Combined supplementation of vanadium and 1alpha,25-dihydroxyvitamin D3 inhibit placental glutathione S-transferase positive foci in rat liver carcinogenesis

The combined effects of vanadium (V) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in inhibiting diethylnitrosamine (DEN)-induced and phenobarbital (PB) promoted hepatocarcinogenesis were examined in male Sprague-Dawley rats. All the rats were subjected to 70% partial hepatectomy (PH) at week 4 and 24h later were administered either solvent trioctanoin (Group B, D, F and H) or 10 mg DEN/kg (Group A, C, E and G) by gavage. Briefly after two weeks of DEN administration, PB were administered (0.05% in basal diet) to all the DEN-treated rats and continued till the completion of the experiment. Supplementary V at the dose of 0.5 ppm in drinking water ad libitum (Group C and D), 1,25(OH)2D3 at the dose of 3 microg/ml in propylene glycol per os twice a week (Group E and F) or both V and 1,25(OH)2D3 at the same above given doses (Group G and H) were started 4 weeks prior to DEN administration (week 0) and continued thereafter till week 15. The expression of the number and area of altered hepatocyte foci (AHF) positive for placental glutathione S-transferase (GST-P) was maximum in DEN-treated and PB promoted group (Group A). V (Group C) and 1,25(OH)2D3 (Group E) treatment significantly reduced the expression of GST-P-positive hepatocytes by 36.02% and 45.16% respectively but an additive protective action (61.46%) was found in Group G which received both V and 1,25(OH)2D3 for the entire period of the study. Moreover, histopathological examination and the incidence of hepatic hyperplastic nodules showed that combined action of V and 1,25(OH)2D3 can able to minimize the appearance of nodules as well and maintain the normal cellular architecture than V and 1,25(OH)2D3 when given alone. These results suggest that, when given together V and 1,25(OH)2D3 could be the chemopreventive agents for rat liver carcinogenesis.     

Carcinogen-induced early molecular events and its implication in the initiation of chemical hepatocarcinogenesis in rats: chemopreventive role of vanadium on this process

Carcinogen-induced formation of DNA adducts and other types of DNA lesions are the critical molecular events in the initiation of chemical carcinogenesis and modulation of such events by chemopreventive agents could be an important step in limiting neoplastic transformation in vivo. Vanadium, a dietary micronutrient has been found to be effective in several types of cancers both in vivo and in vitro and also possesses profound anticarcinogenicity against rat models of mammary, colon and hepatocarcinogenesis. Presently, we report the chemopreventive potential of vanadium on diethylnitrosamine (DEN)-induced early DNA damages in rat liver. Hepatocarcinogenesis was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal (i.p.) injection of DEN (200 mg/kg body weight) at week 4. There was a significant induction of tissue-specific ethylguanines, steady elevation of modified DNA bases 8-hydroxy-2'-deoxyguanosines (8-OHdGs) (P<0.0001; 89.93%) along with substantial increment of the extent of single-strand breaks (SSBs) (P<0.0001) following DEN exposure. Supplementation of 0.5 ppm of vanadium throughout the experiment abated the formations of O(6)-ethylguanines and 7-ethylguanines (P<0.0001; 48.71% and 67.54% respectively), 8-OHdGs (P<0.0001; 81.37%), length:width (L:W) of DNA mass (P<0.01; 62.12%) and the mean frequency of tailed DNA (P<0.001; 53.58%), and hepatic nodulogenesis in preneoplastic rat liver. The study indicates that 0.5 ppm vanadium is potentially and optimally effective, as derived from dose-response studies, in limiting early molecular events and preneoplastic lesions, thereby modulating the initiation stage of hepatocarcinogenesis. Vanadium is chemopreventive against DEN-induced genotoxicity and resulting hepatocellular transformation in rats.     

