Assessment of local genotoxic effects of formaldehyde in humans measured by the micronucleus test with exfoliated buccal mucosa cells

Volunteers (10 women, 11 men) were exposed to formaldehyde (FA) vapors for 4h per day over a period of 10 working days under strictly controlled conditions. Exposure varied randomly each day from constant 0.15 ppm up to 0.5 ppm with four peaks of 1.0 ppm for 15 min each (13.5 ppm h cumulative exposure over 10 working days). FA was masked on four days by co-exposure to ethyl acetate. During exposure, subjects had to perform bicycle exercises (about 80 W) three times for 15 min. Buccal smears were prepared 1 week before the start of the study (control 1), at the start of the study before the first exposure (control 2), at the end of the exposure period of 10 days and 7, 14 and 21 days thereafter. Two thousand cells per data point were analyzed for the presence of micronuclei (MN) and the frequency of MN per 1000 cells was determined on slides coded by an independent quality-assurance unit. No significant increase in the frequency of MN was measured at any time point after the end of the exposure. Twenty-one days after the end of the exposure MN frequencies were significantly lower in comparison with control 1. This study, which was performed under GLP-like conditions, clearly indicates that FA does not induce MN in buccal mucosa cells after peak exposures up to 1 ppm and a cumulative exposure of 13.5 ppm h over 2 weeks.     

Cytogenetic evaluation of arsenic trioxide toxicity in Sprague-Dawley rats

Acute exposure to arsenic trioxide has been reported to induce death and/or multiple organ damage with symptoms including nausea, vomiting, diarrhea, gastrointestinal hemorrhage, cerebral edema, tachycardia, dysrhythmias and hypovolemic shock. Its toxic effects are due to its ability to bind to sulfhydryl groups of proteins and to inhibit energy production. Although the chronic exposure to arsenic trioxide has been linked to various types of cancer, such as skin, liver, lung, bladder and kidney neoplasms, studies of its carcinogenic potential in animals have not been conclusive. In this study, we investigated the genotoxic potential of arsenic trioxide in bone-marrow cells obtained from Sprague-Dawley rats; using chromosomal aberrations (CA), mitotic index (MI) and micronuclei (MN) formation as the toxicological endpoints. Four groups of six male rats each, weighing approximately 60+/-2 g per rat, were injected intraperitoneally, once a day for 5 days with doses of 5, 10, 15 and 20 mg/kg body weight (BW) of arsenic trioxide dissolved in distilled water. A control group was also made of six animals injected with distilled water without chemical. All the animals were sacrificed at the end of the treatment period. Chromosome and micronuclei preparation was obtained from bone-marrow cells following standard protocols. Arsenic trioxide exposure significantly increased the number of structural chromosomal aberrations, the frequency of micronucleated cells and decreased the mitotic index in treated groups when compared with the control group. Our results demonstrate that arsenic trioxide has a clastogenic/genotoxic potential as measured by the bone-marrow CA and MN tests in Sprague-Dawley rats.     

In vitro evaluation of the cytotoxic and mutagenic potential of the 5-aminolevulinic acid hexylester-mediated photodynamic therapy

