Cancer Letter~. 5 {1978} t99--204
1[99
© Elsevier/North-Holland Scientific Publishers :Ltd.
T H E N E E D FOt~ A MAMMALIAN TEST SYSTEM FOR MUTAGENS ACTION Olr SOME REDUCING AGENTS
:
H.F. ST.~CH,I,. WEI and P. LAM B.C. Caneer R.:search C,~,ntre, Unit in Enuironmenta! Careirmgenesis, Vaneou uer (Canada)
(Received 25 Aprll i978) (Revised version.received 22 June 1978) (Accepted 23 June 1878)
SUMMARY Reducing atents and cysteine, cysteamine, glutathione, ascorbic acid a~d H~.O2 with and without the addition o f Cu 2+ did n o t increase significantly the frequency o f mutations in the Salmonella test at non-toxic c o n c e n ~ i t i o n s but triggered a n~arked DNA repair synthesis and induced a relatively high frequency of chromosome aberrations in cultured mammalian cells. Both IatVer effects were reduced by' the addition 0'~ catalase to solutions of the reducing agents plus Cu 2.. To avoid 'False Negatives' in mutagenicity screening the use o f several t~,,t subjects including mammalim.~ cells seems to be required.
INTRODUCTION
After a thorough validation o f several short.te)'m bioassays for mutagens and carci.nogel"Lsit was generally assumed that only a battery o f tests can reduce the number of 'false negatives.' Unresolved, however., is the question as to ~he type o f bioa~says which should be chosen for routine monitoring programs. I]~ this paper we would like to show the necessity o£ complementing the well established $almonellg/mic~osome mutagenicity test [1:6] with assays using mammalian cells [~1,12] and would like to exemplify this proposal on ~Le differing responses of ratio,us subjects tc, the action of reducing agents including cysteamine, cysteine, gllutathione and ascorbic acid. MATERLkLS AND METHODS Mutagenici~z test
The histidme-requidng s r;rains TA98 ml~d T A t 0 0 of Sai.monella typhi.murium were used ~ indicator orgaaisms for mutagenic activits;~ Assays for his + feverAbbrev~a~'ion: IJNNG, N-methyl-N~-nitroso~N~.niLzoguanidive.
200 sion frequency were perfozvned bF exposing at 37°C bacteria in suspension for
60 rain to the reducing a~,en~=and Cuz*, pelle~lg them by centrifugatiol~, washing with PBS, resuspending in top agar, and overlayktg the bacteria-agar mix ~ r e on minimal agar plat~es. F o r cell sur;ival Studies the bacterial suspeasion was d~uted with 0.85% NaC1 and plated on nutrient agar plates [6.,7]. The fin~d concentration of Cu ~+ was 10 -5 M, prepared by dfluti~.g the aqueous s~ock solution of CuSO~ (0.1 M) and glycine (1 M).
D2~A repair tezt To avoid confusion be~,ween DNA repair synthesis measured as unscheduled incolporation of 3HTdR and DNA replication ,during S-phase, all experiments were perfolnned on non-dividir,g populations of human fibrobtasts [ 10]. The non-dividingcell populations were exposed for 3 h to the reducing agents ~md Cu 2+ followed by 3 h of 3HTdR ( ! 0 uCi/ml). The incorporation of 3HTdR was detected by autoradiog:zaphic analysis. The final concentrations of Cu ~+ were ].0-'* M and 10 "s M. Chromosome aberrations Chromosome aberrations were estimated, on colchicine-arrested (4 h l metaphase plates of CHO ceils followed by the usual fixation staining procedure [.10]. The cultured CHO cells were exposed to the various reducing ;iLgen~ and H..O.~ for 3 h and the samples were taken 16 h post-treatment. The f~aal concentration of Cu 2+ was l 0 -4 M. RESULTS
At non,toxic concentrations the thiol compounds (cysteamine, cysb~ine, and glutathione), sodium ascorbate and H202 failed to increase significantly the frequency of h/s+ rew~.,rtants when added for 60 rain to a suspension, of TA98 or TA100 strains orS. typhimurium (Table 1). These compounds ~lso failed to elevate significantly the mutation rate when incubated with Cu 2+ which accelerates their oxidation [4,5,9] and liberation of H~O~ [3]. Whether cysteine and cyste?mine show a significant elevation of the mutation r~te at high toxic ,closes remains questl,mable (Table 1). For comparison, the mutagen and carcinogen N:raethyl-N-m~roso-Ntnitroguanidine (MNNG) induced 340 revertants/10 v survivors for a dose of 10 -s M MNNG and about 250 revertant~ for 10 ~4 M MNNC under similar incubation conditions. The reducing agents and H202 were also c.orlsidered ~o be non-mutagenic in the 'spot test' when the criteria of McCann and Ames were applied [6]. The capacity of the 4 exarrdned reducing agents and H20~ to induce an unscheduled incorporation of 3HTdR becomes evident from the dose-respcmse curves of Fig. 1. Cu 2+ has bee" added to ~J1 reducing agents to increase their capacity to trigger a DNA repair synthesis. The addition of catalase to the mixtures containing reducing agents and Cu 2+ virtually abolishes their capacity to eilcll, a DNA repair synthesis.