Protective effects of Ginkgo biloba against rat liver carcinogenesis

Ginkgo biloba (EGb) has been proposed as a promising candidate for cancer chemoprevention and has shown protective effects on the liver against chemically induced oxidative injury and fibrosis. The potential beneficial effects of EGb were investigated in two rat liver carcinogenesis bioassays induced by diethylnitrosamine (DEN). In a short-term study for anti-initiating screening, male Wistar rats were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb and initiated 14 days later with a single dose of DEN (100 mg/kg i.p.). The respective groups were killed 24h or 2 weeks after DEN-initiation. Liver samples were collected for the analysis of proliferating cell nuclear antigen (PCNA), transforming growth factor alpha (TGF-alpha), p53, apoptosis and induction of single hepatocytes and minifoci positive for the enzyme glutathione S-transferase P-form (GST-P). In a medium-term study for anti-promoting screening, the animals received a single dose of DEN (200 mg/kg i.p.) and, 2 weeks later, were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb for 6 weeks. All animals underwent 70% partial hepatectomy (PH) at week 3 and killed at week 8. Liver samples were collected to analyze development of preneoplastic foci of altered hepatocytes (FAH) expressing GST-P. In the short-term study, pretreatment of rats with 1000 ppm EGb significantly reduced the rates of cell proliferation, apoptosis and p53, TGF-alpha immunoreactivity and the number of GST-P-positive hepatocytes. In the medium-term study, EGb treatment during the post-initiation stage failed to reduce the development of DEN-induced GST-P-positive foci. Thus, EGb presented inhibitory actions during initiation but not promotion of rat liver carcinogenesis induced by DEN.     

The inhibitory effect of sodium selenite on N-nitrosodiethylamine-induced and phenobarbital promoted liver tumourigenesis in rats based on the modulation of polyamine levels

In the present study, we have evaluated the effects of dietary selenite (Se) on polyamine levels and its influence on N-nitrosodiethylamine (DEN) initiated and Phenobarbital (PB) promoted in rat liver carcinogenesis. Dietary selenite at a concentration of 4 ppm (through drinking water) was administered in rats either before initiation (4 weeks), or during promotion (16 weeks) and entire experimental period (20 weeks). Male Wistar strain of albino rats was treated with single intra peritoneal dose of DEN (200 mg kg⁻¹ body weight), after 2 weeks the carcinogenic effect was promoted by PB (0.05%; through diet). Alpha fetoprotein (AFP) was investigated after the 20th-week of experimental period. Selenite-treated animals markedly reduced the AFP during the time of pre-selenite [before initiation (4 weeks)] and entire experimental period (20 weeks), administration rather than the promotion period. This infers that anticancer property of selenite depends on the stage of carcinogenesis, rather than duration of treatment. Evaluation of polyamine levels in hepatoma and surrounding liver tissue showed significant difference in the selenite-treated groups compared with pair-fed control groups. Furthermore, histopathological examination showing remarkable difference between control and treated groups. These results demonstrate that selenite can modulate the development of DEN-induced and PB-promoted rat liver carcinogenesis through a polyamine-dependent mechanism. (Mol Cell Biochem xxx: 165–172, 2005)     

Chemomodulatory effect of Ficus racemosa extract against chemically induced renal carcinogenesis and oxidative damage response in Wistar rats

Ferric nitrilotriacetate (Fe-NTA) is a well-known renal carcinogen. In this communication, we show the chemopreventive effect of Ficus racemosa extract against Fe-NTA-induced renal oxidative stress, hyperproliferative response and renal carcinogenesis in rats. Fe-NTA (9 mg Fe/kg body weight, intraperitoneally) enhances renal lipid peroxidation, xanthine oxidase, gamma-glutamyl transpeptidase and hydrogen peroxide (H(2)O(2)) generation with reduction in renal glutathione content, antioxidant enzymes, viz., glutathione peroxidase, glutathione reductase, catalase, glucose-6-phosphate dehydrogenase and phase-II metabolising enzymes such as glutathione-S-transferase and quinone reductase. It also enhances blood urea nitrogen, serum creatinine, ornithine decarboxylase (ODC) activity and thymidine [(3)H] incorporation into renal DNA. It also enhances DEN (N-diethylnitrosamine) initiated renal carcinogenesis by increasing the percentage incidence of tumors. Treatment of rats orally with F. racemosa extract (200 and 400 mg/kg body weight) resulted in significant decrease in gamma-glutamyl transpeptidase, lipid peroxidation, xanthine oxidase, H(2)O(2) generation, blood urea nitrogen, serum creatinine, renal ODC activity, DNA synthesis (P<0.001) and incidence of tumors. Renal glutathione content (P<0.01), glutathione metabolizing enzymes (P<0.001) and antioxidant enzymes were also recovered to significant level (P<0.001). Thus, our data suggests that F. racemosa extract is a potent chemopreventive agent and suppresses Fe-NTA-induced renal carcinogenesis and oxidative damage response in Wistar rats.     

