Detection of micronuclei in haemocytes of zebra mussel and great ramshorn snail exposed to pentachlorophenol

The frequency of micronuclei (MN) induced by pentachlorophenol (PCP) in haemocytes of zebra mussel, Dreissena polymorpha Pall. and great ramshorn snail, Planorbarius corneus L. was determined over a 14 days of exposure (sampling after 4, 7 and 14 days) under laboratory conditions. PCP doses for zebra mussel ranged from 10 to 150 microg/l, and for ramshorn snail from 10 to 450 microg/l. Micronuclei were detected after bisbenzimide fluorescent staining. Positive responses were observed in both species. The mean MN frequencies in treated mussels ranged between 0.69 and 7.50 per thousand, and between 2.07 and 13.80 per thousand in treated snails. The spontaneous MN levels in mussels averaged from 0.5 to 2.75 per thousand, and in snails from 1.56 to 2.00 per thousand. Our results suggest that haemolymph of both species represent an appropriate test tissue in environmental genotoxicity assessment.     

Assessment of the genotoxic potential of benzo(a)pyrene and pp'-dichlorodiphenyldichloroethylene in Zebra mussel (Dreissena polymorpha)

The single-cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test were carried out with haemocytes of Zebra mussel (Dreissena polymorpha) specimens to evaluate the potential genotoxicity of benzo(a)pyrene (BaP) and pp'-dichlorodiphenyldichloroethylene (pp'-DDE, a metabolite of pp'-DDT). Mussels were exposed to three different concentrations (0.1 microg/L, 2 microg/L, 10 microg/L) of each chemical in water during 168 h (SCGE assay) and 96 h (MN test) of exposure under laboratory conditions. These levels correspond to nominal molar concentrations of 0.4 nM, 7.9 nM and 40 nM for BaP and 0.3 nM, 6.2 nM and 31 nM for pp'-DDE, respectively. Concurrently, the levels of toxicants were measured in soft tissues of the mussels by gas-chromatographic analyses, to evaluate their temporal trends and the dose/response relationships. Significant increases of the ratio between the comet length and the diameter of the comet head (LDR) and of micronucleus frequencies in comparison with baseline levels were observed not only for all concentrations of BaP, but also for pp'-DDE (except 0.3 nM). The concentration above which DNA damage starts to be significantly increased was 0.8 nmol/g lipids for BaP and 1.6 nmol/g lipids for pp'-DDE, respectively. The results of these experiments show a clear genotoxic effect on this non-target organism not only for the well-known genotoxicant BaP, but also for the final metabolite of pp'-DDT at soft-tissue concentrations that have been found in several aquatic ecosystems worldwide.     

Effects of temperature on baseline and genotoxicant-induced DNA damage in haemocytes of Dreissena polymorpha

The potential application of the Comet assay for monitoring genotoxicity in the freshwater mussel Dreissena polymorpha was explored and a preliminary investigation was undertaken of the baseline levels of DNA damage in mussel haemocytes of animals kept at different temperatures. In addition, in vitro cell sensitivity against genotoxicants was assessed in relation to increasing temperatures. The mussels were kept at four different constant temperatures (4, 18, 28 and 37 degrees C) for 15 h. The haemocytes withdrawn were treated in vitro with melphalan, as a model genotoxic compound, or sodium hypochlorite, a common water disinfectant capable of producing mutagenic/carcinogenic by-products, at the established temperatures for 1h. The data obtained in vivo, in cells directly withdrawn from the mussels showed a significant (P<0.001, Student's t test) inter-individual variability, probably due to genetic and epigenetic factors and an increasing amount of DNA damage at increasing temperature. Mussel haemocytes showed a clear dose-response effect after in vitro melphalan treatment. Hypochlorite treatment also significantly increased DNA migration: the damage was temperature dependent, with a similar increase at 4 and 28 degrees C and a minimum level at 18 degrees C. This study demonstrates the potential application of the Comet assay to haemocytes of D. polymorpha. However, these findings suggest that temperature could alter both DNA damage baseline levels in untreated animals and cell sensitivity towards environmental pollutants in in vitro conditions. Therefore, more information is needed about seasonal variations and the natural background levels of DNA damage in mussels living in the wild, before they are used for the monitoring of genotoxic effects in aquatic environments.     

Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 microg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 microg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

Impact of low doses of tritium on the marine mussel, Mytilus edulis: genotoxic effects and tissue-specific bioconcentration

Despite growing scientific, public and regulatory concern over the discharge of radioactive substances, no serious attempts have been made to develop a rationale to evaluate the impact of environmentally relevant radionuclides in the aquatic environment. In this study, we have evaluated the genotoxic effects and tissue-specific concentration of tritium (added as tritiated water, HTO) in the adult life stage of the edible mussel, Mytilus edulis. The genotoxic effects were quantified in terms of the induction of: (a) micronuclei (MN), and (b) DNA single-strand breaks/alkali-labile sites using alkaline single-cell gel electrophoresis (Comet assay) in the haemocytes of exposed animals. The assays were optimised and validated using a range of concentrations (18-56 mgl(-1)) of ethylmethane sulfonate (EMS), a direct-acting reference genotoxic agent, over different exposure periods. Mussels were exposed to a series of concentrations of HTO equivalent to a dose range from 12 to 485 muGyh(-1) for 96 h, and different tissues and organs were then extracted and analysed. The study revealed a dose-dependent increase in the response for both the MN test and the Comet assay and for both EMS and HTO. In addition, HTO delivering dose rates below 500 muGyh(-1) was shown to be capable of inducing genetic damage in the haemocytes of these bivalves. The study also showed that inorganic tritium accumulated differentially in mussel tissues in a dose-dependent manner, with the gut accumulating the highest amount of radioactivity, followed by the gill, mantle, muscle, foot and byssus thread. The faeces and pseudo-faeces accumulated least radioactivity over the exposure period. Differential accumulation of radionuclides has significant implications for biomonitoring programmes, for this and other aquatic organisms. The study also suggests that the generic dose limits recommended by the International Atomic Energy Agency for the protection of aquatic biota might not be applicable to all aquatic organisms.     

Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[a]P and a combination of B[a]P and Cd

The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10μg/L), B[a]P (10μg/L) or the two combined chemicals (10μg/L+1μg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl(2) or CdCl(2)+B[a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10μg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[a]P (10μg/L) induced an early increase of DNA damage in gill cells (after 10h and 1 day), while in both gill cells and haemocytes, B[a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd+B[a]P mixture decreased the DNA-damaging effect of Cd and B[a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[a]P did not induce any cytogenetic effect. B[a]P, Cd and Cd+B[a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[a]P, which makes them more suitable for future bio-monitoring studies.     

Generation of free radicals in haemocytes of mussels after exposure to low molecular weight PAH components: immune activation, oxidative and genotoxic effects

Polycyclic aromatic hydrocarbons (PAHs) constituents, such as phenanthrene (PH) and anthracene (AN), are considered toxic for marine organisms, including bivalve mollusks. The present study showed that the perturbation of health status in mussels, as well as their inability to survive in air (stress on stress response), following exposure to either PH or AN (at a final concentration of 0.1 mg/L respectively), and a mixture of them (ration 1:1, at a final concentration of 0.2 mg/L) for 7 days, is probably related with alterations occurred in their haemocytes. According to the present study, PH and AN, either alone or in a mixture, could induce elevated levels of superoxide anions (^•O₂⁻) within haemocytes of mussels, thus leading to immune susceptibility as indicated by the increased levels of cell death and the elevated levels of lysosomal membrane acid phosphatase (AcP) activity, probably occurred via its overproduction or its release into the cytosol after lysosomal membrane destabilisation. PAH-mediated oxidative and genotoxic effects, indicated by the increased levels of both lipid peroxidation (MDA content) and nuclear abnormalities (MN test) could be related with haemocytes' inability to overcome free radical generation, thus leading to attenuation of general health status, before other disturbances, such as death, occur.     

