Induced Micronucleus Frequency Research Articles

Safety assessment of high fructose corn syrup and fructose used as sweeteners in foods

High Fructose Corn Syrup (HFCS) and Fructose (FR) are widely used sweeteners in many foods and beverages. This study aimed at investigating the cytotoxic effects of HFCS (5%–30%) and FR (62.5–2000 μg/mL) using MTT assay in Human Hepatocellular Carcinoma (HepG2) cells, and genotoxic effects of using Chromosome Aberrations (CAs), Sister Chromatid Exchanges (SCEs), Micronuclei (MN) and comet assays in human lymphocytes. HFCS significantly reduced the cell viability in HepG2 cells at between 7.5% and 30% for 24 and 48 h. 30% HFCS caused a very significant toxic effect. FR had a cytotoxic effect in HepG2 cells at all treatments. However, as fructose concentration decreased, the cell viability decreased. HFCS (10%–20%) and FR (250–2000 μg/mL) decreased the mitotic index at higher concentrations. IC50 value was found to be a 15% for 48 h. IC50 value of FR was detected as 62.5 μg/mL for 24 h and 48 h. HFCS significantly increased CAs frequency at 15% and 20%. FR significantly increased the frequency of CAs at 250, 1000, and 2000 μg/mL for 48 h. Both sweeteners increased the frequency of SCEs at all concentrations. HFCS (15% and 20%) and FR (250, 1000, and 2000 μg/mL) induced MN frequency at higher concentrations. HFCS caused DNA damage in comet assay at 10% −30%. FR increased tail intensity and moment at 125-2000 μg/mL and tail length at 62.5, 250 and 500 μg/mL. Therefore, HFCS and FR are clearly seen to be cytotoxic and genotoxic, especially at higher concentrations.

Role of melatonin mediated G-CSF induction in hematopoietic system of gamma-irradiated mice

AimsHematopoietic acute radiation syndrome (H-ARS) can cause lethality, and therefore, the necessity of a safe radioprotector. The present study was focused on investigating the role of melatonin in granulocytes colony-stimulating factor (G-CSF) and related mechanisms underlying the reduction of DNA damage in hematopoietic system of irradiated mice. Main methodsC57BL/6 male mice were exposed to 2, 5, and 7.5Gy of whole-body irradiation (WBI), 30 min after intra-peritoneal administration of melatonin with different doses. Mice were sacrificed at different time intervals after WBI, and bone marrow, splenocytes, and peripheral blood lymphocytes were isolated for studying various parameters including micronuclei (MN), cell cycle, comet, γ-H2AX, gene expression, amino acid profiling, and hematology. Key findingsMelatonin100mg/kg ameliorated radiation (7.5Gy and 5Gy) induced MN frequency and cell death in bone marrow without mortality. At 24 h of post-WBI (2Gy), the frequency of micronucleated polychromatic erythrocytes (mnPCE) with different melatonin doses revealed 20 mg/kg as optimal i.p. dose for protecting the hematopoietic system against radiation injury. In comet assay, a significant reduction in radiation-induced % DNA tail (p ≤ 0.05) was observed at this dose. Melatonin reduced γ-H2AX foci/cell and eventually reached to the control level. Melatonin also decreased blood arginine levels in mice after 24 h of WBI. The gene expression of G-CSF, Bcl-2-associated X protein (BAX), and Bcl2 indicated the role of melatonin in G-CSF regulation and downstream pro-survival pathways along with anti-apoptotic activity. SignificanceThe results revealed that melatonin recovers the hematopoietic system of irradiated mice by inducing G-CSF mediated radioprotection.

