Proflavin and microwave radiation: absence of a mutagenic interaction

The potential ability of radiofrequency electromagnetic radiation (RFR) in the microwave range to induce mutagenesis, chromosomal aberrations, and sister chromatid exchanges in mammalian cells is being explored in our laboratories. In addition, we have also been examining the ability of simultaneous exposure to RFR and chemical mutagens to alter the genotoxic damage induced by chemical mutagens acting alone. We have performed experiments to determine whether there is an interaction between 2.45-GHz, pulsed-wave, RFR and proflavin, a DNA-intercalating drug. The endpoint studied was forward mutation at the thymidine kinase locus in L5178Y mouse leukemic cells. Any effect on the size distribution of the resulting colonies of mutated cells was also examined. The exposures were performed at net forward powers of 500 or 600 W, resulting in a specific absorption rate (SAR) of approximately 40 W/kg. The culture-medium temperature reached a 3 degrees C maximal increase during the 4-h exposure; appropriate 37 degrees C and convection-heating temperature controls (TC) were performed. In no case was there any indication of a statistically significant increase in the induced mutant frequency due to the simultaneous exposure to RFR and proflavin, as compared with the proflavin exposures alone. There was also no indication of any change in the colony-size distribution of the resulting mutant colonies, neither, and there was no evidence in these experiments of any mutagenic action by the RFR exposure alone.     

Enhancement by methylxanthines of sister-chromatid exchange frequency induced by cytostatics in normal and leukemic human lymphocytes

The effect of theobromine (TB) and diphylline (DP) or (1,2-dihydroxy-3-propyl)theophylline on SCE rates induced in vitro by mitomycin C (MMC), and the effect of caffeine on SCE rates induced in vitro by cytosine arabinoside (Ara-C) was studied. The combined treatments with MMC plus TB or DP showed the potentiating ability of the latter drugs. Caffeine also enhanced SCEs induced by Ara-C in cultured human lymphocytes. Caffeine and adriamycin (ADR) did not act synergistically on induction of SCEs. In a combined study, in vivo and in vitro, lymphocytes taken from 2 leukemic patients who had been given chlorambucil (CBC) or Ara-C by injection 3 h before, and then treated with caffeine in vitro, were found to have synergistically increased exchange frequencies.     

Radiofrequency (microwave) radiation exposure of mammalian cells during UV-induced DNA repair synthesis

The effect of continuous-wave (CW) and pulsed-wave (PW) radiofrequency radiation (RFR) in the microwave range on UV-induced DNA repair has been investigated in MRC-5 normal human diploid fibroblasts. RFR exposure at power densities of 1 (or 5) and 10 mW/cm2 gave a maximum specific absorption rate (SAR) (at 10 mW/cm2) of 0.39 +/- 0.15 W/kg for 350 MHz RFR, 4.5 +/- 3.0 W/kg for 850 MHz RFR, and 2.7 +/- 1.6 W/kg for 1.2 GHz RFR. RFR exposures for 1 to 3 h at 37 degrees C, in either continuous-wave or pulsed-wave modes, had no effect on the rate of repair replication label incorporated into preexisting UV-damaged DNA. RFR exposures (PW), with a constant medium temperature of 39 degrees C at 350 and 850 MHz during the repair period after UV damage, also had no effect. Assay for induction of repair synthesis by RFR exposure alone in non-UV irradiated cells was negative for the 350-, 850-, and 1200-MHz CW and PW RFR at 37 degrees C and the 350- and 850-MHz PW RFR at 39 degrees C. RFR does not induce DNA repair under these exposure conditions. In preliminary experiments--with the tissue culture medium maintained at 39 degrees C and RFR exposures (PW) at the frequencies of 350, 850, and 1200 MHz--no effect on incorporation of [3H]thymidine into DNA undergoing semiconservative synthesis was observed.     

