Genetic Damage In Human Lymphocytes Research Articles

Effects of tartrazine on proliferation and genetic damage in human lymphocytes

The color additive, tartrazine (TRZ), is widely used in food products, drugs and cosmetics. Genotoxicity of TRZ and its metabolites has not been investigated in detail in the presence and absence of a metabolic activator (S9 mix) in human. Therefore, the aim of this study is to investigate the cytotoxic and genotoxic effects of TRZ and its metabolites on cultured human lymphocytes by using chromosome aberration (CA) and micronucleus (MN) tests. Cultures were treated with 625, 1250 and 2500 μg/ml of TRZ in the presence and absence of S9 mix. TRZ showed cytotoxic activity at the highest concentration due to significant decrease in mitotic index (MI) in the absence of S9 mix when compared with solvent control. TRZ and metabolites significantly increased the CAs and aberrant cells in the presence and absence of S9 mix at the higher concentrations. Increased MN values in cultures with and without S9 mix were found to significantly at the highest concentration when tested. Our results indicated that while both TRZ and its metabolites have genotoxic potential on human lymphocyte cultures with and without S9 mix, TRZ can induce cytotoxicity at the highest concentration in culture without S9 mix under the experimental conditions.

In vitro Protective Effect of Rutin and Quercetin against Radiation-induced Genetic Damage in Human Lymphocytes.

Purpose of the Study:Rutin (RUT) and quercetin (QRT) which are dietary compounds were investigated for their ability to protect against ionizing radiation (IR)-induced genotoxicity in human lymphocytes.Materials and Methods:The radiation antagonistic potential of RUT and QRT was assessed by alkaline comet and cytokinesis-block micronucleus (CBMN) assay.Results:Treatment of lymphocytes with RUT and QRT (25 μg/ml) prior exposure to 2 Gy gamma radiation resulted in a significant reduction of frequency of micronuclei as compared to the control set of cells evaluated by CBMN assay. Similarly, treatment of lymphocytes with RUT and QRT before radiation exposure showed significant decrease in the DNA damage as assessed by comet parameters, such as percent tail DNA and olive tail moment.Conclusion:The study demonstrates the protective effect of RUT and QRT against IR-induced DNA damage in human lymphocytes, which may be partly attributed to scavenging of IR-induced free radicals and also by the inhibition of IR-induced oxidative stress.

In vitro studies on chemoprotective effect of borax against aflatoxin B1-induced genetic damage in human lymphocytes

A common dietary contaminant, aflatoxin B1 (AFB1), has been shown to be a potent mutagen and carcinogen in humans and many animal species. Since the eradication of AFB1 contamination in agricultural products has been rare, the use of natural or synthetic free radical scavengers could be a potential chemopreventive strategy. Boron compounds like borax (BX) and boric acid are the major components of industry and their antioxidant role has recently been reported. In the present report, we evaluated the capability of BX to inhibit the rate of micronucleus (MN) and sister chromatid exchange (SCE) formations induced by AFB1. There were significant increases (P<0.05) in both SCE and MN frequencies of cultures treated with AFB1 (3.12ppm) as compared to controls. However, co-application of BX (1, 2 and 5ppm) and AFB1 resulted in decreases of SCE and MN rates as compared to the group treated with AFB1 alone. Borax gave 30-50% protection against AFB1 induced SCEs and MNs. In conclusion, the support of borax was especially useful in aflatoxin-toxicated blood tissue. Thus, the risk on target tissues of AFB1 could be reduced and ensured early recovery from its toxicity.

Avocado extract induces chromosomal aberrations in cultured human lymphocytes

Avocado fruit is consumed in many parts of the world due to its high nutritional value, while extracts from avocado fruit and leaves have been shown to exhibit pharmacological activity against diabetes, hypertension, infections and cancer. We carried out a chromosomal aberration assay in metaphasic preparations of cultured human lymphocytes to evaluate the potential genotoxicity of 50% methanolic extracts of avocado fruits and leaves. Exposure of cells to both leaf and fruit extracts, led to a significant concentration‐dependent increase in chromosomal aberrations as compared to untreated control. The lymphocytes exposed to fruit extract exhibited lower frequency of all types of aberrations at equal concentrations as compared to those exposed to leaf extract. Acrocentric associations and premature centromeric division, which are thought to indicate chromosomal instability and are potential harbingers of non‐disjunction and translocation mutations, were the two most common abnormalities observed in both the exposed groups. In conclusion, the present study suggests that the extracts of both avocado fruit and leaves can potentially induce significant genomic instability and some genetic damage in human lymphocytes in vitro. However, it is essential to carry out in vivo studies before making a final remark on the genotoxicity of the plant extract. This work was funded by JNCHRC, Bhopal, India.

