Evaluation of genotoxic effects in male Wistar rats following microwave exposure

Wistar rats (70 days old) were exposed for 2 h a day for 45 days continuously at 10 GHz [power density 0.214 mW/cm2, specific absorption rate (SAR) 0.014 W/kg] and 50 GHz (power density 0.86 microW/cm2, SAR 8.0 x10(-4) W/kg). Micronuclei (MN), reactive oxygen species (ROS), and antioxidant enzymes activity were estimated in the blood cells and serum. These radiations induce micronuclei formation and significant increase in ROS production. Significant changes in the level of serum glutathione peroxidase, superoxide dismutase and catalase were observed in exposed group as compared with control group. It is concluded that microwave exposure can be affective at genetic level. This may be an indication of tumor promotion, which comes through the overproduction of reactive oxygen species.     

Osteosarcoma development following single inhalation exposure to americium-241 in beagle dogs

Young, mature Beagle dogs underwent single inhalation exposure to respirable aerosols of 241AmO2 to determine the radiation dose distribution to tissues. The dogs were serially sacrificed to assess the clearance of 241Am from the lung, the rate of translocation to internal organs, the pattern of retention in the organs, and the rates and modes of excretion. Americium dioxide was relatively soluble in the lung, leading to the translocation of significant quantities of 241Am to bone and liver, thus delivering radiation doses to these tissues nearly equal to that received by the lung. Osteoblastic osteosarcomas developed in four dogs surviving more than 1000 days after exposure. Histologically, all of the osteosarcomas were associated with areas of radiation osteodystrophy characterized by bone infarction, peritrabecular new bone formation, marrow fibrosis, and microresorptive cavities. The retention and translocation of inhaled 241Am in dogs is similar to that of man, indicating that 241Am inhaled by humans may potentially result in significant risk of bone tumor development.     

Occupational exposure to genotoxic agents.

Millions of workers in the United States are potentially exposed each year to hazardous chemicals, dusts, or fibers in occupational settings. Some of these agents are genotoxic and may cause genetic alterations in the somatic or germ cells of exposed workers. Such alterations, if they occur in proto-oncogenes or tumor suppressor genes, which are involved in controlling cell growth or differentiation, may lead to the development of cancer. Genetic alterations in germ cells may also lead to reproductive failure or genetic disorders in subsequent generations. It has been estimated that occupational exposure accounts for 4% of all human cancers and up to 30% of cancer among blue-collar workers. Approximately 20,000 cancer deaths each year are attributable to occupational exposure in the United States. Occupational cancer and reproductive abnormalities have been listed on the National Occupational Research Agenda master list of research priorities as major occupational diseases and injuries.     

Measurement of plasma or urinary metabolites and Hprt mutant frequencies following inhalation exposure of mice and rats to 3-butene-1,2-diol

Studies were performed to determine if the detoxification pathway of 1,3-butadiene (BD) through 3-butene-1,2-diol (BD-diol) is a major contributor to mutagenicity in BD-exposed mice and rats. First, female and male mice and rats (4-5 weeks old) were exposed by nose-only for 6h to 0, 62.5, 200, 625, or 1250 ppm BD or to 0, 6, 18, 24, or 36 ppm BD-diol primarily to establish BD and BD-diol exposure concentrations that yielded similar plasma levels of BD-diol, and then animals were exposed in inhalation chambers for 4 weeks to BD-diol to determine the mutagenic potency estimates for the same exposure levels and to compare these estimates to those reported for BD-exposed female mice and rats where comparable blood levels of BD-diol were achieved. Measurements of plasma levels of BD-diol (via GC/MS methodology) showed that (i) BD-diol accumulated in a sub-linear fashion during single 6-h exposures to >200 ppm BD; (ii) BD-diol accumulated in a linear fashion during single or repeated exposures to 6-18 ppm BD and then in a sub-linear fashion with increasing levels of BD-diol exposure; and (iii) exposures of mice and rats to 18 ppm BD-diol were equivalent to those produced by 200 ppm BD exposures (with exposures to 36 ppm BD-diol yielding plasma levels approximately 25% of those produced by 625 ppm BD exposures). Measurements of Hprt mutant frequencies (via the T cell cloning assay) showed that repeated exposures to 18 and 36 ppm BD-diol were significantly mutagenic in mice and rats. The resulting data indicated that BD-diol derived metabolites (especially, 1,2-dihydroxy-3,4-epoxybutane) have a narrow range of mutagenic effects confined to high-level BD (>or=200 ppm) exposures, and are responsible for nearly all of the mutagenic response in the rat and for a substantial portion of the mutagenic response in the mouse following high-level BD exposures.     

