Epinephrine, DNA integrity and oxidative stress in workers exposed to extremely low-frequency electromagnetic fields (ELF-EMFs) at 132 kV substations.

There is apprehension about widespread use of electrical and electromagnetic gadgets which are supposed to emit electromagnetic radiations. Reports are controversy. These electromagnetic fields (EMFs) have considerable effect on endocrine system of exposed subjects. This study was focused to assess the possible bioeffects of extremely low-frequency (ELF)-EMFs on epinephrine level, DNA damage and oxidative stress in subjects occupationally exposed to 132 kV high-voltage substations. The blood sample of 142 exposed subjects and 151 non-exposed individuals was analyzed. Plasma epinephrine was measured by enzyme-linked immunosorbent assay, DNA damage was studied by alkaline comet assay along with oxidative stress. Epinephrine levels of sub-groups showed mean concentration of 75.22 ± 1.46, 64.43 ± 8.26 and 48.47 ± 4.97 for high, medium and low exposed groups, respectively. DNA damage ranged between 1.69 µm and 9.91 µm. The oxidative stress levels showed significant increase. The individuals employed in the live-line procedures were found to be vulnerable for EM stress with altered epinephrine concentrations, DNA damage and increased oxidative stress.

Immunohistochemical evaluation of p53, FHIT, and IGF2 gene expression in esophageal cancer.

The aim of the study was to evaluate the expression of tumor suppressor genes p53, fragile histidine triad gene (FHIT), and an oncogene insulin-like growth factor 2 (IGF2) as prognostic markers in the etiology of esophageal cancer. Immunohistochemistry (IHC) was performed in 39 archival tissue samples of different esophageal pathologies for the three genes. Abnormal p53 expression was maximum in all the cases of squamous cell carcinoma, while IGF2 expression was enhanced in squamous cell carcinoma (81%), adenocarcinoma (100%), and dysplasia of squamous epithelium (75%) samples when compared with normals (50%). To our surprise, 75% of normal tissues did not show FHIT expression, which was also not seen in 40% of dysplasias of squamous epithelium, 33.3% of adenocarcinoma, and 41% of squamous cell carcinoma. To the best of our knowledge, this is the first study evaluating IGF2 by IHC, as well as, correlating it with the expression of the two tumor suppressor genes, p53 and FHIT, in esophageal tissue. p53 expression was threefold higher than normal in dysplasias of squamous epithelium and adenocarcinoma, while it was eightfold higher in squamous cell carcinoma. IGF2 expression was low in normal and dysplasia tissue but was increased 1.97-fold in both types of malignancy. FHIT and p53 expression were well correlated in squamous cell carcinoma, supporting the observation that FHIT regulates and stabilizes p53. Altered/lowered FHIT levels may be a result of exposure to various exogenous agents; however, this could not be assessed in the present study as it was carried out on archival samples. A larger prospective study is warranted to establish the role of exogenous factors in FHIT expression.

Combinative exposure effect of radio frequency signals from CDMA mobile phones and aphidicolin on DNA integrity.

The aim of present study is to assess DNA integrity on the effect of exposure to a radio frequency (RF) signal from Code Division Multiple Access (CDMA) mobile phones. Whole blood samples from six healthy male individuals were exposed for RF signals from a CDMA mobile phone for 1 h. Alkaline comet assay was performed to assess the DNA damage. The combinative exposure effect of the RF signals and APC at two concentrations on DNA integrity was studied. DNA repair efficiency of the samples was also studied after 2 h of exposure. The RF signals and APC (0.2 microg/ml) alone or in synergism did not have any significant DNA damage as compared to sham exposed. However, univariate analysis showed that DNA damage was significantly different among combinative exposure of RF signals and APC at 0.2 microg/ml (p < 0.05) and at 2 microg/ml (p < 0.02). APC at 2 microg/ml concentration also showed significant damage levels (p < 0.05) when compared to sham exposed. DNA repair efficiency also varied in a significant way in combinative exposure sets (p < 0.05). From these results, it appears that the repair inhibitor APC enhances DNA breaks at 2 microg/ml concentration and that the damage is possibly repairable. Thus, it can be inferred that the in vitro exposure to RF signals induces reversible DNA damage in synergism with APC.

