Struggles for equivalence: In vitro developmental toxicity model evolution in pharmaceuticals in 2006

Our group has been using the ECVAM Embryonic Stem Cell assay to predict developmental toxicity. In order to improve the separation of non-teratogens from weak teratogens, we have employed measures of gene expression, and different statistical methods from those originally used to develop the test. These approaches have fundamentally not improved the discrimination of ‘weaks’ from ‘nons’. A realization that a very low value for cytotoxicity IC50 would drive a final result for the test in ways that were inappropriate for pharmaceuticals has led us to re-examine the cytotoxicity component. Our current efforts are focused on other, perhaps more sensitive, measures of cytotoxicity, combined with gene expression changes in mouse stem cells in an attempt to correctly identify weak teratogens and non-teratogens.     

Comparing three novel endpoints for developmental osteotoxicity in the embryonic stem cell test

Birth defects belong to the most serious side effects of pharmaceutical compounds or environmental chemicals. In vivo, teratogens most often affect the normal development of bones, causing growth retardation, limb defects or craniofacial malformations. The embryonic stem cell test (EST) is one of the most promising models that allow the in vitro prediction of embryotoxicity, with one of its endpoints being bone tissue development. The present study was designed to describe three novel inexpensive endpoints to assess developmental osteotoxicity using the model compounds penicillin G (non-teratogenic), 5-fluorouracil (strong teratogen) and all-trans retinoic acid (bone teratogen). These three endpoints were: quantification of matrix incorporated calcium by (1) morphometric analysis and (2) measurement of calcium levels as well as (3) activity of alkaline phosphatase, an enzyme involved in matrix calcification.To evaluate our data, we have compared the concentration curves and resulting ID₅₀s of the new endpoints with mRNA expression for osteocalcin. Osteocalcin is an exclusive marker found only in mineralized tissues, is regulated upon compound treatment and reliably predicts the potential of a chemical entity acting as a bone teratogen. By comparing the new endpoints to quantitative expression of osteocalcin, which we previously identified as suitable to detect developmental osteotoxicity, we were ultimately able to illustrate IMAGE analysis and Ca²⁺ deposition assays as two reliable novel endpoints for the EST. This is of particular importance for routine industrial assessment of novel compounds as these two new endpoints may substitute previously used molecular read-out methods, which are often costly and time-consuming.     

Individual and combined developmental toxicity assessment of bisphenol A and genistein using the embryonic stem cell test in vitro

The potential developmental toxicity of environmental estrogenic endocrine disruptors have become a great concern in recent years. In this study, two typical environmental oestrogen, namely, bisphenol A (BPA) and genistein (GEN) were investigated for potential embryotoxicity using the embryonic stem cell test model. Afterwards, a 4 × 4 full factorial design and the estimated marginal means plot were performed to assess the combined effects of these two compounds.According to the linear discriminant functions and classification criteria, bisphenol A and genistein were classified as weakly embryotoxic and strongly embryotoxic respectively. As for combined effects, the overall interaction between BPA and GEN on embryonic stem cells (ESCs) differentiation was synergistic at low dosages, however, on ESCs and 3T3 cell proliferation, the predominate action was additive. Considering the actual daily intake of these chemicals, it is concluded that BPA alone might not have adverse reproductive or developmental effects on human being. However, given that BPA and GEN do have synergistic effect at low concentration, they may disturb normal embryo development together, which could result in birth defect and behavioral alterations later in life.     

Transferability of a modified embryonic stem cell test using a new endpoint for developmental neurotoxicity

