Mutagenesis Advance Access originally published online on September 13, 2006
Mutagenesis 2006 21(5):335-342; doi:10.1093/mutage/gel040
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Increased cytogenetic damage in a zone in transition from agricultural to industrial use: comprehensive analysis of the micronucleus test in peripheral blood lymphocytes
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México México city, DF, México 1 Centro Fray Julián Garcés, Derechos Humanos y Desarrollo Local AC Tlaxcala, México
A complex situation of chemical exposure has been described in México in a zone that is in transition from rural activities towards intensive industrialization, which has brought environmental pollution: chloroform, methylene chloride, indigo and toluene are some of the pollutants found in the Atoyac and Xochiac rivers. A biomonitoring study was planned in order to establish whether there was a biological effect due to the environmental situation. Communities where leukaemia and thrombocytopenic purpura cases have been reported were included in the study, as well as other communities where such cases have not been described. Three occupations were distinguished, according to chemical exposure: industrial workers, agricultural workers and workers in households, education and commerce. A comprehensive analysis in the micronucleus (MN) test was used to study genotoxicity biomarkers. Two metabolic polymorphisms were determined, namely, glutathione transferase mu1 (GSTM1) and theta1 (GSTT1), which are relevant when oxidative responses are involved. Increased genotoxic damage was found, including cells with >1 MN, >1 chromatin bud, and nucleoplasmic bridges. The genotoxic damage was differentially distributed in the regions studied, being more affected those that are closer to the Atoyac and Xochiac rivers, indicating an effect due to environmental exposure to the contaminants present in the rivers. Further characterization of the exposure regimes in these communities will be done in order to contribute to the alleviation of the health risks that environmental pollution is posing on the inhabitants of this area.
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Introduction |
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A complex situation of chemical exposure was detected in a zone in the México state of Tlaxcala, which is in transition from rural activities towards intensive industrialization, where demands on water use have risen and where deterioration of the resource due to chemical pollution might be posing a health hazard to several communities. Chloroform, methylene chloride, indigo, aniline and toluene (1
) are some of the pollutants found in the Atoyac and Xochiac rivers, mixed with other contaminants such as oils and grease in addition to organic matter originating from households (Table I); the main concern for the presence of these contaminants in the rivers is that they enter into the air, reaching the inhabited areas close to the river or through the drains, from the sewage systems. Complaints about odours and irritating air affecting eyes, throat and causing headaches, are a constant among the people living in the communities; in fact, very intensive and irritant emanations are produced at places where the industrial discharges reach the rivers and come into contact with the air; this happens at several points along the Atoyac and Xochiac rivers (Figure 1).
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Simultaneously, the same people living in these towns work in the industries of the zone, including a petrochemical plant and numerous blue denim manufacturers. Working conditions might also contribute to the health hazards, particularly in the textile industry, where workers are exposed to cotton dust, heat and the vapours of numerous toxic agents, without adequate protective equipment or adequate ventilation; they are in contact with chlorine bleachers, sodium hydroxide, indigo and aniline. Some of these are clastogenic, while aniline, has been related with haemolytic anaemia (2
–6) (ATSDR-ToxFAQsTM, http://www.atsdr.cdc.gov/tfacts174.html; ATSDR-MMG, http://www.atsdr.cdc.gov/MHMI/mmg171.html). Working conditions in the petrochemical plant are different: workers are provided with protective equipment and have medical attention; however, toxic chemicals are handled in the plant and the workers complain of vapours and toxins causing occasional discomfort; at present, methanol is produced, whereas toluene and benzene (IARC group 1 carcinogen) (7–9) (ATSDR-ToxFAQsTM, http://www.atsdr.cdc.gov/tfacts3.html; NIOSH, http://www.cdc.gov/niosh/npg/npgd0397.html) are also handled in the plant, as reported by the workers. Finally, agriculturers in the fields use pesticides, the most hazardous of which are paraquat and 2,4-D, both clastogenic and the first one considered as a group 1 carcinogen (IARC classification) (10,11) (CDC Chemical emergencies, http://www.bt.cdc.gov/agent/paraquat/basics/facts.asp; EPA, http://www.epa.gov/safewater/dwh/c-soc/24-d.html). Use of these pesticides is seasonal, once or twice a year, and workers protect themselves with gloves, boots, but not masks. Also, these workers are exposed to the sun and to the watering channels that carry water from the rivers Atoyac and Xochiac. Given this scenario, a biomonitoring study was planned as a first approach to the problem in order to establish whether any detectable biological effects due to the situation described could be found. From among the persons who answered a questionnaire and signed a letter agreeing to participate, a selection was made of workers: Group (1) teachers, students, housekeepers or employed in commercial activities, Group (2) workers in agriculture, and Group (3) workers in the industries. Communities where leukaemia and thromobocytopenic purpura cases have been reported were included in the study, as well as other communities where such cases had not been described; the former ones are towns close to the rivers (Regions 1 and 2, Figure 1) and the latter are at a greater distance (Region 3). Our assumption was that people not occupationally exposed to chemicals should have a lower frequency of genotoxic damage; however, as there was a possibility that non-occupationally exposed people could also have increased genotoxic damage due to the environment, an additional group was included in the study, consisting of residents in México City, of similar socio-economic level, mainly students and workers in the University, comparable to occupational Group 1; they constitute the reference group.
