Telomere length has been proposed as a marker of mitotic cell age and as a general index of human... more Telomere length has been proposed as a marker of mitotic cell age and as a general index of human organism aging. Recent studies have documented the role of telomere dysfunction in breast carcinoma [1] the presence of parabens were detected in the human breast at concentrations up to the microgram per gram tissue range [2]. The impact of telomere status on the response to chemical genotoxic agents has not been studied to any great extent. In our Porcheville facility1, we evaluated the effects of ethyl 4-hydroxybenzoate (antibacterial/antifungal agent used in cosmetic preparations) in a battery of screening and Regulatory in vitro genotoxicity tests. We also investigated the potential correlation of Ethyl 4-hydroxybenzoate (Ethyl Paraben) exposure in the presence and in the absence of metabolic activation (S-9), with the mutagenic effect and telomere dysfunction
Since March 2004, publication date of Directive 2004/24/EC [1] establishing an European legislati... more Since March 2004, publication date of Directive 2004/24/EC [1] establishing an European legislation for traditional herbal medicinal products (THMPs), new common quality, safety and efficacy standards for licensing HMPs have been progressively implemented including the assessment of genotoxic potential. HMPs present a number of characteristics that clearly differentiate them from other medicinal products, including a complex composition with known and unknown constituents. Genotoxic carcinogens present in food or medicinal plants are well known such as safrole, estragole and methyleugenol. Questions dealing with non-clinical safety of herbal medicines were highlighted for the first time in a guideline issued by the EMA in 2006, stressing that “the genotoxic potential of herbal preparations should be assessed”[2]. Guideline EMEA/HMPC/107079/2007 [3] is used for testing the potential genotoxicity of THMPs. This is a stepwise procedure for assessing (T)HMPs genotoxicity outlined in the...
F.Finot1, L.Morat2, A.Essahli1, N.Prigent1, O.Cariou1, F.Paillard1, L.Heidingsfelder3, M.Ricoul2,... more F.Finot1, L.Morat2, A.Essahli1, N.Prigent1, O.Cariou1, F.Paillard1, L.Heidingsfelder3, M.Ricoul2, W.Hemplel2, R.M’kacher2, L.Sabatier2 1 Covance Laboratory SAS, Porcheville, France 2Radiobiology and Oncology laboratory, CEA, DSV/iRCM, Fontenay-aux-Roses, France 3MetaSystems GmbH, Robert-Bosch-Str. 6 D-68804. Altlussheim, Germany A new tool for genotoxic risk: Quantification of Micronucleus and telomere length following telomere and centromere FISH painting. Distinguishing between clastogens and aneugens is necessary in order to evaluate genotoxic risk. The cytokinesis-block micronucleus (CBMN) assay is often used for determining the genotoxic potential of a substance. Micronuclei (MN) can be formed in dividing cells that contain either acentric or whole chromosomes. The introduction of simultaneous staining of telomeres and centromeres using peptide nucleic acid (PNA) probes to shorten hybridization time and to increase signal intensity, has rendered the scoring of different types o...
The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated l... more The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated liver injuries while apoptosis and proliferation remain unexplored in this culture model. Here, we show a transient increase in LDH release and caspase activities indicating an ischemic injury during the slicing procedure. Then, caspase activities decrease and remain low in cultured slices demonstrating a low level of apoptosis. The slicing procedure is also associated with the G0/G1 transition of hepatocytes demonstrated by the activation of stress and proliferation signalling pathways including the ERK1/2 and JNK1/2/3 MAPKinases and the transient upregulation of c-fos. The cells further progress up to mid-G1 phase as indicated by the sequential induction of c-myc and p53 mRNA levels after the slicing procedure and at 24 h of culture, respectively. The stimulation by epidermal growth factor induces the ERK1/2 phosphorylation but fails to activate expression of late G1 and S phase markers...
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2020
BACKGROUND The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized metho... more BACKGROUND The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.
Cosmetic products need to have a proven efficacy combined with a comprehensive toxicological asse... more Cosmetic products need to have a proven efficacy combined with a comprehensive toxicological assessment. Before the current Cosmetic regulation N°1223/2009, the 7th Amendment to the European Cosmetics Directive has banned animal testing for cosmetic products and for cosmetic ingredients in 2004 and 2009, respectively. An increasing number of alternatives to animal testing has been developed and validated for safety and efficacy testing of cosmetic products and cosmetic ingredients. For example, 2D cell culture models derived from human skin can be used to evaluate anti-inflammatory properties, or to predict skin sensitization potential; 3D human skin equivalent models are used to evaluate skin irritation potential; and excised human skin is used as the gold standard for the evaluation of dermal absorption. The aim of this manuscript is to give an overview of the main in vitro and ex vivo alternative models used in the safety testing of cosmetic products with a focus on regulatory re...
