Laboratory of Environmental and Occupational Toxicology
(Head Dr. Luisa Campagnolo e Prof. Antonio Pietroiusti)
The Laboratory of Environmental and Occupational Toxicology was created about seven years ago by the active collaboration between the Histology and Embryology Section and the Occupational Medicine Section of the Department of Biomedicine and Prevention. The laboratory offers a series of in vitro tests, validated by the European Committee for the Validation of Alternative Methods (ECVAM), for the determination of the toxic potential of different substances and particulate matter, including engineered nanoparticles. The intensive research work that characterizes the lab is aimed at the development and upgrading of toxicological tests currently available. That is why our highly qualified staff is dedicated to the development of further tests for “high-throughput screening” of potentially embryotoxic substances.
The advantage of developmental toxicology tests lies in their high sensitivity. Embryonic cells are, in fact, more sensitive to xenobiotic substances than adult cells, thus allowing a more precise assessment of the toxic potential of a specific substance than differentiated cell testing.
The laboratory has all the necessary equipment and employs highly specialized staff, with years of experience in this field. The skills acquired so far have allowed the laboratory to participate in various European projects of the Seventh Framework Program (EU-FP7), including the NANoREG (A Common European Approach to the Regulatory Testing of Manufactured Nanomaterials) project, which was aimed at providing information regarding the biological impact of nanomaterials for regulatory purposes.
The services offered by the Laboratory of Environmental and Occupational Toxicology include:
Embryonic Stem Cell Test
The Embryonic Stem Cell Test (EST) is an ECVAM validated test and is normally used in the pharmaceutical and industrial field to predict the embryotoxic potential of chemical substances. Although it was recently recognized that this test alone cannot be used for regulatory purposes, it allows for the preliminary screening of a large number of substances, allowing the identification of those that require further in-depth study with in vitro and in vivo tests. The EST is based on the determination of the most important embryotoxic parameters: the inhibition of cell differentiation is combined with the study of the different sensitivity between embryonic and adult tissues to cytotoxic damage. In particular, EST uses two cell lines, mouse embryonic stem cells (mES to represent embryonic tissue) and 3T3 cells (mouse fibroblasts to represent adult tissue), and evaluates three endpoints: inhibition of mES differentiation in embryoid bodies (in vitro structures that roughly ricapitulate the embryonic development), and inhibition of the proliferation of mES and 3T3 cells. The numerical values obtained from these experiments are then combined into an algorithm that determines the degree of embryotoxicity (strong, weak, and non-embryotoxic). Complete results about the embryotoxic potential of a particular substance can be obtained in about 15 to 30 days.
The EST was recently developed and successfully used for the evaluation of the embryotoxicity of particles with a diameter shorter than 0.1 micron (Pietroiusti et al., 2011; Campagnolo et al., 2013; Farcal et al., 2015).
Modified Embryonic Stem Cell Test
It has been recognized that during the in vitro differentiation of embryonic stem cells (ES) into embryoid bodies (EB), genes are expressed in a controlled manner in space and time, reproducing what happens in the embryo in vivo (Leahy et al., 1999). The modified EST is based on the analysis of the possible temporal modification in the expression of markers of differentiation, caused by a specific substance.
The test consists of a screening platform using Multiplex Real-Time PCR that allows the evaluation in EBs, either exposed or not to potentially toxic substances, of the differential expression of a selected genes panel, that are expressed in a time-controlled manner during the differentiation. The analysis includes the study of the expression of genes commonly used as early markers for the specification of the three germ layers and of genes identifying the differentiation of the various cell lines. As marker genes of the three germ layers, oct-3 and Fgf-5 (ectodermic markers; Haub and Goldfarb, 1991), GATA-4 (primer endoderm marker and endodermic tissues markers; Arceci et al., 1993), nodal (which, in the embryo, marks the primitive streak; Zhou et al., 1993), and Brachyury T (early mesoderm marker; Herrmann, 1991) are analyzed. As markers of the various lines, flk-1, as markers of endothelial cells and their precursors (Yamaguchi et al., 1993), Nkx-2.5, cardiogenic mesoderm marker (Lints et al., 1993), EKLF, erythroid line marker (Southwood et al., 1996), and Msx3, which marks the neural tube (Wang et al., 1996) will be analyzed. Comparison of gene expression analysis during the EBs differentiation (from day 3 to day 15) in the study, either in presence or not of a substance, allows to understand whether this can disrupt normal embryonic development.
Examination of Embryotoxicity by Embryotoxicity Assay (ETA)
This test, based on the in vitro culture of pre-implant embryos (blastocysts) of the mouse, evaluates the ability of a substance to interfere with the normal blastocyst release process from the pellucid region (shedding), the subsequent adhesion to the culture plate and the outgrowth. The test was originally developed to evaluate the presence of embryotoxins in the serum of patients affected by recurrent miscarriages, and was subsequently used in a wider toxicological range. Blastocysts are harvested, after hormonal stimulation, from animals at 3.5 days into the pregnancy, are randomly divided into groups of at least 20 and cultured in the presence of the substances which are being studied. The cultures are checked and photographed at regular intervals for the next 72 hours. In our laboratory, this test has been applied to the screening of embryotoxic potential of engineered nanoparticles, in combination with EST.
