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/m(2). 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 gamma 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 gamma 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.

Primary DNA damage in human blood lymphocytes exposed in vitro to 2450 MHz radiofrequency radiation.

Human peripheral blood samples collected from three healthy human volunteers were exposed in vitro to pulsed-wave 2450 MHz radiofrequency (RF) radiation for 2 h. The RF radiation was generated with a net forward power of 21 W and transmitted from a standard gain rectangular antenna horn in a vertically downward direction. The average power density at the position of the cells in the flask was 5 mW/cm(2). The mean specific absorption rate, calculated by finite difference time domain analysis, was 2.135 (+/-0.005 SE) W/kg. Aliquots of whole blood that were sham-exposed or exposed in vitro to 50 cGy of ionizing radiation from a (137)Cs gamma-ray source were used as controls. The lymphocytes were examined to determine the extent of primary DNA damage (single-strand breaks and alkali-labile lesions) using the alkaline comet assay with three different slide-processing schedules. The assay was performed on the cells immediately after the exposures and at 4 h after incubation of the exposed blood at 37 +/- 1 degrees C to allow time for rejoining of any strand breaks present immediately after exposure, i.e. to assess the capacity of the lymphocytes to repair this type of DNA damage. At either time, the data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to the comet tail length, fluorescence intensity of the migrated DNA in the tail, and tail moment. The conclusions were similar for each of the three different comet assay slide-processing schedules examined. In contrast, the response of lymphocytes exposed to ionizing radiation was significantly different from RF-radiation- and sham-exposed cells. Thus, under the experimental conditions tested, there is no evidence for induction of DNA single-strand breaks and alkali-labile lesions in human blood lymphocytes exposed in vitro to pulsed-wave 2450 MHz radiofrequency radiation, either immediately or at 4 h after exposure.

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.

Effect of melatonin on mitotic and proliferation indices, and sister chromatid exchange in human blood lymphocytes.

Cells from human peripheral blood were cultured in vitro in the presence of 0.05 to 1.00 mM melatonin, 10(-7) M mitomycin C (positive control) and 0.5% ethanol (solvent control) for 72 h at 37 +/- 1 degree C. Lymphocytes were examined for mitotic and proliferation indices, and for the incidence of sister chromatid exchange. The results indicate that the lymphocytes which were cultured in the presence of > or = 0.20 mM concentrations of melatonin exhibited a significant and concentration-dependent decrease in mitotic index and alteration in proliferation kinetics. This was demonstrated by an increase in the frequency of lymphocytes in their first division, with a concomitant decrease in the second and third or later division cells. The incidence of sister chromatid exchange was similar in the lymphocytes exposed to 0.05 to 1.00 mM melatonin and untreated controls. Exposure of the cells to ethanol, the solvent used, did not alter either the mitotic or proliferation indices, or the frequency of sister chromatid exchange. The lymphocytes treated with mitomycin C showed the expected decrease in mitotic and proliferation indices, and an increased incidence of sister chromatid exchange. These observations indicate that melatonin, when continuously present in the cultures for 72 h at the concentrations tested, while not genotoxic as indicated by the sister chromatid exchange assay, inhibits the proliferation of mitogen stimulated (and proliferating) human blood lymphocytes at supraphysiological concentrations.

Variability in adaptive response to low dose radiation in human blood lymphocytes: consistent results from chromosome aberrations and micronuclei.

The frequencies of chromosome aberrations and micronuclei were evaluated to assess the induction of adaptive response to low dose ionizing radiation in each of the blood samples collected from eight different individuals. Following stimulation with phytohemagglutinin, the cells were exposed to an adaptive dose of 1 cGy X-radiation at 24 hours and a challenge dose of 150 cGy gamma radiation at 48 hours. Lymphocytes were fixed at 54 hours to examine the incidence of chromosome aberrations and at 72 hours to examine the frequency of micronuclei in cytokinesis-blocked binucleated cells. Lymphocytes from five donors, i.e., "responders", exhibited the induction of adaptive response; their lymphocytes, which were pre-treated with 1 cGy had significantly fewer chromosome aberrations and micronuclei induced by the challenge dose of 150 cGy gamma radiation, as compared to the cells which did not receive the pre-treatment with 1 cGy. Such an induction of adaptive response was not observed in the remaining three donors, i.e., "non-responders"; the incidence of chromosome aberrations and micronuclei induced by the challenge dose of 150 cGy was not significantly different between the cells which were pre-exposed and un-exposed to 1 cGy. In all eight individuals, there was a strong positive correlation between the incidence of chromosome aberrations and micronuclei. Hence, whether or not an individual is a 'responder' or 'non-responder' could be assessed using either chromosome aberrations or micronuclei as the end-point. The overall pattern of response confirms the heterogeneity in adaptive response between individuals to ionizing radiation, which may in part be genetically controlled. Because of the simplicity of the technique and rapid assessment of the binucleated cells, we suggest the use of the micronucleus test as an alternative procedure in large scale population studies related to the adaptive response.

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's inhibitory effect on growth of ME-180 human cervical cancer cells is not related to intracellular glutathione concentrations.