Tumour necrosis factor-α (TNF-α) enhances dietary carcinogen-induced DNA damage in colorectal cancer epithelial cells through activation of JNK signaling pathway

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazol [4,5-b] pyridine (PhIP) present in cooked meat are pro-carcinogens and considered to be potential risk factors for CRC. Their carcinogenic and mutagenic effects require metabolic activation primarily by cytochrome P450 1 family enzymes (CYPs); the expression of these enzymes can be modulated by aryl hydrocarbon receptor (AhR) activation and the tumour microenvironment, involving mediators of inflammation. In this study, we hypothesized that tumour necrosis factor-α (TNF-α), a key mediator of inflammation, modulates BaP- and PhIP-induced DNA damage in colon cancer epithelial cells. Importantly, we observed that TNF-α alone (0.1–100 pg/ml) induced DNA damage (micronuclei formation) in HCT-116 cells and co-treatment of TNF-α with BaP or PhIP showed higher levels of DNA damage compared to the individual single treatments. TNF-α alone or in combination with BaP or PhIP did not affect the expression levels of CYP1A1 and CYP1B1 (target genes of AhR signaling pathways). The DNA damage induced by TNF-α was elevated in p53 null HTC-116 cells compared to wild type cells, suggesting that TNF-α-induced DNA damage is suppressed by functional p53. In contrast, p53 status failed to affect BaP and PhIP induced micronucleus frequency. Furthermore, JNK and NF-κB signaling pathway were activated by TNF-α treatment but only inhibition of JNK significantly reduced TNF-α-induced DNA damage. Collectively, these findings suggest that TNF-α induced DNA damage involves JNK signaling pathway rather than AhR and NF-κB pathways in colon cancer epithelial cells.

Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay

Purpose In suspected radiation exposures, cytokinesis-block micronucleus (CBMN) assay is used for biodosimetry by detecting micronuclei (MN) in binucleated (BN) cells in whole blood and isolated peripheral blood mononuclear cell (PBMC) cultures. Standardized harvest protocols for whole blood were published by the International Atomic Energy Agency (IAEA) in 2001 (Technical report no. 405) and 2011 (EPR-Biodosimetry). For isolated PBMC harvest, cytocentrifugation of fresh cells is recommended to preserve cytoplasmic boundaries for MN scoring. However, cytocentrifugation utilizes specialized equipment and long-term cell suspension storage is difficult. In this study, an alternative CBMN harvest protocol is proposed for laboratories interested in culturing PBMCs and storing fixed cells with routine biodosimetry methods. Materials and methods Peripheral blood from 4 males (24, 34, 41, 51 y.o.) and females (26, 37, 44, 56 y.o.) was irradiated with 0 and 2 Gy X-rays. For cells harvested with IAEA 2001 and 2011 protocols, whole blood was used. For cells harvested with our protocol (CRG), isolated PBMCs were used. CRG protocol was validated in DAPI, acridine orange and Giemsa stain, and in three other laboratories. Cytoplasm status, nuclear division index (NDI) and induced MN frequency (MN frequency at 2 Gy – background MN frequency at 0 Gy) (MN/1000 BN) of Giemsa-stained BN cells were compared in IAEA 2001, IAEA 2011, IAEA 2011 + formaldehyde (FA) and CRG protocols. Effects of low and high humidity spreading were evaluated. Results >94% of 1000 BN cells were scorable with clear cytoplasmic boundaries in all donors harvested with CRG protocol. FA addition in IAEA 2011 protocol reduced cell rupture in whole blood cultures, but cell rupture was affected by age, sex and humidity. Almost all cells harvested with IAEA 2001 protocol had cytoplasm loss. PBMCs harvested with CRG protocol stained well in DAPI, acridine orange and Giemsa, and showed high scorable BN frequency in all laboratories. A higher NDI and a lower induced MN frequency were seen in 2 Gy isolated PBMC than whole blood cultures. Conclusion This quick CBMN harvest protocol for isolated PBMCs is a viable alternative to cytocentrifugation, as many scorable BN cells were obtained with routine biodosimetry reagents and equipment. IAEA 2011 + FA protocol should be used to improve CBMN harvest in whole blood cultures. Humidity during spreading should be optimized depending on the harvest protocol. NDI and MN frequency should be separately evaluated for whole blood and isolated PBMC cultures.

