Induced Micronucleus Formation Research Articles

Induction of micronuclei by zinc in human leukocytes: a study using cytokinesis-block micronucleus assay.

In the present study, we report the results of the capability of zinc chloride for the induction of micronuclei in cultured human leukocytes using cytokinesis-block micronucleus assay. Two concentrations of zinc chloride (1.5 x 10(-4) M and 3.0 x 10(-4) M) were used to evaluate the potential of this zinc salt to induce micronucleus formation. This effect was compared with positive (mitomycin C treated) and negative controls (no salt added). Our results show a significant (p < or = 0.001) increase of micronucleated cytokinesis-blocked cells (MNCBs) in zinc-chloride-treated cells compared to the negative control. Induction of MNCBs was not in a dose-dependent manner for zinc chloride concentrations tested. This report is the first to describe the efficiency of cytokinesis-block micronucleus assay to evaluate the genotoxic effects of zinc salt.

Oxidant Stress as a Marker for Cardiovascular Events

During the past decade, numerous experimental and clinical studies have demonstrated that many common conditions predisposing to atherosclerosis, such as hypercholesterolemia, hypertension, diabetes, and smoking, are associated with a reduced vascular availability of nitric oxide (NO•). Nitric oxide not only produces vasodilation but also has potent antiatherogenic properties. These properties include inhibition of platelet aggregation, prevention of smooth muscle cell proliferation, reduction of lipid peroxidation, and inhibition of adhesion molecule expression. Thus, the loss of NO• observed in these various conditions not only alters vascular tone but also may explain in part why these conditions are risk factors for atherosclerosis. See p 2673 Given this apparent link between loss of nitric oxide and atherosclerosis, several groups have been interested in the concept that endothelium-dependent vasodilation, a surrogate for NO• bioavailability, may predict cardiovascular events. Indeed, Suwaidi et al1 followed 157 patients with mildly diseased coronary arteries for an average of 28 months and observed cardiac events only in the patients with the lowest tertile of coronary vasodilation to acetylcholine. Similarly, in a study of 147 patients, Schachinger et al2 used 3 different stimuli for endothelial release of NO: acetylcholine, cold pressor testing, and increased blood flow. The authors showed that responses to each of these stimuli were independent predictors of cardiovascular events during a follow-up period of ≈8 years. Perticone et al3 also demonstrated that endothelial dysfunction in the forearm circulation predicts cardiovascular events in hypertensive subjects. There have been several explanations for why the various risk factors impair endothelial function. One that has received substantial attention is increased production of reactive oxygen species within the vessel.4 In particular, superoxide (O2•-) reacts rapidly with NO•, resulting in the formation of the peroxynitrite anion and loss of NO• …

The role of adrenal corticosteroids in induction of micronuclei by morphine

It has previously been shown that morphine can increase the frequency of micronucleated splenocytes when administered to mice, but not when cells are exposed to the opiate in vitro. Morphine treatment is also known to increase circulating levels of glucocorticosteroids, which have been reported to produce genetic damage in vivo and in vitro. In order to determine whether adrenal hormones might mediate the genotoxic effects of morphine, adrenalectomized and sham-operated mice were treated with morphine sulfate. In sham-operated animals administration of morphine produced a dose-related increase in the frequency of micronucleated cells, whereas adrenalectomy abolished the effect. When plasma from morphine-treated mice was used to supplement growth medium of untreated splenocytes, the frequency of micronucleated cells increased, an effect partially blocked by the steroid antagonist RU 486. The N-methylmorphine, which does not stimulate the release of corticosterone from adrenal glands, induced micronuclei formation in splenocytes, and administration of metyrapone, an inhibitor of corticosterone biosynthesis, blocked the morphine-induced increase in corticosterone secretion, but had no effect on the frequency of micronuclei formation. These results indicate that basal levels of glucocorticosteroids are required for induction of micronuclei by morphine in murine splenocytes, but activation of the hypothalamo–pituitary–adrenal (HPA) axis by morphine does not contribute to the observed response.

