Mojgan Najafzadeh

The protective effect of the flavonoids on food-mutagen-induced DNA damage in peripheral blood lymphocytes from colon cancer patients

The food mutagens IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) are heterocyclic amines (HCA), generated when heating proteinaceous food. This study investigates the protective potential of the flavonoids quercetin (Q) and rutin (R) against oxidative stress induced in vitro by IQ and PhIP in lymphocytes from healthy individuals and untreated, newly diagnosed colon cancer patients using the Comet assay. In the presence of up to 500μM Q and R, the DNA damage resulting from a high dose of PhIP (75μM) or IQ (150μM) was significantly reduced (P<0.001) to levels comparable to six times lower IQ or 7.5 times lower PhIP doses. Lymphocytes from colon cancer patients had greater baseline DNA damage than those from healthy individuals (P<0.01) and this higher level of damage was also observed throughout in vitro treatment. Except for the >50years of age group and male gender, confounding factors such as smoking, drinking and/or dietary habits were not found to be significant. In conclusion, flavonoids reduced oxidative stress caused by food mutagens in vitro in lymphocytes of healthy individuals and colon cancer patients. Thus, dietary supplementation with flavonoid-rich vegetables and fruits may prove very effective in protecting against oxidative stress.

Flavonoids inhibit the genotoxicity of hydrogen peroxide (H2O2) and of the food mutagen 2-amino-3-methylimadazo[4,5-f]-quinoline (IQ) in lymphocytes from patients with inflammatory bowel disease (IBD)

Inflammatory bowel disease (IBD) including Crohns disease (CD) and ulcerative colitis (UC) is a chronic inflammatory gastrointestinal autoimmune condition with an inappropriate immune response. We investigated DNA damage induced in vitro in lymphocytes from IBD patients caused by oxidative stress through H(2)O(2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and whether the plant flavonoids, quercetin and epicatechin, found in fruits, tea and soybeans could effectively reduce such stress. Lymphocytes from IBD patients and healthy volunteers were treated with 50 microg/ml H(2)O(2) or IQ in the presence of quercetin (0-250 microg/ml) or epicatechin (0-100 microg/ml). Flavonoid supplementation (250 microM quercetin or 100 microM epicatechin) caused an overall significant decrease of induced DNA damage resulting in a 48.6% (P < 0.001) reduction of H(2)O(2)-induced and a 43% (P < 0.001) reduction of IQ-induced DNA damage within the patient groups; for the control groups, reductions in DNA damage were 35.2 and 57.1%, respectively (both, P < 0.001). There was less induced DNA damage within lymphocytes from UC patients compared to CD patients for both series of experiments (H(2)O(2) and quercetin, IQ and epicatechin). In conclusion, flavonoids dramatically reduced oxidative stress in vitro in lymphocytes from IBD patients and healthy individuals. Thus, flavonoids could be very effective in the treatment of oxidative stress and encouraged in the diet of IBD patients.

Zinc oxide nanoparticles affect the expression of p53, Ras p21 and JNKs: an ex vivo/in vitro exposure study in respiratory disease patients

Zinc oxide (ZnO) nanoparticles are the mostly used engineered metal oxide nanoparticles in consumer products. This has increased the likelihood of human exposure to this engineered nanoparticle (ENPs) through different routes. At present, the majority of the studies concerning ZnO ENPs toxicity have been conducted using in vitro and in vivo systems. In this study, for the first time we assessed the effect of ZnO ENPs on the major cellular pathways in the lymphocytes of healthy individuals as well as in susceptible patients suffering from lung cancer, chronic obstructive pulmonary disease (COPD) and asthma. Using the differential expression analysis, we observed a significant (P < 0.05) dose-dependent (10, 20 and 40 µg/ml for 6h) increase in the expression of tumour suppressor protein p53 (40, 60 and 110%); Ras p21 (30, 52 and 80%); c-Jun N-terminal kinases; JNKs) (28, 47 and 78%) in lung cancer patient samples treated with ZnO ENPs compared to healthy controls. A similar trend was also seen in COPD patient samples where a significant (P < 0.05) dose-dependent increase in the expression of tumour suppressor protein p53 (26, 45 and 84%), Ras p21 (21, 40 and 77%), JNKs (17, 32 and 69%) was observed after 6h of ZnO ENPs treatment at the aforesaid concentrations. However, the increase in the expression profile of tested protein was not significant in the asthma patients as compared to controls. Our results reiterate the concern about the safety of ZnO ENPs in consumer products and suggest the need for a complete risk assessment of any new ENPs before its use.