Genotoxic Consequences in Common Grass Carp (Ctenopharyngodon idella Valenciennes, 1844) Exposed to Selected Toxic Metals.

The primary aim of the current study was to recognize the biomarker approach as the finest tool to identify the geno-toxicological effects of copper, chromium, and lead inside the blood of grass carp using micronucleus test and comet assay. The induced micronuclei frequency in response to the administered concentrations of LC50 metals was discovered in the erythrocytes of metal-exposed fish at four-time intervals. The genotoxic effect of these metals with respect to the formation of micronuclei was ranked as chromium > lead > copper. Percentages of other cellular and nuclear abnormalities were also determined in the exposed blood films. Equally, the genotoxic studies in terms of comet assay in fish blood revealed significant deviations p < 0.05 against each of the studied metal at their respective time interval as compared with the healthy fish group. However, induced frequency of micronuclei and the calculated DNA damage were not found to be duration dependent. Consequently, copper, chromium, and lead have been explored as cytotoxic elements that can be responsible for inducing genotoxic effects in fish existing aquatic habitats.

Genoprotective effects of gallic acid against cisplatin induced genotoxicity in bone marrow cells of mice.

Chemotherapeutic drugs are used for the treatment of cancer. However, the use of these drugs is limited due to their side effects on normal cells. One of the measures to detect chemoprotection of plant extracts is to evaluate their anticlastogenic effects. In this study, we report the anticlastogenic effect of gallic acid (GA) against cisplatin (Csp), a chemotherapeutic drug, in Swiss albino mice. Three different doses of GA (100, 200 and 400 mg per kg bw) were administered orally to the experimental animals in 0.2 mL quantity for 5 days at 24 hour intervals. Cisplatin, the positive control agent (10 mg per kg bw), was administered intraperitoneally (i.p.) in 0.1 mL quantity. Overall, the results showed that the pretreatment of GA caused a remarkable decrease in Csp induced micronucleus frequency and DNA damage in bone marrow cells of mice. The results suggest that GA showed potent antigenotoxic effects against Csp induced damage in mice bone marrow cells.

The aflatoxin B1‐fumonisin B1 toxicity in BRL‐3A hepatocytes is associated to induction of cytochrome P450 activity and arachidonic acid metabolism

Human oral exposure to aflatoxin B1 (AFB1 ) and fumonisin B1 (FB1 ) is associated with increased hepatocellular carcinoma. Although evidence suggested interactive AFB1 -FB1 hepatotoxicity, the underlying mechanisms remain mostly unidentified. This work was aimed at evaluating the possible AFB1 -FB1 interplay to induce genetic and cell cycle toxicities in BRL-3A rat hepatocytes, reactive oxygen species (ROS) involvement, and the AFB1 metabolizing pathways cytochrome P450 (CYP) and arachidonic acid (ArAc) metabolism as ROS contributors. Flow cytometry of stained BRL-3A hepatocytes was used to study the cell cycle (propidium iodide), ROS intracellular production (DCFH-DA, HE, DAF-2 DA), and phospholipase A activity (staining with bis-BODIPY FL C11-PC). The CYP1A activity was assessed by the 7-ethoxyresorufin-O-deethylase (EROD) assay. Despite a 48-h exposure to FB1 (30μM) not being genotoxic, the AFB1 (20μM)-induced micronucleus frequency was overcome by the AFB1 -FB1 mixture (MIX), presumably showing toxin interaction. The mycotoxins blocked G1/S-phase, but only MIX caused cell death. Overall, the oxidative stress led these alterations as the pretreatment with N-acetyl-l-cysteine reduced such toxic effects. While AFB1 had a major input to the MIX pro-oxidant activity, with CYP and ArAc metabolism being ROS contributors, these pathways were not involved in the FB1 -elicited weak oxidative stress. The MIX-induced micronucleus frequency in N-acetyl-l-cysteine pretreated cells was greater than that caused by AFB1 without antioxidants, suggesting enhanced AFB1 direct genotoxicity probably owing to the higher CYP activity and ArAc metabolism found in MIX. The metabolic pathways modulation by AFB1 -FB1 mixtures could raise its hepatocarcinogenic properties.