Effect of low power ultrasonic radiation on anaerobic biodegradability of sewage sludge

The effect of low power ultrasonic radiation on anaerobic biodegradability of sewage sludge was investigated. For this purpose, soluble substances and variation of microbial system of sewage sludge subjected to low power ultrasonic radiation were tested. The well known hydromechanical shear forces and heating effect of low frequency ultrasound plays a major role in the sludge pre treatment process. More, the increase of soluble substance may partly result from the destruction of microbial cell by excess ultrasonic pretreatment, which will inhibit the anaerobic process. By orthogonal tests, the optimal parameters were found to be an exposure time of 15 min, ultrasonic intensity of 0.35 W/cm² and ultrasonic power density of 0.25 W/ml. Under the optimal condition, anaerobic biodegradability of sewage sludge (R_(vss/ss) %) was increased by 67.6%. Consequently, it can be concluded that low power ultrasonic pretreatment is a valid method for improving anaerobic biodegradability of sewage sludge.     

Sodium selenite enhances glutathione peroxidase activity and DNA strand breaks in hepatoma induced by N-nitrosodiethylamine and promoted by phenobarbital

An element/compound that acts as an antioxidant as well as, can increase the oxidative stress offers a new approach in differentiation therapy. Experiments were carried out to determine the effect of selenite on DNA damage and glutathione peroxidase (GPx) activity in N-nitrosodiethylamine (DEN) induced, phenobarbital promoted rat hepatoma. Supra-nutritional level of selenite (4 ppm) was supplemented at either, before-initiation/after-initiation and/or during entire period of the study. At the end of experiment period (20 weeks), extent of DNA damage (alkaline comet assay), selenium concentration, and GPx activity were assessed on nodular tissue (NL) cells, surrounding liver (SL) cells, and whole liver tissue (control) cells. Hepatic selenium level and GPx activity were decreased in DEN and PB-administered animals, whereas the DNA damage was found to be increased in both NL and SL cells compared with control group. However, the DNA damage is more in SL cells than in NL cells. Pre-supplementation of selenite did not show any difference in DNA (strand breaks) damage, selenium, and GPx activity. Increased hepatic selenium concentration and GPx activity were observed in both NL and SL cells in post-supplementation and entire period of selenite supplemented animals compared to DEN + PB treated animals. However, DNA damage was increased in NL but decreased in SL cells. Supplementation of selenite alone for 16 or 20 weeks had shown increased DNA damage, selenium concentration, and GPx activity compared to normal control animals. In summary, cancer bearing animals increased DNA damage and decreased Se level and GPx activity in NL and SL cells and other organs in cancer bearing animals, supplementation of Se further provoked DNA damage (no change in pretreatment) in NL cells, however it decreased DNA damage SL cells and other organs (kidney, lungs, and spleen). On the other hand Se levels and GPx activity were increased in NL and SL cells and other organs of Se-supplemented rats (no difference in group 3 animals). These results demonstrate that, in addition to chemopreventive and chemotherapeutic role of selenite, it also prevents cellular DNA damage induced in cancerous condition.     

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.     

Effects of different sewage sludge applications on heavy metal accumulation,growth and yield of spinach (Spinacia oleracea L.)

In this study, we present the response of spinach to different amendment rates of sewage sludge (0, 10, 20, 30, 40 and 50 g kg^?1) in a greenhouse pot experiment, where plant growth, biomass and heavy metal uptake were measured. The results showed that sewage sludge application increased soil electric conductivity (EC), organic matter, chromium and zinc concentrations and decreased soil pH. All heavy metal concentrations of the sewage sludge were below the permissible limits for land application of sewage sludge recommended by the Council of the European Communities. Biomass and all growth parameters (except the shoot/root ratio) of spinach showed a positive response to sewage sludge applications up to 40 g kg^?1 compared to the control soil. Increasing the sewage sludge amendment rate caused an increase in all heavy metal concentrations (except lead) in spinach root and shoot. However, all heavy metal concentrations (except chromium and iron) were in the normal range and did not reach the phytotoxic levels. The spinach was characterized by a bioaccumulation factor <1.0 for all heavy metals. The translocation factor (TF) varied among the heavy metals as well as among the sewage sludge amendment rates. Spinach translocation mechanisms clearly restricted heavy metal transport to the edible parts (shoot) because the TFs for all heavy metals (except zinc) were <1.0. In conclusion, sewage sludge used in the present study can be considered for use as a fertilizer in spinach production systems in Saudi Arabia, and the results can serve as a management method for sewage sludge.     

