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P XI A.18 Mutagenic specificity of acrolein and crotonaldehyde in the supF shuttle vector system
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S-XI : Mutational signatures of knawn environmental carcinogens
Ip XI
A.ISI
Prollies of chemIcally Induced nucleotIde subslltulions obtained by reversion of IDcZ poInt mulallons In a set of Escherlcbla coli tester strains
Fatima Garganta, Gerhard Scherer, GOOter Krause. ABF. Ana(ytiscJobiologisches Forschungslabor Miinchen. Germany A set of lactose-auxotropic E. coli tester strains bad been improved for determining the proportion of the six possible transitions and transversions after exposure to directly acting environmental mutagens (Lu et al. 1995: Mutat. Res. 343, 219--227). Adapting this system, we investigated mutagenesis by additional chemicals, including some that require metabolic activation. Revertants were selected by means of lactose-containing minimal medium. In addition , the survival after mutagen exposure was evaluated by plating dilutions of identically treated bacterial suspensions on glucose plates. N-Methyl·N·nitrosoguanidine (MNNG) caused G>A-uansitions about 100 times more frequently than the other base changes. culminating in a 4000-fold increas e of revertant numbers at 4 Ilg/plate. Benzo[a]pyrenediolepoxide (BPDE, 0.1-3 .0 Ilg/plate) induced 20-130 G>T-revertants/plate, which was the prevailing reversion. A dose of 10 Ilg/plate of the DNAintercalating dye ethidium bromide resulted in 20% bacterial survival and doubling of revertant numbers in all six strains, which is consistent with the action of a frame shift-inducing mutagen. Three chemicals were investigated in the presence of an external metabolic activation system with rat liver microsomes. Benzo[a]pyrene elicited a mutation profile and mutagenic response similar to its ultimate mutagen BPDE , but at 1000fold higher doses. After exposure with 5--20 mglplatc of N-nitrosodimethylamine, reversion of the strain respond ing to A>Ctransvers ions prevailed. After treatment w ith benzo[c]phenanthrene (5--30 Ilglplate) , A> T transversions dominated the mutat ion profile, which corresponds to the known adduct formation specifity for adenosine residues. The present bantry of tester stra ins provided a convenient means to establish the distribution of induced base changes by counting revertant colonies instead of molecular analysis.
Ip XI
A.171
Zsolt Kelecsenyi ', Diane L. Spenc~, William J. Caspuy2. INatioMI Institute of Public Health. Budapest , 1966. Hungary; lNIH. RTp, NC 27709 USA • Previously we demonstrated that the cytidine analog, S-azacytidine (SAC) is capable of quantitative induction of 6-thioguanine resistance (6TG') in ASS2 cells. For molecular analyses. a total of 148 SAC-induced 610' clones were isolated . PCR analyses suggested that 133 were putative point mutants and IS were appareDl deletion mutants. The putative point mutants were sequenced and S2I133 showed sequence alterations, mostly GC>CG transversions. In the other 81 mutant clones no mutations in the gpt structural gene were found. We have further characterized the genomic DNA surrounding the gpr gene in these clones . By Southern blotting, we have examined 37 independent 6TG' clones using the restriction enzymes. None of the II clones analyzed showed an aberrant blot pattern as compared to wild-type (WI) nonmutant ASS2 genomic DNA. These data suggest that for these clones, the 6TG' phenotype is not due to rearrangements or deletions of genomic DNA within a range of about 9 kb surrounding the gpt mtegration site . We have also used Southern bloning to characterize the genomic methylation status of the gpr gene in 32 of these 37 clones using methylation sensitiverestriction enzymes. The data show no difference from WI ASS2 cells in the studied 6TG' clones with the used methylation sensitive enzymes. These data demonstrate that SAC is a potent inducer of GC>CG transversions in mammalian cells . In addition, SAC may also be inducing 6TG resistance in ASS2 cells via a mechanism other than genomic rearrangement or direct alterations of the gpt structural gene . Keyword(s): ASS2 cells ; Mutagenesis
