Martin L. Meltz

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.

Melatonin and radioprotection from genetic damage: In vivo/in vitro studies with human volunteers

Peripheral blood samples were collected from human volunteers at 0 (5-10 min before), and at 1 and 2 h after a single oral dose of 300 mg of melatonin. At each time point, (i) the concentration of melatonin in the serum and in the leukocytes was determined, and (ii) the whole blood was exposed in vitro to 150 cGy of 137Cs gamma radiation, and the lymphocytes were cultured with mitogenic stimulation to determine the extent of radiation-induced genetic damage, viz, chromosome aberrations and micronuclei. For each volunteer, the results showed a significant increase in the concentration of melatonin in the serum and in the leukocytes at 1 h after the oral dose of melatonin, as compared to the sample collected at 0 h. The lymphocytes in the blood samples collected at 1 and 2 h after melatonin ingestion and exposed in vitro to 150 cGy gamma radiation exhibited a significant decrease in the incidence of chromosome aberrations and micronuclei, as compared with similarly irradiated lymphocytes from the blood sample collected at 0 h; the frequencies observed in the cells sampled at 2 h after the ingestion of melatonin were consistently lower when compared with those collected at 1 h. The data may have important implications for the protection of human lymphocytes from the genetic damage induced by free radical-producing mutagens and carcinogens.

Unscheduled DNA synthesis tests: A report of the U.S. environmental protection agency gene-tox program☆

The utility of unscheduled DNA synthesis (UDS) testing for screening potentially hazardous chemicals was evaluated using the published papers and technical reports available to the UDS Work Group. A total of 244 documents were reviewed. Based on criteria defined in advance for evaluation of the results, 169 were rejected. From the 75 documents accepted, results were reviewed for 136 chemicals tested using autoradiographic approaches and for 147 chemicals tested using liquid scintillation counting (LSC) procedures; 38 chemicals were tested by both approaches to measure UDS. Since there were no documents available that provided detailed recommendations of UDS screening protocols or criteria for evaluating the results, the UDS Work Group presents suggested protocols and evaluation criteria suitable for measuring and evaluating UDS by autoradiography in primary rat hepatocytes and diploid human fibroblasts and by the LSC approach in diploid human fibroblasts. UDS detection is an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs, because it measures the repair of DNA damage induced by many classes of chemicals over the entire mammalian genome. However, for this system to be utilized effectively, appropriate metabolic activation systems for autoradiographic measurements of UDS in human diploid fibroblasts must be developed, the nature of hepatocyte-to-hepatocyte variability in UDS responses must be determined, and the three suggested protocols must be thoroughly evaluated by using them to test a large number of coded chemicals of known in vivo mutagenicity and carcinogenicity.

Melatonin and protection from whole-body irradiation: survival studies in mice.

The radioprotective ability of melatonin was investigated in mice exposed to an acute whole-body gamma radiation dose of 815 cGy (estimated LD50/30 dose). The animals were observed for mortality over a period of 30 days following irradiation. The results indicated 100% survival for unirradiated and untreated control mice, and for mice treated with melatonin or solvent alone. Forty-five percent of mice exposed to 815 cGy radiation alone, and 50% of mice pretreated with solvent and irradiated with 815 cGy were alive at the end of 30 days. Irradiated mice which were pretreated with 125 mg/kg melatonin exhibited a slight increase in their survival (60%) (p=0.3421). In contrast, 85% of irradiated mice which were pretreated with 250 mg/kg melatonin were alive at the end of 30 days (p=0.0080). These results indicate that melatonin (at a dose as high as 250 mg/kg) is non-toxic, and that high doses of melatonin are effective in protecting mice from lethal effects of acute whole-body irradiation.

Marked Reduction of Radiation-Induced Micronuclei in Human Blood Lymphocytes Pretreated with Melatonin

Human peripheral blood lymphocytes which were pretreated in vitro with melatonin, an endogenously synthesized pineal hormone, for 20 min at 37 +/- 1 degree C exhibited a significant and concentration-dependent reduction in the frequency of gamma-radiation-induced micronuclei compared with irradiated cells which did not receive the pretreatment. The extent of the reduction observed with 2.0 mM melatonin was similar to that found in lymphocytes pretreated for 20 min with 1.0 M dimethylsulfoxide, a known free radical scavenger. These observations indicate that melatonin may have an active role in protection of humans against genetic damage due to endogenously produced free radicals, and also may be of use in reducing damage due to exposure to physical and chemical mutagens and carcinogens which generate free radicals.