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1[99
© Elsevier/North-Holland Scientific Publishers :Ltd.
T H E N E E D FOt~ A MAMMALIAN TEST SYSTEM FOR MUTAGENS ACTION Olr SOME REDUCING AGENTS
:
H.F. ST.~CH,I,. WEI and P. LAM B.C. Caneer R.:search C,~,ntre, Unit in Enuironmenta! Careirmgenesis, Vaneou uer (Canada)
(Received 25 Aprll i978) (Revised version.received 22 June 1978) (Accepted 23 June 1878)
SUMMARY Reducing atents and cysteine, cysteamine, glutathione, ascorbic acid a~d H~.O2 with and without the addition o f Cu 2+ did n o t increase significantly the frequency o f mutations in the Salmonella test at non-toxic c o n c e n ~ i t i o n s but triggered a n~arked DNA repair synthesis and induced a relatively high frequency of chromosome aberrations in cultured mammalian cells. Both IatVer effects were reduced by' the addition 0'~ catalase to solutions of the reducing agents plus Cu 2.. To avoid 'False Negatives' in mutagenicity screening the use o f several t~,,t subjects including mammalim.~ cells seems to be required.
INTRODUCTION
After a thorough validation o f several short.te)'m bioassays for mutagens and carci.nogel"Lsit was generally assumed that only a battery o f tests can reduce the number of 'false negatives.' Unresolved, however., is the question as to ~he type o f bioa~says which should be chosen for routine monitoring programs. I]~ this paper we would like to show the necessity o£ complementing the well established $almonellg/mic~osome mutagenicity test [1:6] with assays using mammalian cells [~1,12] and would like to exemplify this proposal on ~Le differing responses of ratio,us subjects tc, the action of reducing agents including cysteamine, cysteine, gllutathione and ascorbic acid. MATERLkLS AND METHODS Mutagenici~z test
The histidme-requidng s r;rains TA98 ml~d T A t 0 0 of Sai.monella typhi.murium were used ~ indicator orgaaisms for mutagenic activits;~ Assays for his + feverAbbrev~a~'ion: IJNNG, N-methyl-N~-nitroso~N~.niLzoguanidive.
200 sion frequency were perfozvned bF exposing at 37°C bacteria in suspension for
60 rain to the reducing a~,en~=and Cuz*, pelle~lg them by centrifugatiol~, washing with PBS, resuspending in top agar, and overlayktg the bacteria-agar mix ~ r e on minimal agar plat~es. F o r cell sur;ival Studies the bacterial suspeasion was d~uted with 0.85% NaC1 and plated on nutrient agar plates [6.,7]. The fin~d concentration of Cu ~+ was 10 -5 M, prepared by dfluti~.g the aqueous s~ock solution of CuSO~ (0.1 M) and glycine (1 M).
D2~A repair tezt To avoid confusion be~,ween DNA repair synthesis measured as unscheduled incolporation of 3HTdR and DNA replication ,during S-phase, all experiments were perfolnned on non-dividir,g populations of human fibrobtasts [ 10]. The non-dividingcell populations were exposed for 3 h to the reducing agents ~md Cu 2+ followed by 3 h of 3HTdR ( ! 0 uCi/ml). The incorporation of 3HTdR was detected by autoradiog:zaphic analysis. The final concentrations of Cu ~+ were ].0-'* M and 10 "s M. Chromosome aberrations Chromosome aberrations were estimated, on colchicine-arrested (4 h l metaphase plates of CHO ceils followed by the usual fixation staining procedure [.10]. The cultured CHO cells were exposed to the various reducing ;iLgen~ and H..O.~ for 3 h and the samples were taken 16 h post-treatment. The f~aal concentration of Cu 2+ was l 0 -4 M. RESULTS
At non,toxic concentrations the thiol compounds (cysteamine, cysb~ine, and glutathione), sodium ascorbate and H202 failed to increase significantly the frequency of h/s+ rew~.,rtants when added for 60 rain to a suspension, of TA98 or TA100 strains orS. typhimurium (Table 1). These compounds ~lso failed to elevate significantly the mutation rate when incubated with Cu 2+ which accelerates their oxidation [4,5,9] and liberation of H~O~ [3]. Whether cysteine and cyste?mine show a significant elevation of the mutation r~te at high toxic ,closes remains questl,mable (Table 1). For comparison, the mutagen and carcinogen N:raethyl-N-m~roso-Ntnitroguanidine (MNNG) induced 340 revertants/10 v survivors for a dose of 10 -s M MNNG and about 250 revertant~ for 10 ~4 M MNNC under similar incubation conditions. The reducing agents and H202 were also c.orlsidered ~o be non-mutagenic in the 'spot test' when the criteria of McCann and Ames were applied [6]. The capacity of the 4 exarrdned reducing agents and H20~ to induce an unscheduled incorporation of 3HTdR becomes evident from the dose-respcmse curves of Fig. 1. Cu 2+ has bee" added to ~J1 reducing agents to increase their capacity to trigger a DNA repair synthesis. The addition of catalase to the mixtures containing reducing agents and Cu 2+ virtually abolishes their capacity to eilcll, a DNA repair synthesis.
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