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Participation of cytochrome P450 in niclosamide bioactivation
S72
Abstracts / Toxicology Letters 211S (2012) S43–S216
P09-26 Dual role of ascorbate in chromium genotoxicity Anatoly Zhitkovich 1 , Mindy Reynolds 2 , Susan Armknecht 1 Brown University, United States, 2 Washington College, United States
1
Purpose: Hexavalent chromium (CrVI) is a potent human carcinogen that requires cellular reduction to cause DNA damage. Ascorbate (ASC) acts as a principal reducer of Cr-6 in vivo but it is present at very low concentrations in cultured cells, which are typically used for mechanistic studies on Cr(VI) genotoxicity. Therefore, it is important to determine a genotoxic profile of Cr-6 under normal concentrations of ASC. Methods: Cultured human (IMR90, H460) and rodent cells (V79, CHO) were incubated with dehydroascorbic acid to restore physiological ASC levels. Cr(VI)-induced genotoxicity was evaluated by the micronucleus assay, formation of single-strand and double-strand DNA breaks, production of CrDNA adducts and mutagenicity at the Hprt locus. Cytotoxic effects were analyzed by measurements of apoptosis, cell cycle progression and clonogenic survival. Results and conclusions: Restoration of ASC levels strongly increased genotoxicity of Cr(VI), as evidenced by its dramatically higher mutagenicity and greater yields of clastogenic micronuclei and DNA double-strand breaks. The overall production of Cr-DNA adducts was not affected and the formation of single-strand breaks was suppressed at normalized ASC levels. ASC restoration also increased all cytotoxic responses to Cr(VI). Toxic processing of a subgroup of ASC-Cr-DNA adducts was the cause of greater Cr(VI) geno- and cyto-toxicity in ASC-restored cells. Thus, a severe ASC deficiency of cells in culture grossly underestimates the toxicological potency of Cr(VI) and generates a distorted profile of its genotoxic properties. Cr(VI) metabolism by ASC suppresses oxidative damage and shifts genotoxicity to nonoxidative mechanisms mediated by DNA adducts. doi:10.1016/j.toxlet.2012.03.278 P09-27 A novel cell-based model using HCI for toxicological evaluation of genotoxic compounds Yasmin Dietz, Philip G. Hewitt, Simone Graeber, Stefan O. Mueller Merck Serono/Merck KGaA, Germany Genotoxicity is an important issue during pharmaceutical development and chemical risk assessment. In vivo carcinogenicity studies are animal, time and cost-intensive and standard in vitro tests have relatively low specificity. The high content imaging (HCI) technology offers an approach to improve throughput compared to the standard testing battery. In addition to that, a cell-based model using HCI has the advantage of generating data rapidly with small compound needs. Therefore, HCI-based assays would enable screening in an early phase of pharmaceutical drug discovery. This study aimed to develop a novel, specific and sensitive high content imaging-based test system for mutagens and promutagens in vitro using HepG2 cells. Due to their limited metabolic capacity we established a combined system of HepG2 cells and a metabolic activation system (MAS – rat liver S9) for promutagen testing. Nine different proteins involved in DNA-damage response served as putative markers for compound-induced genotoxicity. The protein expression -or activation changes were quantified 48 h after treatment with (pro-)genotoxins (Cyclophosphamide, 7,12Dimethylbenzanthracene, Aflatoxin B1, 2-Acetylaminofluorene,
Actinomycin D, Methyl methanesulfonate, Etoposide) and nongenotoxins (d-mannitol, Phenfomin HCl, Progesterone) using the HCI technology. The best classification was achieved using five out of these nine putative marker proteins. These five proteins are potential candidates for the identification of potential genotoxicants early in the drug discovery process. Our ultimate goal is to combine the specific marker set with sensitive genotoxicity endpoints like micronucleus assay to generate a highly predictive screening system for a broad range of potentially genotoxic compounds. doi:10.1016/j.toxlet.2012.03.279