We developed and analyzed a new surrogate endpoint of the mouse embryonic stem cell test (EST) for developmental neurotoxicity. To determine the sensitivity, specificity, and transferability of the new endpoint, a pre-validation team from three independent laboratories optimized and standardized the protocol for neuronal differentiation of mouse embryonic stem cells (mESCs) by measuring the neuronal differentiation rates of mESCs under different culture conditions, such as the presence or absence of basic fibroblast growth factor (bFGF) in the growth media and varying lengths of culture. In addition, a component ratio of neuronal cells was measured by using flow cytometry analysis of β-III tubulin (Tuj1)-positive cells and real-time polymerase chain reaction analysis of microtubule-associated protein 2 (MAP2) mRNA. Our results showed that the best growth was achieved by culturing mESCs for 12 d in N2B27 medium without bFGF or ascorbic acid. Lead (II) acetate and aroclor 1254 were used to test the usefulness of the new endpoint. When we used the known ID₅₀ values for lead (II) acetate in the EST model, it was classified as non-embryotoxic; however, when we used the new ID₅₀ values that we determined in this study, it was classified as weakly embryotoxic. Aroclor 1254 and penicillin G were also classified as weakly embryotoxic and non-embryotoxic compounds, respectively, when cardiac and neuronal differentiation ID₅₀ values were used. Therefore, our new surrogate endpoint for developmental neurotoxicity is not only sensitive and specific but also transferable among laboratories.     

应用胚胎干细胞试验模型对碱性成纤维细胞生长因子发育毒性的初步评价

目的利用小鼠胚胎干细胞试验(EST)模型,初步评价bFGF的发育毒性。方法体外培养小鼠胚胎干细胞(ESC),通过形态学观察、碱性磷酸酶染色和RT-PCR方法进行ESC未分化鉴定;MTT法检测不同浓度bFGF对ESC和3T3细胞的毒性,RT-PCR半定量分析法检测bFGF对未分化基因Sox-2表达的影响。结果 bFGF对ESC和3T3的半数抑制浓度和ESC的半数抑制分化浓度分别为IC50ESC=15.2 mg.L-1,IC50 3T3=24.2 mg.L-1,ID50 ESC=1.7 mg.L-1。结论 bFGF发育毒性的判定为弱胚胎毒性。     

Embryonic stem cells and the next generation of developmental toxicity testing

Introduction: The advent of stem cell technology has seen the establishment of embryonic stem cells (ESCs) as molecular model systems and screening tools. Although ESCs are nowadays widely used in research, regulatory implementation for developmental toxicity testing is pending.

Areas Covered: This review evaluates the performance of current ESC, including human (h)ESC testing systems, trying to elucidate their potential for developmental toxicity testing. It shall discuss defining parameters and mechanisms, their relevance and contemplate what can realistically be expected. Crucially this includes the question of how to ascertain the quality of currently employed cell lines and tests based thereon. Finally, the use of hESCs will raise ethical concerns which should be addressed early on.

Expert Opinion: While the suitability of (h)ESCs as tools for research and development goes undisputed, any routine use for developmental toxicity testing currently still seems premature. The reasons for this comprise inherent biological deficiencies as well as cell line quality and system validation. Overcoming these issues will require collaboration of scientists, test developers and regulators. Also, validation needs to be made worthwhile for academia. Finally we have to continuously rethink existing strategies, making room for improved testing and innovative approaches.     

Disentangling cellular proliferation and differentiation in the embryonic stem cell test, and its impact on the experimental protocol

The mouse embryonic stem cell test (EST) was designed to predict embryotoxicity based on the inhibition of the differentiation of embryonic stem cells (ESC) into beating cardiomyocytes in combination with cytotoxicity data in monolayer ESC cultures and 3T3 cells. In the present study, we have tested a diverse group of chemicals in the EST, applying different exposure durations, in an attempt to discriminate between effects on proliferation and differentiation within the EST protocol. Chemicals tested were monobutyl phthalate (MBP), 6-aminonicotinamide (6-AN), 5-fluorouracil (5-FU) and 5-bromo-2′-deoxyuridine (BrdU). We showed that 5-FU and BrdU behaved principally different from MBP and 6-AN. 5-FU and BrdU specifically affected cell proliferation during the first three days of the EST protocol, as shown by EB size, protein concentration and cell cycle stage analysis. In addition, we studied the differentiation state of cells in the EST protocol with time to elucidate the transition of pluripotent ESC to more differentiated cell types. Analysis by flow cytometry of the pluripotency marker SSEA-1 in EST showed that although total SSEA-1 positive cells remained unchanged up to and including day 5, the signal intensity already decreased from day 3 onwards. Furthermore, RT-PCR data showed an upregulation of the mesodermal marker T at day 3, whereas the cardiac muscle marker Myh6 was upregulated from day 5 onwards. These findings confirm that proliferation and differentiation of ESC in the EST are highly intertwined processes. Based on these findings we suggest an amended EST protocol which could more clearly discriminate between proliferation and differentiation effects of chemicals within the same EST differentiation protocol. This proposal includes a cytotoxicity assessment in EB at day 3 of the EST after day 0–3 exposure, and cardiac muscle foci counts after exposure from day 3–10 in the EST.     

