SI24 S-XIV: New mutagenicity tests and their evaluation

Ip XIV A.21 Salmonella spiral assay: Manually countlnt: and dataanalysis

Lone Lillemarlc, Mona-Lise Binderup, Birgitte Plesing Moller, VivianJorgensen. National Food Agency of Denmark, Murkhe} Bygade /9, DK­2860 Seborg, Denmark

A method for manually counting of colonies in the Spiral Salmonella Assayby the use of a template has been established, and a model and a spreadsheetfor data analysis have been set up. Upon analysis, each spiral plate yieldsa dose-response curve consisting of 6 points with a concentrallon range of13-fold.

In the literature the order of application of bacteria. test substance andS9-mix have been discussed as well as the possibility of pouring bacteriaandlor S9-mix onto the plate with top agar after spiral plating of the testsubstance.

For evaluation of the method four different concentrations of two knownmutagens 2-nitrofluorene (2-NF) and 2-aminoanthracene (2-AA) dissolvedin dimethyl sulfoxide (OM SO) were tested. To establish the optimum testconditions the application order of bacteria, test substance and S9-mix(only 2-AA) were varied. Also the application of bacteria and S9-mix wereperformed with two different techniques: spiral plating and top agar.

As it appeared that the relatively high volume of DMSO plated in the firstpart of the spiral was toxic for the bacteria. the test was repeated with themore volatile solvents diethyl ether and ethanol. The results will be givenand discussed.

Keyword(s): Spiral Salmonella Assay; Ames test; Volatile solvents

Ip XIV A.31 Deteetlon ofl:enomle DNA damage (ndueed by geno­toxins In mammalian eell lines by a ehemllumlnes­eenee mleroplate DNA repair assay

R.-Y. u', C. Provot l,l.-P'laeg1, C. Bozzato", B. Salles1. IS.F.R./.. Bergan­ton, 33127 Saint Jean d'J1Jac, France; 2IPBS. UPR 9062 CNRS. 205 routede Narbonne. 3/077 Toulouse. France

To easily screen genotoxic compounds, we developed a Damaged DNADetection assay (3D assay). The 3D-assay is an adaptation of the repair assayinitially reported by Wood et al, (I) which takes advantage of an adsoptionstep of plasmid DNA in sensitized microplate wells. After DNA damagingtreatments, digoxigenylated-modified nucleotides were incorporated duringthe repair synthesis step and their presence was detected by an ELISA-likereaction with chemiluminescence detection (2).

Here, we show that this assay allows the analysis of damaged genonucDNA Isolated from mammalian cells following treatment with chemicals.thus making this method interesting for the following applications: (i)detection of DNA damage, (ii) determination of the repair rate and (iii)evaluation of protective agents.

(i) DNA alkylation provoked by EMS and MNNG or oxidative damageinduced by H101 were detected. Moreover, genotoxicity of pro-drugs inmetabolically competent human cell line expressing cytochromes P450s (3)was tested. Oi) After a post-treatment incubation step in drug free medium,kinetics of DNA repair were determined in different cells line by analysingthe decrease amount of DNA damage. (iii) Following H101 treatment theprotective effects of different antioxidants (NAC, silymarin ...) was shown.

(I) Wood, R. D., Robins, P.& Lindahl, T. (1988) Cell. 53, 97-106.(2) B. Salles, C. Provot et al. (1995) Anal. Biochem., 132,37-42.(3) Crespi (1991) Chern. Res. Toxicol 4, 566-572.

Ip XIV A.41 Evaluation of the restriction site mutallon assay us­Inl: murine P53 Intron sequenees

Gareth 1.S. Jenkins, James M. Parry. Centre Jor molecular genetics andtoxicology. University of Wales Swansea. Singleto" Parle, Swansea. UK

The restriction site mutation assay, which detects DNA mutations in re­striction enzyme sites in target regions, was employed to study induced invivo mutagenesis. The agents used in this study were: N-ethyl-N-nitrosourea,2-acetylaminotluorene and 1,2-dimethylhydrazine. Mutations were analYsedin four regions of the murine pS3 gene: exon 4, exon 5, intron 6 andintron 8. The results demonstrated that the two intron regions possessedapproximately tenfold more mutations compared with the exon regions. Thisdata was supported by mutation frequency calculations, made possible bythe inclusion of an internal standard molecule in the RSM assay, whichalso showed the higher mutability of the intron regions. Intron regions weresufficiently sensitive such that a number of spontaneous mutations weredetected. This is the first time that we have detected spontaneous in vivomutations in the RSM assay. The mechanism of this increased mutation inintron sequences remains unclear, but may be due to differential DNA repairor due to selection differences between the coding exon regions and thenoncoding intron regions.

