On the mutagenic action of some enzyme immunoassay substrates.

The mutagenic action of six compounds used in ELISA, EMIT and EIA assays, was investigated by means of the fluctuation test, with Klebsiella pneumoniae as a test organism, and the Ames' plate incorporation test using Salmonella typhimurium TA 98, TA 100 and TA 1537. It appears that 2,2'-azino-di(3-ethyl-benzthiazoline sulphonic acid (6)) or ABTS exerts mutagenic action on Klebsiella pneumoniae at a concentration of 11 g/l, on Salmonella typhimurium TA 100 at a top agar concentration of 0.1 g/l, on Salmonella typhimurium TA 98 at 0.2 g/l and on Salmonella typhimurium TA 1537 at 10 g/l. With umbelliferone, mutagenic action was found only with Klebsiella pneumoniae at a concentration of 0.8 g/l or higher. With o-phenylenediamine, strong mutagenic activity was found only with strain TA 98 and metabolic activation at a top agar concentration of 0.001 g/l. With 5-aminosalicylic acid, beta-methylumbelliferone and p-nitrophenyl-phosphate, no mutagenic action was observed.

The need for a sufficient number of low level sera in comparisons of different serum vitamin B12 assays.

Eight radiochemical methods for the assay of vitamin B12 in serum were compared with the microbiological assay with Lactobacillus leichmannii ATCC 7830 using 198 individual sera of patients. There was a good agreement between the results of most samples with some kits and the microbiological assay. However, especially in the sera of vitamin B12-deficient patients large discrepancies between the results could occur. These variations were due to both the kits used and the performance of the assays in different laboratories. A sufficient number of non-pooled sera of vitamin B12-deficient patients should be included in investigations to validate radiochemical methods.

Azathioprine, a genotoxic agent to be considered non-genotoxic in man.

Azathioprine, an immunosuppressive drug, has been used for 25 years. Azathioprine is rapidly converted into a number of metabolites after absorption. Maximum blood levels in experimental animals (mice) were 11.3 micrograms/ml after a dosage of 33.3 mg/kg. Generally, levels of less than 1 microgram/ml are found. As azathioprine is ineffective in hypoxanthine guanine phosphoribosyltransferase (HPRT)-deficient patients, it will be clear that for immunosuppressive activity azathioprine must be metabolised. Regarding mutagenic activity, its mutagenicity for bacteria seems irrelevant for man because the nitroimidazole moiety can be reduced by bacteria but not or hardly at all by mammalian tissues. So 6-mercaptopurine (a metabolite of azathioprine) and its metabolites should be regarded as the active compounds. In vitro azathioprine can induce chromosome aberrations and other cytogenetic events at high, non-physiological doses. However, in view of the low blood levels it is unlikely that azathioprine can induce chromosome aberrations in kidney transplant patients. It is more probable that azathioprine inhibits the elimination of such aberrant cells through its immunosuppressive activity. It should be pointed out that in microbial mutagenicity systems also, azathioprine concentrations that are not reached in patients are needed to obtain an increased mutation rate.

On the mutagenicity of nitroimidazoles.

Regarding mutagenicity, metronidazole is one of the best-investigated compounds of the nitroimidazoles. This drug is mutagenic on bacteria, especially if base-pair tester strains are used and bacterial nitroreductases are present. The serum levels attained in man after intake of this drug are sufficient to cause mutations in bacteria. Furthermore, interaction with and binding to DNA occurs under anaerobic conditions and sometimes DNA breaks are observed. However, metronidazole does not show mutagenic activity in mammalian cells in vitro; the micronucleus test is negative and chromosome aberrations are only found under anaerobic conditions. With microbial systems the mutagenicity of 47 nitroimidazoles has been investigated. Only 4 compounds were always negative in the applied test systems. Because with base-pair tester strains mutagenicity was assessed, this class of compounds should be regarded as a base-pair mutagen. In fungi, some compounds (e.g. ZK 26173 and azathioprine) are potent mutagens, whilst with most investigated nitroimidazoles only a weak or no mutagenic activity could be detected. Somewhat similar observations have been made in tests with Drosophila melanogaster, a test for gene mutations in mammalian cells, the micronucleus test, cytogenic tests and the dominant lethal test. The reduction products of metronidazole, misonidazole and 1-methyl-2-nitro-5-vinylimidazole, cause DNA damage if the nitro group is reduced in the presence of DNA. Reduction products are formed by microbes in the gut or by mammalian cells under anaerobic conditions. No teratological effect due to metronidazole or most other nitroimidazoles has been observed. Metronidazole is carcinogenic in mice and rats, and dimetridazole in rats. Up to the present, no carcinogenic effects have been observed in man. Azathioprine is probably carcinogenic for man. It is unlikely that the therapeutic applications of the presently used nitroimidazoles, except for azathioprine, will cause an increase in the tumor incidence in man or will cause other genotoxic effects, although such effects cannot be excluded with certainty.