新生小鼠神经干细胞体外培养、分化及鉴定

目的 简化神经干细胞原代培养的取材及步骤,明确体外定向诱导分化条件,为进一步神经干细胞移植相关实验提供基础条件。方法 新生24 h昆明种小鼠无菌条件下冰上取大脑组织,经机械分离加胰酶消化吹打后,加入含有碱性成纤维细胞生长因子、表皮细胞生长因子和B27的DMEM/F12培养基中培养;加入不同浓度胎牛血清诱导其分化,应用免疫荧光技术行巢蛋白、胶质纤维酸性蛋白、微管相关蛋白2染色,对培养细胞鉴定。结果 新生小鼠提取神经干细胞可形成神经球,并稳定增殖传代,经诱导可定向分化为神经元及星形胶质细胞。结论 新生小鼠较胎鼠取材简单,可培养高质量神经干细胞,血清浓度同向星型胶质细胞方向的分化概率呈正相关。     

Defined culture medium for stem cell differentiation: Applicability of serum-free conditions in the mouse embryonic stem cell test

The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST.     

Acute toxicity test of CuO nanoparticles using human mesenchymal stem cells

Despite the growing interest in nanoparticles (NPs), standardized procedures for the evaluation of their toxicity have not been defined. The risk of human exposure is rapidly increasing and reliable toxicity test systems are urgently needed. In vitro methods are ideal in toxicology research because they can rapidly provide reproducible results while preventing the use of animals. Recently, a new test for acute toxicity based on the use of human bone marrow mesenchymal stem cells (hBMMSCs) has been developed and successfully tested in our laboratory following the Interagency Coordinating Committee on the Validation of Alternative Methods guidelines. Along these lines, the aim of this study is to evaluate the acute cytotoxicity of copper oxide (CuO) NPs using the new toxicity test based on hBMMSCs. Our results show that CuO NPs are much more toxic compared to micrometer ones. Specifically, CuO NP exposure exhibits a significant cytotoxicity at all the concentrations used, with an IC₅₀ value of 2.5?±?0.53?µg/ml. On the other hand, CuO microsized particle exposure exhibits a very low cytotoxicity at the same concentrations, with an IC₅₀ value of 72.13?±?16.2?µg/ml.     

Detecting the developmental toxicity of bFGF in the embryonic stem cell test using differential gene expression of differentiation-related genes

Basic fibroblast growth factor (bFGF) is a mitogenic cytokine that can stimulate mesoderm-and neuroectoderm-originated cell proliferation. This study was performed to investigate the effects of bFGF on cell differentiation and the expression of specific markers at different embryonic developmental stages. We firstly evaluated the embryotoxic potential of bFGF in vitro using a modified EST protocol. Sequentially, we further investigated how bFGF impact the different tissue-special genes and proteins expressions during the differentiation of murine ES cells in vitro and attempt to reveal the effects of bFGF on differentiation processes. This analysis was focused on key tissue- and stage-specific genes involved in ectodermal, mesodermal, and endodermal differentiation, including ectodermal-specific gene Nestin, Oligo2 and Syn, mesodermal-specific gene MHC and MyoD, and endodermal-specific gene GATA6, TTR and ALB, as well as undifferentiated gene Sox-2 and Oct-4. The results demonstrate that bFGF could promote expression of ectodermal-specific genes and protein, but suppress the expressions of endoderm-specific and some mesoderm-specific gene and protein. A conclusion can be drawn that bFGF exhibits weak embryotoxicity and mainly promotes ES cell differentiation towards the ectodermal lineages but suppress differentiation into endoderm lineages. These opposing effects of bFGF on the embryonic development of the three germ layers may be related to its weak embryotoxic potential. More specifically, inhibition of expression of the endodermal-specific markers transthyretin (TTR), and albumin (ALB) by bFGF may be of more value in detecting the embryotoxic potential of bFGF.     