The micronucleus (MN; abbreviation also used for micronuclei) test was used to study genotoxicity biomarkers. This test is widely used in biomonitoring, since the information that can be obtained from it is related to mechanisms of action as well as to important steps in the carcinogenic process. The primary damage in genotoxicity is chromosome breakage, which is readily analyzed in the form of MN. Alternatively, a MN may be formed from missing chromosomes during anaphase, due to alterations in the mitotic spindle or in the kinetochore; this event leads to cells lacking or having additional chromosomes of any given pair (12). Secondary damage due to clastogenicity could be gene amplification, chromosome translocation, inversions and the induction of apoptosis (13). Theoretically, all of these can be analyzed in the form of chromatin or nuclear buds (CHB), nucleoplasmic bridges (NPB) and apoptotic figures (AF). In particular, nucleoplasmic bridges are of interest since they have been related with the loss of genomic stability (14). Two metabolic polymorphisms were also studied, in order to determine whether this population would be more susceptible to DNA damage due to a genetic predisposition; we chose two enzymes of the glutathione-S-transferase family which have been described as relevant in the metabolism of volatile organic compounds, and aromatic and polycyclic aromatic hydrocarbons (PAH) (15,16), namely, glutathione transferase mu1 (GSTM1) and theta1 (GSTT1).
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Material and methods |
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Selection of donors for biological testing
Donors were selected from people who responded to a questionnaire that was applied in nine communities, and who had signed a letter consenting to participate after receiving information about the objectives of the study. The questionnaires were studied to make a selection of the people that could participate in a biomonitoring study of genotoxic damage in peripheral lymphocytes. Inclusion criteria were: age range from 19 to 80 years old; they should not have diabetes, cancer or a parasitic infection, or have familial antecedents with cancer. Three categories of occupation were selected: housewives, students, teachers and employees in the commerce, who were considered our controls (Group 1); agricultural workers (Group 2); workers occupationally exposed to toxic chemicals in industries (Group 3) such as the petrochemical plant, in the blue denim laundries, and other industries producing adhesives and paints, and automotive breaks. A personal message was sent to each selected donor, inviting them to donate a blood sample; place and hour of sampling was specified and only those who attended were included in the study. 105 persons agreed to donate a blood sample. The nine communities are distributed in an area of 50 ha in the middle of which the Atoyac river flows (Figure 1). The samples obtained were also grouped according to three regions inside this zone: Region 1 was constituted by the towns situated at the banks of the Atoyac river where the petrochemical plant discharges into the environment. Region 2 is down the course of this river, after it flows through a second discharge by a blue denim laundry industry; two of the communities of this Region 2 are proximal to three discharges by the petrochemical plant and other blue denim laundries (Xalmimilulco and Moyotzingo) and the third community is located down the confluence of the rivers Atoyac and Xochiac (Michac) (Figure 1). Region 3 was constituted by two towns where neither of these rivers flows through and their sewage systems are not connected to the same system as the other communities are (Figure 1). As already stated, an additional control group was included; samples from donors living in México City were used, selected under the same criteria for the individuals in the area. With respect to ethnicity, the donors constitute a homogeneous group of Mestizos, including the reference group. All of them were born in México, as well as their parents, grandparents and great-grandparents (we did not inquire about earlier generations).