P09-28 Participation of cytochrome P450 in niclosamide bioactivation Evelyn Beristain-Castillo, Rafael Camacho-Carranza, Sandra Luz Hernández-Ojeda, Jesús Javier Espinosa-Aguirre Universidad Nacional Autónoma de México, Mexico Niclosamide is an anthelmintic drug, which is frequently used to control snail and lamprey pests. Niclosamide shows mutagenic activity in the presence of hepatic postmitochondrial fractions (S9), suggesting that such mutagenicity is mediated, at least in part, by cytochrome P450 enzymes (CYP). The main objective of this study is to identify the specific CYPs involved in the production of mutagenic metabolites derived from niclosamide. To achieve this goal, we firstly applied Ames tests to confirm the niclosamide mutagenicity. Secondly, we carried out experiments to make a preliminary identification of the CYPs involved in niclosamide bioactivation. For this purpose, we used S9 fractions from rats treated with CYP inducers (phenobarbital/naphthoflavone, benzo[a]pyrene and cyclohexanol). In addition, we inhibited CYP1A and CYP2E using ␣-naphthoflavone and diethyldithiocarbamate to identify the CYP involved. The presence of inhibitors for CYP1A1 (␣-naphthoflavone) significantly decreased niclosamide mutagenicity (F5,29 = 11.1708; p < 0.0001). CYP1A1 supersomeR and cofactors markedly increased niclosamide mutagenicity (F3,8 = 13.5389; p = 0.0017), suggesting CYP1A1 is responsible for the mutagen activation. The 2B1 supersomeR did not show a significant increase in mutagenesis at doses of 150 mcg per plate. The results obtained so far indicate that a metabolite derived from the action of the CYP1A subfamily has a key role in the mutagenic effect of niclosamide. doi:10.1016/j.toxlet.2012.03.280
P09-29 Effects of smoking during pregnancy on DNA damage in maternal and newborns’ blood Emre Durmaz 1 , Ümit Aydogan 2 , Cihangir Mutlu Ercan 2 , Onur Kenan Ulutas 1 , Serkan Kavuk 2 , Ismet C¸ok 1 Gazi University, Faculty of Pharmacy, Turkey, 2 Gulhane Military Medical Academy, Turkey
1
Smoking during pregnancy, due to exposure to a large number of genotoxic and carcinogenic substances contained in cigarette smoke threatens the health of the newborn. Various studies have shown that some of these substances or metabolites are capable of passing through the placenta and harming the newborns’ health. Although many studies demonstrated the possible genotoxic risks of smoking for adults, genotoxic effects for infants with pre-
Abstracts / Toxicology Letters 211S (2012) S43–S216
P09-26 Dual role of ascorbate in chromium genotoxicity Anatoly Zhitkovich 1 , Mindy Reynolds 2 , Susan Armknecht 1 Brown University, United States, 2 Washington College, United States
1
Purpose: Hexavalent chromium (CrVI) is a potent human carcinogen that requires cellular reduction to cause DNA damage. Ascorbate (ASC) acts as a principal reducer of Cr-6 in vivo but it is present at very low concentrations in cultured cells, which are typically used for mechanistic studies on Cr(VI) genotoxicity. Therefore, it is important to determine a genotoxic profile of Cr-6 under normal concentrations of ASC. Methods: Cultured human (IMR90, H460) and rodent cells (V79, CHO) were incubated with dehydroascorbic acid to restore physiological ASC levels. Cr(VI)-induced genotoxicity was evaluated by the micronucleus assay, formation of single-strand and double-strand DNA breaks, production of CrDNA adducts and mutagenicity at the Hprt locus. Cytotoxic effects were analyzed by measurements of apoptosis, cell cycle progression and clonogenic survival. Results and conclusions: Restoration of ASC levels strongly increased genotoxicity of Cr(VI), as evidenced by its dramatically higher mutagenicity and greater yields of clastogenic micronuclei and DNA double-strand breaks. The overall production of Cr-DNA adducts was not affected and the formation of single-strand breaks was suppressed at normalized ASC levels. ASC restoration also increased all cytotoxic responses to Cr(VI). Toxic processing of a subgroup of ASC-Cr-DNA adducts was the cause of greater Cr(VI) geno- and cyto-toxicity in ASC-restored cells. Thus, a severe ASC deficiency of cells in culture grossly underestimates the toxicological potency of Cr(VI) and generates a distorted profile of its genotoxic properties. Cr(VI) metabolism by ASC suppresses oxidative damage and shifts genotoxicity to nonoxidative mechanisms mediated by DNA adducts. doi:10.1016/j.toxlet.2012.03.278 P09-27 A novel cell-based model using HCI for toxicological evaluation of genotoxic compounds Yasmin Dietz, Philip G. Hewitt, Simone