Norma Viviana González

Genotoxic and cytotoxic effects of carbofuran and furadan on Chinese hamster ovary (CHOK1) cells.

The in vitro geno- and cytotoxicity exerted by the N-methylcarbamate pesticide carbofuran (CF) and its commercial formulation furadan (F) were studied in Chinese hamster ovary (CHO(K1)) cells by several bioassays for both genotoxicity (e.g., the sister chromatid exchange (SCE) and micronuclei (MNi) frequencies), and cytotoxicity (e.g., cell-cycle progression, mitotic index (MI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR)). Both CF and F activities were tested within the range of 5-100 microg/ml. CF within a 10-100 microg/ml concentration-range induced a significant dependent increase of SCE frequency and MNi over control values. At the same concentration-range, F increased significantly the SCE frequencies over control values although in a non-dependent manner while only an enhanced frequency of MNi was found in those 50 microg/ml-treated cultures. No binucleated cytokinesis-block cells were found in 100 microg/ml F-treated cultures. The NDI index revealed a delay in the onset of cell-division with 50 and 100 microg/ml of CF and F, respectively. The delayed rate of nuclear division induced by 100 microg/ml of F was higher than that induced by an equal concentration of CF. CF and F induced both a significant concentration-dependent delay in cell-cycle progression and a decrease in the proliferative replication index within 5-100 microg/ml and 50-100 microg/ml concentration-range, respectively. Decreased cell viability was found in up to 26% and 47% in 100 microg/ml CF- and F-treated cultures, respectively. The NR and MTT assays revealed a clear cell growth inhibition when concentrations of 50 and 100 microg/ml of either CF or F were employed. Accordingly, the results highlight that CF by itself and F, even in a greater extend exerts both genotoxicity and cytotoxicity in mammalian cells in culture, at least in CHOK1 cells.

Dicamba-induced genotoxicity in Chinese hamster ovary (CHO) cells is prevented by vitamin E

In the present study the cytogenetic and genotoxic effect of benzoic herbicide dicamba and its Argentinean commercial formulation banvel (57.71% dicamba) was evaluated and whether this effect is mediated through oxidative damage or not. The protective role of vitamin E was also studied. Sister chromatid exchange (SCE) frequency, cell-cycle progression, and cell viability analyses in CHO cells were used as in vitro end-points. Treatments with the test compounds were performed either during 24h (Protocol A) or 12h (Protocol B) before harvesting. Protocol A showed that vitamin E decreased pesticide SCE induction, corrected the cell-cycle delay and partially protected cell-death only in 500 microg/ml dicamba-treated cultures. A similar trend was found in banvel-treated cultures. Protocol B revealed similar protective role of vitamin E only for dicamba-induced geno- and cytotoxicity. Based on these observations it could be suggested that dicamba injures DNA by delivering reactive oxygen species rather than by another type of mechanism/s. Although banvel mimics the effect observed by dicamba, its formulation contains other xenobiotic/s agents able to induce cellular and DNA damage by a different mechanism/s. Further investigations are needed to acquire a comprehensive knowledge of the possible mechanism/s through dicamba and banvel exert their toxic effects.

Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4‐D and its commercial derivative 2,4‐D DMA were studied by measuring sister chromatid exchange (SCE), cell‐cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 μg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10–50 μg 2,4‐D/ml and 25–100 μg 2,4‐D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 μg 2,4‐D/ml and 50 and 100 μg 2,4‐D DMA/ml. In PLC, only 100.0 μg 2,4‐D/ml altered cell‐cycle progression. For both chemicals, a progressive dose‐related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4‐D and 2,4‐D DMA into human lymphocytes in vitro as well as both 2,4‐D and 2,4‐D DMA were more potent genotoxic agents in the presence of human red cells.

A combination of the cytokinesis-block micronucleus cytome assay and centromeric identification for evaluation of the genotoxicity of dicamba.

