Genotoxicity in child populations exposed to polycyclic aromatic hydrocarbons (PAHs) in the air from Tabasco, Mexico.

The economy of the state of Tabasco is based on oil extraction. However, this imposes major effects to the environment and communities. Examples are the Polycyclic Aromatic Hydrocarbons (PAHs) that may be found in the soil, water and sediment of the region. Their volatility makes them available to living beings and results in genotoxic activity. The purpose of this study was to quantify the levels of PAHs in the air at several points in the state, and to analyze their relationship with possible damage to DNA on local inhabitants. Single Cell Gel Electrophoresis Assay (Comet Assay) was applied to peripheral blood lymphocytes of five groups of children between six and 15 years of age. PAH samples were analyzed following US/EPA TO-13-A method. Results indicated the presence in the air of most of the 16 PAHs considered as high priority by EPA, some of which have been reported with carcinogenic activity. Differences (p<0.05) were found between PAHs concentration in the gaseous component and in the particulate component of air samples, with the greatest values for the gaseous component. Greatest PAH concentrations were detected in areas with high oil extraction activities. Children groups from high oil activity areas presented genotoxic damage labeled from moderate to high according to DNA migration from nuclei (Tail Length: 14.2 - 42.14 microm and Tail/Head: 0.97 - 2.83 microm) compared with control group (12.25 and 0.63 microm, respectively). The group with greatest cell damage was located in the area with the greatest oil activity. We conclude that the presence of PAHs in the air may represent a health risk to populations that are chronically exposed to them at high oil activity regions.

Proapoptotic role of novel gene-expression factors.

The mechanisms that control cellular proliferation, as well as those related with programmed cell death or apoptosis, require precise regulation systems to prevent diseases such as cancer. Events related to cellular proliferation as well as those associated with apoptosis involve the regulation of gene expression carried out by three basic genetic expression regulation mechanisms: transcription, splicing of the primary transcript for mature mRNA formation, and RNA translation, a ribosomal machinery-dependent process for protein synthesis. While development of each one of these processes requires energy for recognition and assembly of a number of molecular complexes, it has been reported that an increased expression of several members of these protein complexes promotes apoptosis in distinct cell types. The question of how these factors interact with other proteins in order to incorporate themselves into the different transduction cascades and stimulate the development of programmed cell death, although nowadays actively studied, is still waiting for a clear-cut answer. This review focuses on the interactions established between different families of transcription, elongation, translation and splicing factors associated to the progression of apoptosis.

mRNA expression of MAGE-A3 gene in leukemia cells.

Leukemia-associated antigens such as proteins encoded by MAGE genes might provide tools for immunotherapy of leukemia. Positive and negative results of MAGE-A gene expression in hematological malignancies have been reported. This led us to study MAGE-A gene expression in human leukemias using RT-PCR. Among 115 leukemias from various subtypes, 14/34 (41.17%) AML were positive for one of the three genes analyzed (MAGE-A1 1/32; MAGE-A3 10/32; MAGE-B2 3/12). Expression was also detected in 23/76 (30.26%) B-cell ALL patients (MAGE-A1 2/53; MAGE-A3 20/53; MAGE-B2 1/32). One of these patients expressed both MAGE-A1 (weak signal) and -A3 (strong signal) genes. Other patient with CML were positive for MAGE-B2 (1/5, 20%). MAGE-A3 expression data were corroborated by real time RT-PCR through determination of MAGE-A3 transcript levels. We concluded that the MAGE-A3 gene is expressed at the mRNA level in a proportion of human leukemias.

Role of P53 functionality in the genotoxicity of metronidazole and its hydroxy metabolite.

P53 mediates several biological processes for preservation of genetic stability such as the induction of cell cycle arrest, DNA repair or apoptosis in response to DNA damage. The antiparasitic drug, 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole, MTZ) is able to increase lymphocyte proliferation inducing at the same time chromosomal aberrations. Trying to understand this unexpected event we used cell lines with different P53 functionality, determining the proliferation capacity and the induction of micronuclei (MN) after the treatment with MTZ or its hydroxy metabolite. Our results show that MTZ increased proliferation in a dose response manner in all P53 functional cell lines without inducing changes on the levels of P53 nor MN. However, MTZ hydroxy metabolite induced a dose response increase of P53 and MN, while cell proliferation was not increased. Several studies have shown that the hydroxy metabolite is more potent than MTZ itself. Only in cell lines that do not have a functional P53, MTZ and its metabolite increased both cell proliferation and MN. MTZ use is increasing and its carcinogenicity has not been discarded. Our data indicate that MTZ hydroxy metabolite is potentially a carcinogen and needs to be further studied.

