Jawahar Singh Adhikari

Flow-Cytometric Analysis of Reactive Oxygen Species in Peripheral Blood Mononuclear Cells of Patients with Thyroid Dysfunction

Thyroid hormones are major regulators of energy metabolism and increased levels of the hormones (hyperthyroidism) results in an increase in the metabolic rate. Thyroid dysfunction causing alteration in hormone secretion leads to perturbations in the metabolic status. The hypermetabolic state may cause increased generation of reactive oxygen species (ROS), leading to oxidative stress in these patients. This study was carried out to verify our proposition by measuring the ROS in the terminally differentiated cells like the peripheral blood mononuclear cells of the patients.

Acetoxy-4-methylcoumarins confer differential protection from aflatoxin B1-induced micronuclei and apoptosis in lung and bone marrow cells

The ability of various acetoxy derivatives of 4-methylcoumarins to inhibit the genotoxic changes due to aflatoxin B(1) (AFB(1)) is reported here. Several 4-methylcoumarins (test compounds), such as 7,8-diacetoxy-4-methylcoumarin (DAMC), monoacetoxy-4-methylcoumarin (MAC), 5-N-acetyl-6-acetoxy-4-methylcoumarin (NAMC) and 7,8-dihydroxy-4-methylcoumarin (DHMC) were separately administered intraperitoneally (i.p.) to male wistar rats followed by AFB(1) administration i.p. or intratracheally (i.t.) (2-8 mg/kg b.wt.) and another dose of the test compound. The animals were sacrificed 26h after AFB(1) administration. From animals receiving AFB(1) i.p., bone marrow (BM) cells were isolated and stained with Mayers haematoxylin and eosin. Micronuclei (MN) in BM were scored by light microscopy. From animals receiving AFB(1) i.t., bronchoalveolar lavage (BAL) was obtained, lung cells (LG) were isolated and stained with fluorochrome 6-diamidino-2-phenylindole (DAPI) for the analysis of MN, apoptotic bodies (AP) and cell cycle variations. Rats were separately treated with the vehicle DMSO to serve as the proper control. AFB(1) caused significant dose-dependent induction of MN in BM as well as LG. AP were observed in LG of rats receiving AFB(1) and was found to correlate with MN induction. DAMC injection caused significant decrease in AP due to AFB(1) in LG and MN in both BM and LG. The effectiveness of MAC was approximately half that of DAMC, thereby indicating that number of acetoxy groups on the coumarin molecule determine the efficacy. The fact that NAMC had no effect either on MN or AP indicate that neither acetoxy group at C-6 nor the N-acetyl group at C-5 facilitate the transfer of acetyl group to P-450 required for inhibition of AFB(1)-epoxidation. DHMC, the deacetylated product of DAMC had no normalizing effect on the induction of MN and AP. These findings confirm our earlier hypothesis that DAMC-mediated acetylation of microsomal P-450 (catalysing epoxidation of AFB(1)) through the action of microsomal transacetylase is responsible for the protective action of DAMC. The relative number and position of acetoxy groups on the coumarin nucleus determine the specificity to the transacetylase necessary for the chemopreventive action.

Low-dose radiation therapy of cancer: role of immune enhancement

The efficacy of conventional radiation therapy, one of the most widely used treatment modalities of cancer, is limited by resistance of tumors as well as normal tissue toxicity. In the last decade, several studies have shown that protocols using low-dose radiation (LDR) are more effective in providing local tumor control with negligible normal tissue toxicity. LDR stimulates antioxidant capacity, repair of DNA damage, apoptosis and induction of immune responses, which might be collectively responsible for providing effective local tumor control. This article focuses on the immunostimulatory effects of LDR in in vivo models and its clinical efficacy, supporting the use of LDR regimens (alone or as adjuvant) as an anticancer treatment.

Tumoricidal effects of etoposide incorporated into solid lipid nanoparticles after intraperitoneal administration in Dalton's lymphoma bearing mice

The tumoricidal effects of etoposide incorporated into lipid nanoparticles after single-dose administration were investigated in Daltons lymphoma ascites bearing mice. Etoposide and its nanoparticle formulations were administered intraperitoneally, and the cell cycle perturbation, cytogenetic damage, cell death (apoptosis), tumor regression, and animal survival were investigated as parameters of response with time. The tumor burden of mice treated with etoposide and its nanoparticle formulations decreased significantly (P<.001) compared with the initial up to 4 to 6 days, followed by an increase at later time intervals. Of the 3 different formulations, the survival time of mice was higher when treated with etoposide-loaded tripalmitin (ETP) nanoparticles, followed by etoposide-loaded glycerol monostearate (EGMS) (27.3%) and etoposide-loaded glycerol distearate (EGDS) (27.3%) compared with free etoposide. Cell cycle analysis revealed the hypodiploid peak (sub G0/G1 cell population) as well as G2 arrest in mice treated with etoposide and its nanoparticle formulations. The frequency of dead cells treated with the nanoparticle formulations remained high even after 8 days of treatment compared with free etoposide. The mice treated with nanoparticle formulations exhibited hypodiploid peaks and reduced S phase even 8 days after treatment, whereas the free etoposide-treated mice showed decrease in apoptosis after 3 days of treatment. The apoptotic frequency in cells 17 days after treatment was in the order of ETP>EGMS>EGDS> etoposide. The experimental results indicated that among the 3 nanoparticle formulations studied, the ETP nanoparticles showed greater and prolonged apoptotic induction properties, resulting in the higher increase in survival time of tumor bearing mice.