The effect of chlorpyrifos on protein content and acid DNase activity in the mussel,Mytilus galloprovincialis

We evaluated the effects of short-term exposure to an organophosphate pesticide chlorpyrifos on the digestive gland and gills of the mussel Mytilus galloprovincialis. We studied metabolic activity by quantifying protein content and physiological function responses using acid DNase activity. The increase in protein content was observed in both the target tissues of mussels exposed to 0.03 μg/L chlorpyrifos when compared with control mussels. The pattern of acid DNase activity in digestive gland and gills indicated a tissue-specific response, although the lowest concentration of chlorpyrifos caused changes in acid DNase activity in both tissues. In the digestive gland, the increase of acid DNase activity was observed in mussel exposed to 0.03 μg/L chlorpyrifos, followed by decrease up to 100 μg/L chlorpyrifos. Enzyme activity in the gills showed a dose response effect. The results support the use of acid DNase activity in the digestive gland as a sensitive response to an environmentally relevant range of pesticide concentrations. It may also indicate an effect on mussel physiological status.     

Enhancement of benzene clastogenicity by praziquantel in mice

Praziquantel (PQ) is a commonly used drug to treat patients with schistosomiasis. Previous studies using cells in vitro have shown that PQ can enhance the mutagenic activities of known mutagens. We have conducted a cytogenetic - urine metabolite study to determine the in vivo clastogenic and co-clastogenic potential of PQ with a ubiquitous environmental contaminant, benzene (BZ). 16 groups of adult male ICR mice (5 animals per group) were used. They were negative control, solvent controls (cremophore E1 3%, olive oil and combined), positive control (BZ 440 mg/kg b.w.) and 11 exposed groups. To test for clastogenicity of PQ, mice were treated orally with 100, 400, 800 and 1200 mg/kg b.w. PQ and sacrificed 30 h later for determination of micronuclei (MN) frequency in bone-marrow polychromatic erythrocytes (PCE). None of these PQ does induced an increase of MN frequency. On the other hand, BZ induced, as expected, a high frequency of MN (46.4 +/- 6.34/1000 PCE). The enhancement effect of PQ was tested in 7 groups of mice using 3 different protocols. Mice were treated with 440 mg/kg b.w. BZ and 1 h later with 0, 100, 200, 400, 800 and 1200 mg/kg b.w. PZ. In another group, 800 mg/kg PQ was administered at 3 h after BZ exposure. In the last group, PQ (800 mg/kg) was administered at 1 h prior to BZ exposure. Results from the first combined exposure group showed a significant PQ dose-dependent increase in the frequency of MN in PCE (p less than 0.05). The increase with the two high doses of praziquantel is significantly higher (p less than 0.05) than the MN frequencies in the benzene control and the expected value based on the additive effects of the two agents. Studies with other combined treatment groups showed that the induction of MN was highest when PQ was administered at 1 h before BZ exposure. Moreover, the presence of BZ metabolites (muconic acid, phenol, catechol and hydroquinone) in urine was studied in 6 of the combined treatment groups. This metabolite study revealed that PQ enhanced the metabolism of BZ towards the pathway to form muconaldehyde which is converted to muconic acid in urine. In conclusion, our study showed that PQ is not a clastogen but can enhance the clastogenic activity of BZ in vivo by shifting the metabolic pathways of BZ towards formation of muconaldehyde which may be responsible for the enhancement effect.     