In vitro effects of albendazole and its metabolites on the cell proliferation kinetics and micronuclei frequency of stimulated human lymphocytes.

BACKGROUND: Albendazole (ABZ) is an antiparasitic drug used for the treatment of several helminthiases. After its oral administration, this compound is metabolized to sulfoxide (SOABZ) and sulfone (SO(2)ABZ), SOABZ being the active metabolite. The antiparasitic activity of ABZ has been associated with its capacity to bind with tubulin, altering microtubule formation. Although some studies indicate that ABZ modified microtubule structure in host cells, data concerning the consequences of this phenomenon in human cells are scant. METHODS: In this study we evaluated the effects of ABZ and its metabolites on cell proliferation, as well as on the frequency of micronucleated cells in cultured human lymphocytes. RESULTS: ABZ and SOABZ arrested cell proliferation in metaphase and increased the frequency of micronuclei in treated lymphocytes. Contrariwise, SO(2)ABZ, the inactive metabolite, did not produce any significant effect. CONCLUSIONS: The formation of micronuclei may ultimately result in aneuploidy induction, an effect that could have severe consequences in humans. However, the doses of ABZ and SOABZ at which these effects were observed are several orders of magnitude higher than those found in the plasma of treated individuals. Because there are other mechanisms by which aneuploidy can be induced at even lower doses than micronuclei, i.e., chromosome nondisjunction, it is necessary to evaluate this effect in exposed individuals.

[Chemotherapy of cysticercosis. Review about its pharmacokinetics and toxicology]. / La quimioterapia de la cisticercosis. Revisión acerca de su farmacocinética y toxicología.

Neurocysticercosis (NCC) is the most common parasitic infection of the central nervous system. Praziquantel and albendazole are the two cestocide drugs currently used for the treatment of NCC. The present article reviews the studies on the pharmacokinetics of these compounds, both in animals and humans, that have led to more accurate, precise and short treatment schedules for NCC. Toxicological data indicate that both praziquantel and albendazole do not have severe secondary effects in the short term, however, there is still not sufficient information about their long term effects on human health, mainly with respect to albendazole, for which few studies on its effects on human cells are available. These two drugs constitute an effective treatment not only for NCC but also for several helminthiosis. To keep this advantageuos situation, health care professionals should be aware of the necessity of a more rational use of both anthelminthics, since the potentially adverse long term effects could be related to time and dose of exposure as well as to individual susceptibility. In addition, there is always the possibility that the misuse of these compounds could give rise to resistant species, that may represent a significant problem for public health in countries where parasitic diseases are endemic.

Evaluation of DNA damage in exfoliated tear duct epithelial cells from individuals exposed to air pollution assessed by single cell gel electrophoresis assay.

The search for relevant target cells for human monitoring purposes has increased during the last few years. Cells such as sperm, buccal or nasal and gastric epithelium are being used. In this study, we report the use of exfoliated tear duct epithelial cells as a potential material for human biomonitoring studies, since these cells are a target for environmental pollutants. We employed the alkaline single cell gel electrophoresis (SCGE) assay to evaluate for differences in the basal level of DNA damage between young adults from the south (exposed mainly to high levels of ozone) and from the north (exposed principally to hydrocarbons) regions of Mexico City. We found an increase in DNA migration in tear duct epithelial cells from individuals who live in the southern part of the city compared to those living in the northern part. Moreover, young people who live in the southwest part of the city with the highest values of ozone presented the highest values of DNA damage. These results show the feasibility of using exfoliated tear duct epithelial cells in human biomonitoring studies.

Possible association between Taenia solium cysticercosis and cancer: increased frequency of DNA damage in peripheral lymphocytes from neurocysticercosis patients.