Melatonin and protection from genetic damage in blood and bone marrow: Whole-body irradiation studies in mice

Abstract: The objective of this study was to examine the potential radioprotective properties of pharmacological doses of melatonin in whole‐body irradiated mice. CD2‐FI male mice were treated with melatonin. a secretory product of the pineal gland, and then whole‐body irradiated with an acute dose (150 cGy) of 137Cs gamma rays. Peripheral blood and bone marrow cells were examined for genetic damage, which was determined by comparing the incidence of micronuclei (MN) in both melatonin pre‐treated and non‐treated irradiated animals (and control mice). The percentages of polychromatic erythrocytes (PCEs) in unirradiated mice ranged between 3.1 ± 0.23 and 3.2 ± 0.19 in the peripheral blood and between 51.0 ± 2.03 and 52.8 ± 2.00 in the bone marrow. Whole‐body irradiation resulted in a significant decrease in the percentages of PCEs in the peripheral blood and bone marrow cells. In both tissues, irradiated mice that were pre‐treated with melatonin (5 or 10 mg/kg) exhibited a dose‐dependent increase in the observed incidence of PCEs relative to the expected incidence. The incidence of MN in unirradiated mice ranged between 4.2 ± 0.92 and 4.6 ± 0.97 in the peripheral blood and between 5.0 ± 1.05 and 5.5 ± 1.08 in the bone marrow. Whole‐body irradiation resulted in a significant increase in the incidence of MN in both tissues. In both tissues, irradiated mice that were pre‐treated with melatonin exhibited a significant and dose‐dependent reduction in the observed incidence of MN (relative to the expected incidence). Under the experimental conditions tested, the data indicate that melatonin has the ability to protect the genetic material of hematopoietic cells of mice from the damaging effects of acute whole‐body irradiation.

Cytogenetic Studies in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (835.62 MHz, FDMA)

Abstract Vijayalaxmi, Pickard, W. F., Bisht, K. S., Leal, B. Z., Meltz, M. L., Roti Roti, J. L., Straube, W. L. and Moros, E. G. Cytogenetic Studies in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (835.62 MHz, FDMA). Freshly collected peripheral blood samples from four healthy human volunteers were diluted with RPMI 1640 tissue culture medium and exposed in sterile T-75 tissue culture flasks in vitro for 24 h to 835.62 MHz radiofrequency (RF) radiation, a frequency employed for customer-to-base station transmission of cellular telephone communications. An analog signal was used, and the access technology was frequency division multiple access (FDMA, continuous wave). A nominal net forward power of 68 W was used, and the nominal power density at the center of the exposure flask was 860 W/m2. The mean specific absorption rate in the exposure flask was 4.4 or 5.0 W/kg. Aliquots of diluted blood that were sham-exposed or exposed in vitro to an acute dose of 1.50 Gy of γ radiation were used as negative or positive controls. Immediately after the exposures, the lymphocytes were stimulated with a mitogen, phytohemagglutinin, and cultured for 48 or 72 h to determine the extent of genetic damage, as assessed from the frequencies of chromosomal aberrations and micronuclei. The extent of alteration in the kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to mitotic indices, incidence of exchange aberrations, excess fragments, binucleate cells, and micronuclei. In contrast, the response of the lymphocytes exposed to γ radiation was significantly different from both RF-radiation- and sham-exposed cells for all of these indices. Thus, under the experimental conditions tested, there is no evidence for the induction of chromosomal aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 835.62 MHz RF radiation at SARs of 4.4 or 5.0 W/kg.

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.

Chromosome Damage and Micronucleus Formation in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (847.74 MHz, CDMA)

Abstract Vijayalaxmi, Bisht, K. S., Pickard, W. F., Meltz, M. L., Roti Roti, J. L. and Moros, E. G. Chromosome Damage and Micronucleus Formation in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (847.74 MHz, CDMA). Radiat. Res. 156, 430–432 (2001). Peripheral blood samples collected from four healthy nonsmoking human volunteers were diluted with tissue culture medium and exposed in vitro for 24 h to 847.74 MHz radiofrequency (RF) radiation (continuous wave), a frequency employed for cellular telephone communications. A code division multiple access (CDMA) technology was used with a nominal net forward power of 75 W and a nominal power density of 950 W/m2 (95 mW/cm2). The mean specific absorption rate (SAR) was 4.9 or 5.5 W/kg. Blood aliquots that were sham-exposed or exposed in vitro to an acute dose of 1.5 Gy of γ radiation were included in the study as controls. The temperatures of the medium during RF-radiation and sham exposures in the Radial Transmission Line facility were controlled at 37 ± 0.3°C. Immediately after the exposures, lymphocytes were cultured at 37 ± 1°C for 48 or 72 h. The extent of genetic damage was assessed from the incidence of chromosome aberrations and micronuclei. The kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation-exposed and sham-exposed lymphocytes with respect to mitotic indices, frequencies of exchange aberrations, excess fragments, binucleate cells, and micronuclei. The response of γ-irradiated lymphocytes was significantly different from that of both RF-radiation-exposed and sham-exposed cells for all of these indices. Thus there was no evidence for induction of chromosome aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 847.74 MHz RF radiation (CDMA) at SARs of 4.9 or 5.5 W/kg.