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Interest of immunohistochemistry in the evaluation of tumorigenic potential of advanced therapy medicinal products
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Abstracts / Toxicology Letters 229S (2014) S40–S252
Acknowledgements: This study was supported by the Czech Science Foundation project No. 14-22016S and by the Czech Ministry of Education, Youth and Sports (project No. CZ.1.07/2.3.00/30.0030). http://dx.doi.org/10.1016/j.toxlet.2014.06.524 P-3.16 Modulation of cytochrome P450 1A1-mediated oxidation of benzo[a]pyrene by NADH and cytochrome b5 Radek Indra 1,∗ , Michaela Moserova 1 , Arlt M. Volker 2 , Marie Stiborova 1 1 Department of Biochemistry, Faculty of Science, Charles University, Prague 2, Czech Republic, 2 Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King’s College London, London, United Kingdom
Benzo[a]pyrene (BaP) is a carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP). CYP1A1 is the most important enzyme in BaP bioactivation. Human and rat CYP1A1 metabolize BaP to phenols, diols and diones. Addition of cytochrome b5 (cyt b5 ) increases the amounts of BaP metabolites formed by CYP1A1. Using several in-vitro systems, the mechanism of this phenomenon was investigated. NADPH as a cofactor of NADPH:cytochrome P450 reductase (POR) serves as a donor of electrons to CYPs in its reaction cycle. However, the second electron might also be provided by the system of NADH:cytochrome b5 reductase, cyt b5 and NADH. Here, we investigated the potency of NADH to mediate BaP oxidation by CYP1A1. Liver microsomes of rats, in which CYP1A1 was induced, formed in the presence of NADH the same BaP metabolites as microsomes with NADPH. The amounts of metabolites were also comparable. Human recombinant CYP1A1 expressed with POR in SupersomesTM oxidized BaP to its metabolites even when NADH was present instead of NADPH. In contrast, no BaP metabolites were formed by CYP1A1 expressed with POR in Escherichia coli (Bactosomes). However, BaP incubated ex vivo with the bactosomal CYP1A1, NADH:cytochrome b5 reductase, cyt b5 and NADH without NADPH was also oxidized. The same metabolites as those formed by CYP1A1, POR and NADPH were formed in this system. Our results suggest that NADH can, to some extent, substitute NADPH as an electron donor for the CYP1A1 reaction cycle of BaP oxidation. Acknowledgements: Supported by grants P301/10/0356, 640712 and UNCE204025/2012. http://dx.doi.org/10.1016/j.toxlet.2014.06.525 P-3.17 CYP2E1, GSTO1 and TP53 polymorphisms, response to chemotherapy and survival in advanced non-small cell lung cancer patients Mumtaz Iscan 1,∗ , Karacaoglan Volkan 1 , Ahmet O. Ada 1 , Serdar Bilgen 1 , Semih C. Kunak 2 , Meral Gülhan 3 1 Department of Toxicology, Faculty of Pharmacy, University of Ankara, Ankara, Turkey, 2 Department of Pharmacology, Faculty of Medicine, University of Ordu, Ordu, Turkey, 3 Atatürk Pulmonary Diseases and Thoracic Surgery Hospital, Sanatoryum, Ankara, Turkey
Several gene polymorphisms of xenobiotic/drug metabolizing enzymes and TP53 have been studied for the possible association
with response to chemotherapy and survival rates of patients with non-small cell lung cancer (NSCLC). However, the studies in this regard are limited and the results are contradictory. In this study, CYP2E1*5B, CYP2E1*6 and CYP2E1*7B, GST01 (A140D) and TP53 (Arg72Pro) polymorphisms and response to platinum based chemotherapy and survival in 137 (125 men and 12 women) advanced stage NSCLC patients have been investigated. Although no significant associations were noted between the gene polymorphisms alone and response to chemotherapy, patients with combined variant genotypes of TP53 (Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) responded significantly better than those carrying wild type genotypes to platinum based chemotherapy, p = 0.40. However, we observed that only the patients who had both variant genotypes of TP53 (Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) had shorter survival (median, 17.9 months) compared to wild type genotypes (median, 28.1 months) with marginal significance (p = 0.086). Multivariate analysis also revealed that adjusted hazard ratio of death (HR) of only the combined variant genotypes of TP53 (Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) increased 51-fold, although not significant, as compared to wild type genotypes (HR, 50.61; 95% CI, 0.44-5789.77, p = 0.105). These results show that, among the studied genotypes, only the combination of TP53(Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) is likely to be associated with response to chemotherapy and survival in the patients with advanced NSCLC. Acknowledgements: Supported by the grants from Research Fund of Ankara University (Nos.: 10A3336002 and 2008-08-03-006HPD). http://dx.doi.org/10.1016/j.toxlet.2014.06.526 P-3.18 Interest of immunohistochemistry in the evaluation of tumorigenic potential of advanced therapy medicinal products Katharina Weidmann 1 , Anne-Laure Leoni 1,∗ , Klaus Weber 2 1 BSL BIOSERVICE Scientific Laboratories GmbH, Planegg, Munich, Germany, 2 AnaPath GmbH, Oberbuchsiten, Switzerland
Advanced Therapy Medicinal Products (ATMPs) consist of therapeutical groups including gen therapeutics, somatic cell therapeutics, and engineered tissues containing living cells or tissues. The clinical application approval of cell-based medicinal products requires special procedures with regard to their efficacy and safety testing. The tumorigenic potential of cells present on an implant material can be assessed after subcutaneous implantation in SCIDmouse after 20 weeks. Immunohistochemistry is useful to assess the cell transformation, and tumorigenicity or ectopic engraftment in non-target tissues related to de-differentiation or loss of cell function. In the present study, positive control implants with human cell line HT-1080 developed sarcomas with mitotic figures confirmed by positivity with antibodies against Ki67. However, distant metastases were not found. The tumor cells expressed human vimentin. Positive control RCS implants developed chondrosarcomas that metastasized into the lungs. There were a high number of mitotic figures confirmed by positivity with antibodies against Ki67. At test item (chondrocytes) implant sites, scaffold structures encapsulated by fibrous capsules were visible histologically. There was multifocal mineralization at minor severity degrees. Most samples showed in hematoxylin and eosin-stained sections minor infiltration with cells resembling fibrocyts/fibroblasts. They were detected within the scaffold structures only but not outside the
Abstracts / Toxicology Letters 229S (2014) S40–S252
fibrous capsule. By Ki67, no proliferation was noted at the implantation sites. These cells reacted positively with anti-human vimentin antibodies. The human chondrocytes differentiated in their environment morphologically towards a more mesenchymal cell type but functional chondrocytic aspects remained. Such differentiation would cause wrong conclusions in hematoxylin–eosin stained sections only. http://dx.doi.org/10.1016/j.toxlet.2014.06.527 P-3.19 Effects of biologically active tributyl-/triphenyl-organometal ligands of retinoid X receptors in human breast cancer cell line Dana Macejová ∗ , Lucia Bialeˇsová, Lucia Toporová, Július Brtko Institute of Experimental Endocrinology SAS, Bratislava, Slovakia Retinoid X receptor (RXR)-selective retinoids (“rexinoids”) are useful candidate therapeutic agents of cancer, as they may display less toxicity than retinoic acid receptor (RAR)-selective retinoids (Miller et al., 1997). RXRs play unique modulatory and integrative roles across multiple regulatory systems. Organotin compounds, from which several of them represent pollutants of organic origin (biocides, fungicides exploitable predominantly in agriculture, disinfectants exploitable for adjusting of cooling liquids in industry) (Baldi and Mantovani, 2008) with capability to disrupt endocrine system in both invertebrates and vertebrates – are important agonists of RXR subtypes of nuclear receptors acting in nanomolar concentrations (Nakanishi, 2008). The aim of the present study was to investigate the in vitro effects of triphenyl-/tributyl-substituted organometal compounds from 4A subgroup of periodic table (Sn, Ge) (100 nM) on gene expression (Bcl2, BAX, p53, RXRs, RARs) in MCF-7 cell line (48 h) and on the RXR binding