Radiobiologic significance of apoptosis and micronucleation in quiescent cells within solid tumors following γ-ray irradiation

Purpose: To determine the frequency of apoptosis in quiescent (Q) cells within solid tumors following γ-ray irradiation, using four different tumor cell lines. In addition, to assess the significance of detecting apoptosis in these cell lines. Methods and Materials: C3H/He mice bearing SCC VII or FM3A tumors, Balb/c mice bearing EMT6/KU tumors, and C57BL mice bearing EL4 tumors received 5-bromo-2′-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received γ-ray irradiation at a dose of 4–25 Gy while alive or after tumor clamping. Immediately after irradiation, the tumors were excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 hours after irradiation, tumor cell suspensions obtained in the same manner were fixed. The apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU. Results: In total cells, SCC VII, FM3A, and EMT6/KU cells showed reasonable relationships between MN frequency and surviving fraction (SF). However, fewer micronuclei were induced in EL4 cells than the other cell lines. In contrast, a comparatively close relationship between apoptosis frequency and SF was found in total cells of EL4 cell line. Less apoptosis was observed in the other cell lines. Quiescent tumor cells exhibited significantly lower values of MN and apoptosis frequency probably due to their large hypoxic fraction, similar to total tumor cells on clamped irradiation. Conclusion: γ-ray irradiation induced MN formation in SCC VII, FM3A, and EMT6/KU tumor cells, and the apoptosis was marked in EL4 cells compared with the other cell lines. Our method for detecting the Q cell response to γ-ray irradiation using P cell labeling with BrdU and the MN frequency assay was also applicable to apoptosis detection assay.

Influence of metabolic activation on the induction of micronuclei by antihypertensive drugs in L929 cells.

The influence of metabolic activation on the genotoxic activity of the antihypertensive drugs hydralazine and dihydralazine was investigated. An in vitro micronucleus test for estimating the genotoxic activity of these drugs was used. The results obtained indicated that hydralazine and dihydralazine induce micronuclei formation in L929 cells. When L929 cell cultures were treated with drugs together with liver membrane fraction (S9 fraction) from polychlorinated biphenyl (Aroclor 1254) induced rat liver, the number of micronucleated cells decrease, however, almost to the level found in control cultures. The experiments with modified S9 mix allow the conclusion that the antioxidant enzymes catalase and superoxide dismutase present in S9 liver fraction play a role in the protection of cells from the genotoxic action of hydralazine and dihydralazine.

Effect of dehydrated carrot on the antioxidant enzymes and micronuclei formation in rats challenged with dimethylhydrazine

The modulation of detoxifying enzymes; catalase, glutathione peroxidase (GSH-Px), glutathione reductase (GSSGR), glutathione-S-transferase (GST) in liver and γ-glutamyl transpeptidase (GGT) in kidney elicited by pre feeding of dehydrated carrot at 10 and 20% levels with or without the treatment with dimethylhydrazine (DMH) was studied in rats. The pre-feeding of rats with carrot was found to elevate the hepatic GSH content with concomitant increase in activities of GSH-Px, GSSGR and GST of rats injected with DMH. The DMH — induced increase in TBARS, GGT and micronuclei (MN) was significantly reduced by the ingestion of carrot. It is suggested that short term consumption of carrot has variable effects on the modulation of different detoxifying enzymes and on the DMH — induced micronuclei formation.

In Vivo Genotoxicity of Crystalline Silica as Evidenced by Micronuclei in Pulmonary Alveolar Macrophages: Low-Dose Study

