Mutation Research, 29 (I975) 235-24o ,© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
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29 CARERE,A., G. MORPURGO,G. CARDAMONE,M. BIGNAMI,F. AULICINO,G. DI GIUSEPPE AND G. CONTI,Istituto Superiore di Sanita, Roma (Italy).
Point mutations induced by pharmaceutical drugs To rapidly test the mutagenic activity of pharmaceutical drugs two genetic systems have been set up in the filamentous bacterium Streptomyces coelicolor. The first test in based on the back-mutation of two histidine-requiring strains, hisA z and hisA z2o (leaky mutant), previously characterized (CAREREet al., Mol. Gen. Genet., 123 (1973)). The second is based on the resistance to low levels ot streptomycin. Both genetic systems were checked with methyl-nitro-nitrosoguanidine (MNNG). The technique used was the so-called "plate test" which consists in adding the suspected product directly to a lawn of spores in a petri dish. About one hundred pharmaceutical specialities have been tested so far. Six were found to be mutagenic. Four (Mexase, Enterovioformio, Enterum and Locorten) contain quinolines; one (Lipopil) contains phentermine and the last (Neo Borocillina) contains 2,4-dichlorobenzyl alcohol. The relative mutagenic activity was in decreasing order: MNNG, Mexase, Lipopil, Enterovioformio, Neo-Borocillina, Enterum and Locorten. The same drugs are now being tested on a set of histidine-requiring strains of Salmonella tvphimurium (AMEs et al., Proc. Natl. Acad. Sci. (U.S.A.), 7° (1973)). Up to now quinolines and phentermine seem to be mutagenic in Salmonella producing both frameshift and base substitution mutations. Finally it has been observed that the same drugs are unable to induce point mutations in Aspergitlus nidulans; seventeen of them however strongly increase the incidence of non-disjunction (BIGNAMI et al., Mutation Res., 21 (1973)). The complete composition of the specialities may be found on "L'Informatore Farmaceutico-Organizzazione Editoriale Medico Farmaceutica, Milano, via Edolo 42, 197o" or in tbe "Mercks Index".
3O ELLENBERGER, J., AND G. MOHN,Zentrallaboratorium ffir Mutagenit~tspriifung der DFG, Freiburg i. Br. (West Germany).
More about intrasanguineous mutagenicity testing Since their description in 1973, animal-mediated mutagenicity tests involving intravenous injection of bacteria into laboratory mammals and their recovery from the liver have been developed to routine methods and compared with other techniques. The usefulness of the multi-purpose strain E. coli K-I2/galRSs/arg56/nadm has been further investigated. Forward mutations leading to 5-methyl-tryptophan resistance are of at least three penotypically distinguishable types (e.g. by colony morphology, excretion of tryptophan). Furthermore, in the galRS8 forward mutation system it is possible to detect mutations extending to genes adjacent to the galactose regulatory gene, namely those of the phenotype gal+/lys -. In principle several species of bacteria can be used intravenously. A comparison of the retention by the liver between a deep rough strain of S. typhimurium (TA 1535, kindly supplied by Prof. B. N. AMES)
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4TH ANNUAL MEETING, HEIDELBERG
and tile above-mentioned E. coli strain has shown that both strains can be used in mutagenicity tests; however, the recovery of viable E. cell cells from the liver is approximately ten times higher than that of the Salmonella cells. The sensitivities o~ the following methods to dialkylnitrosalnines as reference mutagens have been compared using the E. coli strain: incubation of bacterial cells together with liver extracts, inoculation of bacterial cells into the peritoneal cavity and after injection into the blood stream. Tile results of these experimeuts will he discussed from tile point of view of metabolic activation.
3t KAPPAS, A., AND S. G. GEORGOI'OULOS, Nuclear Research Center "Democritus",
Biology Department, Athens (Greece).
Fungicides causing mitotic segregation in Aspergillus diploids Tile aromatic hydrocarbon fungicides, Botran (2,6-dichloro-4-nitroaniline), Chloroneb (I,4-dichloro-2,5-dimethoxybenzene), PCNB (pentachloronitrobenzene) and SOPP (sodium orthophenylphenate) affected the stability of Aspergillus nidulans diploid strains. They increased the number of white and yellow segregants in colonies grown from conidia of heterozygous green diploids. Growth inhibition increased with increasing concentration of the fungicide., proportionally to the total number of induced segregants. The contribution of haploidization to induced segregation was greater as fungicide concentration increased, indicating that the fungicides cause non-disjunction.
32 KOLAR, G. F., Institute for Experimental Toxicology and Chemotherapy, German Cancer Research Centre, Heidelberg (West Germany).
Chemical mechanisms and biotransformations involved in triazene mutagenesis The mutagenic activity of alkarvltriazenes in different test systems can be correlated with the chemical reactivity and expected biotransformation of individual compounds (KOLAR et al., in preparation). It is generally accepted that the biological activity of this class of compounds is due to two principal molecular mechanisms which are not only responsible for tile initiation of neoplastic transformation in a living cell (PREUSSMANNet al., Ann. N . Y . Acad. Sci., 163 (1069) 697; KOLAR AND PREUSSMANN, Z. Nalurforsch., 26b (1971) 95o), but probably also for the genetic activity, as follows. Gr) The liberation of reactive arenediazonium cations from labile triazenes which undergo ready cleavage under physiological conditions is responsible for the induction of local tumours in rats and also for their potent genetic activity in test systems t h a t lack the activating enzymes, e.g. in Saccharomyces. (e) Enzvmic hydroxylation of stable compounds at an a-C atom, followed by elimination of the oxygenated side-chain, and ultimately yielding a potential alkylating intermediate, leads to the induction of resorptive tumours and to nmtations in systems containing
235
29 CARERE,A., G. MORPURGO,G. CARDAMONE,M. BIGNAMI,F. AULICINO,G. DI GIUSEPPE AND G. CONTI,Istituto Superiore di Sanita, Roma (Italy).