Protection against radiation-induced hematopoietic damage in bone marrow by hepatocyte growth factor gene transfer

Purpose: To investigate whether adenovirus-mediated delivery of the human hepatocyte growth factor (HGF) gene could prevent radiation-induced hematopoietic damage.Materials and methods: Thirty C57BL/6 mice were randomized into three groups, in which phosphate buffer saline (PBS), mock adenovirus vector (Ad-null) or adenovirus vector containing HGF (Ad-HGF) were injected into the tail vein of each group, respectively. After 48 hours, the mice received a single irradiation dose of 6.5 Gy 60Co gamma rays. Blood samples were extracted via the tail vein at day 0, 4, 7, 10, 14, 21, 24 and 30 after irradiation, for red blood cell (RBC) and white blood cell (WBC) and cluster of differentiation4 (CD4)/cluster of differentiation8 (CD8) ratio assessment. At weekly intervals following irradiation, serum erythropoietin (EPO), Interleukin-6 (IL-6) and Interferon-gamma (IFN-γ) levels were measured using enzyme-linked immunosorbent assay (ELISA). On post-irradiation day 30, the mice were autopsied and erythroid burst-forming units (BFU-E) were evaluated.Results: Adenovirus-mediated HGF gene transfer could increase human HGF level in serum and have a significant elevation in RBC and WBC count. Ad-HGF increased EPO and IL-6 levels and prompted BFU-E formation. Ad-HGF decreased radiation- induced micronucleus frequency in the mouse bone marrow (BM). Most evidence of radiation-induced hematopoietic damage was observed morphologically in bone marrow specimen four weeks after irradiation. Ad-HGF protected against radiation-induced BM failure and increased survival. Finally, Ad-HGF increased the thymic index and enhanced immune function in the irradiated C57BL/6 mice.Conclusions: This is the first report to date that demonstrates the potential of HGF gene transfer to prevent radiation-induced hematopoietic damage.

Chromium (VI) induced acute toxicity and genotoxicity in freshwater stinging catfish, Heteropneustes fossilis

Acute toxicity and genotoxicity of Chromium(VI) as K2Cr2O7 were evaluated in freshwater stinging catfish, Heteropneustes fossilis. Fish showed behavioral alterations after K2Cr2O7 exposure and 96h-LC50 was 35.724mg/L in semi-static bioassay. Fish were exposed to three sub-lethal concentrations (concentration I=1/4th of 96h-LC50, concentration II=1/10th of 96h-LC50 and concentration III=environmental concentration of Cr reported in the river Buriganga). Blood, liver and gill samples were collected after 48h, 96h and 192h. Micronucleus (MN) assay was conducted in blood erythrocytes and DNA damage was evaluated by comet assay in whole blood, gill and liver tissues. Cr(VI) significantly (p<0.05) induced MN frequency and tail DNA (percent) which increased in a concentration depended manner in all types of tissues. Frequency of MN and tail DNA (percent) increased after 48 and 96h of exposure which decreased after 192h of exposure. The liver was the most sensitive to chromium (VI) exposure among the tissues with highest tail DNA (33.70±0.68 percent) at 9.0mg/L after 96h. This study found MN and comet assays in combination as an adequate approach for ecotoxicological monitoring and Cr(VI) as potential genotoxic agent.