Variable mutagenic activity of commercial preparations of ABTS (2,2'-azino-di(3-ethyl-benz-thiazoline) sulphonic acid.

Different batches of ABTS obtained from the same commercial source varied in their capacity to effect direct mutation in the strains of Salmonella typhimurium used routinely in the incorporation test of Ames. One batch, obtained in 1976, and another obtained early in 1979, both exhibited direct base-pair substitution and frame-shift activities. These activities, however, were absent from each of two batches obtained after 1979, and also from a highly purified preparation from a different source. The possible presence of the unsulphonated immediate precursor of ABTS as a mutagenic impurity is an unlikely explanation for the activity of the mutagenic preparations. It is more probable that the commercial synthesis generated other, mutagenic, impurities which remained in the batches obtained in 1976 and early in 1979, but were absent or were removed from later batches. The identity of these active impurities is unknown. Pure ABTS is neither a direct nor an indirect mutagen.

Are econazole, miconazole and clotrimazole mutagenic to bacteria?

The fungistatic drugs econazole, miconazole and clotrimazole were investigated as to mutagenic properties in the fluctuation test with Klebsiella pneumoniae and Escherichia coli K12 as test organisms and in the plate-incorporation test, with and without metabolic activation, with Salmonella typhimurium strains TA98 and TA100. No mutagenic activity of the 3 compounds on these microorganisms was found.

The mutagenic action of quindoxin, carbadox, olaquindox and some other N-oxides on bacteria and yeast.

The mutagenic action of 3 coccidiostatic chinoxaline-N-oxide derivatives, quindoxin, carbadox and olaquindox, was investigated by Luria and Delbrück's fluctuation test, with Klebsiella pneumoniae and Escherichia coli K12 as test organisms. These compounds were mutagenic at very low concentrations (2 X 10(-5)--500 X 10(-5) mmole/l). In the Ames test they showed a mutagenic action without metabolic activation with Salmonella typhimurium TA98 and TA100 at concentrations of 0.001-0.1 mmole/l in the top agar. Hence, these compounds cause both base-pair substitutions and frame-shift mutations. When Saccharomyces cerevisiae D4 was cultivated in the presence of the compounds, an increase in the mitotic gene conversions was observed. Certain other N-oxides also showed a mutagenic action in the fluctuation test. With Klebsiella pneumoniae, 4-nitroquinoline 1-oxide was mutagenic at a concentration of 0.005 mmole/l, quinoline 1-oxide at 10 mmole/l and benzofuroxan at 0.01 mmole/l. In this test no mutagenic action was found with 4-nitropyridine 1-oxide, pyridine 1-oxide or 4-picoline 1-oxide. With Salmonella typhimurium TA98 and TA100, 4-nitroquinoline 1-oxide, benzofuroxan and 4-nitropyridine 1-oxide were mutagenic, whereas quinoline 1-oxide, pyridine 1-oxide and 4-picoline 1-oxide were not. In contrast, with the fluctuation test, 4-nitroquinoline 1-oxide appeared to be more mutagenic than quindoxin, carbadox and olaquindox in the plate incorporation test.

The mutagenic action of nitroimidazoles. II. Tinidazole, ipronidazole, panidazole and ornidazole.

The 5-nitroimidazoles tinidazole (Fasigyn), ipronidazole (Ro-7-1554), panidazole and ornidazole (Tiberal, Ro-7-0207) in concentrations of 0.02--1 mM per liter increased the mutation frequency of Klebsiella pneumoniae. Escherichia coli K12 and Citrobacter freundii to streptomycin resistance, including streptomycin dependence, in Luria and Delbrück's fluctuation test. At low concentration (0.1 mM), the increase of the mutation frequency caused by each compound was nearly the same, i.e. 3--4 times the spontaneous mutation frequency. At higher concentrations, considerable differences between the mutagenic activities of the compounds occurred.