BCNU-induced sister chromatid exchanges are increased by X irradiation

We have studied the effect on sister chromatid exchange (SCE) induction in 9L rat brain tumor cells caused by combination treatment with BCNU and X rays. Over the dose and concentration ranges used in these experiments, BCNU induced relatively large numbers of SCEs, while X rays induced few SCEs. When cells were X irradiated immediately after BCNU treatment, the number of SCEs induced was greater than the number of SCEs expected by adding the number of SCEs induced by each agent alone; the number of SCEs induced as a result of this BCNU-X-ray interaction increased as the concentration of BCNU and/or dose of X rays increased. When the addition of bromodeoxyuridine was delayed from 0 to 16 hr after BCNU treatment, the number of SCEs induced declined to control levels by 16 hr. If X irradiation was delayed for up to 16 hr after BCNU treatment the same pattern of decrease was observed; the number of SCEs induced at each time point, however, was greater than that induced by BCNU and X rays alone. X irradiation from 0-16 hr before BCNU treatment produced the same number of SCEs as that produced by BCNU alone. Thus the SCE assay is capable of detecting a drug-X-ray interaction in mammalian cells and provides a sensitive means of studying the sequencing and timing that leads to the interaction.     

Influence of radiofrequency radiation on chromosome aberrations in CHO cells and its interaction with DNA-damaging agents

A limited number of contradictory reports have appeared in the literature about the ability of radiofrequency (rf) radiation to induce chromosome aberrations in different biological systems. The technical documentation associated with such reports is often absent or deficient. In addition, no information is available as to whether any additional genotoxic hazard would result from a simultaneous exposure of mammalian cells to rf radiation and a chemical which (by itself) induces chromosome aberrations. In the work described, we have therefore tested two hypotheses. The first is that rf radiation by itself, at power densities and exposure conditions which are higher than is consistent with accepted safety guidelines, can induce chromosome aberrations in mammalian cells. The second is that, during a simultaneous exposure to a chemical known to be genotoxic, rf radiation can affect molecules, biochemical processes, or cellular organelles, and thus result in an increase or decrease in chromosome aberrations. Mitomycin C (MMC) and Adriamycin (ADR) were selected because they act by different mechanisms, and because they might put normal cells at risk during combined-modality rf radiation (hyperthermia)-chemotherapy treatment of cancer. The studies were performed with suitable 37 degrees C and equivalent convection heating-temperature controls in a manner designed to discriminate between any thermal and possible nonthermal action. Radiofrequency exposures were conducted for 2 h under conditions resulting in measurable heating (a maximum increase of 3.2 degrees C), with pulsed-wave rf radiation at a frequency of 2450 MHz and an average net forward power of 600 W, resulting in an SAR of 33.8 W/kg. Treatments with MMC or ADR were for a total of 2.5 h and encompassed the 2-h rf radiation exposure period. The CHO cells from each of the conditions were subsequently analyzed for chromosome aberrations. In cells exposed to rf radiation alone, and where a maximum temperature of approximately 40 degrees C was achieved in the tissue culture medium, no alteration in the frequency from 37 degrees C control levels was observed. Relative to the chemical treatment with MMC alone at 37 degrees C, for two different concentrations, no alteration was observed in the extent of chromosome aberrations induced by either simultaneous rf radiation exposure or convection heating to equivalent temperatures. At the ADR concentration that was used, most of the indices of chromosome aberrations which were scored indicated a similar result.(ABSTRACT TRUNCATED AT 400 WORDS)     

Some factors affecting sister-chromatid differentiation (SCD) and sister-chromatid exchange (SCE) in Hordeum vulgare.

A study of some factors affecting sister-chromatid differentiation (SCD) and sister-chromatid exchanges (SCE) in Hordeum vulgare is reported. After we studied the influence of 5-fluorodeoxyuridine (FdU) and growth temperature on SCE in barley cells, and the effect of FdU, growth temperature, the growth time of plant cells in 5-bromo-2-deoxyuridine (BrdU) solution on SCD, we found an experimental condition under which the frequency of SCE is lower, but the percentage of SCD is higher. Our data show that ascorbic acid, mitomycin C, adriamycin, and maleic hydrazide induce SCEs in cells of Hordeum vulgare by means of free radicals. This can be shown from the two observations: (1) sulfhydryl compounds such as cysteine and glutathione can completely or partially inhibit the SCEs induced by ascorbic acid, mitomycin C, adriamycin and maleic hydrazide; (2) the amounts of free radicals in root tips correlate with the frequencies of SCE in root tip cells.     

