UV-light induced sister chromatid exchanges in xeroderma pigmentosum lymphocytes

Cultured lymphocytes from 9 patients with clinically different types of xeroderma pigmentosum were exposed to ultraviolet light at 24 h. An increased rate of sister chromatid exchanges were observed in 6 patients (128--148% increase in three, 34--51% in three), but not in three patients with deSanctis-Cacchione syndrome (xeroderma pigmentosum with mental defect), compared to simultaneously cultured controls. A positive result could be useful as preliminary cytogenetic diagnostic test. The results are interpreted as an expression of UV-light induced chromosomal instability due to impaired DNA repair.     

Sister chromatid exchanges in Vicia faba induced by arsenic-contaminated drinking water from Zimapan, Hidalgo, Mexico

Sister chromatid exchanges (SCE) in Vicia faba root tips were used to examine well water containing high levels of arsenic. The increased amount of arsenic was contained in well water from different towns of Zimapan, Hidalgo, Mexico. Treatments of 3 h were applied followed by the differential staining technique of Tempelaar et al. (Mutation Res. 103 (1982) 321-326). Concentrations of arsenic from 0.267 up to 1.070 mg/l were determined by colorimetry in the polluted samples used for this study. These values were above the permissible limit of 0.05 mg/l in drinking water. In all cases, except one in which the As concentration was 0.021, the arsenic-contaminated water produced significant increases of SCE compared with the control (p < 0.001) and a concentration-response relationship was observed. The SCE potency factor of 33 per mg/l of arsenic was calculated as the slope of a common regression line, pooling data previously obtained in the Comarca Lagunera and the results observed in Zimapan.     

Dose dependent of sister chromatid exchanges in humans lymphocytes induced by in vitro alpha-particle irradiation

AIM: To study the effect of (-)-stepholidine (SPD) on serum prolactin (PRL) level and elucidate its pharmacological action on dopamine D2 receptors. METHOD: After i.p. administration of dopamine receptor agonist, antagonist, or SPD, the serum PRL levels were determined by radioimmunoassay. RESULTS: SPD (24 mg.kg-1, i.p.) caused a rapid rise in serum PRL level, lasting more than 1 h. SPD 0.2-40 mg.kg-1 raised serum PRL level in a dose-dependent manner with ED50 of 3.7 mg.kg-1 (95% confidence limits, 2.6-4.3 mg.kg-1) and PRL maximal level of 448 +/- 64 micrograms.L-1. Pergolide 2 mg.kg-1 i.p. caused a decrease (P < 0.01 vs saline) of PRL level, which was partially attenuated by SPD of 5 mg.kg-1 and completely abolished by 10 mg.kg-1. CONCLUSION: SPD is a dopamine D2 receptor antagonist.     

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.     

Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine

Sections from human jejunum were stained histochemically for aminopeptidase and alkaline phosphatase and the aldolase isozymes were detected with the mixed aggregation immuno-cytochemical technique. All enzyme concentrations increased from the bottom to the upper part of the crypt. The concentration of aldolase-A per cell was the same in the upper part of the crypt and the villus, whereas the concentration of the other three enzymes was still higher. Therefore, high amounts of aldolase-B, aminopeptidase and alkaline phosphatase are present in cells highly active in absorption in a fashion similar to that found in the proximal tubule cells of kidney. The relatively undifferentiated cells of the crypts contained both aldolase-A and aldolase-B. Alkaline phosphatase gains its full activity later than aminopeptidase. The synthesis of microvillar membrane enzymes comes to an end earlier than that of the cytosol enzymes.     

Sister chromatid exchange and micronucleus frequency in human lymphocytes of 1,650 subjects in an Italian population: II. Contribution of sex, age, and lifestyle

Sister chromatid exchange (SCE) and micronuclei (MN) analysis was carried out on 1,650 healthy individuals living in Pisa and in two nearby small cities, Cascina and Navacchio (Ca-Na). The effect of smoking on SCEs was linearly correlated with the number of cigarettes per day, and an increase of 7.3% SCEs was detectable for as few cigarettes as 1-10/day. Ex-smokers showed intermediate mean values of SCEs (8.09 +/- 1.88) in comparison with never smokers (7.54 +/- 1.61) and current smokers (8.45 +/- 1.94). Mean values of SCEs of ex-smokers decreased linearly with time of smoking cessation, reaching the mean values of never smokers within 8 years. The extent of SCE decrease was inversely proportional to the number of cigarettes previously smoked. No interaction between smoking habits and coffee or alcohol drinking on SCEs was observed. A borderline (P = 0.053) increase in mean SCE values in coffee drinkers (more than 3 cups/day) was found. The age effect on SCEs was remarkable in Ca-Na, but not in Pisa donors. Job type was not associated with significant modification of mean values of SCEs. Multiple logistic regression analysis revealed a statistically significant association between the proportion of high frequency cells (HCF) outliers and coffee consumption. Age and sex appeared to be by far the most important variables associated with modifications in MN frequency, which increased by 0.04 per thousand and 0.02 per thousand per year in males and females, respectively. Children and young donors (age < or = 40 years) showed lower MN frequency regardless of sex, whereas sex appeared to determine a significantly higher increase of MN only in females older than 40 years. In contrast, in males the MN rate by age tended to level off after the age of 30-50. MN frequencies of Pisa blue- and white-collar workers were statistically significantly higher than in students (+0.71 and +0.55 per thousand, respectively). Smoking did not determine any increase of MN frequency. A total lack of correlation (P = 0.913) between MN and SCEs was observed.     