Telomere length has been proposed as a marker of mitotic cell age and as a general index of human... more Telomere length has been proposed as a marker of mitotic cell age and as a general index of human organism aging. Recent studies have documented the role of telomere dysfunction in breast carcinoma [1] the presence of parabens were detected in the human breast at concentrations up to the microgram per gram tissue range [2]. The impact of telomere status on the response to chemical genotoxic agents has not been studied to any great extent. In our Porcheville facility1, we evaluated the effects of ethyl 4-hydroxybenzoate (antibacterial/antifungal agent used in cosmetic preparations) in a battery of screening and Regulatory in vitro genotoxicity tests. We also investigated the potential correlation of Ethyl 4-hydroxybenzoate (Ethyl Paraben) exposure in the presence and in the absence of metabolic activation (S-9), with the mutagenic effect and telomere dysfunction
Since March 2004, publication date of Directive 2004/24/EC [1] establishing an European legislati... more Since March 2004, publication date of Directive 2004/24/EC [1] establishing an European legislation for traditional herbal medicinal products (THMPs), new common quality, safety and efficacy standards for licensing HMPs have been progressively implemented including the assessment of genotoxic potential. HMPs present a number of characteristics that clearly differentiate them from other medicinal products, including a complex composition with known and unknown constituents. Genotoxic carcinogens present in food or medicinal plants are well known such as safrole, estragole and methyleugenol. Questions dealing with non-clinical safety of herbal medicines were highlighted for the first time in a guideline issued by the EMA in 2006, stressing that “the genotoxic potential of herbal preparations should be assessed”[2]. Guideline EMEA/HMPC/107079/2007 [3] is used for testing the potential genotoxicity of THMPs. This is a stepwise procedure for assessing (T)HMPs genotoxicity outlined in the...
F.Finot1, L.Morat2, A.Essahli1, N.Prigent1, O.Cariou1, F.Paillard1, L.Heidingsfelder3, M.Ricoul2,... more F.Finot1, L.Morat2, A.Essahli1, N.Prigent1, O.Cariou1, F.Paillard1, L.Heidingsfelder3, M.Ricoul2, W.Hemplel2, R.M’kacher2, L.Sabatier2 1 Covance Laboratory SAS, Porcheville, France 2Radiobiology and Oncology laboratory, CEA, DSV/iRCM, Fontenay-aux-Roses, France 3MetaSystems GmbH, Robert-Bosch-Str. 6 D-68804. Altlussheim, Germany A new tool for genotoxic risk: Quantification of Micronucleus and telomere length following telomere and centromere FISH painting. Distinguishing between clastogens and aneugens is necessary in order to evaluate genotoxic risk. The cytokinesis-block micronucleus (CBMN) assay is often used for determining the genotoxic potential of a substance. Micronuclei (MN) can be formed in dividing cells that contain either acentric or whole chromosomes. The introduction of simultaneous staining of telomeres and centromeres using peptide nucleic acid (PNA) probes to shorten hybridization time and to increase signal intensity, has rendered the scoring of different types o...
The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated l... more The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated liver injuries while apoptosis and proliferation remain unexplored in this culture model. Here, we show a transient increase in LDH release and caspase activities indicating an ischemic injury during the slicing procedure. Then, caspase activities decrease and remain low in cultured slices demonstrating a low level of apoptosis. The slicing procedure is also associated with the G0/G1 transition of hepatocytes demonstrated by the activation of stress and proliferation signalling pathways including the ERK1/2 and JNK1/2/3 MAPKinases and the transient upregulation of c-fos. The cells further progress up to mid-G1 phase as indicated by the sequential induction of c-myc and p53 mRNA levels after the slicing procedure and at 24 h of culture, respectively. The stimulation by epidermal growth factor induces the ERK1/2 phosphorylation but fails to activate expression of late G1 and S phase markers...
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2020
BACKGROUND The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized metho... more BACKGROUND The cytokinesis-block micronucleus (CBMN) assay is an internationally recognized method for measuring DNA damage after exposure to genotoxic agents, as well as a biomarker for DNA repair and chromosomal instability. The high baseline level of micronuclei (MN) in the healthy population has limited the sensitivity and application of the CBMN assay for the follow-up of exposed populations. We reevaluated the sensitivity of the CBNM assay using semi-automated MN scoring following telomere and centromere (TC) staining after in vitro exposure to genotoxic agents (mitomycin or radiation) or aneugenic agents (vinblastine). MATERIALS AND METHODS Blood samples from 12 healthy donors were exposed to 137Cs at seven doses from 0.1-4 Gy and cultured for 72 h. Cytochalasin B was added at 46 h of culture. The exposure of chemical agents (mitomycin or vinblastine) was performed after 48 h of culture for 3 h. Cytochalasin B was added after treatment and slides were prepared 24 h after. MN was semi-automatically scored following TC staining. Nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) were assessed in a human cell line after TC staining. RESULTS The introduction TC staining to the scoring of MN not only renders MN scoring more efficient and robust, but also permits discrimination between exposure to clastogenic (MN with only telomere signals) and aneugenic agents (MN with both TC signals). The resulting improvement of MN detection led to an increase in the sensitivity of the CBMN assay following low-dose radiation exposure (0.3 versus 0.1 Gy). Hyperradiosensitivity phenomenon was observed after low dose exposure. A dose-response curve was obtained for up to 4 Gy. In addition, TC staining permits assessment of the nature of NPBs and NBUDs as biomarkers for genotoxicity and chromosomal instability. CONCLUSION These approaches can be potentially used to follow-up populations exposed to genotoxic agents and assess cancer risk.
Cosmetic products need to have a proven efficacy combined with a comprehensive toxicological asse... more Cosmetic products need to have a proven efficacy combined with a comprehensive toxicological assessment. Before the current Cosmetic regulation N°1223/2009, the 7th Amendment to the European Cosmetics Directive has banned animal testing for cosmetic products and for cosmetic ingredients in 2004 and 2009, respectively. An increasing number of alternatives to animal testing has been developed and validated for safety and efficacy testing of cosmetic products and cosmetic ingredients. For example, 2D cell culture models derived from human skin can be used to evaluate anti-inflammatory properties, or to predict skin sensitization potential; 3D human skin equivalent models are used to evaluate skin irritation potential; and excised human skin is used as the gold standard for the evaluation of dermal absorption. The aim of this manuscript is to give an overview of the main in vitro and ex vivo alternative models used in the safety testing of cosmetic products with a focus on regulatory re...
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