Changes in the number of DNA-protein cross links in spleen lymphocytes of mice exposed to low intensity low dose gamma irradiation

Levels of DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB) in spleen lymphocytes were studied in mice exposed to low-intensity gamma-radiation (1.7 mGy/day) for 1, 4, 10, 20, and 30 days. The spleen mass and count of lymphocytes isolated from this organ also has been investigated. The significant increase in the DPC level as compared to the control occurred on the 10-th and 30-th days of irradiation at doses of 1.7 and 5.1 cGy, accordingly. The number of spleen lymphocytes normalized to organ mass significantly decreased on the 4-th and 30-th days of the experiment. No increase was found in levels of alkali-labile sites and SSB. In contrast, the increase in the amount of duplex form DNA was recorded on the 4-th and 30-th days of the experiment. Our indicate that DPC formation after irradiation at low doses represents some form of cellular response to the damaging agent.     

Multi-endpoint biological monitoring of phosphine workers

5-Aminosalicylic acid (5ASA), a prescribed drug for ulcerative colitis, is a potent scavenger of oxygen-derived free radicals. The present study was undertaken to ascertain its ability to protect against radiation-induced damage. The drug dose-dependent effect, optimum time of drug administration and radiation dose-dependent effect (0-4 Gy) on in vivo radiation protection against micronuclei induction in polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were studied in the bone marrow of mice. Intraperitoneal injection of 10-125 mg/kg of the drug 30 min before whole body irradiation with 3 Gy produced a significant reduction in the frequency of micronucleated erythrocytes at 24 h after exposure. The optimum dose for protection without drug toxicity was 25 mg/kg body weight. Injection of 25 mg/kg of the drug 60 or 30 min before or within 15 min after 3 Gy whole body gamma-irradiation resulted in a significant decrease in the radiation-induced PCE and NCE with micronuclei (MPCE and MNCE) and an increase in the ratio of PCE to NCE (P/N), at 24 h post-irradiation. Maximum effect was seen when the drug was administered 30 min before irradiation. Therefore, to study the radiation dose-response, mice were pre-treated with 25 mg/kg of 5ASA 30 min before 1-4 Gy of gamma-irradiation. Radiation increased the MN frequency linearly (r(2)=0.99) with dose. Pre-treatment with 5ASA significantly reduced the MN counts to 40-50% of the radiation (RT) alone values, giving a dose modification factor (DMF) of 2.02 (MPCE) and 2.53 (MNCE). Irradiation resulted in a dose-dependent decline in the P/N ratio at all the doses of radiation studied. 5ASA produced a significant increase in the P/N ratio from that of irradiated controls, at all doses of radiations tested. These results show that 5ASA protect mice against radiation-induced MN formation and mitotic arrest.     

The genetic toxicity of 3,3',4,4'-tetrachloroazobenzene and 3,3',4, 4'-tetrachloroazoxybenzene: discordance between acute mouse bone marrow and subchronic mouse peripheral blood micronucleus test results

3,3',4,4'-Tetrachloroazobenzene (TCAB) and 3,3',4, 4'-tetrachloroazoxybenzene (TCAOB) are dioxin-like chemicals that were investigated for toxicity in 13-week gavage studies in male and female B6C3F(1) mice and F344N rats by the National Toxicology Program. As part of the comprehensive toxicological investigation of these chemicals, peripheral blood smears from mice treated 5 days per week for 13 weeks with 0.1-30mg/kg/day TCAB or TCAOB were analyzed for the frequency of micronucleated (MN) normochromatic erythrocytes (NCE). Both chemicals produced significant increases in MN-NCE in male and female mice. In contrast to these positive results in subchronic exposure studies, no significant increases were seen in acute bone marrow MN tests in male mice administered three daily injections of 50-200mg/kg/day TCAB and TCAOB. The results with TCAB and TCAOB suggest that the routine integration of MN tests with subchronic toxicity studies may allow detection of mutagenic activity for some chemicals that fail to elicit responses in short-term, high dose tests. In addition, the integration of mutagenicity tests into general toxicity tests reduces the use of laboratory animals and the cost of the testing.     

