Nuclear translocation and DNA-binding activity of NFKB (NF-kappaB) after exposure of human monocytes to pulsed ultra-wideband electromagnetic fields (1 kV/cm) fails to transactivate kappaB-dependent gene expression.

The objective of this study was to investigate whether exposure of human monocytes to a pulsed ultra-wideband electromagnetic field (EMF) of 1 kV/cm average peak power triggers a signaling pathway responsible for the transcriptional regulation of NFKB (NF-kappaB)-dependent gene expression. Human Mono Mac 6 (MM6) cells were exposed intermittently to EMF pulses for a total of 90 min. The pulse width was 0.79+/-0.01 ns and the pulse repetition rate was 250 pps. The temperature of the medium was maintained at 37 degrees C in both sham- and EMF-exposed flasks. Total NFKB DNA-binding activity was measured in the nuclear extracts by the electrophoretic mobility shift assay. Cells exposed to the EMFs and incubated for 24 h postexposure showed a 3.5+/-0.2-fold increase in the NFKB DNA-binding activity. Since activation of NFKB was observed, the possibility of kappaB-dependent gene expression in response to exposure to the EMFs was investigated using NFKB signal-specific gene arrays. The results revealed no difference in the NFKB-dependent gene expression profiles at 8 or 24 h postexposure, indicating that activated NFKB does not lead to the differential expression of kappaB-dependent target genes. To determine whether the absence of the kappaB-dependent gene expression was due to compromised transcriptional regulation of NFKB, the functional activity of NFKB was examined in cells transiently transfected with Mercury Pathway constructs containing 4x NFKB binding sites associated either with the luciferase reporter system or a control vector. Pulsed EMF exposure did not induce NFKB-driven luciferase activity in these cells, indicating that the activation of NFKB at 24 h after the 1 kV/cm EMF exposure is functionally inactive. From these results, it is clear that the EMF-induced NFKB activation is only a transient response, with minimal or no downstream effect.

Post-translational modification of I-kappa B alpha activates NF-kappa B in human monocytes exposed to 56Fe ions.

The objective of this study was to investigate whether heavy ion (56Fe) radiation exposure activates one of the key transcriptional regulators, nuclear factor-kappa B (NF-kappa B), in normal human monocytes (Mono Mac 6 cells: MM6). The study revealed that the exposure of MM6 cells to 56Fe ions resulted in increased NF-kappa B DNA-binding activity. The activation was both dose- and time-dependent, with a maximum response at the 2 h time point after a 0.7 Gy dose. Cells pre-incubated with inhibitors of the phosphorylation and proteasome signaling pathway, completely blocked heavy ion-induced activation of NF-kappa B. These results clearly indicate that 56Fe ions can induce NF-kappa B DNA-binding activity in normal human monocytes, that the activation is rapid and persistent, and that the heavy ion-induced activation of NF-kappa B is mediated through phosphorylation of I-kappa B alpha and the subsequent proteasome-dependent degradation pathway. Since activation of NF-kappa B by extracellular stimuli is implicated in inflammation, infection and cancer induction, as well as in protection of cells against insult, it will be important in subsequent studies to elucidate whether heavy ion-induced NF-kappa B activation is involved in downstream gene expression.

Micronuclei in the peripheral blood and bone marrow cells of rats exposed to 2450 MHz radiofrequency radiation.

PURPOSE: To determine the incidence of micronuclei in peripheral blood and bone marrow cells of rats exposed continuously for 24h to 2450 MHz continuous wave radiofrequency radiation (RFR) at an average whole-body specific absorption rate (SAR) of 12W/kg. MATERIALS AND METHODS: Eight adult male Sprague-Dawley rats were exposed to 2450 MHz RFR in circularly polarized waveguides. Eight sham-exposed rats were kept in similar waveguides without the transmission of RFR. Four rats were treated with mitomycin-C (MMC) and used as positive controls. All rats were necropsied 24h after the end of RFR and sham exposures, and after the 24h treatment with MMC. Peripheral blood and bone marrow smears were examined to determine the frequency of micronuclei (MN) in polychromatic erythrocytes (PCE). RESULTS: The results indicated that the incidence of MN/2000 PCE were not significantly different between RFR- and sham-exposed rats. The group mean frequencies of MN in the peripheral blood were 2.3+/-0.7 in RFR-exposed rats and 2.1+/-0.6 in sham-exposed rats. In bone marrow cells, the average MN incidence was 3.8+/-1.0 in RFR-exposed rats and 3.4+/-0.7 in sham-exposed rats. The corresponding values in positive control rats treated with MMC were 23.5+/-4.7 in the peripheral blood and 33.8+/-7.4 in bone marrow cells. CONCLUSION: There was no evidence for the induction of MN in peripheral blood and bone marrow cells of rats exposed for 24h to 2450 MHz continuous wave RFR at a whole body average SAR of 12 W/kg.

