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New study design considerations for somatic and germ cell analysis in the Big Blue® transgenic rodent mutation assay
S130
Abstracts / Toxicology Letters 221S (2013) S59–S256
Hamster Ovary (CHO) cells and the in vitro genotoxic and antigenotoxic effects of VA were assessed by cytokinesis-blocked micronucleus and alkaline comet assays in human lymphocytes. At all tested concentrations (2–400 M) VA was found to have an antioxidant activity and also at the concentrations of VA showed no cytotoxic effects on CHO cells. No genotoxic effect was observed at all tested concentrations (5–1000 M) of VA both in micronucleus and comet assays in human lymphocytes. VA caused a significant reduction in the frequency of micronuclei and the extent of DNA damage induced by H2 O2 especially at high concentrations when compared to cultures treated with H2 O2 only. These results have suggested the protective effects of vanillic acid against H2 O2 induced DNA damage in human lymphocytes. http://dx.doi.org/10.1016/j.toxlet.2013.05.241
P10-17 New study design considerations for somatic and germ cell analysis in the Big Blue® transgenic rodent mutation assay Robert R. Young a,∗ , David Bruning a , Marilyn J. Aardema b a BioReliance Corporation, Rockville, MD, USA, b Marilyn Aardema Consulting, LLC, Chief Scientific Advisor BioReliance Corp., USA
The Big Blue® Transgenic Rodent Mutation (TRM) assay is an in vivo test for somatic and germ cell mutagenicity. The recently finalized OECD Test Guideline 488 for TRM assays, ECHA’s use of TRM assays to investigate in vivo mutagenicity for REACH registrations, and ECHA’s preference for TRM over UDS assays (ECHA-13-R-01-EN, 2013) have led to renewed focus on TRM assays. A recent ECHA review (ECHA/2011/217, 2012) indicated that there are a limited number of laboratories available to conduct TRM assays. To meet the scientific and regulatory need for this assay, BioReliance, co-developer of the Big Blue® assay has obtained the rights to the Big Blue® mouse and rat lines and is requalifying the cII Big Blue mutation assay to OECD TG 488 standards. There are recent scientific revisions proposed to OECD TG 488 (G. Douglas, Personal communication) that include using cauda epididymis instead of vas deferens and using seminiferous tubules and cauda at both 3 day sampling and 49 day sampling time instead of 42 days for mouse germ cells. This new study design is being used in BioReliance’s qualification studies. To date, the background cII mutation frequency in liver in our laboratory is 27.8 ± 12.6 × 10−5 which is in line with other laboratories (M. Manjanatha, FDA NCTR, Personal Communication, 2013). Results for vehicle and N-ethyl-Nnitrosourea (ENU) treatment in liver, bone marrow, seminiferous tubules and cauda epididymis will be presented following proposed OECD 488 study design changes. http://dx.doi.org/10.1016/j.toxlet.2013.05.242
P10-18 Noncanonical activation of ATR-p53 axis by DNA–protein crosslinks controls cell death responses to formaldehyde Anatoly Zhitkovich ∗ , Victor Wong, Jessica Morse Brown University, Providence, RI 02921, USA We examined how human cells respond to DNA–protein crosslinks (DPC) formed by carcinogenic formaldehyde (FA). We found that FA caused a rapid activation of the DNA damage-
responsive ATR kinase that preferentially targeted the p53 transcription factor at moderate damage levels (bell-shaped response) and CHK1 kinase at higher stress levels (sublinear response). Inhibition of mismatch repair, nucleotide and base excision repair did not impact p53 activation, arguing against the role of small adducts in this response. The p53-targeting signaling occurred exclusively in S phase of cycling populations. DPC-activated p53 played a major role in the fate determination of FA-treated cells by inducing senescence in lung fibroblasts and promoting apoptosis in lung epithelial cells. S phase-arrested cells tested negative for ssDNA and p53 phosphorylation was TopBP1-independent, pointing to a novel mode of ATR activation. FA was also found to disrupt ATR association with the replicative helicase MCM. These results argue that unlike typical DNA adducts that stall elongating DNA polymerases, superbulky DPC block duplex unwinding by MCM helicase, which prevents accumulation of ssDNA that is required for the canonical route of ATR activation. Overall, our findings indicate that S phase-specific, ssDNA-independent activation of the ATR-p53 axis is the main stress signaling response to FA-DPC in human cells. Chronic exposure to cytotoxic doses of FA can lead to a selection of resistant cells with deficient p53, providing an explanation for the origin of p53 mutations by this weakly mutagenic carcinogen. http://dx.doi.org/10.1016/j.toxlet.2013.05.243