Induction of sister-chromatid exchanges by styrene analogues in cultured human lymphocytes

Styrene and 11 styrene analogues were tested for their ability to induce sister-chromatid exchanges (SCEs) by a 48-h treatment in human whole-blood lymphocyte cultures (72 h). Styrene, its methyl-substituted derivatives (substituted at 2-, 3-, 4-, 3,5- or beta-position) and two styrene oxides (3,5-dimethylstyrene-7,8-oxide and 4-nitrostyrene-7,8-oxide) induced a distinct dose-dependent increase in SCEs. Also, 4-methylstyrene-7,8-oxide and alpha-methylstyrene showed a positive effect, but were not able to double the mean number of SCEs/cell at the concentration ranges available. Ethylbenzene had a marginal effect on SCEs at the highest dose tested. 2-Phenylethanol did not increase SCEs. The results indicate that styrene and methylstyrenes are converted into reactive metabolites in the whole-blood lymphocyte cultures. The negative or weak effects of styrene analogues without a double bond in the side-chain (ethylbenzene and 2-phenylethanol) suggest that the reactive metabolites are derived from the conversion of the vinyl group and are styrene-7,8-oxides.     

Sister chromatid exchange studies for monitoring DNA damage in lymphocytes of malignant lymphoma patients under cytostatic therapy

Sister chromatid exchange (SCE) frequencies were studied in lymphocytes from 45 patients with malignant lymphoma. Fifteen patients were untreated when studied. The mean SCE frequency for these patients was 8.70 +/- 0.99 per mitosis. The mean score for 35 controls was 4.37 +/- 1.19. SCE mean scores were significantly higher in the untreated patients than in the controls (p < 0.001). Nine patients were treated with radiotherapy alone. The mean SCE frequency (6.80 +/- 0.87) they demonstrated was significantly lower (p < 0.01) than that found in untreated patients. Twelve patients received cyclophosphamide 1 month before the study was started. They demonstrated a mean SCE frequency (12.00 +/- 1.31) significantly higher (p < 0.05) than that found in patients who had received regimens that did not contain cyclophosphamide (9.72 +/- 1.32). From these findings we suggest that untreated patients with malignant lymphoma have elevated SCE frequencies, which may be further increased by chemotherapeutic agents.     

Chromosome breaks and sister chromatid exchanges in albinos in Nigeria

The prevalence of squamous cell carcinoma of the skin in albinos reaches approximately 90% in patients over 20 years of age in the vicinity of Enugu, Nigeria. Chromosome breaks and sister chromatid exchanges (SCE) were evaluated in tyrosinase-positive oculocutaneous albinos and pigmented controls of Ibo extraction who were life-long residents of Nigeria. No difference in the frequency of chromosomal breaks in 14 albinos compared to 6 pigmented controls, and no differences in the frequency of SCE in 9 albinos compared to 3 controls could be detected. Increased chromosomal abnormalities in lymphocytes do not appear to be assoicated with albinism or fulminating skin cancer present in albinos in the tropics.     

Pig plasma modulates cell cycle kinetics but not the baseline frequency of sister-chromatid exchanges in human lymphocytes

The effect of human and pig plasma on the baseline frequency of sister-chromatid exchanges (SCEs) in human and porcine plasma leukocyte cultures was studied. Human plasma leukocyte cultures, but not porcine plasma leukocyte cultures, showed at least a twofold increase in SCE frequency over whole blood culture values. Addition of pig plasma to human plasma leukocyte cultures and human plasma to pig plasma leukocyte cultures did not modify the baseline SCE frequencies. In both human and porcine cultures, cell proliferation was slower in plasma leukocyte cultures than in whole blood cultures. The addition of pig plasma to human plasma leukocyte cultures, but not the incorporation of human plasma in pig plasma leukocyte cultures, accelerated the cell cycle progression of lymphocytes. With 10% pig plasma in the plasma leukocyte culture medium, lymphocyte proliferation was similar to that in whole blood cultures. Smaller concentrations of pig plasma rendered cell cycle progression intermediate between the basal plasma leukocyte culture and whole blood culture values. Exchanging fetal calf serum for human AB serum in human plasma leukocyte cultures did not affect the cell cycle kinetics of lymphocytes but it did decrease their baseline SCE frequency.     

Chromosomal aberrations induced in human lymphocytes by high-LET irradiation

The affinity of a DNA sequence for the histone octamer in a core nucleosome depends on the intrinsic flexibility of the DNA. This parameter can be affected both by the sequence-dependent conformational preferences of individual base steps and by the nature and location of the exocyclic groups of the DNA bases. By adopting highly preferred conformations particular types of base step can influence the rotational positioning of the DNA on the surface of the histone octamer. The asymmetry of the next higher order of chromatin structure is determined in part by the asymmetric binding of the globular domain of histone H5 to the core nucleosome.     

