Thrombosis and malignancy: pathogenesis and prevention

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine, were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchange (SCE) and chromosome aberration (CA) assays in bone marrow cells of mice. These are the most commonly used antimalarial drugs available at present throughout the world. The results of the bacterial mutagenicity assays showed a very weak mutagenic effect of all three drugs in Salmonella strains TA97a and TA100 both with and without S9 mix and in TA104 only with S9 mix. The results of the in vivo SCE and CA assays indicate that these three drugs are genotoxic in bone marrow cells of mice.     

Mutagenicity assay in Salmonella and in vivo sister chromatid exchange in bone marrow cells of mice for four pyrazolone derivatives

Phenylbutazone (PB), oxyphenbutazone (OPB), antipyrine (AP) and dipyrone (DP) are four important pyrazolone derivatives mainly used as anti-inflammatory, antipyretic and analgesic drugs. At present these are the most widely used pyrazolone derivatives throughout the world. The widespread use of these drugs are of great concern for human health problems. In the present study these four drugs were tested in mutagenicity assays in Salmonella strains TA97a, TA98, TA100 and TA102 using a plate incorporation assay both with and without S-9 mix and for in vivo sister chromatid exchanges (SCE) in bone marrow cells of mice. The first three drugs were negative in all the tester strains but dipyrone showed a weak mutagenic activity at higher concentrations in all four strains both with and without metabolic activation. In the in vivo SCE assay in male mice, all four drugs showed a statistically significant increase in SCE in bone marrow cells when compared with control.     

Dialkylquinoneimine metabolites of chloroacetanilide herbicides induce sister chromatid exchanges in cultured human lymphocytes

Some of the most widely-used herbicides are the chloroacetanilides exemplified by alachlor and butachlor (derived from 2,6-diethylaniline) and metolachlor and acetochlor (synthesized from 2-ethyl-6-methylaniline). This investigation tests the hypothesis that the previously-observed oncogenicity of these herbicides is due to genotoxic intermediates such as diethylbenzoquinoneimine, a purported alachlor metabolite. Syntheses are reported here for the corresponding 2,6-dialkylbenzoquinoneimines, selected chloroacetyldialkylbenzoquinoneimines and several other candidate or known metabolites. The possible mutagenicity of diethylbenzoquinoneimine was tested in Salmonella typhimurium strains TA98 and TA100 with a weakly-positive response in the TA100 strain indicating induction of base-pair substitution mutations. The frequency of sister chromatid exchange (SCE) in Chinese hamster ovary cells was increased by alachlor at 10 microM and diethylaniline but not ethylmethylaniline at 30 and 3 microM. Isolated and cultured peripheral lymphocytes (mostly T cells) were used from two human donors to study the effects of the chloroacetanilides and their metabolites on primary human cells. In tests at 10 microM, the SCE frequency was increased by alachlor and possibly acetochlor but not by butachlor, metolachlor, dimethachlor (a 2,6-dimethyl analog) and dimethenamid (an analog based on 2,4-dimethyl-3-thienylamine). At 0.3 microM in cultured human lymphocytes, alachlor, the corresponding chloroacetanilide (N-dealkyl-alachlor) and aniline metabolites (and their 4-hydroxy derivatives), and diethylbenzoquinone were inactive or active in only one of the two donors whereas at 0.1-0.3 microM the SCE ratio for treated cells divided by the controls was always higher for diethylbenzoquinoneimine than for ethylmethyl- and dimethylbenzoquinoneimines. All the tested compounds were toxic to lymphocytes, but the depression of the mitotic index and increased duration of the cell cycle were not directly linked with SCE induction. Previous investigations have suggested that chloroacetanilide herbicides such as alachlor derived from 2,6-dialkylanilines are metabolized to 2,6-dialkylbenzoquinoneimines and the present study provides the first direct evidence that these metabolites are genotoxic in human lymphocytes.     

