Genetic damage in humans exposed to extremely low-frequency electromagnetic fields.

The classification of extremely low-frequency magnetic fields by the International Agency for Research on Cancer in the group of 'possible human carcinogens' (group 2B) is essentially based on epidemiologic evidence showing an association between MF exposures and childhood leukaemia. Despite many in vitro and in vivo investigations, there is no established causal relationship yet. However, human cytogenetic biomonitoring studies that were conducted in the past show predominantly positive results, i.e. increased cytogenetic damage in peripheral blood lymphocytes or buccal cells of ELF-MF-exposed subjects. This is important given the established link between observed cytogenetic damage in cells of people and an increased cancer risk. We here conducted an evaluation of the published investigations and found that many of the studies clearly have shortcomings, which often prevent any firm conclusion. As a matter of fact, there are reasons to believe that effects are not that impressive. However, the totality of the studies cannot simply be disregarded warranting further caution and the application, to a certain extent, of the precautionary principle.

Investigation into the genotoxicity of water extracts from Hypoxis species and a commercially available Hypoxis preparation.

We performed an in vitro evaluation of the genotoxic potential of water extracts from four Hypoxis species (Hypoxis hemerocallidea, H. colchicifolia, H. rigidula, H. acuminata) and a commercial preparation thereof using the neutral red uptake (NRU) assay, the alkaline comet assay and the cytome assay in human hepatoma HepG2 cells. The relative cytotoxicity of these samples was established by determining their NI50 values (50% inhibition of NRU), and these results were used for dose-finding in genotoxicity tests. None of the tested extracts were identified as genotoxic in both the alkaline comet assay and cytome assay.

1,4-diarylpiperazines and analogs as anti-tubercular agents: synthesis and biological evaluation.

Despite progress in modern chemotherapy to combat tuberculosis, the causative pathogen Mycobacterium tuberculosis (M.tb.) is far from eradicated. Bacillary resistance to anti-mycobacterial agents, bacillary persistence and human immunodeficiency virus (HIV) co-infection hamper current drug treatment to completely cure the infection, generating a constant demand for novel drug candidates to tackle these problems. A small library of novel heterocyclic compounds was screened in a rapid luminometric in vitro assay against the laboratory M.tb. strain H37Rv. A group of amidines was found to have the highest potency and was further evaluated for acute toxicity against C3A hepatocytes. Next, the most promising compounds were evaluated for activity against a multi-drug resistant clinical isolate. The group of amidines was also tested for their ability to kill intracellular M.tb. residing in mouse J774A.1 macrophages. Finally, we report on a correlation between the structural differences of the compounds and their anti-mycobacterial activity.

In vitro and in vivo genotoxicity of radiofrequency fields.

There has been growing concern about the possibility of adverse health effects resulting from exposure to radiofrequency radiations (RFR), such as those emitted by wireless communication devices. Since the introduction of mobile phones many studies have been conducted regarding alleged health effects but there is still some uncertainty and no definitive conclusions have been reached so far. Although thermal effects are well understood they are not of great concern as they are unlikely to result from the typical low-level RFR exposures. Concern rests essentially with the possibility that RFR-exposure may induce non-thermal and/or long-term health effects such as an increased cancer risk. Consequently, possible genetic effects have often been studied but with mixed results. In this paper we review the data on alleged RFR-induced genetic effects from in vitro and in vivo investigations as well as from human cytogenetic biomonitoring surveys. Attention is also paid to combined exposures of RFR with chemical or physical agents. Again, however, no entirely consistent picture emerges. Many of the positive studies may well be due to thermal exposures, but a few studies suggest that biological effects can be seen at low levels of exposure. Overall, however, the evidence for low-level genotoxic effects is very weak.

Mutagenic and antimutagenic properties of extracts from South African traditional medicinal plants.

