loss, decreased gestation survival and two cases of dystocia, while litter size, pup survival/weight were affected at 10 and 40 mg/kg/day. The NOEL for parental toxicity could not be determined, while the NOEL for reproductive toxicity was 2.5mg/kg/day. The maternal thyroid and reproductive effects in this study were consistent with iodine toxicity.

Isotretinoin exposure during pregnancy: assessment of spontaneous reports in France.

BACKGROUND: In three previous studies, we have shown that pregnant women were still being exposed to isotretinoin and that compliance with recommendations was incomplete. The relaxation of these recommendations (summary of product characteristics 2004), combined with the release of generic brands, encouraged us to carry out a fourth study. OBJECTIVE: To assess isotretinoin exposure during pregnancy following the application of less stringent recommendations and the marketing of generic isotretinoin brands. METHODS: All cases of isotretinoin exposure during pregnancy, between 1 January 2003 and 31 December 2006, spontaneously reported to pharmacovigilance centres, the Teratogenic Agent Information Centre, and pharmaceutical companies in France were assessed. Cases were classified for analysis into the following groups: 'conception <1 month after isotretinoin discontinuation', 'conception during isotretinoin treatment' and 'patient already pregnant when isotretinoin was started'. The rate of spontaneously reported isotretinoin exposure during pregnancy was estimated by dividing the number of isotretinoin-exposed pregnancies by the number of women of childbearing age treated with isotretinoin. RESULTS: Over 4 years, 147 cases of isotretinoin exposure during the teratogenic risk period were spontaneously reported, i.e. 'conception <1 month after isotretinoin discontinuation' (23%), 'conception during isotretinoin treatment' (61%), and 'patient already pregnant when isotretinoin was started' (16%). Nineteen percent of the patients did not use any form of contraception. In 23% of the patients, the method of contraception used did not comply with recommendations, while in 86% of the cases, isotretinoin was prescribed by a dermatologist. Among the 44 pregnancies with available data on fetuses or neonates, there were two (4.5%) malformations compatible with the time of exposure and with isotretinoin embryopathy. The rate of spontaneously reported isotretinoin exposure during pregnancy has increased by approximately 30%, from 0.32 (95% CI 0.26, 0.38) to 0.41 (95% CI 0.34, 0.49) per 1000 women of childbearing age treated since 1999-2002. CONCLUSIONS: We suggest that recommendations be tightened, with specific information regarding the most effective contraceptive method combined with compulsory monthly pregnancy testing during treatment. The French Drug Agency has informed the European Medicines Agency of the need for measures aimed at improving compliance.

Overview of developmental and reproductive toxicity research in China: history, funding mechanisms, and frontiers of the research.

Reproductive and developmental toxicology (DART) is the discipline that deals with adverse effects on male and female resulting from exposures to harmful chemical and physical agents. DART research in China boasted a long history, but presently has fallen behind the western world in education and research. The funding mechanisms for DART research in China were similar to that for other toxicological disciplines, and the funding has come from research grants and fellowships provided by national, ministerial, and provincial institutions. Finally, the frontiers of DART research in China could be summarized as follows: (1) use of model animals such as the zebrafish and roundworm, and use of cutting-edge techniques such as stem cell culture, as well as transgenic, metabonomic, and virtual screening to study the mechanisms of developmental toxicity for some important toxicants in China; (2) use of model animals and other lower-level sentinel organisms to evaluate and monitor the developmental toxicogical risk of environmental chemicals or pollutants; (3) epidemiological studies of some important reproductive hazards; (4) in-depth studying of the reproductive and developmental toxicity of some important environmental chemicals; and (5) evaluation and study of the reproductive and developmental toxicity of traditional Chinese medicines.

An overview of developmental and reproductive toxicity risk assessment in China.

Development and reproductive toxicology (DART) studies in animals are integral parts of nonclinical safety evaluation of drugs. The State Food and Drug Administration (SFDA) of the People's Republic of China developed a new guideline on DART studies in 2006. This guideline is in broad agreement with ICH guideline S5A (1994), "Detection of Toxicity to Reproduction for Medical Products," and M3, "Nonclinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals." However, the new guideline on DART testing differs from ICH in some detailed requirements. This overview reviews some main specifications in China's guidelines on DART testing, and also briefly introduces the recommendations for conducting human clinical trials and marketing authorization in China.

Developmental toxicity assessment of thermoresponsive poly(N-isopropylacrylamide-co-acrylamide) oligomers in CD-1 mice.

BACKGROUND: Although polymers and hydrogels are used successfully in biomedical applications, including implants and drug delivery devices, smaller molecular weight oligomers, such as those investigated here, have not been extensively studied in vivo. Poly(N-isopropylacrylamide-co-acrylamide), or P(NIPAAm-co-AAm), has a unique thermoresponsive behavior and is under investigation as a novel drug delivery system for metastatic cancer treatment. To date, no studies have been published regarding the safety of P(NIPAAm-co-AAm) to the conceptus. METHODS: From gestation days (GD) 6-16, pregnant CD-1 mice were dosed via i.p. injection with aqueous solutions containing 500, 750, or 1,000 mg/kg/d P(NIPAAm-co-AAm). Dams were sacrificed on GD 17 and their litters were examined for abnormalities. RESULTS: P(NIPAAm-co-AAm) caused no statistically significant difference in maternal weight gain or percent resorbed or dead fetuses compared to control values, but fetal weight was significantly decreased in the two highest dosage groups. CONCLUSIONS: At the highest dosages employed, maternal exposure to P(NIPAAm-co-AAm) was associated with decreased fetal weight. However, as the estimated human exposure levels for persons using this system would be some 1,500-fold lower than the lowest dosage administered in this study, the authors feel that this oligomer was not shown to pose a biologically significant risk at relevant human dosages.