characteristics in rat liver. The results indicate that all-trans retinoic acid (1 mM), tributyltin chloride (TBT-Cl) and tributyltin bromide significantly reduced expression of anti-apoptotic Bcl2 mRNA, when compared to mock treated cells and 9-cis retinoic acid (100 nM) (p < 0.01). However, triphenyltin chloride (TPT-Cl) did not affect this expression. Moreover, TBT-Cl has shown capability to bind to RXRs with the similar characteristics as a natural RXR ligand – 9cRA when compared to TPT-Cl. Acknowledgements: Supported by the grants APVV-0160-11, APVV-0290-10, Vega 2/0171/14 and CEMAN grants. http://dx.doi.org/10.1016/j.toxlet.2014.06.528 P-3.20 Exudative inflammatory lesions in the nasal cavity in 28-day dose range finding studies in CByB6F1 mice and in 26-week carcinogenicity studies in Tg.rasH2 mice Marie McKeon ∗ , Reem H. Elbekai, Mahdav Paranjpe, David Bruning BioReliance Corporation, Rockville, MD, USA
the route of administration. This lesion whenever noted either in the vehicle or test article treated mice is mainly due to the low pH of the vehicle or test article formulation. The lesion is characterized by the presence of eosinophilic protein material, fibrin, sparse mucin, sloughed cells and degenerate neutrophils within the cavities. With lesions of advancing severity there is often degeneration, necrosis and erosion of the underlying mucosa. Occasionally, there is hyperplasia as well as squamous metaplasia of the mucosa. Retrospective analysis of our data pertaining to the 28 day dose range finding and 26 week carcinogencity studies revealed that in spite of inflammatory changes with necrosis and hyperplasia there is no progression to tumor formation in the nasal cavities. http://dx.doi.org/10.1016/j.toxlet.2014.06.529 P-3.21 Aryl hydrocarbon receptor ligands benzo[a]pyrene, ellipticine and Sudan I are potent inducers of cytochrome b5 in rats Iveta Mrízová 1,∗ , Michaela Moserová 1 , Helena Dracínská 1 , Volker M. Arlt 2 , Eva Frei 3 , Marie Stiborová 1 1 Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic, 2 Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health King’s, London, UK, 3 Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center, Heidelberg, Germany
Cytochrome b5 influences the hydroxylation of various xenobiotics catalyzed by some cytochrome P450 (CYP) enzymes. Therefore, its expression levels are crucial for such CYP-mediated reactions. This is also true for the oxidation of the anticancer drug ellipticine and the carcinogens benzo[a]pyrene (BaP) and Sudan I (1-phenylazo-2-hydroxynaphtalene); their oxidation by CYP1A1, dictating their biological effects, is strongly influenced by cytochrome b5. We found that these compounds were inducers of cytochrome b5 in liver and kidney of rats. Here, the mechanism of such induction was investigated. Beside a potency of BaP, ellipticine and Sudan I to induce cytochrome b5, they also induced enzymes that are regulated by activation of the aryl hydrocarbon receptor (AHR), CYP1A1 and NAD(P)H:quinone oxidoreductase 1 (NQO1), both at mRNA and protein levels, corresponding to their ability to act as AHR ligands. Up to 5-, 10- and 100-fold increases in NQO1, cytochrome b5 and CYP1A1 protein expression levels, respectively, were caused by treatment of rats with these compounds. The increase in protein levels was paralleled by an increase in mRNA expression in most cases. Because of the induction of cytochrome b5 by the tested xenobiotics in parallel to that of CYP1A1 and NQO1, an analogous induction mechanism via the activation of AHR might be suggested. Since BaP, ellipticine and Sudan I induced the expression levels of the enzymes essential for their oxidation dictating their biological activities (CYP1A1 and cytochrome b5), they exert concerted regulatory control on their own genotoxic and/or pharmacological effects. Acknowledgement: Supported by P301/10/0356, UNCE204025/ 2012 and 640712. http://dx.doi.org/10.1016/j.toxlet.2014.06.530
Three levels of nasal cavity are routinely examined as a protocol required tissue in 28 day dose range finding and 26 week carcinogenicity studies involving CByB6F1 and Tg.rasH2 mice, respectively. Exudative inflammation of the nasal cavity, which is not a background lesion, is noted in both types of studies with oral gavage as