Photodynamic therapy (PDT) of tumors with 5-aminolevulinic acid hexylester (h-ALA) causes photo-oxidative reactions in treated tissues. In order to study cytotoxic and/or mutagenic effects, cells of the tumor cell line RPMI 2650 as well as fibroblasts of the cell line WS 1 were given photodynamic treatment in vitro. The cells were photosensitized with a 1mM h-ALA-medium solution for 5h and illuminated with different light doses (0.5, 1.0, 1.5 and 2.0 J/cm2) using red light (633+/-20 nm). PDT-induced cytotoxic effects were determined by measurement of the mitotic index (MI) and the nuclear division index (NDI). Chromosome aberrations (CA) and micronuclei (MN) were recorded to study mutagenicity. After treatment of the photosensitized RPMI 2650 cells with a light dose of 2.0 J/cm2, the MI was significantly decreased to 16.9 per thousand in comparison with that of the h-ALA control (33.8 per thousand ). In photosensitized WS 1 cells, light doses up to 2.0 J/cm2 showed no significant effect. The NDI of photosensitized RPMI 2650 cells was significantly decreased by light doses from 1.0 to 2.0 J/cm2, whereas no significant effect was seen in WS 1 cultures. Thus, h-ALA-PDT only induced desirable cytotoxic effects in tumor cells, but not in the fibroblasts. After application of light doses from 0.5 to 2.0 J/cm2, photosensitized RPMI 2650 cultures showed CA in 7.0-7.5% of the metaphases, which was not a significant increase (h-ALA control: 5.5%). In WS 1 cultures metaphases containing CA varied non-significantly from 5.0 to 7.5%. The MN rates were approximately the same in illuminated RPMI 2650 cultures and in the corresponding h-ALA control (4.4-4.9 per thousand ). The MN rates of the illuminated WS 1 cultures also varied non-significantly from 4.5 to 5.0 per thousand in comparison with the h-ALA control (5.5 per thousand ). In the mutagenicity tests the h-ALA-PDT had no significant effect, neither on the tumor cells nor on the fibroblasts. In addition to the cytogenetic analysis, spectral karyotyping (SKY) was used to characterize the cell lines and gain more detailed information on possibly PDT-induced CA. The SKY evaluation also showed no significant increase of the CA rate, but confirmed the result of the CA test. Thus, within the scope of the experiments performed, a mutagenic potential of the h-ALA-PDT can be excluded.     

Formation of micronuclei and of clastogenic factor(s) in patients receiving therapeutic doses of iodine-131

The micronucleus (MN) assay in peripheral blood lymphocytes was applied to assess the genotoxic potential of a single dose of iodine-131 (131I) given to six patients for ablation of thyroid remnants after total thyroidectomy. Lymphocytes were taken at various times after 131I therapy (from 2 to 180 days), and evaluated for the presence of MN in the binucleated cells identified after blocking cytokynesis with cytochalasin B. The presence of ultrafiltered, low-molecular weight, clastogenic factor(s) (CFs) in the plasma of 11 patient undergoing 131I therapy was also sequentially assessed.A significantly increased MN frequency was observed in lymphocytes of patients as soon as the first sampling time (2 days after 131I therapy), multifactor analysis of variance (MANOVA): P<0.0001, peaking at day 7 at almost four-fold the spontaneous frequency observed in the pre-therapy samples. MN frequency slowly declined thereafter, reaching the baseline levels at the 180-day time point. When tested against peripheral blood lymphocytes from a healthy donor, the ultrafiltered CFs obtained from 11 patient's plasma induced a significant increase of the MN frequency peaking at day 15. Thereafter, a slow MN frequency decline was observed and the baseline frequency was reached after 180 days. A significant relationship was found between the MN frequency observed in lymphocytes of patients after 131I therapy and the genotoxic CFs activity present in their plasma (P=0.019).These findings suggest that 131I induces a significant increase in the MN frequency of peripheral blood lymphocytes, as well as the formation of transferable CFs which persist for at least 60 days after administration of the radionuclide. The presence of these CFs might be responsible of chromosome aberrations often observed in cultured lymphocytes following X-ray exposure. The possibility of reducing the genotoxic activity of radionuclide therapy by chemoprevention of CFs with antioxidant drugs remains to be explored.     