AIM OF THE STUDY: The aim of this paper was to summarize the results of our investigations on the in vitro genotoxic as well as antigenotoxic effects of a great number of selected South African traditional medicinal plants. MATERIALS AND METHODS: Investigations of methanol and dichloromethane extracts of selected plants were conducted with the bacterial Ames, Umu-C and VITOTOX tests, and with the cytochalasin B micronucleus test and alkaline comet assay in human white blood cells. RESULTS: A number of extracts were found to have genotoxic properties. Amongst the genotoxic plant extracts, especially methanol extracts of Helichrysum simillimum DC. (Asteraceae) should be highlighted. On the other hand, some plant extracts also showed antimutagenic potential. Here Bauhinia galpinii N.E.Br. (Fabaceae) and especially Chlerodendrum myricoides (Hochst.) Vatke (=Rotheca myricoides (Hochst.) Steane & Mabb.; Lamiaceae) appear to have antimutagenic properties. CONCLUSION: The safe use of Helichrysum similimum should be questioned and further investigations on its mutagenicity and overall biological properties should be encouraged. Antimutagenic properties of especially Bauhinia galpinii and Rotheca myricoides are considered of particular interest as it may be assumed that these antimutagenic natural substances are able to lower the cancer risk from everyday exposures to environmental mutagens as well as to mutagenic pharmaceuticals.

Nucleotide excision repair impairment by nodularin in CHO cell lines due to ERCC1/XPF inactivation.

The problem of toxicity of cyanobacterial toxins is of increasing concern, as the incidence of such blooms grows. Among the toxins, the most abundant in the environment are hepatotoxins known as nodularins and microcystins. These toxins are responsible for almost all known cases of fresh and brackish water intoxication and are responsible for recurrent episodes of human and animal illness and death. Moreover, they are believed to be potent tumor promoters and initiators. However, the mechanisms by which these toxins induce liver cancer are not well understood. The aim of the present study was to determine the effect of nodularin on the kinetics of nucleotide excision repair (NER) in Chinese hamster ovary (CHO) cells exposed to UV radiation. The first set of experiments was performed to define the optimal treatment conditions for nodularin to avoid the possibility of encountering false positive signals in the comet assay due to the apoptogenic activity of nodularin. Based on the analysis of apoptosis, the 6-h treatment time of cells with nodularin (1mug/ml, 10mug/ml and 20mug/ml) was chosen for the alkaline comet assay. The kinetics of NER was determined in CHO cell lines: AA8 (wild-type) and mutant cell lines: UV135 (XPG(-)), UV41 (XPF(-)) and UV20 (ERCC1(-)) exposed to 20J/m(2) UV radiation. The micronucleus assay was performed to determine a residual DNA damage in four cell lines treated with nodularin (10mug/ml) and exposed to equitoxic doses UV radiation. Radiation doses of UV producing 50% of survival for AA8, UV135, UV20 and UV41 cell lines were calculated from UV survival curves. The results show that nodularin impairs the incision/excision step of NER in CHO cells by the ERCC1/XPF inactivation and leads to an increased level of UV-induced cytogenetic DNA damage.

Cytogenetic analysis of tannery workers in Morocco.

This study investigated the possible genetic effects in blood lymphocytes of tannery workers from Morocco being professionally exposed to multiple chemical agents. It was shown that the frequencies of cells with chromosome aberrations and micronuclei were significantly increased in the lymphocytes of the workers compared with the frequencies found in an unexposed control population.

Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo.

We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole-body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 microg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation.

Genotoxicity of the Musi River (Hyderabad, India) investigated with the VITOTOX test.

The bacterial VITOTOX genotoxicity test was used to screen water samples collected from three different stations along the banks of the river Musi, in Hyderabad, India. Water was collected at three stations that differed from each other in the nature of the surrounding industrial and other activities. A number of different pollutants were also measured in water, soil and air samples. The three stations were found highly polluted and different with regard to the genotoxicity and toxicity of their samples. These results demonstrate the need for further biological studies in this area to generate valuable data on genomic instability, risk assessment of cancer, and to provide avenues for risk management.

Genetic effects of radiofrequency radiation (RFR).

