Mutagenesis, Vol. 15, No. 3, 203-205,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
The bleomycin amplification assay in V79 cells predicts frameshift mutagenicity of intercalative agents
Abbott Laboratories, D-468, AP13A, 100 Abbott Park Road, Abbott Park, IL 60064, USA
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Abstract |
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We have recently reported on the use of a cell-based bleomycin amplification assay for the detection of DNA intercalating agents. In order to further validate this assay, two series of proprietary compounds were evaluated for frameshift mutagenesis in the Ames bacterial reversion system and for bleomycin amplification in the Chinese hamster V79 micronucleus system. It is shown that 10 of 11 frameshift-positive compounds were bleomycin amplifiers. These studies indicate that positive frameshift mutagenicity findings are consistent with expectations from the results of the bleomycin amplification assay, providing additional validation of the amplification assay for the detection of DNA intercalating agents. The studies also demonstrate that intercalation is necessary but not sufficient for frameshift mutagenesis since bleomycin amplifiers lacking frameshift mutagenic activity were also identified.
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Introduction |
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Frequently, compounds not expected to be genotoxic on the basis of structure produce unexpected genotoxicity in standard genetic toxicology assays. The underlying molecular bases for such responses are often not understood and, therefore, rational synthesis to decrease or eliminate genotoxicity cannot be carried out. We have proposed that some unexpected genotoxicities arise from non-covalent drug–DNA interactions, particularly intercalation, and we have recently developed a Chinese hamster V79 cell-based assay system for detecting such events (Snyder, 1998
; Snyder and Strekowski, 1999). The assay is a modification of a naked DNA system in which the ability of putative DNA intercalating agents to amplify bleomycin-induced DNA strand breakage was assessed (for a review see Strekowski, 1992). In the cell-based assay, bleomycin-dependent DNA damage is quantified using micronucleus formation as the end-point. We have conducted bleomycin amplification studies with a wide range of structural classes of known intercalating and non-intercalating compounds (Snyder and Strekowski, 1999). In some cases intercalative ability has been confirmed with the ethidium displacement assay (Snyder, 1998) or viscometry (Snyder and Strekowski, 1999). However, since DNA intercalation is only one way in which bleomycin amplification can occur (Snyder and Strekowski, 1999), additional studies are essential in order to further demonstrate the link between intercalation and bleomycin amplification. To better address this, we examined the bleomycin amplification responses of two series of proprietary compounds for which Ames frameshift mutagenesis data are available. Because frameshift mutagenesis is generally considered to arise from DNA intercalation, it would be anticipated that a concordance would exist between Ames frameshift mutagenesis and bleomycin amplification results.
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Materials and methods |
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Chemicals
Bleomycin sulfate and cytochalasin B were obtained from Sigma Chemical Co. (St Louis, MO). Proprietary compounds were obtained from the chemical inventories of Abbott Laboratories.
Cells and in vitro micronucleus/bleomycin amplification assay
Chinese hamster lung V79 cells were grown and maintained in EMEM supplemented with 10% fetal bovine serum. All studies were conducted by seeding 400 000 cells/well in a 6-well tissue culture dish and growing for 24 h prior to any drug treatment. Drug treatments (5 µg/ml bleomycin ± test compound) were for 3 h in fresh medium in the absence of an S9 metabolic activation system. The test compound was always added to cultures at least 1min prior to addition of bleomycin as this schedule appeared to enhance the amplification response. Treatment was followed by removal of the drug(s) and addition of fresh medium containing 3 µg/ml cytochalasin B for ~16 h. This incubation time resulted in 70–80% of the cells having undergone one round of replication. Cells were then harvested with trypsin/EDTA, hypotonically swollen with 0.075 M KCl, affixed to glass slides, fixed with methanol, stained with Dif-Quik (Dade Diagnostics, Aguado, PR) and coverslipped. Slides were examined for numbers of binucleate and mononucleate cells in order to assess the antiproliferative effect of the drug treatment. In addition ~300–500 binucleate cells were examined for the presence of micronuclei indicative of chromosome breakage.
Test compounds were evaluated at concentrations up to the limits of solubility and/or toxicity. For chemicals testing positive as amplifiers, an approximate least effective concentration was determined. Enhancement of bleomycin-induced micronucleus formation is interpreted as indicating that the test article interacted with DNA in such a way as to enhance accessibility of the DNA to bleomycin. An arbitrary 2-fold rule was applied as a minimal requirement for a positive amplification response (Snyder and Strekowski, 1999). Additional testing will help to further define appropriate criteria for a positive response in this assay.