The effects of various concentrations of melatonin on the growth of ME-180 human cervical cancer cells in vitro was examined. Melatonin at a concentration of 2 mM inhibited the growth of the cells after 48 h of melatonin treatment. At concentrations of 2 microM or 0.1 mM melatonin had no effect on cell proliferation. To determine whether the inhibitory effect of melatonin on the growth of the cervical cancer cells was linked to intracellular glutathione concentrations, experiments were performed in which intracellular glutathione levels were depressed by the addition of buthionine sulfoximine to the incubation medium 24 h before the addition of melatonin. The results show that 2 mM melatonin treatment still inhibits the growth of cells when glutathione levels are depressed by 95%. Even with depressed glutathione levels, 0.1 mM melatonin still had no effect on cell growth. Thus, melatonin's ability to inhibit ME-180 cervical cell growth in vitro may be independent of intracellular glutathione concentrations. It was also found that during one passage the intracellular glutathione levels of cervical cancer cells gradually decreases. When 4.5-day-old medium was replaced with new medium, intracellular glutathione levels partially recovered within 36 h. This suggests that the observed gradual reduction of cellular glutathione during incubation was a result of a reduction of some constituent in the medium after prolonged culture of the cells.

Neurobehavioral effects of repeated sublethal soman in primates.

Juvenile male baboons were trained to perform a match-to-sample discrimination task; effects of repeated sublethal exposure to the organophosphate nerve gas, soman, upon task performance were then explored. Both acute and subchronic exposure schedules were employed, and soman potency was verified by assay of soman-induced inhibition of acetylcholinesterase activity in whole blood, plasma, and erythrocytes. A characteristic profile of behavioral effects encompassing immediate, persistent, and delayed effects was observed. Immediate dose-related effects of soman included: increases in mean session response time, increases in errors, and decreases in extra responses. Seizures were also observed at the highest dose of soman employed (5 micrograms/kg). The increase in mean session response time was due to intermittent lapses in responding to stimuli (attentional deficits). Both the attentional deficits and intermittent generalized seizures were also persistent effects, with both occurring randomly after acute exposure to 5 micrograms/kg soman. Preliminary evidence suggests that occurrence of attentional deficits was associated with the occurrence of generalized and/or focal seizures; and that these effects may reflect irreversible lesions which become more threatening to the animal with increasing time. An additional, delayed effect was a sudden marked increase in the incidence of extra inconsequential responses which occurred several weeks after cessation of soman exposures.

Acute soman effects in the juvenile baboon: effects on a match-to-sample discrimination task and on total blood acetylcholinesterase.

Male juvenile baboons, trained on a match-to-sample operant discrimination task, were given acute intramuscular injections of soman (methyl pinacolyl phosphonofluoridate) at 1.0, 2.0, 3.0, 4.0, and 5.0 micrograms/kg. The different doses were given in a mixed order just before a behavioral test session. Just prior to administration of each soman dose and immediately following the 2-hr behavioral test session, a sample of blood (0.5 ml) was drawn from the baboon and analyzed for inhibition of acetylcholinesterase activity. Thereafter, blood sampling was accomplished at weekly intervals and soman was administered again only when whole blood acetylcholinesterase reached at least 80% of pre-soman control level. Behavioral effects of soman included a slowing of response times, a decrease in extra inconsequential responses, a decrease in responsiveness to the visual stimuli and an increase in errors. These effects were observed when acetylcholinesterase (AChE) levels fell to 25 mumoles/hr/ml blood or less. The threshold dose for behavioral effects was very close to the dose of soman which induced seizures.

Behavioral effects of drugs as a function of maternal polybrominated biphenyl body burden.

Prior to breeding, female rats were dosed orally for 20 days with either 0.5 mg/kg polybrominated biphenyl (PBB), 5.0 mg/kg PBB, or the lecithin liposome vehicle. Male offspring of these 3 treatment groups did not differ with respect to the learning of an operant discrimination task. However, administration of phenobarbital or d-amphetamine impaired the behavior of the offspring of low-dose PBB dams less than that of the offspring of controls. The behavioral effects of the drugs were generally inversely related to levels of liver PBB and activities of liver aryl hydrocarbon hydroxylase (AHH).

Correlation of systemic and biochemical effects of PBB with behavioral effects.

Rats were given PBB orally at 1, 3, and 6 mg/kg (or vehicle as control) daily for 20 days. Some animals were sacrificed immediately while the food intake of remaining animals was limited to attain and maintain 80% of normal body weight. No effect of PBB upon body weight was observed for any of the dose levels employed. Immediately after dosing, liver/body weight ratios were 110% of controls for the 1 mg/kg group and 152% for the 3 and 6 mg/kg groups; after weight reduction for 2-6 months liver/body weight ratios for all 3 dose groups were 160-170% of controls. In the absence of body fat, most tissues exhibited dose-dependent retention of PBB 2-6 months after dosing with highest levels in liver followed by kidney. In 6 mg/kg rats weight-reduced for 6 months, liver AHH activity was 613% of controls; in rats sacrificed immediately after dosing, liver AHH activity was dose-related but appeared to reach maximal value at the 3 mg/kg dose. Both calcium binding to synaptic plasma membranes and calcium uptake by intact synaptosomes was significantly reduced in the brains of 1 mg/kg PBB rats, but not affected in preparations from 3 and 6 mg/kg animals.