Evaluation of delayed apoptotic response in lethally irradiated human melanoma cell lines

To assess the lethal doses of gamma radiation and corresponding apoptotic response in new established human melanoma cell lines we exposed exponentially growing cultures to 8-100 Gy gamma radiation. The apoptosis and cell survival were determined by trypan blue exclusion, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction, agarose gel electrophoresis, colony forming assay, and long-term survival assay. The maximal DNA fragmentation 3 days after irradiation was observed in cultures irradiated with 20 Gy (36.9% TUNEL positive cells). The cultures irradiated with 50 and 100 Gy contained 18.7% and 16.4% TUNEL positive cells, respectively. Cultures exposed to 8 and 20 Gy gamma radiation recovered by week 3-4. Lethally irradiated (50 and 100 Gy) cultures which contained less apoptotic cells by day 3 died by week 5. A detectable increase in melanoma cell pigmentation after irradiation was also observed. The survival of human melanoma cell cultures after exposure to gamma radiation does not correlate with the level of apoptotic cells by day 3. At high radiation doses (> 50 Gy) when the radiation induced cell pigmentation is not inhibited the processes of apoptotic DNA fragmentation might be preferentially inactivated.     

Dioxin induces genomic instability in mouse embryonic fibroblasts

Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI), i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mouse embryonic fibroblasts (C3H10T1/2) were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN) and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days). For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.     

Cadmium exposure on day 12 of gestation in the Wistar rat: distribution, uteroplacental blood flow, and fetal viability

The effects of cadmium exposure (40 mumole CdCl2/kg, s.c.) on day 12 of gestation were evaluated in the Wistar rat. At 16-18 hours following such cadmium exposure, blood flow (as determined by radiolabeled microspheres) to the chorioallantoic placenta (CAP) was significantly reduced by 35%; at 24-26 hours, blood flow to the CAP had returned to control levels and was still unaffected at 38-43 hours. Uterine blood flow was not significantly altered at any of these timepoints. Between 16-18 and 24-26 hours after cadmium exposure, the concentration of cadmium in the placenta decreased significantly, while total cadmium content did not change. By 38-43 hours after cadmium exposure, total cadmium content of the placenta had increased significantly, although cadmium concentration was unchanged. There were no adverse effects on fetal viability or growth, as determined on day 20 of gestation. In sharp contrast, near-term (day 18) exposure to 40 or 50 mumole CdCl2/kg (s.c.) resulted in 53% and 82% mean incidences of fetolethality, respectively, within 24 hours. Administration of 50 mumole CdCl2/kg (sc) on day 12 also had no effect on fetal growth but resulted in increased fetolethality (12%). Thus midgestational cadmium exposure and its accompanying alterations in placental blood flow do not compromise fetal viability or growth. The differential response to cadmium at mid- and late gestation, in terms of fetolethality, is not due to maternal cadmium dose.     

Chemoprevention of genotoxic damage in lung cells of rats exposed to cigarette smoke]

Male Sprague-Dawley rats were exposed to cigarette smoke (CS) according to a complex protocol which lasted 40 days. Some of the groups were pre-treated with N-acetyl-L-cysteine (NAC) by gavage (1 g/kg b.w.) 5 h before each exposure. Bronchoalveolar lavage was performed in each animal at the end of each exposure period in order to recover pulmonary alveolar macrophages (PAM). Cells were identified and counted under the microscope, and the number of micronucleated (MN) and binucleated (BN) PAM was registered. The results showed an increase in the number of MN PAM, which was already evident after 8 days of CS exposure; this increase remained constant after 28 and 40 days of exposure. A significant decrease in the number of MN PAM was observed in the animals pre-treated with NAC. BN PAM were significantly increased after 28 and 40 days of exposure; again, a slight yet not significant decrease was detected in NAC-pre-treated animals. On the whole, this study demonstrates that CS is clearly clastogenic to alveolar macrophages and that NAC can efficiently prevent this cytogenetic damage.     

The genetic toxicity of 3,3′,4,4′-tetrachloroazobenzene and 3,3′,4,4′-tetrachloroazoxybenzene: discordance between acute mouse bone marrow and subchronic mouse peripheral blood micronucleus test results

3,3′,4,4′-Tetrachloroazobenzene (TCAB) and 3,3′,4,4′-tetrachloroazoxybenzene (TCAOB) are dioxin-like chemicals that were investigated for toxicity in 13-week gavage studies in male and female B6C3F₁ mice and F344N rats by the National Toxicology Program. As part of the comprehensive toxicological investigation of these chemicals, peripheral blood smears from mice treated 5 days per week for 13 weeks with 0.1–30 mg/kg/day TCAB or TCAOB were analyzed for the frequency of micronucleated (MN) normochromatic erythrocytes (NCE). Both chemicals produced significant increases in MN-NCE in male and female mice. In contrast to these positive results in subchronic exposure studies, no significant increases were seen in acute bone marrow MN tests in male mice administered three daily injections of 50–200 mg/kg/day TCAB and TCAOB. The results with TCAB and TCAOB suggest that the routine integration of MN tests with subchronic toxicity studies may allow detection of mutagenic activity for some chemicals that fail to elicit responses in short-term, high dose tests. In addition, the integration of mutagenicity tests into general toxicity tests reduces the use of laboratory animals and the cost of the testing.     

