Sister chromatid exchanges and cell proliferative abilities in cultured peripheral blood lymphocytes of patients with rheumatoid and reactive arthritis.

OBJECTIVE: Analysis of cytogenetic alterations in peripheral blood lymphocytes (PBL) of patients with acute and chronic reactive arthritis (AcReA and ChrReA) and rheumatoid arthritis (RA). METHODS: The frequencies of sister chromatid exchanges (SCE) and cell proliferative abilities were analysed in PBL from 69 patients with arthritis and 30 healthy controls. The analyses were done on metaphase chromosomes from PBL grown in cell culture for 72 hours. Cytogenetic parameters were compared among study groups and correlations with different clinical, immune and demographic characteristics were analysed. RESULTS: No significant increases in the frequencies of SCE were detected in PBL from patients with AcReA, ChrReA and RA as compared to controls. However, marked impairment of cell proliferative abilities was detected in cultured lymphocytes from patients with arthritis as compared to healthy controls. Significant associations between measures of disease activity and proliferative abilities of PBL were established. Parameters of lymphocyte proliferation were also influenced by concentration of anti-inflammatory cytokine interleukin-10 in the blood of patients. CONCLUSION: No increased risk of genetic alterations as measured by the rate of SCE was found in patients with RA and ReA. It is most likely that impaired proliferative abilities of peripheral blood lymphocytes are related to disease activity and could reflect systemic changes in cytokines production and intracellular signal transduction.

Cytogenetic analysis of peripheral blood lymphocytes of children treated with nitrofurantoin for recurrent urinary tract infection.

The objective of this study was to determine whether nitrofurantoin, used for long-term antimicrobial prophylaxis of urinary tract infection, may induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) in lymphocytes of treated children. Ninety-nine blood samples were taken from 69 children aged from 0.2 to 13 years and suffering from urinary tract infection. The treatment consisted of oral administration of nitrofurantoin at doses of 5-8 mg/kg/day for the first 7 days and at doses of 1-2 mg/kg/day for the rest of the treatment period. Blood was sampled before the start of the nitrofurantoin therapy and after 1, 3, 6 and 12 months of the therapy. Analysis of variance showed no statistically significant increase in CA and SCE frequencies in lymphocytes of children treated with nitrofurantoin for 1-12 months. However, a significant increase in SCE rates was determined in lymphocytes of those patients whose blood samples were available both before and after treatment with nitrofurantoin (6.21 +/- 0.28 and 7.30 +/- 0.39 SCE/cell, respectively, P = 0.0315, Student's paired t-test). Moreover, there was a statistically significant correlation (r = 0.6603, P = 0.0270) between cumulative dose of nitrofurantoin and SCE frequency in lymphocytes of children after 1 month of the therapy. Also, in vitro experiments indicated that nitrofurantoin was able to induce both CAs and SCEs in human lymphocytes. Positive findings with chromosome aberrations and SCEs in vitro and suggestive results with SCEs in vivo indicate that further, much larger follow-up studies are needed to elucidate the genetic safety of the therapeutic use of nitrofurantoin.

Cytogenetic analysis of children under long-term antibacterial therapy with nitroheterocyclic compound furagin.

Cytogenetic analysis of chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) was performed in 109 blood samples from 95 pediatric patients with urinary tract infections (UTIs). Children were exposed to diagnostic levels of X-rays during voiding cystourethrography and subsequently treated for one to 12 months with low doses of furagin - N-(5-nitro-2-furyl)-allylidene-1-aminohydantoin. Furagin is 2-substituted 5-nitrofuran, chemically and structurally similar to well-known antibacterial compound nitrofurantoin. Increased frequencies of CAs were found in children undergoing voiding cystourethrography as compared with the unexposed, acentric fragments being the most frequent alteration (2.03 versus 0.88 per 100 cells, P=0.006). However, a significant decrease in the frequency of acentric fragments was determined with the time elapsed since X-ray examination was performed. A time-independent increase in SCE frequency was found in lymphocytes of children treated with furagin. Total CA frequency did not differ significantly between groups of children with various duration of furagin treatment. However, frequency of chromatid exchanges (triradials and quadriradials) increased significantly with duration of treatment.

Genotoxicity of dill (Anethum graveolens L.), peppermint (Menthaxpiperita L.) and pine (Pinus sylvestris L.) essential oils in human lymphocytes and Drosophila melanogaster.

