• Aspirin and ibuprofen, in bulk and nanoforms: effects on DNA damage in peripheral lymphocytes from breast cancer patients and healthy individuals

      Dandah, Osama M.M.; Najafzadeh, Mojgan; Isreb, Mohammad; Linforth, R.; Tait, C.; Baumgartner, Adolf; Anderson, Diana (2018-02)
      Regular use of non-steroidal anti-inflammatory drugs (NSAIDs) may be protective against tumours, including breast cancer. We have studied the effects of ibuprofen and aspirin on DNA damage in lymphocytes obtained from breast cancer patients and healthy female controls. Both nanoparticle (NPs) and bulk formulations were used in the comet and micronucleus (MN) assays. Non-toxic doses (250 ng/ml ibuprofen; 500 ng/ml aspirin) were tested. Aspirin, both bulk and nano formulations, significantly reduced DNA damage measured with the comet and micronucleus assays; the nano formulation was more effective. Ibuprofen was not effective in the comet assay but showed a significant reduction in MN frequency, with the nano formulation being more effective. NPs may have better penetration through the nuclear membrane relative to the bulk formulation. NSAIDs such as aspirin and ibuprofen may have a promising role in cancer prevention and treatment.
    • Comparison of DNA damage in human lymphocytes from healthy individuals and asthma, COPD and lung cancer patients treated in vitro / ex vivo with the bulk nano forms of aspirin and ibuprofen

      Najafzadeh, Mojgan; Ali, Aftab H.M.; Jacobe, B.; Isreb, Mohammad; Gopalan, Rajendran C.; Shang, Lijun (2015)
      Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzyme activity, a significant mechanism of action of NSAIDs. Inflammation is associated with increasing cancer incidence. Recent pre-clinical and clinical studies have shown that NSAID treatment could cause an anti-tumour effect in cancers. Such studies are lengthy and expensive. The present study, however, examined DNA damage in the Comet and micronucleus assays in peripheral blood lymphocytes of patients with respiratory diseases and healthy individuals using the nanoparticle (NP) and bulk versions of the NSAIDs, aspirin and ibuprofen. Lymphocytes are suitable surrogate cells for cancers and other disease states. DNA damage decreased in lymphocytes from healthy individuals, asthma, COPD and lung cancer patient groups after treatment with aspirin nano-suspension (ASP N) and ibuprofen nano-suspension (IBU N) compared to their bulk version (micro-suspension) in both assays. However, when ASP N was compared to untreated lymphocytes in all groups in the Comet assay, DNA damage significantly decreased in all groups, except the asthma group. When IBU N was compared to untreated lymphocytes, in healthy individuals and the lung cancer group, DNA damage decreased, but increased in asthma and COPD groups. Similarly, micronuclei (MNi) increased after ASP N and IBU N in the healthy individual and lung cancer groups, and decreased in asthma and COPD groups. Also shows that whilst there are basic similarities with different genetic endpoints in terms of nano and bulk forms, but highlights some differences between the disease states examined. Furthermore, lymphocyte responses after IBU N and ibuprofen bulk were investigated by patch-clamp experiments demonstrating that IBU N inhibited ion channel activity by 20%. This molecular epidemiology approach mirrors pre-clinical and clinical findings, and provides new information using nanoparticles.
    • Development and evaluation of nanoemulsion and microsuspension formulations of curcuminoids for lung delivery with a novel approach to understanding the aerosol performance of nanoparticles

      Al Ayoub, Yuosef; Gopalan, Rajendran C.; Najafzadeh, Mojgan; Mohammad, Mohammad A.; Anderson, Diana; Paradkar, Anant R.; Assi, Khaled H. (2019-02-25)
      Extensive research has demonstrated the potential effectiveness of curcumin against various diseases, including asthma and cancers. However, few studies have used liquid-based vehicles in the preparation of curcumin formulations. Therefore, the current study proposed the use of nanoemulsion and microsuspension formulations to prepare nebulised curcuminoid for lung delivery. Furthermore, this work expressed a new approach to understanding the aerosol performance of nanoparticles compared to microsuspension formulations. The genotoxicity of the formulations was also assessed. Curcuminoid nanoemulsion formulations were prepared in three concentrations (100, 250 and 500 µg/ml) using limonene and oleic acid as oil phases, while microsuspension solutions were prepared by suspending curcuminoid particles in isotonic solution (saline solution) of 0.02% Tween 80. The average fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD) of the nebulised microsuspension formulations ranged from 26% and 7.1 µm to 40% and 5.7 µm, for 1000 µg/ml and 100 µg/ml respectively. In a comparison of the low and high drug concentrations of the nebulised nanoemulsion, the average FPF and MMAD of the nebulised nanoemulsion formulations prepared with limonene oil ranged from 50% and 4.6 µm to 45% and 5.6 µm, respectively; whereas the FPF and MMAD of the nebulised nanoemulsion prepared with oleic acid oil ranged from 46% and 4.9 µm to 44% and 5.6 µm, respectively. The aerosol performance of the microsuspension formulations were concentration dependent, while the nanoemulsion formulations did not appear to be dependent on the curcuminoids concentration. The performance and genotoxicity results of the formulations suggest the suitability of these preparations for further inhalation studies in animals.
    • DNA Damage in Healthy Individuals and Respiratory Patients after Treating Whole Blood In vitro with the Bulk and Nano Forms of NSAIDs

