Natarajan Mohan

Induction of Nuclear Factor κB after Low-Dose Ionizing Radiation Involves a Reactive Oxygen Intermediate Signaling Pathway

Reactive oxygen intermediates (ROIs) have been found to be the messengers in the activation of the kappa B transcription regulator in mitogen- or cytokine-stimulated cells, operating in conjunction with or independently of various other mechanisms; these include Ca(++)-dependent and PKC-dependent cytoplasmic signaling pathways. We have recently reported that low-dose ionizing radiation induces NF-kappa B in human lymphoblastoid 244B cells. Since ionizing radiation generates free radicals in cells, we have investigated whether the ROIs generated by ionizing radiation induce NF-kappa B activity, and also whether they do so by a similar mechanism as in cells treated with PMA or H2O2. The results not only confirm a previous observation from our laboratory that low-dose ionizing radiation (0.1-2.0 Gy) activates kappa B transcription factor transiently with a maximal induction at 0.5 Gy exposure, but also demonstrate mechanistically that the activation of NF-kappa B by low-dose ionizing radiation can be inhibited considerably by the antioxidant N-acetyl-L-cysteine, indicating that at least the major part of the activation process is mediated by ROIs. These findings support the idea that ROIs can regulate the kappa B elements which in turn can serve as response elements for oxidant stress.

EFFECT OF MELATONIN ON NF-κB DNA-BINDING ACTIVITY IN THE RAT SPLEEN

It was recently demonstrated that the pineal neurohormone melatonin is a hydroxyl radical scavenger and antioxidant, and that it plays an important role in the immune system. In studies reported herein, we have investigated the relationship of the melatonin level and the NF‐κ B DNA binding activity in the spleen of Sprague—Dawley rats. These in vivo results indicate that NF‐ κB DNA binding activity in the spleen is lower at night, when endogenous melatonin levels are elevated, than during the day, when endogenous melatonin levels are lower. Furthermore, exogenously administered melatonin (10mg/kg) was shown to cause a significant decrease in NF‐κB DNA binding activity in the spleen at 60min after intraperitoneal injection (as compared with vehicle‐treated rats). These new findings suggest that the normal night time rise which can be expected for melatonin may be associated with increased NF‐κB DNA binding activity in the spleen. The melatonin, therefore, could potentially act to modulate spleen function and/or the immune system by regulating the NF‐κB DNA binding activity in the spleen.

Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion

We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kappaB (NF-kappaB). Here we investigate the hypothesis that low shear-induced activation of NF-kappaB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-kappaB, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kappaB activation. However, cytokine-induced NF-kappaB activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-kappaB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.

Activation of nuclear factor κB in human lymphoblastoid cells by low-dose ionizing radiation

Nuclear factor kB (NF-kB) is a pleiotropic transcription factor which is involved in the transcriptional regulation of several specific genes. Recent reports demonstrated that ionizing radiation in the dose range of 2-50 Gy results in expression of NF-kB in human KG-1 myeloid leukemia cells and human B-lymphocyte precursor cells; the precise mechanism involved and the significance are not yet known. The present report demonstrates that even lower doses of ionizing radiation, 0.25-2.0 Gy, are capable of inducing expression of NF-kB in EBV-transformed 244B human lymphoblastoid cells. These results are in a dose range where the viability of the cells remains very high. After exposure to {sup 137}Cs {gamma} rays at a dose rate of 1.17 Gy/min, a maximum in expression of NF-kB was seen at 8 h after a 0.5-Gy exposure. Time-course studies revealed a biphasic time-dependent expression after 0.5-, 1- and 2-Gy exposures. However, for each time examined, the expression of NF-kB was maximum after the 0.5-Gy exposure. The expression of the p50 and p65 NF-kB subunits was also shown to be regulated differentially after exposures to 1.0 and 2.0 Gy. 32 refs., 3 figs.

Proliferation and cytogenetic studies in human blood lymphocytes exposed in vitro to 2450 MHz radiofrequency radiation

Aliquots of human peripheral blood collected from two healthy human volunteers were exposed in vitro to continuous wave 2450 MHz radiofrequency radiation (RFR), either continuously for a period of 90 min or intermittently for a total exposure period of 90 min (30 min on and 30 min off, repeated three times). Blood aliquots which were sham-exposed or exposed in vitro to 150 cGy gamma radiation served as controls. The continuous wave 2450 MHz RFR was generated with a net forward power of 34.5 W and transmitted from a standard gain rectangular antenna horn in a vertically downward direction. The mean power density at the position of the cells was 5.0 mW/cm2. The mean specific absorption rate calculated by Finite Difference Time Domain analysis was 12.46 W/kg. Immediately after exposure, lymphocytes were cultured for 48 and 72 h to determine the incidence of chromosomal aberrations and micronuclei, respectively. Proliferation indices were also recorded. There were no significant differences between RFR-exposed and sham-exposed lymphocytes with respect to; (a) mitotic indices; (b) incidence of cells showing chromosome damage; (c) exchange aberrations; (d) acentric fragments; (e) binucleate lymphocytes, and (f) micronuclei, for either the continuous or intermittent RFR exposures. In contrast, the response of positive control cells exposed to 150 cGy gamma radiation was significantly different from RFR-exposed and sham-exposed lymphocytes. Thus, there is no evidence for an effect on mitogen-stimulated proliferation kinetics or for excess genotoxicity within 72 h in human blood lymphocytes exposed in vitro to 2450 MHz RFR.

Interaction of hyperthermia with Taxol in human MCF-7 breast adenocarcinoma cells

Hyperthermia treatments (43 degrees C, 1 h) were performed on exponentially growing MCF-7 breast adenocarcinoma cells at the beginning, middle, or end of 24 h incubations of the cells in vitro with Taxol (paclitaxel). When the cells were heated at the beginning or middle of the Taxol incubation, the hyperthermia treatment protected against the toxic effect of each of the Taxol concentrations examined (5, 10 and 100 nM). Consistent with earlier studies, Taxol treatment at 37 degrees C resulted in an accumulation of greater than 94% of the cells in G2/M at 24 h. Heating the cells at the middle or end of the Taxol treatment resulted in a similar accumulation. However, heat treatment during the first hour of Taxol exposure resulted in a significantly smaller percentage of cells (approximately 50%) in G2/M. HPLC analysis showed that at 37 degrees C, Taxol uptake into MCF-7 cells approached maximum within 0.25 h and increased only slightly more over the next 11.75 h. The parental Taxol level was markedly lower by 24 h. In contrast, 1 h hyperthermia treatments at the beginning or middle of the Taxol incubation resulted in higher Taxol concentrations at 12 and 24h, and higher intracellular concentrations overall than at 37 degrees C. These results indicate that hyperthermia inhibits Taxol related cell cycle effects and cytotoxicity, in spite of causing higher concentrations of Taxol to be present in heated cells.