Influence of herbicide, 2,4-dichlorophenoxy acetic acid, on haemocyte DNA of in vivo treated mussel

The influence of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) on haemocyte DNA of in vivo treated mussels Mytilus galloprovincialis has been investigated by flow cytometry and epifluorescence microscopy. Haemocyte proliferation and atypical flow cytometric DNA histograms were observed in mussels treated with 20 and 100 μg/g of 2,4-D. The stimulation of proliferation by 2,4-D was also obvious by DNA labelling with BrdU followed by FITC conjugated anti-BrdU MoAb visualised by epifluorescence microscopy. An apoptotic sub-G₁ peak resulted in mussels that were exposed to higher doses of herbicide at 100 and 500 μg/g as well as subpopulation could be detected by flow cytometric analysis. In these experiments morphological changes characteristic for apoptotic cells were looked for by fluorescence microscopy. A low percentage of cells in S as well as in G₂M phase indicating G1 arrest were detected in haemocytes from these mussels that had survived 4 days of 20 μg/g 2,4-D exposure. In addition, sister-chromatid exchanges (SCE) could be seen with the immunolabelling BrdU method. Thus, in vivo treatment and the subsequent uptake of 2,4-D causes serious genetic consequences and raises concerns regarding the potential overall fitness and health effects in mussel populations.     

Effect of 910-MHz electromagnetic field on rat bone marrow

Aiming to investigate the possibility of electromagnetic fields (EMF) developed by nonionizing radiation to be a noxious agent capable of inducing genotoxicity to humans, in the current study we have investigated the effect of 910-MHz EMF in rat bone marrow. Rats were exposed daily for 2 h over a period of 30 consecutive days. Studying bone marrow smears from EMF-exposed and sham-exposed animals, we observed an almost threefold increase of micronuclei (MN) in polychromatic erythrocytes (PCEs) after EMF exposure. An induction of MN was also observed in polymorphonuclear cells. The induction of MN in female rats was less than that in male rats. The results indicate that 910-MHz EMF could be considered as a noxious agent capable of producing genotoxic effects.     

Changes of Blue Mussels Mytilus edulis L. Lipid Composition Under Cadmium and Copper Toxic Effect

The lipid and fatty acid composition of the blue mussels Mytilus edulis L. gills and digestive glands was evaluated after 24 and 72 h of cadmium (Cd) and copper (Cu) exposure. Mussels were exposed to different cadmium (10, 100, and 500 μg/L) and copper (5, 50, and 250 μg/L) concentrations. Similar stress response of predominant membrane phospholipids level as well as polyenoic and non-methylene interrupted (NMI) fatty acids content was observed in mussel gills under both cadmium and copper effects. Increased NMI fatty acids level after 24 h, the metal ions treatment suggests that these acids contribute to the protective response to the membrane oxidative stress caused by accumulation of the metals. The content of cholesterol, some minor membrane phospholipids, and storage lipids (triacylglycerols, TAG) in the mussels’ organs alter significantly under the cadmium and copper effect. A two-step response at the digestive glands TAG level depends on the duration of the cadmium and copper treatments (24 and 72 h) on the mussels. The results demonstrate that Cd and Cu impact has adverse effects on gills and digestive glands lipid and fatty acids composition. The type of observed effects varies with the nature and concentration of the metal ions and depends on the role of the metals in the mussels’ life activity.     

P-glycoprotein induction: an antidotal pathway for paraquat-induced lung toxicity

The widespread use of the nonselective contact herbicide paraquat (PQ) has been the cause of thousands of deaths from both accidental and voluntary ingestion. The main target organ for PQ toxicity is the lung. No antidote or effective treatment to decrease PQ accumulation in the lung or to disrupt its toxicity has yet been developed. The present study describes a procedure that leads to a remarkable decrease in PQ accumulation in the lung, together with an increase in its fecal excretion and a subsequent decrease in several biochemical and histopathological biomarkers of toxicity. The administration of dexamethasone (100 mg/kg ip) to Wistar rats, 2 h after PQ intoxication (25 mg/kg ip), decreased the lung PQ accumulation to about 40% of the group exposed to only PQ and led to an improvement in tissue healing in just 24 h as a result of the induction of de novo synthesis of P-glycoprotein (P-gp). The involvement of P-gp in these effects was confirmed by Western blot analysis and by the use of a competitive inhibitor of this transporter, verapamil (10 mg/kg ip), which, given 1 h before dexamethasone, blocked its protective effects, causing instead an increase in lung PQ concentration and an aggravation of toxicity. In conclusion, the induction of P-gp, leading to a decrease in lung levels of PQ and the consequent prevention of toxicity, seems to be a new and promising treatment for PQ poisonings that should be further clinically tested.     