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Abstracts / Toxicology Letters 229S (2014) S40–S252
Acknowledgements: This study was supported by the Czech Science Foundation project No. 14-22016S and by the Czech Ministry of Education, Youth and Sports (project No. CZ.1.07/2.3.00/30.0030). http://dx.doi.org/10.1016/j.toxlet.2014.06.524 P-3.16 Modulation of cytochrome P450 1A1-mediated oxidation of benzo[a]pyrene by NADH and cytochrome b5 Radek Indra 1,∗ , Michaela Moserova 1 , Arlt M. Volker 2 , Marie Stiborova 1 1 Department of Biochemistry, Faculty of Science, Charles University, Prague 2, Czech Republic, 2 Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King’s College London, London, United Kingdom
Benzo[a]pyrene (BaP) is a carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP). CYP1A1 is the most important enzyme in BaP bioactivation. Human and rat CYP1A1 metabolize BaP to phenols, diols and diones. Addition of cytochrome b5 (cyt b5 ) increases the amounts of BaP metabolites formed by CYP1A1. Using several in-vitro systems, the mechanism of this phenomenon was investigated. NADPH as a cofactor of NADPH:cytochrome P450 reductase (POR) serves as a donor of electrons to CYPs in its reaction cycle. However, the second electron might also be provided by the system of NADH:cytochrome b5 reductase, cyt b5 and NADH. Here, we investigated the potency of NADH to mediate BaP oxidation by CYP1A1. Liver microsomes of rats, in which CYP1A1 was induced, formed in the presence of NADH the same BaP metabolites as microsomes with NADPH. The amounts of metabolites were also comparable. Human recombinant CYP1A1 expressed with POR in SupersomesTM oxidized BaP to its metabolites even when NADH was present instead of NADPH. In contrast, no BaP metabolites were formed by CYP1A1 expressed with POR in Escherichia coli (Bactosomes). However, BaP incubated ex vivo with the bactosomal CYP1A1, NADH:cytochrome b5 reductase, cyt b5 and NADH without NADPH was also oxidized. The same metabolites as those formed by CYP1A1, POR and NADPH were formed in this system. Our results suggest that NADH can, to some extent, substitute NADPH as an electron donor for the CYP1A1 reaction cycle of BaP oxidation. Acknowledgements: Supported by grants P301/10/0356, 640712 and UNCE204025/2012. http://dx.doi.org/10.1016/j.toxlet.2014.06.525 P-3.17 CYP2E1, GSTO1 and TP53 polymorphisms, response to chemotherapy and survival in advanced non-small cell lung cancer patients Mumtaz Iscan 1,∗ , Karacaoglan Volkan 1 , Ahmet O. Ada 1 , Serdar Bilgen 1 , Semih C. Kunak 2 , Meral Gülhan 3 1 Department of Toxicology, Faculty of Pharmacy, University of Ankara, Ankara, Turkey, 2 Department of Pharmacology, Faculty of Medicine, University of Ordu, Ordu, Turkey, 3 Atatürk Pulmonary Diseases and Thoracic Surgery Hospital, Sanatoryum, Ankara, Turkey
Several gene polymorphisms of xenobiotic/drug metabolizing enzymes and TP53 have been studied for the possible association
with response to chemotherapy and survival rates of patients with non-small cell lung cancer (NSCLC). However, the studies in this regard are limited and the results are contradictory. In this study, CYP2E1*5B, CYP2E1*6 and CYP2E1*7B, GST01 (A140D) and TP53 (Arg72Pro) polymorphisms and response to platinum based chemotherapy and survival in 137 (125 men and 12 women) advanced stage NSCLC patients have been investigated. Although no significant associations were noted between the gene polymorphisms alone and response to chemotherapy, patients with combined variant genotypes of TP53 (Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) responded significantly better than those carrying wild type genotypes to platinum based chemotherapy, p = 0.40. However, we observed that only the patients who had both variant genotypes of TP53 (Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) had shorter survival (median, 17.9 months) compared to wild type genotypes (median, 28.1 months) with marginal significance (p = 0.086). Multivariate analysis also revealed that adjusted hazard ratio of death (HR) of only the combined variant genotypes of TP53 (Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) increased 