The effect of pretreatment with metallothionein (MT) inducers (bismuth nitrate or zinc chloride) on clastogenicity of anticancer drugs was investigated. Bismuth nitrate (50 mumol/kg) or zinc chloride (400 mumol/kg) was administered s.c. to mice once a day for two days prior to treatment with 3.3 mumol/kg of cis-diamminedichloroplatinum(II) (cis-DDP), 3.4 mumol/kg of adriamycin (ADR), 72 mumol/kg of cyclophosphamide (CPA) or 0.41 mumol/kg of L-phenylalanine mustard (L-PAM). The frequency of occurrence of erythrocytes with micronuclei in bone marrow was increased by each anticancer drug at 24 h after treatment. Micronucleus formation was significantly prevented by pretreatment with either bismuth nitrate or zinc chloride. MT concentration in bone marrow cells of mice at the time of treatment with anticancer drugs increased to 2- and 3.5-fold by pretreatment with bismuth nitrate and zinc chloride, respectively. These results indicate that MT induction in bone marrow cells effectively prevents micronucleus induction of anticancer drugs.
Background radiation is likely to constitute one of the factors involved in biological evolution since radiations are able to affect biological processes. Therefore, it is possible to hypothesize that organisms are adapted to environmental background radiation and that this adaptation could increase their ability to respond to the harmful effects of ionizing radiations. In fact, adaptive responses to alkylating agents and to low doses of ionizing radiation have been found in many organisms. In order to test for effects of adaptation, cell susceptibility to treatments with high doses of radiomimetic chemical agents has been studied by growing them in a reduced environmental radiation background. The experiment has been performed by culturing yeast cells (Saccharomyces cerevisiae D7) in parallel in a standard background environment and in the underground Gran Sasso National Laboratory, with reduced environmental background radiation. After a conditioning period, yeast cells were exposed to recombinogenic doses of methyl methanesulfonate. The yeast cells grown in the Gran Sasso Laboratory showed a higher frequency of radiomimetic induced recombination as compared to those grown in the standard environment. This suggests that environmental radiation may act as a conditioning agent.
The V-E5 cell line, a mutant V79 Chinese hamster cell line, was used to study the effect of chromosomal instability on the spectrum of gene mutations and chromosome aberrations induced by the anthracycline antibiotic adriamycin (AM). V-E5 cells showed hypersensitivity to the cytotoxic effects of AM when compared to the parental cell line. AM caused both, chromosome-type aberrations and chromatid-type aberrations in V-E5 cells. Under the same experimental conditions, gene mutations were induced at the hprt locus which mainly represented deletion mutations. The spectrum of AM-induced chromosomal aberrations and gene mutations did not show any peculiarities in comparison to normal V79 cells. It is concluded that the genomic instability in V-E5 cells does not influence the pathways leading to chromosome aberrations and gene mutations after AM treatment.
We have previously established an immunoblastic B lymphoma cell line, designated HOB1. This cell line is hypersensitive to a wide spectrum of chemotherapeutic agents. Two co-regulated polypeptides around 64 kDa (termed p64) were induced 10-30-fold in response to adriamycin and some other drugs at the IC50 (the concentration inhibiting cell growth by 50%). These inducible proteins are localized as monomeric forms in the cytosolic fraction, with isoelectric points of pH = 6.2 (major protein) and pH = 7.0 (minor protein). An adriamycin-resistant cell line was established from HOB1 cells. The p64 inducibility was dramatically reduced in resistant HOB1 cells or unrelated cell lines which show phenotypic resistance to adriamycin toxicity. The loss of p64 inducibility in resistant cells is not due to a failure of cells to take up adriamycin since drug accumulation kinetics remained the same as in the parental cells.
Fluorescence in situ hybridization with chromosome-specific DNA probes is being increasingly utilized for the detection of chromosome aberrations induced in vitro and in vivo by chemical and physical agents. Although potentially a powerful technique, FISH studies for aneuploidy can be heavily influenced by cellular phenomena and hybridization artifacts which make the performance and interpretation of the results difficult. As a consequence, frequently hyperdiploid frequencies are reported in the literature which are substantially higher than one would expect based upon frequencies seen in conventional metaphase analyses. In this article, a number of the potential pitfalls that we have encountered while performing FISH analyses for aneuploidy are discussed and their potential impact on the observed hybridization frequencies is described. After considering these factors, the frequencies of lymphocyte nuclei containing 3 and 4 chromosome copies are compared between metaphase values obtained from published human population studies and interphase values obtained from similar studies using FISH. It is concluded that by using caution in the evaluation of slides, interphase studies using FISH to detect hyperdiploidy and polyploidy can provide estimates of numerical alterations which closely reflect those seen during metaphase analysis using either FISH or conventional approaches. However, due to the inability of interphase analysis to distinguish hyperdiploidy from polyploidy as well as other potential problems, frequencies of aneuploid nuclei obtained using single label FISH should only be considered approximations of absolute frequencies. For additional accuracy, multi-color FISH with two or more different probes should be performed.
