A comparative analysis of chromosomal aberrations in cultured human lymphocytes due to fluoroquinolone drugs at different expression periods.

Fluoroquinolones (FQ) are broad-spectrum antibacterial agents widely used for the treatment of infections with various types of gram negative and gram positive bacteria. Specifically, gatifloxacin (GFX) is under development as a component in a new antituberculosis fixed-dose drug combination. In the context of this project, GFX was also tested for genotoxic activity in human peripheral lymphocytes, and the induction of chromosomal aberrations by GFX in PHA-M stimulated cultured human lymphocytes, investigated under conditions of conventional and increased expression times, was further compared to the analogous effects induced by some other second- and third-generation FQ antibacterial agents, namely ofloxacin (OFX), ciprofloxacin (CFX) and sparfloxacin (SFX). OFX did not induce any significant chromosomal aberrations in human lymphocytes. CFX and SFX exhibited slight to moderate clastogenic potential at cytotoxic concentrations (150, 175, 200 and 225 microg/ml), and GFX, a third-generation FQ, induced a clear, concentration-dependent increase in the frequency of chromosomal aberrations at cytotoxic concentrations (150, 200 and 250 microg/ml). These effects were not apparent when metaphases were analysed at the conventionally used sampling time of 24 h, but only after prolongation of the expression time between treatment and harvesting to a sampling time of 36 h (4 h exposure and 32 h expression period). Also, an increased incidence of numerical aberrations (polyploidy and endoreduplication) was seen with GFX at non-cytotoxic concentrations (12.5, 25, 50 and 75 microg/ml). These effects can be attributed to the slight cross-reactivity of FQs between their inhibitory activity towards their intended targets, the prokaryotic type II topoisomerase enzymes DNA gyrase and topoisomerase IV, and the analogous mammalian enzyme topoisomerase II. We have also observed the formation of polycentrics, i.e., chromosomes with five to six centromeres, a rarely reported structural aberration, in GFX-treated cells. The significance of these observations with respect to the conventional conduct of such studies and to the interpretation of the effects is discussed.

The toxicology of iodate: a review of the literature.

Because it is more stable than iodide, most health authorities preferentially recommend iodate as an additive to salt for correcting iodine deficiency. Even though this results in a low exposure of at most 1,700 microg/d, doubts have recently been raised whether the safety of iodate has been adequately documented. In humans and rats, oral bioavailability of iodine from iodate is virtually equivalent to that from iodide. When given intravenously to rats, or when added to whole blood or tissue homogenates in vitro or to foodstuff, iodate is quantitatively reduced to iodide by nonenzymatic reactions, and thus becomes available to the body as iodide. Therefore, except perhaps for the gastrointestinal mucosa, exposure of tissues to iodate might be minimal. At much higher doses given intravenously (i.e., above 10 mg/kg), iodate is highly toxic to the retina. Ocular toxicity in humans has occurred only after exposure to doses of 600 to 1,200 mg per individual. Oral exposures of several animal species to high doses, exceeding the human intake from fortified salt by orders of magnitude, pointed to corrosive effects in the gastrointestinal tract, hemolysis, nephrotoxicity, and hepatic injury. The studies do not meet current standards of toxicity testing, mostly because they lacked toxicokinetic data and did not separate iodate-specific effects from the effects of an overdose of any form of iodine. With regard to tissue injury, however, the data indicate a negligible risk of the small oral long-term doses achieved with iodate-fortified salt. Genotoxicity and carcinogenicity data for iodate are scarce or nonexisting. The proven genotoxic and carcinogenic effects of bromate raise the possibility of analogous activities of iodate. However, iodate has a lower oxidative potential than bromate, and it did not induce the formation of oxidized bases in DNA under conditions in which bromate did, and it may therefore present a lower genotoxic and carcinogenic hazard. This assumption needs experimental confirmation by proper genotoxicity and carcinogenicity data. These in turn will have to be related to toxicokinetic studies, which take into account the potential reduction of iodate to iodide in food, in the intestinal lumen or mucosa, or eventually during the liver passage.

Pentachlorophenol.