Stem cell test: a practical tool in toxicogenomics.

During early embryonic development, at blastocyst stage, the embryo has an outer coat of cells and an inner cell mass (ICM). ICM is the reservoir of embryonic stem (ES) cells, which are pluripotent, i.e., have the potential to differentiate into all cell types of the body. Cell lines have been developed from ES cells. In addition, there are embryonic germ (EG) cell lines developed from progenitor germ cells, and embryonic carcinoma (EC) cell lines developed from teratomas. These cell lines are being used for the study of basic and applied aspects in medical therapeutics, and disease management. Another potential of these cell lines is in the field of environmental mutagenesis. In addition to ES cells, there are adult stem cells in and around different organs and tissues of the body. It is now possible to grow pure populations of specific cell types from these adult stem cells. Treating specific cell types with chemical or physical agents and measuring their response offers a shortcut to test the toxicity in various organ systems in the adult organism. For example, to evaluate the genotoxicity of a chemical (e.g., drug or pesticide) or a physical agent (e.g., ionizing radiation or non-ionizing electromagnetic radiation) during embryonic development, a large number of animals are being used. As an alternative, use of stem cell lines would be a feasible proposition. Using stem cell lines, efforts are being made to standardize the protocols, which will not only be useful in testing the toxicity of a chemical or a physical agent, but also in the field of drug development, environmental mutagenesis, biomonitoring and other studies.

Evaluation of micronucleus frequencies and DNA damage in glass workers exposed to arsenic.

Arsenic (As) is a known human carcinogen; however, very little is known about the health consequences of occupational exposure to As. In the present study, we assessed the genotoxic damage in the blood cells and in the buccal cells of south Indian glass factory workers who are occupationally exposed to As. The As content in the whole blood of 200 workers and 165 controls was evaluated with inductively coupled plasma mass spectrometry. Blood leukocytes from the subjects were monitored for the level of DNA damage using the Comet assay (mean comet tail length); buccal cells were used to determine the frequency of micronuclei (MN). The mean As concentration was significantly higher in the workers (56.76 microg/L) than in the controls (11.74 microg/L) (P < 0.001). The workers also had increased frequencies of MN in the buccal cells and increased levels of DNA damage in leukocytes compared to the controls (P < 0.001). There were significant correlations between the genotoxicity endpoints that were evaluated and blood As concentration, smoking, age, and the duration of working in the factory. Also, a significant correlation was observed between the frequency of MN and comet tail-length for the worker samples. Our findings indicate that chronic occupational exposure to As is genotoxic and that the Comet assay and micronucleus test are useful assays for evaluating genotoxicity in humans occupationally exposed to As.

Melatonin in pathogenesis and therapy of cancer.

Melatonin is a neuroendocrine hormone secreted by the pineal gland to transduce the body's circadian rhythms. An internal 24 hour time keeping system (biological clock) regulated by melatonin, controls the sleep-wake cycle. Melatonin production is a highly conserved evolutionary phenomenon. The indole hormone is synthesized in the pinealocytes derived from photoreceptors. Altered patterns and/or levels of melatonin secretion have been reported to coincide with sleep disorders, jetlag, depression, stress, reproductive activities, some forms of cancer and immunological disorders. Lately, the physiological and pathological role of melatonin has become a priority area of investigation, particularly in breast cancer, melanoma, colon cancer, lung cancer and leukemia. According to the 'melatonin hypothesis' of cancer, the exposure to light at night (LAN) and anthropogenic electric and magnetic fields (EMFs) is related to the increased incidence of breast cancer and childhood leukaemia via melatonin disruption. Melatonin's hypothermic, antioxidant and free radical scavenging properties, attribute it to an immunomodulator and an oncostatic agent as well. Many clinical studies have envisaged the potential therapeutic role of melatonin in various pathophysiological disorders, particularly cancer. A substantial reduction in risk of death and low adverse effects were reported from various randomized controlled trials of melatonin treatment in cancer patients. This review summarizes the physiological significance of melatonin and its potential role in cancer therapy. Furthermore, the article focuses on melatonin hypothesis to represent the cause-effect relationship of the three aspects: EMF, LAN and cancer.

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.

