Toxicological Characterization of a Novel in Vivo Benzo[ a ]pyrene Metabolite, 7-Oxo-benz[ d ]anthracene-3,4-dicarboxylic Acid Anhydride (original) (raw)
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Mutagenesis, 2005
The DNA damage induced by 7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-Noxide hydrochloride (Q-85 HCl) in Caco-2 cells under hypoxic and well-oxygenated conditions has been studied by using the comet assay. This compound has shown a good in vitro profile of high selective toxicity in hypoxia, but its mechanism of action is unknown. The DNA damage has been evaluated by performing the comet assay after a 2-h treatment with Q-85 HCl (0.1, 0.2, 0.4 mM in hypoxia; 20, 40 mM in well-oxygenated conditions). The number of cells in apoptosis has also been assessed by flow cytometry analysis of Annexin V-FITC staining. The capability of the cells to repair the DNA damage and the proliferation rate was evaluated at different times after the treatment (24-168 h). Under hypoxic conditions, a clear dose-dependent increase in the number of nuclei with a comet was observed (comet score: 132 6 13, 343 6 30 and 399 6 1; control comet score: 42 6 14). Under well-oxygenated conditions, the number of nuclei with comet increased significantly with respect to the control (comet score: 273 6 14 and 312 6 9; control comet score: 27 6 4). Cells in apoptosis were not detected by the comet assay nor by flow cytometry. The recovery from DNA damage was time-and concentration-dependent in hypoxia (cells treated with the highest concentration still showed DNA damage after 72 h) and rather time-dependent in well-oxygenated conditions (DNA was completely repaired after 24 h). In conclusion, Q-85 HCl acts by DNA damage and not only the reduced intermediate is genotoxic but also some other derivatives and Q-85 HCl itself may be acting.
Cancer Research, 1982
To analyze the distribution of radioactive carcinogens and [3H]thymidine between nuclear DNA (nDNA) and mitochondria! DMA (mtDNA), we have developed a simple and rapid method for the separation of nDNA and mtDNA using gel electrophoresis of cell lysates. Using this method, we found that, when C3H10T1/2 cells are exposed to either 0.5 fiM [3H]-(±)-7/S,8adihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene ([3H]BPDE) or 1 ¡IM [3H]benzo(a)pyrene, the mtDNA contains a major fraction of the total adducts formed with cellular DNAs. Deoxynucleoside adducts formed between benzo(a)pyrene and mtDNA in intact C3H10T1/2 cells or between BPDE and isolated rat liver mtDNA were analyzed by high-performance liquid chromatography, and were found to be much more heterogeneous than those present in nDNA of C3H1OT1/2 cells. The extensive modification of mtDNA with BPDE in C3H10T'/2 cells is associated with preferential inhibition of the incorpora tion of [3H]thymidine into mtDNA, when compared to incorpo ration of [3H]thymidine into nDNA. To analyze the factors re sponsible for the extensive modification of mtDNA by BPDE, we investigated the role of a lipid phase utilizing liposome:DNA complexes as a model system. We found that the liposomes protect BPDE from spontaneous hydrolysis and enhance the extent of DNA modification at low DNA concentrations. These findings extend previous evidence suggesting that the mito chondria may be important cellular targets in the process of chemical carcinogenesis.