Pentachlorophenol (PCP) is a substance whose widespread use, mainly in wood protection and pulp and paper mills, has led to a substantial environmental contamination. This in turn accounts for a significant exposure of the general human population, with rather high exposure levels being attained in occupational settings. Investigations on the genotoxic activity of PCP have given rise to divergent results which would seem to make an evaluation difficult. By grouping them into 3 categories a somewhat clearer picture, allowing finally an (admittedly tentative) assessment, can be obtained. PCP does seem to be at most a weak inducer of DNA damage: it produces neither DNA-strand breaks nor clear differential toxicity to bacteria in rec-assays in the absence of metabolic activation. Also in SCE induction no increase can be observed in vivo, while PCP is found marginally active in a single in vitro experiment. Metabolic activation, however, leads to prophage induction and to DNA strand breaks in human lymphocytes, presumably through the formation of oxygen radicals. A possible further exception in this area might be the positive results in the yeast recombination tests, although their inadequate reporting makes a full evaluation difficult. PCP does not seem to induce gene (point) mutations, as most bacterial assays, the Drosophila sex-linked recessive lethal test and in vitro assays with mammalian cells did not demonstrate any effects. Marginally positive results were obtained in the mammalian spot test in vivo and in one bacterial test; the positive result in the yeast assay for cycloheximide resistance is fraught somewhat with its questionable genetic basis. PCP does, however, induce chromosomal aberrations in mammalian cells in vitro and in lymphocytes of exposed persons in vivo. Those in vivo results that were unable to provide evidence of chromosomal damage are hampered either by methodological inadequacies or by too low exposure levels. The (rodent) metabolite tetrachlorohydroquinone might be a real genotoxic agent, capable of binding to DNA and producing DNA strand breaks; this activity is probably due to semiquinone radical formation and partly mediated through active oxygen species. Since this compound has not been tested in the common bacterial and mammalian mutagenicity assays, the few ancillary results on this substance cannot be used in a meaningful human risk assessment of PCP. Furthermore, this metabolite has only been produced by human liver microsomes in vitro, but has not been detected in exposed humans in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Chirality-dependent DNA reactivity as the possible cause of the differential mutagenicity of the two components in an enantiomeric pair of epoxides.

Chemical compounds containing an epoxy group are very reactive substances and, in many cases, they therefore exhibit strong mutagenic properties. Very often such epoxides contain an asymmetric C atom and thus exist as racemic mixtures of optical isomers, the so-called R- and S-enantiomers. It is well known that in many cases a biological activity resides completely in one of the two enantiomeric forms of a molecule. Also, the R- and S-enantiomeric forms of epoxystyrene exhibit different mutagenic activities in Salmonella typhimurium TA100, although their chemical reactivity does not differ to a recognizable extent. Neither could the higher mutagenic activity of the R-epoxystyrene be attributed to a slower enzymatic hydrolysis reaction. Thus, the intrinsic potential for eliciting mutagenic responses may not be the same for the two enantiomers, as there is evidence of qualitative differences in the binding to DNA, pointing strongly to an intrinsic difference in mutagenic activity of the two enantiomers.

The mutagenic activity of sodium perborate.

Sodium perborate (CAS No. 1333-73-9, 10486-00-7, or 13517-20-9, depending on the structural formula given) is produced in huge amounts mainly for its use as a bleaching agent in laundry detergents. Its action involves the liberation of active oxygen species at elevated temperatures. In view of the widespread use of this compound it is surprising to note that no mutagenicity test data yet exist. The investigations reported in this paper have shown that sodium perborate is indeed capable of producing mutagenic changes in a number of in vitro test systems. Its potential for inflicting damage to DNA could be demonstrated in an assay which is tailored to probe for oxidative damage induced by a chemical agent. As expected, sodium perborate proved to be able to oxidize thymidine to an appreciable extent at an incubation temperature of 80 degrees C, but even at 40 degrees C thymidine oxidation was measurable. The compound induced point mutations in the Salmonella typhimurium strains TA100 and TA102, while TA98 did not respond. Also, incubation in the presence of a mammalian auxiliary metabolic system (rat liver S9) abolished the mutagenic activity completely. Finally, Chinese hamster ovary cells (strain CHO-K1) were shown to undergo extensive chromosomal damage when treated with sodium perborate. The rather unusual prevalence of chromosome rearrangements was especially noted. Sodium perborate is thus to be regarded as a direct-acting in vitro mutagen.

Chemical impurity as the possible cause of nitrofen mutagenicity.

The results of Salmonella mutagenicity tests with the herbicide nitrofen (2,4-dichlorophenyl-4-nitrophenyl ether, CAS 1836-75-5) reported in the literature seem to vary according to source or lot of the technical product. This behaviour could be reproduced in our experiments, and it could be traced to the varying content of bis(4-nitrophenyl) ether in nitrofen. Nitrofen samples with the highest bis(4-nitrophenyl) ether concentrations proved to exhibit also the highest mutagenic activities, while the one containing no bis(4-nitrophenyl) ether was also devoid of mutagenic activity towards Salmonella typhimurium TA 100.

Uptake, metabolism and mutagenic activity of aromatic glycidyl compounds.