Modulatory Effects of Shape Pluchea Lanceolata Against Chemically Induced Oxidative Damage, Hyperproliferation And Two-Stage Renal Carcinogenesis in Wistar Rats

Ferric nitrilotriacetate (Fe-NTA) is a well-established renal carcinogen. Here, we have shown that Pluchea lanceolata (PL) belonging to the family Asteraceae. PL attenuates Fe-NTA induced renal oxidative stress, hyperproliferative response and renal carcinogenesis in rats. It promoted DEN (N-diethyl nitrosamine) initiated renal carcinogenesis by increasing the percentage incidence of tumors and induces early tumor markers viz. ornithine decarboxylase (ODC) and renal DNA synthesis. Fe-NTA (9 mg Fe/kg body weight, intraperitoneally) also enhances renal lipid peroxidation (LPO), xanthine oxidase (XO) and hydrogen peroxide (H₂O₂) generation with reduction in renal glutathione content (GSH), antioxidant enzymes, viz., glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), glucose-6-phosphate dehydrogenase and phase-II metabolizing enzymes such as glutathione-S-transferase and quinone reductase (QR). It also enhances blood urea nitrogen (BUN) and serum creatinine. Oral treatment of rats with PL extract (100 and 200 mg/kg body weight) resulted in significant decrease in lipid peroxidation (LPO), xanthine oxidase (XO), H₂O₂ generation, blood urea nitrogen (BUN), serum creatinine, renal ODC activity, DNA synthesis (p < 0.001) and incidence of tumors. Renal glutathione content (p < 0.01), its metabolizing enzymes (p < 0.001) and antioxidant enzymes were also recovered to significant level (p < 0.001). Thus, present study supports PL as a potent chemopreventive agent and suppresses Fe-NTA-induced renal carcinogenesis and oxidative damage response in Wistar rat.     

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9% NaCl. All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci.     

Chemoprevention of lung cancer by lycopene

An investigation was conducted to assess the chemopreventive potential of lycopene (LP), a naturally occurring hydrocarbon carotenoid found in tomatoes and their products, administered during the post-initiation stage in a multiorgan carcinogenesis model. One hundred eighteen B6C3F1 mice of both sexes were subjected to combined treatment with diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU) and 1,2-dimethylhydrazine (DMH) from day 11 after birth to week 9 (DMD treatment) (groups 1 and 2) or given their vehicles (group 3). Then group 1 received LP (25 or 50 ppm in drinking water) for 21 weeks from weeks 11 to 32. Group 2 served as a carcinogen alone control and group 3 was given only LP (25 or 50 ppm). The incidences and multiplicities of lung adenomas plus carcinomas combined in male mice in group 1 receiving LP 50 ppm were significantly decreased as compared to the DMD alone or DMD and LP 25 ppm group values (75.0 vs 18.8%, P < 0.02; 0.94+/-0.17 v.s 0.25+/-0.14, P < 0.001). While hepatocellular carcinomas were lacking in the DMD and LP groups, two cases were found in the DMD alone group (not statistically significant). The values for aberrant crypt foci (ACF) and tumors in the colon and kidney did not show any significant variation among the carcinogen-treated subgroups. The results of this study provide evidence that the tomato carotenoid, lycopene, may have potential as a chemopreventive agent against carcinogenesis in the male lung.     

Chemopreventive effect of vanadium in a rodent model of chemical hepatocarcinogenesis: reflections in oxidative DNA damage, energy-dispersive X-ray fluorescence profile and metallothionein expression

In the present study, we investigated the antitumour efficacy of vanadium in a defined rodent model of experimental hepatocarcinogenesis. Hepatic preneoplasia was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal injection of diethylnitrosamine (DEN) (200 mg/kg body weight) followed by promotion with phenobarbital (PB). The levels of modified DNA bases 8-hydroxy-2′-deoxyguanosine (8-OHdG), a potential marker involved in the initiation of carcinogenesis, were measured by high-performance liquid chromatography, whereas tissue trace element status and expression of metallothionein (MT), a Cu–Zn metalloprotein associated with neoplastic cell growth and subsequent development of premalignant phenotype of the cell, were studied by energy-dispersive X-ray fluorescence spectrometry and enzyme-coupled immunohistochemistry, respectively. There was a significant and steady elevation of modified bases (8-OHdG) along with substantial increase in MT immunoexpression and disturbance in trace element homeostasis following DEN exposure. Supplementation of vanadium at a dose of 0.5 ppm for four consecutive weeks strictly abated the formation of 8-OHdG (P < 0.0001; 81.28%) in preneoplastic rat liver. In a long-term DEN plus PB regimen, vanadium was able to limit in situ MT expression with a concomitant decrease in MT immunoreactivity (P < 0.05). Furthermore, vanadium treatment throughout the study restored hepatic levels of essential trace elements and decreased nodular incidence (58.34%) and nodule multiplicity (P < 0.001; 66.89%) in rats treated with DEN plus PB. Taken together, the study provides evidence in support of the chemopreventive potential of vanadium in limiting neoplastic transformation during the preneoplastic stages of hepatocarcinogenesis in rats.     