Ip XI
A.ISI
Keyword{s): Mutational spectrum; Mutagenesis; ~galactosidase
Ip XI
A.161
Studies on the InacllvaUon ofcarclDOl:enlc MNNG by the additIon of soluble vitamIns and SH compounds
Teruh isa Hirayama, Yasushi Katayama, Terue Kasai, Tetsushi Watanabe. Kyoto Pharmaceutical University. Kyoto 607. Japan The relationship between the inhibitory effects of water soluble vitamins and SH compounds on the formation of 06-methyldeoxyguanosine (MedGuo) and on the mutagenicity of N-methyl-N' -nitre-Nsnitresoguanidine (MNNG) was investigated. Vitamin BI, B2, B6, nicotinamide, ascorbic acid (AsA) and dehydroascorbic acid (DHA) were selected as water soluble vitamins, and cysteine (Cys) and glutathione were also selected as SH compounds. In the experiments of inhibitory effects of the compounds on the formation of 06-MedGuo, MNNG, 2' -decxyguanosine and each of the above compounds were incubated at 37 {I! {JC for 6 h, and the result ing 06-MedGuo was derivatized to the fluorescent compounds. Then, the fluorophore was determined by HPLc, and the formation ratio (%) of06-MedGuo was calculated and compared to that in the reaction without vitamin or SH compound. By the addition of SH compounds (I00-folds molar of MNNG), the amounts of the formed 06-MedGuo was depressed to less thao 10";'. The inhibitory effects of AsA and DHA on the formation of 06-MedGuo were greater than those of other vitamins. The desmutagenic effects of AlA and Cys on the mutagenicity of MNNG in Salmonella typhimurium TAlOO were observed. and the mutagenicity was reduced to 10% or less by the equimolar AsA and CystoMNNO. The desmutagenic effects of vitamin B I, B2 and B6 on MNNO were not observed. Keyword(s): 06-methyldcoxyguanosine; desmutagenicity ; vitam ins
Molecular aDalysls of mutations In ASSl eells Induced by 5-azacytldlne
Mlltagenlc speclllclty or acrolein and crotonaldehYde ID the lupF shuttle vector system
Masanobu Kawaaishi", Tomonari Matsuda', Go Sasaki', Talcashi Vagi] 1 Saburo Matsui , Hira1ru Take~ . I Researclt Center fer ElXJironment~ Quality Control. Kyoto University. 1-2 lUmihama, Otsu, Shiga $20. Japan; J Faculty of Science, Graduate School of Medicine. Kyoto University. Sa9>oku Kyoto 60rHJl. Japan ; JDepartment of Radiation Genet ics. GlUduate School of Medicine, Kyoto Uniuersity. ~ku Kyoto 606-01, Japan Mutation induction in the supF gene of the shuttle vector plasmid pMYI89 by acrolein and crotonaldehyde was exam ined . These muta_ genic/carcinogenic compounds are present in our environment as conunonly_ used industrial chemicals, natural products of widespread occurrence, enVironmental contaminants and products of endogenous metabolism in human beings. The pMYI89, which was derived from pZI89, was treated with acrolein or crotonaldehyde. The plasmids were replicated and mutagenized in normal human fibroblast WI38-VAI3. With both compounds. 76-8S% of the mutations induced were base substitutions and the remainder of the mutations were framesh ift mutations. About half of the mutant plasimids (46--47%) had a single base substitution, while 18-2S% had multiple base substitutions and interestingly 12-13% had tandem (adjacent two) base substitutions . Of the base subst itution mutat ions, 44-SO"/. were G :C to T :A transversions and 23-24% were G:C to A:T transitions. Keyword{s): Acrole in; Crotonaldehyde; Mutation spectrum; supF
Ip XI
A.191
DNA and cell ultrastructure damage following mium admlolstratlon on the skin Z
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Caroline Fasanya 1, Lema Gilliard , Menard , Akinyela AbduJlah2 Glenn Sponholtr , Christopher Ikediobi • Lekan M. Latinwo 2 • I Colleg~ of Pharmacy and Pharmaceutical Sciences. Florida A & M University, Tallahassee. FL 32307. USA; J Department of Biology. Florida A. & AI University. Tallahassee. FL 32307. USA; J Department ofChemistry. Florida A &: M University. TalIDltassee. FL 32307. USA