Modulatory effects of Thymbra spicata L. different extracts against the mercury induced genotoxicity in human lymphocytes in vitro

Mercury, a xenobiotic metal, is a highly deleterious environmental pollutant. Moreover, in any form mercury is reported to be toxic. On the other hand, Thymbra spicata L., a member of the Lamiaceae family, has long been investigated popularly of biological roles; mainly antimicrobial and antioxidant activities. However, there are very scarce data on the cytogenetic effects of thyme species. The purpose of this study was to investigate the genetic safety of different extracts from T. spicata (water extract, methanol extract, and ethanol extract) and the effects of T. spicata on mercury (as HgCl(2)) induced genotoxicity. Sister chromatid exchange (SCE) and micronucleus (MN) assays were performed to assess DNA damages in cultured human lymphocytes (n=5). Our results clearly revealed that, the SCE and MN rates induced by HgCl(2) were alleviated by the presence of T. spicata. As conclusion, this study demonstrated for the first time that the T. spicata provided increased resistance of DNA against HgCl(2) induced genetic damage in human lymphocytes. Based on the results of this study, it may be concluded that the T. spicata is a nontoxic material that could be used as a suppressor of heavy metal-induced genotoxicity.

Micronuclei levels in mothers and their newborns from regions with different types of air pollution

The aim of this study was to analyze genetic damage in human lymphocytes measured using automated image analysis of micronuclei (MN) in a group of 178 mothers and their newborns from two locations in the Czech Republic. The concentrations of benzo[a]pyrene (B[a]P), particulate matter of aerodynamic diameter <2.5 μm (PM2.5) and benzene were measured by stationary monitoring in the winter season of 2008/2009 in the capital city of Prague and in Ceske Budejovice, a regional city in a rural area. The 3-month mean concentration of B[a]P before delivery was lower in Prague in comparison with Ceske Budejovice: 1.9 ± 0.5 ng/m 3 vs. 3.2 ± 0.2 ng/m 3 ( p < 0.001). The opposite trend was found for PM2.5 and benzene: 27.0 ± 2.5 μg/m 3 and 2.5 ± 0.5 μg/m 3 vs. 24.5 ± 0.7 μg/m 3 and 2.1 ± 0.8 μg/m 3 ( p < 0.001) for Prague vs. Ceske Budejovice, respectively. The average age of the mothers was 31 years (range, 18–49 years). The frequencies of MN per 1000 binucleated cells were 8.35 ± 3.06 vs. 6.47 ± 2.35 ( p < 0.001) for mothers from Prague and Ceske Budejovice, respectively, and 2.17 ± 1.32 vs. 3.82 ± 2.43 ( p < 0.001) for newborns from Prague and Ceske Budejovice, respectively. Other factors, including vitamin intake, exposure to tobacco smoke, body mass index (BMI) before pregnancy, the education level of the mothers and the impact of the mothers’ and fathers’ ages were analyzed in our study. The results suggest that the different sensitivity of the study groups to various mixtures of carcinogenic pollutants could be affected by significant differences in lifestyle factors. Possible higher genetic damage was analyzed in newborns of smoking mothers, and the birth weight of this group was 7.4% lower ( p < 0.05) in comparison with the newborns of nonsmoking mothers. No impact of the age of the mothers or fathers on MN frequency in the newborns was observed.