Alkylation-induced genotoxicity as a predictor of DNA repair deficiency following experimental oral carcinogenesis

The aim of this study was to evaluate alkylation induced genotoxicity as a result of DNA repair deficiency during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis by means of single cell gel (comet) assay. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. Blood samples and oral mucosa cells collected from all animals were divided into two aliquots of 20 μL each to study basal DNA damage and DNA damage due to genotoxin sensitivity. The first aliquot was processed immediately for comet assay to assess basal DNA damage. The second aliquot was treated with a known genotoxin, methylmetanesulfonate. Significantly greater DNA damage was noticed to oral mucosa cells from 4, and 12 weeks post-treatment. Peripheral blood cells did show statistically significant differences (P < 0.05) after 20 weeks-group (squamous cell carcinoma). In conclusion, alkylation induced genotoxicity as a result of DNA repair deficiency is present in oral mucosa cells following oral experimental carcinogenesis.     

Solubilization profiles of metal ions from bioleaching of sewage sludge as a function of pH

Two patterns of solubilization of metal ions resulting from bioleaching of sewage sludge by sulphur-oxidizing Thiobacillus spp. were established as a function of pH. Chromium and copper ions required a pH of 2–3 to initiate their solubilization, whereas nickel and zinc ions had their solubilization initiated at pH 6–6.5. The patterns obtained were independent of the sludge solids concentrations investigated (10, 17, 25, 32.5 and 40 g l^–1).     

Phytoextraction of heavy metals from municipal sewage sludge by Rosa multiflora and Sida hermaphrodita

The aim of the study was to evaluate the efficacy of the multiflora rose var. “Jatar” (Rosa multiflora Thunb. ex Murray) and the Virginia fanpetals (Sida hermaphrodita Rusby) to phytoextract heavy metals from municipal sewage sludge. The 6-year field experiment involved four levels of fertilization with sewage sludge at doses of 0, 10, 20, 40, and 60 Mg DM (Dry Mass) sludge ha^?1. The increasing doses of sewage sludge were found to significantly increase the yield of multiflora rose and Virginia fanpetals biomass. They also significantly increased the content of heavy metals in these plants. The highest uptake of heavy metals by the multiflora rose and Virginia fanpetals crops was recorded at the fertilization dose of 60 Mg DM ? ha^?1. Our investigations show that the Virginia fanpetals was more efficient in the phytoextraction of Cr, Ni, Cu, Zn, and Cd from the sewage sludge than the multiflora rose, due to the greater yields and higher heavy metal uptake by the former plant. In turn, the multiflora rose phytoextracted greater amounts of Pb from the sewage sludge. The analyses indicate that the Virginia fanpetals can be used for phytoremediation (phytoextraction) of heavy metals contained in sewage sludge.     

Genotoxicity assessment in Eisenia andrei coelomocytes: a study of the induction of DNA damage and micronuclei in earthworms exposed to B[a]P- and TCDD-spiked soils

Earthworms are useful indicators of soil quality and are widely used as model organisms in terrestrial ecotoxicology. The assessment of genotoxic effects caused by environmental pollutants is of great concern because of their relevance in carcinogenesis. In this work, the earthworm Eisenia andrei was exposed for 10 and 28 days to artificial standard soil contaminated with environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50ppm) and 2,3,7,8-tetrachloro-dibenzo-para-dioxin (TCDD) (1×10(-5), 1×10(-4), 2×10(-3)ppm). Micronucleus (MNi) induction was evaluated in earthworm coelomocytes after DNA staining with the fluorescent dye DAPI. In the same cells, the DNA damage was assessed by means of the alkaline comet assay. Induction of MNi in coelomocytes, identified according to standard criteria, was demonstrated. B[a]P exposure for 10 and 28 days induced a significant increase in MNi frequency. In TCDD-treated earthworms, a significant effect on chromosomal damage was observed at all the concentrations used; surprisingly, greater effects were induced in animals exposed to the lowest concentration (1×10(-5)ppm). The data of the comet assay revealed a significant increase in the level of DNA damage in coelomocytes of earthworms exposed for 10 and 28 days to the different concentrations of B[a]P and TCDD. The results show that the comet and MN assays were able to reveal genotoxic effects in earthworms exposed even to the lowest concentrations of both chemicals tested here. The combined application in E. andrei of the comet assay and the micronucleus test, which reflect different biological mechanisms, may be suggested to identify genotoxic effects induced in these invertebrates by environmental contaminants in terrestrial ecosystems.     