The possible effects of radiofrequency (RF) exposure on the genetic material of cells are considered very important since damage to the DNA of somatic cells can be linked to cancer development or cell death whereas damage to germ cells can lead to genetic damage in next and subsequent generations. This is why the scientific literature reports many investigations on the subject. According to a number of review papers, the conclusion so far is that there is little evidence that RFR is directly mutagenic and that adverse effects that were reported in some of the papers are predominantly the result of hyperthermia. Yet, some subtle indirect effects on DNA replication and/or transcription of genes under relatively restricted exposure conditions cannot be ruled out. Furthermore, the possibility of combined effects of RFR with environmental carcinogens/mutagens merits further attention. The present paper takes into account more recent investigations but the conclusion remains the same. A majority of studies report no increased (cyto)genetic damage but yet, a considerable number of investigations do. However, many studies were not sufficiently characterized, are therefore difficult to replicate and cannot be compared to others. Experimental protocols were very different from one study to another and investigations from a single laboratory were very often limited in the sample size or number of cells investigated, preventing a robust statistical analysis. Subtle, but significant differences between RFR-exposed and sham-exposed cells cannot be found in such conditions. For the above reasons, it was concluded at a workshop in Löwenstein (November 2002) that further investigations by individual laboratories most probably will not add much to the discussion of radiofrequency radiation (RFR) genotoxicity. Large, well coordinated, international collaborative studies involving participation of several experienced scientists are considered an alternative of uttermost importance. One such study is now being planned.

Effect of coexposure to 50 Hz magnetic fields and an aneugen on human lymphocytes, determined by the cytokinesis block micronucleus assay.

Interference of 50 Hz extremely low frequency magnetic fields (ELF-MF) with the known aneugen vinblastine (VBL) on micronucleus formation was tested with the in vitro cytokinesis block micronucleus assay in human lymphocyte cultures. Isolated lymphocyte cultures were prepared from 18 individuals. Three groups of quadruplicate cultures from six unrelated individuals were exposed to 50 Hz ELF-MF of background (bkg), 80 and 800 microT, respectively, during the complete incubation period (72 h). Twenty-four hours after culture initiation, one replicate culture from each individual within each ELF-MF group was exposed to 0, 5, 10, or 15 ng/ml VBL. The isolated lymphocyte cultures were scored for the presence of micronuclei, the nuclear division index (NDI), and apoptosis. As expected, increased VBL concentration resulted in an increased micronucleus and apoptosis frequency and in a decreased NDI. In the presence of VBL, there was a systematic tendency for increased micronucleus and apoptosis frequency in the ELF-MF exposed groups compared to the bkg group. In the absence of VBL, we observed no statistically significant effect of ELF-MF on micronucleus induction or apoptosis frequency, but the NDI was significantly higher in the 800 microT group compared to the other groups, suggesting an effect of ELF-MF on cell proliferation. An interaction between ELF-MF and VBL on NDI was observed. This interaction reflected the drastic decrease in NDI due to coexposure to VBL.

Haemopoietic cell proliferation in murine bone marrow cells exposed to extreme low frequency (ELF) electromagnetic fields.

As leukemia is one of the health hazards that is sometimes associated with exposure to extreme low frequency fields, we studied the in vitro effects of ELF fields on haemopoietic cell proliferation. First, the cytotoxic effect of 80 microT, 50 Hz magnetic fields on 3T3 cell proliferation was investigated using the neutral red test. Many chemicals are believed to cause damage because they interfere with basal or "housekeeping" cell functions. The basal cell functions are present in every cell. Non-specialized, actively dividing cells are suitable for measuring cytotoxic effects. Cytotoxic doses can be identified by exposing actively dividing cells in vitro and measuring growth inhibition caused by interference with these basal cell functions. 80 microT, 50 Hz magnetic fields caused no cytotoxicity: we were not able to demonstrate any interference with essential cell functions in the non-differentiated 3T3 cell line. Furthermore, the in vitro effects of ELF fields on murine haemopoietic and stromal stem cell proliferation were studied. Haemopoiesis is a continuous process, where mature blood cells are replaced by the proliferation and differentiation of more primitive progenitor and stem cells. Blood formation is tightly regulated by the stromal micro-environment. Exposure of murine bone marrow cells, from male and female mice, to 80 microT (50 Hz) magnetic fields showed a reduction in the proliferation and differentiation of the granulocyte-macrophage progenitor (CFU-GM) compared to non-exposed bone marrow cells. The results on the effect of the ELF-field on stromal stem cell proliferation (CFU-f) are somewhat equivocal at the moment. CFU-f from female mice showed a reduction while CFU-f from male mice were not decreased.

