Genotoxicity of 15-deoxygoyazensolide in bacteria and yeast

Sesquiterpene lactones (SLs) present a wide range of pharmacological activities. The aim of our study was to investigate the genotoxicity of 15-deoxygoyazensolide using the Salmonella/microsome assay and the yeast Saccharomyces cerevisiae. We also investigated the nature of induced DNA damage using yeast strains defective in DNA repair pathways, such as nucleotide excision repair (RAD3), error prone repair (RAD6), and recombinational repair (RAD52), and in DNA metabolism, such as topoisomerase mutants. 15-deoxygoyasenzolide was not mutagenic in Salmonella typhimurium, but it was mutagenic in S. cerevisiae. The hypersensitivity of the rad52 mutant suggests that recombinational repair is critical for processing lesions resulting from 15-deoxygoyazensolide-induced DNA damage, whereas excision repair and mutagenic systems does not appear to be primarily involved. Top 1 defective yeast strain was highly sensitive to the cytotoxic activity of 15-deoxygoyazensolide, suggesting a possible involvement of this enzyme in the reversion of the putative complex formation between DNA and this SL, possibly due to intercalation. Moreover, the treatment with this lactone caused dose-dependent glutathione depletion, generating pro-oxidant status which facilitates oxidative DNA damage, particularly DNA breaks repaired by the recombinational system ruled by RAD52 in yeast. Consistent with this finding, the absence of Top1 directly affects chromatin remodeling, allowing repair factors to access oxidative damage, which explains the high sensitivity to top1 strain. In summary, the present study shows that 15-deoxygoyazensolide is mutagenic in yeast due to the possible intercalation effect, in addition to the pro-oxidant status that exacerbates oxidative DNA damage.     

Genotoxicity of diphenyl diselenide in bacteria and yeast

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. This may increase the risk of human exposure to the chemical at the workplace. We have determined its mutagenic potential in the Salmonella/microsome assay and used the yeast Saccharomyces cerevisiae to assay for putative genotoxicity, recombinogenicity and to determine whether DNA damage produced by DPDS is repairable. Only in exponentially growing cultures was DPDS able to induce frameshift mutations in S. typhimurium and haploid yeast and to increase crossing over and gene conversion frequencies in diploid strains of S. cerevisiae. Thus, DPDS presents a behavior similar to that of an intercalating agent. Mutants defective in excision-resynthesis repair (rad3, rad1), in error-prone repair (rad6) and in recombinational repair (rad52) showed higher than WT-sensitivity to DPDS. It appears that this compound is capable of inducing single and/or double strand breaks in DNA. An epistatic interaction was shown between rad3-e5 and rad52-1 mutant alleles, indicating that excision-resynthesis and strand-break repair may possess common steps in the repair of DNA damage induced by DPDS. DPDS was able to enhance the mutagenesis induced by oxidative mutagens in bacteria. N-acetylcysteine, a glutathione biosynthesis precursor, prevented mutagenesis induced by DPDS in yeast. We have shown that DPDS is a weak mutagen which probably generates DNA strand breaks through both its intercalating action and pro-oxidant effect.     

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.     

Genotoxicity of selenite in diploid yeast

Selenite, a chemical of industrial importance and also an antimutagenic/anticarcinogenic agent, was tested for mutagenic and recombinogenic effects in 2 diploid yeast strains, Saccharomyces cerevisiae BZ 34 and D7. Selenite induced gene conversion and toxicity in BZ 34 and a variety of genetic events, viz. back-mutation, gene conversion, mitotic crossing-over, aberrant colony formation and also toxicity in the D7 strain. In both strains, the genetic effects of selenite showed a peak and a decline during 5 h of treatment while its toxicity increased marginally during 1-5 h. In the BZ 34 strain, the presence of glutathione (GSH) during selenite treatment greatly enhanced the convertogenic and toxic effects of selenite.     

Genotoxicity assay of oil dispersants in bacteria (mutation, differential lethality, SOS DNA-repair) and yeast (mitotic crossing-over)