Micronucleus test with cyclophosphamide using mouse peripheral blood reticulocytes.

The usefulness of the micronucleus test using supravital staining of peripheral blood reticulocytes with acridine orange was evaluated in two laboratories after administering cyclophosphamide (CYP) as a model chemical by intraperitoneal injection (i.p.) to CD-1 mice. The frequencies of micronucleated peripheral reticulocytes (MNRETs) increased dose-dependently at each sampling time. There were no significant differences in the results obtained with this new method by the two laboratories. Although the induction of MNRETs was delayed by about 24 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optical sampling time, 24 h for MNPCEs and 48 h for MNRETs. Therefore, it is concluded that this method is a suitable alternative to that using femoral marrow cells.     

Protective<Emphasis Type="Italic">in vivo</Emphasis> effect of curcumin on copper genotoxicity evaluated by comet and micronucleus assays

Curcumin is a phytochemical with antiinflammatory, antioxidant and anticarcinogenic activities. Apparently, curcumin is not genotoxic in vivo, but in vitro copper and curcumin interactions induce genetic damage. The aim of this study was to test if in vivo copper excess induces DNA damage measured by comet and micronucleus assays in the presence of curcumin. We tested 0.2% curcumin in Balb-C mice at normal (13 ppm) and high (65, 130 and 390 ppm) copper ion concentrations. The comet and micronucleus assays were performed 48 hr after chemical application. Comet tail length in animals treated with 0.2% curcumin was not significantly different from the control. Animals exposed to copper cations (up to 390 ppm) exhibited higher oxidative DNA damage. Curcumin reduced the DNA damage induced by 390 ppm copper. We observed statistically significant increase in damage in individuals exposed to 390 ppm copper versus the control or curcumin groups, which was lowered by the presence of curcumin. Qualitative data on comets evidenced that cells from individuals exposed to 390 ppm copper had longer tails (categories 3 and 4) than in 390 ppm copper + curcumin. A statistically significant increase in frequency of micronucleated erythrocytes (MNE/10000TE) was observed only in 390 ppm copper versus the control and curcumin alone. Also cytotoxicity measured as the frequency of polychromatic erythrocytes (PE/1000TE) was attributable to 390 ppm copper. The lowest cytotoxic effect observed was attributed to curcumin. In vivo exposure to 0.2% curcumin for 48 hr did not cause genomic damage, while 390 ppm copper was genotoxic, but DNA damage induced by 390 ppm copper was diminished by curcumin. Curcumin seems to exert a genoprotective effect against DNA damage induced by high concentrations of copper cations. The comet and micronucleus assays prove to be suitable tools to detect DNA damage by copper in the presence of curcumin.     

DNA adducts and liver DNA replication in rats during chronic exposure to N-nitrosodimethylamine (NDMA) and their relationships to the dose-dependence of NDMA hepatocarcinogenesis

Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drinking water) for up to 180 days resulted in rapid accumulation of N7- and O6-methylguanine in liver and white blood cell DNA, maximum adduct levels being reached within 1-7 days, depending on the dose. The levels of both adducts remained constant up to treatment day 28, subsequently declining slowly to about 40% of maximal levels for the liver and 60% for white blood cells by day 180. In order to elucidate the role of DNA replication in NDMA hepatocarcinogenesis, changes in liver cell labeling index (LI) were also measured on treatment days 21, 120 and 180. Although the time- and dose-dependence of the observed effects were complex, a clear trend towards increased rates of hepatocyte LI, as indicated by BrdU incorporation, with increasing NDMA doses was evident, particularly above 1 ppm, a concentration above which NDMA hepatocarcinogenicity is known to increase sharply. In contrast, no increase in Kupffer cell DNA replication was found at any of the doses employed, in accordance with the low susceptibility of these cells to NDMA-induced carcinogenesis. No significant increase in the occurrence of necrotic or apoptotic cells was noted under the treatment conditions employed. These results suggest that, in addition to the accumulation of DNA damage, alterations in hepatocyte DNA replication during the chronic NDMA exposure may influence the dose-dependence of its carcinogenic efficacy.