Genotoxic effects of carbon black particles, diesel exhaust particles, and urban air particulates and their extracts on a human alveolar epithelial cell line (A549) and a human monocytic cell line (THP-1).

The possible genotoxicity of small particulate matter has been under investigation for the last 10 years. Diesel exhaust particles (DEP) are considered as "probably carcinogenic" (IARC group 2A) and a number of studies show genotoxic effects of urban particulate matter (UPM). Carbon black (CB) is carcinogenic in rats. In this study the cytotoxic and genotoxic potency of these three particle types was investigated by exposing human cells (A549 and THP-1 cell lines) in vitro to CB, DEP (SRM 1650, NIST), and UPM (SRM 1648, NIST) for 48 hr. Cytotoxicity was assessed using the Alamar Blue assay, whereas genotoxicity was assessed using the single-cell gel electrophoresis (comet assay). The particles were characterized with regard to their mean diameter in tissue culture medium (CB 100 nm, DEP 400 nm, UPM 2 microm), their total carbon content (CB 99%, DEP 85%, UPM 15%), and their acid-soluble metal composition (UPM >> CB approximately DEP). The concentrations ranged from 16 ng/ml to 16 microg/ml for cytotoxicity tests and from 16 ng/ml to 1.6 microg/ml for genotoxicity tests. In both assays, paraquat was used as a reference chemical. The CB, DEP, and UPM particles showed no significant cytotoxicity. However, all three particles were able to cause significant DNA damage, although to a different extent in the two cell lines. The genotoxicity of washed particles and dichloromethane extracts was also investigated. In THP-1 cells CB washed particles and DEP extracts caused significant DNA damage. This difference in effect may be related to differences in size, structure, and composition of the particles. These results suggest that CB, DEP, and UPM are able to cause DNA damage and, therefore, may contribute to the causation of lung cancer. More detailed studies on influence of size, structure, and composition of the particles are needed.

Protective effect of the bile salt hydrolase-active Lactobacillus reuteri against bile salt cytotoxicity.

Bacterial bile salt hydrolysis is considered a risk factor for the development of colon cancer because of the risk of forming harmful secondary bile salts after an initial deconjugation step. In this study, the influence of enhanced bacterial bile salt transformation by the bile salt hydrolase-active Lactobacillus reuteri was studied in batch culture using the microbial suspension of the Simulator of the Human Intestinal Microbial Ecosystem; (SHIME), which was supplemented with oxgall at 5 g/l or 30 g/l. Changes in the fermentative capacity of the microbial ecosystem and the (geno)toxic properties of the SHIME supernatants were investigated. Increasing concentrations of oxgall inhibited the fermentation. Transient cell toxicity was observed for samples supplemented with 5 g oxgall/l, while samples with 30 g oxgall/l exhibited toxicity. The results of the haemolysis test suggest that the detrimental effects were probably due to the membrane-damaging effects of bile salts. In all cases, the adverse effects could be counteracted by the addition of 7.5 +/- 0.5 log10 CFU L. reuteri/ml. Plausible mechanisms for the protective properties of L. reuteri could involve a precipitation of the deconjugated bile salts and a physical binding of bile salts by the bacterium, thereby making the harmful bile salts less bioavailable.

The comet assay: a tool to study alteration of DNA integrity in developing plant leaves.