Ames bacterial reversion assays
Ames Salmonella mutagenesis assays were conducted in frameshift strains TA-1538, TA-98 and/or TA-1537 and in some cases in the non-frameshift strain TA-100 essentially according to standard procedures. An Aroclor-induced rat liver S9 fraction was obtained from Moltox. Positive responses were defined as at least 2-fold increases over background colony counts for TA-98 and TA-100 and at least 3-fold increases over background for TA-1535 and TA-1538.
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Results and discussion |
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Figure 1
shows the chemical structures of the series 1 compounds used in the present studies. As shown in Table I, the naphthylpropenoic acid derivatives 1 and 5, the benzoxazole 3 and the phenylpyridazine 7 tested negative in Ames frameshift strains, whereas the naphthylpropenoic acid derivatives 2 and 6, the benzthiazole 4, the phenylpyridazine 8 and the quinoxaline 9 were positive frameshift mutagens. None of these compounds were positive in Ames base pair strains (not shown). Compounds 2, 4, 8 and 9 required Aroclor-induced S9 mix for mutagenicity, whereas compound 6 was equally mutagenic with or without activation. The Ames-positive compounds all have in common a potentially reactive amine, while this function is lacking in all Ames-negative compounds with the exception of compound 5. Cytochrome p450-driven metabolism of compounds 2, 4, 8 and 9 is expected to generate DNA-reactive hydroxylamines. Compound 6 is already a reactive molecule and needs no further metabolism for mutagenicity. None of these compounds tested positive in the in vitro micronucleus assay in the absence of bleomycin (not shown).
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Table I
demonstrates that with the exception of compound 4, positive Ames frameshift mutagens are intercalators (bleomycin amplification positive) and negative Ames frameshift compounds are not intercalators (bleomycin amplification negative). Compound 4, despite testing positive in the Ames system, did not appear to intercalate in V79 cells. The reason for this is not clear but may relate to poor cellular uptake since no antiproliferative effects of this compound were noted when tested alone in V79 cells (not shown). It may also relate to the absence of S9 in the amplification assay although it seems unlikely that the S9 fraction would substantially modify the intercalating ability of this compound. The structural similarity between compounds 2 and 5, in the light of their opposing mutagenicities, is interesting. The fact that compound 2 intercalates based on amplification results, while compound 5 does not, may provide the explanation for these results. For this entire series, prediction of intercalation based on structure alone would be difficult as there is no obvious relationship between the rather subtle structural variations and intercalation/frameshift mutagenesis. Thus for series 1 compounds, frameshift mutagenicity and bleomycin amplification results are consistent with designation of these compounds as DNA intercalating agents even though the intercalative structure–activity relationship (SAR) of members of this series is not obvious.
The proprietary nature of series 2 compounds precludes publication of their structures. All 18 compounds are structurally related heterocyclic aromatic amines comprised of two fused rings and two or three unfused rings. A clear SAR for Ames frameshift mutagenicity was determined and found to relate to a particular ring system. The compounds reported herein comprise six frameshift-positive compounds, i.e. 1, 2, 3, 7, 10 and 12, and 12 frameshift-negative compounds (Table II). Compound 14 was positive in the base pair strain TA-100, but was not a frameshift mutagen. As would be expected for aromatic amines, these compounds were only mutagenic in the presence of an S9 metabolic activation system.
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Using an arbitrary 2-fold criterion for a positive amplification response (Snyder and Strekowski, 1999
), only four series 2 compounds, i.e. 4, 14, 15 and 18, are considered to lack intercalation activity. None of these four were frameshift mutagens. Compound 4 had limited solubility; compound 14 had a somewhat different ring configuration and compound 18 was a member of a structurally dissimilar class of compounds. Compound 15 was very similar in structure to compound 6, which produced only a very weak bleomycin amplification response. Of the remaining 14 amplifying compounds, six were frameshift mutagens and the other eight were not. All six of the mutagens carried a cationic N-alkyl substitution, whereas this function was not found in any of the intercalating but non-mutagenic compounds. This apparent requirement for a cationic terminus on an intercalating agent in order to confer genotoxicity was also previously observed for antihistamines (Snyder, 1998). The cationic function may serve to stabilize intercalative binding, prolonging the residence time of the molecule on the DNA and, in this way, enhancing genotoxic potential. Thus within the antihistamine series and both chemical series reported herein, it may be concluded that intercalation is necessary but not sufficient for genotoxicity.