      Najafzadeh, Mojgan; Normington, Charmaine; Jacob, B.K.; Isreb, Mohammad; Gopalan, Rajendran C.; Anderson, Diana (2016-09-28)
      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.
    • DNA damage in lymphocytes from healthy individuals and respiratory disease patients, treated ex vivo/in vitro with aspirin and ibuprofen nanoparticles compared to their bulk forms

      Anderson, Diana; Najafzadeh, Mojgan; Ali, Aftab H.M.; Jacobe, B.; Isreb, Mohammad; Gopalan, Rajendran C.; Shang, Lijun (2014)
    • 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)

      Habas, Khaled S.A.; Abdulmwli, Mhamoued; Demir, E.; Jacob, B.K.; Najafzadeh, Mojgan; Anderson, Diana (2018-10)
      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.
    • Effect of drinking water disinfection by-products in human peripheral blood lymphocytes and sperm

      Ali, Aftab H.M.; Kurzawa-Zegota, Malgorzata; Najafzadeh, Mojgan; Gopalan, Rajendran C.; Plewa, M.J.; Anderson, Diana (2014-12)
      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. 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. 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. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses.
    • Electrophysiological changes of the ion channels in human lymphocytes after nanoparticle exposure

      Shang, Lijun; Najafzadeh, Mojgan; Anderson, Diana (2014)
      Lymphocytes have many ion channels. These ion channels contribute to T cell-mediated autoimmune and/or inflammatory responses and therefore are targets for pharmacological immune modulation [1]. Lymphocytes are also suitable surrogate cells for cancer [2] and other diseases states [3] where inflammation is associated with increasing disease incidence. Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, have been associated with anti-tumour effects in cancers [4]. We recently compared DNA damage caused by the nanoparticle forms (NPs) of the NSAIDs, aspirin and ibuprofen and their bulk forms in peripheral blood lymphocytes of patients with respiratory diseases and healthy individuals in the Comet and micronucleus assays [5]. In this present study, we investigate electrophysiological changes from lymphocytes after NP exposure and compare these results with their DNA damage. 10 ml peripheral blood was collected from patients and healthy control individuals. Ethical permission was obtained from the Bradford Ethics Committee REC ref no: 09/H1313/37, ReDA no: 1202, and the University of Bradford ref no: 0405/8. Ibuprofen USP was purchased from Albermarle Europe sprl (Belgium). Pharmcoat 606 (HPMC) was kindly donated by Shinetsu (Japan). Aspirin and sodium lauryl sulphate were purchased from Sigma. Kollidon 30 (PVP K-30) was purchased from BASF (UK). Bulk and nano compound suspensions of aspirin and ibuprofen (IBU) were kindly prepared by Lena Nanoceutics (Bradford, UK). Whole blood collected from healthy individuals and cancer patients were treated for 30 mins with 500µg/ml of IBU bulk and nano forms separately. Whole-cell currents were recorded with normal patch clamping technique. The extracellular solution contained (in mM) the following: NaCl 125; KCl 5; MgCl2 1; CaCl2 2.5; HEPES 10; pH 7.4. The electrode internal solution contained (in mM) the following: KF 120; MgCl2 2; HEPES 10; EGTA 10; and CaCl2 1, pH 7.4. All experiments were carried out at room temperature. Compared with untreated cells, lymphocytes treated with IBU in NP form had lower whole-cell currents and the activities of ion channels were inhibited by 20% compared to those in bulk form. This result is mirrored by the DNA damage which occurred in lymphocytes after exposure to nanoparticles [5]. Although the intracellular biochemical mechanisms and ion channels involved in our nanoparticle toxicity remain to be determined, this study provides direct evidence that 500 μg/ml IBU in nano form can cause membrane damage to lymphocytes after a relatively short exposure. Such cytotoxicity of nanoparticles in lymphocytes may be associated with early membrane damage. Further detailed investigation is needed to explain the changes of lymphocytes in response to different concentrations of NPs in real time.
    • An evaluation of DNA damage in human lymphocytes and sperm exposed to methyl methanesulfonate involving the regulation pathways associated with apoptosis