The mutagenic action of nitroimidazoles. II. Effects of 2-nitroimidazoles.

The 2-nitroimidazoles Ro-71051 (N-benzyl-2-nitro-1-imidazole-acetamide) and Ro-5-9963 (3(2-nitro-1-imidazolyl)-1,2-propanediol) increased the mutation rate of a Klebsiella pneumoniae mutant to streptomycin-resistance including streptomycin-dependence in Luria and Delbruck's fluctuation test in concentrations of 0.05-1 mM and 0.02-0.2 mM respectively. The 2-nitroimidazole, azomycin, (Ro-5-9129/001) failed to increase the mutation rate. The results are compared to those obtained with the 5-nitroimidazoles methronidazoles metronidazole, nimorazole and dimetridazole, which caused a degree of increase similar to Ro-7-1051 and Ro-59963.

The capacity of some nitro- and amino-heterocyclic sulfur compounds to induce base-pair substitutions.

The capacity of 27 heterocyclic sulfur compounds to induce base-pair substitutions was investigated with Klebsiella pneumoniae ur- pro- and Salmonella typhimurium TA100 as test organisms. Among the compounds tested, all sulfur compounds with nitro groups and some thiazoles with an amino group were mutagenic. Among the nitrothiazoles, the most potent mutagen was niridazole, followed by 2-acetamido-5-nitrothiazole, 2-bromo-5-nitrothiazole, N-(5-nitrothiazol-2-yl)benzamide, and 2-amino-5-nitrothiazole. Of the nitrothiophenes, 2-nitrothiophene was more mutagenic than 3-nitrothiophene and 2,4-dinitrothiophene. 4-Nitroisothiazole was also mutagenic. Of the aminothiazoles, 2-amino-5-bromothiazole and 2-amino-5-chlorothiazole were mutagenic to both test organisms. With 2-amino-5-(p-nitrophenylsulfonyl)thiazole, a mutagenic action was only found with Salmonella typhimurium TA100, whereas 2-aminothiazole and 2-amino-4-methylthiazole were only mutagenic with Klebsiella pneumoniae. With the other 13 compounds, no mutagenic activity was observed. Of the coccidiostatics, 2-acetamido-5-nitrothiazole was also mutagenic on Escherichia coli K12 and Saccharomyces cerevisiae D4 but non-mutagenic on Salmonella typhimurium TA1530, TA1535, TA1537 and TA98, while 2-amino-5-nitrothiazole was mutagenic on Escherichia coli K12, Salmonella typhimurium TA1530, TA1535 and TA98, and non-mutagenic on strain TA1537 and on Saccharomyces cerevisiae D4.

Comparison of the mutagenic potency of 2-chloroethanol, 2-bromoethanol, 1,2-epoxybutane, epichlorohydrin and glycidaldehyde in Klebsiella pneumoniae, Drosophila melanogaster and L5178Y mouse lymphoma cells.

A series of 2 haloethanols and 3 epoxides was investigated in 3 mutagenicity test systems, namely (1) the fluctuation test in Klebsiella pneumoniae, (2) the sex-linked recessive lethal test in Drosophila melanogaster, and (3) the HGPRT test with L5178Y mouse lymphoma cells. The order of mutagenic potency was, in Klebsiella: glycidaldehyde greater than 2-bromoethanol = epichlorohydrin greater than 1,2-epoxybutane greater than 2-chloroethanol; in Drosophila: glycidaldehyde = epichlorohydrin greater than 1,2-epoxybutane; in mouse lymphoma cells: epichlorohydrin greater than 1,2-epoxybutane. The haloethanols were non-mutagenic in Drosophila. 2-Chloroethanol and glycidaldehyde were negative in mouse lymphoma cells. The high mutagenic potency of epichlorohydrin as compared with 1,2-epoxybutane was consistent in all systems, and with published data.

The mutagenic action of nitroimidazoles. IV. A comparison of the mutagenic action of several nitroimidazoles and some imidazoles.

