Micronucleus Frequency In Human Lymphocytes Research Articles

Protective effects of curcumin against genotoxicity induced by 131-iodine in human cultured lymphocyte cells.

Background:131-radioiodine has been widely used as an effective radionuclide for treatment of patients with thyroid diseases. The purpose of the present study is to investigate the radioprotective effects of curcumin as a natural product that protects against the genotoxic effects of 131I in human cultured lymphocytes.Materials and Methods:Whole blood samples from human volunteers were incubated with curcumin at doses of 5, 10, and 50 μg/mL. After 1-hour incubation, the lymphocytes were incubated with 131I (100 μCi/1.5 ml) for 2 hours. The lymphocyte cultures were then mitogenically stimulated to allow for evaluation of the number of micronuclei in cytokinesis-blocked binucleated cells.Results:Incubation of lymphocytes with 131I at dose 100 μCi/1.5 mL induced genotoxicity shown by increase in micronuclei frequency in human lymphocytes. Curcumin at 5, 10, and 50 μg/mL doses significantly reduced the micronuclei frequency. Maximal protective effects and greatest decrease in micronuclei frequency were observed when whole blood was incubated with 50 μg/mL dose of curcumin with 52%.Conclusion:This study has important implications for patients undergoing 131I therapy. Our results indicate a protective role for curcumin against the genetic damage and side effects induced by 131I administration.

Potential chemoprotective effects of selenium on diazinon-induced DNA damage in rat peripheral blood lymphocyte

The purpose of this study was to investigate the protective effects of selenium (Se) against genotoxicity induced by diazinon (DZN) in rat peripheral blood lymphocytes by micronucleus (MN) test. Animals were concurrently administered intraperitoneally with DZN in proper solvent (20mg/kg body weight (b.w.)) and Se at three different doses (0.5, 1, and 2mg/kg b.w.) for 30 consecutive days. The positive control group received DZN at the same dose without Se. After 24h of last injection, 0.5ml blood of each rat was received and cultured in culture medium for 44h. The lymphocyte cultures were mitogenically stimulated with cytochalasin B to allow the evaluation of number of MNs in cytokinesis-blocked binucleated cells. Incubation of lymphocytes with DZN induced additional genotoxicity and is shown by increase in MNs frequency in human lymphocytes. Se at low dose of 0.5mg/kg had a maximum effect and significantly reduced the MNs frequency in cultured lymphocytes (p<0.0001) that reduced the frequency of MN from 12.78±0.24% for DZN group to 4.40±0.36. The present study revealed that Se particularly at low doses has a potent antigenotoxic effect against DZN -induced toxicity in rats, which may be due to the scavenging of free radicals and increased antioxidant status.

A Decreased Micronucleus Frequency in Human Lymphocytes after Folate and Vitamin B12 Intervention: a Preliminary Study in a Yunnan Population

Micronucleus (MN) is a validated biomarker for DNA damage in peripheral blood lymphocytes (PBL) and is a reflection of the changes of human nutritional status. Folate (FA) and vitamin B(12) are one-carbon metabolism-related micronutrients, which play important roles in maintaining genomic stability. To investigate the correcting effects of FA and B(12) intervention on DNA damage in PBL. One hundred fifty-six volunteers without history of cancer were divided into 5 age groups (20 - 69 y, 47.4 % male) for establishing the baseline of chromosomal damage by means of cytokinesis-block micronucleus assay. Twelve individuals whose MN frequency was higher than the median value in each age group were selected for a four-month FA (200 - 400 μg/day)-B12 (3.125 - 25 μg/day) intervention dosed as to age and MTHFR genotypes. There were significantly positive correlations between age and MN frequency in all groups (p < 0.01). Among all age groups, the baseline MN frequencies were higher in females than that in males. The MN frequencies from 10 volunteers were reduced by 33.5 % after the intervention with the two micronutrients (p < 0.01), and two individuals did not show any changes. Dietary supplement intake of FA and B(12) based on MTHFR genotypes could protect the genome from damage and benefit genome health.