      Habas, Khaled S.A.; Najafzadeh, Mojgan; Baumgartner, Adolf; Brinkworth, Martin H.; Anderson, Diana (2017-10)
      Exposure to DNA-damaging agents produces a range of stress-related responses. These change the expression of genes leading to mutations that cause cell cycle arrest, induction of apoptosis and cancer. We have examined the contribution of haploid and diploid DNA damage and genes involved in the regulation of the apoptotic process associated with exposure, The Comet assay was used to detect DNA damage and quantitative RT-PCR analysis (qPCR) to detect gene expression changes in lymphocytes and sperm in response to methyl methanesulfonate. In the Comet assay, cells were administered 0–1.2 mM of MMS at 37 °C for 30 min for lymphocytes and 32 °C for 60 min for sperm to obtain optimal survival for both cell types. In the Comet assay a significant increase in Olive tail moment (OTM) and % tail DNA indicated DNA damage at increasing concentrations compared to the control group. In the qPCR study, cells were treated for 4 h, and RNA was isolated at the end of the treatment. qPCR analysis of genes associated with DNA stress responses showed that TP53 and CDKN1A are upregulated, while BCL2 is downregulated compared with the control. Thus, MMS caused DNA damage in lymphocytes at increasing concentrations, but appeared not to have the same effect in sperm at the low concentrations. These results indicate that exposure to MMS increased DNA damage and triggered the apoptotic response by activating TP53, CDKN1A and BCL2. These findings of the processing of DNA damage in human lymphocytes and sperm should be taken into account when genotoxic alterations in both cell types are produced when monitoring human exposure.
    • Ex vivo/in vitro protective effect of myricetin bulk and nano-forms on PhIP-induced DNA damage in lymphocytes from healthy individuals and pre-cancerous MGUS patients

      Akhtar, Shabana; Najafzadeh, Mojgan; Isreb, Mohammad; Newton, L.; Gopalan, Rajendran C.; Anderson, Diana (2020-07)
      2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) is a central dietary mutagen, produced when proteinaceous food is heated at very high temperatures potentially causing DNA strand breaks. This study investigates the protective potential of a well-researched flavonoid, myricetin in its bulk and nano-forms against oxidative stress induced ex vivo/in vitro by PhIP in lymphocytes from pre-cancerous monoclonal gammopathy of undetermined significance (MGUS) patients and those from healthy individuals. The results from the Comet assay revealed that in the presence of myricetin bulk (10 µM) and myricetin nano (20 µM), the DNA damage caused by a high dose of PhIP (100 µM) was significantly (P < 0.001) reduced in both groups. However, nano has shown better protection in lymphocytes from pre-cancerous patients. Consistent results were obtained from the micronucleus assay where micronuclei frequency in binucleated cells significantly decreased upon supplementing PhIP with myricetin bulk (P < 0.01) and myricetin nano (P < 0.001), compared to the PhIP treatment alone. To briefly determine the cellular pathways involved in the protective role of myricetin against PhIP, we studied gene expression of P53 and ATR kinase (ATM- and Rad3-related), using the real-time PCR technique.
    • Genotoxic Effects in Peripheral Blood and Sperm in Humans in Healthy Individuals and Those with Disease States

      Anderson, Diana; Baumgartner, Adolf; Najafzadeh, Mojgan (2018-05-01)
      The Comet assay is one of the most versatile tools in toxicology today and can be used to measure responses in both diploid (peripheral blood lymphocytes) and haploid (sperm) primary cells in humans. This chapter will discuss how these cells are employed to determine if they have differential responses to chemical and physical agents in healthy and disease-affected individuals and how such information can be of use to man.
    • An in vitro investigation into the protective and genotoxic effects of myricetin bulk and nano forms in lymphocytes of MGUS patients and healthy individuals

