Cytogenetic effects of chemotherapy with three combinations of anti-tubercular drugs involving isoniazid,thiacetazone, para-aminosalicylic acid and streptomycin on human lymphocytes: chromosome aberrations,sister chromatid exchanges and mitotic index

Cytogenetic effects of three combinations of anti-tubercular drugs were evaluated on human lymphocytes in vivo and were compared with controls of two types: (1) newly diagnosed tuberculosis patients before starting therapy and (2) individuals from the general population. The drugs used were: isoniazid (INH), thiacetazone (TAZ), para-aminosalicylic acid (PAS), and streptomycin (SM). These drugs were tested in the following combinations: (a) INH + TAZ + SM, (b) INH + PAS + SM, (c) INH + SM. The frequency of chromosome aberrations was significantly increased in patients treated with both the triple drug combinations, i.e., with INH + TAZ + SM and INH + PAS + SM, whereas patients treated with INH + SM did not exhibit an increase in the frequency of chromosome aberrations as compared to the controls. Although both the triple drug combinations were clastogenic, none of the three drug combinations tested induced an increase in the frequency of sister chromatid exchanges (SCEs). In other words, the mechanisms leading to SCEs and chromosome aberrations may be different. SM appeared to depress the mitotic index in patients treated with INH + SM and INH + PAS + SM, though it was found to possess a mild anti-clastogenic effect. INH + TAZ + SM, on the other hand, enhanced the mitotic index. This enhanced mitotic index was probably due to the presence of TAZ.     

Chromosome-damaging action of isoniazid and thiacetazone on human lymphocyte cultures in vivo

Summary Antitubercular drugs in general are given in various combinations, one being isoniazid and thiacetazone. In the present study, was evaluated the in vivo chromosome-damaging effects of a combination of these two drugs in 72 h lymphocyte cultures.Chromosome aberrations were significantly increased in the patients treated with INH and thiacetazone as compared with two types of controls: (1) tuberculosis patients before starting the drug treatment and (2) individuals from the general population. The most frequently observed aberrations were chromatid breaks and gaps.It has been shown that individually, isoniazid may not be clastogenic on human chromosomes in therapeutic doses. The effects of thiacetazone on human chromosomes are not known. Consequently, the enhancement in chromosomal aberrations in the drug-exposed patients may be due to a synergistic effect of isoniazid and thiacetazone or to the clastogenic effects of thiacetazone alone.     

In Canada: A Trial of Chemoprophylaxis in Inactive Tuberculosis

One thousand five hundred and thirty-six patients with inactive tuberculosis were given a course of preventive treatment consisting of either INH alone or INH and PAS while 840 similar patients served as a control group. Discontinuation of the treatment was frequent and was usually caused by development of complaints which the patients ascribed to the drugs they were taking.The annual reactivation rate among controls was 4.9 per 1000. During the period of taking drugs the treated group suffered a reactivation rate of 0.7 per 1000 and those who had taken the medication for at least six months suffered a subsequent annual reactivation rate of 1.3 per 1000. The rate for those who discontinued treatment in the first six months was 5.1 per 1000. There were no reactivations in patients who took INH and PAS for over six months. Bacilli from two of the patients with reactivations who were treated for a prolonged period with INH alone showed resistance to this drug.Chemoprophylaxis of inactive cases is a potent weapon in tuberculosis control; however, it requires thorough motivation and supervision.     

Transcendental meditation.

A trial of chemoprophylaxis to prevent reactivation of tuberculosis in persons with inactive disease who had never had adequate chemotherapy was conducted in Canada in the mid-1960s. Preventive drug treatment consisted of either isoniazid (INH) alone or INH plus para-aminosalicylic acid (PAS), for a maximum of 18 months. Long-term evaluation in 1974 of 1571 treated patients and 834 control patients demonstrated clearly the substantial and sustained value of adequate chemoprophylaxis in reducing the risk of reactivation. Among those who took INH alone for 6 months or more the annual reactivation rate was 1.2 per 1000 persons, while among those who took INH plus PAS the rate was 0.38/1000. These rates were, respectively, 70 and 90% less than the average rate in the controls, 3.9/1000. Among those who underwent chemoprophylaxis for less than 6 months the annual reactivation rate was 3.7/1000, similar to that in the controls. Cost-benefit analysis showed chemoprophylaxis to be economically sound. Despite the recent increasing application of this preventive measure, there are still many persons living in Canada who could benefit substantially from a course of chemoprophylaxis.     