Keyword(s): restriction site mutation assay; intran; exon; mutation

Ip XIV A.51 Metabolle aellvatlon of promutagenle carelnogenswitb human hepatoma (Hep G2) eells

F. Darroudil,l, S. Knasrniillef, R. Sanyal", C.M. Meijers", A.T. Natarajan l ,2 .

JMGC, Department of Radiation Genetics and Chemical Mutagenesis,University ofLeiden, Leiden, The Netherlands; 2J.A. Cohen Institute. IRS.Leiden; The Netherlands; j Institute ofTumor Biology and cancer Research,Uniuersity of Vienna, Vienna, Austria

We have shown that Hep 02 cells can be used as metabolic activation systemas well as target for DNA damage. We have isolated S9-fraction from HepG2 cells and mvestigated its ability to activate promutagenic carcinogensusing Chinese hamster ovary (CHO) cells (in vitro) and Ames Salmonellaassay.

DIfferent cytogenetic assays, i.e. sister-chromatid exchanges (SCEs), mi­cronuclei (MN) 10 binucleated cells as well as aneuploidy (using MN in com­binatio~ ,:"ith a pancentromeric probe and tluorescene in situ hybridization),CytotOXICIty andlor gene mutanoa (at HPRT locus) were used as biologicalend-points.

The mutagenic potential of different types ofheterocyclic aromatic aminescooked food mutagens such as IQ, MeIQ. PHIP and Trp-P-l, as well ~hexamethylphosphoramide (HMPA) and safrole were studied.

Usmg a banery consisting of different test systems i.e., human HepG2, CHO cells as well as Ames test, positive results were found with allchemicals used in this study.

This is the first report concerning the mutagenic potential of HMPA andsafrole in in vitro eukaryotic test systems as well as in Ames Salmonellaassay.

The present data indicate that Hep G2 cells are more efficient in activatingdifferent classes of indirectly-acting promutagens than the one derived fromrat liver S9-fraction.

Keyword(s): Bioactivation of mutagens; Hep G2 cell system

Ip XIV A.61 Mutagenle action of myeotoxtns at moleeular level

Marcela Aranda1, Manuel Ellahueile 1. IDep. Cs. Biologicas, Universidad

de Santiago de Chile. Casilla 40 Correo 33. Santiago. Chile; 1Laboratoriode Genetica Toxicologica, Fac. Cs. Qcas. y Farm., Uniuersidad de Chile,Casilla 123 Santiago I. Chile

It has become evident that many human cancers are associated with changesin p53 gene. The inactivation of its tumor-suppressor activity is an almostuniversal step in the development of human cancers. Cells without workingpS3 arc more susceptible to the mutagenic effects of DNA-damaging agents.In the past few years, researchers have found that some carcinogens leavecharacteristic fingerprints in p53. Atlatoxin BI, causes a serine substitutionat a particular spot on the pS3 protein, some carcinogens in cigarette smokeand oxy radicals also cause a charaeteristic replacement. Such mutational

S-XIV: New mutagenicity tests and their evaluation SI25

fingerprints can offer clues to a cancer's origin . The spectrum of mutationsin p53 induced in human cancer can help to identify particular carcinogens.Fumon isin BI (FBI), a mycotoxin found on com and other grains, can inter­feres with the synthesis of sphingol ipids, causing intracellular accumulationof sphinganine and sphingosine. The genotoxic ity of FBI was evaluated bymeans of the Comet assay. FBI (15 and 1.25 mglKg) produced a 17% and8% of tailed cells respectively, in mice CFI after 14 h treatment.

The mutagenic action of FBI on p53 gene in human cells was determinedusing SSCP-PCR analysis and nucleotide sequencing analysis . Base inser­tions in exons 7-9 produced a stop codon (TAA) and a truncated proteinwas obtained .

what it does suggest is that the spontaneous mutation rate in the naturalenvironment may differ significantly from that shown in the laboratory.

Keyword(s): Spontaneous rnutanon; Mixed and Pure Culture

Ip XIV A.91 Bioluminescent detection of DNA damage viaumuC' -luxAB gene fusion

Tamara Justus', Susan M. Tbomas" , The Flinders University of SouthAustralia, Adelaide S.A.• $042. Australia

Keyword(s): mutagenic ity; umuDC; lraAB

Keyword(s): mutagenicity test; in vitro RAPDs

Concepc ion Becerril" , Mar Ferrero', Argelia Castailo. Dluision of Envi­ronmental Toxicology. CISA-INIA . E-28130 JfIldeolmoJ. Madrid . Spain;t Toxicology Unit. CNAyN. I. Carlos III. E-28220 Majadahonda, Madrid,Spa",

Increasing concern about environmental mutagens has lead to continuouslydevelop new in vivo and in vitro lest in order to elucidate DNA damage.Recent methodologies in molecular biology can be used to improve thismutagenic ity test, among this, the use of DNA or RNA fingerprint ing byarbitrarily primed polymerase chain reaction.

Random Amplified Polymorphic DNA (RAPDs) is a modification of thepolymerase chain reaction that generate an information rich of genomic DNAand can be used in the detection of genomic alterations, such as losses orgains in anonymous sequences and deletions or. insertions of one or a fewnuelcotides.

We have been recently characterised the DNA pattern of the RTG-2cells, an established fibroblastic-like cell line derived from rainbow trout(Oncorrynchus mykiss), by RAPDs using several arbitrarily primers and itspair combinations. In this work we show the results obtained after exposethis cells to 0.5 (g/ml of Mitomicin C at different exposure length (4, 6, 8, 24and 48 h.), Differences in DNA fingerprint were obtained between controland treated cells. All exposure periods, except for 48 h shows in two of theprimers used .