Micronucleus frequency in human lymphocytes after exposure to diphenylamine in vitro

Diphenylamine (DPA) is an antioxidant compound that occurs naturally in several vegetables. It is widely applied in agriculture for preservation of the quality of apples and pears, and used for controlling superficial scald, a disorder that renders fruits of a number of apple cultivars unfit for the market. Because of its anti-oxidative properties, DPA also has several industrial applications. The potential genotoxic effect of DPA on human lymphocytes has previously been investigated in only two studies, which focused on detection of chromosome aberrations and sister chromatid exchange, respectively. In the present analysis, we evaluated micronucleus (MN) formation in freshly isolated human peripheral lymphocytes exposed to different concentrations (0.625, 1.25, 2.50, 5.0 and 10.0μg/ml) of DPA. Peripheral venous blood was collected from ten healthy subjects, and a total of 10,000 bi-nucleated cells were analyzed. Results indicated that DPA significantly increased the micronucleus frequency at concentrations of 1.25μg/ml and higher. Significant differences in the MN frequency were also found between the lower dose (0.625μg/ml) and all other doses tested, with the exception of 1.25μg/ml. Our results indicate a potential cytogenetic effect of DPA on human cells in vitro and require further in vivo studies to clarify the actual genotoxicity of this compound and the consequent risks for human health.

Effects of ochratoxin A on micronucleus frequency in human lymphocytes

Effects of ochratoxin A on micronucleus frequency in human lymphocytes

Protective effects of hesperidin against genotoxicity induced by 99mTc-MIBI in human cultured lymphocyte cells

Radiopharmaceuticals have been widely used as nuclear tracers for myocardial perfusion imaging. The purpose of this study was to investigate the radioprotective effects of hesperidin as a flavonoid which protects against the genotoxic effects of (99m)Tc-MIBI in human cultured lymphocytes. Whole blood samples from human volunteers were incubated with hesperidin at doses of 10, 50 and 100 micromol. After 1 h of incubation, the lymphocytes were incubated with (99m)Tc-MIBI (200 microCi/2 ml) for 3 h. The lymphocyte cultures were then mitogenically stimulated to allow for evaluation of the number of micronuclei in cytokinesis-blocked binucleated cells. Incubation of lymphocytes with (99m)Tc-MIBI at this high dose induces additional genotoxicity and shown by increases in micronuclei frequency in human lymphocytes. Hesperidin at these doses significantly reduced the micronuclei frequency in cultured lymphocytes. The maximum protective effect and greatest decrease in micronuclei frequency occurred when cultures were incubated with a 100-micromol dose of 65% hesperidin. This study has important implications for patients undergoing nuclear medicine procedures. The results indicate a protective role for hesperidin against the genetic damage and side effects induced by radiopharmaceutical administration.

The azo dyes Disperse Red 1 and Disperse Orange 1 increase the micronuclei frequencies in human lymphocytes and in HepG2 cells

The use of azo dyes by different industries can cause direct and/or indirect effects on human and environmental health due to the discharge of industrial effluents that contain these toxic compounds. Several studies have demonstrated the genotoxic effects of various azo dyes, but information on the DNA damage caused by Disperse Red 1 and Disperse Orange 1 is unavailable, although these dyes are used in dyeing processes in many countries. The aim of the present study was to evaluate the mutagenic activity of Disperse Red 1 and Disperse Orange 1 using the micronucleus (MN) assay in human lymphocytes and in HepG2 cells. In the lymphocyte assay, it was found that the number of MN induced by the lowest concentration of each dye (0.2 μg/mL) was similar to that of the negative control. At the other concentrations, a dose response MN formation was observed up to 1.0 μg/mL. At higher dose levels, the number of MN decreased. For the HepG2 cells the results were similar. With both dyes a dose dependent increase in the frequency of MN was detected. However for the HepG2, the threshold for this increase was 2.0 μg/mL, while at higher doses a reduction in the MN number was observed. The proliferation index was also calculated in order to evaluate acute toxicity during the test. No differences were detected between the different concentrations tested and the negative control.