Morpho-functional changes in the ovaries of the sea urchin Strongylocentrotus intermedius after exposure to cadmium

The ovaries were studied in the sea urchins kept in a sea water added with 1, 50 and 100 micrograms/l cadmium chloride for 5, 15, 40, 72 and 130 days. The gland reaction depended on the drug dose and exposure. A short exposure (5 and 15 days) stimulated the development of a larger, as compared with the control, number of oogonia and raised the activity of acid and alkaline phosphatases. A long exposure decreased the number of germ cells, decelerated their growth, destroyed gametes and accessory cells, inhibited the activity of alkaline phosphatase. The cadmium accumulation in the ovaries was noted only on the 130th day at concentrations of 50 and 100 micrograms/l. The monitoring of morphological and biochemical indices allowed to conclude that cadmium exerted a toxic effect on the sea urchin ovaries.     

Protective effect of zinc on cadmium-induced micronuclei in V79 cells

The induction of micronuclei (MN) in mitotically active cells has been widely used and promoted as a biological marker of exposure to environmental toxins. In our study the effect of zinc on cadmium genotoxicity was investigated in V 79 cells. The results indicate that cadmium chloride exposure for 24 h increased micronucleus frequency and the percentage of binucleated cells in dose-dependent manner. At the highest concentration of cadmium (50 microM Cd) 23 MN were found in 1000 cells. The protective effect of zinc on cadmium genotoxicity was investigated at lower concentrations (5-25 microM CdCl2). At 50 microM Cd, the number of MN increased significantly (16 MN).     

Induction of micronuclei in wild-type and p53(+/-) transgenic mice by inhaled bromodichloromethane

Bromodichloromethane (BDCM) is commonly present in trace amounts in drinking water as a disinfection by-product. BDCM has been shown to be carcinogenic in mice and rats when given by gavage at relatively high doses. Genotoxic activity as well as induced regenerative cell proliferation may contribute to the carcinogenic potential of BDCM. The purpose of the current studies was to evaluate the ability of BDCM to induce micronuclei (MN) in bone marrow and blood of wild-type and p53(+/-) mice on the C57BL/6 and FVB/N genetic backgrounds using the inhalation route of exposure. Toxicity studies were being conducted in this laboratory with inhaled BDCM to select doses for longer-term cancer bioassays using wild-type and p53(+/-) transgenic mice on different genetic backgrounds. Bone marrow samples from these experiments were evaluated for the induction of MN after 1 and 3 weeks of exposure. Accumulation of MN in the peripheral blood was also evaluated at the 13-week time point of a cancer study with the p53(+/-) mice. For the 1-week time point, male C57BL/6 wild-type and p53(+/-) mice and FVB/N wild-type and p53(+/-) mice were exposed daily for 6h per day for 7 consecutive days to atmospheric BDCM concentrations of 0, 1, 10, 30, 100, or 150 ppm. In a second experiment, mice were exposed daily for 6h per day for 3 weeks to atmospheric BDCM concentrations of 0, 0.5, 1, 3, 10, or 30 ppm. Resulting levels of polychromatic erythrocytes (PCE) containing MN were assessed in the bone marrow. For all of the 1- and 3-week exposure groups, the only statistically significant increase in the percentage of bone marrow PCE cells containing MN was in the 1-week 100 ppm BDCM exposure group in the FVB/N wild-type mice (control 0.26% versus exposed 1.16%). C57BL/6 p53(+/-) mice and FVB/N p53(+/-) mice were exposed daily for 6 h per day for 13 weeks to atmospheric BDCM concentrations of 0, 0.5, 3, 10, or 15 ppm. MN were quantified in samples of peripheral blood. Statistically significant increases in the percentage of peripheral blood NCE cells containing MN were seen at the highest BDCM exposure group of 15 ppm in both the C57BL/6 p53(+/-) strain (control 0.36% versus exposed 0.67%) and the FVB/N p53(+/-) strain (control 0.36% versus exposed 0.86%). These data indicate weak induction of MN by BDCM, but only at high atmospheric concentrations relative to normal environmental exposures and with extended periods of exposure. Although comparisons are difficult because responses were negative or marginal, the p53 genotype or the genetic background did not appear to substantially alter susceptibility to the genotoxic effects of BDCM.     