Effect of melatonin on cell growth, metabolic activity, and cell cycle distribution.

We have recently demonstrated that the pineal secretory product melatonin inhibits the key transcriptional regulator nuclear factor-kappa B (NF-kappa B). As the activation of NF-kappa B is known to regulate the expression of cellular genes associated with cell cycle progression, cell growth, and differentiation, we investigated the effect of melatonin treatment on several cellular processes. These include cell viability, metabolic activity, and cell cycle phase distribution. Human embryonic kidney (293S) cells were treated with melatonin at concentrations of 0.02, 0.2, or 2 mM. When cell viability was measured 24, 48, and 72 hr after continuous exposure to melatonin using the trypan blue dye exclusion method, no significant cell death was observed. Even after exposure to 2 mM melatonin for 72 hr, cell viability remained at 98%. In contrast, another antioxidant compound, pyrrolidine dithiocarbomate (PDTC), at a 2 mM concentration reduced cell viability to 80.7+/-2.1% as early as 24 hr compared with untreated controls (P<0.05). When the metabolic activity was determined at 24, 48, and 72 hr using the colorimetric MTT assay, no significant changes in metabolic activity were observed. Even if the cells were treated with 10 mM melatonin for 72 hr, the metabolic activity was similar to that of the control cells. When cell cycle analysis was performed by flow cytometry, no marked difference in cell cycle distribution was observed. Melatonin at a concentration of 2 mM, however, did slightly alter the cell cycle (percentage of S phase cells) at 48 hr. This study revealed that when 293S cells are treated with concentrations of melatonin up to 2 mM, no significant alterations in three important cellular functions occurred. Exogenously added melatonin appeared to have a limited influence on the normal functioning of the cells even when the exposure continued for 72 hr.

Chromosome damage and micronucleus formation in human blood lymphocytes exposed in vitro to radiofrequency radiation at a cellular telephone frequency (847.74 MHz, CDMA).

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/m(2) (95 mW/cm(2)). 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 gamma 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 degrees C. Immediately after the exposures, lymphocytes were cultured at 37 +/- 1 degrees 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 gamma-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.

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.

Antioxidant compounds interfere with the 3.

Antioxidants are often added to culture media as cytoprotective agents. We examined the effects of antioxidants on the results and interpretation of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability. Without cells, the thiol-containing antioxidant compounds beta-mercaptoethanol, dithiothreitol, pyrrolidine-dithiocarbamate, and N-acetyl-L-cysteine (acetylcysteine) reduced MTT tetrazolium salts to a blue formazan product in a dose-dependent manner. Addition of the compounds L-ascorbic acid and (+)-alpha-tocopherol acid succinate had different effects. In contrast, addition of the antioxidants N-acetyl-5-methoxytryptamine and (-)-2-oxo-4-thiazolidine carboxylic acid, which do not contain reactive thiol groups, did not result in the development of blue formazan product. These results showed that antioxidants, and potentially other chemotherapeutic compounds that contain free thiol groups or other reducing equivalents, readily reduce MTT to produce the blue formazan, irrespective of the viability of the cells present. This undescribed reaction can, therefore, significantly influence the results and interpretation of cell-viability experiments.

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.

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

The objective of this study was to examine the potential radioprotective properties of pharmacological doses of melatonin in whole-body irradiated mice. CD2-F1 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.

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.

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.

Melatonin: possible mechanisms involved in its 'radioprotective' effect.

Peripheral blood samples were collected from four human volunteers before, and at 1 and 2 h after a single oral dose of 300 mg of melatonin. From each sample, separate aliquots of whole blood were exposed to 100-150 cGy gamma radiation in vitro to determine the extent of genetic damage. Irradiated lymphocytes from all volunteers which were collected after melatonin ingestion exhibited a significantly decreased extent of primary DNA damage and reduced frequencies of chromosomal aberrations and micronuclei, as compared with similarly irradiated cells collected before the oral dose of melatonin. The extent of the melatonin-associated decrease in primary DNA damage was less than the corresponding decrease observed in the incidence of chromosomal aberrations and micronuclei; the latter assays required an additional post-irradiation incubation of the cells at 37+/-1 degreesC for 48 and 72 h, respectively. The possible mechanisms involved in the radioprotective influence of melatonin are discussed.

Melatonin reduces gamma radiation-induced primary DNA damage in human blood lymphocytes.