Expression of common fragile sites in untreated non-Hodgkin's lymphoma with aphidicolin and folate deficiency.

The frequency and distribution of aphidicolin induced and folate sensitive common fragile sites on chromosomes of peripheral blood lymphocytes in untreated non-Hodgkin's lymphoma patients and healthy controls showed a considerable overlap in the expression of common fragile sites between the two groups. However, a significant increase in the expression of 16 aphidicolin induced common fragile sites was seen in untreated lymphoma patients. In the folate deficient cultures only, the common fragile sites 2q22, 8q24, 11q13, 12q21, 16q22, 17p12 and 20p12 were found in both the groups under study. The fragile sites at 8q22, 8q24, 11q13 and 18q21 in patients showed an increased expression over the control group. Interestingly these fragile sites were located in the same chromosomal bands as the oncogenes, MOS, MYC, BCL-1 and BCL-2 as well as cancer breakpoints specifically associated with non-Hodgkin's lymphoma, suggesting the possibility that fragile sites may play a critical role in the pathogenesis of non-Hodgkin's lymphoma.

Guidelines for the preparation and analysis of the fragile X chromosome in lymphocytes.

The following guidelines were adopted by an Ad Hoc Committee convened at the Fourth International Workshop on the Fragile X Syndrome and X-Linked Mental Retardation to establish minimum cytogenetic standards for the preparation and analysis of the fragile X chromosome. The intention of the committee was to develop and provide practical standards for the routine cytogenetic detection of the fragile X. The guidelines describe reasonable criteria for effective tissue culture methods for eliciting the Xq27.3 fragile site in vitro and for the analysis of such chromosome preparations.

Genotoxicity evaluation of the tricyclic antidepressants amitriptyline and imipramine using human lymphocyte cultures.

Two tricyclic antidepressants, amitriptyline and imipramine, were evaluated for their in vitro cytogenetic effects in human lymphocyte cultures. Peripheral blood cultures from three normal healthy donors were set up for 72 hr for each of the drugs. The drugs were added at the start (72-hr exposure), 24 hr (48-hr exposure), and 48 hr (24-hr exposure) after initiation of the cultures. The concentrations evaluated at each exposure time were 50, 250, 1,000, and 10,000 ng/ml for amitriptyline and 25, 500, and 5,000 ng/ml for imipramine. The first two concentrations correspond to the plasma levels of the respective drugs after therapeutic doses. All treatments for a donor were given at the same time. Untreated cultures served as controls for the baseline frequency of the parameters assayed. The parameters assayed were chromosome aberrations, mitotic index, and sister chromatid exchanges (SCEs). Amitriptyline was found to be nongenotoxic at plasma levels by all the parameters assayed. However, frequencies of chromosome aberrations and SCEs were significantly increased at concentrations 4 and 40 times the plasma level (1,000 and 10,000 ng/ml) although the actual increases was small. The mitotic index was not affected at any concentration. Through imipramine showed a significant increase in chromosome damage at the upper plasma level and at concentrations higher than that, SCE frequency was significantly increased only at concentration higher than the plasma level (5,000 ng/ml), the actual increase being small for both these parameters. The mitotic index was not affected at any concentration. These results suggest that amitriptyline may be a slightly safer drug than imipramine from a genetic point of view.

In vivo genotoxic effect of arsenic trioxide in mice using comet assay.

Although arsenic has been the subject of toxicological research, acute in vivo genotoxic studies using relevant animal models and uniform methodology are lacking. Hence, the present study aims to study DNA damage caused by arsenic trioxide in mice in in vivo using alkaline single cell gel electrophoresis (Comet) assay. Mice were administered orally 0,0.13,0.27,0.54,1.08,2.15,4.3 and 6.45 mg/kg body weight of arsenic trioxide dissolved in distilled water. The samples of whole blood were collected at 24,48,72 h, first and second week post-treatment and the assay was carried out to determine DNA damage as represented by comet tail-length. All the doses induced significant increase in comet tail-length at 24 h post-treatment (P<0.05) showing a clear dose dependent increase from 0.13 to 2.15 mg/kg b.wt. and a dose dependent decrease in higher doses (4.3-6.45 mg/kg b.wt). At 48 h post-treatment all the doses showed a significant increase (P<0.05) in comet tail-length when compared to 24 h post-treatment. A gradual decrease in the comet tail-length was observed for all the doses from 72 h post-treatment onwards indicating a gradual repair in DNA damage. This indicates a non-linear dose and time response between DNA damage and different doses of arsenic trioxide at different time-intervals. A significant increase in comet tail-length at all the doses clearly gives evidence that arsenic trioxide cause DNA damage effectively. The study indicates that the alkaline comet assay is a reliable and effective method to detect DNA damage caused by metals.