Helminths, particularly some Schistosoma species, have been associated with cancer in humans. Neurocysticercosis, produced by cysticerci of the helminth Taenia solium, has been associated with the emergence of brain tumours and haematological malignancies. Local tumours, such as glioblastoma, could be explained by the induction of DNA damage in cells surrounding the cysticercus and chronically exposed to an inflammatory host response. However, systemic effects such as haematological malignancies are not easy to understand. The present work was conducted in Mexico to find out whether DNA damage arises in peripheral lymphocytes in patients with neurocysticercosis. We utilized a highly sensitive technique to analyse chromosomal aberrations, in-situ hybridization with probes against chromosomes 1, 2 and 4, and in addition the blocked-cytokinesis technique was used to determine the formation of micronuclei, a peculiar form of DNA damage. The study was made in lymphocytes from 8 patients before and after the administration of praziquantel, 1 of the 2 drugs used for neurocysticercosis treatment. The frequencies of chromosome aberrations and micronuclei in peripheral blood lymphocytes were higher in the infected patients as compared to those observed both in healthy donors and in the group of patients after praziquantel therapy. Our results suggest that chromosome aberrations induced in peripheral cells during neurocysticercosis could be associated with the development of haematological neoplasias.

Structural improvement of higher education in environmental toxicology in Latin America and Europe.

Industrial development has resulted in an increased release of chemicals and other agents into the environment, resulting in damage to the environment as well as increasing the risk of adverse effects on human health. Environmental toxicology (ET) is the discipline responsible for assessing the risks to human health and the environment from the effects of new chemicals and those already present in the environment. The development of human resources in toxicology is therefore a priority in both Latin America (LA) and the European Union (EU), although LA professionals are more involved in risk evaluation than in risk assessment compared to their EU colleagues. A solid background in general toxicology will enable those interested in environmental issues to tackle local problems. Moreover, the increasing globalization of markets and, therefore, of the necessary regulations, requires harmonisation of postgraduate programmes to ensure that risk assessment and management related to the environment are dealt with uniformly and by highly qualified scientists. The Inaugural Meeting of the ALFA-OMET Toxicology', a 2-year programme supported by the European Commission, offered the opportunity to discuss a number of these issues. The present status of existing ET courses in the EU and LA and the corresponding professional profiles in the two regions were examined, and a harmonized academic curriculum for a postgraduate professional profiles in the two regions were examined, and a harmonized academic curriculum for a postgraduate course in environmental toxicology was developed. Finally, a course programme for toxicology and a specialization in environmental toxicology designed by a panel of experts was discussed, and its relevance as a model for other specialisation programmes was analysed. Exercises such as those performed by ALFA-OMET may be useful not only in promoting discussion for the implementation of national and international professional registers in LA, but also in encouraging the same, ongoing process in the EU.

Are metals dietary carcinogens?

Humans have been in contact with metals almost since the beginning of our existence. In fact, one cannot even think on human evolution without considering the great role played by metals in mankind's development. Metals are common moieties of molecules involved in a wide variety of biological processes, and hence are found in virtually all living organisms. Some metals are essential for human nutrition; others are found as contaminants in foodstuffs. One feature of the normal human diet which is frequently found is the simultaneous presence of both essential and toxic metals. Other factors important in the risk-evaluation analysis of metals are their pharmacokinetics, interactions among them and with other major components of the diet, and, especially, the great differences in the dietary habits of different populations and in the regional distribution of metals. In attempting to understand the role which dietary metals could play in human carcinogenesis, we found that the many factors involved and the lack of specific information made it difficult to reach firm conclusions on the hazards of dietary metals. We hope that this paper will raise the interest of genetic toxicologists in the subject and will consequently facilitate a risk analysis of the carcinogenic potential of dietary metals.

The application of single cell gel electrophoresis or Comet assay to human monitoring studies.

OBJECTIVE: In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. MATERIAL AND METHODS: This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. RESULTS: Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. CONCLUSIONS: We discuss the assay feasibility as a genotoxic biomarker.

Analysis of the DNA damage induced by praziquantel in V-79 Chinese hamster fibroblasts and human blood cells using the single-cell gel electrophoresis assay.

The analysis of the genotoxicity of praziquantel, an effective antihelminthic widely used in countries where parasitic infections are still serious public health problems, has been extensively performed using diverse in vitro and in vivo assays and endpoints. However, results are not conclusive, since reports to date indicate either praziquantel is mutagenic, comutagenic, or even antimutagenic. In the present work, the clastogenic potential of praziquantel was investigated in V-79 Chinese hamster fibroblasts and human peripheral blood using a sensitive technique such as the single-cell electrophoresis assay. Results indicate that even though praziquantel induced DNA single-strand breaks both in V-79 cells and unstimulated human leukocytes, this effect was not translated into persistent DNA damage, since neither SCE nor HPRT mutations were induced. This suggests that the effect observed in the SCGE assay is an early event not closely related to praziquantel mutagenicity, because this DNA damage could be efficiently repaired.

