Carmen Estevan

An integrated approach for detecting embryotoxicity and developmental toxicity of environmental contaminants using in vitro alternative methods

The main available alternatives for testing embryotoxicity are cellular tests with stem cells and in vitro-ex vivo tests with embryos. In cellular tests, the most developed alternative is the embryonic stem cell test, while the most developed tests involving embryos are the zebrafish and whole embryo culture test. They are technically more complex than cellular tests, but offer the advantage of determining the expectable phenotypic alteration caused by the exposure. Many efforts are currently being made, basically through proteomic and genomic approaches, in order to obtain improvements in predictivity of these tests. Development is a very complex process, and it is highly unlikely that a single alternative test can yield satisfactory performance with all types of chemicals. We propose a step-wise approach where model complexity, and consequently technical skills and economical costs, gradually increase if needed. The first level would be run short cellular assays to detect effects in early differentiation stages. The second level would involve longer cellular embryotoxicity tests to search embryotoxicants that have an effect on late differentiation stages. The third stage would consider tests with embryos because they allow the determination of hazards based on molecular and morphological alterations, and not only on differentiating cells.

Chlorpyrifos and its metabolites alter gene expression at non-cytotoxic concentrations in D3 mouse embryonic stem cells under in vitro differentiation: considerations for embryotoxic risk assessment.

The effects of organophosphate insecticide chlorpyrifos (CPF) on development are currently under discussion. CPF and its metabolites, chlorpyrifos-oxon (CPO) and 3,5,6-trichloro-2-pyridinol (TClP), were more cytotoxic for D3 mouse embryonic stem cells than for differentiated fibroblasts 3T3 cells. Exposure to 10 μM CPF and TClP and 100 μM CPO for 12 h significantly altered the in vitro expression of biomarkers of differentiation in D3 cells. Similarly, exposure to 20 μM CPF and 25 μM CPO and TClP for 3 days also altered the expression of the biomarkers in the same model. These exposures caused no significant reduction in D3 viability with mild inhibition of acetylcholinesterase and neuropathy target esterase by CPF and severe inhibition by CPO. We conclude that certain in vivo exposure scenarios are possible, which cause inhibition of acetylcholinesterase but without clinical symptoms that reach high enough systemic CPF concentrations able to alter the expression of genes involved in cellular differentiation with potentially hazard effects on development. Conversely, the risk for embryotoxicity by CPO and TClP was very low because the required exposure would induce severe cholinergic syndrome.

Organophosphorus pesticide chlorpyrifos and its metabolites alter the expression of biomarker genes of differentiation in D3 mouse embryonic stem cells in a comparable way to other model neurodevelopmental toxicants.

There are discrepancies about whether chlorpyrifos is able to induce neurodevelopmental toxicity or not. We previously reported alterations in the pattern of expression of biomarker genes of differentiation in D3 mouse embryonic stem cells caused by chlorpyrifos and its metabolites chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol. Now, we reanalyze these data comparing the effects on these genes with those caused in the same genes by retinoic acid, valproic acid, and penicillin-G (model compounds considered as strong, weak, and non-neurodevelopmental toxicants, respectively). We also compare the effects of chlorpyrifos and its metabolites on the cell viability of D3 cells and 3T3 mouse fibroblasts with the effects caused in the same cells by the three model compounds. We conclude that chlorpyrifos and its metabolites act, regarding these end-points, as the weak neurodevelopmental toxicant valproic acid, and consequently, a principle of caution should be applied avoiding occupational exposures in pregnant women. A second independent experiment run with different cellular batches coming from the same clone obtained the same result as the first one.

Cytotoxic effect against 3T3 fibroblasts cells of saffron floral bio-residues extracts.

For every kilogram of saffron spice produced, about 63 kg of floral bio-residues (FB) (tepals, stamens and styles) are thrown away. Extracts of these bio-residues in water (W1), water:HCl (100:1, v/v) (W2), ethanol (E3), ethanol:HCl (100:1, v/v) (E4), dichloromethane (D5) and hexane (H6) were prepared. Their composition in flavonols and anthocyanins, and their effect on cell viability were determined. W1 was the richest in kaempferol 3-sophoroside (30.34 mg/g dry FB) and delphinidin 3,5-diglucoside (15.98 mg/g dry FB). The highest tested concentration (900 μg/ml) of W1, W2, E4, D5 and H6 did not significantly decrease the cell viability. Only E3 at that concentration caused a significant decrease of 38% in the cell viability. Therefore, all extracts studied are not cytotoxic at concentrations lower than 900 μg/ml, and W1 is proposed as the optimal for food applications due to its greater contribution of phenolic compounds.