Rat embryo culture test
The post-implant rat embryo test was developed to identify substances inducing malformations and consequent embryotoxicity; Given its complexity, this test cannot be used as a large-scale screening method, but rather as a secondary approach to a limited number of substances identified as potentially toxic through other tests, to assess the priority for in vivo studies. The test is based on the use of rat embryos at a level of 1-5 somites, which are maintained in rotating cultures for 48 hours, during which the major organogenic events occur (e.g. neural tube closure, eye and ear development). At the end of the growth, the comparison between the controls and embryos cultivated in the presence of the substances being studied allows to point out any alterations in the development.
The advantage of developmental toxicology tests lies in their high sensitivity. Embryonic cells are, in fact, more sensitive to xenobiotic substances than adult cells, thus allowing a more precise assessment of the toxic potential of a specific substance than differentiated cell testing.
The laboratory has all the necessary equipment and employs highly specialized staff, with years of experience in this field. The skills acquired so far have allowed the laboratory to participate in various European projects of the Seventh Framework Program (EU-FP7), including the NANoREG (A Common European Approach to the Regulatory Testing of Manufactured Nanomaterials) project, which was aimed at providing information regarding the biological impact of nanomaterials for regulatory purposes.
The services offered by the Laboratory of Environmental and Occupational Toxicology include:
Embryonic Stem Cell Test
The Embryonic Stem Cell Test (EST) is an ECVAM validated test and is normally used in the pharmaceutical and industrial field to predict the embryotoxic potential of chemical substances. Although it was recently recognized that this test alone cannot be used for regulatory purposes, it allows for the preliminary screening of a large number of substances, allowing the identification of those that require further in-depth study with in vitro and in vivo tests. The EST is based on the determination of the most important embryotoxic parameters: the inhibition of cell differentiation is combined with the study of the different sensitivity between embryonic and adult tissues to cytotoxic damage. In particular, EST uses two cell lines, mouse embryonic stem cells (mES to represent embryonic tissue) and 3T3 cells (mouse fibroblasts to represent adult tissue), and evaluates three endpoints: inhibition of mES differentiation in embryoid bodies (in vitro structures that roughly ricapitulate the embryonic development), and inhibition of the proliferation of mES and 3T3 cells. The numerical values obtained from these experiments are then combined into an algorithm that determines the degree of embryotoxicity (strong, weak, and non-embryotoxic). Complete results about the embryotoxic potential of a particular substance can be obtained in about 15 to 30 days.
The EST was recently developed and successfully used for the evaluation of the embryotoxicity of particles with a diameter shorter than 0.1 micron (Pietroiusti et al., 2011; Campagnolo et al., 2013; Farcal et al., 2015).
Modified Embryonic Stem Cell Test
It has been recognized that during the in vitro differentiation of embryonic stem cells (ES) into embryoid bodies (EB), genes are expressed in a controlled manner in space and time, reproducing what happens in the embryo in vivo (Leahy et al., 1999). The modified EST is based on the analysis of the possible temporal modification in the expression of markers of differentiation, caused by a specific substance.
The test consists of a screening platform using Multiplex Real-Time PCR that allows the evaluation in EBs, either exposed or not to potentially toxic substances, of the differential expression of a selected genes panel, that are expressed in a time-controlled manner during the differentiation. The analysis includes the study of the expression of genes commonly used as early markers for the specification of the three germ layers and of genes identifying the differentiation of the various cell lines. As marker genes of the three germ layers, oct-3 and Fgf-5 (ectodermic markers; Haub and Goldfarb, 1991), GATA-4 (primer endoderm marker and endodermic tissues markers; Arceci et al., 1993), nodal (which, in the embryo, marks the primitive streak; Zhou et al., 1993), and Brachyury T (early mesoderm marker; Herrmann, 1991) are analyzed. As markers of the various lines, flk-1, as markers of endothelial cells and their precursors (Yamaguchi et al., 1993), Nkx-2.5, cardiogenic mesoderm marker (Lints et al., 1993), EKLF, erythroid line marker (Southwood et al., 1996), and Msx3, which marks the neural tube (Wang et al., 1996) will be analyzed. Comparison of gene expression analysis during the EBs differentiation (from day 3 to day 15) in the study, either in presence or not of a substance, allows to understand whether this can disrupt normal embryonic development.
Examination of Embryotoxicity by Embryotoxicity Assay (ETA)
This test, based on the in vitro culture of pre-implant embryos (blastocysts) of the mouse, evaluates the ability of a substance to interfere with the normal blastocyst release process from the pellucid region (shedding), the subsequent adhesion to the culture plate and the outgrowth. The test was originally developed to evaluate the presence of embryotoxins in the serum of patients affected by recurrent miscarriages, and was subsequently used in a wider toxicological range. Blastocysts are harvested, after hormonal stimulation, from animals at 3.5 days into the pregnancy, are randomly divided into groups of at least 20 and cultured in the presence of the substances which are being studied. The cultures are checked and photographed at regular intervals for the next 72 hours. In our laboratory, this test has been applied to the screening of embryotoxic potential of engineered nanoparticles, in combination with EST.
Rat embryo culture test
The post-implant rat embryo test was developed to identify substances inducing malformations and consequent embryotoxicity; Given its complexity, this test cannot be used as a large-scale screening method, but rather as a secondary approach to a limited number of substances identified as potentially toxic through other tests, to assess the priority for in vivo studies. The test is based on the use of rat embryos at a level of 1-5 somites, which are maintained in rotating cultures for 48 hours, during which the major organogenic events occur (e.g. neural tube closure, eye and ear development). At the end of the growth, the comparison between the controls and embryos cultivated in the presence of the substances being studied allows to point out any alterations in the development.