Tea phenols in bulk and nanoparticle form modify DNA damage in human lymphocytes from colon cancer patients and healthy individuals treated in vitro with platinum-based chemotherapeutic drugs

BACKGROUND Tea catechin epigallocatechin-3-gallate (EGCG) and other polyphenols, such as theaflavins (TFs), are increasingly proving useful as chemopreventives in a number of human cancers. They can also affect normal cells. The polyphenols in tea are known to have antioxidant properties that can quench free radical species, and pro-oxidant activities that appear to be responsible for the induction of apoptosis in tumor cells. The bioavailability of these natural compounds is an important factor that determines their efficacy. Nanoparticle (NP)-mediated delivery techniques of EGCG and TFs have been found to improve their bioavailability to a level that could benefit their effectiveness as chemopreventives. AIM The present study was conducted to compare the effects of TFs and EGCG, when used in the bulk form and in the polymer (poly[lactic-co-glycolic acid])-based NP form, in oxaliplatin- and satraplatin-treated lymphocytes as surrogate cells from colorectal cancer patients and healthy volunteers. MATERIALS & METHODS NPs were examined for their size distribution, surface morphology, entrapment efficiency and release profile. Lymphocytes were treated in the Comet assay with oxaliplatin and satraplatin, washed and treated with bulk or NP forms of tea phenols, washed and then treated with hydrogen peroxide to determine single-strand breaks after crosslinking. RESULTS The results of DNA damage measurements by the Comet assay revealed opposite trends in bulk and NP forms of TFs, as well as EGCG. Both the compounds in the bulk form produced statistically significant concentration-dependent reductions in DNA damage in oxaliplatin- or satraplatin-treated lymphocytes. In contrast, when used in the NP form both TFs and EGCG, although initially causing a reduction, produced a concentration-dependent statistically significant increase in DNA damage in the lymphocytes. DISCUSSION These observations support the notion that TFs and EGCG act as both antioxidants and pro-oxidants, depending on the form in which they are administered under the conditions of investigation.

In vitro sensitivities to UVA of lymphocytes from patients with colon and melanoma cancers and precancerous states in the micronucleus and the Comet assays

To use lymphocytes as surrogate cells to investigate their in vitro sensitivities to ultraviolet (UV) treatment in different cancers and precancerous states by comparison with lymphocytes from healthy control individuals was the main aim of this research. UV light induces precise cellular and genomic mutations. In this study, the effect of ultraviolet A (UVA) (320-400 nm) was used as a generic mutagen to evaluate in vitro different sensitivities from lymphocytes of patients with suspected melanoma (SM), malignant melanoma (MM), polyposis coli (PC) and colorectal cancer (CRC). DNA damage was evaluated by two different methods: the micronucleus (MN) assay and the Comet assay. The baseline frequency of MNs was significantly increased in lymphocytes from all patients (SM, MM, PC and CRC) when compared to healthy individuals. After UV irradiation, MN frequencies were significantly increased in lymphocytes of all groups, both patients and healthy individuals. However, the MN frequency in all patient groups was significantly higher than in the healthy individual group. Similar results for the induction of genomic DNA damage were obtained for the Comet assay. Also for the Comet assay, UVA-induced DNA damage for all four patient groups was significantly increased when compared to healthy individuals (SM, MM, PC and CRC groups: P < 0.001). Conclusively, peripheral lymphocytes from patients with cancers MM and CRC or precancerous states SM and PC are more sensitive to a generic mutagen such as UVA than lymphocytes from healthy individuals. This feature may be used as an essential biomarker to screen and diagnose precancerous states and cancers in early stages.

Sensitivity and specificity of the empirical lymphocyte genome sensitivity (LGS) assay: implications for improving cancer diagnostics

Lymphocyte responses from 208 individuals: 20 with melanoma, 34 with colon cancer, and 4 with lung cancer (58), 18 with suspected melanoma, 28 with polyposis, and 10 with COPD (56), and 94 healthy volunteers were examined. The natural logarithm of the Olive tail moment (OTM) was plotted for exposure to UVA through 5 different agar depths (100 cell measurements/depth) and analyzed using a repeated measures regression model. Responses of patients with cancer plateaued after treatment with different UVA intensities, but returned toward control values for healthy volunteers. For precancerous conditions and suspected cancers, intermediate responses occurred. ROC analysis of mean log OTMs, for cancers plus precancerous/suspect conditions vs. controls, cancer vs. precancerous/suspect conditions plus controls, and cancer vs. controls, gave areas under the curve of 0.87, 0.89, and 0.93, respectively (P<0.001). Optimization allowed test sensitivity or specificity to approach 100% with acceptable complementary measures. This modified comet assay could represent a stand‐alone test or an adjunct to other investigative procedures for detecting cancer.—Anderson, D., Najafzadeh, M., Gopalan, R., Ghaderi, N., Scally, A. J., Britland, S. T., Jacobs, B. K., Reynolds, P. D., Davies, J., Wright, A. L., Al‐Ghazal, S., Sharpe, D., Denyer, M. C., Sensitivity and specificity of the empirical lymphocyte genome sensitivity (LGS) assay: implications for improving cancer diagnostics. FASEB J. 28, 4563–4570 (2014). www.fasebj.org

Effect of drinking water disinfection by-products in human peripheral blood lymphocytes and sperm.

BACKGROUND Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. OBJECTIVES To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. METHODS The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. RESULTS In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. CONCLUSION These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses.