Embryonic Stem Cells in Toxicological Studies

1.1 Toxicity assays For most of their lives, humans are exposed to a variety of chemicals from different sources. The characterization of chemicals’ toxicity implies the necessity to have available reliable testing methods to assess their capability to produce adverse effects on living organisms. Thus, it is necessary to acquire a battery of relevant internationally agreed methods to be employed by governments, industry and independent laboratories to assess the safety of chemicals. Presently, a set of validated tests is widely used to analyze the toxicity of chemicals and pharmaceutical drugs and to ensure their safety, and is recognized for this purpose (OECD, 2008; Stummann et al., 2009). OECD Guidelines require in vivo studies to comply with the human health risk assessment process before registering and authorizing the use of chemicals. Toxicity guidelines characterize the adverse effects against the target organism that are attributable to exposure to the tested chemical with two main purposes in mind: the hazard identification and risk assessment deriving from exposure to the assessed chemical (Estevan et al., 2011). Currently, the OECD has at least one in vivo validated guideline for testing the following endpoints: skin sensitization, skin and eye irritation and corrosion, mutagenicity, acute toxicity, target organ systemic toxicity (repeated daily dose for 28 days, 90 days, and for more than 12 months), carcinogenicity and toxicity to reproduction (screening, teratogenicity and two generations). All these OECD Guidelines have to be usually applied for a safe and consistent risk assessment, even when the chemical’s hazard is not related to the tested target system. In this way, some of the most relevant tests are reproduction/developmental studies because the endpoints assessed in these protocols and the non-observed-adverse-effect levels (NOAELs) obtained are typically taken into account not only for reproductive toxicity testing, but also for the risk assessment of general systemic effects. For example, the NOAELs of systemic maternal toxicity in the teratogenicity study are frequently considered for risk characterization in short-term exposure scenarios. Guidelines for testing toxicity to reproduction are also highly relevant because the effects of chemicals or drugs on germ cells or early embryos may lead to infertility or impaired development of pre-implantation embryos, and might result in embryotoxic or teratogenic

Roles of NTE protein and encoding gene in development and neurodevelopmental toxicity

Neuropathy Target Esterase (NTE) is a membrane protein codified by gene PNPLA6. NTE was initially discovered as a target of the so-called organophosphorus-induced delayed polyneuropathy triggered by the inhibition of the NTE-associated esterase center by neuropathic organophosphorus compounds (OPs). The physiological role of NTE might be related to membrane lipid homeostasis and seems to be involved in adult organisms in maintaining nervous system integrity. However, NTE is also involved in cell differentiation and embryonic development. NTE is expressed in embryonic and adult stem cells, and the silencing of Pnpla6 by interference RNA in D3 mouse cells causes significant alterations in several genetic pathways related to respiratory tube and nervous system formation, and in vasculogenesis and angiogenesis. The silencing of gene PNPLA6 in human NT2 cells at the beginning of neurodifferentiation causes severe phenotypic alterations in neuron-like differentiated cells; e.g. reduced electrical activity and the virtual disappearance of markers of neural tissue, synapsis and glia. These phenotypic effects were not reproduced when NTE esterase activity was inhibited by neuropathic OP mipafox instead of being silenced at the genetic level. Neuropathic OP chlorpyrifos seems able to induce neurodevelopmental alterations in animals. However, the effects of chlorpyrifos in the expression of biomarker genes of differentiation in D3 cells differ considerably from the effects induced by Pnpla6 silencing. In conclusion, available information suggests that PNPLA6 and/or the NTE protein play a role in early neurodifferentiation stages, although this role is not dependent upon the esterase NTE center. Therefore, impairments caused by OPs, such as chlorpyrifos, on neurodevelopment are not due to inhibition of NTE esterase enzymatic activity.

Validated and Nonvalidated Mechanism-Based Methods for Testing Developmental Toxicity

There are several methods alternative to animal experimentation for testing developmental toxicity. Among the array of available methods only three have overcome a blind validation study. These are the cellular methods called embryonic stem cell tests and micromass test and the test with embryos called whole embryo culture test. Other nonvalidated alternative embryotoxicity tests using embryos are the frog embryo teratogenesis assay and specially the developmental toxicity assay with zebrafish, which is the most promising so far. There are also many promising but nonvalidated cellular tests, all of them based on testing the impairments induced by chemicals on stem cells from different biological sources on cellular differentiation. A very important part of these cellular methods is devoted to testing developmental neurotoxicity. The main advantage of methods using embryos is that they allow determining the morphological impairment caused by the toxicant, whereas cellular methods are usually more appropriate for determining mechanisms of toxicity.