5 oil dispersants and a sample of paraffin were devoid of mutagenic activity in the Ames reversion test, with and without S9 mix, using 7 his- S. typhimurium strains (TA1535, TA1537, TA1538, TA97, TA98, TA100, TA102). However, 3 dispersants produced direct DNA damage in E. coli WP2, which was not repairable in repair-deficient strains (WP2uvrA, CM871, TM1080), as shown by two different DNA-repair test procedures. The uvrA excision-repair system was in all cases the most important mechanism involved in repairing the DNA damage produced by oil dispersants, while the combination of uvrA with other genetic defects (polA, recA, lexA) decreased the efficiency of the system. The observed genotoxic effects were considerably lowered in the presence of S9 mix containing liver S9 fractions from Aroclor-treated rats. The sample of oil dispersant yielding the most pronounced DNA damage in repair-deficient E. coli failed to induce gene sfiA in E. coli (strain PQ37), using the SOS chromotest, or mitotic crossing-over in Saccharomyces cerevisiae (strain D5). The direct toxicity of the oil dispersant to both bacterial and yeast cells was markedly decreased in the presence of rat-liver preparations. These two short-term tests were effective in detecting the genotoxicity of both direct-acting compounds (such as 4-nitroquinoline N-oxide and methyl methanesulfonate) and procarcinogens (such as cyclophosphamide, 2-aminoanthracene and 2-aminofluorene). Moreover, the SOS chromotest was successfully applied to discriminate the activity of chromium compounds as related to their valence (i.e. Cr(VI) genotoxic and Cr(III) inactive). Combination of oil dispersants with Cr(VI) compounds did not affect the direct mutagenicity to S. typhimurium (TA102) of a soluble salt (sodium dichromate) nor did it result in any release of a water-soluble salt (lead chromate), as also confirmed by analytical methods. On the other hand, exposure to sunlight tended to decrease, to a slow rate, the direct genotoxicity of an oil dispersant in the bacterial DNA-repair test.     

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride

Ribonucleotide reductase (RNR) of the yeast Saccharomyces cerevisiae is a tetrameric protein complex, consisting of two large and two small subunits. The small subunits Y2 and Y4 form a heterodimer and are encoded by yeast genes RNR2 and RNR4, respectively. Loss of Y4 in yeast mutant rnr4Delta can be compensated for by up-regulated expression of Y2, and the formation of a small subunit Y2Y2 homodimer that allows for a partially functional RNR. However, rnr4Delta mutants exhibit slower growth than wild-type (WT) cells and are sensitive to many mutagens, amongst them UVC and photo-activated mono- and bi-functional psoralens. Cells of the haploid rnr4Delta mutant also show a 3- to 4-fold higher sensitivity to the oxidative stress-inducing chemical stannous chloride than those of the isogenic WT. Both strains acquired increased resistance to SnCl2 with age of culture, i.e., 24-h cultures were more sensitive than cells grown for 2, 3, 4, and 5 days in liquid culture. However, the sensitivity factor of three to four (WT/mutant) did not change significantly. Cultures of the rnr4Delta mutant in stationary phase of growth always showed higher frequency of budding cells (budding index around 0.5) than those of the corresponding WT (budding index <0.1), pointing to a delay of mitosis/cytokinesis.     

Interaction of stannous chloride leads to alteration in DNA, triphosphate nucleotides and isolated bases

Stannous chloride (SnCl₂) is a reducing chemical agent used in several man-made products. SnCl₂ can generate reactive oxygen species (ROS); therefore, studies have been carried out in order to better understand its damaging action in biological systems. In this work, calf thymus DNA, triphosphate nucleotides and isolated bases were incubated with SnCl₂ and the results were analyzed through UV spectrophotometry. The presence of stannous ions altered the absorption spectra of all three isolates. The amount of stannous ions associated to DNA was measured by atomic absorption spectrophotometry. Data showed that more than 40% of the initial SnCl₂ concentration was present in the samples. Our results are in accordance with the damaging potential of this salt and present evidence that stannous ions can complex with DNA, inducing ROS in its vicinity, which may be responsible for the observed lesions. (Mol Cell Biochem xxx: 173–179, 2005)     

Toxicity and accumulation of thallium in bacteria and yeast

Thallium sulphate inhibited microbial growth, withBacillus megaterium KM, more sensitive to the metal thanSaccharomyces cerevisiae andEscherichia coli. Inhibition ofB. megaterium KM andS. cerevisiae, but not ofE. coli, was alleviated by increasing the potassium concentration of the medium; inhibition of respiration ofS. cerevisiae, but not ofE. coli, was similarly alleviated. Thallium was rapidly bound, presumably to cell surfaces, byS. cerevisiae andE. coli, and was progressively accumulated by energy-dependent transport systems (probably concerned primarily with potassium uptake) with both organisms. Thallium uptake kinetics suggested more than one transport system operated in yeast, possibly reflecting a multiplicity of potassium transport systems. ApparentK _m andK _i values for competitive inhibition of thallium uptake by potassium indicatedS. cerevisiae to have a higher affinity for thallium uptake than for potassium, whileE. coli had a transport system with a higher affinity for potassium than for thallium. The likely systems for thallium transport are discussed. A mutant ofE. coli with tenfold decreased sensitivity to thallium was isolated and apparently effected surface binding of thallium in amounts equivalent to the wild type organism, but showed no subsequent uptake and accumulation of the metal from buffer, even though it was able to accumulate potassium to normal intracellular concentrations during growth. Abbreviations: Metal are referred to by their recognised atomic symbols (e.g. TI = Thallium; K = potassium; Co = cobalt)     