P10-19 Preclinical safety assessment of aqueous Gentiana lutea radix extracts Claudia Turek ∗ , Nora Mörbt, Jennifer Felenda, Kerstin Link, Beckmann Christiane, Margit Müller, Peter Vögele, Florian Stintzing WALA Heilmittel GmbH, Bad Boll, Germany Gentiana lutea L. (yellow gentian) is a perennial herb with long roots and yellow flowers, indigenous to mountainous regions of Europe. Broadly applied as pharmaceutically active substance, preparations from the underground organs are indicated for the treatment of several digestive disorders due to bitter substances present in Gentianae radix, such as the secoiridoid glycoside gentiopicroside. However, in its assessment report on Gentiana lutea L., radix, the Committee on Herbal Medicinal Products of the European Medicines Agency (EMA/HMPC/578322/2008, 12th November 2009, pp. 1–19) raised concern about a potential mutagenicity of this herbal substance. Owing to positive results in Ames tests with isolated components of gentian root, the committee requested to initiate in vitro studies on mammalian cells and assessment of potential genotoxic compounds. Therefore, in the present study, aqueous extracts of Gentiana lutea L., radix were analysed for the content of gentiopicroside as well as the xanthones gentisine and isogentisine using high performance liquid chromatography/diode-array detection. In a second step, extracts were subjected both to a bacterial and a mammalian mutation assay according to the “Guideline on the assessment of genotoxicity of herbal medicinal substances/preparations” (EMEA/HMPC/107079/2007, 21st May 2008, pp. 1–12). In order to assess the mutagenic potential, the experiments were conducted with and without exogenous metabolic activation using different concentration levels of the test items in compliance with current OECD guidelines.
Abstracts / Toxicology Letters 221S (2013) S59–S256
Hamster Ovary (CHO) cells and the in vitro genotoxic and antigenotoxic effects of VA were assessed by cytokinesis-blocked micronucleus and alkaline comet assays in human lymphocytes. At all tested concentrations (2–400 M) VA was found to have an antioxidant activity and also at the concentrations of VA showed no cytotoxic effects on CHO cells. No genotoxic effect was observed at all tested concentrations (5–1000 M) of VA both in micronucleus and comet assays in human lymphocytes. VA caused a significant reduction in the frequency of micronuclei and the extent of DNA damage induced by H2 O2 especially at high concentrations when compared to cultures treated with H2 O2 only. These results have suggested the protective effects of vanillic acid against H2 O2 induced DNA damage in human lymphocytes. http://dx.doi.org/10.1016/j.toxlet.2013.05.241
P10-17 New study design considerations for somatic and germ cell analysis in the Big Blue® transgenic rodent mutation assay Robert R. Young a,∗ , David Bruning a , Marilyn J. Aardema b a BioReliance Corporation, Rockville, MD, USA, b Marilyn Aardema Consulting, LLC, Chief Scientific Advisor BioReliance Corp., USA
The Big Blue® Transgenic Rodent Mutation (TRM) assay is an in vivo test for somatic and germ cell mutagenicity. The recently finalized OECD Test Guideline 488 for TRM assays, ECHA’s use of TRM assays to investigate in vivo mutagenicity for REACH registrations, and ECHA’s preference for TRM over UDS assays (ECHA-13-R-01-EN, 2013) have led to renewed focus on TRM assays. A recent ECHA review (ECHA/2011/217, 2012) indicated that there are a limited number of laboratories available to conduct TRM assays. To meet the scientific and regulatory need for this assay, BioReliance, co-developer of the Big Blue® assay has obtained the rights to the Big Blue® mouse and rat lines and is requalifying the cII Big Blue mutation assay to OECD TG 488 standards. There are recent scientific revisions proposed to OECD TG 488 (G. Douglas, Personal communication) that include using cauda