Monserrat Sordo

Mitotic index and cell proliferation kinetics for identification of antineoplastic activity

The mitotic index (MI) and cell proliferation kinetics (CPK) of human blood lymphocyte cultures were determined to evaluate the effects of six antineoplastic drugs with well known cytostatic activity: cisplatin, melphalan, bleomycin, methotrexate, 5-fluorouracil and 6-mercaptopurine. All six drugs showed a clear effect on the inhibition of MI. The first three drugs interact directly with DNA showing a dose-related retardation of CPK. Methotrexate, 5-fluorouracil and 6-mercaptopurine, which act on ribonucleotide biosynthesis, showed no significant effects on CPK. The results suggest that CPK and MI measurements are useful for the prescreening of drugs with potential cytostatic activity.

Arsenite reduces insulin secretion in rat pancreatic β-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25

An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic beta-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca2+]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5-2 microM). The global activity of calpains increased with 2 microM arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 microM arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the beta cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca(2+)]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion.

Relationship between micronuclei formation and p53 induction.

Human exposure to multiple chemicals compromises the integrity of genetic material. Hence, it is essential to determine the extent of DNA damage induced by xenobiotics. In cell lines, the induction of p53 expression in response to treatments with DNA-damaging agents has been proposed as a tool for the detection of genotoxic damage, although a direct correlation between a marker of chromosomal damage and p53 expression has not previously been studied. The micronucleus assay is a widely used genotoxicity test that has been shown to detect structural and numerical chromosomal damage. The present study was designed to characterize the relationship between micronuclei and p53 induction. RKO cells were cultured and treated with non-cytotoxic concentrations of colchicine, vinblastine, bleomycin or arsenic. Mannitol and clofibrate, which are non-genotoxic chemicals, were also included. The frequency of micronuclei was evaluated using the cytokinesis-block assay, and p53 induction was measured by Western blot assay. Our data showed that a significant induction of micronuclei and of p53 protein occurred only with the genotoxic chemicals. No differences in p53 induction were associated with the clastogenic or aneuplodogenic potential of the chemical exposure. The linear regression analysis revealed a direct relationship between p53 levels and the induction of micronuclei (p=0.0001, r(2)=0.9372), indicating that the level of p53 is associated with chromosomal damage.

Arsenic Exposure and Calpain-10 Polymorphisms Impair the Function of Pancreatic Beta-Cells in Humans: A Pilot Study of Risk Factors for T2DM

The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function.

A Permethrin/Allethrin Mixture Induces Genotoxicity and Cytotoxicity in Human Peripheral Blood Lymphocytes

Two pyrethroids, permethrin and allethrin, are often combined for large-scale use in public health programs to control vector-borne diseases. In this study, the genotoxic potential of a commercial formulation of permethrin and allethrin was examined using cultured human peripheral blood lymphocytes (PBL). Genotoxicity was evaluated using the cytokinesis-block micronucleus cytome (CBMN cyt) assay by measuring the frequency of micronuclei (MN), nuclear division index (NDI), formation of nucleoplasmic bridges (NPB) and nuclear buds (NBUD), as well as apoptotic and necrotic cells. Human PBL were treated with different concentrations of a permethrin/allethrin mixture (1/0.01, 5/0.07, and 10/0.14 μg/ml) for 24 or 36 h. The highest concentration (10/0.14 μg/ml) of permethrin/allethrin mixture significantly increased MN frequency and percent apoptotic cells after incubations for 24 or 36 h. The NDI was markedly decreased in response to treatment with 5/0.07 or 10/0.14 μg/ml permethrin/allethrin for both 24 and 36 h. Exposure to the permethrin/allethrin mixture did not significantly alter formation of NBUD, NPB, or percent necrotic cells. The MN frequency was significantly correlated with the number of apoptotic and necrotic cells but inversely correlated with NDI. Data demonstrated that a mixture of permethrin and allethrin induced concentration- and time-dependent cytotoxic and genotoxic damage to human PBL in vitro.

p53 Expression in circulating lymphocytes of non-melanoma skin cancer patients from an arsenic contaminated region in Mexico. A pilot study

Arsenic is a common environmental toxicant and epidemiological studies associate arsenic exposure with various pathologic disorders and several types of cancer. Skin cancers are the most common arsenic-induced neoplasias and the prevalence of skin lesions has been reported to be significantly elevated in individuals exposed to arsenic via drinking water in México. Being lymphocytes the main cells used for human monitoring, we evaluated the expression of p53 protein in the lymphocytes from 44 healthy individuals and 19 samples from individuals living in a chronic arsenicism endemic region. Of the latter group, 12 individuals had non-melanoma skin cancer and 9 of them expressed p53 in the circulating lymphocytes, whereas only one of the 7 non-cancer arsenic exposed individuals expressed it. In the healthy non-arsenic exposed group only one from 44 individuals expressed the protein. These results suggest a clear relationship between non-melanoma skin cancer and p53 expression in circulating lymphocytes. p53 expression in circulating lymphocytes should be evaluated as a potential biomarker of effect or susceptibility.