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.     

Sister chromatid exchange frequencies in Escherichia coli analyzed by recombination at the dif resolvase site

Sister chromatid exchange (SCE) in Escherichia coli results in the formation of circular dimer chromosomes, which are converted back to monomers by a compensating exchange at the dif resolvase site. Recombination at dif is site specific and can be monitored by utilizing a density label assay that we recently described. To characterize factors affecting SCE frequency, we analyzed dimer resolution at the dif site in a variety of genetic backgrounds and conditions. Recombination at dif was increased by known hyperrecombinogenic mutations such as polA, dut, and uvrD. It was also increased by a fur mutation, which increased oxidative DNA damage. Recombination at dif was eliminated by a recA mutation, reflecting the role of RecA in SCE and virtually all homologous recombination in E. coli. Interestingly, recombination at dif was reduced to approximately half of the wild-type levels by single mutations in either recB or recF, and it was virtually eliminated when both mutations were present. This result demonstrates the importance of both RecBCD and RecF to chromosomal recombination events in wild-type cells.     

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.     

Analysis of estrone, estradiol and estriol in serum and urine by mass fragmentography using GC-MS (author's transl)

The first successful analysis of estrogens in serum and urine was performed by mass fragmentography using a GC-MS combined system. The equipment used was Shimadzu LKB 9000 GC-MS (MID-PM). The TMSi derivative of the compounds were analyzed using the GC-MS system equipped with a 3 ft x 3mm column packed with 1.5% OV-1 and the temperature was programed from 200 degrees to 260 degrees C at 10 degrees C/min increments. The mass spectrum showed molecular ions at m/e 342, 416 and 504 which correspond to the TMSi derivatives of Estrone (E-1), Estradiol (E-2) and Estriol (E-3) respectively. Molecular ion peak of each compound was applied to establish the precise quantitative evaluation of E-1, E-2 and E-3. 1) The minimum detectable limits of the compounds injected into the column were ca. 2 pg for E-1, E-2 and 5 pg for E-3 respectively. 2) The sensitivity was of the order of ng or pg which enables quantitation with 2 ml of human serum and urine samples obtained from normally ovulating women. 3) This method was very convenient for extracting the compounds from biological fluids, and the procedure can be carried out easily in a short time. 4) Mass fragmentography makes possible the simultaneous measurements of E-1, E-2 and E-3 in samples obtained from pregnant women.     

Rate of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by co-cultivation with normal fibroblasts

Six strains of Bloom syndrome (BlS) fibroblasts responded to co-cultivation with normal fibroblasts at a 1:2 ratio by a reduced rate of sister chromatid exchanges (SCE's) from a mean of 67.5 (range = 59--78) to 28.4 (range = 21--35). The response was dose-dependent in one strain tested at 1:2, 1:1, and 2:1 ratios. In addition, quadriradial exchange figures and other signs of increased chromosomal instability were not found in BlS cells following co-cultivation with control cells. Control cells did not respond to BlS cells and maintained a normal rate of SCEs. Culture medium conditioned for 48 hrs by normal fibroblasts could also reduce the rate of SCEs in BlS fibroblasts, but less than in co-cultivation. We suggest that the reduced rate of SCEs and the lack of chromosomal instability in BlS cells following co-cultivation represent a corrective effect that is related to the basic defect and not dependent on cell-to-cell contact.     

Long-term exposure to fluoride in drinking water and sister chromatid exchange frequency in human blood lymphocytes

The genetic toxicity of fluoride has been investigated extensively by various test systems. However, results obtained have been inconsistent. Fluoride has been reported to be non-genotoxic, genotoxic, and synergistic or antagonistic with certain mutagens. To date, there are no published human studies on the genotoxicity of fluoride. The purpose of this investigation was to determine genotoxic risks of long-term exposure to various concentrations of fluoride in drinking water in humans with normal or inadequate nutrition. Six groups of subjects with either normal or inadequate nutritional intakes were selected from areas of approximately 0.2, 1.0, or 4.8 ppm (10.5, 52.6, or 252.6 mumol/L) fluoride in water. The subjects had been continuous residents in the area for at least 35 years. Samples of drinking water, plasma, and urine were analyzed for fluoride content. Blood lymphocytes were examined to determine the frequency of sister chromatid exchange (SCE). Blood chemistry and electrolytes were also analyzed. The results showed that average daily fluoride intake as well as urine and plasma fluoride levels increased with increase in the fluoride content of the drinking water. The blood chemistry and electrolyte values were within the normal range for all populations, but several parameters were significantly different. While the numerical differences were small, the subjects with low fluoride in the water (0.11 and 0.23 ppm or 5.8 and 12.1 mumol/L) had significantly higher SCE frequencies than those with higher fluoride exposures.(ABSTRACT TRUNCATED AT 250 WORDS)