Effect of Free Radicals and Temperature on Sister Chromatid Exchanges in Hordeum vulgare L.

The clastogenic (chromosome-damaging) effect of many chemical and physical agents is believed to be mediated by reactive oxygen-detived radicals. The interaction of these free radicals with DNA and the significance of the radical-induced DNA lesions in mutagenesis and carcinogenesis have been the subjects of increasing interest during recent years. Sister chromatid exchange (SCE) reflects an interchange between DNA molecules at homologous loci within a replicating chromosome. SCE analysis was found to have increased use for monitoring the exposure of cell to mutagenic carcinogens. The authors found that the induction of SCEs in cells of Hordeum vulgare L. by ascorbic acid, mitomycin C, adriamycin and maleic hydrazid was through the action of free radicals. They also studied the influence of growth temperature on average generation time(AGT) and SCEs. and disclosed a close correlation between AGT and SCEs. The Brdu-Giemsa techniques were used for the detection of SCEs and AGT in cytological preparations of metaphase chromosomes.     

Induction of chromosome damage by aniline and its C-hydroxylated metabolites in the root cells of Vicia faba

The induction of sister-chromatid exchanges (SCEs) and chromosome aberrations (CAs) by aniline hydrochloride (AH) and its C-hydroxylated metabolites, o-, m- and p-aminophenol, in the root cells of Vicia faba was examined. AH induced CAs, but not SCEs. All the C-hydroxylated metabolites of aniline induced both SCEs and CAs. However, the treatment of cells with these metabolites at concentrations that did not cause significant increases in CAs resulted in significant increases in SCEs. These results seem to suggest that the substance that induced CAs in root cells treated with AH was not the C-hydroxylated metabolites of aniline.     

Cytological characterization of repair-deficient CHO cell line 43-3B. I. Induction of chromosomal aberrations and sister-chromatid exchanges by UV and its modulation with 3-aminobenzamide

The induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) by short-wave ultraviolet (UV) and X-irradiation was studied in Chinese hamster ovary (CHO) wild-type (WT) cells and one of its UV-hypersensitive mutants, 43-3B. The results indicate that CHO 43-3B show high levels of spontaneously occurring chromosomal aberrations and SCEs; these levels are, respectively, approximately 4 and 1.7 times those found in WT CHO. Treatment with UV produced a considerable delay in the cell-cycle progression of the mutant cells compared to the WT cells. Doses of UV that had no effect on WT cells, significantly induced chromosomal alterations in the mutant in a dose-dependent manner. An approximately 5-fold increase in the induced frequencies of SCEs was obtained in 43-3B cells after UV treatment. No synergistic effect was observed with UV irradiation and the inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), in either cell type. The frequency of SCEs in the mutant cell lines was lower than would be expected if the effects of UV and the inhibitor were additive. X-Ray alone in G1 and in combination with 3AB in G2 did not induce increased frequencies of chromosomal aberrations in mutant cells in comparison to the WT cells.     

Contribution of incorporated 5-bromodeoxyuridine in DNA to the frequencies of sister-chromatid exchanges induced by inhibitors of poly-(ADP-ribose)-polymerase

3-Aminobenzamide and benzamide, two potent inhibitors of poly-(ADP-ribose)-polymerase increase the frequencies of SCEs in Chinese hamster ovary cells in a dose-dependent manner. SCEs were studied in cells in which the inhibitors were present either during the first cell cycle or the second cell cycle or both. Most of the induced SCEs were found to be formed during the second cell cycle in which BU-containing DNA was used as template for DNA synthesis. In cells which were pregrown for 4 cell cycles in the presence of BrdUrd, in order to obtain both sister chromatids bifiliarly substituted with BU in their DNA, it was found that the presence of inhibitor even in the first cell cycle increased the frequencies of SCEs. It is concluded that the incorporated BrdUrd plays an important role in the origin of spontaneous and induced SCEs. 3-Aminobenzamide alone or benzamide in the presence of BrdUrd during culture, did not increase the frequencies of mutations to HGPRT^? in these cells.     