Inter-individual variance in micronuclei frequency in lymphocytes of patients with cervical insufficiency

The aim of this work was to explain the inter-individual variance in baseline and induced micronuclei (MN) frequency in peripheral blood lymphocytes (PBLs) of females with uterine cervical insufficiency. The baseline and induced MN response of the PBLs in 32 patients were investigated using the cytokinesis-block micronucleus (CBMN) method. The obtained mean values of induced MN frequency (11.31 ? 4.44/1000 BN cells), were significantly higher (p &lt; 0.001) in comparison to their baseline MN frequencies (7.81 ? 3.33 MN/1000 BN cells). The analysis of variance (ANOVA) showed statistically significant difference between-group vs. within-group variance in baseline (p &lt; 0.05) and induced MN frequency (p &lt; 0.001), in professionally exposed patients when compared to the non-exposed. Multivariate logistic regression analysis confirmed that patients with high baseline MN frequency (cut-off 8.5 MN/1000 MN), had significantly increased (odds ratio OR = 5.875; CI = 1.28 - 26.795, p &lt; 0.05) induced MN frequency (&gt;12 MN/1000 BN cells). Our results suggest that the baseline MN frequency in PBLs directly influences the scope of the induced MN response.

The protective role of curcumin on perfluorooctane sulfonate-induced genotoxicity: Single cell gel electrophoresis and micronucleus test

Perfluorooctane sulfonate (PFOS) is a man-made fluorosurfactant and global pollutant. PFOS a persistent and bioaccumulative compound, is widely distributed in humans and wildlife. Therefore, it was added to Annex B of the Stockholm Convention on Persistent Organic Pollutants in May 2009. Curcumin is a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric. It is commonly used as a dietary spice and coloring agent in cooking and anecdotally as an herb in traditional Asian medicine. In this study, male rats were treated with three different PFOS doses (0.6, 1.25 and 2.5 mg/kg) and one dose of curcumin, from Curcuma longa (80 mg/kg) and combined three doses of PFOS with 80 mg/kg dose of curcumin by gavage for 30 days at 48 h intervals. Here, we evaluated the DNA damage via single cell gel electrophoresis or comet assay and micronucleus test in bone marrow in vivo. PFOS induced micronucleus frequency and decreased the ratio of polychromatic erythrocyte to normochromatic erythrocyte in bone marrow. Using the alkaline comet assay, we showed that all doses of the PFOS strongly induced DNA damage in rat bone marrow and curcumin prevented the formation of DNA damage induced by PFOS.

Influence of DNA-repair gene variants on the micronucleus frequency in thyroid cancer patients

The role of different DNA-repair genes (OGG1, XRCC1, XRCC2 and XRCC3) on both the spontaneous and the induced frequency of micronuclei (MN) has been studied in the lymphocytes of a group of 114 patients with differentiated thyroid cancer (DTC). Induction of MN was achieved by treatment of the lymphocytes with 0.5Gy of gamma-radiation.The selected genes are involved in base-excision repair (BER) (OGG1, Ser326Cys; XRCC1, Arg280His and Arg399Gln), and in homologous recombination repair (HRR) (XRCC2, Arg188His and XRCC3, IVS5-14G). Genotyping was carried out by use of the iPLEX (Sequenom) technique.Results indicate that only the OGG1-Ser326Cys polymorphism was able to modulate the MN frequency. This effect was only observed in the spontaneous MN frequency (P=0.016), but not in the MN frequency induced after irradiation. In addition, a strong correlation was observed between spontaneous and induced MN frequency, which would suggest an underlying genetic background.

The effect of aging on micronuclei frequency and proliferation in human peripheral blood lymphocytes

INTRODUCTION:Increase in the instability of cellular genome with an increasing age is the result of an accumulation of cellular damage and mutations. This instability which might be observed as chromosome damage or chromosome losses can be measured by the micronucleus technique.AIM:The aim of this study was to investigate the effect of aging and oxidative stress induced by non-toxic levels of H2O2 on micronuclei induction and their relationship to cell proliferation in human peripheral blood lymphocytes.MATERIALS AND METHODS:Healthy volunteers with different ages were choosen. Spontaneous and H2O2 induced micronuclei frequencies were measured in peripheral blood lymphocytes of 30 volunteers by the micronucleus method.RESULTS:Spontaneous micronuclei frequencies increased first then started to decrease after 50 years of age. This biphasic response was significantly higher than micronucleus (MN) frequencies induced by H2O2 (P < 0.05), which followed the similar shape of response to increasing ages with lower frequencies. Proliferative capacity of cells either treated with H2O2 or not did not differ with an increasing age giving similar responses.CONCLUSION:These results indicate biphasic character of chromosome damage; first increase and decrease after 50 years with an increasing age. But this change pattern was not correlated with the steady state of proliferation capacity of cells through an increasing age. Decreases in H2O2-induced MN frequencies compared to spontaneous MN frequencies may be inducing an apoptosis by H2O2 treatment leading to underscoring damaged cells.