      Akhtar, Shabana; Najafzadeh, Mojgan; Isreb, Mohammad; Newton, L.; Gopalan, Rajendran C.; Anderson, Diana (2020-07)
      The present study investigated the genoprotective and genotoxic effects of myricetin bulk (10 μM) and nano forms (20 μM) in the lymphocytes from pre-cancerous, monoclonal gammopathy of unknown significance (MGUS) patients and healthy individuals using the Comet and micronucleus assays. The study also evaluated the effect of myricetin on P53 expression levels, using the Western blot technique. Results showed that throughout the in-vitro treatment, lymphocytes from the patients group had higher levels of baseline DNA damage compared to the healthy group. Myricetin in both forms induced significant DNA damage, only at higher concentrations (>40 μM). The micronucleus assay showed a significant reduction (P < 0.01) in the frequency of micronuclei in mono-nucleated cells in the patient group treated with the nano form of myricetin at the non-toxic dose of 20 μM. There was a significant increase in both gene and protein P53 levels in lymphocytes isolated from healthy individuals and pre-cancerous patients. These results suggested a protective effect of myricetin and indicated its nutritional supplement potential for protection against cancer development among patients suffering from MGUS.
    • Inhibition of survivin expression after using oxaliplatin and vinflunine to induce cytogenetic damage in vitro in lymphocytes from colon cancer patients and healthy individuals

      Alotaibi, Amal; Najafzadeh, Mojgan; Davies, J.; Baumgartner, Adolf; Anderson, Diana (2017-10-17)
      Chemotherapy drugs usually inflict a lethal dose to tumour cells with the consequence that these cells are being killed by cell death. However, each round of chemotherapy also causes damage to normal somatic cells. The DNA cross-linking agent oxaliplatin which causes DNA double-strand breaks and vinflunine which disrupts the mitotic spindle are two of these chemotherapy drugs which were evaluated in vitro using peripheral lymphocytes from colorectal cancer patients and healthy individuals to determine any differential response. Endpoints examined included micronucleus (MN) induction using the cytokinesis-blocked micronucleus (CBMN) assay and pancentromeric fluorescence in situ hybridisation. Also, survivin expression was monitored since it regulates the mitotic spindle checkpoint and inhibits apoptosis. Oxaliplatin produced cytogenetic damage (MN in binucleated cells) via its clastogenic but also previously unknown aneugenic action, possibly through interfering with topoisomerase II, whilst vinflunine produced MN in mononucleated cells because of incomplete karyokinesis. Survivin expression was found to be significantly reduced in a concentration-dependent manner by not only oxaliplatin but surprisingly also vinflunine. This resulted in large numbers of multinucleated cells found with the CBMN assay. As survivin is upregulated in cancers, eliminating apoptosis inhibition might provide a more targeted chemotherapy approach; particularly, when considering vinflunine, which only affects cycling cells by inhibiting their mitotic spindle, and alongside possibly other pro-apoptotic compounds. Hence, these newly found properties vinflunine – the inhibition of survivin expression - might demonstrate a promising chemotherapeutic approach as vinflunine induces less DNA damage in normal somatic cells compared to other chemotherapeutic compounds.
    • Nanoparticles in Biomedicine and Medicine, and Possible Clinical Toxicological Application of Peripheral Lymphocytes in the Risk Assessment Process for Susceptible Disease State Individuals

      Najafzadeh, Mojgan; Anderson, Diana (2017-11-13)
      Nanoparticle usage has emerged in the medical field as a technology well-suited to the diagnosis and treatment of a variety of disease states. The distinctive characteristics of engineered nanoparticles (ENPs) such as higher surface-area-to-volume ratios find various applications in personal care products, food packaging, drug-delivery systems, therapeutics, biosensors and others. The exponential increase in ENP-containing consumer products in the last decade has also increased their inadvertent release into the environment and the debate relating to their adverse effects on human and environmental health. The use of NPs for different functions in human studies has significantly increased the application of NPs in biomedicine, for instance, imaging of cell and tissues, drug delivery and sensing of target molecules. These nanomaterials have been investigated for the treatment and detection of various pathological conditions. There are suitable biological systems now available in man using peripheral blood lymphocytes to determine the effect of NPs in various disease states.
    • ROS-induced Oxidative Damage in Lymphocytes Ex Vivo/in Vitro From Healthy Individuals and MGUS Patients: Protection by Myricetin Bulk and Nanoforms