The results obtained allow us to propose this system as a promis ing toolfor detection of genotox ic pollutants.

Detection of mltomlcln C In vitro effects by DNARngerprlntlng on rainbow trout cells

There ISa direct link between the mutagenic effect of chemicals in bacteriaand their carcinogen ic activity in humans . As bacteria are simple, rapidlygrowing organisms they can be used in inexpensive, rapid mutagenic itydetection tests. Prokaryotes have elaborate cellular mechanisms which re­spond to !)NA damage and repair. In Escherichia coli it is the umu operonwhich participates in 80S induced mutagenic DNA repair. Previous studieshave developed umu gene fusions using lacZ as a reporter gene . This studyhas Shown that umu can also be successfully fused to the biolum inescentreporter gene IraAB for the purpose of mutagen detection . The lux genesof the genera Vibrio and Photobacterium have been well characterised andexplo ited as sensitive reporter genes for measuring gene expression and thedetection of genetically engineered microorganisms. In this test system arelationship was seen between increasing mutagen dose and an increase inbioluminescence. This provided an effective system for mutagenicdetectionvia lummescence . The test was also rapid, easy to perform and sensitive.The minimum dose which produced a doubling in response above that ofbackground level was 0.3 JIM· for uv, 0.33 ug/ml N-Methyl-N' -nitro-N­nitrosoguanid ine and 10 nl methylmethanesulphonate. Further appl icationsof this system are currently under investigation for routine testing of complexenvironmental samples as well as validation in the detection of a broad rangeof potentially mutagenic chemicals.

Ip XIV A.tOI

Samantha Mead, Susan M. Thomas. The Flinders University of SouthAustralia , Adelaide, S.A., $042. Australia

Akihiro Araki, Rie Ishida, Toshiaki Sasaki, Taijiro Matsushima. JapanBioassay Research Center; Hadano, Kanagawa, 2$7, Japan

Keyword(s) : E. coli WP2 tester strain; tolqmtcB) mutation; Deep rough likestrain

We have compared the sensitivity of new tester strains having tolC cytoplas­mic membrane mutation (deep rough like strains) Escherichia coli WP2tolC ,WP2toIClpKMIOI , WP2uvrA, tolC and WP2uvrA, tolClpKMIOI and strainscarrying normal cytoplasmic membrane (non-deep rough tester stra ins) E.coli WP2, WP2!pKMIOI, WP2uvrA and WP2uvrAlpKMIOI by measuringthe specific activity (revertants/mg) of mutagens using preincubation method(at 37C for 20 min.). The tolC deep rough like strains were more sensitiveto polycyclic and heterocyclic compounds such as 2-arninoanthracene, 2­nitrofluorene, Glu-P-I , benzo{a)pyrene, mitomycin C, streptonigrin and dox­orubicin than non deep rough tester strains . Mutagenic ity of 2-nilrofluorenewere not detected by WP2, WP2/pKMIOI and WP2uvrA, but detected byWP2toIC, WP2tolClpKMIOI, WP2uvrA, tolC. However, deep rough likestrains were not sensitive enough to detect the mutagenicity ofstreptozotoein,Ara-A and cisplatin. Mutagenicity of Ara-A was not detected by WP2uvrA,tolClpKMIOI, but detected by WP2uvrAJpKMIOI. Enhancing effect ofsensitivity to detecting mutagenicity by tole cytoplasmic membrane mutationwere clearly observed on low sensitivity strain WP2, but less clearly on highsensitivity strain WP2uvrAlpKM 10I. E. coli WP2 tester strains having deeprough like tolC mutation were of great advantage in detecting mutagens.

Keyword(s) : Fumonis in; p53 gene; Mutagenesis

Previously experiments using bacteria in pure culture have been used to pre­dict mutation rates in the environment. These methods provide informationon the mutation rate of the species when exposed to a mutagen, Bacteria livein an environment associated with multi-species competition and are rarelyfound in pure culture, yet spontaneous mutation continues to be measuredusing organisms cultivated in pure culture. Recently a collection of bacteriaiodigenous to subsurface caves in Naracoone, South Australia were isolated.The aim of the study was to examine the impact of increasing anthropogenicinllux into the cave, on the genetic stability of the caves major inhabitants,bacteria. To determine if the spontaneous mutation rate as measured in thelaboratory was an accurate reflection ofwhat was occurring naturally, parallelcultures, one containing a reporter strain of bacteria in pure culture andanother with the reporter strain as well as bacteria from the cave were setup. The results show that the mutation rate in the reporter strain differeddepend ing on whether the organism was grown in pure as opposed to mixedculture . There are several explanations for why this may be the case but

Ip XIV A.81 An evaluation of spontaneous mutation In the envi­ronment

Ip XIV A.7/ Comparison ofsensltlvlty and ulillty of tolC (mtcB)deep rough Uke mutant of E. coli WP2 tester strainsand WP2 tester strains