An investigation of the relationshıp between coronary atherosclerosis and chromosome damage in human peripheral blood lymphocytes

Background: Coronary atherosclerosis is a disease of the coronary arteries and is a result of cumulative processes that are mostly seen in old-age population. Progressive accumulation of oxidative damage to cellular macromolecules, especially DNA, over a lifetime is one of the important causes of aging. This accumulation of damage may result in chromosome instability and this can be measured by micronucleus (MN) technique. Materials and Methods: In this study, MN technique was applied to observe the relationship between chromosome instability and proliferative effectiveness of lymphocytes and coronary atherosclerosis. Non-toxic levels of hydrogen peroxide were also administered to observe any existing differential effect due to this stress inducing oxidizing agent. Spontaneous and H 2 O 2 stress induced MN frequencies and proliferation state of peripheral lymphocytes were measured from both 30 patients diagnosed as coronary atherosclerosis and 30 healthy control individuals of the same age range. Results: There was no significant difference between spontaneous MN frequencies of patient and control groups (P = 0.08). Also, probability index (PI) values between these groups reflected no difference (P = 0.13). Hydrogen peroxide addition did not change spontaneous MN frequencies either in control (P = 0.23) or in patient group (P = 0.81). This non-toxic H 2 O 2 level failed to show any different effect on patients compared to controls (P = 0.41). Although increasing PI values were observed as a result of H 2 O 2 administration only in patient group, the comparisons between the two groups showed no significant difference (P = 0.34). Conclusion: Chromosome instability and proliferation index that were measured by MN technique in this study have no role in discriminating coronary atherosclerosis patients from healthy individuals.

Investigating micronucleus assay applicability for prediction of normal tissue intrinsic radiosensitivity in gynecological cancer patients

BackgroundPelvic organs morbidity after irradiation of cancer patients remains a major problem although new technologies have been developed and implemented. A relatively simple and suitable method for routine clinical practice is needed for preliminary assessment of normal tissue intrinsic radiosensitivity. The micronucleus test (MNT) determines the frequency of the radiation induced micronuclei (MN) in peripheral blood lymphocytes, which could serve as an indicator of intrinsic cell radiosensitivity. AimTo investigate a possible use of the micronucleus test (MNT) for acute radiation morbidity prediction in gynecological cancer patients. Materials and methodsForty gynecological cancer patients received 50Gy conventional external pelvic irradiation after radical surgery. A four-field “box” technique was applied with 2D planning. The control group included 10 healthy females.Acute normal tissue reactions were graded according to NCI CTCAE v.3.0. From all reaction scores, the highest score named “summarized clinical radiosensitivity” was selected for a statistical analysis.MNT was performed before and after in vitro irradiation with 1.5Gy. The mean radiation induced frequency of micronuclei per 1000 binucleated cells (MN/1000) and lymphocytes containing micronuclei per 1000 binucleated cells (cells with MN/1000) were evaluated for both patients and controls.An arbitrary cut off value was created to pick up a radiosensitive individual: the mean value of spontaneous frequency of cells with MN/1000±2SD, found in the control group. ResultsBoth mean spontaneous frequency of cells with MN/1000 and MN/1000 were registered to be significantly higher in cancer patients compared to the control group (t=2.46, p=0.02 and t=2.51, p=0.02). No statistical difference was registered when comparing radiation induced MN frequencies between those groups.Eighty percent (32) of patients developed grade 2 summarized clinical radiosensitivity, with great variations in MNT parameters. Only three patients with grade 2 “summarized clinical radiosensitivity” had values of cells with MN/1000 above the chosen radiosensitivity threshold. ConclusionThe present study was not able to confirm in vitro MNT applicability for radiosensitivity prediction in pelvic irradiation.