Individual sensitivity to cytogenetic effects of 1,2:3,4-diepoxybutane in cultured human lymphocytes: influence of glutathione S-transferase M1, P1 and T1 genotypes

Although some blood parameters have been suggested to modulate in-vitro induction of sister chromatid exchanges by 1,2:3,4-diepoxybutane (DEB), a metabolite of 1,3-butadiene, the increased sensitivity has largely been assigned to a homozygous deletion of glutathione S-transferase T1 gene (GSTT1 null genotype). However, some DEB-sensitive individuals have been shown to be GSTT1 positive (having at least one undeleted GSTT1 allele). To examine potential causes for this overlap, we evaluated the effect of GSTM1, GSTP1, and GSTT1 genotypes, together with various life-style and blood parameters, on the DEB induction of sister chromatid exchanges and cells with chromosomal aberrations (aberrant cells) in lymphocyte cultures of 115 and 62 human donors, respectively. Our results supported the important role of the GSTT1 genotype in DEB sensitivity; 76% of cultures from GSTT1 null donors but only 4% of those from GSTT1 positive donors were DEB-sensitive, as defined by sister chromatid exchange measurements. The GSTT1 genotype also clearly affected DEB-induced aberrant cells, 92% of GSTT1 null and 8% of GSTT1 positive donors being sensitive to DEB. All individuals showing a high response to DEB in both sister chromatid exchange and aberrant cell analyses were GSTT1 null. Baseline aberrant cell measurements but not sister chromatid exchange measurements were marginally higher among GSTT1 null donors compared with GSTT1 positive donors. GSTM1 and GSTP1 genotypes had no influence on these cytogenetic end-points. Blood transaminases, gamma-glutamyl transferase, urea, creatinine and white blood cell count showed a clear negative association with DEB-induced aberrant cells, whereas wine drinkers had more aberrant cells than non-drinkers. A higher sister chromatid exchange-response to DEB was observed in lymphocytes from women and smokers than from men and non-smokers, respectively. Erythrocyte count correlated negatively with DEB-induced sister chromatid exchanges. Thus, a variety of parameters seemed to modulate the individual DEB-sensitivity together with the GSTT1 genotype. Although the known contributing factors accounted for a considerable part of individual variability in sister chromatid exchanges (59.4%) and aberrant cells (46.7%) in DEB treatment, they did not, however, fully explain the overlap in cytogenetic response between GSTT1 positive and null individuals.     

Relationship between cognitive and social dysfunction in schizophrenia

Cytogenetic monitoring was carried out on a group of 38 nurses who reconstitute antineoplastic drugs in order to determine the extent of chromosomal damage. Genotoxic activities of antineoplastic drugs are studied by chromosome aberration assay, micronucleus assay, sister chromatid exchange (SCE) frequency high frequency cells (HFC) analysis, and mitotic activity of peripheral lymphocytes. Results confirmed that occupational exposure to a mixture of antineoplastic drugs may cause genome damages. The results of this study show that biomonitoring after exposure to a mixture of antineoplastic drugs which express clastogenic and aneugenic activity should involve a battery of cytogenetic methods.     

Cytogenetic investigations on patients with oral submucous fibrosis

Lymphocyte cultures were set up from venous blood samples collected from 23 patients of submucous fibrosis (SMF) and 10 normal controls. Slides, thus prepared, were processed and screened for G-, C-banding and sister chromatid exchange (SCE) frequency analysis. No gross chromosomal anomalies except that a few breaks and gaps were observed to be randomly distributed throughout the genome. However, a proportionate increase in SCE frequency in SMF patients as compared to the normal control individuals was observed. An attempt has been made to correlate the period of betel leaves, nuts, quid and tobacco chewing with the incidence of chromosomal anomalies and increase in SCE frequency and its sexwise distribution in these patients.     

Ambient, biological, and biological effect monitoring of exposure to polycyclic aromatic hydrocarbons (PAHs)

A novel strategy was utilised to assess the risk to health from exposure to polycyclic aromatic hydrocarbons (PAHs). Ambient monitoring was carried out by personal sampling. Urinary thioethers (UTh) and urinary 1-hydroxypyrene (1-HP) were utilised for biological monitoring. Urinary d-glucaric acid (UDGA) and sister chromatid exchange (SCE) in peripheral blood lymphocytes were used as biological effect markers. The population was categorised into exposed and control groups according to the external dose of PAHs. The excretion of 1-HP in the controls over the 3-day period showed a relatively stable baseline, while the exposed showed a significant increase over the same period of time. SCE frequency in the exposed population was significantly different from controls.     

About the nomination and training of family physicians

OBJECTIVE: To study sister chromatid exchange (SCE) frequency in lymphocytes of pregnant women with intrauterine growth retardation (IUGR) infants. METHODS: The frequency of SCE in peripheral blood lymphocytes of 14 pregnant women with IUGR babies and 90 normal pregnant women as well as the umbilical blood of their infants were investigated. RESULTS: The mean frequency of SCE in maternal and umbilical blood with normal pregnancy were 7.58 +/- 0.32 and 5.05 +/- 0.29 respectively, while they were 10.53 +/- 2.69 and 7.25 +/- 1.34 in IUGR group respectively. The difference of SCE levels between the 2 groups of mother was statistically significant (P < 0.01), so was that of the umbilical blood group. CONCLUSION: One of the causes of IUGR may be related to heredity injury. Thus, SCE will be a new diagnostic index to predict IUGR.     