Escherichia coli and Salmonella typhimurium strains deficient in the OxyR-regulated adaptive response to oxidative stress were used to study the mode in which spontaneous SOS-dependent mutations are generated in a distressed bacterial population. When assayed on supplemented selective medium, the E. coli strain IC3821 (trpE65), carrying the Delta oxyR30 mutation and containing the plasmid pRW144 (mucA/B), showed a frequency of spontaneous Trp(+) revertants similar to that of the oxyR(+) control. Instead, the IC3821 strain exhibited an enhancement in the clonal occurrence of spontaneous revertants arising at random during growth on a nonselective medium. A similar enhancement was observed for the S. typhimurium strain TA4125 (hisG428 Delta oxyR2). The mutator effect observed in oxyR(-) cells would be induced by an increased background of reactive oxygen species; it provides a model for studying the mutability of a cell population constantly exposed to mutation-inducing agents. In the IC3821 strain, revertants were induced by t-butyl hydroperoxide with higher efficiency than in oxyR(+). We suggest that strain IC3821 could be useful for the detection of SOS-dependent mutagenesis induced by chemical oxidants.
The glycophorin A (GPA) mutation assay was used to examine the risk of in vivo somatic mutation in infants following neonatal administration of vitamin K. The assay assesses damage to erythroid stem cells by measuring the frequency of NO and NN variant red cells of MN blood group heterozygotes using FACS analysis. Blood samples were obtained from 178 infants aged between 10 and 183 days. Twenty-six children were excluded from study having received a blood transfusion. Sixty-four of the remaining 152 infants were of the MN phenotype, samples from whom were analysed using the assay system, providing the first data of NO and NN variant frequencies (vfs) in children aged less than 1 year. Twenty of these 64 infants received vitamin K orally (group A), 17 intramuscularly (group B) and 25 intravenously (group C). Results were compared with those from a reference population of children aged 1-15 years. There were no significant differences in NO, NN and total vf between any of groups A, B and C. For all groups both NO and total vf were significantly lower than those for the control population. This result is of some interest and clearly warrants further investigation. NN and total vfs were greater than the 95th percentile for the pooled data from groups A, B and C in three instances, one in each group. It was thus not possible to demonstrate an association between the route of vitamin K administration and an increase in mutation at the GPA locus.
Four compounds commonly found in the human diet, allyl isothiocyanate (AITC), phenethyl isothiocyanate (PEITC) and their parent glucosinolates sinigrin and gluconasturtiin, were tested for cytotoxic and genotoxic effects in a Chinese hamster ovary cell line (CHO). The isothiocyanates were found to be more than one thousand times more cytotoxic than the glucosinolates, showing significant cytotoxic activity at concentrations below 1.0 microgram/ml. AITC was unable to induce either chromosome aberrations or sister chromatid exchanges (SCEs) even at highly cytotoxic doses. In contrast, PEITC was found to induce both aberrations and SCE at concentrations of 0.9-1.2 micrograms/ml whilst sinigrin and gluconasturtiin induced aberrations at concentrations above 2 mg/ml.
Treatment of HeLa cells with cadmium chloride and zinc chloride increases the survival rate of nitrous acid-treated adenovirus 2 (ade2). This increase is maximal if the time interval between cell treatment and virus infection is delayed by 36 h. The induction process requires protein synthesis only during the 3-h period immediately following treatment; cycloheximide does not prevent the expression of enhanced reactivation if added to the cells after this time.
The antimutagenic effect of eugenol on the mutagenicity of cyclophosphamide (CP), mitomycin C (MMC), ethyl methanesulfonate (EMS) and benzo[a]pyrene (B[a]P) was assessed in the rodent bone marrow micronucleus test using male Swiss mice. Oral administration of eugenol (0.4% in the diet) for 15 days was found to decrease significantly the frequency of micronucleated polychromatic erythrocytes (MPEs) elevated by CP. No effect was found on the frequency of MPEs elevated by MMC, EMS and B[a]P. The results provide some support for antimutagenic potency of eugenol in vivo.
This paper introduces a new parameter, derivable from dose-response data for induced mutagenesis in bacteria, that can be used to quantify mutational responses in short-term tests. We called this parameter the mutational response of the bipartite experimental system (agent plus cells). We define it as being jointly proportional to the efficiency of the mutagen and the sensitivity of the test. We show how this quantity can be used to rank order chemical carcinogens on the basis of their mutagenicity and to determine the strength of any quantitative correlation that may exist between mutagenicity in bacteria and carcinogenicity in rodents. We find that this particular measure of mutational response for 10 direct-acting monofunctional alkylating agents correlates remarkably well with the rodent carcinogenicity of these chemicals measured in terms of their reciprocal TD50 values.