S-XI : Mutational signatures of knawn environmental carcinogens
Ip XI
A.ISI
Prollies of chemIcally Induced nucleotIde subslltulions obtained by reversion of IDcZ poInt mulallons In a set of Escherlcbla coli tester strains
Fatima Garganta, Gerhard Scherer, GOOter Krause. ABF. Ana(ytiscJobiologisches Forschungslabor Miinchen. Germany A set of lactose-auxotropic E. coli tester strains bad been improved for determining the proportion of the six possible transitions and transversions after exposure to directly acting environmental mutagens (Lu et al. 1995: Mutat. Res. 343, 219--227). Adapting this system, we investigated mutagenesis by additional chemicals, including some that require metabolic activation. Revertants were selected by means of lactose-containing minimal medium. In addition , the survival after mutagen exposure was evaluated by plating dilutions of identically treated bacterial suspensions on glucose plates. N-Methyl·N·nitrosoguanidine (MNNG) caused G>A-uansitions about 100 times more frequently than the other base changes. culminating in a 4000-fold increas e of revertant numbers at 4 Ilg/plate. Benzo[a]pyrenediolepoxide (BPDE, 0.1-3 .0 Ilg/plate) induced 20-130 G>T-revertants/plate, which was the prevailing reversion. A dose of 10 Ilg/plate of the DNAintercalating dye ethidium bromide resulted in 20% bacterial survival and doubling of revertant numbers in all six strains, which is consistent with the action of a frame shift-inducing mutagen. Three chemicals were investigated in the presence of an external metabolic activation system with rat liver microsomes. Benzo[a]pyrene elicited a mutation profile and mutagenic response similar to its ultimate mutagen BPDE , but at 1000fold higher doses. After exposure with 5--20 mglplatc of N-nitrosodimethylamine, reversion of the strain respond ing to A>Ctransvers ions prevailed. After treatment w ith benzo[c]phenanthrene (5--30 Ilglplate) , A> T transversions dominated the mutat ion profile, which corresponds to the known adduct formation specifity for adenosine residues. The present bantry of tester stra ins provided a convenient means to establish the distribution of induced base changes by counting revertant colonies instead of molecular analysis.
Ip XI
A.171
Zsolt Kelecsenyi ', Diane L. Spenc~, William J. Caspuy2. INatioMI Institute of Public Health. Budapest , 1966. Hungary; lNIH. RTp, NC 27709 USA • Previously we demonstrated that the cytidine analog, S-azacytidine (SAC) is capable of quantitative induction of 6-thioguanine resistance (6TG') in ASS2 cells. For molecular analyses. a total of 148 SAC-induced 610' clones were isolated . PCR analyses suggested that 133 were putative point mutants and IS were appareDl deletion mutants. The putative point mutants were sequenced and S2I133 showed sequence alterations, mostly GC>CG transversions. In the other 81 mutant clones no mutations in the gpt structural gene were found. We have further characterized the genomic DNA surrounding the gpr gene in these clones . By Southern blotting, we have examined 37 independent 6TG' clones using the restriction enzymes. None of the II clones analyzed showed an aberrant blot pattern as compared to wild-type (WI) nonmutant ASS2 genomic DNA. These data suggest that for these clones, the 6TG' phenotype is not due to rearrangements or deletions of genomic DNA within a range of about 9 kb surrounding the gpt mtegration site . We have also used Southern bloning to characterize the genomic methylation status of the gpr gene in 32 of these 37 clones using methylation sensitiverestriction enzymes. The data show no difference from WI ASS2 cells in the studied 6TG' clones with the used methylation sensitive enzymes. These data demonstrate that SAC is a potent inducer of GC>CG transversions in mammalian cells . In addition, SAC may also be inducing 6TG resistance in ASS2 cells via a mechanism other than genomic rearrangement or direct alterations of the gpt structural gene . Keyword(s): ASS2 cells ; Mutagenesis