Pion-nucleus single charge exchange induced by stopped negative pions

The early genotoxic action of oral exposure to UICC crocidolite asbestos fibres was studied in different short-term tests. Fischer-344 rats were gavaged with 50 mg/b.w.kg untreated asbestos fibres and fibres which had been allowed to adsorb benzo(a)pyrene molecules from extremely low concentration (0.25-2.5 microg/ml) aqueous solutions. This system can be considered a model for the drinking of potable water contaminated by asbestos fibres together with biologically active organic micro-pollutants. The Ames Salmonella mutagenicity assay was performed on concentrated urine and serum samples of treated animals. The formation of micronuclei and sister chromatid exchanges was also studied in the bone marrow of the exposed rats. The micronucleus analysis indicated marginal genotoxic activity only upon treatment with crocidolite prepared from the solution of 1 microg/ml. A dose-dependent increase was, however, demonstrated in the sister chromatid exchange frequency upon treatment with benzo(a)pyrene coated fibres. These experiments suggest the acute cogenotoxic activity of such fibres in orally exposed animals.     

Malignancies of the musculoskeletal system

The induction of sister chromatid exchanges (SCEs) was investigated in mice after a ten min exposure, in vivo, to 2 MHz focused, pulse-wave ultrasound with a pulse repetition rate of 1000 Hz, pulse duration of 10 microseconds. The bone marrow cells of the pregnant female mice and the fetal liver cells were analyzed. The cell cycle specific metaphase patterns were additionally evaluated. In the bone marrow cells, the mean frequencies of SCEs were 2.77 in control, 3.56 in the cells exposed to ultrasound at 586.2 mW/cm2 (spatial average temporal average, SATA); in the fetal liver cells, 2.64 in control, 3.84 in the cells exposed. The frequencies of SCEs significantly were increased by the treatment. Faster cell kinetics was observed in fetal liver cells than bone marrow cells of pregnant female. But there was no cell-growth inhibitory effect of ultrasound on both bone marrow and fetal liver cells. In fetal liver cells, the critical acoustic power was 160.0-278.9 mW/cm2 (SATA).     

Characterisation of genotoxic properties of 2',2'-difluorodeoxycytidine

The genotoxic properties of 2',2'-difluorodeoxycytidine (dFdC) were characterised using diploid, mortal low-passage fibroblasts (LPF cells) and the spontaneously transformed fibroblast cell line V79. In both cell types, incorporation of dFdC into the DNA led to an increase of DNA single-strand breaks evaluated by an in situ nick translation assay and to an accumulation of cells in the S-phase of the cell cycle. At concentrations below those leading to cell cycle arrest, dFdC neither induced sister chromatid exchange (SCE) nor structural chromosome aberrations in LPF cells, whereas V79 cells accumulated SCEs as well as chromosome breaks over a broad dose range. In LPF cells treated with dFdC, chromosomal alterations were detected by the micronucleus assay within a narrow concentration range, whereas in V79 cells, a dose-dependent increase in the appearance of micronuclei was seen up to cytotoxic concentrations. In addition, V79 cells went into apoptosis, as evaluated by nuclear fragmentation and condensation, whereas this phenomenon was not detectable in LPF cells.     

Drosophila melanogaster, Vicia faba and Arabidopsis thaliana short-term bioassays in genotoxicity evaluation of air and soil samples from sites surrounding two industrial factories in the Czech Republic

The Somatic Mutation and Recombination Test (SMART) in wing cells of Drosophila melanogaster, the Vicia faba cytogenetic tests-Sister Chromatid Exchange (SCE) and Micronucleus Test (MN), and the Müller test for gametic mutations in Arabidopsis thaliana were used for genotoxicity testing of environmental samples of pollutants from the surroundings of LACHEMA chemical factory (Brno, Czech Republic) and DEZA factory in Valasské Mezirící (Moravia, Czech Republic). Tested soil and air samples were taken from the near vicinity of both factories. The surroundings of both sites are heavy loaded by exhalation of chemicals from the factories. Chemical analyses of the 16 Polycyclic Aromatic Hydrocarbons (PAHs) according to the United States Environmental Protection Agency (US EPA) list of priority pollutants and heavy metals were performed in both soil and air samples. The Drosophila wing spot test was positive in 70.6% of the tested samples, the Vicia sister chromatid exchange test in 62.5%, and the Arabidopsis Müller test in 58.9%. The micronucleus Vicia faba test was quite insensitive in tested environmental samples. The concordance between SMART and SCE was 62.5%, between SMART and Müller test 76.5%, and between Müller test and SCE 100%. Total concordance of these three tests was 79.7%. Müller test for gametic mutation in Arabidopsis thaliana and cytogenetic SCE test in Vicia faba seem to be quite sensitive and convenient plant bioassays for assessing the mutagenic potential of environmental agents, when compared to the SMART test in Drosophila melanogaster.     