Reactive oxygen species formation is not enhanced by exposure to UMTS 1950 MHz radiation and co‐exposure to ferrous ions in Jurkat cells

This study was designed to assess if radiofrequency (RF) radiation induces oxidative stress in cultured mammalian cells when given alone or in combination with ferrous ions (FeSO₄). For this purpose the production of reactive oxygen species (ROS) was measured by flow cytometry in human lymphoblastoid cells exposed to 1950 MHz signal used by the third generation wireless technology of the Universal Mobile Telecommunication System (UMTS) at Specific Absorption Rate of 0.5 and 2.0 W/kg. Short (5–60 min) or long (24 h) duration exposures were carried out in a waveguide system under strictly controlled conditions of both dosimetry and environment. Cell viability was also measured after 24 h RF exposure using the Resazurin and Neutral Red assays. Several co‐exposure protocols were applied to test if RF radiation is able to alter ROS formation induced by FeSO₄ (RF given before or concurrently to FeSO₄). The results obtained indicate that non‐thermal RF exposures do not increase spontaneous ROS formation in any of the experimental conditions investigated. Consistent with the lack of ROS production, no change in cell viability was observed in Jurkat cells exposed to RF radiation for 24 h. Similar results were obtained when co‐exposures were considered: combined exposures to RF radiation and FeSO₄ did not increase ROS formation induced by the chemical treatment alone. In contrast, in cultures treated with FeSO₄ as positive control, a dose‐dependent increase in ROS formation was recorded, validating the sensitivity of the method employed. Bioelectromagnetics 30:525–535, 2009. © 2009 Wiley‐Liss, Inc.     

Induction of sister-chromatid exchanges in ICR 2A frog cells exposed to 265-313 nm monochromatic ultraviolet wavelengths and photoreactivating light

Exposure of ICR 2A frog cells to 265 nm, 289 nm, 302 nm or 313 nm monochromatic ultraviolet (UV) wavelengths induced the formation of sister-chromatid exchanges (SCEs). However, treatment of cells with photoreactivating light (PRL) following the UV irradiations resulted in a lower level of SCEs compared with cells incubated in the dark. Hence, it can be concluded that pyrimidine dimers are the principal photoproducts responsible for the induction of SCEs in cells exposed to 265-313 nm UV due to the specificity of DNA photolyase for the light-dependent monomerization of dimers in DNA. It was also found that the maximum yield of induced SCEs in 313 nm-irradiated cells was only about 7 SCEs per cell whereas the plateau values for the shorter wavelengths were approximately 15-20 SCEs per cell. In addition, treatment of cells with 313 nm plus 265 nm light resulted in a lower level of SCEs than in cells exposed to 265 nm UV alone. These results can be interpreted in the context of a replication model for SCE, in which the high level of non-dimer damages produced in the DNA of 313 nm-irradiated cells inhibits the induction of SCEs by the pyrimidine dimers that are also produced by this wavelength.     

Induction of the fragile X on BrdU-substituted chromosomes with direct visualization of sister chromatid exchanges on banded chromosomes

Summary After incorporation of BrdU for one or more replication cycles, the fragile site at Xq27 [fra(X)] was induced by a late pulse with excess thymidine (dT), resulting in the simultaneous visualization of G bands and differentially stained sister chromatids. The degree of BrdU substitution (uni- vs bifilarly substituted DNA) did not affect the expression of the fra(X). Without addition of dT, expression was the same in M1, M2, and M3 cells. With the addition of dT, expression was reduced in M1 cells and increased in M2 and M3 cells. One way to explain this fact would be an increased repair of the fragile site in M1 cells by illegitimate G:BrdU pairing under dCTP-deficient conditions. A preferential depletion of M3 cells, and to a lesser extent also M2 cells, could suggest a synergistic toxic effect of BrdU substitution and dCTP depletion. With this technique, sister chromatid exchanges (SCEs) could be directly localized at band level, facilitating a more detailed study of SCEs at the Xq27 fragile site.     

Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells

Possible induction of sister-chromatid exchanges by butachlor, paraquat, phorate and monocrotophos was examined in primary rat tracheal epithelial (RTE) and Chinese hamster ovary (CHO) cells. At dose levels that killed less than 50% of the cell population, monocrotophos induced SCEs positively in CHO and RTE cells, while paraquat was positive only in RTE cells. In two trials of the same experiment, paraquat and butachlor in CHO cells, and phorate in either RTE or CHO cells failed to induce a significant number of SCEs at any dose level within the ranges assayed. On the other hand, in RTE cells, butachlor induced a significant number of SCEs at a dose level of 5 micrograms/ml in one trial, but was insignificant in another. The inductions in these assays were, however, dose-dependent. The addition of S9 mixture did not alter the results of SCE induction by these 4 pesticides in CHO cells. RTE cells were more vulnerable to paraquat in cytotoxicity and SCE assays than CHO cells. Cytotoxicities were ranked as butachlor greater than phorate greater than paraquat greater than monocrotophos to CHO cells and paraquat greater than butachlor greater than phorate greater than monocrotophos to RTE cells. Significant cell cycle delays were only found in the treatments with the highest dose levels of butachlor, paraquat and phorate in CHO cells. In addition, this is the first report on SCE induction in RTE cells.     

Spontaneous and induced sister chromatid exchanges in the blood lymphocytes of healthy persons and of xeroderma pigmentosum patients exposed to the inhibitors of DNA repair and replication caffeine, 3-methoxybenzamide and novobiocin

The frequency of sister chromatid exchanges (SCEs), both spontaneous and induced by UV-light, X-rays, mitomycin C and ethylmetansulphonate (EMS), has been investigated in cultured human peripheral blood lymphocytes. Besides, frequency of spontaneous and induced SCEs was studied under the action of the inhibitors of topoisomerase II, polymerase poly(ADP-ribose), and DNA repair, i. e. novobiocin, 3-metoxybenzamide, and caffeine, respectively. It is shown that the base-line SCEs in lymphocytes of the patient with xeroderma pigmentosum II (XP2LE) is dramatically higher compared to that in normal and pigmented xerodermoid cells (XP3LE). The above inhibitors of DNA synthesis and repair enhance the rate of spontaneous SCEs in normal, XP2LE and XP3LE cells. UV-, X-ray and chemical mutagens induced an increased frequency of SCEs in these cells. Simultaneous treatment with mutagenes and inhibitors of DNA synthesis and DNA repair enhanced the rate of SCEs in lymphocytes of healthy donors and in the XP3LE patient. The frequency of the XP2LE cells. Novobiocin, 3-MBA and caffeine significantly decreased the frequency of SCEs in mitomycin C- and EMS-treated XP2LE lymphocyte, which nevertheless was much higher than that in normal cells treated with the same agents.     

Chemotherapy cytotoxicity of human MCF-7 and MDA-MB 231 breast cancer cells is altered by osteoblast-derived growth factors

One-third of women with breast cancer will develop bone metastases and eventually die from disease progression at these sites. Therefore, we analyzed the ability of human MG-63 osteoblast-like cells (MG-63 cells), MG-63 conditioned media (MG-63 CM), insulin-like growth factor I (IGF-I), and transforming growth factor beta 1 (TGF-beta1) to alter the effects of adriamycin on cell cycle and apoptosis of estrogen receptor negative (ER-) MDA-MB-231 and positive (ER+) MCF-7 breast cancer cells, using cell count, trypan blue exclusion, flow cytometry, detection of DNA fragmentation by simple agarose gel, and the terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method for apoptosis (TUNEL assay). Adriamycin arrested MCF-7 and MDA-MB-231 cells at G2/M phase in the cell cycle and inhibited cell growth. In addition, adriamycin arrested the MCF-7 cells at G1/G0 phase and induced apoptosis of MDA-MB-231 cells. Exogenous IGF-I partially neutralized the adriamycin cytotoxicity/cytostasis of cancer cells. MG-63 CM and TGF-beta1 partially neutralized the adriamycin cytotoxicity of MDA-MB-231 cells but enhanced adriamycin blockade of MCF-7 cells at G1/G0 phase. MG-63 osteoblast-like cells inhibited growth of MCF-7 cells while promoting growth and rescued MDA-MB-231 cells from adriamycin apoptosis in a collagen co-culture system. These data suggest that osteoblast-derived growth factors can alter the chemotherapy response of breast cancer cells. Conceivably, host tissue (bone)-tumor cell interactions can modify the clinical response to chemotherapy in patients with advanced breast cancer.     