51-fold, although not significant, as compared to wild type genotypes (HR, 50.61; 95% CI, 0.44-5789.77, p = 0.105). These results show that, among the studied genotypes, only the combination of TP53(Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) is likely to be associated with response to chemotherapy and survival in the patients with advanced NSCLC. Acknowledgements: Supported by the grants from Research Fund of Ankara University (Nos.: 10A3336002 and 2008-08-03-006HPD). http://dx.doi.org/10.1016/j.toxlet.2014.06.526 P-3.18 Interest of immunohistochemistry in the evaluation of tumorigenic potential of advanced therapy medicinal products Katharina Weidmann 1 , Anne-Laure Leoni 1,∗ , Klaus Weber 2 1 BSL BIOSERVICE Scientific Laboratories GmbH, Planegg, Munich, Germany, 2 AnaPath GmbH, Oberbuchsiten, Switzerland
Advanced Therapy Medicinal Products (ATMPs) consist of therapeutical groups including gen therapeutics, somatic cell therapeutics, and engineered tissues containing living cells or tissues. The clinical application approval of cell-based medicinal products requires special procedures with regard to their efficacy and safety testing. The tumorigenic potential of cells present on an implant material can be assessed after subcutaneous implantation in SCIDmouse after 20 weeks. Immunohistochemistry is useful to assess the cell transformation, and tumorigenicity or ectopic engraftment in non-target tissues related to de-differentiation or loss of cell function. In the present study, positive control implants with human cell line HT-1080 developed sarcomas with mitotic figures confirmed by positivity with antibodies against Ki67. However, distant metastases were not found. The tumor cells expressed human vimentin. Positive control RCS implants developed chondrosarcomas that metastasized into the lungs. There were a high number of mitotic figures confirmed by positivity with antibodies against Ki67. At test item (chondrocytes) implant sites, scaffold structures encapsulated by fibrous capsules were visible histologically. There was multifocal mineralization at minor severity degrees. Most samples showed in hematoxylin and eosin-stained sections minor infiltration with cells resembling fibrocyts/fibroblasts. They were detected within the scaffold structures only but not outside the
Abstracts / Toxicology Letters 229S (2014) S40–S252
fibrous capsule. By Ki67, no proliferation was noted at the implantation sites. These cells reacted positively with anti-human vimentin antibodies. The human chondrocytes differentiated in their environment morphologically towards a more mesenchymal cell type but functional chondrocytic aspects remained. Such differentiation would cause wrong conclusions in hematoxylin–eosin stained sections only. http://dx.doi.org/10.1016/j.toxlet.2014.06.527 P-3.19 Effects of biologically active tributyl-/triphenyl-organometal ligands of retinoid X receptors in human breast cancer cell line Dana Macejová ∗ , Lucia Bialeˇsová, Lucia Toporová, Július Brtko Institute of Experimental Endocrinology SAS, Bratislava, Slovakia Retinoid X receptor (RXR)-selective retinoids (“rexinoids”) are useful candidate therapeutic agents of cancer, as they may display less toxicity than retinoic acid receptor (RAR)-selective retinoids (Miller et al., 1997). RXRs play unique modulatory and integrative roles across multiple regulatory systems. Organotin compounds, from which several of them represent pollutants of organic origin (biocides, fungicides exploitable predominantly in agriculture, disinfectants exploitable for adjusting of cooling liquids in industry) (Baldi and Mantovani, 2008) with capability to disrupt endocrine system in both invertebrates and vertebrates – are important agonists of RXR subtypes of nuclear receptors acting in nanomolar concentrations (Nakanishi, 2008). The aim of the present study was to investigate the in vitro effects of triphenyl-/tributyl-substituted organometal compounds from 4A subgroup of periodic table (Sn, Ge) (100 nM) on gene expression (Bcl2, BAX, p53, RXRs, RARs) in MCF-7 cell line (48 h) and on the RXR binding characteristics in rat liver. The results indicate that all-trans retinoic acid (1 mM), tributyltin chloride (TBT-Cl) and