Combined repeated-dose toxicity study of silver nanoparticles with the reproduction/developmental toxicity screening test

Combined repeated-dose toxicity study of citrate-capped silver nanoparticles (7.9 ± 0.95 nm) with reproduction/developmental toxicity was investigated in rats orally treated with 62.5, 125 and 250 mg/kg, once a day for 42 days for males and up to 52 days for females. The test was performed based on the Organization for Economic Cooperation and Development test guideline 422 and Good Laboratory Practice principles. No death was observed in any of the groups. Alopecia, salivation and yellow discolouration of the lung were observed in a few rats but the symptoms were not dose-dependent. Haematology, serum biochemical investigation and histopathological analysis revealed no statistically significant differences between control group and the treated groups. Toxicity endpoints of reproduction/developmental screening test including mating, fertility, implantation, delivery and foetus were measured. There was no evidence of toxicity.     

Evaluation of the developmental toxicity of diallyl phthalate administered orally to rats

The objective of this study was to evaluate the developmental toxic potential of diallyl phthalate (DAP) in rats. Pregnant Sprague-Dawley rats were given DAP at doses of 0 (olive oil), 100, 150, 200, and 250 mg/kg/day, by gavage (5 ml/kg), on Gestational Days (GD) 6 through 20. Gross examination at necropsy revealed liver lesions in dams given 150 mg/kg/day or higher doses. In addition, maternal weight gain and food consumption were significantly reduced at 200 and 250 mg/kg/day. There was no significant increase in the incidence of resorptions, or malformations, at any dose. Fetal body weight was significantly reduced at 200 and 250 mg/kg/day. There was a significant increase in the incidence of fetuses with skeletal variations at 250 mg/kg/day. Retarded ossification of certain bones (i.e. forelimb and hindlimb phalanges, metatarsals, and caudal vertebrae) was also observed. There was no sign of developmental toxicity at 100 and 150 mg/kg/day.

Thus, DAP caused fetal toxicity at doses which also produced maternal effects, but no embryolethality or teratogenicity.     

In vitro toxicity assessment of three hydroxylated fullerenes in human skin cells

Carbon fullerenes possess unique properties and their interactions with biomolecules have widespread applications. Functionalization of fullerenes with hydroxyl groups (fullerenols) can increase the solubility and potential for cellular interaction, but the health and safety effects of varying degrees of fullerene hydroxylation in biological systems is poorly understood. Existing reports regarding the toxicity and inflammatory potential of fullerenols give conflicting conclusions. To further elucidate the potential for toxicity of fullerenols, human epidermal keratinocytes (HEK) were exposed to fullerenols (low (C₆₀(OH)₂₀), medium (C₆₀(OH)₂₄), and high (C₆₀(OH)₃₂)) at concentrations ranging from 0.000544–42.5 μg/ml for 24 and 48 h. A statistically significant (p < 0.05) decrease in viability with alamar Blue (aB) was noted only with C₆₀(OH)₃₂ at 42.5 μg/ml after 24 h. Nanoparticle (NP) controls showed minimal NP/assay interference of the three fullerenols with the aB viability assay. Normalized IL-8 concentration for C₆₀(OH)₂₀ was not significantly different from control, while C₆₀(OH)₂₄ and C₆₀(OH)₃₂ showed a significant decrease at 24 and 48 h. These results suggest that different hydroxylation of fullerenes caused no cytotoxicity or inflammation up to 8.55 μg/ml. These findings suggest that extrapolation across similar NP will be dependent upon surface chemistry and concentration which may affect the degree of agglomeration and thus biological effects.     