Genotoxic properties of the essential oils extracted from dill (Anethum graveolens L.) herb and seeds, peppermint (Menthaxpiperita L.) herb and pine (Pinus sylvestris L.) needles were studied using chromosome aberration (CA) and sister chromatid exchange (SCE) tests in human lymphocytes in vitro, and Drosophila melanogaster somatic mutation and recombination test (SMART) in vivo. In the CA test, the most active essential oil was from dill seeds, then followed essential oils from dill herb, peppermint herb and pine needles, respectively. In the SCE test, the most active essential oils were from dill herb and seeds followed by essential oils from pine needles and peppermint herb. Essential oils from dill herb and seeds and pine needles induced CA and SCE in a clear dose-dependent manner, while peppermint essential oil induced SCE in a dose-independent manner. All essential oils were cytotoxic for human lymphocytes. In the SMART test, a dose-dependent increase in mutation frequency was observed for essential oils from pine and dill herb. Peppermint essential oil induced mutations in a dose-independent manner. Essential oil from dill seeds was almost inactive in the SMART test.

Acute toxicity and genotoxicity of aquatic hydrophobic pollutants sampled with semipermeable membrane devices.

Triolein-filled semipermeable membrane devices (SPMDs) were deployed for 4 weeks in polluted water sources in Lithuania. The mixtures of pollutants sampled by the SPMDs were fractionated by size-exclusion chromatography (SEC). The fraction containing average molecular weight compounds such as polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides was screened by gas chromatography and mass spectrometry. The whole (non-fractionated) samples and their SEC fractions were tested in bioassays including Microtox, Mutatox, Daphnia pulex immobilization assay and the sister chromatid exchange (SCE) in human lymphocytes in vitro test. The Microtox test was most sensitive with the estimated EC(50) values in the range of milligrams or even micrograms per milliliter based on the amount of the SPMD triolein. Part of the observed toxicity was caused by elemental sulfur co-sampled by the SPMDs from sediments. The sum of toxicity equivalents of the SEC fractions was smaller than the relative toxicity of the whole samples indicating the presence of synergistic interactions in the complex mixtures of chemical pollutants. The toxic or genotoxic response induced by the chemical mixtures and their fractions was smaller in the D. pulex, Mutatox and SCE tests. In Mutatox, a positive response was only detected without the S9 metabolic activation which indicates the presence of mainly direct-acting mutagens in the samples. Interpretation of the Mutatox data was difficult due to the complexity of dose-response and time-response relationships. The study has demonstrated the potential as well as some limitations of SPMDs in the monitoring of biological effects of bioavailable organic pollutants in the aquatic environment.

Chromosomal aberrations and sister-chromatid exchanges in Lithuanian populations: effects of occupational and environmental exposures.

Cytogenetic analysis of chromosomal aberrations (CA) in 175,229 cells from 1113 individuals, both unexposed and occupationally or environmentally exposed to heavy metals (mercury and lead), organic (styrene, formaldehyde, phenol and benzo(a)pyrene) and inorganic (sulfur and nitrogen oxides, hydrogen and ammonium fluorides) volatile substances and/or ionizing radiation was performed. In addition, 11,250 cells from 225 individuals were scored for the frequency of sister-chromatid exchanges (SCE). Increased frequencies of CA were found in all occupationally exposed groups. A principal difference between the exposure to heavy metals and organic substances was found: increase in the CA frequency was dependent on duration of exposure to mercury but not dependent on duration of exposure to styrene, formaldehyde and phenol. A higher CA incidence was found in lymphocytes of children living in the vicinity of a plant manufacturing phosphate fertilizers. This indicates that children are a sensitive study group for the assessment of environmental exposure. However, the results of SCE analysis in these children were inconclusive. Exposure to ionizing radiation was found to cause chromosome breaks and chromatid exchanges in Chernobyl clean-up workers and chromatid breaks, chromatid exchanges, dicentric chromosomes and chromosome translocations in workers from the Ignalina Nuclear Power Plant. The increased frequency of chromatid exchanges in individuals exposed to ionizing radiation was quite unexpected. This may be attributed to the action of some unrecognized life-style or occupational factors, or to be a result of radiation-induced genomic instability. Also an increased SCE frequency was found in lymphocytes of Chernobyl clean-up workers.

High titers of antibodies to two human polyomaviruses, JCV and BKV, correlate with increased frequency of chromosomal damage in human lymphocytes.

Associations of antibody titers to the JC and BK human polyoma viruses and the frequency of chromosome aberrations (CA) in human peripheral blood lymphocytes were studied. Study group consisted of 33 workers occupationally exposed to low doses of ionizing radiation and 11 control persons. There were no statistically significant differences in the JC and BK virus titer values between two groups of donors. It was found that JC and BK virus titers explained approximately 6% of total inter-individual variation in CA frequency. Such factors as alcohol abuse, age and, in this special group, exposure to ionizing radiation explained an additional 53% of the total variation in CA frequency. In six clean-up workers and one control, rogue cell (cells with multiple chromosome-type aberrations) were found. The incidence of rogue cells correlated significantly with JC and BK virus titers as well as a history of recent acute respiratory disease.