The formation of predatory aggressiveness in the ontogeny of rats

In tests on outbred male rats the problem was studied of the existence of "sensitive periods" in the formation of rats predatory aggression. Breeding of small rats together with mice from the first till 60-th postnatal day considerably inhibits their muricidity in the adult age, what is manifest in tests of predatory aggressiveness till the 270-th day of rats life. Breeding of mice together with rats in the age of 1-20, 20-40, 40-60 and 20-60 postnatal days does not influence significantly their muricidity. Antiaggressive effect of breeding of mice with adult rats from the 150-th to the 210-th day is a short-term one, and breeding of mice with old rats in the age of 540-600 postnatal days does not significantly effect the muricidity.     

Extended exposure of adult and fetal mice to 50 Hz magnetic field does not increase the incidence of micronuclei in erythrocytes.

The flow cytometer-based micronucleus assay was used to study the effects on chromosomes in erythroid cells of CBA/Ca mice after extended exposure to 50 Hz magnetic field (MF), 14 microT, peak-to-peak (p-p). The study included two different experiments: (a) mice exposed in utero during 18 days of their prenatal stage, and (b) adult mice exposed for 18 days. In experiment (a) 35 days after exposure was terminated, peripheral blood was drawn from the mice exposed in utero to determine whether the exposure had a genotoxic effect on the pluripotent erythroid stem cells. About 200000 polychromatic erythrocytes (PCE) and 200000 normochromatic erythrocytes (NCE) were analysed from each of 20 exposed mice. The EMF exposure did not significantly change the frequency of micronucleated PCE or NCE in comparison with 20 sham-irradiated mice. There was no difference in the proportion of PCE between exposed and unexposed animals. Similarly, in experiment (b) no differences were seen between EMF exposed and unexposed adult mice when samples of peripheral blood were taken at the end of exposure and analyzed for micronuclei in PCE and NCE. The proportion of PCE was the same in both groups. The results indicate that exposure to EMF does not induce direct or indirect effects on chromosomes in erythroid cells expressed as increased levels of micronucleated erythrocytes of mice. No indications of delayed genetic effects were found.     

In vivo radioprotective potential of thymol, a monoterpene phenol derivative of cymene

The radioprotective and anticlastogenic potential of a phenol derivative monoterpene thymol(TOH), against whole-body gamma radiation was studied in Swiss albino mice. Acute toxicity of TOH, with an LD(50(14)) of 1134.03mg/kgbwt., was observed when administered intra-peritoneally (i.p.). The radioprotective potential of TOH was evaluated using the optimal dose of 10mg/kgbwt. TOH, which increased the LD(50/30) by 2.17Gy and resulted in a dose reduction factor (DRF) of 1.25. A significant (p<0.01) reduction in micronucleated, polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE), and an increased PCE/NCE ratio was also observed after administration of 10mg/kg.b.wt. TOH prior to gamma radiation, indicating its antigenotoxic effect. TOH pre-treatment significantly (p<0.01) elevated reduced glutathione, glutathione-S-transferase, catalase, and superoxide dismutase levels and decreased lipid peroxidation levels in mouse liver homogenates at 24 and 48h after exposure to 4.5Gy of radiation. Further, TOH treatment before exposure to 7.5Gy of gamma radiation resulted in a significant (p<0.01) increase in hematological parameters at various post-treatment time points, with increased numbers of endogenous spleen colonies as well. The histological observations indicated a decline in villus heights and crypt numbers in mouse jejunum and were accompanied by a significant decrease in bone marrow nucleated cells in the irradiated group, which was almost normalized by pre-treatment with TOH. Our study clearly documents the antioxidant, anticlastogenic and radioprotective potentials of TOH, which may be attributed to several possible mechanisms, such as normalization of intracellular antioxidant levels and free radical scavenging activities by TOH.     

Combined action of gamma and UHF radiation on conditioned reflex behavior of rats

Dynamics of the conditioned behaviour of rats in a shuttle box was studied after combined exposure to Cs-gamma radiation (1 Gy, 1.32 Gy/min) and microwave radiation (2450 MHz, 1 mW/cm2, 3 h). The number of conditioned and interstimulus responses was found to decrease on day 5 after microwave + gamma irradiation and to increase on day 30 after gamma + microwave irradiation.     