Comments from the Developmental Neurotoxicology Committee of the Japanese Teratology Society on the OECD Guideline for the Testing of Chemicals, Proposal for a New Guideline 426, Developmental Neurotoxicity Study, Draft Document (October 2006 version), and on the Draft Document of the Retrospective Performance Assessment of the Draft Test Guideline 426 on Developmental Neurotoxicity.

In October 2006, a new revision of the draft guideline (OECD Guideline for the Testing of Chemicals, Proposal for a New Guideline 426. Developmental Neurotoxicity Study) and Draft Document of the Retrospective Performance Assessment (RPA) of the Draft Test Guideline 426 on Developmental Neurotoxicity were distributed following incorporation of the results of the Expert Consultation Meeting in Tokyo on May 24-26, 2005. The draft guideline consists of 50 paragraphs and an appendix with 102 references; and the draft RPA consists of 37 paragraphs with 109 references. National coordinators were requested to arrange for national expert reviews of these draft documents in their member countries. Members of the Developmental Neurotoxicology (DNT) Committee of the Japanese Teratology Society (JTS) reviewed, discussed, and commented on the draft Test Guideline Proposal. The DNT Committee of the JTS also commented on the draft document of the RPA. These comments were sent to the OECD Secretariat. The DNT Committee of the JTS expects the comments to be useful for the finalization of these draft documents.

Assessment of teratogenic risk.

Although the association between prenatal exposure to old generation antiepileptic drugs (AEDs) and major congenital malformations has been studied for many years, it is not clear whether any specific AED, or AED combination, is more harmful than others, or whether any pattern of malformations can be considered specific for any given drug. Relationships between dosage and plasma concentrations of AEDs and the risk of malformations also need to be clarified. The greatest limitation of all studies performed to date is the fact that none included a sufficiently large number of pregnancies. For newer generation AEDs the teratogenic risk, if any, is unknown. Large prospective studies are needed. The best approach is the establishment of registries through international collaboration. Inclusion of non-epileptic controls and untreated women is not strictly necessary to evaluate the comparative teratogenic risk of AEDs. The modalities of data collection should be pre-defined; common protocols, sufficiently exhaustive but at the same time easy to perform, should be shared from the beginning. Information on the presence or absence of major malformations or prenatal growth retardation, and on all major factors that may affect the teratogenic endpoints should be obtained. Study designs should ensure high quality data recording, and adequate quality assurance and auditing procedures. Further requisites are a clear definition of congenital malformation and prolonged follow-up to detect the occurrence of congenital malformations not detected at birth.

[A novel in vivo 31P-nuclear magnetic resonance technique for assessment of teratogenicity induced by environmental endocrine disrupters in mice].

Novel in vivo 31P-nuclear magnetic resonance spectoroscopy(NMR) and NMR-imaging techniques for accurate and noninvasive assessment of teratogenicity induced by environmental endocrine disrupters(EDs) were developed. Mice with pregnancy were administered ED at extremely low dose, and then in vivo 31P-NMR spectra of embryos were acquired noninvasively and quantitatively to evaluate the energy metabolism. A significant decrease in embryo ATP level was seen, but no significant changes were detected by conventional histological and biochemical methods. In conclusion, in vivo NMR techniques are highly sensitive(at least 100-fold more sensitive than conventional methods) and are useful for toxicological assessment of environmental pollutants.

Physiologic assessment of fetal compromise: biomarkers of toxic exposure.

Understanding the physiologic and endocrinologic basis of fetal development is a major goal of perinatal biology. During the past decade a number of technological developments have allowed more precise evaluation of the fetus in utero and diagnosis of abnormalities. Despite these methodological achievements, however, there are no specific biological markers currently available to indicate that exposure to a given xenobiotic is associated with a cellular, subcellular, or pharmacodynamic event. This paper evaluates the following issues: What are some of the unique physiologic and endocrinologic features of the fetal milieu intérieur? What problems are peculiar to fetal assessment? Of what value are techniques such as ultrasonography, amniocentesis, chorionic villus sampling, fetoscopy, and fetal blood and tissue sampling for obtaining appropriate biomarkers? What are some examples of validated biomarkers and their applicability? What promising biomarkers are on the horizon? What are some of the promising techniques such as the evaluation of fetal body movements, breathing activity, electronic heart rate monitoring, and nuclear magnetic resonance? How may molecular probes be of value as biological markers of fetal compromise? What are some of the major research gaps and needs, and how should research priorities be set? Some of these topics are addressed. Moreover, the more general role(s) that various diagnostic methods and biological markers can have in an understanding of the regulation of fetal growth and differentiation and the role of xenobiotics in affecting the normal course of events are discussed.