Asynchronous DNA replication detected by fluorescence in situ hybridisation as a possible indicator of genetic damage in human lymphocytes

The present study aimed to correlate the DNA replication timing of different genes with genetic damage and frequency of cancer. Using a fluorescence in situ hybridisation (FISH) approach, the replication timing of three loci, two human genes possessing transcriptional capability and involved in both the cellular response to genetic damage and cancer development (TP53 and RB1) and the non-coding locus D22S163, was evaluated. The data obtained show that normal human lymphocytes exposed in vitro to known DNA-damaging agents, e.g. H2O2, ionizing radiation and mitomycin C, exhibit an asynchronous replication of the genes TP53 and RB1. In vivo studies were performed in three different populations from Kazakhstan. In two of these populations that are living in polluted areas and have higher cancer mortalities than people living in a control area, a DNA replication behaviour similar to that observed in human lymphocytes exposed in vitro to known genotoxic agents was detected. The results obtained further indicate that DNA damage hampers replication and FISH represents a fast and accurate method of assessing asynchronous replication by providing an important tool to evaluate DNA damage at a populational level.

β-Carotene Reduces Bleomycin-Induced Genetic Damage in Human Lymphocytes

We had previously shown in a human feeding study that ingestion of tomato and carrot juices decreases DNA breaks and oxidized pyrimidine bases in peripheral lymphocytes and enhances expression of glutathione S-transferase (GST) in a subpopulation of the volunteers. The aim of this study was to determine how the major carotenoids of these juices (β-carotene or lycopene) could contribute to the observed antigenotoxicity. Physiological concentrations (2 μM) of water-soluble β-carotene and lycopene were incubated for 18–24 h with lymphocytes and then treated with bleomycin or H2O2. Strand breaks, oxidized DNA bases, and persistence of damage (DNA repair) were measured by single-cell microgelelectrophoresis. GST–protein (GSTP1) was determined using an immunoassay and by measuring enzyme activity. HPLC analysis showed that β-carotene was taken up by the cells after 24 h, and this was associated with a reduction of bleomycin-induced damage (29.11 ± 1.86% tail intensity without versus 21.54 ± 2.36% with β-carotene). Lycopene was ineffective. The carotenoids did not modulate repair of bleomycin- and H2O2-induced damage and did not alter levels of oxidized pyrimidine bases nor GST expression. The results indicate that β-carotene can enter the cell and protect against strand breaks but not against oxidized DNA bases. Therefore, β-carotene accounts for only part of the protection observed in vivo with carotenoid-rich vegetable juices.

Induction of genetic damage in human lymphocytes and mutations in Salmonella by trihalomethanes: role of red blood cells and GSTT1-1 polymorphism.

The brominated trihalomethanes (THMs) are mutagenic and carcinogenic disinfection by-products frequently found in chlorinated drinking water. They can be activated to mutagens by the product of the glutathione S-transferase-Theta (GSTT1++-1) gene in Salmonella RSJ100, which has been transfected with this gene. To evaluate this phenomenon in humans, we have examined the genotoxicity of a brominated THM, bromoform (BF), using the Comet assay in human whole blood cultures exposed in vitro. No differences were found in the comet tail length between cultures from GSTT1-1(+) versus GSTT1-1(-) individuals (1.67 +/- 0.40 and 0.74 +/- 0.54 microm/mM, respectively, P = 0.28). The high variability was due to the relatively weak induction of comets by BF. Combining the data from both genotypic groups, the genotoxic potency of BF was 1.20 +/- 0.34 microm/mM (P = 0.003). GSTT1-1 is expressed in red blood cells but not in the target cells (lymphocytes), and expression within the target cell (as in Salmonella RSJ100) may be necessary for enhanced mutagenesis in GSTT1-1(+) relative to GSTT1-1(-) cultures. To examine this, we exposed Salmonella RSJ100 and a control strain not expressing the gene (TPT100) to the most mutagenic brominated THM detected in Salmonella, dibromochloromethane (DBCM), either in the presence or absence of S9 or red blood cells from GSTT1-1(+) or GSTT1-1(-) individuals. S9 did not activate DBCM in the non-expressing strain TPT100, and it did not affect the ability of the expressing strain RSJ100 to activate DBCM. As with S9, red cells from either genotypic group were unable to activate DBCM in TPT100. However, red cells (whole or lysed) from both genotypic groups completely repressed the ability of the expressing strain RSJ100 to activate DBCM to a mutagen. Such results suggest a model in which exposure to brominated THMs may pose an excess genotoxic risk in GSTT1-1(+) individuals to those organs and tissues that both express this gene and come into direct contact with the brominated THM, such as the colon. In contrast, those organs to which brominated THMs would be transported via the blood might be protected by erythrocytes. Such a proposal is reasonably consistent with the organ specificity of drinking water-associated cancer in humans, which shows slightly elevated risks for cancer of the colon and bladder but not of the liver.