Assessment of the genotoxicity of propineb in mice bone marrow cells using micronucleus assay

Propineb, a dithiocarbamate fungicide, is commonly used for the control of disease in a wide range of crops in agriculture. The genotoxic effects of commercial formulation of propineb in bone marrow cells of mice was investigated in vivo by micronucleus (MN) assay. The three different concentrations of propineb (12.5, 25 and 50 μg/mL; 0.01 mL per gram) were injected intraperitoneally (i.p.) to mice for 24 and 48 h. The results of the MN assay indicated that propineb induced a significant increase in frequency of micronucleated polychromatic erythrocytes (MNPCE) at 25 and 50 μg/mL concentrations for 24 h and at the highest (50 μg/mL) concentration for 48 h when compared with negative control. Also significant reduction for the polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio which is indicative for bone marrow cytotoxicity was observed at the same concentrations for 24 and 48 h. These results lead us to the conclusion that propineb may have genotoxic and cytotoxic potential due to induction in the frequency of MN and a reduction in PCE/NCE ratio in the bone marrow cells of mice.     

Increased micronucleus frequency after oral administration of cadmium in dogs

Cadmium (Cd) is a toxic heavy metal that has been classified as a human carcinogen by the International Agency for Research on Cancer. The genotoxic effects of cadmium oxide (CdO) were investigated in cultured dog lymphocytes after a short-term oral CdO administration by the micronucleus (MN) test. The dogs were given 10 mg CdO/kg body weight per day for 3 and 28 d, respectively group I (n=7) and group II (n=6). Blood samples were collected at the beginning of feeding and at 4 and 29 d after Cd administration and cultured for 72 h. Whereas no significant increase in the MN frequency in group I was observed (p=0.398), a significant MN induction with CdO was found in group II (p=0.028) when compared with initial MN frequencies of dogs in both groups. Our results suggest that CdO might be directly and/or indirectly genotoxic after a monthly oral administration of CdO in dogs.     

Modulating effects of humic acids on genotoxicity induced by water disinfectants in Cyprinus carpio

The use of chlorinated disinfectants during drinking-water production has been shown to generate halogenated compounds as a result of interactions of humic acids with chlorine. Such chlorinated by-products have been shown to induce genotoxic effects and consumption of chlorinated drinking-water has been correlated with increased risk for cancer induction in human populations. The aim of this work was to test the potential genotoxic effects on circulating erythrocytes of the fish Cyprinus carpio exposed in vivo to well-waters disinfected with sodium hypochlorite (NaClO), chlorine dioxide (ClO2) or peracetic acid (CH3COO2H, PAA), in the absence or presence of standard humic acids (HA). The effects were measured by use of the micronucleus (MN) and the single-cell gel electrophoresis (Comet) assays at different sampling times after a 3-day exposure period. The exposure to chlorine disinfectants without the addition of HA produced a clear toxic effect. Significant cytogenetic damage (i.e. MN induction) was detected in fish populations exposed to both NaClO and ClO2 with humic acids. In the Comet assay, a significant decrease of DNA migration was observed in erythrocytes of specimens after exposure to NaClO-disinfected water without HA. No effects were observed in any other experimental condition.     