Genetic toxicity of cytokines.

Review of the literature shows that such cytokines as human interferons alpha and gamma, tumor necrosis factor alpha, epidermal growth factor and interleukin-2 may exhibit genotoxic properties in human peripheral blood lymphocyte cultures. For all above cytokines, except interleukin-2, parabolic-like relationship between the dose and the frequency of sister chromatid exchanges was found. Although the mechanisms of these genotoxic actions remain largely unknown, generation of free radicals or interaction with enzymes such as DNA topoisomerase II may be suspected. Human interferon alpha also may be considered as an antimutagenic compound in human cells. Human tumor necrosis factor alpha has been reported to enhance cytotoxicity and DNA fragmentation produced by DNA topoisomerase II-targeted anticancer drugs. At the same time, it has some radio- and chemoprotective properties in vitro and in vivo. Despite these facts, the question about genotoxicity of cytokines is not answered. Some problems must be resolved before receiving the final answer. First, much more cytokines must be tested for their genotoxic activity. Second, appropriate test-systems must be designed. Third, genotoxicity studies of cytokines must account for cytokine interaction in the cytokine network as well as for such cytokine-induced effects as cytotoxicity and apoptosis. Fourth, in each case, it is necessary to have experimental evidence that observed genotoxic effects were caused by cytokine under investigation and not by the other factors.

Chromosome aberrations and rogue cells in lymphocytes of Chernobyl clean-up workers.

A cytogenetic analysis was performed on peripheral blood lymphocytes from 183 Chernobyl clean-up workers and 27 control individuals. Increased frequencies of chromosome aberrations were associated with exposure to radiation at Chernobyl, alcohol abuse and a history of recent influenza infection. However, only approximately 20% of Chernobyl clean-up workers had an increased frequency of dicentric and ring chromosomes. At the same time, an increased frequency of acentric fragments in lymphocytes of clean-up workers was characteristic. The use of multivitamins as dietary supplement significantly decreased the frequency of chromosome aberrations, especially of chromatid breaks. Rogue cells were found in lymphocytes of 28 clean-up workers and 3 control individuals. The appearance of rogue cells was associated with a recent history of acute respiratory disease (presumably caused by adenoviral infection) and, probably, alcohol abuse. Dicentric chromosomes in rogue cells were distributed according to a negative binomial distribution. Occurrence of rogue cells due to a perturbation of cell cycle control and abnormal apoptosis is suggested.

Increased frequency of sister chromatid exchanges in lymphocytes of Chernobyl clean-up workers.

Sister chromatid exchanges (SCEs) were analysed in lymphocytes from 12 control persons and 33 Chernobyl clean-up workers. The group of Chernobyl clean-up workers consisted of civilians who were forced to go to Chernobyl to clean up environmental contamination caused by Chernobyl disaster. On average, they received 0.13 (range 0.04-0.249) Gy of external irradiation before returning to home. Cytogenetic analyses were performed 6-8 years after the irradiation. Standard cytogenetic techniques were used. Mean SCE frequency was 7.45 +/- 0.69 SCE/cell in controls and 10.30 +/- 0.31 SCE/cell in clean-up workers (p < 0.05). Analysis of variance showed that exposure to radiation explained 19.6%, occupational exposure to various chemical substances, 11.9%, coffee consumption, 8.3%, smoking, 4.2%, interaction between smoking and coffee consumption, 3.6%, and alcohol abuse, 3.4% of total variation in SCE frequency. Effects of all above confounding factors were significant (p < 0.05). In addition, increased frequencies of chromosome aberrations due to exposure at Chernobyl and alcohol consumption were observed. However, there was no correlation between external dose of irradiation and the frequency of chromosome aberrations. Thus, even 6-8 years after the irradiation, cytogenetic effects in lymphocytes of Chernobyl clean-up workers are still significant.

Sister-chromatid exchanges and their distribution in human lymphocytes in relation to age, sex and smoking.

The effects of age, sex and smoking on sister-chromatid exchange (SCE) frequency and distribution in human lymphocytes were assessed by means of multiple linear regression. Differences in SCE scores were associated with all above variables: SCE increased with age and cigarette smoking intensity, and higher SCE frequencies were observed in females. Changes in SCE distribution were associated with age and smoking: the ratio of sample variance to sample mean (heterogeneity index) increased with age and smoking intensity. Cell proliferation kinetics, as measured by replication index, inversely correlated with age. Monte Carlo methods were used to show that in the occupational study, analysis of 20-50 persons per group and 25 cells per person may be recommended.