The mutagenic action of 51 imidazoles was investigated. The fluctuation test of Luria and Delbrück was used, with Klebsiella pneumoniae as test organism. 8 compounds, including 5 with a weak mutagenic action in the fluctuation test, were also investigated by the Ames test in which Salmonella typhimurium TA100 was used. Of the 51 imidazoles examined, 33 were nitroimidazoles. 31 of the latter appeared to be mutagenic, whereas out of the 18 other imidazoles without a nitro group only 2 were mutagenic. Several of the substances tested for mutagenicity showed an antimicrobial activity. No direct relationship between antimicrobial action, growth inhibition and mutagenicity was established. With methyl-nitroimidazoles a relationship was found between the chemical structure and mutagenic action. However, when the nitroimidazoles had a more complex chemical structure, a relationship between this structure and mutagenicity could not be established.

The mutagenic action of aliphatic epoxides.

The mutagenic action of 45 epoxides was investigates in Luria and Delbrück's fluctuation test with Klebsiella pneumoniae as test organism. In this test, 36 of the 45 epoxides appeared to be mutagenic. The mutagenicity of 1,2-epoxides decreased with increasing length of the carbon chain. The mutagenic activity of compounds with a non-terminal epoxide group appeared to be less than that of substances with a terminal one. Generally 1,2-epoxide compounds with electronegative groups were more mutagenic than 1,2-epoxypropane. Of the diepoxides, 1,2,3,4-diepoxybutane appeared to be more mutagenic than 1,2,7,8-diepoxyoctane, while the ring compounds 1,2,5,6-diepoxycyclooctane was hardly mutagenic. The ring compound 4-vinylcyclohexenedioxide, used in electron microscopy that the antibiotic fosfomycin is among the more potent mutagenic substances investigated in this study.

Mutagenicity of methyl bromide in a series of short-term tests.

Methyl bromide is commonly used as a soil fumigant in greenhouses. In the framework of a toxicological evaluation, it was tested for possible genotoxic properties in two bacterial test systems (the fluctuation test using Klebsiella pneumoniae and the plate test using Salmonella typhimurium TA100 and TA98), two systems using mammalian cells in vitro (forward mutations at the TK and HPRT loci in L5178Y mouse lymphoma cells and unscheduled DNA synthesis in primary rat-liver cells) and in the sex-linked recessive lethal test using Drosophila melanogaster. Methyl bromide was active in all tests except the DNA-repair assay. The results indicate a relatively low mutagenic efficiency of the compound, as expected from its alkylating properties.

Increased mutagenicity of some nitroimidazoles by non-mutagenic nitrotoluene on Klebsiella pneumoniae (fluctuation test).

In the fluctuation test the mutation frequency of Klebsiella pneumoniae by the 5-nitroimidazoles panidazole and dimetridazole was increased by adding the non-mutagenic substances 4-nitrotoluene or toluene-4-sulfonamide. This effect was not found with 1-methyl-5-nitroimidazole, metronidazole, ronidazole, nimorazole and 1-methyl-2-hydroxymethyl-5-nitroimidazole. It is suggested that the molecules of panidazole or dimetridazole form some association, which is destroyed by 4-nitrotoluene or toluene-4-sulfonamide, thus increasing the concentration of mutagenic particles.

[Resistance to antibiotics in Salmonella]. / Resistentie tegen antibiotica bij Salmonella.

Approximately 20,000 strains of Salmonella were screened annually for resistance to tetracycline, chloramphenicol, kanamycin and ampicillin since 1959, and also to trimethoprim since 1978. Tetracycline-resistant strains increased in human subjects and pigs from 1961. After the ban on incorporation of tetracycline in animal feeds for nutritive purposes in 1974, the proportion of tetracycline-resistant strains in pigs and human subjects decreased. In veal calves, the number of strains of S. typhimurium and S. dublin resistant to multiple drugs increased from 1972. Strains resistant to multiple antibiotics in man were mainly isolated from adoptive children from Indonesia. No further spread of these strains was observed. So far, strains similar to those in calves resistant to multiple drugs were only incidentally isolated from human patients.

[Reduction of the number of tetracycline-resistant strains of Salmonella in the Netherlands (author's transl)]. / Afname van het aantal tetracycline-resistente Salmonella stammen in Nederland.

Since 1974, tetracycline resistance in salmonellae of human and porcine origin has decreased nation-wide in the Netherlands. This decrease had coincided with the ban on incorporation of tetracycline in animal feeds for growth-promotion.