Blood sampling
Blood samples were collected with Sarstedt (México) syringes, with heparin for lymphocyte cultures and with EDTA for DNA extraction. Codes were assigned beforehand to each set of syringes and culture tubes in order to process the samples blindly. The same was done with the cryotubes. One person took note of the sample identification and kept the codes. Samples with heparin were maintained in a cool environment during the transportation to the laboratory to be processed; these samples were of 3 ml vol. and were used for lymphocyte cultures. Samples with EDTA were placed in contact with a cooling cushion. Transportation was done by car and took 3–6 h between sampling and processing. EDTA samples were separated in two aliquots and frozen in liquid nitrogen on arrival at the laboratory; these samples were of 3 ml vol. and were used for DNA extraction. Sampling was done in three visits to the zone; each time, blood samples of donors living in México City were taken and processed; these samples were coded and analysed blindly with the other samples.
Lymphocyte cultures
Three whole blood cultures per donor were done for MN evaluation with a duration of 48 h. Bromodeoxyiuridine (BrdU; Sigma, México) incorporation was used to label cells that proliferated in culture, according to the immunohistochemical method reported elsewhere (17). A label index was estimated as a control of proliferation of cultures; it was estimated as the number of immunostained cells in 2000 cells counted in slides stained with anti-BrdU-DAB and counterstained with Giemsa. A comprehensive analysis of cytogenetic damage was done, that included the analysis of MN, as well as CHB and NPB; the number of cells with >1 MN or >1 CHB were registered separately. In addition, AF which are a measure of cell toxicity (18), were also evaluated (Figure 2). Two cultures per donor were evaluated and 2000 cells per culture were counted. Frequencies for each biomarker were calculated according to the following formula:
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DNA extraction
A BIO-RAD AquaPure Genomic DNA Blood kit was used to extract DNA from each frozen blood sample and the method was followed according to the manufacturer's instructions, including RNase treatment.
GSTM1 polymorphism
The method described by Hirvonen et al. (19) was used to determine the presence or absence of the GSTM1 gene. A PCR was conducted with primers amplifying the exons 4 and 5 region of the GSTM1 gene, along with a specific primer to amplify a sequence of the closely related gene, GSTM4, which is always present. Details are given elsewhere (20). Bands of 231 and 158 bp appeared in an agarose gel (1%) when the GSTM1 gene was present; a band of 231 bp was missing when the GSTM1 gene was absent.
GSTT1 polymorphism
GSTT1 amplification was performed according to Pemble et al. (21) and co-amplified with a beta-globin gene sequence taken from Bell and Pitman (22). Primers used were: GSTT1 forward primer: 5'-ttc ctt act ggt cct cac atc tc; GSTT1 reverse primer: 5'-tca ccg gat cat ggc cag ca. Beta-globin forward: 5'-caa ctt cat cca cgt tca cc and Beta-globin reverse primer: 5'-gaa gag cca agg aca ggt ac. PCR was conducted according to standard conditions and genotypes were determined by the presence or the absence of a 480 bp band in a 1% agarose gel; beta-globin appeared at 270 bp size.
Statistical analysis
Program Microsoft Excel was used to organize data. Program Stata 7.0 was used for statistical analyses. Normality tests revealed that data did not distribute normally; so a logN transformation was made to perform a stepwise multivariate regression analysis with the whole dataset, including the referents outside the zone of study; backward elimination and a P-level of 
0.05 were used to retain variables in the model. The variables included were: regions studied (four categories explained in the Introduction); socio-economical activity (four categories also explained in the Introduction); gender, smoking, drinking, GSTM1 and GSTT1 polymorphisms (two categories each); age and body mass index (BMI; as continuous variables). Interactions suggested by the results with each genotoxicity parameter were studied in new models.
The whole data set was also used to analyze differences due to the GSTM1 and GSTT1 polymorphisms, with the one-way ANOVA test.
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Results |
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126 individuals were studied, aged 19–80 years old. The variables considered for the analyses are summarized in Table II; means, standard deviations and range refer to the donors of the area and the same data for the referents are presented in the lower part of the table. These variables were used in the stepwise multivariate regression analysis to find the predictors for each of the biomarkers evaluated in the MN test. The results of this analysis are presented in Table III. Three of these Table III analyses were performed: on data of referents alone, on the data of donors in the zone of study and both together. Region and socio-economical activity were not used in the analysis of the referent group, since they were constant. According to this analysis, no predictor variables were found in the referent group; conversely, in the group living in the zone, cells with >1 MN were significantly higher in women and in smokers. The total frequency of MN in the zone was higher in persons with a low BMI and with the GSTT1-null genotype. Cells with >1 CHB were more frequent among non-smokers and men, whereas NPB were increased among men living in Region 2 and non-smokers.