Disruption of microtubule assembly and spindle formation as a mechanism for the induction of aneuploid cells by sodium arsenite and vanadium pentoxide.

Arsenic and vanadium are important environmental and industrial pollutants. Due to their widespread occurrence and potential genotoxicity, we studied the aneuploidy-inducing effects of these elements in cultured human lymphocytes using a variety of techniques including fluorescence in situ hybridization (FISH) with DNA probes for chromosomes 1 and 7, immunostaining of the lymphocyte spindle apparatus, and an in vitro assay measuring the polymerization and depolymerization of tubulin. Dose-related increases in hyperdiploidy were seen in lymphocyte cultures treated with sodium arsenite (NaAsO2) or vanadium pentoxide (V2O5) over concentrations ranging from 0.001 to 0.1 microM. NaAsO2-treated cells from different donors exhibited similar hyperdiploid frequencies, whereas substantial inter-individual variability was seen in the V2O5-treated cells. Examination of the spindle apparatus using an anti-beta-tubulin antibody indicated that these compounds might disrupt spindle formation by interacting with microtubules. Additional in vitro assays using purified tubulin indicated that both compounds inhibited microtubule assembly and induced tubulin depolymerization. These results indicate that in vitro exposure to both NaAsO2 and V2O5 can induce aneuploidy in human lymphocytes, and that this effect may occur through a disruption of microtubule function.

DNA damage in leukocytes and buccal and nasal epithelial cells of individuals exposed to air pollution in Mexico City.

There is an increased interest in using biological markers to monitor individuals for possible exposure to environmental toxicants. Test systems which permit the sensitive detection of DNA damage and DNA repair are critically important in such studies. The single cell gel electrophoresis (SCG) assay is a rapid and a sensitive method for the evaluation of DNA damage at the single cell level, providing information on the occurrence of DNA single-strand breaks and alkali labile sites using alkaline conditions. In this study, the differences in the basal level of DNA damage between young adults from the south (exposed principally to high levels of ozone) and young adults from the north (exposed principally to hydrocarbons and particles) of Mexico City were investigated by the SCG assay using three different cell types (leukocytes and nasal and buccal epithelial cells). We found an increased DNA migration in blood leukocytes and nasal cells from individuals who live in the southern part of the city compared to those living in the northern part; however, no differences were observed for buccal epithelial cells. These results show the feasability of using the SCG assay to evaluate DNA damage in different tissues and its great potential for use in the monitoring of humans potentially exposed to genotoxic pollutants.

Altered profile of urinary arsenic metabolites in adults with chronic arsenicism. A pilot study.

Relationships between alterations in the profile of urinary arsenic (As) species and the presence of cutaneous signs of arsenicism were studied in Region Lagunera, Mexico. The use of urinary concentrations of putative substrates and products of the As metabolism pathway, as indicators of metabolic efficiency is also discussed. Arsenic was determined by hydride generation atomic absorption spectrophotometry and separation of As species was performed by ion exchange chromatography. The exposed group had an average of 0.408 mg As/l of total As (TAs) in their drinking water, whereas "control' individuals had 0.031 mg/l. Urinary concentrations of arsenic species and TAs were 20 to 95 times higher in the exposed group. Significant increases in the relative proportions of inorganic arsenic (Asi) and monomethylarsonic acid (MMA), accompanied by decreases of dimethylarsinic acid (DMA) were also found in exposed individuals. Therefore, significant decreases in the value of the MMA/Asi, DMA/MMA and DMA/ Asi ratios were observed, suggesting a decreased As methylating ability. Exposed individuals bearing cutaneous signs had a significantly longer time of exposure, higher urinary concentrations and proportions of MMA and MMA/Asi values, and significantly lower DMA/ MMA than exposed individuals without cutaneous signs. Further research is needed to identify better parameters for assessing the efficiency of As metabolism in chronically exposed populations and to confirm the potential relationship between metabolic alterations and overt signs of As toxicity.

Induction of p53 protein expression by sodium arsenite.