Heritability of baseline and induced micronucleus frequencies

The scoring of micronuclei (MN) is widely used in biomonitoring and mutagenicity testing as a surrogate marker of chromosomal damage inflicted by clastogenic agents or by aneugens. Individual differences in the response to a mutagenic challenge are known from studies on cancer patients and carriers of mutations in DNA repair genes. However, it has not been studied to which extent genetic factors contribute to the observed variability of individual MN frequencies. Our aim was to quantify this heritable genetic component of both baseline and radiation-induced MN frequencies. We performed a twin study comprising 39 monozygotic (MZ) and 10 dizygotic (DZ) twin pairs. Due to the small number of DZ pairs, we had to recruit controls from which 38 age- and gender-matched random control pairs (CPs) were generated. For heritability estimates, we used biometrical modelling of additive genetic, common environmental, and unique environmental components (ACE model) of variance and direct comparison of variance between the sample groups. While heritability estimates from MZ to DZ comparisons produced inconclusive results, both estimation methods revealed a high degree of heritability (h(2)) for baseline MN frequency (h(2) = 0.68 and h(2) = 0.72) as well as for the induced frequency (h(2) = 0.68 and h(2) = 0.57) when MZ were compared to CP. The result was supported by the different intraclass correlation coefficients of MZ, DZ and CP for baseline (r = 0.63, r = 0.31 and r = 0.0, respectively) as well as for induced MN frequencies (r = 0.79, r = 0.74 and r = 0.0, respectively). This study clearly demonstrates that MN frequencies are determined by genetic factors to a major part. The strong reflection of the genetic background supports the idea that MN frequencies represent an intermediate phenotype between molecular DNA repair mechanisms and the cancer phenotype and affirms the approaches that are made to utilise them as predictors of, for example, cancer risk.

In vitro micronucleus test in HepG2 transformants expressing a series of human cytochrome P450 isoforms with chemicals requiring metabolic activation

It is known that many genotoxic chemicals require oxidative metabolism to elicit genotoxicity. Induced rat liver S9 fraction has been employed as a ‘metabolite factory’ in in vitro genotoxicity testing. However, the relevance of the induced rat liver S9 fraction has been called into question due to the differences in the rat and human cytochrome P450 (CYP) activities. In the present study, we used a series of ten transformants expressing major human CYP isoforms such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in HepG2 cells. To elucidate the usefulness and feasibility of these transformants, genotoxicity was tested without using rat S9. Among these transformants, benzo(a)pyrene-induced or cyclophosphamide-produced micronucleus (MN) frequency was markedly increased in transformants expressing CYP1A2 or CYP2C9, respectively. To explore the possibility that these transformants can be used for screening the possible genotoxicity of newly developed drugs, a chemical which is known to enhance genotoxicity in the presence of external metabolic activation system, okadaic acid (OA), was investigated. OA-induced MN frequency was significantly induced in transformants expressing CYP1A2 compared with the other CYP isoforms. The induced MN frequency was suppressed by treatment with a CYP1A2 specific inhibitor and CYP1A2 to siRNA. In control HepG2 cells harboring an empty vector, OA was treated with microsomes expressing CYP1A2 to induce MN. These results demonstrated that this screening system worked well and OA was found to be metabolically activated by CYP1A2 to induce MN. Based on the results obtained in the present study, this system of transformants is useful to elucidate the genotoxicity involving human CYP metabolism in the process of drug discovery.