The use of the same image analysis system to detect genetic damage in human lymphocytes treated with doxorubicin in the Comet and fluoresence in situ hybridisation (FISH) assays

Two assays, the alkaline single cell gel electrophoresis (Comet) assay and the fluorescence in situ hybridisation (FISH) of a whole chromosome or `chromosome painting' assay have gained importance in recent years as witnessed by the increasing yield of scientific literature using these techniques. Thus, it would be useful to have one system to measure both endpoints. In the present communication, a cost-effective electronic imaging system developed by Kinetic Imaging Ltd., UK, has been used to measure, after treatment of human lymphocytes with doxorubicin, DNA damage in the Comet assay (using software package KOMET) and chromosome damage with the FISH technique (using software package KROMASCAN). The chromosome damage has been detected using chromoprobe™-M for chromosome 1 and compared with chromosome damage measured using the conventional Giemsa staining technique. In all three assays, cycling cells were treated, after phytohaemagglutinin stimulation, at 48 h for about 20 h, which resulted in statistically significant dose-related responses in each assay. In non-cycling cells there was no increase in damage in the Comet assay, but there was in the chromosome assays. The FISH assay was only conducted in cycling cells, since the probe used was metaphase-specific. At the highest doses of doxorubicin used, FISH and conventional chromosome assays had similar sensitivities.

The relationship between micronuclei in human lymphocytes and plasma levels of vitamin C, vitamin E, vitamin B12 and folic acid.

The cytokinesis-block micronucleus assay is increasingly being applied to the study of spontaneous or induced genetic damage in human lymphocytes, but little is known about dietary and other lifestyle factors that could influence this index. As part of a larger study investigating the role of dietary factors on baseline genetic damage in human lymphocytes from 152 non-smoking females and 113 non-smoking males evenly distributed between the ages of 20 and 87 years, we have measured (a) the micronucleus (MN) frequency and (b) the plasma level of the anti-oxidant vitamins C and E and the B vitamins folic acid and B12. Multiple regression analysis indicated that age (beta value = 0.598, P < 0.0001) was the most important factor influencing the variance of micronucleus frequency in females, while micronutrient levels had no apparent significant effects on genetic damage. In males age was also the predominant factor (beta value = 0.505, P < 0.0001) influencing genetic damage, but vitamin-C level also contributed positively and significantly to the observed MN frequency (beta value = 0.220, P < 0.0228). To avoid the potential confounding effect of collinearity between variables we also performed separate simple regression analysis for each plasma micronutrient in relation to age-adjusted micronucleus frequency; the results from this analysis again showed a significant and positive effect of plasma vitamin C on age-adjusted micronucleus frequency in males only (beta value = 0.188, P = 0.0503), while no effect was observed for the other micronutrients in both sexes. In view of the predominant age effect, we also focused on the data obtained in the youngest age groups of both sexes. In view of the predominant age effect, we also focused on the data obtained in the youngest age groups of both sexes (i.e. 20-30 years olds) and found (a) that the MN frequency in young males is significantly and positively correlated with plasma vitamin C levels (r = 0.823, P < 0.001) but negatively correlated with plasma vitamin B12 status (r = -0.799, P < 0.001) and (b) in females the only significant correlation was an inverse relationship between MN frequency and the combined folate and vitamin B12 plasma levels (r = -0.4632, P < 0.030).(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of nicotinic acid supplementation in vivo on oxygen radical-induced genetic damage in human lymphocytes

The ability of nicotinic acid to protect human lymphocytes in vivo against oxygen radical-induced DNA strand breakage was tested. NAD + concentrations rose progressively in lymphocytes to nearly 5 times baseline levels in human volunteers ingesting nicotinic acid (100 mg/day) for 8 weeks. Strand breaks decreased proportionately to NAD + concentrations over this time period in lymphocytes exposed to oxygen radicals. After 8 weeks of supplementation with nicotinic acid, radical-treated lymphocytes incubated for 24 h evidenced significantly less DNA damage compared to controls.