Ip XI
A.ISI
Keyword{s): Mutational spectrum; Mutagenesis; ~galactosidase
Ip XI
A.161
Studies on the InacllvaUon ofcarclDOl:enlc MNNG by the additIon of soluble vitamIns and SH compounds
Teruh isa Hirayama, Yasushi Katayama, Terue Kasai, Tetsushi Watanabe. Kyoto Pharmaceutical University. Kyoto 607. Japan The relationship between the inhibitory effects of water soluble vitamins and SH compounds on the formation of 06-methyldeoxyguanosine (MedGuo) and on the mutagenicity of N-methyl-N' -nitre-Nsnitresoguanidine (MNNG) was investigated. Vitamin BI, B2, B6, nicotinamide, ascorbic acid (AsA) and dehydroascorbic acid (DHA) were selected as water soluble vitamins, and cysteine (Cys) and glutathione were also selected as SH compounds. In the experiments of inhibitory effects of the compounds on the formation of 06-MedGuo, MNNG, 2' -decxyguanosine and each of the above compounds were incubated at 37 {I! {JC for 6 h, and the result ing 06-MedGuo was derivatized to the fluorescent compounds. Then, the fluorophore was determined by HPLc, and the formation ratio (%) of06-MedGuo was calculated and compared to that in the reaction without vitamin or SH compound. By the addition of SH compounds (I00-folds molar of MNNG), the amounts of the formed 06-MedGuo was depressed to less thao 10";'. The inhibitory effects of AsA and DHA on the formation of 06-MedGuo were greater than those of other vitamins. The desmutagenic effects of AlA and Cys on the mutagenicity of MNNG in Salmonella typhimurium TAlOO were observed. and the mutagenicity was reduced to 10% or less by the equimolar AsA and CystoMNNO. The desmutagenic effects of vitamin B I, B2 and B6 on MNNO were not observed. Keyword(s): 06-methyldcoxyguanosine; desmutagenicity ; vitam ins
Molecular aDalysls of mutations In ASSl eells Induced by 5-azacytldlne
Mlltagenlc speclllclty or acrolein and crotonaldehYde ID the lupF shuttle vector system
Masanobu Kawaaishi", Tomonari Matsuda', Go Sasaki', Talcashi Vagi] 1 Saburo Matsui , Hira1ru Take~ . I Researclt Center fer ElXJironment~ Quality Control. Kyoto University. 1-2 lUmihama, Otsu, Shiga $20. Japan; J Faculty of Science, Graduate School of Medicine. Kyoto University. Sa9>oku Kyoto 60rHJl. Japan ; JDepartment of Radiation Genet ics. GlUduate School of Medicine, Kyoto Uniuersity. ~ku Kyoto 606-01, Japan Mutation induction in the supF gene of the shuttle vector plasmid pMYI89 by acrolein and crotonaldehyde was exam ined . These muta_ genic/carcinogenic compounds are present in our environment as conunonly_ used industrial chemicals, natural products of widespread occurrence, enVironmental contaminants and products of endogenous metabolism in human beings. The pMYI89, which was derived from pZI89, was treated with acrolein or crotonaldehyde. The plasmids were replicated and mutagenized in normal human fibroblast WI38-VAI3. With both compounds. 76-8S% of the mutations induced were base substitutions and the remainder of the mutations were framesh ift mutations. About half of the mutant plasimids (46--47%) had a single base substitution, while 18-2S% had multiple base substitutions and interestingly 12-13% had tandem (adjacent two) base substitutions . Of the base subst itution mutat ions, 44-SO"/. were G :C to T :A transversions and 23-24% were G:C to A:T transitions. Keyword{s): Acrole in; Crotonaldehyde; Mutation spectrum; supF
Ip XI
A.191
DNA and cell ultrastructure damage following mium admlolstratlon on the skin Z
Rir
z
cad_
Caroline Fasanya 1, Lema Gilliard , Menard , Akinyela AbduJlah2 Glenn Sponholtr , Christopher Ikediobi • Lekan M. Latinwo 2 • I Colleg~ of Pharmacy and Pharmaceutical Sciences. Florida A & M University, Tallahassee. FL 32307. USA; J Department of Biology. Florida A. & AI University. Tallahassee. FL 32307. USA; J Department ofChemistry. Florida A &: M University. TalIDltassee. FL 32307. USA