Chromosomal aberrations in tuberculosis patients before and after treatment with short-term chemotherapy

Cytogenetic effects of 4 common anti-tubercular drugs, isoniazid (H), streptomycin (S), rifampicin (R) and pyrazinamide (Z), in 3 different combinations (2 SHRZ, 2 HRZ and 2 H2R2Z2) were evaluated in the lymphocytes of tuberculosis patients undergoing chemotherapy, in order to estimate their mutagenic potential in combination. All 3 regimens showed an increased frequency of chromosomal aberrations after treatment compared to before treatment. These findings are of significance in the treatment of tuberculosis, as the drugs in question are observed to be mutagenic/clastogenic.     

Clastogenic factors in plasma of HIV-1 infected patients

The objective of this study was to investigate the clastogenic activity of plasma ultrafiltrates from HIV-I infected patients. Clastogenic factors are chromosome-damaging agents with low molecular weight (<10,000 daltons) which cause chromosome aberrations, sister chromatid exchanges, DNA strand breakage, and gene mutation. They have first been described in the plasma of irradiated persons, but they are also found in hereditary breakage syndromes and chronic inflammatory diseases with autoimmune reactions. Their formation and their clastogenic effects are modulated by superoxide anion radicals. We analyzed a total of 22 HIV-1 positive patients in comparison to 20 reference plasma samples from healthy HIV negative blood donors of similar age. The plasma ultrafiltrates (filter cutoff 10,000 daltons) from patients induced a statistically significant increase in chromosomal breakage in the cytogenetic test system (20.5 ± 6.8 aberrations per 100 cells), while no increase was observed in test cultures exposed to plasma ultrafiltrates from healthy blood donors (6.3 ± 2.9 aberrations per 100 cells). The breakage values were slightly, but not significantly, lower in the 10 patients with more than 200 T-helper cells/ml (18 ± 4 aberrations per 100 cells), than in the 12 patients with less than 200 T-helper cells/ml (22.3 ± 7.9 aberrations per 100 cells). HIV patients with high clastogenic activity (induction of more than 20 aberrations per 100 cells, range 20 to 39) showed higher plasma levels for malondialdehyde than those with lower clastogenic activity (less than 20 aberrations per 100 cells, range 12 to 18). However, the difference was statistically not significant. Another lipid peroxidation product, 4-hydroxynonenal, was increased equally in both groups. There were no significant differences in water- and lipid-soluble plasma antioxidants between the low- and high-breakage group. In agreement with previous findings, the clastogenic effects of plasma ultrafiltrates in the test cultures were reduced by the antioxidant enzyme superoxide dismutase. The presence of clastogenic factors in the plasma of HIV patients is further evidence for a prooxidant state in these persons. Since clastogenic factor formation appears to occur at an early stage of the disease, it may be significant for virus release or activation, because of the superoxide anion stimulating effects of clastogenic factors. From a practical standpoint, clastogenic factors may be useful for evaluation of promising drugs.     