The Role of Paraoxonase Polymorphisms in the Induction of Micronucleus in Paraoxon-Treated Human Lymphocytes

Human paraoxonase‐1 (PON1) is a high‐density lipoprotein‐associated enzyme that has a role in the detoxification of organophosphorus compounds by hydrolyzing the bioactive oxons. PON1 polymorphims are responsible, at least in part, for the variation in the catalytic activity and expression of the enzyme and have been associated with susceptibility to organophosphorus pesticide toxicity, mainly neurotoxicity. The aim of this study was to determine whether paraoxon induced micronuclei and to examine the role of PON1 polymorphism in paraoxons genotoxic potential. First, dose finding cytogenetic experiments were performed on lymphocyte cultures from three donors and a range of paraoxon concentration (1–25 μM) were tested. In a second set of experiments, 5 μM paraoxon was added to blood cultures of 11 donors with two different PON1 haplotypes (PON T−108M55Q192 with low activity and haplotype PON C−108L55 R192 with high activity, referred to as PON1QQ and as PON1 RR, respectively). Because PON1 is present in blood, the effect of adding 5 μM paraoxon and 70 μl of autologous plasma to lymphocyte cultures also was examined. Paraoxon had no effect on cell viability, but caused a significant dose‐dependent increase in MN frequency. The basal MN frequencies were similar on QQ and RR genotypes. A significant difference was observed in the MN frequency only in lymphocytes from individuals with the QQ genotype treated with 5 μM paraoxon and the autologous plasma did not modify these effects. The results obtained in this study suggest that PON1 genotype might have an important role in the identification of individuals at risk for cancer development due to occupational exposure to pesticides. Environ. Mol. Mutagen. 2009.

Cytostatic and genotoxic effect of temephos in human lymphocytes and HepG2 cells.

Temephos is an organophosphorus pesticide that is used in control campaigns against Aedes aegypti mosquitoes, which transmit dengue. In spite of the widespread use of temephos, few studies have examined its genotoxic potential. The aim of this study was to evaluate the cytotoxic, cytostatic and genotoxic effects of temephos in human lymphocytes and hepatoma cells (HepG2). The cytotoxicity was evaluated with simultaneous staining (FDA/EtBr). The cytostatic and genotoxic effects were evaluated using comet assays and the micronucleus technique. We found that temephos was not cytotoxic in either lymphocytes or HepG2 cells. Regarding the cytostatic effect in human lymphocytes, temephos (10 μM) caused a significant decrease in the percentage of binucleated cells and in the nuclear division index as well as an increase in the apoptotic cell frequency, which was not the case for HepG2 cells. The comet assay showed that temephos increased the DNA damage levels in human lymphocytes, but it did not increase the MN frequency. In contrast, in HepG2 cells, temephos increased the tail length, tail moment and MN frequency in HepG2 cells compared to control cells. In conclusion, temephos causes stable DNA damage in HepG2 cells but not in human lymphocytes. These findings suggest the importance of temephos biotransformation in its genotoxic effect.

Suppression of p53 and p21CIP1/WAF1 reduces arsenite-induced aneuploidy.

Aneuploidy and extensive chromosomal rearrangements are common in human tumors. The role of DNA damage response proteins p53 and p21(CIP1/WAF1) in aneugenesis and clastogenesis was investigated in telomerase immortalized diploid human fibroblasts using siRNA suppression of p53 and p21(CIP1/WAF1). Cells were exposed to the environmental carcinogen sodium arsenite (15 and 20 microM), and the induction of micronuclei (MN) was evaluated in binucleated cells using the cytokinesis-block assay. To determine whether MN resulted from missegregation of chromosomes or from chromosomal fragments, we used a fluorescent in situ hybridization with a centromeric DNA probe. Micronuclei were predominantly of clastogenic origin in control cells regardless of p53 or p21(CIP1/WAF1) expression. MN with centromere signals in cells transfected with NSC siRNA or Mock increased 30% after arsenite exposure, indicating that arsenite induced aneuploidy in the tGM24 cells. Although suppression of p53 increased the fraction of arsenite-treated cells with MN, it caused a decrease in the fraction with centromeric DNA. Suppression of p21(CIP1/WAF1) like p53 suppression decreased the fraction of MN with centromeric DNA. Our results suggest that cells lacking normal p53 function cannot become aneuploid because they die by mitotic arrest-associated apoptosis, whereas cells with normal p53 function that are able to exit from mitotic arrest can become aneuploid. Furthermore, our current results support this role for p21(CIP1/WAF1) since suppression of p21(CIP1/WAF1) caused a decrease in aneuploidy induced by arsenite, suggesting that p21(CIP1/WAF1) plays a role in mitotic exit.

GENOTOXIC EFFECTS OF KARWINSKIA HUMBOLDTIANA TOXIN T-514 IN PERIPHERAL BLOOD LYMPHOCYTES

Toxicity by Karwinskia humboldtiana, a Rhamnaceae plant, has been assessed in a number of studies. Four dimmeric anthracenones, named T-496, T-514, T-516 and T-544 for their molecular weight, have been Isolated from this plant. T-514, In particular, has been shown to be toxic to liver and lung as well as to tumoral cell lines, preferentially to those from liver tumors. For this reason It has been suggested that the toxin could be used as an antineoplastlc agent. The present study was performed to characterize the biological activity of T-514 as a potential cytostatic and genotoxic agent. Peripheral blood lymphocytes in culture were used as a test system, where chromosomal aberrations and sister chromatid exchanges were scored in order to evaluate genotoxicity, and mitotic index and cell proliferation kinetics were used as parameters for cytostatic and cytotoxic ability. Genotoxicity to lymphocytes was negative. However, proliferation was affected by the toxin, demonstrating a cytostatic activity independent of genotoxic damage.