Growth of rubella virus in primary rabbit kidney cell cultures maintained with and without calf serum

The frequency of BrdU-induced sister chromatid exchanges (SCE) in cultured lymphocytes from patients with ataxia telangiectasia, Werner syndrome, and xeroderma pigmentosum was normal. The rate was increased in xeroderma pigmentosum following exposure to ultraviolet light and spontaneously raised in the Bloom syndrome. Quadriradial exchanges between homologous chromosomes in Bloom syndrome not only involve sister chromatids but also homologous (non-sister) chromatids. This could result in the formation of recombinant chromosomes and is viewed as a genetically determined form of increased somatic recombination in man. Endoreduplicated metaphases showed 'twin' and 'single' exchanges in a 1:2 ratio. This suggests a comparable frequency of exchanges at both divisions and provides evidence for the polarity of the chromatid subunits and the presence of a single chain of DNA.     

Genotoxicity studies on professional hair colorists exposed to oxidation hair dyes

The cytogenic repercussions of occupational exposure to oxidation hair dyes were assessed by using three assays in professional hair colorists. The assays were sister chromatid exchanges (SCE) in circulating lymphocytes to evaluate the interchange of DNA replication products at apparently homologous chromosomal loci, single cell gel electrophoretic (SCGE) assay to detect the presence of DNA strand breaks/alkali-labile damage, and the Ames assay using Salmonella typhimurium strain TA98 to detect the urine mutagenicity. The ability of these assays to detect genetic damage caused by oxidation hair dyes in man compared with closely matched controls produced the following findings. (i) The SCE assay could not detect the mutagenic effect in lymphocytes of exposed subjects from whom complete data were obtained. However, subjects (controls and exposed) with a history of smoking had slightly increased SCEs than the non-smokers in both groups. (ii) The extent of DNA migration (SCGE assay) did not distinguish between the samples in either the exposed or control subjects. Like the SCE results, the exposed and control smoker subjects showed a greater proportion of damaged lymphocytes with apparent migration of DNA. (iii) No clear differences in the mutagenic activity of the urine samples were observed between the exposed and control subjects. But, pooling exposed and controls together, a positive and significant variation in the urinary mutagenic effect was observed with the number of cigarettes smoked per day.     

K-ras mutations in the duodenal fluid of patients with pancreatic carcinoma

This study was conducted to test the hypothesis that the margin of safe fluoride exposure is narrowed in rats that are physiologically compromised by renal dysfunction. The study objective was to determine whether increases in fluoride retention and tissue fluoride levels in rats with surgically induced renal insufficiency result in toxic fluoride effects not ordinarily observed in healthy animals. Uremic and sham-operated control rats received 0 microg/ml, 5 (0.26 mmol/l), 15 (0.79), or 50 microg/ml (2.63 mmol/l) of fluoride in their drinking water for 3 or 6 months. Fluoride retention was monitored, and, following euthanasia, tissue fluoride and biochemical markers of tissue function were analyzed. Selected tissues were saved for histology, and bone marrow cells were harvested for determining the frequency of sister chromatid exchange, a marker of genetic damage. In spite of significantly higher levels of fluoride in the tissues of the animals with renal insufficiency, there were no clinically adverse, fluoride-induced, extraskeletal physiological, biochemical, or genetic effects of chronic exposure to common levels of fluoride in these rats.     