Determination of Drug Resistance of Mycobacterium tuberculosis Cultures

A total of 3,303 strains of Mycobacterium tuberculosis were tested for sensitivity to streptomycin (SM), isoniazid (INH), and p-aminosalicylic acid (PAS) by the Steenken modified minimal inhibitory concentration (MIC) test. A simultaneous double blind comparison was carried out on 277 selected strains by the Steenken MIC test and the Canetti proportion method. Agreement between the results for the two tests was 82% for SM, 95% for INH, and 89% for PAS. A small number of strains appeared to be sensitive when tested by one method but resistant by the other. MIC determinations were carried out on 83 strains by using Steenken-Smith, Lowenstein-Jensen, and Middlebrook 7H10 media containing a more extended range of concentrations of the test drugs. The MIC values for both SM and dihydrostreptomycin increased on Steenken-Smith medium compared with the other two. INH did not show any medium effect, whereas PAS showed increased MIC values in 7H10 agar. The significance of the comparisons of the MIC values on the various media is discussed in terms of possible changes in the drug sensitivity testing methods used at present in this laboratory.     

Drug resistance among Mycobacterium tuberculosis strains isolated in Taiwan during 1975.

455 strains of Mycobacterium tuberculosis were isolated from patients with history of treatment in Taiwan Provincial Tuberculosis Control Bureau and tested for resistance against various antituberculosis agents including streptomycin (SM), paraaminosalicylic acid (PAS), isoniazid (INH), cycloserine (CS), prothionamide (1321TH), kanamycin (KM), ethambutol (EMB), and rifampicin (RFP). In vitro resistance to SM and INH was more frequently found than others and the resistance to a single drug was more common than multiple resistance.     

Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells

The clastogenic activity of hydroquinone (HQ) in germ cells of male mice was evaluated by analysis of chromosomal aberrations in primary spermatocytes and differentiating spermatogonia. In the first experiment with treated spermatocytes the most sensitive stage of meiotic prophase to aberration induction by HQ was determined. Testicular material was sampled for microscopic analysis of cells in diakinesis-metaphase I at 1, 5, 9, 11, and 12 days after treatment with 80 mg/kg of HQ, corresponding to treated diplotene, pachytene, zygotene, leptotene and preleptotene. The frequencies of cells with structural chromosome aberrations peaked at 12 days after treatment (p less than 0.01). This indicates that the preleptotene when DNA synthesis occurred was the most sensitive stage of meiotic prophase. In the second experiment the dose response was determined 12 days post treatment by applying 2 additional doses of 40 mg/kg and 120 mg/kg. The clastogenic effects induced by 40 and 80 mg/kg were significantly different from the controls (p less than or equal to 0.01) and higher than the results obtained with 120 mg/kg of HQ. A humped dose-effect relationship was observed. In a third experiment the same doses were used to analyse chromosomal aberrations in dividing spermatogonia of mice 24 h after treatment with HQ. All the administered doses gave results statistically different from the control values (p less than or equal to 0.01) and the data were fitted to a linear equation. HQ was found to be clastogenic in male mouse germ cells. It is concluded that the clastogenic effect in male germ cells is of the same order of magnitude as in mouse bone marrow cells.     

Chromosome aberrations in lymphocytes and clastogenic factors in plasma detected in Belarus children 10 years after Chernobyl accident

In 1996, 10 years after Chernobyl accident, a cytogenetic analysis was carried out to assess whether chromosome aberrant cells (CA) were still detectable in the lymphocytes and clastogenic factors (CFs) were present in the plasma of children coming from Gomel (Belarus), one of the most heavily contaminated regions. Furthermore, the possible contribution of plasmatic CFs to the amount of CA was investigated. The presence of CA was examined in the lymphocytes from 29 thyroid tumour-affected children and 41 healthy children (local controls). Thirty healthy children living in Pisa (Italy) were enrolled in the study as additional controls from an uncontaminated area. No significant difference was observed between the two control groups, whereas a significantly increased frequency of CA was found in the tumour-affected children, as compared with Gomel and Pisa controls (chi 2-test, p < 0.001). However, when soil contamination level was taken into account, the chromosome type CA frequency observed in tumour-affected children coming from the more contaminated areas (> 4 Ci/km2) resulted significantly higher than that in other children, either affected or not (p = 0.003). The presence of CFs was analyzed on the plasma ultrafiltrate from 41 children. 7/10 (70%) plasma samples from tumour-affected children and 17/23 (74%) Gomel controls resulted to possess clastogenic activity irrespective of soil contamination levels. No activity was detected in the plasma of eight Pisa controls (0%). The difference between both Gomel groups and Pisa controls was highly significant (p = 0.002). A borderline, but not statistically significant correlation (p = 0.08) was observed between basal CA frequency and CF potency, which became significant (p = 0.03) when only chromosome type of aberrations was considered. We conclude that, although the presence of CFs in the plasma of these children might be partly responsible of the cytogenetic effects observed, the main source of damage has considered to be do to the previous and/or continuous exposure to environmental radiocontaminants. Tumour-affected children may represent a subset of the population either more sensitive to clastogenic damage or exposed to higher levels of contaminants.     