We have previously published results showing time-course data (Leigh et at., 1998) and a dose-response relationship between macrophage micronucleus formation and crystalline silica dose in intratracheally instilled rats at 2.5, 7.5, and 22.5 mg dosage, without an inert dust control (Wang et al., 1997). We here extend this study to low dose (0.025, 0.25, and 2.5 mg crystalline silica) with 2.5 mg TiO2 control. Specific-pathogen-free male Wistar rats were intratracheally instilled with 0.5 ml saline, and 0.025 mg, 0.25 mg, or 2.5 mg crystalline silica (Min-U-Sil 5) and 2.5 mg TiO2 suspended in 0.5 ml saline (5 rats in each group). Five days after instillation, rats were sacrificed and 10 ml of bronchial alveolar lavage fluid was obtained. A 100-μ1 volume was placed on slides by Cytospin centrifugation, stained with Diff-Quik, and 1000 macrophages were scored for micronuclei (defined by diameter < half main nucleus; same staining; round shape and complete separation). Micronucleus incidence was significantly elevated (p < .01) at the lowest crystalline silica dose compared with saline control. There was a dose-response relationship with crystalline silica exposure. Numbers (mean ± SEM) of micronucleated macrophages per 1000 macrophages scored were 1.5 ± 0.5 (saline), 3.3 ± 0.3 (0.025 mg crystalline silica), 7.1 ± 0.4 (0.25 mg crystalline silica), 10.1 ± 0.3 (2.5 mg crystalline silica), and 0.9 ± 0.3 (2.5 mg TiO2). We conclude that intratracheal instillation of low doses of crystalline silica can induce micronucleus formation in alveolar macrophages in a dose-related manner. We further believe that this is not a nonspecific effect, consistent with crystalline silica being a genotoxic carcinogen.

Genotoxicity testing of potassium canrenoate in cultured rat and human cells.

Potassium canrenoate (PC), a competitive aldosterone antagonist used as a diuretic and in the treatment of hypertension, was examined for its capacity to produce genotoxic effects in cultured rat and human cells. At subtoxic concentrations (10-90 microM) PC was found to induce a dose-dependent degree of DNA fragmentation, as detected by the Comet assay, and of DNA repair synthesis, as measured by quantitative autoradiography, in primary cultures of hepatocytes from rat and human donors of both genders. In rat hepatocytes both DNA fragmentation and DNA repair were more marked after 3 h than after 20 h exposure and in cultures from females than from males. In human hepatocytes from one male and two female donors, PC caused a similar effect in terms of DNA fragmentation, whereas DNA repair was detected in cultures from only two of the same three donors and was less marked than in rat hepatocytes. A modest but statistically significant increase in micronucleated cells was present in primary cultures of replicating rat hepatocytes exposed to 10 or 30 microM PC for 48 h, the response being, in this case also, more evident in females than in males. In contrast, PC did not induce micronucleus formation in human hepatocytes from two female donors. Any evidence of DNA fragmentation and micronucleus formation was absent in cultured human lymphocytes. Taken as a whole these findings support the hypothesis that hepatocytes activate PC to DNA-damaging reactive species. PC induced the observed genotoxic effects at concentrations close to those produced in humans by the administration of therapeutic doses, but these effects were as a whole more marked in rat than in human hepatocytes. Since PC shares the 17-hydroxy-3-oxopregna-4,6-diene structure with cyproterone acetate, chlormadinone acetate and megestrol acetate, previously found to be genotoxic to both rat and human hepatocytes, the potential carcinogenic hazard of this type of steroids cannot be neglected.

Induction of micronuclei in V79 cells by fractions of roofing asphalt fume condensate

More than 50,000 workers in the United States are exposed to roofing asphalt fumes that may pose genotoxic and potential carcinogenic hazards. The Type III roofing asphalt is most frequently used in roof-application. Results of our previous studies showed that fume condensates of Type III roofing asphalts induced micronuclei (MN) in vitro in cultured V79 cells and DNA adduct formation in vivo in rat lung cells. In this study, the genotoxicity of whole fume condensates (WFC) of Type III roofing asphalt and its five chemical fractions (A, B, C, D and E) was determined by the micronucleus assay using V79 cells. Linear regressions were determined for the dose response of MN frequencies and percent of binucleated and multinucleated cells (MTC) following the treatment. Results showed that the numbers of micronucleated cells in cultures treated with Type III roofing asphalt WFC and its fractions B, C, D and E were significantly higher than that in the control culture, and that the slopes of the linear regression line for fractions B and C were greater than those for the WFC and fractions D and E. A clear dose response of binucleated cells was also induced by the WFC and fractions B and C. These findings indicate that: (1) WFC and all fractions, except fraction A, induced MN formation in cultured V79 cells; (2) fractions B and C possess the highest genotoxic activity; (3) the roofing asphalt WFC contains chemicals or chemical classes that induce not only chromosomal aberrations but also binucleation in V79 cells.