Zinc opposes genotoxicity of cadmium and vanadium but not of lead

Protection by essential metals against the genotoxic effects of toxic elements is an open question. Here, human Hs27 dermal fibroblasts and B-mel melanoblasts were exposed for 10 days to (1 μM) zinc (Zn) or copper (Cu) or selenium (+ 4, Sei; + 6, Sea). Afterwards, cells were exposed for 3 days to subtoxic concentrations of lead (Pb, 100 μM) or vanadium (+ 5, V, 2 μM) or cadmium (Cd, 3 μM), slightly reducing, by themselves, cell proliferation and unaffecting cell viability and apoptosis. Genotoxic damage was evaluated by cytokinesis-block micronucleus assay (CBMN) and single cell gel electrophoresis (Comet assay, CA). CBMN and CA were preliminarly assessed following 3, 10 and 30 days of exposure to the above concentrations of Pb, V and Cd: Pb induced micronuclei (MN) formation in both Hs27 and B-mel cells, without determining direct DNA damage (as shown by CA); V did not reveal genotoxic effects on fibroblasts (as shown by CBMN and CA) but increased the frequency of MN and comets in melanoblasts; Cd induced a great number of MN and comets in fibroblasts but not in melanoblasts; all these effects did not differ after 3, 10 or 30 days of exposure to such elements so that Hs27 and B-mel cells were exposed to Pb,V and Cd for 3 days following pretreatment with (1 μM) Zn, Cu, Sei or Sea. By itself, the 10 day-exposure to (1 μM) Zn, Cu, Sei or Sea did not affect cell proliferation, viability, apoptosis and formation of MN or comets in either Hs27 or B-mel cells. Only Zn significantly reduced the Cd- and V-induced MN and comet formation in fibroblasts and melanoblasts, respectively; in these cells, however, Zn did not affect the Pb-induced MN formation. These results emphasize the role of Zn, in respect to other essential metals, in opposing the genotoxic effects of cancerogenic (Cd) or potentially cancerogenic elements (V).     

Subpopulations of mononuclear leukocytes associated with inhibition of Ehrlich ascites tumor growth by treatment with Bothrops jararaca venom

Snake venoms have been used as antineoplastic substances in several experimental models. We demonstrated in previous studies that Bothrops jararaca venom (BjV) induces inhibition of Ehrlich ascites tumor (EAT) growth accompanied by an increase of mononuclear (MN) leukocytes in all groups inoculated with EAT and/or venom. The objective of the present study was to characterize the subpopulations of MN leukocytes involved in the inhibition of EAT growth by treatment with BjV. Swiss mice were inoculated with 1.0x10(3) EAT cells by the intraperitoneal route and treated with 0.4 mg/kg of BjV by the same route (Group TV). Treatment was started 24 h after tumor cell inoculation and consisted of five intraperitoneal injections performed at 72 h intervals. After 2, 8 and 14 days, groups of animals were sacrificed and the number of B, TCD4 and TCD8 lymphocytes, macrophages and natural killer cells present in the peritoneal cavity was determined by flow cytometry. The control group consisted of animals inoculated with EAT and treated with 0.1 ml of saline under the same conditions as the experimental group (Group T). Two additional control groups consisted of animals not inoculated with EAT and treated with saline or venom. Data were analyzed statistically by the Kruskal-Wallis non-parametric test for independent samples. On the 2nd and 8th day we observed a difference between groups T and TV (group T > group TV) for all cell types, except natural killer cells, that only differed on the 2nd day. However, on the 14th day there was no difference in MN cells among groups. These data suggest that the inhibition of EAT is related to the toxic action of BjV on tumor cells and/or to the proteolytic effect of the venom on the mediators produced by the cells for growth modulation.     

Study of a rat skin in vivo micronucleus test: data generated by mitomycin C and methyl methanesulfonate.

We have developed an in vivo micronucleus (MN) test that uses rat skin as the target organ. Sample preparation involves cold-treating the epidermis with trypsin, peeling it off with a fine forceps, treating it in hypotonic solution, and staining it with acridine orange (A.O.). We evaluated the assay using mitomycin C (MMC) and methyl methanesulfonate (MMS) as model clastogens, applying them as single and repeat treatments. Both chemicals induced a significant, dose-dependent increase in MN frequency in basal cells. One treatment per day for 3 days was optimal for MN induction.     

Response of S 180 murine tumor to bleomycin in combination with radiation and hyperthermia using micronucleus assay: a multimodality approach for therapeutic augmentation