Oxidative stress is induced in the rat brain following repeated inhalation exposure to manganese sulfate

Eight-week-old rats inhaled manganese (Mn) in the form of MnSO₄ at 0, 0.03, 0.3, or 3.0 mg Mn/m³ for 6 h/d for 7 d/wk (14 consecutive exposures). Brain manganese concentrations in these animals were reported by Dorman et al. in 2001, noting the following rank order: olfactory bulb>striatum>cerebellum. We assessed biochemical end points indicative of oxidative stress in these three brain regions, as well as the hypothalamus and hippocampus. Glutamine synthetase (GS) protein levels and total glutathione (GSH) levels were determined for all five regions. GS mRNA and metallothionein (MT) mRNA levels were also evaluated for the cerebellum, hypothalamus, and hippocampus. Statistically significant increases (p<0.05) in GS protein were observed in the olfactory bulb upon exposure to the medium and high manganese doses. In the hypothalamus, statistically significant (p<0.05) but more modest increases were also noted in the medium and high manganese dose. Total GSH levels significantly (p<0.05) decreased only in the hypothalamus (high manganese dose), and MT mRNA significantly increased in the hypothalamus (medium manganese dose). No significant changes were noted in any of the measured parameters in the striatum, although manganese concentrations in this region were also increased. These results demonstrate that the olfactory bulb and hypothalamus represent potentially sensitive areas to oxidative stress induced by exceedingly high levels of inhaled manganese sulfate and that other regions, and especially the striatum, are resistant to manganese-induced oxidative stress despite significant accumulation of this metal.     

Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation

The induction of inherited DNA sequence mutations arising in the germline (i.e., sperm or egg) of mice exposed in utero to diesel exhaust particles (DEPs) via maternal inhalation compared to unexposed controls was investigated in this study. Previous work has shown that particulate air pollutants (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development (in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates of spontaneous mutation, making this endpoint a valuable tool for studying inherited mutation and genomic instability. In the present study, pregnant C57Bl/6 mice were exposed to 19mg/m(3) DEP from gestational day 7 through 19, alongside air exposed controls. Male and female F1 offspring were raised to maturity and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females. In contrast, a statistically significant increase in the mutation frequency of male mice exposed in utero to DEP was observed (2-fold; Fisher's exact p<0.05). Thus, maternal exposure to DEP results in increased mutation in sperm during development.     

Induction of metallothionein in rat tissues following subchronic exposure to mercury shown by radioimmunoassay

Although the analysis of metallothionein (MT) by radioimmunoassay (RIA) is not a common technique, its use is preferred over other methods since it offers the advantages of sensitivity and specificity. In this paper we present data on the basal levels of MT in rat tissues and physiological fluids of female Sprague-Dawley rats. The mean basal MT concentrations of the following organs and fluids were determined by RIA to be: liver (9.8 μg/g), kidney (68 μ/g), brain (0.8 μg/g), spleen (1.0 μg/g), heart (5.4 μg/g), plasma (11 ng/ml), and urine (200–300 μg/g creatinine). Following subcutaneous exposure to inorganic mercury (0.2 μmol/kg/d, 5 d a week for up to 4 wk), the metal accumulated primarily in the kidney. There was also a simultaneous accumulation of zinc in the liver and of zinc and copper in the kidney. Induction of MT did take place in liver, kidney, brain, and spleen. No increases in the MT contents of blood and urine were noted. The excess zinc and copper in the kidney of exposed animals were found to be associated predominantly with MT. No overt signs of mercury toxicity were noted in these animals and the incidence of proteinurea was nil. The data are discussed with reference to methods of MT determination in animal tissues and in relation to mercury metabolism and toxicity.     