Point mutations induced by pharmaceutical drugs To rapidly test the mutagenic activity of pharmaceutical drugs two genetic systems have been set up in the filamentous bacterium Streptomyces coelicolor. The first test in based on the back-mutation of two histidine-requiring strains, hisA z and hisA z2o (leaky mutant), previously characterized (CAREREet al., Mol. Gen. Genet., 123 (1973)). The second is based on the resistance to low levels ot streptomycin. Both genetic systems were checked with methyl-nitro-nitrosoguanidine (MNNG). The technique used was the so-called "plate test" which consists in adding the suspected product directly to a lawn of spores in a petri dish. About one hundred pharmaceutical specialities have been tested so far. Six were found to be mutagenic. Four (Mexase, Enterovioformio, Enterum and Locorten) contain quinolines; one (Lipopil) contains phentermine and the last (Neo Borocillina) contains 2,4-dichlorobenzyl alcohol. The relative mutagenic activity was in decreasing order: MNNG, Mexase, Lipopil, Enterovioformio, Neo-Borocillina, Enterum and Locorten. The same drugs are now being tested on a set of histidine-requiring strains of Salmonella tvphimurium (AMEs et al., Proc. Natl. Acad. Sci. (U.S.A.), 7° (1973)). Up to now quinolines and phentermine seem to be mutagenic in Salmonella producing both frameshift and base substitution mutations. Finally it has been observed that the same drugs are unable to induce point mutations in Aspergitlus nidulans; seventeen of them however strongly increase the incidence of non-disjunction (BIGNAMI et al., Mutation Res., 21 (1973)). The complete composition of the specialities may be found on "L'Informatore Farmaceutico-Organizzazione Editoriale Medico Farmaceutica, Milano, via Edolo 42, 197o" or in tbe "Mercks Index".
3O ELLENBERGER, J., AND G. MOHN,Zentrallaboratorium ffir Mutagenit~tspriifung der DFG, Freiburg i. Br. (West Germany).
More about intrasanguineous mutagenicity testing Since their description in 1973, animal-mediated mutagenicity tests involving intravenous injection of bacteria into laboratory mammals and their recovery from the liver have been developed to routine methods and compared with other techniques. The usefulness of the multi-purpose strain E. coli K-I2/galRSs/arg56/nadm has been further investigated. Forward mutations leading to 5-methyl-tryptophan resistance are of at least three penotypically distinguishable types (e.g. by colony morphology, excretion of tryptophan). Furthermore, in the galRS8 forward mutation system it is possible to detect mutations extending to genes adjacent to the galactose regulatory gene, namely those of the phenotype gal+/lys -. In principle several species of bacteria can be used intravenously. A comparison of the retention by the liver between a deep rough strain of S. typhimurium (TA 1535, kindly supplied by Prof. B. N. AMES)
236
4TH ANNUAL MEETING, HEIDELBERG
and tile above-mentioned E. coli strain has shown that both strains can be used in mutagenicity tests; however, the recovery of viable E. cell cells from the liver is approximately ten times higher than that of the Salmonella cells. The sensitivities o~ the following methods to dialkylnitrosalnines as reference mutagens have been compared using the E. coli strain: incubation of bacterial cells together with liver extracts, inoculation of bacterial cells into the peritoneal cavity and after injection into the blood stream. Tile results of these experimeuts will he discussed from tile point of view of metabolic activation.
3t KAPPAS, A., AND S. G. GEORGOI'OULOS, Nuclear Research Center "Democritus",
Biology Department, Athens (Greece).
Fungicides causing mitotic segregation in Aspergillus diploids Tile aromatic hydrocarbon fungicides, Botran (2,6-dichloro-4-nitroaniline), Chloroneb (I,4-dichloro-2,5-dimethoxybenzene), PCNB (pentachloronitrobenzene) and SOPP (sodium orthophenylphenate) affected the stability of Aspergillus nidulans diploid strains. They increased the number of white and yellow segregants in colonies grown from conidia of heterozygous green diploids. Growth inhibition increased with increasing concentration of the fungicide., proportionally to the total number of induced segregants. The contribution of haploidization to induced segregation was greater as fungicide concentration increased, indicating that the fungicides cause non-disjunction.
32 KOLAR, G. F., Institute for Experimental Toxicology and Chemotherapy, German Cancer Research Centre, Heidelberg (West Germany).
Chemical mechanisms and biotransformations involved in triazene mutagenesis The mutagenic activity of alkarvltriazenes in different test systems can be correlated with the chemical reactivity and expected biotransformation of individual compounds (KOLAR et al., in preparation). It is generally accepted that the biological activity of this class of compounds is due to two principal molecular mechanisms which are not only responsible for tile initiation of neoplastic transformation in a living cell (PREUSSMANNet al., Ann. N . Y . Acad. Sci., 163 (1069) 697; KOLAR AND PREUSSMANN, Z. Nalurforsch., 26b (1971) 95o), but probably also for the genetic activity, as follows. Gr) The liberation of reactive arenediazonium cations from labile triazenes which undergo ready cleavage under physiological conditions is responsible for the induction of local tumours in rats and also for their potent genetic activity in test systems t h a t lack the activating enzymes, e.g. in Saccharomyces. (e) Enzvmic hydroxylation of stable compounds at an a-C atom, followed by elimination of the oxygenated side-chain, and ultimately yielding a potential alkylating intermediate, leads to the induction of resorptive tumours and to nmtations in systems containing