The plant ribosome inactivating protein saporin induces micronucleus formation in peripheral human lymphocytes in vitro

Saporin belongs to the family of plant enzymes known as ribosome inactivating proteins (RIPs) for their property to depurinate the major rRNA, thus leading to inactivation of ribosomes. In this work we analyzed the genotoxic effects of saporin, purified from root cultures of Saponaria officinalis, by evaluating micronucleus formation and by the quantitative determination of cytosolic histone-associated DNA fragments. Saporin induces micronuclei formation in cultured human lymphocytes in a dose dependent manner; treated lymphocytes show a decrease in cell viability and a concomitant increase in the apoptotic response evidenced by the appearance of cytosolic oligonucleosomes. On the other hand saporin treatment failed to induce sister-chromatid exchange (SCE) at any of the doses tested.

Chemically induced micronucleus formation in V79 cells—comparison of three different test approaches

The in vitro micronucleus test (MNT) is a useful assay for the detection of mutagenic events on both the chromosomal and the genomic level. The main disadvantage for introducing the in vitro MNT into official test guidelines seems to be the disparity of existing protocols. To contribute to the aim of standardisation, three different methodological approaches of the in vitro MNT with V79 cells were compared: the standard assay using an asynchronically growing mixed cell population, the cytokinesis block (CB) assay and a modified MNT, the so-called mitotic shake-off (MSO) method. V79 cells were thus treated with two known aneugens (colcemide and griseofulvin) and two clastogens (mitomycin C and cyclophosphamide) over various time periods. The cultures of the CB assay were additionally exposed to cytochalasin B (Cyt-B), an inhibitor of cell, but not of nucleus division. After treatment, the cells were harvested and analysed for the appearance of micronuclei (MN). All three assays yielded positive results for all test substances. These results support the suitability of the MNT with V79 cells with regard to the ability to detect the genotoxic potential of both clastogens and aneugens independent of the test protocol applied. Thus, all three methods are appropriate for MN detection, but due to the fact that the application of Cyt-B has no advantages for a cell line like V79 in which nearly all cells undergo a normal cell cycle, its use is not recommended.

Induction of micronuclei in vitro by organochlorine compounds in beluga whale skin fibroblasts

Beluga whales ( Delphinapterus leucas) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants and have a high incidence of cancer. Environmental contaminants may be partly responsible for the high cancer incidence observed in this population. DNA damage plays an important role in the development of cancer. The micronuclei (MN) assay was used to test the genotoxic potential of organochlorine (OC) pesticides with and without external metabolic factor in skin fibroblasts of an Arctic beluga whale. Toxaphene, chlordane and p, p′-DDT induced significant ( p<0.05) concentration-response increases of micronucleated cells (MNCs). Statistically significant increases in MNCs, ranging from 1.7- to 5-folds when compared to control cultures, were observed for 0.05, 0.5, 5 and 10 μg/ml toxaphene, 2, 5 and 10 μg/ml chlordane and 10 and 15 μg/ml p, p′-DDT. Presence of exogeneous metabolic factor (S9) completely abolished the MN induction potency of chlordane and p, p′-DDT, and toxaphene induced MN formation at higher concentrations (0.5 μg/ml) than without S9 mix. The ecotoxicological significance of MN induction by low concentrations of toxaphene is unknown and do not imply that toxaphene is involved in the etiology of cancer in St. Lawrence beluga whales. However, because of the known genotoxicity of toxaphene and the long lifespan of beluga whales, it cannot be excluded that toxaphene may pose a long-term genetic hazard to the more contaminated whales of this population.

