S150 Abstracts / Toxicology Letters 229S (2014) S40–S252

Acknowledgements: This study was supported by theCzech Science Foundation project No. 14-22016S and by theCzech 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-3.16Modulation of cytochrome P450 1A1-mediatedoxidation of benzo[a]pyrene by NADH andcytochrome 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 SciencesDivision, MRC-PHE Centre for Environment and Health, King’s CollegeLondon, London, United Kingdom

Benzo[a]pyrene (BaP) is a carcinogen that covalently binds toDNA after metabolic activation by cytochrome P450 (CYP). CYP1A1is the most important enzyme in BaP bioactivation. Human andrat CYP1A1 metabolize BaP to phenols, diols and diones. Addi-tion of cytochrome b5 (cyt b5) increases the amounts of BaPmetabolites formed by CYP1A1. Using several in-vitro systems,the mechanism of this phenomenon was investigated. NADPHas a cofactor of NADPH:cytochrome P450 reductase (POR) servesas a donor of electrons to CYPs in its reaction cycle. However,the second electron might also be provided by the system ofNADH:cytochrome b5 reductase, cyt b5 and NADH. Here, weinvestigated the potency of NADH to mediate BaP oxidation byCYP1A1. Liver microsomes of rats, in which CYP1A1 was induced,formed in the presence of NADH the same BaP metabolites asmicrosomes with NADPH. The amounts of metabolites were alsocomparable. Human recombinant CYP1A1 expressed with POR inSupersomesTM oxidized BaP to its metabolites even when NADHwas present instead of NADPH. In contrast, no BaP metaboliteswere formed by CYP1A1 expressed with POR in Escherichia coli(Bactosomes). However, BaP incubated ex vivo with the bactosomalCYP1A1, NADH:cytochrome b5 reductase, cyt b5 and NADH withoutNADPH was also oxidized. The same metabolites as those formedby CYP1A1, POR and NADPH were formed in this system. Ourresults suggest that NADH can, to some extent, substitute NADPHas an electron donor for the CYP1A1 reaction cycle of BaP oxidation.

Acknowledgements: Supported by grants P301/10/0356, 640712and UNCE204025/2012.

http://dx.doi.org/10.1016/j.toxlet.2014.06.525

P-3.17CYP2E1, GSTO1 and TP53 polymorphisms,response to chemotherapy and survival inadvanced 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 ofAnkara, Ankara, Turkey, 2 Department of Pharmacology, Faculty ofMedicine, University of Ordu, Ordu, Turkey, 3 Atatürk PulmonaryDiseases and Thoracic Surgery Hospital, Sanatoryum, Ankara, Turkey

Several gene polymorphisms of xenobiotic/drug metabolizingenzymes and TP53 have been studied for the possible association

with response to chemotherapy and survival rates of patientswith non-small cell lung cancer (NSCLC). However, the studiesin this regard are limited and the results are contradictory. Inthis study, CYP2E1*5B, CYP2E1*6 and CYP2E1*7B, GST01 (A140D)and TP53 (Arg72Pro) polymorphisms and response to platinumbased chemotherapy and survival in 137 (125 men and 12women) advanced stage NSCLC patients have been investigated.Although no significant associations were noted between the genepolymorphisms alone and response to chemotherapy, patientswith combined variant genotypes of TP53 (Arg72Pro, Pro72Pro)and CYP2E1*6 (*1A/*6) responded significantly better than thosecarrying wild type genotypes to platinum based chemotherapy,p = 0.40. However, we observed that only the patients who hadboth variant genotypes of TP53 (Arg72Pro, Pro72Pro) and CYP2E1*6(*1A/*6) had shorter survival (median, 17.9 months) comparedto wild type genotypes (median, 28.1 months) with marginalsignificance (p = 0.086). Multivariate analysis also revealed thatadjusted hazard ratio of death (HR) of only the combined variantgenotypes of TP53 (Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6)increased 51-fold, although not significant, as compared to wildtype genotypes (HR, 50.61; 95% CI, 0.44-5789.77, p = 0.105). Theseresults show that, among the studied genotypes, only the combi-nation of TP53(Arg72Pro, Pro72Pro) and CYP2E1*6 (*1A/*6) is likelyto be associated with response to chemotherapy and survival inthe patients with advanced NSCLC.

Acknowledgements: Supported by the grants from Research Fundof Ankara University (Nos.: 10A3336002 and 2008-08-03-006HPD).

http://dx.doi.org/10.1016/j.toxlet.2014.06.526

P-3.18Interest of immunohistochemistry in theevaluation of tumorigenic potential ofadvanced 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 oftherapeutical groups including gen therapeutics, somatic cell ther-apeutics, and engineered tissues containing living cells or tissues.The clinical application approval of cell-based medicinal productsrequires special procedures with regard to their efficacy and safetytesting. The tumorigenic potential of cells present on an implantmaterial can be assessed after subcutaneous implantation in SCID-mouse after 20 weeks.

Immunohistochemistry is useful to assess the cell transfor-mation, and tumorigenicity or ectopic engraftment in non-targettissues related to de-differentiation or loss of cell function.

In the present study, positive control implants with human cellline HT-1080 developed sarcomas with mitotic figures confirmedby positivity with antibodies against Ki67. However, distant metas-tases were not found. The tumor cells expressed human vimentin.Positive control RCS implants developed chondrosarcomas thatmetastasized into the lungs. There were a high number of mitoticfigures confirmed by positivity with antibodies against Ki67.

At test item (chondrocytes) implant sites, scaffold structuresencapsulated by fibrous capsules were visible histologically. Therewas multifocal mineralization at minor severity degrees. Mostsamples showed in hematoxylin and eosin-stained sections minorinfiltration with cells resembling fibrocyts/fibroblasts. They weredetected within the scaffold structures only but not outside the