Response of a transplantable tumor, S180, grown intradermally in inbred Balb/c mice, was assessed by using micronucleus assay after treating the solid tumors with bleomycin (BLM), radiation (RT) and hyperthermia (HT) vis-a-vis multimodality approach. The frequency of micronuclei (MN) though did not vary greatly during the one week of observation in untreated tumors, it significantly increased in the drug and RT groups at 24 hr post-treatment. However, MN frequency was non-significant in the HT group from the control. A drug dose dependent linear increase in the frequency of MN induction was evident in 10, 15 and 20 mg/kg body weight BLM alone treated groups. Combination of radiation with BLM or HT further increased the MN counts in the bimodality groups. But, MN induction at 24 hr post-treatment in the trimodality group (BLM + RT + HT) was non-significant from that of the bimodality treatments. However, the tumors treated with trimodality treatment presented severe tumor necrosis, indicating increased cell loss, and resulting in immediate tumor regression. In all the bi-modality groups MN counts though declined 3 or 5 days post-treatment, the values remained significantly higher than the control, on day 7 post-treatment. Micronucleus assay could be used as a predictive parameter for the assessment of post-irradiation tumor regression response. However, the tumor response assessment with MN assay alone may not be sufficient and the role of other parameters, such as apoptosis and necrosis, in immediate tumor regression, especially radiosensitive/thermosensitive tumors can not be ignored while taking multimodality approach into consideration for cancer therapy.     

Hormone replacement therapy: one-year follow up of DNA damage

Although hormone replacement therapy (HRT) may offer considerable benefits for menopausal women, the potential cancer risk may limit its use. This work aimed at assessing whether HRT is able to induce DNA damage in postmenopausal women monitored by the micronucleus (MN) test, which provides a reliable biomarker of genotoxicity and cancer risk assessment. A group of 16 healthy women (non-smokers) in spontaneous menopause were given oral estradiol (2 mg oral micronized 17-beta estradiol daily) for 1 month, followed by a 30-day wash-out period and a transdermal treatment with 17-beta estradiol (1.5 mg gel daily) during 1 month. Oral intake of dihydrogesterone (10 mg/day for 12 days/month) was cyclically combined with oral or transdermal estradiol during the next 9 months. Venous blood samples were collected before the treatment, and after 1, 3, 6 and 12 months of therapy. Slides were scored blind and MN frequency was evaluated as number of micronuclei per 1000 binucleated cells. The baseline plasma levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) were simultaneously measured. The means of MN frequency were 18.2+/-1.6, 18.6+/-2.1, 14.8+/-1.5, 15.9+/-1.0 and 17.7+/-1.3 for samples collected before and at 1, 3, 6 and 12 months, respectively. The MN frequencies at every sampling time did not statistically differ from the basal values. In addition, no statistically significant associations between MN values and hormone levels of E2 and FSH were observed throughout the entire study. This study shows the absence of any significant increase of MN frequencies in women undergoing oral and/or transdermal HRT, sequentially monitored for up to 12 months of therapy.     

Stage-dependent effects of 20-hydroxyecdysone on DNA synthesis of corpus allatum cells in the silkworm,Bombyx mori

DNA synthesis in cells of the corpus allata (CA) of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU); developmental changes during the 3rd, 4th, and last larval instars and effects of 20-hydroxyecdysone treatment were examined. During both the 3rd and 4th larval instars, the number of DNA-synthesizing cells fluctuated, and relatively low levels were observed during the middle stages. On day 0 of the last larval instar, the number of DNA-synthesizing cells per gland was 9.2, which then increased on day 1 and remained at levels ranging from 12.9 and 16.9 cells per gland. A major peak level (28 BrdU-labeled cells per gland) occurred on day 8, two days after larvae entered the wandering stage. When last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 0 or 1, the number of DNA-synthesizing cells dramatically decreased to very low levels and these low levels were maintained throughout the remainder of the instar. However, no effect was observed when last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 3, indicating the stage-specific action of 20-hydroxyecdysone. The mechanism by which 20-hydroxyecdysone treatment inhibits DNA synthesis of CA cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. It was found that the decrease in responsiveness of DNA synthesis of CA cells of 20-hydroxyecdysone-treated larvae to stimulation by growth factors from hemolymph may have been, at least in part, responsible for the indirect inhibitory effects of 20-hydroxyecdysone.     