In vivo genotoxic effects of smoking and occupational lead exposure in printing press workers.

The objective of the present study was to evaluate the genotoxicity of a combination exposure to lead and smoking in workers from the printing industry and also to examine the possible interaction between the two agents. Individuals were classified into 4 different groups: control group, lead-exposed group, smokers and the double-exposure group. Chromosomal analysis was carried out according to conventional methods. Our preliminary study shows that lead-exposed individuals had a significantly increased frequency of sister chromatid exchanges. Further, double exposure to smoking and lead inhibits mitosis.

Are rogue cells an indicator of cancer risk due to the action of bacterial restriction endonucleases?

Cytogenetic surveys in normal individuals have occasionally shown the occurrence of cells with multiple chromosome-type aberrations in some of the subjects. These cells, which are rare, have been termed as rogue cells. Rogue cells, which have been observed worldwide, have a mysterious nature. It has been suggested that they may give rise to cancer. Various mechanisms have been considered for the causation of the rouge-cell phenomenon in the past but none of them appears to be fully justified. In this paper we propose their occurrence due to the action of bacterial restriction endonucleases.

Effect of cephaloridine on human chromosomes in vitro in lymphocyte cultures.

Cephaloridine is a semi-synthetic broad-spectrum bactericidal antibiotic derived from cephalosporin C. The drug was tested for its clastogenic effects on human chromosomes in vitro at various concentrations and for various periods of treatment (24, 48, 72 h) in 72-h lymphocyte cultures. 2 concentrations of the drug used in the tests (20 and 60 micrograms/ml) were similar to those found in the plasma of individuals after receipt of therapeutic doses of 500 mg and 2 g, respectively. 2 other concentrations tested were either below (10 micrograms/ml) or above (120 micrograms/ml) the plasma level. Control cultures were set up simultaneously for each batch of experimental cultures. The aberration frequency was increased with increasing concentration of the drug, irrespective of the duration of the exposure. In other words, there was a dose-effect relationship. No increase in chromosomal aberrations was observed with lower doses of 10 and 20 micrograms/ml. Significant increases were seen with higher doses of 60 and 120 micrograms/ml. Aberrations were mostly of the chromatid type. The results show that cephaloridine is clastogenic at the upper levels of permissible therapeutic doses.

Genotoxicity evaluation of individuals working with photocopying machines.

Photocopying machines are a common sight in the cities of India. There is ample evidence showing that the components of toners individually or in the form of a complex mixture are genotoxic. Toxic components of the photocopiers are from their emissions, toners and extremely low frequency electromagnetic fields (ELF-EMFs). In the present study micronucleus test (MNT) on buccal epithelial cells, cytokinesis block micronucleus (CBMN) assay and chromosomal aberration analysis on peripheral blood mononuclear cells was performed on 98 workers occupationally involved in photocopying and 90 age and sex matched controls. The results showed a significant increase in the frequency of MN in buccal epithelial cells and peripheral blood lymphocytes, as well as chromosomal aberrations in the exposed as compared to the control subjects.

Detection of influenza virus induced DNA damage by comet assay.