Effects of lifestyle on micronuclei frequency in human lymphocytes in Japanese hard-metal workers

Objective The risks of cardiovascular disease, cancer, and other major causes of mortality are largely attributable to lifestyle factors such as smoking, alcohol drinking, hours of working and sleeping, physical activity, diet, and stress. Earlier studies have suggested that an unhealthy lifestyle is also associated with increased lymphocyte sensitivity to mutagens, oxidative DNA damage level, and leukocyte DNA damage. In order to explore the genotoxicity of unhealthy lifestyle, we evaluated the effect of overall lifestyle as well as some individual lifestyle factors on micronuclei (MN) frequency in cultured human lymphocytes. Method The study was conducted among 208 healthy adult (19 to 59 years) male Japanese hard-metal workers. The subjects were divided into groups according to their self-reported good, moderate, and poor lifestyles based on their responses to a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, sleeping hours, working hours, physical exercise, eating breakfast, balanced nutrition, and mental stress), the presence or absence of each of which was summed to obtain a health practice index (HPI: range 0–8). Peripheral blood was taken and the cytokinesis-block micronuclei (CBMN) assay was performed. Results Total lifestyle quality as measured by the HPI was strongly negatively associated with MN frequency in cultured human lymphocytes ( p < 0.01). Nutritional imbalance, lack of regular exercise (< 2 times per week), insufficient sleep ( ≤ 6 h per day), and overtime working (≥ 9 h per day) each contributed significantly to higher MN frequency (all p < 0.05). In the smoker group, a significantly higher MN frequency was only found in heavy smokers ( p < 0.05). On the other hand, mental stress, eating breakfast, and alcohol drinking had no effect on MN frequency. Conclusions Taken together, these findings indicate that poor lifestyle habits significantly increase MN frequency in human lymphocytes.

HOGG1326, XRCC1399 and XRCC3241 polymorphisms influence micronucleus frequencies in human lymphocytes in vivo

A pooled analysis of five biomonitoring studies was performed to assess the influence of hOGG1(326), XRCC1(399) and XRCC3(241) gene polymorphisms on micronuclei (MN) frequency in human peripheral blood lymphocytes, as measured by the ex vivo/in vitro cytokinesis-block micronucleus (CBMN) assay. Each study addressed a type of occupational exposure potentially able to induce DNA strand breakage (styrene, ionising radiation, cobalt/hard metal, welding fumes and inorganic arsenite compounds), and therefore MN, as a result of base excision repair and double-strand break repair deficiencies. The effect of genotype, age, exposure to genotoxic agents and smoking habit on MN induction was determined using Poisson regression analysis in 171 occupationally exposed male workers and in 132 non-exposed male referents. The analysis of genotype-genotype, genotype-smoking and genotype-exposure interactions by linear combinations of parameters showed significantly higher MN frequencies in the following subsets: (i) occupationally exposed workers carrying either the Thr/Thr or the Thr/Met XRCC3(241) genotypes compared to their referent counterparts (P < 0.001) and (ii) carriers of the Met/Met XRCC3(241) genotype compared to Thr/Thr XRCC3(241) carriers, as far as they are non-exposed and bear the variant (Ser/Cys or Cys/Cys) hOGG1(326) genotype (P < 0.01). Significantly lower MN frequencies were observed in carriers of the variant hOGG1(326) genotype compared to Ser/Ser hOGG1(326) carriers in the subgroup of non-smokers with Thr/Thr XRCC3(241) genotype (P < 0.01). Stratified analysis by occupational exposure showed a significant MN increase with smoking in occupationally exposed carriers of the Arg/Gln XRCC1(399)genotype (P < 0.001). In contrast, a significant MN decrease with smoking was observed in referents carrying the Ser/Ser hOGG1(326) genotype (P < 0.01). These findings provide evidence that the combination of different DNA repair genes and their interaction with environmental genotoxic agents may modulate MN induction. Understanding the complexity of the relationships between exposure, DNA repair and MN frequencies require larger scale studies and complementary biomarkers.

Basal and induced micronucleus frequencies in human lymphocytes with different GST and NAT2 genetic backgrounds

Basal and induced frequencies of genetic damage can be modulated by different host factors, including genes involved in phase II metabolism. Since polymorphic variants in the glutathione S-transferase (GST) and N-acetyl transferase (NAT) genes have been associated with cancer risk, we explored the possible links between GSTM1, GSTP1, GSTT1 and NAT2 variants and the frequency of micronuclei (MN) in human lymphocytes. This exploratory study was carried out in 30 thyroid cancer patients, before and after receiving an average dose of 109.9±1.3mCi radioactive iodine as a co-adjuvant therapy. The results indicate that none of the polymorphisms studied show any kind of association with the basal level of micronuclei. When the same patients were followed after radioiodine exposure, a significant increase in the frequency of MN was observed in practically all of them (28/30), indicating the genotoxic activity of the ionising radiation exposure. The increase in MN frequency was not associated with any of the GST polymorphisms evaluated. Nevertheless, the presence of slow acetylator phenotypes and, in particular, the presence of the NAT2*7 allele was significantly associated with a lower increase of the MN frequency after radioiodine treatment.