Anticlastogenic effects of Aevitum intake in a group of chemical industry workers.

The incidence of chromosome aberrations (CAs) was investigated in cultured lymphocytes of 109 styrene-, formaldehyde-, and phenol-exposed workers in comparison with 64 controls. There was a marked increase in the incidence of the structural chromosome aberrations in the first mitotic division metaphases of occupationally exposed workers (3.59 +/- 0.26 CAs/100 cells vs 1.47 +/- 0.14 in controls (P < 0.01). 22 occupationally-exposed workers were selected for the trial including 1-month administration of a drug Aevitum (100,000 U of retinol palmitate plus 0.1 g of alpha-tocopherol acetate dissolved in 0.2 ml of oil) at a daily dose of 1-2 capsules for 5 days a week. The frequency of chromosome aberrations before, after the administration of a cumulative Aevitum dose of 2.0, 4.0 and 8.0 ml, and 6 weeks after the cessation of vitamin intake was 5.68 +/- 0.63, 4.33 +/- 0.45, 2.67 +/- 0.34, 2.00 +/- 0.25, and 2.64 +/- 0.21 per 100 cells, respectively. Thus, Aevitum was found to cause a significant decrease in occupationally-induced chromosome damage in human lymphocytes.

Modulation by novobiocin of sister-chromatid exchanges induced by tumor necrosis factor in human lymphocytes.

The effect of the antibiotic novobiocin on human recombinant tumor necrosis factor (TNF)-induced sister-chromatid exchanges (SCEs) were examined in human peripheral blood lymphocytes. TNF, when introduced in a dose range of 10-1000 U/ml at the initiation of culture, was found to cause a significant increase in SCE frequency. The simultaneous addition of TNF and novobiocin (25 micrograms/ml) in the assay resulted in no increase of SCE frequency. Delayed (for 24 h) addition of novobiocin suppressed the induction of SCEs by 50, 100 and 500 U/ml but not by 1000 U/ml of TNF.

Sister chromatid exchanges in lymphocytes of normal and alcoholic subjects.

The effects of alcohol consumption, cigarette smoking and age on sister chromatid exchange (SCE) frequency in human lymphocytes were assessed by means of multiple linear regression. An increase in SCE rates was associated with alcohol consumption (p = 0.0001), smoking (p = 0.0231), and, to a small extent (p = 0.057), age. These three confounding factors explain 48% of the inter-personal variation in SCE rates among subjects studied.

Effect of tumor necrosis factor on cell proliferation kinetics and sister chromatid exchange frequency in human lymphocytes.

Recombinant human tumor necrosis factor alpha (TNF) was found to cause a significant increase in cell proliferation rates and sister chromatid exchange (SCE) frequency in phytohemagglutinin-stimulated human lymphocytes in vitro. The cells were treated for the entire cultivation time with 10, 50, 100, 500, 1000 and 5000 U/ml TNF. Cell proliferation rates, as measured by the replication index, increased significantly (P less than 0.01) in a concentration-independent manner. The maximal extent of SCE induction (15.18 +/- 0.57 versus 10.26 +/- 0.45 SCEs/cell in control, P less than 0.001) was observed with 50 U/ml TNF.

Replication index in cultured human lymphocytes: methods for statistical analysis and possible role in genetic toxicology.

The replication index (RI) is an index of cytokinetics in cultured cells. This paper presents statistical methods for the analysis of RI data. It also proposes formulas for the calculation of RI and its standard error. The proposed statistical analysis may be applied only in cases when replicate cultures are used. If duplicate or repeated cultures are used, the interculture variation may provide a more suitable error term for RI. Determining the RI in human blood cultures may be useful in genotoxicity testing, because inhibition of DNA synthesis will result in a decrease of the RI. Monte Carlo methods are used to show that the sensitivity of the proposed method is roughly equivalent to that of a chi 2 test. Power studies indicate that for the study sample, no less than 200 cells should be analyzed when assessing the RI.

Persistence of cytogenetic damage induced by alkylating antineoplastic drug phopurinum in human lymphocytes in vivo and in vitro.

Sister chromatid exchanges (SCEs) and structural chromosome aberrations (CAs) induced by cytostatic drug phopurinum in vivo and in vitro were studied in human lymphocytes. Phopurinum was found to cause a significant increase of CAs in lymphocytes of patients undergoing cytostatic therapy. Increased CA rates, however, declined rapidly after the cessation of treatment. This decline observed in vivo is in agreement with experimental results obtained in vitro, where it is found that the induction of SCEs and CAs occur only during the 1st cell cycle after pulse-treatment as G1 stage with phopurinum. Thus, phopurinum induced short-lived DNA damage both experimentally and in vivo.