Induction of unscheduled DNA synthesis in liver and micronucleus in bone marrow of rats exposed in vivo to the benzidine-derived azo dye, Direct Black 38

The genotoxic activity of the benzidine-derived azo dye, Direct Black 38 (DB38), was studied in vivo, using two different genetic end-points: unscheduled DNA synthesis in liver (UDS) and bone marrow micronucleus (MN). Exposure times were 12, 24 or 36 h. Both assays were performed in the same rat, except for the 24-h exposure when only MN was investigated. For the liver UDS assay, the rat hepatocarcinogen, 6-dimethylaminophenylazobenzthiazole (6BT), was used as positive control and for the MN assay, cyclophosphamide (CP). In agreement with earlier results, 6BT gave rise to a dose-related increase in liver UDS after 12-h exposure to 25 or 50 mg/kg bw. After 36-h exposure, there was still an indication of a weak dose-response effect between 0 and 5 net nuclear grains (NG). DB38 induced liver UDS at the higher dose levels used (500 and 1000 mg/kg), and after both 12- and 36-h exposure. With the longer exposure time, a weak induction of UDS was also observed at 100 mg/kg. The strongest UDS induction (12.2 NG), was obtained in one rat after 36-h exposure to 500 mg/kg. DB38 also had a weak effect on the MN induction, which was statistically significant at the higher concentrations used. A dose-related response was observed at all exposure times used.     

Dl-α-tocopherol enhances the herbicide 1,1'-dimetyl-4,4'-bipyridium dichloride (paraquat, PQ) genotoxicity in cultured anuran leukocytes

This cytogenetic and pharmacological study attempts to clarify genotoxicity-enhancement-effect of dl-α-tocopherol (one form of vitamin E) in combination with the herbicide 1,1'-dimetyl-4,4'-bipyridium dichloride (paraquat, PQ) on cultured anuran leukocytes using the superoxide dismutase-mimic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (Mn(III)TMpyP), the hydrogen peroxide-scavenger catalase and the electron donor nicotinamide adenine dinucleotido phosphate (NADPH). PQ only was found to induce structural chromosomal damage in cultured anuran leukocytes in a dose-dependent manner. PQ plus NADPH, which served as positive control, enhanced the genotoxic effect of PQ. Dl-α-tocopherol only did not induce any structural chromosomal damage in the leukocytes. PQ plus dl-α-tocopherol, however, enhanced the genotoxic effect of PQ. PQ plus Mn(III)TMpyP, PQ plus catalase and PQ plus Mn(III)TMpyP plus catalse suppressed the genotoxic effect of PQ. Furthermore, PQ plus dl-α-tocopherol-enhanced chromosomal damage was also inhibited by Mn(III)TMpyP plus catalase. These results suggest that dl-α-tocopherol in combination with PQ functions as an electron donor to PQ.     

Genotoxic and Mutagenic Assessment of Hexavalent Chromium in Fish Following In Vivo Chronic Exposure

Chromium is a well-documented carcinogen. To evaluate the genotoxic potential of hexavalent chromium on an aquatic bio-system, freshwater murrel fish (Channa punctatus) were exposed to potassium dichromate. The 96-h LC₅₀ for potassium dichromate was 61.80 mg/L for the test fish in a static system. On the basis of the 96-h LC₅₀, fish were exposed to sublethal concentrations of the test chemical. Fish exposed to the test chemical were sampled on days 1, 7, 14, 21, and 28 post-exposure and blood and gill cells were collected. Significantly (p < .05) higher DNA damage in both lymphocyte and gillcells and micronuclei formation in whole blood was observed at different test concentrations and sampling times of the test chemical as compared to control fish. The mean% tail DNA in the comet tail assay showed a concentration-dependent increase and the maximum% tail DNA was observed on day 7 of exposure in both cells. A similar trend was also observed in micronuclei induction in blood with maximum induction on day 21. Hexavalent chromium showed genotoxic potential in chronic exposure of C. punctatus, and the micronucleus test and the comet assay are the methods for sensitive and rapid detection of the genetic effects.     

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.