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.     

Influence of chronic exposure to low doses of space ionizing radiation on the character of formation of microbial assemblage in the habitat of orbital station

Statistically valid relations between radiation conditions in compartments of MIR station and the micromicete population (CFU number) on the surface of the equipment and the interior have been established. It was found that in conditions of a chronic exposure to space radiation the number of CFU increased in one thousand and more times with increasing of absorbed dose rate from 200 up to 1000 microGy/day. The results of land-based model experiments confirmed morphological changes in the "flight" strains of funguses under exposure to low doses of gamma (100-800 microGy/day) and neutron (0.2-2 neutron/cm2.s) radiation. It was found that the morphological changes in the control (museum) cultures of funguses of the same species, which were expressed in the weak increase of vegetative mycelium, were detected only after repeated gamma- and gamma + neutron irradiation.     

Effect of exposure of pregnant rats to cadmium on prenatal and postnatal development of the young

Cadmium chloride in doses of 2, 12 and 40 mg Cd/kg was administered per os to pregnant rats from the 7th to 16th day of pregnancy. In another experiment female rats were exposed to cadmium oxide at a concentration of 0.02 mg Cd/m3 or 0.16 mg Cd/m3 for 5 hours a day and 5 days weekly for a period of 5 months or 1 mg Cd/m3 for 4 months. The exposure was then continued during mating and from the 1st to 20th day of pregnancy. A decrease in fertility was only observed in females exposed by inhalation to cadmium oxide at a concentration of 1 mg Cd/m3, at which concentration cadmium exhibits a considerable toxic effect on the whole organism. The young of females orally treated with CdCl2 in a dose of 40 mg Cd/kg displayed congenital defects in the form of sirenomelia or amelia, as well as raised cadmium levels in tissues. A retardation of intrauterine development manifested by lower body weight and slowed down osteogenesis was observed in the other groups. A cadmium concentration increase was not found in the tissues of the young in these groups. Inhalation exposure to 0.16 mg Cd/m3 of females prior to and during pregnancy induced in their young a decrease in viability, lower body weight gain, prolongation of latency in the negative-geotaxis test, lower locomotor activity and deteriorated development of the conditioned-reflex response. The offspring of females exposed to 0.02 mg Cd/m3 displayed lowered locomotor activity and worsened consolidation of the conditioned-reflex response.     

Activities of superoxide dismutase and catalase in erythrocytes and plasma transaminases of goldfish (Carassius auratus gibelio Bloch.) exposed to cadmium

Four groups of goldfish were exposed to cadmium in a concentration of 20 mg Cd/l water under aquarium conditions. The duration of exposure was 1, 4, 7 and 15 days. It was shown that the activity of superoxide dismutase (SOD) in the red blood cells (RBC) significantly decreased after the first day of cadmium exposure. However, the SOD activity increased after 7 and 15 days of cadmium treatment. Elevated activity of catalase (CAT) was found in erythrocytes of cadmium-treated fishes after 15 days, whereas plasma GOT levels was increased after 7 and 15 days and GPT levels after 1, 4, 7 and 15 days of cadmium treatment. This was accompanied by a significant decrease of blood hemoglobin concentrations (after 15 days) and hematocrit values (after 7 and 15 days). However, the concentration of blood glucose significantly increased after 1, 4, 7 and 15 days of cadmium exposure. These results indicate that cadmium causes oxidative stress and tissue damage in the exposed fishes.     

The Estimation of Molecular and Cytogenetic Effects in Mice Exposed to Chronic Low Dose-Rate Gamma-Radiation

Molecular and cytogenetic parameters were estimated in male CBA/lac mice exposed to chronic low dose-rate -radiation (62 cGy/year) for 40, 80, 120, 210, and 365 days. After 40 days of exposure (6.7 cGy), spleen lymphocyte susceptibility to hydrogen peroxide was shown to increase. However, beginning from the day 120 of the treatment (20.4 cGy), the opposite effect was observed. An increase in number of the DNA–protein crosslinks was recorded in spleen lymphocytes only on day 40 of the experiment. The number of DNA breaks increased significantly beginning from day 120 of the experiment, as shown by the DNA-comet method. On the day 210 of irradiation, the frequency of abnormal sperm heads in the mice significantly increased. The number of normochromatic micronucleated erythrocytes of the peripheral blood remained unchanged.