The relation between chemically induced sister-chromatid exchanges and chromatid breakage.

Studies of classical chromosome aberrations and sister-chromatid exchanges (SCES) suggest independent mechanisms for the two events despite some common features. Examination of chromosome breakage caused by X-rays, visible light, and viruses has shown that few chromatid breaks are accompanied by SCEs at the sites of breaks. No similar observations were available for chemically induced breaks, but it has been reported that rat chromosomes exposed to dimethylbenzanthracene (DMBA) contained a preponderance of both aberrations and SCEs in certain specific regions, implicating a common process in their formation. These conclusions were drawn from a comparison of breaks induced in vivo with SCEs induced in vitro. However, we used 7 chemical mutagens to induce both chromatid breaks and SCEs in "harlequin" chromosomes of cultured rat and Chinese hamster ovary (CHO) cells and found that 25% of the 914 breaks scored were associated with SCEs. The proportion of breaks accompanied by SCEs is related to the overall SCE frequency and falls into the range predicted on the basis that breaks and SCEs occur independently. The reported association between sites for SCEs and aberrations also reflects secondary factors, such as induction of SCEs and aberrations during DNA synthesis in late replicating regions of the chromosomes.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

Sister-chromatid exchanges in human lymphocytes induced by dimethoate, omethoate, deltamethrin, benomyl and their mixture.

Dimethoate and omethoate, two common organophosphorus insecticides, induced a dose-related increase in the frequency of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro (P of the regression lines less than 0.01). Two other common pesticides, the pyrethroid insecticide deltamethrin and the systemic fungicide benomyl, induced a modest increase in SCEs which bordered on statistical significance (P = 0.053 and 0.055, respectively). Mixtures of the four pesticides at total concentrations of 41.5 and 83 micrograms/ml (composed of 43% dimethoate, 43% omethoate, 12% deltamethrin and 1.2% benomyl) induced a dose-dependent increase in SCEs (P less than 0.01). The effects of these mixtures of pesticides were variable using lymphocytes from different individuals, although these differences did not attain statistical significance. Moreover, low concentrations of the four pesticides that did not increase SCEs significantly when tested alone, were positive for SCE induction when tested as a mixture. The experiments show that sub-threshold doses of pesticides may increase SCEs when present in a mixture.     

Effect of recombinant interferon-alpha on streptonigrin-induced chromosome aberrations and sister-chromatid exchanges in hamster cells

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the radiomimetic antibiotic streptonigrin (SN, 250 ng/ml, 20 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-45,000 IU/ml) was added to the cell cultures 30 min before SN and left in the culture medium until the end of SN treatment or until cell harvesting. A statistically significant increase in the frequency of CAs and SCEs was observed following treatment with SN (P < 0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs and SCEs over control values. Low rIFN-alpha-2a doses produced a reduction in the frequency of CAs and an increase in the yield of SCEs induced by SN, while high doses of the cytokine caused an increase in the yield of CAs and a reduction in the frequency of SCEs induced by the antibiotic. In addition, rIFN-alpha-2a caused a marked inhibition (around 50%) on the yield of SN-induced chromatid-type aberrations in the G(2) phase of the cell cycle. It is suggested that the inhibitory effect of rIFN-alpha-2a on the SN-induced chromosome damage is due to the stimulation of DNA synthesis and repair by the cytokine. On the other hand, our results give further support to our previous hypothesis that the induction of CAs and SCEs by SN is based on different mechanisms.