Cytogenetic studies in leprosy patients before and after chemotherapy

Summary The frequencies of chromosome aberrations and sister chromatid exchanges (SCEs), cell proliferation kinetics and mitotic indices were studied in peripheral blood lymphocyte cultures of leprosy patients both before and after chemotherapy. The differences in the frequencies of chromosome aberrations and SCEs between controls, paucibacillary and multibacillary patients were found to be statistically highly significant (P < 0.001). The extent of cytogenetic damage seemed to depend on the severity of the disease. Lymphocytes of untreated leprosy patients showed a low mitotic index and a slow rate of cell proliferation. Following combined treatment with dapsone and rifampicin there was an increase, but to a lesser degree (P < 0.01), in the frequency of SCEs and chromosome aberrations while the drug combination of dapsone, rifampicin and clofazamine had a non-mutagenic effect on chromosomes of the patient. Furthermore, after drug treatment, the cell proliferation rate and mitotic indices in paucibacillary patients were comparable to that of controls. These results indicate the clastogenic potency of Mycobacterium leprae and the remedial effects that follow therapeutic drug treatment.     

Effects of pH in the in vitro chromosomal aberration test

The relation between the pH of the medium and clastogenic activity was studied in Chinese hamster ovary (CHO) K1 cells in vitro. The pH was adjusted with NaOH, KOH, HCl or H2SO4. No clastogenic activity was observed over the initial pH range of 7.3-10.9 without S9 mix, but a few chromosomal aberrations were induced at pH 10.4 with S9 mix. The frequency of aberrations increased with the increase in amount of S9. At acidic pH, many chromatid breaks were induced at initiatial pH 5.5 or below without S9 mix, and aberrations such as chromatid breaks and chromatid exchanges were induced at initial pH 6.2 or below with S9 mix. Using MES and Bis-Tris as buffers instead of sodium bicarbonate, we observed that aberrations of the chromatid break type were inducible at pH 6.2 or below. These results show that the combination of strong alkalinity and S9 is clastogenic to CHO-K1 cells, and also that weakly acidic media are genetically active. The results indicate that incubations at non-physiological pH might give false-positive responses.     

Chromosome-damaging activity of a ruthenium radiosensitizer, RuCl2 (DMSO)2 (4-nitroimidazole)2, in Chinese hamster ovary cells in vitro

The metal complex, RuCl2 (DMSO)2 (4-nitroimidazole)2, 1, which has hypoxic radiosensitizing properties, was examined for genotoxic activity, as measured by the in vitro induction of chromosome aberrations (chromatid breaks and chromatid exchanges) in Chinese hamster ovary (CHO) cells. A dose-dependent increase in the frequencies of metaphases with chromatid aberrations was observed for 1. Addition of S9 liver microsomal mixture and 1 to the cultured CHO cells did not alter the clastogenic activity noted for the complex itself. The clastogenic (chromosome damaging) activity of a precursor complex, cis-RuCl2(DMSO)4 and the ligand, 4-nitroimidazole (4-NO2-Im) were found to be less than that of 1 at corresponding concentrations. A comparison with two drugs used clinically with radiation, cis-dichlorodiammineplatinum(II) (cis-DDP) and misonidazole (miso), indicated that the clastogenic activity of 1 was similar to miso and much less than that of cis-DDP.     