Chemoprevention of benzene-induced bone marrow and pulmonary genotoxicity.

Our earlier studies documented the ability of 7,8-diacetoxy-4-methylcoumarin (DAMC) to cause irreversible inhibition of cytochrome P-450 linked mixed function oxidases (MFO) mediated by membrane bound DAMC: protein transacetylase. Since P-450 catalyzed oxidation of benzene is crucial to its toxic effects, the action of DAMC and related analogues were considered promising in preventing the genotoxicity due to benzene. For this purpose rats were pretreated with various acetoxy-4-methylcoumarins (test compounds), which was followed by the administration of benzene either intratracheally (IT) or intraperitoneally (IP), and sacrificed 26 h after the injection of benzene. The incidence of micronuclei (MN) in bone marrow (BM) and lung (LG) were assessed by light and fluorescent microscopy, respectively. A dose-dependent induction of MN in BM and LG cells was observed in rats administered with benzene. A significant reduction in benzene-induced MN in BM and LG was observed as a result of DAMC administration to rats; a higher dose of DAMC resulted in greater inhibition of clastogenic action of benzene as revealed by MN incidence. 7,8-dihydroxy-4-methylcoumarin (DHMC), the deacetylated product of DAMC, demonstrated relatively lesser potency to inhibit the clastogenic action of benzene. This observation is consistent with the ability of DAMC to inhibit the formation of benzene oxide as well as to scavenge the oxygen radicals formed during the course of benzene metabolism. The fact that DHMC can only scavenge the oxygen radicals and is ineffective in inhibiting benzene oxidation in vivo explains the reduced capability of dihydroxy coumarin to prevent MN due to benzene. 7-Acetoxy-4-methylcoumarin (MAC) inhibits the MN due to benzene being roughly 50% of that produced by DAMC. DAMC is also effective in normalizing the cell cycle alterations produced by benzene in BM and LG. These observations further substantiate our hypothesis that the biological effects of acetoxy coumarins are mediated by the action of membrane bound transacetylase that catalyzes the acetylation of concerned proteins. Teratogenesis Carcinog. Mutagen. 21:181-187, 2001.

Sulforaphane mitigates genotoxicity induced by radiation and anticancer drugs in human lymphocytes

Sulforaphane, present in cruciferous vegetables such as broccoli, is a dietary anticancer agent. Sulforaphane, added 2 or 20 h following phytohemaglutinin stimulation to cultured peripheral blood lymphocytes of individuals accidentally exposed to mixed γ and β-radiation, reduced the micronucleus frequency by up to 70%. Studies with whole blood cultures obtained from healthy volunteers confirmed the ability of sulforaphane to ameliorate γ-radiation-induced genotoxicity and to reduce micronucleus induction by other DNA-damaging anticancer agents, such as bleomycin and doxorubicin. This reduction in genotoxicity in lymphocytes treated at the G(0) or G(1) stage suggests a role for sulforaphane in modulating DNA repair. Sulforaphane also countered the radiation-induced increase in lymphocyte HDAC activity, to control levels, when cells were treated 2 h after exposure, and enhanced histone H4 acetylation status. Sulforaphane post-irradiation treatment enhanced the CD 34(+)Lin(-) cell population in culture. Sulforaphane has therapeutic potential for management of the late effects of radiation.

Polarization of macrophages towards M1 phenotype by a combination of 2-deoxy-d-glucose and radiation: Implications for tumor therapy.

2-Deoxy-d-glucose (2-DG) has been found to enhance the cytotoxicity of ionizing radiation and chemotherapeutic drugs in several tumor cell lines in vitro. Systemic administration of 2-DG together with localized irradiation of the tumor leads to tumor regression and cure (disease free survival), which correlate with the differential levels of anti-tumor immunity observed in Ehrlich ascites tumor (EAT) bearing mice. Macrophages being a major player of the innate immune system, we investigated the activation status of splenic macrophages during radio-sensitization of EAT in mice as well as in peritoneal macrophages ex vivo and macrophagic cell line (Raw 264.7) in vitro. Results suggest that under in vivo conditions, the combined treatment (2-DG+radiation) restores the M1 phenotype in spleen that correlated with the tumor response. However, 2-DG neither induced significant cytotoxicity nor enhanced radiation-induced cell death in peritoneal macrophages and the macrophage cell line (Raw 264.7). Further, increased arborization and enhanced functional status (expression of MHC class II, CD80, CD86 and phagocytosis) were observed after the combined treatment. Besides this activation, the combined treatment also skewed the macrophages towards M1 phenotype as evidenced by the enhanced secretion of IL-12, IL-2, TNF-α and IFN-γ. These observations suggest that 2-DG not only preserves the survival of normal macrophages during irradiation, but also activates macrophages by polarizing towards M1 phenotype, which is known to be tumoricidal in nature. This study for the first time sheds light on a potential antitumor immune activation by 2-DG involving macrophagic stimulation during in vivo radio-sensitization of tumors, besides the other known antitumor effects of this glucose analogue.