Chromosomal aberration and micronucleus assays were used to investigate the extent of cytogenetic damage in peripheral blood lymphocytes from four patients in two unrelated families with hereditary megaduodenum. The frequencies of total chromosomal aberrations, which significantly correlated with those of micronuclei, were higher in the patients than in sex- and age-matched controls, with no overlapping between the two groups. The considerable chromosomal fragility in patients with hereditary megaduodenum may be a genotypic marker for preclinical diagnosis predictive of increased cancer risk.
Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per microgram. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per micrograms. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.
The T-cell-cloning assay was established to determine the frequency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutant lymphocytes in the presence of the selective agent 6-thioguanine in peripheral blood from a human control population. We investigated 44 healthy adults (blood donors) and found a mean mutant frequency of 7.2 x 10(-6) (geometric mean 5.6 x 10(-6)). An elevated mean mutant frequency occurred in smokers as compared to non-smokers. However, a statistically significant increase was only observed between female smokers and female non-smokers while there was only a slight difference in the male group. A significant difference in mutant frequency could be found between individuals younger than 35 years and those above 35. But the difference of the mutant frequency with age showed up only among smokers. No significant effect of the gender was observed. Mutant frequency was inversely related to the cloning efficiency.
The first 26 families of complex chromosome-type exchanges (from three breaks in two chromosomes to five breaks in five chromosomes) have been evaluated, and the 15 060 exchanges resulting from unrestricted restitution or rejoining of the break ends yields 203 distinctive patterns, if a single participating chromosome is FISH-painted. If we assume that any exchange that produces an acentric fragment of any sort (compound-terminal or interstitial) will be ultimately eliminated in a continuously dividing cell population, then, irrespective of family origin, only 17 of these patterns (approximate to 8%) will be transmitted, long-term.The 17 are illustrated, and the implications briefly discussed.
A new gene, designated dinP, was found during E. coli genomic sequencing around the 5.5 min region. Its coding region is preceded by a sequence similar to the consensus binding sequence for LexA, the so-called SOS box sequence. The amino acid sequence of DinP (351 amino acid residues) has a strong similarity to the C. elegans hypothetical protein F22B7.6 and weaker similarities to the UmuC homologues in E. coli and Salmonella typhimurium and also to REV1 of Saccharomyces cerevisiae. Another SOS operon (dinJ1 and dinJ2 genes) found in this region is also described.
Ortho-meta-and para-nitrobenzyl bromides alcohols ethers and esters were synthesized and tested for their mutagenicity toward Salmonella typhimurium TA100, TA100NR (nitroreductase deficient) and TA98 in absence of S9 mix and in TA100 with S9 mix. Compounds of the ortho-and meta-series were non mutagenic with and without S9 mix. Except for the alcohol and ether, the compounds of the para-series were mutagenic in TA100 with activity sequence propionate > butyrate > benzoate > acetate > bromide and this specific activity was reduced considerably by S9 mix. The Ames Salmonella test system does not seem to be an appropriate model to evaluate mutagenicity of o-nitrobenzyls. However, further work is in progress to test all the compounds for mutagenicity in mammalian system
We have molecularly characterized the SNG1 gene that confers hyper-resistance to the mutagen N-methyl-N'nitro-N-nitrosoguanidine (MNNG) in Saccharomyces cerevisiae when overexpressed on a multi-copy plasmid. This hyper-resistance to MNNG is not due to depletion of glutathione pools since multi-copy SNG1 containing yeast transformants contain at least wild type levels of glutathione; DNA repair seems unaffected in these transformants as the multi-copy SNG1-mediated MNNG hyper-resistance is also seen in DNA repair mutants belonging to each of the three epistasis groups of yeast repair mutants. It could be shown that SNG1 is not under control of the YAP1 encoded transcription activator that controls expression of at least two genes involved in MNNG metabolism in yeast. sng1 null mutants are viable but exhibit only slight sensitivity to MNNG, indicating that SNG1 does not encode a protein involved in a major detoxification step of this mutagen. Sequencing of the HYR-mediating passenger DNA revealed that SNG1 encodes a 547 a polypeptide containing seven transmembrane-spanning regions that may be membrane-bound. Comparison of the DNA sequence with established gene databanks revealed that SNG1 is a novel yeast gene.
Chromosome analyses were carried out on peripheral blood lymphocytes of semi-domestic reindeer in Norway which had been exposed to varying amounts of radiocesium emanating from the Chernobyl accident. The sampling was done in the period 1987-1990. The material included 192 reindeer, originating from four herds in central Norway, an area considerably affected by fallout from the Chernobyl accident, and from three herds in northern Norway which was unaffected by fallout from the accident. Significant heterogeneity in the distribution of chromosome aberrations between herds was observed. The pattern of chromosome aberration frequencies between herds was not related to the variation in radiocesium exposure from the Chernobyl accident. Other factors than the Chernobyl accident appear therefore to be of importance for the distribution of aberration frequencies found among present herds. Within the most contaminated area the reindeer born in 1986 showed significantly more chromosome aberrations than those born both before and after 1986. This could suggest that the Chernobyl accident fallout created an effect particularly among calves, during the immediate post-accident period in the most exposed areas.
