Horst Spielmann

The validated embryonic stem cell test to predict embryotoxicity in vitro

In the embryonic stem cell test (EST), differentiation of mouse embryonic stem cells (mESCs) is used as a model to assess embryotoxicity in vitro. The test was successfully validated by the European Center for the Validation of Alternative Methods (ECVAM) and models fundamental mechanisms in embryotoxicity, such as cytotoxicity and differentiation. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. To predict the embryotoxic potential of a test substance, three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts. A special feature of the EST is that it is solely based on permanent cell lines so that primary embryonic cells and tissues from pregnant animals are not needed. In this protocol, we describe the ECVAM-validated method, in which the morphological assessment of contracting cardiomyocytes is used as an endpoint for differentiation, and the molecular-based FACS-EST method, in which highly predictive protein markers specific for developing heart tissue were selected. With these methods, the embryotoxic potency of a compound can be assessed in vitro within 10 or 7 d, respectively.

A new model for embryotoxicity testing: teratogenicity and pharmacokinetics of valproic acid following constant-rate administration in the mouse using human therapeutic drug and metabolite concentrations

Abstract The limitations of a conventional testing procedure for embryotoxicity-multiple dosing throughout the organogenesis period of the mouse or rat-are discussed for drugs such as valproic acid (VPA) which exhibit pharmacokinetic properties in these species which are very different from those in the human. Administration of VPA to the mouse, once each day, resulted in peak plasma drug concentrations 10 fold higher than human therapeutic plasma levels, after which the drug levels rapidly decreased to insignificant levels which persisted until the time of the next dose. Dose-dependent growth retardation, a sharp increase in the resorption rate and a high incidence of exencephaly were observed with this dosing regimen. A new mouse model was developed where constant-rate application of the drug with osmotic minipumps resulted in drug levels throughout the organogenesis period which were similar to those observed during human therapy. Such therapeutic drug levels produced a slight but significant fetal growth retardation and increased resorption rates, but not exencephaly. It is suggested that maintaining plasma concentrations in the experimental animal comparable to human therapeutic drug levels should offer a more realistic model for drug-embryotoxicity testing.

Simple assay of 0.1–1.0 pmol of ATP, ADP, and AMP in single somatic cells using purified luciferin luciferase☆

Abstract The sensitivity of ATP determinations with crude firefly luciferin luciferase is limited by contaminating ATP converting enzymes, which cause a rapid decrease of the ATP level during the assay. Purified luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration. Sensitivity and specificity of the ATP assay are, therefore, considerably increased when purified enzyme is used instead of crude extracts of the enzyme. ATP, 0.1–1.0 pmol as well as higher amounts can be determined with commercial preparations of purified and stabilized luciferase. In ADP and AMP measurements with the luciferase assay, problems are arising from the enzymes required for the conversion to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides. Exclusion of contaminated enzymes and removal of ammonium sulfate from adenylate kinase were the only prerequisites for determinations of 0.1–1.0 pmol of ADP and AMP with purified luciferase. The application of the assay in determinations of ATP, ADP, and AMP in single preimplantation mouse embryos is described.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Reconsidering pluripotency tests: Do we still need teratoma assays?

The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.

Differential Sensitivity of Preimplantation Mouse Embryos to UV Irradiation in Vitro and Evidence for Postreplication Repair

The sensitivity of various stages of preimplantation mouse embryos to uv irradiation in vitro was determined by two criteria of development during in vitro culture after uv irradiation: development throughout the following 24 h, and the ability to form a blastocyst. In these studies the uv sensitivity was the highest in 1-cell embryos and it decreased with development towards the morula stage as indicated by the ED/sub 50/ values of the rate of blastulation following uv irradiation: 2-cell embryos 90 erg/mm/sup 2/, 4- and 8-cell embryos 100 erg/mm/sup 2/, and morulae 140 erg/mm/sup 2/. After uv irradiation the in vitro development of the embryos was further impaired by the presence of caffeine in the culture medium at concentration levels which did not interfere with the development of nonirradiated embryos. In 2-cell embryos caffeine treatment was only effective during the first 24 hr after irradiation. These results suggest that early mammalian embryos are able to repair uv-induced DNA lesions by the post replication repair mechanism.

A modular one-generation reproduction study as a flexible testing system for regulatory safety assessment.

The European Unions Registration, Evaluation and Authorisation of Chemicals (REACH) legislation mandates testing and evaluation of approximately 30,000 existing substances within a short period of time, beginning with the most widely used high production volume (HPV) chemicals. REACH testing requirements for the roughly 3000 HPV chemicals specify three separate tests for reproductive toxicity: two developmental toxicity studies on different animal species (OECD Test Guideline 414) and a two-generation reproduction toxicity study (OECD TG 416). These studies are highly costly in both economic and animal welfare terms. OECD TG 416 is a fertility study intended to evaluate reproductive performance of animals in the P and F1-generations following repeated exposure to a test substance. It can also be used to detect adverse effects on structural and functional development. Thus, it has conventionally been preferred to the one-generation study (OECD TG 415). Recently, the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) proposed that routine two-generation studies could in most cases be replaced with an enhanced one-generation study (Reuter et al. [1]). The flexible design proposed by HESI-ACSA allows for the addition of one or more specialised modules, if triggered (e.g. production of a second generation or the investigation of classical developmental toxicity or developmental neuro- or immunotoxicity). Significantly, however, the HESI-ACSA proposal was designed for use in the safety assessment of pesticidal, as opposed to industrial, chemicals. Thus for the purposes of REACH, a streamlined one-generation study that also examines structural development would be the most efficient means of addressing current information requirements for HPV chemicals. This study represents a flexible testing system that can be modified to meet regulatory needs in a variety of sectors.

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B4) but no trace of LDH-5 (A4) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.

The effects of carbon-13 incorporation into preimplantation mouse embryos on development before and after implantation

Abstract Preimplantation mouse embryos were cultured in vitro for 48 hours from the 8-cell to the blastocyst stage in media containing uniformly labelled 13 C-glucose. The 13 C content of the blastocysts was 20 atom % according to incorporation studies with 14 C-glucose. No embryotoxic effects of carbon-13 incorporation could be detected on the basis of these criteria of normal development: the percentage of embryos reaching the blastocyst stage during the culture period; the number of cells in these blastocysts; and the development after transplantation to pseudopregnant foster mothers.

Cyclophosphamide treatment prior to implantation: the effects on embryonic development

Research in teratology usually focuses on the period of organogenesis, because this is the most sensitive period for the induction of malformations in mammals1,2. Attempts to study drug actions on earlier periods of pregnancy have not been very successful, because the preimplantation embryo is remarkably resistant to teratogens. In the earliest study of this problem the treatment of cleaving rabbit eggs with purine analogues in vivo 3 has no effect on development until at or after implantation. Investigations on the effects of X-irradiation4 during the first days of pregnancy in the rat increased neither gross congenital malformations nor fetal growth retardation at the end of pregnancy. There is, furthermore, no indication for an increased rate of abnormalities among the offspring from embryos cultured in vitro in the presence of various teratogens during the preimplantation period and subsequently transferred to foster mothers5. Additionally the fetuses from transplanted preimplantation mouse embryos on which different kinds of microsurgery had been performed6 or which had been frozen to -269 °C for up to one year7 never showed an increased malformation rate at term. The effect of teratogens on embryos during the preimplantation period, therefore, has been explained by Austin8 as follows: ‘The effect of teratogens on the cleavage embryo depends on the number of cells killed or inhibited: above a certain portion, the embryo dies; below that figure, the remaining cells multiply to replace those lost and subsequent development is essentially normal.’