Mutation Research-genetic Toxicology and Environmental Mutagenesis, 2002
Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32 P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides Abbreviations: ara-C, cytosine arabinoside; B[a]P, benzo[a]pyrene; BNF, -naphthoflavone; C3H10T1/2, C3H10T1/2CL8; trans-B[a]P-4,5-diol, (±) trans-4,5-dihydro-4,5-dihydroxyB[a]P; BPDE, anti-trans-B[a]P-7,8-diol-9,10-epoxide; MMS, methyl methanesulfonate; PAH, polycyclic aromatic hydrocarbon; ROS, reactive oxygen species ଝ The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. (S. Nesnow). 1383-5718/02/$ -see front matter. Published by Elsevier Science B.V. PII: S 1 3 8 3 -5 7 1 8 ( 0 2 ) 0 0 2 1 8 -8
Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations
Mutagenesis, 2004
The alkaline Comet assay was used to investigate the in vivo genotoxicity of 17 compounds. Altogether 21 studies were conducted with these compounds. The investigations were triggered for various reasons. The main reason for performing the studies was to evaluate the in vivo relevance of in vitro genotoxicity ®ndings with 10 compounds. Eight of these compounds showed no effects in the in vivo Comet assay while two compounds induced altered DNA migration patterns in speci®c organs. The remaining seven compounds were tested to follow up on neoplastic/preneoplastic or chronic toxicity changes as detected in speci®c target organs identi®ed in rodent studies, to investigate the possibility of site-ofcontact genotoxicity and to test the liver as a target organ for a suspected reactive metabolite. For the studies, various organs of rodents were analyzed, depending on the suspected properties of the compounds, including liver, jejunum, leukocytes, stomach mucosa, duodenum, lung and kidney. All tissues were amenable to investigation by gel electrophoresis after simple disaggregation of organs by means of mincing or, in the case of epithelial cells from the gastrointestinal tract, scraping off cells from the epithelium. In conclusion, the Comet assay was found to be a reliable and robust test to investigate in vivo genotoxicity in a variety of rodent organs. Therefore, it is concluded that in vivo Comet assay data are useful for elucidating positive in vitro genotoxicity ®ndings and to evaluate genotoxicity in target organs of toxicity.
DNA BINDING AND MUTAGENICITY OF 7-SUBSTITUTED DERIVATIVES OF BENZ[A]ANTHRACENE
The b i n d i n g o f c a r c i n o g e n i c 7-hydroxyniethylbenz[a]anthracene (7-HOCHz-B[a]A) and 7-acetoxymethylbenz[a]anthracene (7-AcOCH2-B[a]A) t o c a l f thymus DNA was s t u d i e d i n t h e p r e s e n c e o r a b s e n c e of microsonial enzymes o r n u c l e o s i d e p h o s p h a t e s . I n t h e a b s e n c e of microsomes o r ATP l i t t l e o r no b i n d i n g w a s d e t e c t e d f o r e i t h e r h y d r o c a r b o n (HC). Microsomal enzymes s i g n i f i c a n t l y enhanced t h e b i n d i n g o f b o t h H C ' s t o DNA when compared t o c o n t r o l as measured by r a d i o a c t i v i t y bound t o DNA. When 7-HOCH2-B[a]A was i n c u b a t e d a t 37" w i t h ATP t h e r e w a s a l i n e a r i n c r e a s e i n b i n d i n g o v e r a s i x h r p e r i o d . Of t h e n u c l e o s i d e p h o s p h a t e s t e s t e d , ATP, and t o a lesser e x t e n t ADP and CTP, m e d i a t e d b i n d i n g o f 7-HOCH2-B[a]A s u g g e s t i n g f o r m a t i o n of a reactive p h o s p h a t e e s t e r . M u t a g e n i c i t y s t u d i e s w i t h 7-acetoxymethyl-, 7-hydroxymethyl-, 7-formyl-, 7methyl-, and 7-methoxymethyl-B[a]A were c o n d u c t e d u s i n g t h e Salmon e l l a r e v e r s e m u t a t i o n a s s a y . A l l compounds e x h i b i t e d m u t a g e n i c a c t i v i t y i n t h e p r e s e n c e o f S-9; o n l y 7-AcOCH2-B[a]A w a s a c t i v e w i t h o u t S-9 i n d i c a t i n g t h i s compound t o b e a n u l t i m a t e mutagen. INTRODUCTION Recent s t u d i e s have shown t h a t p o l y c y c l i c h y d r o c a r b o n s are m e t a b o l i z e d t o r e a c t i v e i n t e r m e d i a t e s c a p a b l e of c o v a l e n t b i n d i n g t o DNA r e s u l t i n g i n c a r c i n o g e n e s i s a n d / o r m u t a g e n e s i s . i m e n t s of G e l b o i n [ l ] and o t h e r s [ 2 , 31 have l e d t o t h e c o n c e p t The e x p e rt h a t a microsomal enzyme s y s t e m may b e r e s p o n s i b l e f o r t h e & I 383 Copyright 0 1979 hy Marcel Dekker, lnc
Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay
Mutagenesis, 1996
The single cell gel electrophoresis (SCGE) or comet assay, which measures DNA strand breaks in individual cells, was used to analyse DNA damage and repair induced by the SN r type alkylating carcinogens N-ethyl-iV'-nitro-iVnitrosoguanidine and N-ethyl-N-nitrosourea in CHO cells. The comet assay was comparable in sensitivity to the alkaline elution assay. The alkyl-adducts detected as DNA single-strand breaks (ssb) by this technique were completely repaired within 24 h after treatment. These data indicate that long-lived lesions, such as alkylphosphotriesters, are not converted into ssb under the standard SCGE alkaline conditions (pH 13.5). The lesions revealed by the comet assay are mainly apurinic/apyrimidinic (AP) sites and breaks formed as intermediates in the base excision repair process of N-alkylpurines. When SCGE was performed at pH 12.5 instead of pH 13.5 a lower level of ssb was detected and these breaks were completely resealed within 2 h after treatment These data suggest that different subsets of lesions are detected under different pH conditions. The SCGE combined with inclusion within the cells of endonuclease III revealed that a high portion of AP sites induced by alkylation damage were not converted into ssb by alkali. The level of endonuclease ITi-sensitive sites decreased as a function of the repair time and by 24 h after treatment no sites were left on the DNA. The use of this modified SCGE assay allows the estimation of the total amount of unrepaired AP sites present on DNA. Alkylation-induced ssb as detected by the comet assay should be regarded as an indicator of repair rate and balance more than a measure of actual DNA damage.
The genotoxic potential of methapyrilene using the alkaline Comet assay in vitro and in vivo
Toxicology, 2011
The genotoxicity of methapyrilne (MP) has been evaluated in a number of assays since it was found to be a rat hepatocarcinogen with subsequent withdrawal as an over-the-counter antihistamine. Whilst it has not been classified as a genotoxin, there are reports of positive findings from mammalian cell gene mutation and transformation assays. To investigate further the genotoxic potential of MP, the alkaline Comet assay was used to evaluate DNA damage both in primary hepatocytes in culture and in vivo in the rat. To confirm bioactivation was required to induce the hepatotoxic mechanism, aminobenzotriazole, a broad spectrum cytochrome P450 enzyme inhibitor was used as a pre-treatment. The levels of glutathione and glutathione disulfide were determined in both hepatocytes in culture and in the liver following in vivo exposure. MP showed significant increases in DNA damage in freshly isolated male rat hepatocyte suspensions that could be significantly reduced by pre-incubation of aminobenzotriazole (ABT). DNA damage showed a marked sex difference, with male hepatocytes being more susceptible, and showing a concurrent depletion of glutathione (GSH) compared with female hepatocytes. Modulation of the GSH levels by diethylmaleate and ␥-glutamylcysteinylethyl ester, elevated and reduced the levels of DNA damage, respectively. In the in vivo Comet assay, there was no evidence of DNA damage following MP (150 mg/kg p.o) treatment for three consecutive days, although histological and liver enzyme changes were seen. Total protein GSH content was elevated in MP-treated animals and superoxide dismutase levels were increased specifically in periportal regions. Taken together, these data support the potential for MP to induce oxidative stress. The differences in DNA damage detected by the Comet assay in vitro, and in rat liver in vivo, could be attributed to differences in metabolism and response to oxidant insult or the inability of the assay to discriminate damage in a small number of individual cells in the whole liver.