Oxidative stress response and its role in sensitivity to isoniazid in mycobacteria: characterization and inducibility of ahpC by peroxides in Mycobacterium smegmatis and lack of expression in M. aurum and M. tuberculosis.

Mycobacterium tuberculosis is a natural mutant with inactivated oxidative stress regulatory gene oxyR. This characteristic has been linked to the exquisite sensitivity of M. tuberculosis to isonicotinic acid hydrazide (INH). In the majority of mycobacteria tested, including M. tuberculosis, oxyR is divergently transcribed from ahpC, a gene encoding a homolog of the subunit of alkyl hydroperoxide reductase that carries out substrate peroxide reduction. Here we compared ahpC expression in Mycobacterium smegmatis, a mycobacterium less sensitive to INH, with that in two highly INH sensitive species, M. tuberculosis and Mycobacterium aurum. The ahpC gene of M. smegmatis was cloned and characterized, and the 5' ends of ahpC mRNA were mapped by S1 nuclease protection analysis. M. smegmatis AhpC and eight other polypeptides were inducible by exposure to H2O2 or organic peroxides, as determined by metabolic labeling and Western blot (immunoblot) analysis. In contrast, M. aurum displayed differential induction of only one 18-kDa polypeptide when exposed to organic peroxides. AhpC could not be detected in this organism by immunological means. AhpC was also below detection levels in M. tuberculosis H37Rv. These observations are consistent with the interpretation that ahpC expression and INH sensitivity are inversely correlated in the mycobacterial species tested. In further support of this conclusion, the presence of plasmid-borne ahpC reduced M. smegmatis susceptibility to INH. Interestingly, mutations in the intergenic region between oxyR and ahpC were identified and increased ahpC expression observed in deltakatG M. tuberculosis and Mycobacterium bovis INH(r) strains. We propose that mutations activating ahpC expression may contribute to the emergence of INH(r) strains.     

Chromosome aberrations in lymphocytes and clastogenic factors in plasma detected in Belarus children 10 years after Chernobyl accident

In 1996, 10 years after Chernobyl accident, a cytogenetic analysis was carried out to assess whether chromosome aberrant cells (CA) were still detectable in the lymphocytes and clastogenic factors (CFs) were present in the plasma of children coming from Gomel (Belarus), one of the most heavily contaminated regions. Furthermore, the possible contribution of plasmatic CFs to the amount of CA was investigated. The presence of CA was examined in the lymphocytes from 29 thyroid tumour-affected children and 41 healthy children (local controls). Thirty healthy children living in Pisa (Italy) were enrolled in the study as additional controls from an uncontaminated area. No significant difference was observed between the two control groups, whereas a significantly increased frequency of CA was found in the tumour-affected children, as compared with Gomel and Pisa controls (χ²-test, p<0.001). However, when soil contamination level was taken into account, the chromosome type CA frequency observed in tumour-affected children coming from the more contaminated areas (>4 Ci/km²) resulted significantly higher than that in other children, either affected or not (p=0.003). The presence of CFs was analyzed on the plasma ultrafiltrate from 41 children. 7/10 (70%) plasma samples from tumour-affected children and 17/23 (74%) Gomel controls resulted to possess clastogenic activity irrespective of soil contamination levels. No activity was detected in the plasma of eight Pisa controls (0%). The difference between both Gomel groups and Pisa controls was highly significant (p=0.002). A borderline, but not statistically significant correlation (p=0.08) was observed between basal CA frequency and CF potency, which became significant (p=0.03) when only chromosome type of aberrations was considered. We conclude that, although the presence of CFs in the plasma of these children might be partly responsible of the cytogenetic effects observed, the main source of damage has considered to be do to the previous and/or continuous exposure to environmental radiocontaminants. Tumour-affected children may represent a subset of the population either more sensitive to clastogenic damage or exposed to higher levels of contaminants.     