5℃低温对转SOD或POD基因烟草影响的研究

对盆栽9叶龄的3个烟草近等基因系进行5℃低温处理。测定处理2d、4d和6d以及处理6d后恢复生长2d和4d烟草叶片的生理生化指标,观察处理2d、4d和6d后恢复生长7d烟草的生长情况。结果表明随着低温处理时间的延长,细胞质膜透性、丙二醛含量、可溶性蛋白质和糖含量均升高;SOD活性则开始下降随后升高,但始终低于对照;低温造成株高、叶片数和生物量显著下降。     

Electrophysiological and morpho-histochemical study of action of adrenalectomy on hippocampal neurons

Chronic insufficiency of adrenal hormones is a pathology leading to brain dysfunction. By electrophysiological approach there were studied mechanisms of adaptation of neural networks to chronic hormonal deprivation by extracellular recording of the single spike activity of hippocampal neurons (HN), which was caused by high-frequency stimulation of the entorhinal cortex (EC) in rats with unilateral removal of adrenal (adrenalectomy—AE). The balance of excitatory and inhibitory responses recorded in intact rat HN underwent characteristic changes in dynamics of development of neurodegeneration: the inhibitory responses dominating in norm were decreased in all AE terms (from 42% to 25% by 18 weeks). On the contrary, the minimal percentage of excitatory responses in norm was sharply increased at 25–27 days after AE (from 17% to 60%), by indicating a possible increase in cholinergic neurotransmission. The high level of the mean frequency of peristimulus spiking was recorded from the 25–27th day to the 18th week after AE, which indicates the presence of the high level of glutamate or the expressed activation of NMDA receptors. On the whole, the ratio of the excitatory/inhibitory HN responses suggests discrepancy of neural activity in EC HN chains under the AE conditions. Histochemical analysis has shown an increased sensitivity to AE in the CA1 area neurons. After disruption of neuronal structure by the 5th day of AE, 25–27 days after AE, proliferation of cellular elements was observed in the CA1 area, due which the complete filling of the “devastated” areas of hippocampus and a sharp enhancement of phosphatase activity occurred by 8-10 weeks in neuronal nuclei of the dentate gyrus. By 18 weeks after AE, most neurons in the CA1 area were subjected to chromatolysis with a fall of phosphatase activity. The presented make certain contribution to understanding of mechanisms of control of cognitive function and brain plasticity with interconnection with hormonal factors.     

Kinetics and mechanisms of macrophage activation by Corynebacterium anaerobium

Kinetics and mechanisms of macrophage activation by heat-killed Corynebacterium anaerobium (CA) in mice were investigated. The carbon clearance test revealed that the function of the reticuloendothelial system rose markedly on the 4th day after a single intravenous injection of CA and continued in a highly enhanced state until the 14th day. This activity declined gradually and dropped to a normal level around the 21st day. On the other hand, both lysosomal enzymes, beta-glucuronidase and acic phosphatase, of peritoneal macrophages decreased after the CA injection and then recovered, taking an almost inverse course to the function of the reticuloendothelial system. These results might be attributable to possible extracellular secretion of the lysosomal enzymes in accordance with macrophage activation by CA. A remarkable cytotoxicity of peritoneal macrophages, examined in vitro against L 929 cells, was detected on the 4th day following intraperitoneal administration of CA. It was maintained up to the 14th day and then declined rapidly. The mechanisms of macrophage activation by CA were also examined in vitro. CA-homogenate, heat-killed CA disrupted with an ultrasonicator, directly activated thioglycollate-induced macrophages. The macrophages were aslo activated by simultaneous treatment with both CA-homogenate and CA-sensitized spleen cells. Furthermore, the supernatant obtained from the culture of CA-sensitized spleen cells with CA-homogenate was capable of inducing activation of the macrophages. Conversely, the culture supernatant of spleen cells from CA-immunized athymic nude mice with CA-homogenate was unable to activate them. It was ascertained from the above-mentioned results that macrophages are activated initially by direct action of CA in a nonspecific way and subsequently by a soluble factor elaborated by CA-sensitized lymphocytes in an immunological way.     

Analysis of micronuclei, histopathological changes and cell proliferation in nasal epithelium cells of rats after exposure to formaldehyde by inhalation