tributyltin bromide significantly reduced expression of anti-apoptotic Bcl2 mRNA, when compared to mock treated cells and 9-cis retinoic acid (100 nM) (p < 0.01). However, triphenyltin chloride (TPT-Cl) did not affect this expression. Moreover, TBT-Cl has shown capability to bind to RXRs with the similar characteristics as a natural RXR ligand – 9cRA when compared to TPT-Cl. Acknowledgements: Supported by the grants APVV-0160-11, APVV-0290-10, Vega 2/0171/14 and CEMAN grants. http://dx.doi.org/10.1016/j.toxlet.2014.06.528 P-3.20 Exudative inflammatory lesions in the nasal cavity in 28-day dose range finding studies in CByB6F1 mice and in 26-week carcinogenicity studies in Tg.rasH2 mice Marie McKeon ∗ , Reem H. Elbekai, Mahdav Paranjpe, David Bruning BioReliance Corporation, Rockville, MD, USA
the route of administration. This lesion whenever noted either in the vehicle or test article treated mice is mainly due to the low pH of the vehicle or test article formulation. The lesion is characterized by the presence of eosinophilic protein material, fibrin, sparse mucin, sloughed cells and degenerate neutrophils within the cavities. With lesions of advancing severity there is often degeneration, necrosis and erosion of the underlying mucosa. Occasionally, there is hyperplasia as well as squamous metaplasia of the mucosa. Retrospective analysis of our data pertaining to the 28 day dose range finding and 26 week carcinogencity studies revealed that in spite of inflammatory changes with necrosis and hyperplasia there is no progression to tumor formation in the nasal cavities. http://dx.doi.org/10.1016/j.toxlet.2014.06.529 P-3.21 Aryl hydrocarbon receptor ligands benzo[a]pyrene, ellipticine and Sudan I are potent inducers of cytochrome b5 in rats Iveta Mrízová 1,∗ , Michaela Moserová 1 , Helena Dracínská 1 , Volker M. Arlt 2 , Eva Frei 3 , Marie Stiborová 1 1 Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic, 2 Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health King’s, London, UK, 3 Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center, Heidelberg, Germany
Cytochrome b5 influences the hydroxylation of various xenobiotics catalyzed by some cytochrome P450 (CYP) enzymes. Therefore, its expression levels are crucial for such CYP-mediated reactions. This is also true for the oxidation of the anticancer drug ellipticine and the carcinogens benzo[a]pyrene (BaP) and Sudan I (1-phenylazo-2-hydroxynaphtalene); their oxidation by CYP1A1, dictating their biological effects, is strongly influenced by cytochrome b5. We found that these compounds were inducers of cytochrome b5 in liver and kidney of rats. Here, the mechanism of such induction was investigated. Beside a potency of BaP, ellipticine and Sudan I to induce cytochrome b5, they also induced enzymes that are regulated by activation of the aryl hydrocarbon receptor (AHR), CYP1A1 and NAD(P)H:quinone oxidoreductase 1 (NQO1), both at mRNA and protein levels, corresponding to their ability to act as AHR ligands. Up to 5-, 10- and 100-fold increases in NQO1, cytochrome b5 and CYP1A1 protein expression levels, respectively, were caused by treatment of rats with these compounds. The increase in protein levels was paralleled by an increase in mRNA expression in most cases. Because of the induction of cytochrome b5 by the tested xenobiotics in parallel to that of CYP1A1 and NQO1, an analogous induction mechanism via the activation of AHR might be suggested. Since BaP, ellipticine and Sudan I induced the expression levels of the enzymes essential for their oxidation dictating their biological activities (CYP1A1 and cytochrome b5), they exert concerted regulatory control on their own genotoxic and/or pharmacological effects. Acknowledgement: Supported by P301/10/0356, UNCE204025/ 2012 and 640712. http://dx.doi.org/10.1016/j.toxlet.2014.06.530
Three levels of nasal cavity are routinely examined as a protocol required tissue in 28 day dose range finding and 26 week carcinogenicity studies involving CByB6F1 and Tg.rasH2 mice, respectively. Exudative inflammation of the nasal cavity, which is not a background lesion, is noted in both types of studies with oral gavage as
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