Graeber, Stefan O. Mueller Merck Serono/Merck KGaA, Germany Genotoxicity is an important issue during pharmaceutical development and chemical risk assessment. In vivo carcinogenicity studies are animal, time and cost-intensive and standard in vitro tests have relatively low specificity. The high content imaging (HCI) technology offers an approach to improve throughput compared to the standard testing battery. In addition to that, a cell-based model using HCI has the advantage of generating data rapidly with small compound needs. Therefore, HCI-based assays would enable screening in an early phase of pharmaceutical drug discovery. This study aimed to develop a novel, specific and sensitive high content imaging-based test system for mutagens and promutagens in vitro using HepG2 cells. Due to their limited metabolic capacity we established a combined system of HepG2 cells and a metabolic activation system (MAS – rat liver S9) for promutagen testing. Nine different proteins involved in DNA-damage response served as putative markers for compound-induced genotoxicity. The protein expression -or activation changes were quantified 48 h after treatment with (pro-)genotoxins (Cyclophosphamide, 7,12Dimethylbenzanthracene, Aflatoxin B1, 2-Acetylaminofluorene,
Actinomycin D, Methyl methanesulfonate, Etoposide) and nongenotoxins (d-mannitol, Phenfomin HCl, Progesterone) using the HCI technology. The best classification was achieved using five out of these nine putative marker proteins. These five proteins are potential candidates for the identification of potential genotoxicants early in the drug discovery process. Our ultimate goal is to combine the specific marker set with sensitive genotoxicity endpoints like micronucleus assay to generate a highly predictive screening system for a broad range of potentially genotoxic compounds. doi:10.1016/j.toxlet.2012.03.279
P09-28 Participation of cytochrome P450 in niclosamide bioactivation Evelyn Beristain-Castillo, Rafael Camacho-Carranza, Sandra Luz Hernández-Ojeda, Jesús Javier Espinosa-Aguirre Universidad Nacional Autónoma de México, Mexico Niclosamide is an anthelmintic drug, which is frequently used to control snail and lamprey pests. Niclosamide shows mutagenic activity in the presence of hepatic postmitochondrial fractions (S9), suggesting that such mutagenicity is mediated, at least in part, by cytochrome P450 enzymes (CYP). The main objective of this study is to identify the specific CYPs involved in the production of mutagenic metabolites derived from niclosamide. To achieve this goal, we firstly applied Ames tests to confirm the niclosamide mutagenicity. Secondly, we carried out experiments to make a preliminary identification of the CYPs involved in niclosamide bioactivation. For this purpose, we used S9 fractions from rats treated with CYP inducers (phenobarbital/naphthoflavone, benzo[a]pyrene and cyclohexanol). In addition, we inhibited CYP1A and CYP2E using ␣-naphthoflavone and diethyldithiocarbamate to identify the CYP involved. The presence of inhibitors for CYP1A1 (␣-naphthoflavone) significantly decreased niclosamide mutagenicity (F5,29 = 11.1708; p < 0.0001). CYP1A1 supersomeR and cofactors markedly increased niclosamide mutagenicity (F3,8 = 13.5389; p = 0.0017), suggesting CYP1A1 is responsible for the mutagen activation. The 2B1 supersomeR did not show a significant increase in mutagenesis at doses of 150 mcg per plate. The results obtained so far indicate that a metabolite derived from the action of the CYP1A subfamily has a key role in the mutagenic effect of niclosamide. doi:10.1016/j.toxlet.2012.03.280
P09-29 Effects of smoking during pregnancy on DNA damage in maternal and newborns’ blood Emre Durmaz 1 , Ümit Aydogan 2 , Cihangir Mutlu Ercan 2 , Onur Kenan Ulutas 1 , Serkan Kavuk 2 , Ismet C¸ok 1 Gazi University, Faculty of Pharmacy, Turkey, 2 Gulhane Military Medical Academy, Turkey
1
Smoking during pregnancy, due to exposure to a large number of genotoxic and carcinogenic substances contained in cigarette smoke threatens the health of the newborn. Various studies have shown that some of these substances or metabolites are capable of passing through the placenta and harming the newborns’ health. Although many studies demonstrated the possible genotoxic risks of smoking for adults, genotoxic effects for infants with pre-