      Akhtar, Shabana; Najafzadeh, Mojgan; Isreb, Mohammad; Newton, L.; Gopalan, Rajendran C.; Anderson, Diana (2020-04)
      We investigated the protective role of myricetin bulk and nanoforms, against reactive oxygen species (ROS)-induced oxidative stress caused by hydrogen peroxide and tertiary-butyl hydro peroxide in lymphocytes in vitro from healthy individuals and those from pre-cancerous patients suffering with monoclonal gammopathy of undetermined significance (MGUS). The change in intracellular reactive oxygen species was measured once cells were treated with myricetin bulk forms and nanoforms with and without either hydrogen peroxide or tertiary-butyl hydro peroxide co-supplementation. The direct and indirect antioxidant activity of myricetin was spectrofluometrically measured using the fluorescent dye 2',7'-dichlorofluorescin diacetate and using the Comet assay, respectively. Hydrogen peroxide (50 µM) and tertiary-butyl hydro peroxide (300 µM) induced a higher level of reactive oxygen species-related DNA damage and strand breaks. Addition of myricetin nanoform (20 µM) and bulk (10 µM) form could, however, significantly prevent hydrogen peroxide- and tertiary-butyl hydro peroxide-induced oxidative imbalances and the nanoform was more effective. Glutathione levels were also quantified using a non-fluorescent dye. Results suggest that myricetin treatment had no significant effect on the cellular antioxidant enzyme, glutathione. The current study also investigates the effect of myricetin on the induction of double-strand breaks by staining the gamma-H2AX foci immunocytochemically. It was observed that myricetin does not induce double-strand breaks at basal levels rather demonstrated a protective effect.
    • Sensitivity and specificity of the empirical lymphocyte genome sensitivity (LGS) assay: implications for improving cancer diagnostics

      Anderson, Diana; Najafzadeh, Mojgan; Gopalan, Rajendran C.; Ghaderi, Nader; Scally, Andy J.; Britland, Stephen T.; Jacobs, B.J.; Reynolds, P.D.; Davies, J.; Wright, A.L.; et al. (2014-10-28)
      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.
    • 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

      Alotaibi, Amal; Bhatnagar, P.; Najafzadeh, Mojgan; Gupta, K.C.; Anderson, Diana (2013)
      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. 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. 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. 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.
    • TiO2 NPs induce DNA damage in lymphocytes from healthy individuals and patients with respiratory diseases-An Ex vivo/In vitro Study

      Osman, Ilham F.; Najafzadeh, Mojgan; Sharma, Vyom; Shukla, Ritesh K; Jacob, B.K.; Dhawan, A.; Anderson, Diana (2018-01-01)
      Nanotechnology has preceded nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. 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 (ns). 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. All results were achieved in the absence of cytotoxicity.
    • The use of isolated peripheral lymphocytes and human whole blood in the comet assay

      Najafzadeh, Mojgan; Anderson, Diana (2016-10-27)
      The comet assay is a sensitive method used to detect DNA damage, measuring DNA breaks and alkali labile lesions in eukaryotic cells. Here, the use of whole blood in the alkaline gel electrophoresis method is described. Two hundred and seventy blood samples from individuals were examined: 120 healthy individuals, 65 suspected or pre-cancerous individuals and 85 cancer patients. Each sample was divided into two identical volumes in different falcon tubes. The blood was prepared and stored by adding the same amount of RPMI medium and 10% DMSO. Using the Student’s t-Test, the data showed a p value = 0.59 for Olive tail moment (OTM) and 0.16 for % tail DNA, and no statistically significant differences between the two methods, with or without treatment. In conclusion, using whole blood instead of isolated lymphocytes saves time, is still very sensitive and requires less than 20 µL of blood from each individual.
    • Using a Modified Lymphocyte Genome Sensitivity (LGS) Test or TumorScan Test to Detect Cancer at an Early Stage in Each Individual

      Anderson, Diana; Najafzadeh, Mojgan; Scally, Andy J.; Jacob, B.K.; Griffith, John; Chaha, R.; Linforth, R.; Soussaline, M.; Soussaline, F. (2019-01-03)
      Our previous case-control study observed isolated lymphocytes from 208 individuals and determined the differences in the sensitivity to genomic damage of lymphocytes derived from cancer patients, pre/suspect cancer patients and healthy volunteers using the Comet assay (Anderson et al, 2014). We adapted the LGS technique using a slightly different method and examined 700 more blood samples from 598 patients with cancer or suspected cancer and 102 healthy individuals. To help increase the sensitivity of the test and detect cancer at the level of each individual, we joined with the IMSTAR team who analysed our cells with their fully automated Pathfinder™ cell reader-analyser system. With this reading and analysis system 4,000 to 10,000 cells were able to be read per slide. The new test which is called TumorScan is a highly sensitive test to detect any cancer at an early stage through the response of the white blood cells to UV treatment. These patient blood samples have also been collected at the stage before confirming diagnosis and treatment. There were four of these individuals with cancer who had received anti-cancer treatment. The results from these patients showed a reverse pattern compared to non-treated cancer patients and followed the pattern seen in healthy individuals. The results are consistent with the early results as reported in the above 2014 paper. Given the results from these samples were in a particularly challenging subgroup, whose cancer status was difficult to distinguish, the data suggest that the technique using the TumorScan system could exceed the area under the ROC curve >93% obtained in the earlier study on a group basis, whereas this present study was to detect cancer at an early stage in each individual.