DNA integrity of Nicotiana tabacum L. and Vicia faba L. leaves in different stages of growth was analysed with the single cell gel electrophoresis (comet) assay. With this test DNA of individual cells is stretched by electrophoresis and the migration is measured, which gives an image of the nuclear DNA organisation. Nuclei were sampled when the plants had developed an apical bud, five true leaves and cotyledons. To get an idea of the kind of lesions observed, three different comet protocols were used. The neutral protocol with electrophoresis in a neutral buffer and the semi-alkaline or alkaline assay with alkaline unwinding followed by electrophoresis in neutral alkaline buffer, respectively. For V. faba there was a successive increased cellular DNA mobility with age of the leaves. The percentage DNA migration in control cells of fully developed leaves from N. tabacum almost reached the same level than after irradiation of not fully developed leaves with 50 Gy X-rays. The increased stretching of DNA with leaf age was most obvious if the DNA duplex was converted to single strands by alkali treatment before electrophoresis. Therefore, it could be concluded that with the ageing of leaves there is a decrease in DNA integrity, which could be the result of rising amounts of DNA single-strand breaks and 'alkali-vulnerable sites'.

Genetic, carcinogenic and teratogenic effects of radiofrequency fields.

This paper reviews the literature data on the genetic toxicology of radiofrequency (RF) radiation. Whereas in the past most studies were devoted to microwave ovens and radar equipment, it is now mobile telecommunication that attracts most attention. Therefore we focus on mobile telephone frequencies where possible. According to a great majority of the papers, radiofrequency fields, and mobile telephone frequencies in particular, are not genotoxic: they do not induce genetic effects in vitro and in vivo, at least under non-thermal exposure conditions, and do not seem to be teratogenic or to induce cancer. Yet, some investigations gave rather alarming results that should be confirmed and completed by further experiments. Among them the investigation of synergistic effects and of possible mechanisms of action should be emphasised.

Mixed phenotype murine leukemias.

Cell lines were derived from eight individual leukemias induced by X-rays in NFS mice. First typed as null cells (surface immunoglobulin negative, Thy-1 negative), they turned out to have a mixed phenotype with myeloid cytochemical markers, pre-B surface antigens and molecular markers of pro-B lymphocytes. They represent murine models for mixed phenotype (pro-pre-B-myeloid) leukemias.

Influence of blood storage after in vitro exposure on the yield of micronuclei observed in human lymphocytes.

In a cooperative study, four laboratories evaluated the micronucleus test in irradiated and non-irradiated lymphocytes with respect to time of storage, the difference between X-ray and gamma exposure and inter-observer variance. The results were compared with parallel studies on dicentric aberrations. No significant differences between laboratories, with respect to storage time or between gamma and X-irradiation were observed for micronuclei and dicentrics using analysis of variance. However, micronuclei are not suitable for an assessment of exposure inhomogeneity because of a significant overdispersion already in controls and homogeneously irradiated samples.

Stable chromosome aberrations 25 years after severe accidental radiation exposure.

A thorough cytogenetic analysis using G-banding was performed on 100 peripheral blood lymphocytes from an individual who had been accidentally exposed to radiation more than 25 years previously. More than 60% of the analysed cells were found to possess one or more stable chromosome aberrations (e.g. reciprocal translocations). Chromosomes 1 and 11 were more involved in these aberrations than would be expected from the relative chromosome lengths. No identical stable aberrations were found, suggesting that, 25 years after near-lethal exposure, haemopoietic stem cells display substantial diversity.

Suggestive evidence that genes controlling invasion and metastasis of T-cell lymphomas are located on mouse chromosome 3.

Cell lines differing in their malignant potential have been derived from the murine BW5147 T-cell lymphosarcoma. To evaluate the involvement of chromosomal aberrations in tumor progression within this model, we have analyzed the karyotypes and the in vitro invasiveness of 13 related nonmetastatic and metastatic variants. Giemsa banding revealed the presence of several marker chromosomes, one of which was of particular importance. Depending on the cell line, four variants of this marker I were found: Marker Ia corresponds to two translocated chromosomes 3, marker Ib is a deleted Ia marker, marker Ic is a Ib translocated to small unidentified chromosome fragment, and marker Id is a further deleted Ib marker. The Ia and Id markers were characteristic for the noninvasive, nonmetastatic lines, whereas the Ib and Ic markers predominated in the invasive, metastatic variants. The results suggest that metastasis-enhancing genes are located between the D and FI band of mouse chromosome 3 and that metastasis-suppressing genes are located between the FI and H band of the same chromosome.