Experimental methods to study human transplacental exposure to genotoxic agents

Human placenta differs more than any other organ between species. This is the primary reason to develop models utilizing human tissue to study placental functions. There are no major ethical restrictions using human placenta for scientific studies. Also, the size of human placenta enables a great number of different parameters to be studied in one placenta. The most important cell types considering transplacental transfer, are the trophoblasts differentiating into syncytiotrophoblasts facing maternal circulation, and endothelial cells of fetal vessels. Primary trophoblasts are difficult to culture and do not grow in monolayer thus inhibiting studies on the polarized functions of transport. Several cell lines originating from trophoblasts have been developed, of which BeWo cells seem most useful for transport studies, because they grow in a tight monolayer. Placental tissue can also be retained as explant cultures, although the trophoblast viability is very restricted despite of culture conditions. Cotyledons of human placenta can be retained viable in an isolated organ perfusion. Perfused placental tissue stays viable longer than placental tissue in tissue culture. Although human placental perfusion is the most tedious experimental method to study placental functions, there are several good reasons to develop it further: transplacental transfer and molecular mechanisms of genotoxic compounds can be studied. Placental perfusion is the only experimental method that retains fully the structure of placenta for polarized transport. Furthermore, perfusion of placentas from mothers, who smoke, use illegal drugs or have a disease, allows studies on the impact of such factors on fetal exposure to genotoxic agents.     

Evaluation of genotoxic effects in human peripheral blood leukocytes following an acute in vitro exposure to 900 MHz radiofrequency fields

Human peripheral blood leukocytes from healthy volunteers have been employed to investigate the induction of genotoxic effects following 2 h exposure to 900 MHz radiofrequency radiation. The GSM signal has been studied at specific absorption rates (SAR) of 0.3 and 1 W/kg. The exposures were carried out in a waveguide system under strictly controlled conditions of both dosimetry and temperature. The same temperature conditions (37.0 +/- 0.1 degrees C) were realized in a second waveguide, employed to perform sham exposures. The induction of DNA damage was evaluated in leukocytes by applying the alkaline single cell gel electrophoresis (SCGE)/comet assay, while structural chromosome aberrations and sister chromatid exchanges were evaluated in lymphocytes stimulated with phytohemagglutinin. Alterations in kinetics of cell proliferation were determined by calculating the mitotic index. Positive controls were also provided by using methyl methanesulfonate (MMS) for comet assay and mitomycin-C (MMC), for chromosome aberration, or sister chromatid exchange tests. No statistically significant differences were detected in exposed samples in comparison with sham exposed ones for all the parameters investigated. On the contrary, the positive controls gave a statistically significant increase in DNA damage in all cases, as expected. Thus the results obtained in our experimental conditions do not support the hypothesis that 900 MHz radiofrequency field exposure induces DNA damage in human peripheral blood leukocytes in this range of SAR.     

Protection against genotoxic damages following whole body gamma radiation exposure in mice by lipoic acid

Alpha-lipoic acid (LA) protected plasmid pBR 322 DNA, under in vitro conditions from gamma radiation induced strand breaks as evidenced by the prevention of the loss of supercoiled covalently closed circular form upon irradiation. It also protected the membrane lipids of liver homogenates from the oxidative damages. Whole body exposure of mice to gamma-radiation resulted in damage to cellular DNA in various tissues and administration of LA prior to the radiation exposure prevented the radiation induced DNA damage as assessed by alkaline comet assay. Administration of LA to mice prior to the radiation exposure also prevented induction of chromosomal damages in bone marrow cells and formation of micronuclei in blood reticulocytes. Thus taken together, LA a normal cellular constituent could be used as a radioprotector against whole body radiation exposure scenarios.     