Gene–environmental interactions between alcohol-drinking behavior and ALDH2 and CYP2E1 polymorphisms and their impact on micronuclei frequency in human lymphocytes

Ethanol is converted to acetaldehyde by alcohol dehydrogenase (ADH), cytochrome p4502E1 (CYP2E1) and catalase. This metabolite is then detoxified by aldehyde dehydrogenase 2 (ALDH2), a key enzyme in the elimination of acetaldehyde, via further oxidation to acetic acid. The toxic effects of acetaldehyde are well documented and may be partially mediated by genotoxic damage. In the present study, we investigated the effects of alcohol-drinking behavior and genetic polymorphisms in two different genes (ALDH2 and CYP2E1) on the micronuclei (MN) frequency in 248 healthy Japanese men. Genotyping was performed by PCR–RFLP analysis. The ALDH2 variant (deficient type) was significantly associated with an increased MN frequency in subjects drinking more than three times/wk, while habitual drinkers with wild-type CYP2E1 also had a significantly increased MN frequency. Furthermore, when the subjects were divided into eight groups according to their drinking frequency and genotypes of ALDH2 and CYP2E1, we found that habitual drinkers with homozygous CYP2E1 *1/ *1 and heterozygous ALDH2 *1/ *2 or homozygous ALDH2 *2/ *2 showed the highest mean MN frequency. In the present study, we found clear associations among ALDH2 and CYP2E1 gene polymorphisms, alcohol-drinking behavior and genotoxic effects in a healthy Japanese population. Therefore, analysis of the polymorphisms of alcohol-metabolizing enzymes may lead to elucidation of the mechanism(s) for individual susceptibilities to the toxicity of ethanol metabolites.

Evaluation of the cytotoxicity, cytostaticity and genotoxicity of Argentatins A and B from Parthenium argentatum (Gray)

Argentatins A and B are abundant triterpenes present in Parthenium argentatum. Both compounds have shown cytotoxic properties on K562, MCF-7, PC-3, HCT-15 and U251 human cancer cell lines. Furthermore the cytotoxic, cytostatic and genotoxic effects of the argentatins on proliferating lymphocytes were evaluated using cytokinesis-block micronucleus test. Argentatin A had no cytostatic properties, but it was cytotoxic for proliferating lymphocytes at a concentration of 25 μM ( P < 0.005). On the other hand, argentatin B showed significant cytostatic effects ( P < 0.001) at concentrations of 5 to 25 μM and it did not show cytotoxic effects at the same concentrations. Neither argentatin showed genotoxic effects in terms of micronucleus frequency in human lymphocytes. According to these results the argentatins are not able to cause injury on DNA by clastogenic or aneugenic mechanisms.

Evaluation and characterization of micronuclei induced by the antitumour agent ASE [3beta-hydroxy-13alpha-amino-13, 17-seco-5alpha-androstan-17-oic-13, 17-lactam-p-bis(2-chloroethyl)amino phenylacetate] in human lymphocyte cultures.

3beta - Hydroxy - 13alpha - amino - 13, 17 - seco - 5alpha - androstan - 17 -oic-13,17-lactam-p-bis(2-chloroethyl)amino phenylacetate (ASE) is a homo-aza-steroidal ester of p-bis(2-chloroethyl) amino phenyl acetic acid and has been shown to display antineoplastic, mutagenic and genotoxic activity. In the present study an effort has been made to evaluate the ability of ASE to induce micronuclei (MN) in human lymphocytes treated in vitro using the cytokinesis-block assay. Lympocytes were treated with different concentrations of ASE (0.1, 0.25, 0.5, 1, 2.5, 5, 10 and 20 microg/ml) at two different cell culture times, 21 and 41 h after culture initiation. ASE treatment lasted until cell harvest, for 51 and 31 h, respectively. Two types of cultures were used, whole blood and isolated lymphocyte cultures. The content of induced MN was identified by FISH analysis, using an alpha-satellite DNA probe, in binucleate cells. Our results suggest that ASE is capable of increasing MN frequencies in human lymphocytes under both culture conditions. This increase is related to the concentration in a linear dose-dependent manner and is also dependent on the duration of treatment. FISH analysis has shown that the induced MN resulted mainly from breakage events. Additionally, a weak aneugenic effect was found at the higher concentrations in whole blood cultures as well as in isolated lymphocyte cultures. Cytotoxic effects of ASE were observed under both cell culture conditions with a linear dose-dependent relationship according to CBPI evaluation and were more pronounced in isolated lymphocyte cultures.