Aromatic diglycidyl compounds are very active mutagens when assayed in in vitro tests. In vivo, however, resorcinol diglycidyl ether provided no evidence for the clastogenic activity, while diglycidylaniline exhibited definite mutagenic activity in the micronucleus test. Since the only difference between these two compounds lies in the binding mode of the glycidyl groups to the aromatic nucleus (i.e. ether oxygen vs. aminic nitrogen), this apparent discrepancy in mutagenic activity led to the question of the mechanisms involved in such an activity difference. Although no clear signs of differential uptake or excretion could be detected in mice, differences could be seen in the spectrum of urinary metabolites; while resorcinol diglycidyl ether seemed to become fully converted to the genetically inactive bis-diol compound, a sizeable proportion of diglycidylaniline was converted only to the diol-epoxide. In vitro investigations and enzyme kinetic measurements with postmitochondrial supernatant of rat or mouse liver homogenate (S-9) finally yielded the biochemical explanation for this behaviour, as they showed a very low affinity of the diol-epoxide metabolite of diglycidylaniline for the epoxide hydrolase, normally involved in the degradation of such compounds. The diol-epoxide obtained from resorcinol diglycidyl ether, on the other hand, has an affinity to the degradation enzyme similar to, or even higher than, the one measured with the parent substance.

The mutagenicity of mono- and di-functional aromatic glycidyl compounds.

Although epoxides are very important compounds in today's technology, they are not as well investigated for their carcinogenic and mutagenic properties as their economical importance would suggest. The present study tried, on one hand, to bridge the gap between microbial testing of such compounds and the in vivo mammalian test system. On the other hand, the comparative testing of structurally related substances was expected to yield some clues as to the structural determinants of possible in vivo mutagenic activity. Our investigations with 4 compounds of similar structure, namely glycidylmethylaniline (GMA), diglycidylaniline (DGA), phenylglycidylether (PGE), and resorcinol diglycidyl ether (RDGE), first demonstrated that, in bacterial assays, all 4 epoxides acted as potent mutagens. Only the 2 difunctional epoxides, however, were active in an in vitro chromosomal aberration assay with CHO cells. That such in vitro results cannot be supposed to represent the in vivo situation is again demonstrated by discovery that only one of the two in vitro active substances (and in fact it is the less active one) also has a damaging effect in vivo. It, therefore, seems quite justifiable to conclude that two structural features must be combined in an aromatic glycidyl compound if it is to exert chromosome damaging activity in vivo: The substance must possess 2 epoxy functions, and they must be bound to the aromatic nucleus by an aminic nitrogen. The industrial use of aromatic glycidyl ethers might, therefore, be considered not to pose a great systemic risk for mutagenicity or carcinogenicity, without completely excluding the possibility of local effects.

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. III. The influence of the composition and preparation of the minimal medium in the Salmonella mutagenicity test.

The influence of factors connected with the preparation of the minimal medium for the Ames test has been investigated. Faulty sterilization procedures can lead to the generation of toxic and/or mutagenic by-products in the minimal medium. Changes in histidine concentration affect not only the number of spontaneously arising colonies on the plate, but also the number of induced mutants. Although the number of spontaneous mutants increases slightly with increasing histidine concentration, the influence on the number of induced mutants depends on the nature of the mutagen tested.

Dose and effect of methyl-2-benzimidazolylcarbamate in the "mammalian spot test", an in vivo method for the detection of genetic alterations in somatic cells of mice.

In the spot test, mouse embryos which are heterozygous for four different recessive coat-colour genes are treated in utero by injection of a mutagen into the peritoneal cavity of the mother or by other appropriate routes of administration. If this treatment leads in a pigment precursor cell to an alteration of the wild type allele of one of the genes under study or to its loss, a colour spot in the adult coat may be seen. Peroral application of 100-300 mg methyl-2-benzimidazolylcarbamate (MBC)/kg to the mother during the tenth day postconception led to an increase in the frequency of colour spots in the coats of offspring. The data are consistent with the hypothesis that MBC is a point mutagen.

Herbicidal phenylalkylureas as possible mutagens I. Mutagenicity tests with some urea herbicides.

Substituted phenylalkylureas are widely used as herbicides. In the assay system of Friedman and Staub, which measures the inhibition of testicular DNA synthesis (DSI test), most of these substances showed a positive reaction, i.e. they depressed tnymidine incorporation significantly. Bacterial tests demonstrated a weak mutagenic activity, too; but in the micronucleus test the compounds were almost inactive. As one member of this group of chemicals--the herbicide monuron--is a recognized carcinogen, these results seem to indicate a possible hazard, but more investigations are needed to quantify this danger.