Correlation between Mitotic Delay and Micronucleus Assay in Breast cancer patients

Breast cancer has been associated with a variety of mutations in susceptibility genes. These patients also exhibit impaired DNA repair capacity when compared to controls, which has been assessed by different tests measuring the ability to repair DNA damage. Yet unanswered is the question if those tests produce correlated results on an individual basis. For this purpose, micronucleus test (MNT) and mitotic delay assay were performed simultaneously on the same probands. Blood samples were obtained from 46 probands. Micronucleus test was performed with spontaneous or radiation-induced lymphocyte cultures (2Gy at setup). Samples for mitotic delay were irradiated after 54h, 18h prior to measurement. Mitotic delay index was calculated as (ratio [cells G2-phase]/[cells S-phase] of irradiated cultures divided by G2/S ratio of control cultures). There was no correlation of spontaneous MN frequencies to radiation-induced MN frequencies (p=0.38). While there was also no correlation between mitotic delay index and spontaneous MN frequencies (p=0.78), we did observe a statistically significant correlation between mitotic delay index and induced MN-frequencies (p=0.04) As both increased mitotic delay index and radiation induced micronucleus frequencies are known to be associated with defects in DNA damage repair and with breast cancer, the presence of correlation between these both tests indicates that the produced results characterize DNA repair capacity in a similar way. Thus they seem to measure linked aspects of DNA damage response. A combined approach using these tests could prove to be a useful step towards increased significance when screening patients for a phenotype of decreased DNA repair.

Radiation-induced bystander effects enhanced by elevated sodium chloride through sensitizing cells to bystander factors

Radiation-induced bystander effects (RIBE) have been demonstrated to occur widely in various cell lines. However, very little data is available on the genotoxic effects of RIBE combined with other factor(s). We reported previously that with a low dose of α-particle irradiation, the fraction of γ-H2AX foci-positive cells in non-irradiated bystander cells was significantly increased under elevated NaCl culture conditions. In this study, we further investigated the functional role of NaCl in the enhancement of RIBE using a specially designed co-culture system and micronucleus (MN) test. It was shown that the MN frequency was not increased significantly by elevated NaCl (9.0 g/L) alone or by medium exposure. However, with 1.0 cGy α-particle irradiation, the induced MN frequency increased significantly in both irradiated and non-irradiated bystander regions. Additional studies showed that elevated NaCl made the non-irradiated bystander cells more vulnerable to bystander factors. Furthermore, it was found that the induced MN frequency in cells both in irradiated and non-irradiated bystander regions was weakened when the hypertonic medium was changed to normotonic medium for 2 h before irradiation. Such observations were quite similar to the co-effect of NaCl and hydrogen peroxide (H 2O 2), indicating that elevated NaCl might sensitize non-irradiated cells to bystander factors-induced oxidative stress.

Micronuclei frequency in peripheral blood lymphocytes of cancer patients: A meta-analysis

Micronuclei (MN) frequency is a biomarker of chromosomal damage, genome instability, and cancer risk that integrates acquired mutations and genetic susceptibility. To evaluate and summarize the evidence reporting association between cancer and MN formation, we performed a meta-analysis assessing the frequency of this biomarker in cancer patients. Findings from 37 publications were retrieved through an extensive search of the MedLine/PubMed database. Given the heterogeneity of the study design, all studies were re-classified into three groups: (i) baseline MN frequency of untreated cancer patients (25 studies), (ii) induced MN frequency in thyroid cancer patients undergoing radioiodine treatment (9 studies), and (iii) radiosensitivity of lymphocytes (12 studies) in untreated cancer patients. A meta-estimate of the frequency ratio (meta-FR) was computed in each group. A significant increase of MN frequency was found in untreated cancer patients (meta-FR=1.45; 95% Confidence Interval (95% CI): 1.28-1.64) and in thyroid cancer patients after radioiodine treatment (meta-FR=2.26; 95% CI: 1.90-2.68). The third meta-analysis showed a negative trend of meta-FR's when plotted vs. the dose used to study patients' radiosensitivity, possibly associated to a high rate of apoptosis. The results of this review substantiate the existing evidence about a role of MN in various steps of carcinogenesis. The relatively small numbers of papers suitable for the meta-analysis call for new and larger studies, possibly based on high-throughput techniques, to further understand the role of MN formation in the occurrence of genetic instability and cancer.