Endothelin and the airway mucosa

Antimony compounds are widely used in various manufacturing and semiconducting industries. Previously, it has been shown that antimony trichloride (SbCl3) elevates sister chromatid exchange (SCE) rates in V79 cells after a 28-h incubation. However, only limited data on its genotoxic effects are available so far. The present results demonstrate that a 4-h exposure to > 50 microM SbCl3 could induce micronuclei (MN) formation in cultured Chinese hamster ovary (CHO-K1) cells, human bronchial epithelial (BES-6) cells and human fibroblasts (HF). The order of sensitivity to SbCl3 determined by Sulforhodamine B (SRB)-staining survival assay is HF > BES-6 cells > CHO-K1 cells, with LD50 values in these cells being approximately 40, 80 and 180 microM, respectively. Apoptosis and DNA fragmentation was not found in cells immediately following 4-h SbCl3 treatment. However, DNA fragmentation was detected in CHO-K1 cells after 4-h SbCl3 treatment and a 16 h or more post incubation in fresh medium by 1.5% agarose gel electrophoresis. The delayed apoptosis was also observed under microscopic examination in HF, BES-6 and CHO-K1 cells after similar treatment protocol. In addition, an increase in calcium accumulation appeared in CHO-K1 cells and HF immediately after a 4-h SbCl3 treatment, or after a 24-h post incubation in fresh medium. The present results provide important genotoxic and cytotoxic information characterizing the cellular changes induced by short-term SbCl3 exposure in rodent and human cells.     

Effect of 60 Hz magnetic field exposure on c-fos expression in stimulated PC12 cells

Chromatid breaks are thought to result from DNA double-strand breaks (dsb) but the mechanisms are not yet understood. The early (but still prevailing) 'breakage-first' hypothesis fails to explain the large size of chromatid breaks; many of which are estimated to represent the apparent loss of between 15 and 45 Mbp (up to 30% of an average chromatid). The alternative 'exchange' hypothesis of Revell has potential for explaining the large sizes of deletions, but assumes the interaction of two lesions which therefore predicts a quadratic dependence of chromatid breaks on radiation dose. The exchange hypothesis is not tenable for mammalian cells since chromatid breaks are observed to be induced linearly with dose in both human and rodent cells. An alternative 'signal' model of chromatid breaks is outlined whereby a single dsb, occurring within a large looped chromatin domain, is signalled (possibly by molecules such as DNAPK or ATM protein) and triggers the cell to undergo a recombinational exchange, either within a chromatid or between sister chromatids. If incomplete, such recombinational exchanges would appear as chromatid breaks at metaphase. It is suggested that the large looped chromatin domains could be equivalent to one or more likely several replication 'factories' in which the DNA processing enzymes required for exchange formation would be located.     

Evidence for an adaptive DNA repair pathway in CHO and human skin fibroblast cell lines

When Escherichia coli is chronically exposed to very low, nontoxic doses of a monofunctional alkylating agent (notably N-methyl-N'-nitro-nitrosoguanidine, MNNG), the adaptive DNA repair pathway is induced which enables the bacteria to resist the killing and mutagenic effects of further alkylation damage. Mutation resistance in adapted bacteria is achieved, at least partly, by a greatly increased capacity of the cells to eliminate the minor DNA alkylation product O6-methyl-guanine, which has been strongly implicated as premutagenic and precarcinogenic. We now show that the chronic treatment of a Chinese hamster ovary (CHO) and a SV40-transformed human skin fibroblast (GM637) cell line with non-toxic levels of MNNG renders the cells resistant to the induction of sister chromatid exchange (SCE) by further alkylation damage. CHO cells also become resistant to killing (GM637 cells have not yet been tested). Having ruled out explanations such as changes in cell cycle distribution, mutagen permeability and mutagen detoxification, we conclude that resistance is probably achieved by the cells becoming more efficient at repairing alkylation damage, analogous to the adaptive response of E. coli.     

Induction of sister chromatid exchanges and micronuclei by titanium dioxide in Chinese hamster ovary-K1 cells

Titanium dioxide (TiO2) has color properties of extreme whiteness and brightness, is relatively inexpensive, and is extensively used as a white pigment in a variety of materials. TiO2, an effective blocker of ultraviolet light, is frequently added to sunscreens and cosmetic creams. However, the genotoxicity of TiO2 remains to be controversial. In this report, we have demonstrated that TiO2 can be transported into Chinese hamster ovary-K1 (CHO-K1) cells. The effects of TiO2 on induction of sister chromatid exchanges (SCE) and micronuclei (MN) were then studied in these cells. The SCE frequency in CHO-K1 cells treated with TiO2 at a nonlethal dose range (0 to 5 microM) for 24 h was significantly and dose-dependently increased. By the conventional MN assay, TiO2 at the dose ranged from 0 to 20 microM slightly increased the MN frequency in CHO-K1 cells. However, in the cytokinesis-block MN assay, the number of MN per 1000 binucleated cells was significantly and dose-dependently enhanced in CHO-K1 cells treated TiO2 at the same dose range for 24 h. These results suggest that TiO2 is a potential genotoxic agent.