Frameshift mutagenesis is generally thought to arise via intercalation yet the fact that series 2 compounds are aromatic amines and that metabolic activation is required for mutagenesis leads to the question of the actual role of intercalation in frameshift mutagenesis within this series. The intercalative ability of these compounds could conceivably be enhanced by P450-driven metabolic activation, but this seems unlikely since intercalation is primarily a function of planarity. If intercalation simply served the function of bringing a DNA-reactive group (e.g. an activated amine) into stabilized proximity with the DNA, one would expect these compounds to also be base pair mutagens, but for the most part they are not. Studies on heterocyclic aromatic amines in the aminofluorene (Hoffman and Fuchs, 1997) and aminoimidazoazaarene (Schut and Snyderwine, 1999) series suggest that frameshift mutagenesis occurs when covalent adduct formation stabilizes Streisinger `slips' at repetitive base sites (Streisinger and Owen, 1985). This may be a common feature behind all frameshift mutagenesis since even classical intercalators such as acridines appear to act through stabilization of slipped structures (Ferguson and Denny, 1990). Thus any adduct or helical distortion that tends to disproportionately favor the slipped state might be expected to promote frameshift mutagenesis. In the absence of such detectable `slips', mutagenesis would be expected to arise from traditional adducted base mispairing processes. In the present context, since the compounds require metabolic activation for mutagenesis, it is reasonable to assume that stabilization of the slipped state occurs only as a consequence of adduct formation through the activated amine.
The results on the 27 compounds reported here provide additional confidence that bleomycin amplification in V79 cells results from DNA intercalation. In fact, we have now tested other series of novel compounds in the bleomycin amplification assay and have been able to establish strong correlations between positive genetic toxicity findings and bleomycin amplification (intercalation) activity in almost every instance. These findings, along with those published previously on a wide range of DNA intercalating and non-intercalating compounds (Snyder and Strekowski, 1999), further validate this assay. Since not all intercalating agents are frameshift mutagens, the ability to detect intercalation in a second cell-based system should provide a valuable new tool for understanding unexpected genotoxicities and for rational drug design to limit these effects.
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Acknowledgments |
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The authors wish to thank Dr George Hoffman, College of the Holy Cross, for helpful discussions.
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Notes |
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1 To whom correspondence should be addressed at: DuPont Pharmaceuticals, Stine-Haskell Research Center, PO Box 30, H1/1710, Newark, DE 19714-0030, USA. Tel: +1 302 451 4503; Fax: +1 302 451 4827; Email: ronald.d.snyder{at}dupontpharma.com
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Ferguson,L.R. and Denny,W.A. (1990) Frameshift mutagenesis by acridines and other reversibly-binding DNA ligands. Mutagenesis, 5, 529–540.
Hoffmann,G.R. and Fuchs,R.P.P. (1997) Mechanisms of frameshift mutations: insight from aromatic amines. Chem. Res. Toxicol., 10, 347–359.[Web of Science][Medline]
Schut,H.A.J. and Snyderwine,E.G. (1999) DNA adducts of heterocyclic amine food mutagens: implications for mutagenesis and carcinogenesis. Carcinogenesis, 20, 353–368.
Snyder,R.D. (1998) A review and investigation into the mechanistic basis of the genotoxicity of antihistamines. Mutat. Res., 411, 235–248.[Web of Science][Medline]
Snyder,R.D. and Strekowski,L. (1999) Enhancement of bleomycin-induced micronucleus formation in V79 cells as a rapid and sensitive screen for non-covalent DNA-binding compounds. Mutat. Res., 444, 181–192.[Web of Science][Medline]
Streisinger,G. and Owen,J.E. (1985) Mechanisms of spontaneous and induced frameshift mutation in bacteriophage T4. Genetics, 109, 633–659.
Strekowski,L. (1992) Molecular basis for the enhancement and inhibition of bleomycin-mediated degradation of DNA by DNA-binding compounds. Adv. Detailed React. Mech., 2, 61–109.
Received on August 31, 1999; accepted on December 13, 1999.
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