Prevention of Cyclophosphamide-induced Micronucleus Formation in Mouse Bone Marrow by Indole-3-carbinol

Indole-3-carbinol (I3C) is a glucobrassicin derivative isolated from cruciferous vegetables. In this study, the protective effect of I3C is reported against cyclophosphamide (CP)-induced micronuclei formation in mouse bone marrow cells. The three test doses, namely 500, 250 and 125 mg/kg body weight of I3C provided protection when given 48 hr prior to the single ip administration of cyclophosphamide (50 mg/kg). The efficacy of the test doses of I3C was also evaluated using a lower dose of CP (25 mg/kg body weight). A significant inhibition in micronuclei formation was noticed with I3C at 250 and 125 mg/kg body weight dose. I3C could not induce micronuclei formation at the test doses 500 and 250 mg/kg body weight. I3C, therefore seems to have a preventive potential against CP-induced micronuclei formation in Swiss mouse bone marrow cells.

Cleistanthin B causes G1 arrest and induces apoptosis in mammalian cells.

Cleistanthin B is a potential anticancer agent isolated from the tropical plant Cleistanthus collinus. We have previously shown that cleistanthin B is clastogenic and induces micronuclei formation and chromosomal aberrations. We now show that this compound inhibits DNA synthesis in Chinese hamster ovary (CHO) cells and induces apoptosis in cervical carcinoma (SiHa) cells. Flow cytometric analysis of cleistanthin treated CHO cells revealed that they were blocked in G1. Cervical carcinoma (SiHa) cells exposed to cleistanthin B shrank, rounded up and had condensed chromatin and fragmented nuclei. DNA isolated from cleistanthin treated cells exhibited the characteristic apoptotic ladder when electrophoresed in agarose gels. These results were confirmed by flow cytometry. Etoposide, a structurally similar compound also induced apoptosis in these cells although with a difference. Etoposide induced apoptosis after permitting cells to enter into S phase, while cleistanthin B stopped entry of cells into S phase and subsequently drove them to apoptosis.

Genistein inhibits growth of B16 melanoma cells in vivo and in vitro and promotes differentiation in vitro.

Consumption of soy products has been linked to a reduced mortality and morbidity from a number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumour cell lines in vitro; however, a role of genistein in retarding tumour growth in vivo is less well documented. In this study, in addition to examining the effects of genistein on the growth of murine B16 melanoma cells in vitro, we have examined the effects of feeding a genistein-rich diet on s.c. growth of these tumour cells in mice. In vitro, the melanoma cells showed an increase in sensitivity to genistein with increasing time of exposure, culminating in a 50% growth inhibition (IC50) at 12.5 microM after 7 days. Genistein at 25 microM induced micronucleus formation after 24 hr and at concentrations as low as 2.5 microM induced morphological changes indicative of differentiation. Growth of solid tumours implanted into female C57BL/6J mice was inhibited by 50% when mice were fed genistein for 1 week before and for 1 week after inoculation with B16 melanoma cells. Plasma genistein concentrations at the time of tumour removal were 1.1 microM, which is similar to levels reported in humans consuming diets high in soybeans or soybean products, while control animals had no detectable genistein in plasma. Our results provide additional in vivo evidence suggesting that genistein retards the growth of implanted tumours, adding further to studies suggesting that this isoflavonoid is a biologically active component of soy foods.

Assessment of mutagenic potential of thiram

Thiram is a widely used dithiocarbamate fungicide. In this study, the mutagenicity of thiram was investigated using the micronucleus and dominant lethal tests in Swiss albino mice. A single ip injection of 100 mg thiram/kg body weight, which is the maximum tolerated dose (MTD), significantly induced micronucleus formation in bone marrow cells after 30 and 48 hr of exposure; 50% and 25% of the MTD also induced micronucleus formation after the above time periods. A significant number of dead implants were induced when thiram was given to male mice in the diet at 10% of the oral LD 50 during the whole spermatogenesis cycle (8 wk); this post-implantation loss indicates a dominant lethal mutation.

Quinoline-induced chromosome aberrations and sister chromatid exchanges in rat liver.