Erythropoietic activity of human blood following exposure to hyperbaric hyperoxygenation]

Before exposure of man to hyperoxia the blood plasma posessed erythropoietic activity, but 18 to 20 hours after exposure to compressed air condition in the high pressure chamber corresponding to the depth of 100 metres there proved to be a marked fall of the erythropoietic activity. No statistically significant shifts were revealed in the peripheral blood indices by that time.     

Induction of sister-chromatid exchanges and chromosomal aberrations in hematopoietic tissue of a marine fish following in vivo exposure to genotoxic carcinogens

The development of procedures to assess genetic damage in fish exposed in situ to point sources of aquatic pollution can be expected to contribute to the evaluation of the role of genotoxic contaminants in epizootic neoplasia in fish populations. To this end methods have been developed for assessing the in vivo induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in tissues of a marine teleost, the oyster toadfish, which may be applicable to other species. An alternative to the solid tissue and squash techniques for metaphase preparation permits the resolution of more than 100 SCEs/metaphase in toadfish kidney cells, which have moderately large chromosomes (0.122 pg DNA/chromosome). The bleeding of toadfish which have been injected with 5-bromodeoxyuridine (BrdUrd) and the subsequent use of hematopoietic tissue (kidney) for cytogenetic analysis was shown to increase the metaphase yield and provide a more predictable production of second-division metaphases required for SCE analysis. With these methods linear dose-dependent increases in chromatid-type exchange CAs and SCEs were obtained with i.p. exposure to ethyl methanesulfonate (EMS) and cyclophosphamide (CP). The doses required to double the observed control SCE frequencies (least effective doses) were 170 mg/kg for EMS and 7.4 mg/kg for CP. which are comparable to those reported for rodent bone marrow assays. A BrdUrd-sensitive site for chromatid breakage was observed on a pair of apparently homologous acrocentric chromosomes for the toadfish.     

Lactate dehydrogenase activity in human placenta following exposure to environmental pollutants.

The impact of environmental pollution at the place of residence of pregnant women and of their smoking habits on the cellular energy metabolism of placental tissue was investigated. Samples of full-term placentas were randomly collected from two environmentally different regions of Slovakia (Bratislava, Stará Lubovna) and the activity of lactate dehydrogenase (LDH) was measured. Our results showed enhanced LDH activity in the placenta that was dependent on both the type of environmental pollutants at the place of residence and the smoking habits during pregnancy. The enhanced LDH activity may reflect hypoxic conditions due to the accumulation of heavy metals and toxic compounds of tobacco smoke in the placental tissue. A high content of heavy metal particles, found in placental samples from Stará Lubovna in our previous studies, might contribute to the increased LDH activity in placentas from this region. We hypothesize that fine metal particles deposited in the placental tissue might be phagocytozed by the syncytiotrophoblast, thus contributing to the decreased oxygen level in placental tissue.     

Measurement of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase activity in human tissues.

The activity of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was measured in normal and diseased human liver, brain and kidney. Organs from patients with aspartylglucosaminuria show very little activity. Crude homogenates of human organs show a reaction catalysed by a complex enzyme system. With homogenate, the formation of product was linear with time up to about 6 h. Reaction times longer than 6-7h resulted in a decrease in the total concentration of product. This phenomenon was not found with the partially purified enzyme fraction. Linearity of the enzyme activity with different protein concentrations was found, independent of the incubation time. Longer incubation of the crude homogenate resulted in the utilization of the product, N-acetylglucosamine. This phenomenon was not observed with the partially purified enzyme fraction. This amidase from human organs differs from that obtained from other sources and apparently represents a rather complex enzyme system.