The frequencies of micronuclei (MN), histopathological changes and cell proliferation were determined in the nasal epithelium of male Fischer-344 rats after exposure to formaldehyde (FA) by whole-body inhalation for four weeks (6h/day, 5 days/week). Groups of 12 rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15ppm. The micronucleus test (MNT) was performed with nasal epithelial cells prepared from six animals per group. Two thousand cells per animal were analysed for the presence of MN. The other six rats per group were subcutaneously implanted with osmotic pumps containing 5-bromo-2'-deoxyuridine (BrdUrd), three days prior to necropsy. Paraffin sections were made from the nasal cavity (four levels) of these animals for histopathology and cell-proliferation measurements. The frequency of cells with MN was not increased in any of the groups. However, there was also no induction of MN in nasal cells of rats exposed to a single dose of cyclophosphamide (CP, 20mg/kg) by gavage and analysed 3, 7, 14 or 28 days after the treatment. In contrast, nasal epithelial cells from rats exposed to 10 or 15ppm FA vapour showed clear site-specific pathological changes (focal epithelial degeneration, inflammation and squamous metaplasia) in a decreasing gradient (anterior to posterior). Analysis of slides after anti-BrdUrd antibody staining clearly indicated increased cell proliferation (unit length labelling indices, ULLI) after exposure to 6ppm and higher. No treatment-related effects were measured after exposure to 0.5, 1 and 2ppm. When comparing the cell-proliferation rate of normal epithelium with that of directly adjacent metaplastic epithelium, no consistent pattern was found: depending on the location, cell proliferation of normal epithelia was either higher or lower. Our results support previous findings demonstrating local cytotoxic effects in the nose of rats after inhalation of FA. However, induction of MN in the nasal epithelium as an indicator of a mutagenic effect was not seen. Because only limited experience exists for the MNT with rat nasal epithelial cells, this result has to be interpreted with great care. The contribution of mutagenicity to FA's carcinogenicity in rat nasal epithelium remains unclear.     

Cytogenetic effects of extremely low frequency magnetic field on Wistar rat bone marrow

In this study, the genotoxic and cytotoxic potential of extremely low frequency magnetic fields (ELF-MF) was investigated in Wistar rat tibial bone marrow cells, using the chromosomal aberration (CA) and micronucleus (MN) test systems. In addition to these test systems, we also investigated the mitotic index (MI), and the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs). Wistar rats were exposed to acute (1 day for 4h) and long-term (4h/day for 45 days) to a horizontal 50Hz, 1mT uniform magnetic field generated by a Helmholtz coil system. Mitomycin C (MMC, 2mg/kg BW) was used as positive control. Results obtained by chromosome analysis do not show any statistically significant differences between the negative control and both acute and long-term ELF-MF exposed samples. When comparing the group mean CA of long-term exposure with the negative control and acute exposure, the group mean of the long-term exposed group was higher, but this was not statistically significant. However, the mean micronucleus frequency of the longer-term exposed group was considerably higher than the negative control and acutely exposed groups. This difference was statistically significant (p<0.01). The results of the MI in bone marrow showed that the averages of both A-MF and L-MF groups significantly decreased when compared to those in the negative control (p<0.001 and p<0.01, respectively). No significant differences were found between the group mean MI of A-MF exposure with L-MF. We found that the average of PCEs/NCEs ratios of A-MF exposed group was significantly lower than the negative control and L-MF exposed groups (p<0.001 and p<0.01, respectively). In addition, the group mean of the PCEs/NCEs ratios of L-MF was significantly lower than negative control (p<0.01). We also found that the MMC treated group showed higher the number of CA and the frequency of MN formation when compared to those in all other each groups (p-values of all each groups <0.01) and also MMC treated group showed lower MI and the PCEs/NCEs ratios when compared to those in all other each groups (p-values of all groups <0.01). These observations indicate the in vivo suspectibility of mammals to the genotoxicity potential of ELF-MF.     

Role of vitamin E-acetate on cisplatin induced genotoxicity: An <Emphasis Type="Italic">in vivo</Emphasis> analysis

Vitamin E has generated immense interest because of its potential of being an antioxidant, a neuroprotector, and a protector against atherosclerosis, carcinogenesis and cardiovascular disease. However, the prooxidant chemistry of vitamin E cannot be ignored since it is related to the generation of peroxyl radicals. In the present study, 125, 250 and 500 mg/kg of vitamin E-acetate (VE) administered intraperitoneally (i.p.) to Balb/C mice significantly induced 6%, 8% and 11.33% (control value=2.33%) of chromosome aberrations (CA) and 0.88%, 1.39% and 1.81% (control value=0.61%) of micronucleus (MN), following 24 hour of treatment in the bone marrow cells. In the germ cells, VE did not induce any sperm head abnormality (SHA) after 35 days of exposure. Most importantly, it has been observed that pre-treatment with VE significantly reduces CA, MN, and SHA induction by chemotherapeutic drug cisplatin (CIS). Our findings suggest that lone treatment with VE induce genotoxicity in somatic cells after 24 and 48 hours of exposure but not in germ cells after 35 days of exposure, whereas pre-treatment with VE reduces CIS induced genotoxicity as well as cytotoxicity. There exists a thin line of difference on the behavioral transition of VE when acting alone and when acting with a drug.     