The purpose of this study was to further investigate the cytotoxic and genotoxic effects of dicamba and Banvel(®) employing the cytokinesis-block micronucleus cytome (CBMN-cyt) assay estimated by the analysis of the nuclear division index (NDI), the frequency of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs). Besides, for mechanism of MN induction CREST anti-kinetochore antibody analysis was performed. The activities of both compounds were tested within the range of 50-500 μg/ml on Chinese hamster ovary (CHO-K1) cells. Overall, dicamba and Banvel(®) produced a NDI dose-dependent decrease but the response was statistically significant only in cultures treated with Banvel(®) at a 100-500 μg/ml concentration range. A dose-dependent induction of MN was observed after dicamba- and Banvel(®)-treatments within the 50-400 μg/ml and 50-500 μg/ml concentration-ranges, respectively. Induction of NPBs and NBUDs was significantly enhanced by both test compounds. The NPBs/MN ratio values found for dicamba and Banvel(®) were 0.04-0.11 and 0.05-0.18, respectively. Results clearly demonstrated that dicamba and Banvel(®) exerted both cyto- and genotoxic damage on CHO-K1 cells. Furthermore, the CBMN-cyt assay employed confirmed our previous investigations concerning the cellular and DNA damaging capabilities of dicamba and highlights that both clastogenic and aneugenic mechanisms are implicated in the MN induction.

Sex- and age-related temporal variations in intestinal-epithelium proliferation in the suckling mouse.

Intestinal-crypt enterocytes are a cell population undergoing constant renewal in the mouse. Both adult and 28 d old animals have been shown to exhibit circadian rhythms in cell proliferative indices, but there are only scant data on the 24 h mitotic activity in the small and large intestine of younger mice. The present studies were thus undertaken in order to characterize the proliferative pattern of enterocytes in the duodenum and colon of 7 and 14 d old males and females of the C3H/S strain. Animals of each sex and from each age group were sacrificed every 4 h during a 24 h span, with each animal receiving an injection of colchicine 4 h before sacrifice. Samples of duodenum and colon were removed and processed for hematoxylin–eosin staining. Twenty longitudinally sectioned crypts within each sample were analyzed, and the mitotic indices of both cell populations from each animal were estimated. The arithmetic mean±SEM for each experimental group were then calculated and the statistical significance of differences between the means assessed by ANOVA and Student t-tests. We observed a greater daily mitotic activity in the duodenum than the colon, and moreover enterocytic proliferation in both those regions was greater in 14 than 7 d old animals. Twenty-four hour variations in mitotic activity occurred in all the experimental groups and tissues except for the large intestine of 7 d old females. Finally, the temporal profile of epithelium proliferation in the suckling mouse varied with age, sex, and site of the intestine studied.

Testing genotoxicity and cytotoxicity strategies for the evaluation of commercial radiosterilized fetal calf sera.

Effects of 18 commercial lots of fetal calf serum (FCS) after gamma-irradiation and their non-irradiated counterparts were comparatively analyzed on CHO-K1 and MDBK MDL1 cells for genotoxicity [sister chromatid exchange (SCE), micronuclei (MNi), and single cell gel electrophoresis (SCGE)], cytotoxicity [cell-cycle progression (CCP), proliferative replication index (PRI), mitotic index (MI), growth promotion (GP), and plating efficiency (PE)], and microbiological properties (mycoplasma and bovine viral diarrhea virus contamination). SCE and SCGE were the most informative end-points for genotoxicity since significant differences were found in 44.4% (P<0.05-0.001, Students t-test) and 61.1% (P<0.05-0.001, chi(2) test) samples, respectively. MI was the cytotoxicity assay revealing the greatest variation, showing differences in 66.7% (P<0.05-0.001, chi(2) test) samples. Thus, these three end-points for screening bioproducts such as FCS were found most suitable for detecting potential geno-cytotoxicants in biological samples; their simultaneous use could be strongly recommended.

Daily variations in colchicine-induced apoptosis in duodenal crypts.