Influenza virus A2/HK/68 is known to be a biological mutagen and teratogen. Reports are available implicating influenza virus as a causative agent of chromosomal aberrations in cells in culture and also in circulating leukocytes of humans. Also, an increased incidence of abortions, prenatal mortality and congenital abnormalities during the periods of epidemics has also been reported. In view of these reports, it would be worthwhile to screen persons especially pregnant women exposed to influenza virus for possible DNA damage. The present study reports the use of Comet assay to measure influenza virus induced DNA damage. We have carried out in vitro infection experiments using human leukocytes. Our results clearly indicate that influenza virus A2/HK/68 induces DNA damage in leukocytes right from 2-h post-infection. Maximum damage was observed at 24-h post-infection. However, at 48-h post-infection, a slight decrease was observed which can be attributed to the DNA repair occurring in the cells. Thereafter, irreparable damage was noticed. Cell viability results have shown lack of cytotoxicity till 72-h post-infection. However, significant cytotoxicity was observed only at 96-h post-infection. The occurrence of DNA damage without cell death may result in chromosomal aberrations or mutations. Therefore, it is most advisable to get screened for the possible DNA damage especially persons frequently infected with influenza and pregnant women.

Risk assessment in cervical dysplasia patients by single cell gel electrophoresis assay: a study of DNA damage and repair.

Precancerous lesions of cervix, commonly known as dysplasia, present a complex problem because of their biological behavior. Increased genetic instability, either inherent or induced by some external mutagen, is considered as a primary event or a predisposing factor to neoplastic transformation. The relationship between genetic instability and susceptibility towards cervical cancer was evaluated with the comet or single cell gel electrophoresis (SCGE) assay. Among precancerous individuals, genomic instability was observed in cervical epithelial cells and peripheral blood leukocytes. The mean basal DNA damage and mean susceptibility to DNA damage by the mutagen (MNNG) treatment increased whereas repair capacity decreased with progression of the disease in a stepwise manner. Inter and intra individual variability was maximum in cancerous group. Risk was estimated by giving a predictive value for each precancerous individual. In combination with morphological, biochemical, and cytogenetic parameters, the SCGE assay may serve as a novel tool to predict the fate of cervical dysplasia.

DNA damage in leukocytes of mice treated with copper sulfate.

Single stranded DNA breaks induced by copper sulfate (CuSO(4)) in mice has been studied in vivo using Alkaline Single Cell Gel Electrophoresis (Comet assay). Mice were administered orally with doses of 0, 1.25, 2.50, 5.00, 7.50, 10.00 and 12.50 mg/kg body weight (b.wt.) of CuSO4 respectively. The samples of whole blood were collected at 24, 48, 72 h, first week and second week post-treatment and the assay was carried out to determine single strand DNA breaks as represented by comet tail-length. In addition, the sample was used to study the repair efficiency by incubating the samples with RPMI medium for 2 h. Results indicated a significant DNA damage at all the doses after treatment with CuSO4 when compared to controls showing a clear dose-dependent response (p < 0.05). A gradual decrease in the tail-lengths from 48 h post-treatment was observed and by second week, the values returned to control levels at all doses. The study on the repair efficiency indicated that mice treated with all the doses of CuSO4 showed decrease in mean comet tail-length indicating repair efficiency capacity but less when compared to those of controls. The study also reveals that comet assay is a sensitive and rapid method for detecting DNA damage caused by trace metals such as copper (Cu).

Genotoxic effect of monocrotophos to sentinel species using comet assay.

Monocrotophos is the single largest selling agrochemical in India. Sensitive biomarkers to study the genotoxic effects caused by monocrotophos in aquatic organisms, especially fish, are lacking. The fish used in this study are Tilapia mossambica, which are edible, commercially valuable and distributed all over India. The objective of this study was to study DNA strand breaks induced by monocrotophos in T. mossambica in vivo using single-cell micro gel electrophoresis/comet assay. Tilapia were treated orally with 0.313, 0.625, 1.25, 1.875, 2.5, 3.125, 3.75 and 4.375 ppm of monocrotophos and the assay was performed on nucleated erythrocytes after 24, 48, 72 and 96 h. A significant increase in mean comet tail-length (5.21-7.46 microM), indicating DNA damage, was observed at all the doses with monocrotophos when compared to controls (3.36 microM). The mean tail-length showed a dose-related increase and time-dependent decrease. The maximum increase in mean comet tail-length was observed at 24 h. Relative to these effects, reductions in mean comet tail-length were seen at 48 and 72 h. By 96 h, values had returned to control levels at all doses, indicating repair of the damaged DNA and/or loss of heavily damaged cells. The study reveals that the comet assay is a sensitive and rapid method to detect genotoxicity of monocrotophos and other environmental pollutants in sentinel species.