Micronucleus frequency in human lymphocytes is related to plasma vitamin B12 and homocysteine

In a series of studies, we have been able to confirm that the micronucleus index in cytokinesis-blocked lymphocytes is significantly negatively correlated with plasma vitamin B12 (B12) concentration and significantly positively correlated with plasma homocysteine (HC). Furthermore we have shown in a randomised double-blind placebo-controlled dietary intervention study that intake of 3.5 times the RDI of folic acid and B12 significantly reduces the micronucleus index only in those with above average levels of micronucleus frequency. Micronucleus frequency is minimised when plasma HC is below 7.5 μmol/l and plasma B12 is above 300 pmol/l. Therefore, it is important to take account of the effect of B12 and HC when using the micronucleus assay for human biomonitoring studies.

Synthesis of [123I] iodoantipyrine to study the high-LET characteristics of Auger electrons in mammalian cells

4-Iodoantipyrine was synthesised from phenazone, labeled with123I and administered to cells in culture. 4-[123I]iodoantipyrine carries the radionuclide across the cell membrane allowing one to study the biological effects of the very short ranged Auger electrons emitted by this isotope. The formulation used to prepare this compound proves to be free of reagents that may have an adverse effect on the mitotic activity of cells in culture. By observing micronuclei frequencies in human lymphocytes and CHO-K1 cells the high-LET characteristics of Auger electrons could be demonstrated. Compared to extracellular disintegrations from [123I]NaI and123I-human serum albumin, a substantial reduction in the variation in radiosensitivity of these cell types was noted when treated with 4-[123I] iodoantipyrine.

Intra- and inter-individual variation of background and radiation-induced micronucleus frequencies in human lymphocytes.

Serial blood samples were taken from four healthy individuals (three males, one female, aged between 26 and 51 years) in 3-monthly intervals during 1 year. Leucocyte suspensions were prepared and exposed to 3 Gy of 137Cs gamma-rays or left unirradiated as controls. In a cytokinesis-blocked (CB) micronucleus (MN) assay significant inter- and intra-donor variations of background and radiation-induced MN incidences became apparent. The two sources of variation lead to an extra variance sigma I2, in addition to the sample variance sigma e2 of MN incidences. The contributions of the different components to the total variance were estimated by means of a variance component model. The deviation sigma I for the mean background MN level of 1.53 x 10(-2) MN/CB cell was +/- 0.67 x 10(-2) and for the mean radiation-induced MN level of 0.53 MN/CB cell it was +/- 0.10. The contribution of the intra-individual variance to sigma I2 was about 50% for background MN levels and 75% for radiation-induced MN frequencies. With respect to the application of the CB-MN assay as a biological dosimetry system, the consequences of the present findings for calibration purposes and low-dose estimation are discussed. The calculation of the variance components is explained in an appendix, which serves also as an example for the adaptation of analysis of variance techniques to the evaluation of data derived from scoring of MN, as well as from scoring of metaphase chromosomal aberrations.

Preliminary studies on scoring micronuclei by computerised image analysis

Initial studies of the use of computerised image analysis to determine micronucleus frequencies in human lymphocytes that have completed one nuclear division are described. Two methods, based on (a) bromodeoxyuridine incorporation and (b) cytokinesis blocking with cytochalasin-B, were studied. The former method is directly amenable to automation. Cytokinesis-blocked cells could not be automatically recognised by image analysis but it was possible to obtain the correct micronucleus frequency from the integrated optical density histograms by using the mononucleate/binucleate cell ratio obtained by visual analysis. The mean (±1 S.E.) integrated optical density of X-ray-induced micronuclei was 11.2% (±1.1) of that measured for nuclei of G 1 cells.