Apoptotic cell death can be induced by several agents, among them colchicine, a microtubule disrupting‐drug that affects continuously renewing cell populations, such as the intestinal crypt enterocytes. The objectives of this investigation were (1) to confirm in vivo colchicines‐inductive effect and (2) to determine the existence of 24 h variations in the crypt enterocytes apoptotic indices. The study was done on C3H/S male adult mice housed under standardized conditions. Starting at midnight until the end of a circadian period, subgroups of mice were sacrificed after having been injected with colchicine or saline i.p. 4 h beforehand. Duodenal samples were processed for hematoxylin‐eosin staining and TUNEL technique. In order to score the number of apoptosis, the longitudinal sections of the crypts were divided into three regions comprised, respectively, of tiers 1–4, 5–12, and 13–20, proceeding from the bottom to the top of the crypt. Values of each lot were expressed as mean±SEM. A highly significant statistical difference in apoptotic indices was found for colchicine‐treated animals. The 24 h curve for colchicine‐induced apoptosis displayed qualitative and quantitative differences compared to other inducer agents. Highest apoptotic indices were found in the deepest crypt regions. Daily variations were observed in all the crypt sectors of the colchicine‐treated animals and in tiers 5–12 of the saline controls. The present work demonstrates that the colchicine cytotoxicity due to its apoptotic‐inducing effect depends on the dosing time during the 24 h in this mouse strain.

Lectin histochemistry for detecting cadmium-induced changes in the glycosylation pattern of rat placenta

Abstract Cadmium (Cd) is an industrial and environmental pollutant that produces toxic effects on gametogenesis, pre- and post-implantation embryos, and the placenta. Because the effects of acute Cd intoxication on the placenta are not well understood, we investigated changes in its glycosylated components in Cd treated dams at days 4, 7, 10 and 15 of gestation using lectin histochemistry. CdCl2 was administered to pregnant rats; control animals received sterile normal saline. Placentas were processed for DBA, Con A, SBA, PNA, UEA-I, RCA-I and WGA lectin histochemistry to evaluate changes in the carbohydrate pattern of the placenta that might modify cell interactions and contribute to embryonic alterations. Lectin binding was analyzed in the yolk sac; trophoblast giant cells; trophoblast I, II and III; spongiotrophoblast cells and endovascular trophoblast cells in the chorioallantoic placenta. Our lectin binding patterns showed that Cd caused alteration of SBA and DBA labeling of trophoblast-derived cells, which suggested increased expressions of α and β GalNAc. Cd also caused decreased UEA-1 binding affinity, which indicated fewer α-L-Fuc residues in placentas of Cd treated dams. The nonreactivity in trophoblast I of the control placentas incubated with Con-A contrasted with the labeling in placentas of experimental dams, which indicated increased expression of terminal α-D-Man, and α-D-Glc residues. We found that Cd altered the reactivity of placenta to several lectins, which indicated modification of the glycotype presented by the fetal component of the placenta. We report that Cd exerts a deleterious effect on the glycosylation pattern of the placenta.

Antimitotic activity of EA21b mammary-carcinoma extract

Many tumors produce factors that affect cell-cycle and cell proliferation. In the present study we have analyzed the effect of a mammary-tumor extract injection on the mitotic activity of several organs in young male C3H/S mice previously standardized for circadian periodicity. One-half of the animals received an intraperitoneal EA21b tumor extract dose at 16:00 h, while the other half received saline. Animals were sacrificed on the following day at 08:00, 12:00 or 16:00 h. 4 h after receiving an injection of colchicine by the same route. Samples of duodenum, kidney, liver, and submaxillary gland were excised and processed for hematoxylin-eosin staining. Mitotic indices, expressed as the number of colchicine-arrested metaphases per 1,000 nuclei, were assessed in convoluted tubule epithelium, duodenal crypt enterocytes, hepatocytes and submaxillary gland ductal and acinar sialocytes. All values were expressed as mean ± SEM. Statistical analyses were performed by ANOVA, Bonferroni and Student’s t-tests. In contrast to the mitotic indices reductions observed in renal convoluted tubules cells and duodenal crypt enterocytes, neither the submaxillary gland nor the liver were found to contain cell types whose mitotic activity was affected by the tumor extract. We conclude that EA21b mammary carcinoma contains one or more factors that inhibit the proliferation of selected populations of normal cells.