Increased chromosomal radiosensitivity in patients undergoing radioimmunoglobulin therapy

Lymphocytes from individual patients undergoing radiolabeled immunoglobulin therapy have been examined both for chromosome aberrations expressed immediately upon explant, or for chromosome aberrations induced by a subsequent challenge of gamma-rays after PHA-stimulated proliferation. Despite interpatient variation, there is strong correlation between levels of chromosome aberrations observed in the initial mitosis after mitogenic stimulation and levels induced by a challenge dose of radiation in replicate cultures after several cell cycles of growth. These data indicated that even after proliferation, human lymphocytes retain a memory of in vivo exposure to ionizing radiation that can be observed by challenge with a clastogenic agent. This persistent hypersensitivity occurs at high frequency, suggesting that it may be related to initial steps in multistage carcinogenesis.     

Metabolomics of urine for the assessment of microvesicular lipid accumulation in the liver following isoniazid exposure

This study was conducted to develop a noninvasive marker of hepatic microvesicular lipid accumulation (MVLA), a histopathological effect currently diagnosed in humans following liver biopsy. MVLA is detected in animal studies of chemicals and drugs and occurs in some humans exposed to chemicals or pharmaceuticals. Because MVLA is a reversible histopathology, early detection of MVLA using a noninvasive method, could aid clinicians in the treatment of patients taking drugs that are known to induce this injury. Isoniazid (INH) was selected as a model compound for this investigation, because MVLA occurs in tuberculosis (TB) patients treated with a combination therapy, which includes INH. This study used male rats dosed daily with INH at 0, 10, or 300 mg/kg/day for up to 8 days. Urine, blood, and liver were obtained following 1 and 8 days. NMR metabolomics of urine revealed markers that correlated (100%) with the findings of MVLA in the right, left, and median liver lobes in 4/9 rats administered the high dose of INH for 8 days. Metabolomics of liver extracts also revealed markers that correlated with the MVLA injury. Serum enzymes that are clinically used to assess liver injury were not consistently correlated to the findings of MVLA. Metabolite changes consistent with the presence of MVLA correlated with interruptions in inositol, carbohydrate, glycerolipid, and glyoxylate metabolism. This study reveals markers that could find pre-clinical use, provides insights into mechanisms involved in MVLA, and demonstrates the need for the validation of noninvasive MVLA markers in human patients.     

Relative efficacy of short-term tests in detecting genotoxic effects of cadmium chloride in mice in vivo

The ability of intraperitoneally administered cadmium chloride (0.42-6.75 mg/kg) to induce genotoxic damage in somatic and germ cells of mice was evaluated using chromosomal aberrations, sister-chromatid exchanges (SCE), micronuclei and sperm-head abnormalities as end-points. A significant increase in the frequency of chromosomal aberrations and SCEs was observed in almost all treated series when compared to the negative control. Micronucleus formation in polychromatic erythrocytes was not affected significantly except at the highest concentration used (6.75 mg/kg). Significant differences were observed in the frequency of sperm with abnormal head morphology at all concentrations tested except the lowest one. The clastogenic effects of cadmium chloride in both somatic and germinal cells are found to depend directly on the concentrations used.     

Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.     

Clastogenic effect of pesticides in peripheral lymphocytes of cotton-field workers.

We studied clastogenic effects in peripheral lymphocytes of cotton-field workers who were exposed to different pesticides. All the cells were grown in RPMI 1640 medium for 48 and 72 h. The type of aberrations observed in the exposed group are gaps, breaks, dicentrics, exchanges, rings and polyploidy. The frequency of total chromosomal aberrations increased significantly in male pesticide applicators when compared to controls. A significant decrease in mitotic index was observed in the exposed group as compared to the control group. The 48-h cultures showed high incidence of chromosomal aberrations and low mitotic index when compared to 72-h cultures. The difference in chromosomal aberrations between 48- and 72-h cultures was not significant. 24 out of 26 individuals showed ill health effects such as severe giddiness and nervous disorders.