epididymis instead of vas deferens and using seminiferous tubules and cauda at both 3 day sampling and 49 day sampling time instead of 42 days for mouse germ cells. This new study design is being used in BioReliance’s qualification studies. To date, the background cII mutation frequency in liver in our laboratory is 27.8 ± 12.6 × 10−5 which is in line with other laboratories (M. Manjanatha, FDA NCTR, Personal Communication, 2013). Results for vehicle and N-ethyl-Nnitrosourea (ENU) treatment in liver, bone marrow, seminiferous tubules and cauda epididymis will be presented following proposed OECD 488 study design changes. http://dx.doi.org/10.1016/j.toxlet.2013.05.242
P10-18 Noncanonical activation of ATR-p53 axis by DNA–protein crosslinks controls cell death responses to formaldehyde Anatoly Zhitkovich ∗ , Victor Wong, Jessica Morse Brown University, Providence, RI 02921, USA We examined how human cells respond to DNA–protein crosslinks (DPC) formed by carcinogenic formaldehyde (FA). We found that FA caused a rapid activation of the DNA damage-
responsive ATR kinase that preferentially targeted the p53 transcription factor at moderate damage levels (bell-shaped response) and CHK1 kinase at higher stress levels (sublinear response). Inhibition of mismatch repair, nucleotide and base excision repair did not impact p53 activation, arguing against the role of small adducts in this response. The p53-targeting signaling occurred exclusively in S phase of cycling populations. DPC-activated p53 played a major role in the fate determination of FA-treated cells by inducing senescence in lung fibroblasts and promoting apoptosis in lung epithelial cells. S phase-arrested cells tested negative for ssDNA and p53 phosphorylation was TopBP1-independent, pointing to a novel mode of ATR activation. FA was also found to disrupt ATR association with the replicative helicase MCM. These results argue that unlike typical DNA adducts that stall elongating DNA polymerases, superbulky DPC block duplex unwinding by MCM helicase, which prevents accumulation of ssDNA that is required for the canonical route of ATR activation. Overall, our findings indicate that S phase-specific, ssDNA-independent activation of the ATR-p53 axis is the main stress signaling response to FA-DPC in human cells. Chronic exposure to cytotoxic doses of FA can lead to a selection of resistant cells with deficient p53, providing an explanation for the origin of p53 mutations by this weakly mutagenic carcinogen. http://dx.doi.org/10.1016/j.toxlet.2013.05.243
P10-19 Preclinical safety assessment of aqueous Gentiana lutea radix extracts Claudia Turek ∗ , Nora Mörbt, Jennifer Felenda, Kerstin Link, Beckmann Christiane, Margit Müller, Peter Vögele, Florian Stintzing WALA Heilmittel GmbH, Bad Boll, Germany Gentiana lutea L. (yellow gentian) is a perennial herb with long roots and yellow flowers, indigenous to mountainous regions of Europe. Broadly applied as pharmaceutically active substance, preparations from the underground organs are indicated for the treatment of several digestive disorders due to bitter substances present in Gentianae radix, such as the secoiridoid glycoside gentiopicroside. However, in its assessment report on Gentiana lutea L., radix, the Committee on Herbal Medicinal Products of the European Medicines Agency (EMA/HMPC/578322/2008, 12th November 2009, pp. 1–19) raised concern about a potential mutagenicity of this herbal substance. Owing to positive results in Ames tests with isolated components of gentian root, the committee requested to initiate in vitro studies on mammalian cells and assessment of potential genotoxic compounds. Therefore, in the present study, aqueous extracts of Gentiana lutea L., radix were analysed for the content of gentiopicroside as well as the xanthones gentisine and isogentisine using high performance liquid chromatography/diode-array detection. In a second step, extracts were subjected both to a bacterial and a mammalian mutation assay according to the “Guideline on the assessment of genotoxicity of herbal medicinal substances/preparations” (EMEA/HMPC/107079/2007, 21st May 2008, pp. 1–12). In order to assess the mutagenic potential, the experiments were conducted with and without exogenous metabolic activation using different concentration levels of the test items in compliance with current OECD guidelines.