Peripheral blood samples were collected from human volunteers 5-10 min before, and at 1 and 2 h after a single oral dose of 300 mg of melatonin. At each time point: (1) the concentration of melatonin in the serum and in the leukocytes was determined; and (2) the whole blood was exposed in vitro to 100 cGy of gamma radiation. Immediately after exposure to the radiation, the lymphocytes were examined to determine the extent of primary DNA damage, viz., single strand breaks and alkali labile lesions (determined from the length of DNA migration and fluorescence intensity of migrated DNA in the comet tail), using the alkaline comet assay. 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 hour. The lymphocytes in the blood samples collected at 1 and 2 h after melatonin ingestion and exposed in vitro to 100 cGy gamma radiation exhibited a significant decrease in the extent of primary DNA damage, as compared with similarly irradiated lymphocytes from the blood sample collected before melatonin ingestion. The extent of the melatonin-associated decrease in primary DNA damage did not correspond with the decrease reported earlier in the incidence of chromosomal aberrations and micronuclei; the latter assays required an additional postirradiation incubation of the cells at 37 +/- 1 degrees C for 48 and 72 h, respectively.

Frequency of micronuclei in the peripheral blood and bone marrow of cancer-prone mice chronically exposed to 2450 MHz radiofrequency radiation.

C3H/HeJ mice, which are prone to mammary tumors, were exposed for 20 h/day, 7 days/week, over 18 months to continuous-wave 2450 MHz radiofrequency (RF) radiation in circularly polarized wave guides at a whole-body average specific absorption rate of 1.0 W/kg. Sham-exposed mice were used as controls. The positive controls were the sentinel mice treated with mitomycin C during the last 24 h before necropsy. At the end of the 18 months, all mice were necropsied. Peripheral blood and bone marrow smears were examined for the extent of genotoxicity as indicated by the presence of micronuclei in polychromatic erythrocytes (PCEs). The results indicate that the incidence of micronuclei/1,000 PCEs was not significantly different between groups exposed to RF radiation (62 mice) and sham-exposed groups (58 mice), and the mean frequencies were 4.5 +/- 1.23 and 4.0 +/- 1.12 in peripheral blood and 6.1 +/- 1.78 and 5.7 +/- 1.60 in bone marrow, respectively. In contrast, the positive controls (7 mice) showed a significantly elevated incidence of micronuclei/1,000 PCEs in peripheral blood and bone marrow, and the mean frequencies were 50.9 +/- 6.18 and 55.2 +/- 4.65, respectively. When the animals with mammary tumors were considered separately, there were no significant differences in the incidence of micronuclei/1,000 PCEs between the group exposed to RF radiation (12 mice) and the sham-exposed group (8 mice), and the mean frequencies were 4.6 +/- 1.03 and 4.1 +/- 0.89 in peripheral blood and 6.1 +/- 1.76 and 5.5 +/- 1.51 in bone marrow, respectively. Thus there was no evidence for genotoxicity in mice prone to mammary tumors that were exposed chronically to 2450 MHz RF radiation compared with sham-exposed controls.

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.

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.

Effect of melatonin on NF-kappa-B 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-kB DNA binding activity in the spleen of Sprague. Dawley rats. These in vivo results indicate that NF-kB 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 (10 mg/kg) was shown to cause a significant decrease in NF-kB DNA binding activity in the spleen at 60 min 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-kB 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-kB DNA binding activity in the spleen.

Molecular markers of ionizing radiation-induced gene mutations in mammalian cells.

We have isolated independent Chinese hamster ovary (CHO) cell mutants at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus from untreated, 60Co gamma-ray-exposed, and 212Bi alpha-exposed cells and identified the molecular changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. Both the parental CHO-K1 cells and the X-ray-sensitive mutant xrs-5 cells were studied. The radiosensitive xrs-5 cells are defective in DNA double-strand break rejoining ability and in V(D)J recombination, which can be complemented by Ku protein. Of the 71 spontaneous CHO-K1 hprt mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of one to eight exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a high deletion spectrum with 45% of the 20 mutants analyzed showing a loss of one to eight exons and 30% showing total deletion. Exposure to an equitoxic dose of alpha radiation from 212Bi, a 220Rn daughter, resulted in a spectrum similar to the gamma-ray spectrum in that 75% of the 49 mutants analyzed were deletions. To alpha radiation, however, tended to produce larger intragenic deletions than gamma radiation. Of the 92 spontaneous xrs-5 mutants analyzed for deletions, 43% showed a loss of one to eight exons and 14% showed total deletion. This suggests that, in certain regions of the hprt gene, base alterations can be converted into large deletions and alteration in the Ku protein complex can influence this type of mutational process. Exposure to alpha radiation (10% survival) to xrs-5 cells resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed on change in exon number or size, 16% showed a loss of one to eight exons, and 41% showed total deletion. While the defect in xrs-5 cells has a profound effect on spontaneous mutant spectra, this defect does not appear to affect alpha-induced mutation spectra.