In vitro studies on radioprotective efficacy of silymarin against γ-irradiation

Abstract Purpose: Silymarin has been widely exploited for its hepatoprotective activities. This study aimed to evaluate the protective efficacy of silymarin against γ-radiation. Materials and methods: The radioprotective properties of silymarin were studied using different assays. Cytotoxicity of silymarin on Human embryonic kidney (HEK) cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Protective efficacy against γ-radiation was assessed by studying reduction in micronuclei frequency and free radical generation using 2′,7′-dichlorodihydroflurescin diacetate (H2DCFDA). Radiation-induced apoptosis was estimated by Annexin V-PI (propidium iodide) analysis and cell cycle analysis. γ-radiation induced changes in mitochondrial membrane potential (MMP) and DNA damage was estimated employing flow-cytometry and comet assay respectively. Results: MTT assay and Annexin V-PI studies showed that pre-incubation of HEK cells with silymarin protected them from γ-irradiation. Significant reduction in apoptosis (76.36%) was observed. Silymarin also decreased the percentage of radiation-induced micronuclei (> 69%) (p < 0.05 ). Measurement of intracellular reactive oxygen species (ROS) by H2DCFDA revealed a reduction in ROS (21%) at 0.5 h. Cell cycle analysis revealed G1 block in the unirradiated control, which declined in the silymarin pretreated irradiated group (0.5 h). Silymarin treatment resulted in a significant increase in MMP (2 h) against the radiation control. Moreover, the presence of silymarin during irradiation significantly decreased the DNA damage (as measured by comet assay). Conclusions: Protection against radiation-induced cell-death and DNA damage by silymarin could be attributed to a reduction in ROS induced by γ-radiation. In vitro experiments on HEK cells explicitly prove that silymarin is a promising, effective and safe radiation countermeasure agent.

Lymphocyte chromosomal aberration assay in radiation biodosimetry

Exposure to ionizing radiations, whether medical, occupational or accidental, leads to deleterious biological consequences like mortality or carcinogenesis. It is considered that no dose of ionizing radiation exposure is safe. However, once the accurate absorbed dose is estimated, one can be given appropriate medical care and the severe consequences can be minimized. Though several accurate physical dose estimation modalities exist, it is essential to estimate the absorbed dose in biological system taking into account the individual variation in radiation response, so as to plan suitable medical care. Over the last several decades, lots of efforts have been taken to design a rapid and easy biological dosimeter requiring minimum invasive procedures. The metaphase chromosomal aberration assay in human lymphocytes, though is labor intensive and requires skilled individuals, still remains the gold standard for radiation biodosimetry. The current review aims at discussing the human lymphocyte metaphase chromosomal aberration assay and recent developments involving the application of molecular cytogenetic approaches and other technological advancements to make the assay more authentic and simple to use even in the events of mass radiation casualties.

Enhanced Antitumor Immunity Contributes to the Radio-Sensitization of Ehrlich Ascites Tumor by the Glycolytic Inhibitor 2-Deoxy-D-Glucose in Mice

Two-deoxy-D-glucose (2-DG), an inhibitor of glycolysis differentially enhances the radiation and chemotherapeutic drug induced cell death in cancer cells in vitro, while the local tumor control (tumor regression) following systemic administration of 2-DG and focal irradiation of the tumor results in both complete (cure) and partial response in a fraction of the tumor bearing mice. In the present studies, we investigated the effects of systemically administered 2-DG and focal irradiation of the tumor on the immune system in Ehrlich ascites tumor (EAT) bearing Strain “A” mice. Markers of different immune cells were analyzed by immune-flow cytometry and secretary cytokines by ELISA, besides monitoring tumor growth. Increase in the expression of innate (NK and monocytes) and adaptive CD4+cells, and a decrease in B cells (CD19) have been observed after the combined treatment, suggestive of activation of anti-tumor immune response. Interestingly, immature dendritic cells were found to be down regulated, while their functional markers CD86 and MHC II were up regulated in the remaining dendritic cells following the combination treatment. Similarly, decrease in the CD4+ naïve cells with concomitant increase in activated CD4+ cells corroborated the immune activation. Further, a shift from Th2 and Th17 to Th1 besides a decrease in inflammatory cytokines was also observed in the animals showing complete response (cure; tumor free survival). This shift was also complimented by respective antibody class switching followed by the combined treatment. The immune activation or alteration in the homeostasis favoring antitumor immune response may be due to depletion in T regulatory cells (CD4+CD25+FoxP3+). Altogether, these results suggest that early differential immune activation is responsible for the heterogenous response to the combined treatment. Taken together, these studies for the first time provided insight into the additional mechanisms underlying radio-sensitization by 2-DG in vivo by unraveling its potential as an immune-modulator besides direct effects on the tumor.