Genotoxicity of cadmium chloride in human lymphocytes evaluated by the comet assay and cytogenetic tests

Peripheral blood lymphocytes were tested in vitro for genotoxic effects of cadmium chloride. Whole blood samples of four healthy, non-smoking subjects were preincubated with CdCl2 in concentrations of 10(-4), 10(-3), and 5 . 10(-3) mol/L for three hours before the cells were assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay) or cultivated for chromosomal aberrations (CA), sister chromatid exchanges (SCE), and the micronucleus (MN) test. The comet assay showed notable interindividual differences. The results of the cytogenetic tests showed an increase in the frequency of CA, MN, and SCE with CdCl2 in the treated cultures, yet none was able to show a correlation between concentrations of cadmium chloride and the frequency of damages. The MN slides were stained with Giemsa and with DNA fluorochrome 4', 6'-diamidino-2-phenylindole (DAPI). The frequency of MN in slides stained with DAPI was significantly higher than in those stained with Giemsa, which might be due to an underestimation of small micronuclei in Giemsa-stained slides.     

The effect of stannous and stannic (tin) chloride on DNA in Chinese hamster ovary cells

Tin(II) at concentrations up to 500 μM stannous chloride (SnCl₂), produced extensive DNA damage, as detected by alkaline sucrose gradient (ASG) analysis in Chinese hamster ovary (CHO) cells treated for 1 h at 37°C in serum-free minimal essential medium (MEM). However treatment of cells with tin(IV), as stannic chloride (SnCl₄), produced no such DNA damage. There was no loss in colony formation 6 days after either treatment suggesting that the DNA damage induced by the tin(II) was rapidly repaired and/or that DNA synthesis proceeded on the damaged templates permitting cell division to occur. Alternatively, the type of DNA damage caused by tin(II) may not lead to a reduction in colony-forming ability. Tin(II) produced about 200 times more ASG detectable DNA damage on an equi-molar basis than did Cr(VI), a known human carcinogen. This study indicates that tin(II) may be potentially genotoxic.     

Genetic toxicity of procarbazine in bacteria and yeast.

Procarbazine [N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride] is used to treat Hodgkin's disease. This compound was tested in vitro without and with S10 fraction from mice liver (microsomal assay) using Saccharomyces cerevisiae strain D7, Salmonella typhimurium (strains TA98, TA100, TA1535) and in vivo in Swiss albino mice (host-mediated assay) using D7. Procarbazine, without S10 fraction, is highly toxic and induced mitotic crossover, gene conversion, and reverse mutation in D7. It had a toxic effect on all the Salmonella strains; but did not induce reverse mutations at the histidine loci. Procarbazine, with S10 fraction, was less toxic and did not induce genetic effects in yeast or Salmonella. In the host-mediated assay, no genetic effects were seen.     

Gas chromatographic determination of inorganic tin in rat urine after a single oral administration of stannous chloride and mono-, di-, and triphenyltin chloride

A method is described for the determination of inorganic tin by gas chromatography with flame photometric detection. The inorganic tins, stannous and stannic, were extracted with hydrochloric acid and n-hexane—benzene in the presence of 0.05% tropolone, and both inorganic tins were pentylated to tetrapentyltin with a Grignard reagent prior to gas chromatography. The absolute limit of detection for tetrapentyltin was 3 pg as tin. The recovery of stannous chloride added to rat urine samples was 80.2 ± 2.4% (mean ± S.D., n = 8). The application of this method to the study of urinary excretion of inorganic tin and organotin compounds in rats following oral administration of tin compounds is presented. The urinary excretion of tin compounds was observed over a period of 96 h following administration of stannous chloride or phenyltin compounds. Most of the inorganic tin was excreted into urine within 24 h after administration of stannous chloride. In the experiments on organotin administration, the level of the excretion as total tin for monophenyltin reached a maximum ca. 0–24 h after administration, whereas the maxima for di- and triphenyltin were found after 24–48 h and 48–72 h, respectively. The predominant excretion product of these tin compounds found in urine was monophenyltin.