Genotoxic effects of potassium bromate on human peripheral lymphocytes in vitro

The aim of this study was to investigate the genotoxic effects of potassium bromate, which is used as a bleaching agent in flour, on human peripheral blood lymphocytes in vitro by sister chromatid exchange (SCE), chromosomal aberrations (CA) and micronucleus (MN) tests, and also to determine whether it has any genotoxic potential for humans. Cells were treated with 400, 450, 500, 550 microg/ml concentrations of potassium bromate for 24 and 48 h. The SCE frequencies showed an increase after both treatment periods, however, the differences between the treated cells and the control groups were found to be statistically significant only for the 48-h treatment. In addition, potassium bromate statistically significantly induced CA after the 24-h and 48-h treatment periods. Strikingly, potassium bromate induced CA as much as the positive control, mitomycin-C (MMC). Furthermore, potassium bromate decreased both the cell proliferation index (PI) and the mitotic index (MI). Although micronucleus formation was induced by potassium bromate during the 24-h treatment period in a dose-dependent manner, only the doses 500 and 550 microg/ml yielded statistically significant results. In contrast, MN formation was significantly induced at all doses during the 48-h treatment period. These in vitro results provide important evidence about genotoxicity of potassium bromate on a human cell culture system.     

Low persistence of radiation-induced centromere positive and negative micronuclei in cultured human cells

The micronucleus (MN) assay is widely used both in genetic toxicology and in the biomonitoring of human populations. Lymphocytes, cell lines, and bone marrow and epithelial cells are usually employed as target systems in such studies. However, little effort has been done to assess the persistence of MN in highly proliferative cells. To study the behaviour of MN containing whole chromosomes or acentric fragments, we have performed a time course experiment on the persistence of gamma-ray (3 Gy) induced MN in a human lymphoblastoid cell line. The frequency and content of MN were analyzed 1, 3, 7, 14, and 56 days after irradiation by pancentromeric fluorescence in situ hybridization (FISH). We observed a clear induction of both centromere positive and negative MN at completion of the first mitotic division. The frequency of both types of MN drastically declined to basal levels 7 days after irradiation with an identical kinetics. We therefore conclude that centromere positive and negative MN are highly unstable upon cell division, indicating that the MN assay could not be a good biomarker of DNA damage induced by acute treatments in highly proliferative cells. The implication of our findings in biomonitoring and in genotoxicity studies is discussed.     

Investigations on the turnover of adrenocortical mitochondria

The effects of chronic administration of ACTH and dexamethasone on the morphology of mitochondria in zona glomerulosa cells of the rat adrenal cortex were investigated by stereological techniques. It was found that the volume of the mitochondrial compartment as well as the surface of the outer and inner mitochondrial membranes were significantly increased or decreased in relation to the number of days of ACTH- or dexamethasone-treatment. In ACTH-administered rats, the average volume of individual mitochondria decreased significantly up to the 6th day of treatment and then showed a conspicuous increase from the 6th to the 15th day, whereas in dexamethasone administered animals this parameter, after a small increase during the first 6 days of treatment, displayed a significant decrease. The number of mitochondria per cell showed a dramatic increase during the first 6 days of treatment with ACTH and continued to increase, but only slightly, with the subsequent treatment. In contrast, this parameter showed a parabolic decrease as a function of the duration of treatment in animals receiving dexamethasone. In the light of evidence showing that dexamethasone blocks ACTH-release, these findings are discussed and interpreted to indicate that ACTH is involved in the maintenance and stimulation of the growth and proliferative activity of mitochondria in rat adrenal zona glomerulosa.     

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.