Induction of chromosome aberrations and sister chromatid exchanges (SCEs) was studied in hepatocytes of F344 rats exposed in vivo to the hepatocarcinogen quinoline (Q). Hepatocytes were isolated 4-48 hr after a single dose of 200 mg/kg body weight or 24 hr after 28 repeated doses (once a day) of 25-200 mg/kg body weight/day by gastric intubation, and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 hr. A single dose of Q induced chromosome aberrations in up to 22% of metaphase cells, and SCEs with a frequency of up to 1.27 per chromosome 12 hr after the dose, while the control values were 1% and 0.63 per chromosome, respectively. Treatment with 28 repeated doses of Q induced significant chromosome aberrations and SCEs dose-dependently. Cytogenetic damage induced induced in the liver by repeated doses of Q was greater than induced by a single dose. Furthermore, Q induced replicative DNA synthesis in the liver, but failed to induce micronucleus formation in the bone marrow. The noncarcinogen 8-hydroxyquinoline was also examined and found to be essentially non-genotoxic to rat liver. These results show that Q is a genotoxic carcinogen to rat liver and the present method of in vivo cytogenetic assay should be useful for evaluating the genotoxicity of hepatocarcinogens.

Micronucleus formation in V79 cells treated with respirable silica dispersed in medium and in simulated pulmonary surfactant

Chinese hamster lung fibroblasts (V79 cells) were challenged with respirable silica particles using an in vitro genotoxicity assay. Two particle sizes of crystalline quartz and a non-crystalline silica were assayed for induction of micronuclei (MN) in V79 cells. Some of the silica dusts used were pretreated with simulated pulmonary surfactant to model in vivo exposure conditions. The results showed that both crystalline and non-crystalline silica dispered in medium (MEM) induced MN formation in a dose-dependent manner. Crystalline silica was more active in this assay than non-crystalline silica on a mass basis. The results also show that the frequency of micronucleated cells in cultures treated with surfactant-coated silica was not significantly different from that of the non-treated control cultures. These results seem to indicate that silica can cause chromosomal aberrations and/ or aneuploidies in V79 cells; however, pretreatment of silica particles with simulated pulmonary surfactant reduces or delays genotoxicity in this assay.

Combination of the chemotherapeutic agent 5-fluorouracil with an inhibitor of its catabolism results in increased micronucleus induction

The rate limiting step in 5-fluorouracil catabolism is catalyzed by the enzyme dihydropyrimidine dehydrogenase. Since degradation of 5-fluorouracil decreases its efficacy in chemotherapy, the inhibition of its catabolism is a promising tool. We investigated the formation of micronuclei in vitro in mouse L5178Y cells. 5-fluorouracil induced an increase in micronucleus frequency, which could significantly be enhanced by the concurrent application of 2,6-dihydroxypyridine, an inhibitor of dihydropyrimidine dehydrogenase. The 5-fluorouracil concentration necessary to reach maximal genotoxic effects could be reduced to half in the presence of inhibitor 2,6-Dihydroxypyridine alone and the naturally occurring enzyme substrate uracil did not induce micronucleus formation. Combined application of the chemotherapeutic agent 5-fluorouracil and an inhibitor of its could reduce side-effects by lowering the effective dose of the active drug. With this study we provide further support for the usefulness of this concept.

Influence of benzylisothiocyanate and 13- cis-retinoic acid on micronucleus formation induced by benzo[ a]pyrene

The purpose of this study was to investigate the influence of benzylisothiocyanate (BIT) and 13- cisretinoic acid (RA) upon the genotoxic potential of benzo[ a]pyrene (BaP) to induce micronucleus formation in the bone marrow of mice. Eighty-two male mice were divided into 10 groups. One group served as a negative control (olive oil intubation). Four groups received an oral intubation of various concentrations of BIT (15 to 120 mg/kg) and i.p. injections of BaP (185 mg/kg). Another four groups were treated identically, but received RA (20 to 150 mg/kg) in place of BIT. Finally, one group received only i.p. injection of BaP (185 mg/kg). The results showed that both BIT and RA significantly reduced the frequency of micronucleus formation in the bone marrow of the BaP treated animals. BIT was found to be effective at all the tested concentrations. RA was effective only at three of the four tested concentrations (40, 75 and 150 mg/kg). These findings indicate that both BIT and RA may reduce the genotoxic effects of benzo[ a]pyrene in mice under the test conditions utilized.