Arsenic is carcinogen for humans and has been shown to act as an enhancer in initiated animal models. In a previous work we found impairment of lymphocyte proliferation in arsenic-exposed individuals and in vitro we obtained dose-related inhibition of mitotic response and lymphocyte proliferation. Intrigued by these effects and based on the role of p53 on cell proliferation, we tested different concentrations of sodium arsenite for their ability to induce the expression of tumor suppressor gene p53 in different cell lines (HeLa, C-33A. Jurkat) and a lymphoblast cell line transformed with Epstein-Barr virus (LCL-EBV). We also evaluated changes in their viability after 24 h arsenic treatment; C-33A cells showed the higher sensitivity to arsenic treatment while HeLa, Jurkat and LCL-EBV cells showed similar cytotoxicity curves. Immunoblots showed an increased expression of p53 gene with 1 microM sodium arsenite in Jurkat cells and 10 microM sodium arsenite in HeLa and LCL-EBV cells. In addition, we transfected Jurkat cells and human lymphocytes with wild-type and mutated p53 genes; lymphocytes and Jurkat cells that received the mutated p53 showed increased sensitivity to arsenic cytotoxicity. Data obtained indicate that arsenic induces p53 expression and that cells with a functional p53 contend better with damage induced by this metalloid.

Effect of hydroxyurea and normal plasma on DNA synthesis in lymphocytes from Fanconi anemia patients.

Fanconi anemia (FA) is characterized at the cellular level by a high frequency of spontaneous chromosomal aberrations; crosslinking agents cause an abnormal increase in the frequency of chromosomal damage, and semiconservative DNA synthesis is severely inhibited. Deoxyribonucleotides are needed in both semiconservative and repair DNA synthesis. To investigate the involvement of deoxyribonucleotide pools in the inhibition of DNA synthesis in FA, we evaluated the effect on FA lymphocytes of hydroxyurea (HU), an inhibitor of ribonucleotide reductase which is known to alter the intracellular levels of deoxyribonucleotides. To achieve this goal, lymphocyte cultures of 4 FA patients and 4 normal individuals were used. Cultures were treated with HU and/or mitomycin C and normal human plasma. All cultures were processed to detect the number of DNA synthesizing nuclei by autoradiography. Scoring of 2000 nuclei for each kind of culture every 6 h in the last 24 h of incubation showed that, in long incubation periods, DNA synthesis in FA is largely inhibited by HU and this hypersensitivity may be partially decreased by addition of normal human plasma. It is known that recovery from damage induced by HU involves several enzymes such as flavin oxido-reductase, superoxide dismutase and catalase which are involved in the production or scavenging of O2 radicals; FA cells are deficient in the detoxification of oxygen and this could explain the response of FA cells to HU.

Genotoxic effects of metronidazole.

Metronidazole (MTZ) is an effective agent used in the treatment of parasitic infections. Its genotoxic effects have been shown in a variety of prokaryotic systems; however, negative results have been reported in human in vivo studies. Due to its wide spread use, a study was performed to evaluate the chromosomal aberration frequencies in peripheral blood lymphocyte cultures from 10 individuals, before and after metronidazole treatment. A significant increase in the percentage of cells with chromatid and isochromatid breaks was observed after metronidazole treatment (1500 mg per day for 10 days). The percentages of cells with aberrations did not correlate with the levels of MTZ found in plasma. Individual variability was observed with respect to both the induction of aberrations and the concentration of MTZ in plasma. They could represent differences at the metabolic level, since metronidazole is known to be biotransformed by a polymorphic P450 cytochrome, and its metabolites have shown mutagenic activity.

DNA damage in exfoliated buccal cells of smokers assessed by the single cell gel electrophoresis assay.

The alkaline single-cell gel electrophoresis assay or comet assay is a sensitive and rapid method for DNA strand breaks and detection of alkali labile sites at the single cell level, it further provides information on the presence of damage among individual cells. In this paper we explore the use of this technique utilizing exfoliated buccal mucosa cells from non-smokers (9 donors) and smokers (11 donors). The extent of DNA image length was found to be significantly increased in the smoker group (89.30 +/- 16.18 microns) than in the non-smoker group (52.01 +/- 10.43 microns). Our results indicate that the single-cell gel electrophoresis assay could be applied to human monitoring using exfoliated buccal epithelial cells.