应用胚胎干细胞实验模型体系对异硫氰酸盐发育毒性的评价

目的应用胚胎干细胞实验模型体系探讨苯甲基异硫氰酸盐(BITC)和苯乙基异硫氰酸盐(PEITC)的发育毒性。方法小鼠胚胎干细胞(ESC)经体外悬滴和悬浮培养分化获得心肌细胞及经视黄酸5×10-7mol·L-1诱导培养分化获得神经细胞,利用RT-PCR方法分析BITC和PEITC0,1,2,4和8μmol·L-1对ESC定向分化为特异表达肌球蛋白重链(MHC)基因的心肌细胞以及特异表达巢蛋白(巢蛋白)基因的神经细胞的影响,计算BITC和PEITC对ESC定向分化能力的抑制作用。同时应用MTT法测定BITC和PEITC对ESC129品系小鼠D3和BALB/c品系小鼠3T3细胞活性的抑制作用,结合定向分化抑制作用结果评价BITC和PEITC的发育毒性。结果 BITC和PEITC对ESC体外心肌细胞定向分化半数抑制浓度(ID50)为3.56和3.48mg·L-1,BITC和PEITC对ESC体外定向分化神经细胞的ID50分别为3.87和2.43mg·L-1,依据发育毒性评价公式计算得BITC和PEITC均无心肌发育毒性作用,BITC和PEITC的神经发育毒性作用分别为无和弱。结论 BITC和PEITC均无心肌发育毒性作用,但PEITC具有弱神经发育毒性作用。     

Comparison of gene expression regulation in mouse- and human embryonic stem cell assays during neural differentiation and in response to valproic acid exposure

Embryonic stem cell tests (EST) are considered promising alternative assays for developmental toxicity testing. Classical mouse derived assays (mEST) are being replaced by human derived assays (hEST), in view of their relevance for human hazard assessment. We have compared mouse and human neural ESTn assays for neurodevelopmental toxicity as to regulation of gene expression during cell differentiation in both assays. Commonalities were observed in a range of neurodevelopmental genes and gene ontology (GO) terms. The mESTn showed a higher specificity in neurodevelopment than the hESTn, which may in part be caused by necessary differences in test protocols. Moreover, gene expression responses to the anticonvulsant and human teratogen valproic acid were compared. Both assays detected pharmacological and neurodevelopmental gene sets regulated by valproic acid. Common significant expression changes were observed in a subset of homologous neurodevelopmental genes. We suggest that these genes and related GO terms may provide good candidates for robust biomarkers of neurodevelopmental toxicity in hESTn.     

An efficient neuron-astrocyte differentiation protocol from human embryonic stem cell-derived neural progenitors to assess chemical-induced developmental neurotoxicity

Human embryonic stem cell neuronal differentiation models provide promising in vitro tools for the prediction of developmental neurotoxicity of chemicals. Such models mimic essential elements of human relevant neuronal development, including the differentiation of a variety of brain cell types and their neuronal network formation as evidenced by specific gene and protein biomarkers. However, the reproducibility and lengthy culture duration of cell models present drawbacks and delay regulatory implementation. Here we present a relatively short and robust protocol to differentiate H9-derived neural progenitor cells (NPCs) into a neuron-astrocyte co-culture. When frozen-stored NPCs were re-cultured and induced into neuron-astrocyte differentiation, they showed gene- and protein expression typical for these cells, and most notably they exhibited spontaneous electrical activity within three days of culture as measured by a multi-well micro-electrode array. Modulating the ratio of astrocytes and neurons through different growth factors including glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) did not compromise the ability to develop spontaneous electrical activity. This robust neuronal differentiation model may serve as a functional component of a testing strategy for unravelling mechanisms of developmental neurotoxicity.     

Surface charge and dosage dependent potential developmental toxicity and biodistribution of iron oxide nanoparticles in pregnant CD-1 mice

Iron oxide nanoparticles have attracted much attention because of their potential applications, such as drug delivery, biomedical imaging, and photocatalysis. Due to their small size and the potential to cross the placental barrier, the risk to pregnant women and the developing fetus from exposure to nanoparticles is of great concern. The developmental toxicity and biodistribution of a single dose versus multiple doses of iron oxide nanoparticles with positive or negative surface charges were investigated in vivo. Multiple doses of positively-charged nanoparticles given over several days resulted in significantly increased fetal deaths and accumulation of iron in the fetal liver and placenta. These results indicate both positively and negatively charged iron oxide nanoparticles have the ability to cross the placenta and accumulate in the fetus, though greater bioaccumulation and toxicity was observed with a positively-charged surface coating.