DNA Damage in Healthy Individuals and Respiratory Patients after Treating Whole Blood In vitro with the Bulk and Nano Forms of NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzyme activity which affects the inflammatory response. Inflammation is associated with increasing cancer incidence. Pre-clinical and clinical studies have shown that NSAID treatment could cause an anti-tumor effect in cancers. In the present study, blood was taken from healthy individuals (n = 17) and patients with respiratory diseases or lung cancer (n = 36). White blood cells (WBC) were treated with either a micro-suspension, i.e., bulk (B) or nano-suspension (N) of aspirin (ASP) or ibuprofen (IBU) up to 500 μg/ml in the comet assay and up to 125 μg/ml in the micronucleus assay. In this study results were compared against untreated lymphocytes and their corresponding treated groups. The results showed, that NSAIDs in their nano form significantly reduced the DNA damage in WBCs from lung cancer patients in bulk and nano compared to untreated lymphocytes. Also, there was a decrease in the level of DNA damage in the comet assay after treating WBCs from healthy individuals, asthma and COPD groups with aspirin N (ASP N) but not with IBU N. In addition, the number of micronuclei decreased after treatment with NSAIDs in their nano form (ASP N and IBU N) in the healthy as well as in the lung cancer group. However, this was not the case for micronucleus frequency in asthma and COPD patients. These data show that lymphocytes from different groups respond differently to treatment with ASP and IBU as measured by comet assay and micronucleus assay, and that the size of the suspended particles of the drugs affects responses.

TiO2 NPs Induce DNA Damage in Lymphocytes from Healthy Individuals and Patients with Respiratory Diseases—An Ex Vivo/In Vitro Study

Little is known of the effects of nanoparticles in human systems, let alone in diseased individuals and nanotechnology has preceded nanotoxicology. Therefore, the effects of titanium dioxide (TiO2) nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases [lung cancer, chronic obstructive pulmonary disease (COPD) and asthma] were compared with those in healthy Individuals, to determine differences in sensitivity to nanochemical insult. The Comet assay was performed according to recommended guidelines. The micronucleus assay and ras oncoprotein detection were conducted according to published standard methods. The results showed statistically significant concentration-dependent genotoxic effects of TiO2 NPs in both respiratory patient and control groups in the Comet assay. The TiO2 NPs caused DNA damage in a concentration dependent manner in both groups (respiratory and healthy controls) with the exception of the lowest TiO2 concentration (10 μg/ml) which did not induce significant damage in healthy controls (n.s). When OTM data were used to compare the whole patient group and the control group, the patient group had more DNA damage (p > 0.001) with the exception of 10 μg/ml of TiO2 that caused less significant damage to patient lymphocytes (p < 0.05). Similarly, there was an increase in the pattern of cytogenetic damage measured in the MN assay without statistical significance except when compared to the negative control of healthy individuals. Furthermore, when modulation of ras p21 expression was investigated, regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels. Results were achieved in the absence of cytotoxicity.

DNA damage protection by bulk and nano forms of quercetin in lymphocytes of patients with chronic obstructive pulmonary disease exposed to the food mutagen 2-amino-3-methylimidazo [4,5-f]quinolone (IQ)

ABSTRACT Chronic obstructive pulmonary disease (COPD) in humans, describes a group of lung conditions characterised by airflow limitation that is poorly reversible. The airflow limitation usually progresses slowly and is related to an abnormal inflammatory response of the lung to toxic particles. COPD is characterised by oxidative stress and an increased risk of lung carcinoma. The 2‐amino‐3‐methylimidazo [4,5‐f]quinoline (IQ) is one of a number of mutagenic/carcinogenic heterocyclic amines found mainly in well‐cooked meats which are thus part of the regular diet. Antioxidants are very important in order to protect the cells against oxidative damage. The aim of the present study was to assess the effects of IQ on the level of DNA damage and susceptibility to a potent mutagen in peripheral blood cells of COPD patients. DNA damage and the frequency of micronuclei (MNi) were evaluated using the Comet and micronucleus assays, respectively. Differential expressions of both mRNA and protein of the endogenous antioxidant enzyme catalase were evaluated with quantitative polymerase chain reaction (qPCR) and Western blot analysis, respectively. Furthermore, the effect of bulk and nano forms of quercetin and their combination with IQ were examined. Results of the present study clearly demonstrated that MNi frequency in the peripheral blood lymphocytes exhibited a positive correlation with the DNA damage as evident from the different Comet assay parameters. Increase of the endogenous antioxidant catalase also showed there was a stimulation of this enzyme system by IQ. Whereas, the endogenous antioxidant quercetin significantly reduced oxidative stress in COPD patients and healthy individuals. HighlightsIn COPD patients and healthy individuals IQ increased DNA damage and frequency of micronuclei.Induction of catalase indicates a stimulation of the antioxidant enzyme system.Quercetin significantly reduced oxidative stress in COPD patients and healthy individuals.The bulk version of quercetin was compared directly to its nano form which caused greater reduction.