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Predictor variables when the whole dataset was used, behaved slightly differently (Table III); cells with 1 MN were more frequent in Regions 1 and 2 and less frequent in the reference group; the same was true for cells with >1 MN that were found more frequently among donors of Regions 1 and 2. The frequency of MN showed a relationship with the GSTT1-null genotype who had higher frequencies of cells with 1 MN and total MN. Total frequency of MN was also found to be related with a lower BMI and with increasing age. Cells with 1 CHB were more frequent among persons living in Regions 1 and 2, whereas the total frequency of CHB was higher in persons who do not drink. Finally, NPB were predicted by gender, being higher among men who live in Region 2 and among non-smokers.
No predictor variables were found for apoptotic figures (Table III). This parameter is related with cytotoxicity and together with necrosis and a proliferation index it could give information about an additional toxic effect of contaminants. Given the conditions of our cultures, however, we were not able to detect this kind of effect, since no variation in the amount of apoptosis was found, and necrotic cells were not observed, probably due to the hypotonic treatment used in the harvest; furthermore, the proliferation of cultures was in the normal range of 85–95% label index.
The means, standard deviations and range of values of each biomarker, according to regions and activities are shown in Table IV.
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Polymorphisms and genotoxicity
The GSTM1-null frequency was 45%, whereas GSTT1-null was 9.5%. No effects on biomarkers frequencies were observed due to GSTM1 polymorphism, whereas GSTT1-null showed a higher frequency of cells with 1 MN, total frequency of MN and NPB (Table V).
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Associations between the cytogenetic parameters
Pearson correlation was applied to analyse how each parameter related to the others and it was found that cells with 1 MN correlated well with the total number of MN, with cells with 1 CHB and total number of CHB. Finally cells with >1 CHB correlated with apoptotic figures and with NPB. Necrotic cells were not observed; the lack of this type of cell could be due to the fact that hypotonic treatment was used to harvest the whole blood cultures and it is recognized that this treatment might remove necrotic cells together with red blood cells (23
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Discussion |
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Genotoxic damage was found which was not associated with a probable occupational exposure to chemicals, but was better associated with the different regions of the area of study. The greatest number of leukaemia cases have been reported in Villalta, San Baltazar and Sta María, the Region 1. In Sta. Ana, the main health problem reported is asthma; this town is located in Region 2 (Figure 1) (24
). Regions 1 and 2 were the most affected with MN and CHB, and in Region 2 the individuals showed the highest frequencies of NPB. This biomarker constitutes a complex damage, involving a cycle of chromosome breakage, fusion of the extremes and bridge formation during anaphase; it was validated as a biomarker of DNA damage in human WIL2-NS cells treated with hydrogen peroxide, superoxide or after co-incubation with activated human neutrophils (25) and they were related with the production of MN in cells where chromosome breakage occurred with the formation of rearrangement leading to the NPB, and acentric fragments leading to MN (13). The present study is a first report of this damage occurring in vivo in peripheral lymphocytes of clinically healthy individuals.
CHB, on the other hand, have been associated with the expulsion of amplified DNA from the cells (26). This process has been observed in cultures grown under selective conditions (27–29) which induce gene amplification. Shimizu et al. (26,30) showed that amplified DNA is localized selectively to specific sites at the periphery of the nucleus and eliminated via nuclear budding to form MN during the S phase of the cell cycle. Amplification of genes occurs in response to drugs leading to resistance and has been widely documented in transformed cell lines; furthermore, a chronic exposure to toxic chemicals could also lead to the amplification of specific genes related with their metabolism (31). In vitro studies conducted in our laboratory demonstrated an increase of these structures in normal peripheral lymphocytes treated with colcemid and mitomycin-c in the short period of a 24 h treatment (17). Hence the increased amount of CHB and cells with >1 CHB found in our subjects, suggests that this mechanism of cell survival has been induced. The agents presumed to be responsible for the damage found have not been studied for CHB or NPB production, although clastogenic compounds are supposed to induce this effect; according to this, toluene could be causing this effect, even though chloroform and methylene chloride could contribute to the damage found, by inducing an oxidative response (32).
Cells with >1 MN constitute another biomarker of interest, since they were not found in the reference group. They showed a higher frequency in Regions 1 and 2, even though a frequency was also found in Region 3. Aneuploidogenic agents may induce cells with >1 MN, by altering the normal segregation of chromosomes into daughter cells and some pesticides have been recognized as aneugens; however, aneugens would also induce multinucleation and arrest proliferating cells in metaphase and these events were not observed. Furthermore, even the donors non-occupationally exposed to pesticides showed cells with >1 MN; clastogenic agents, on the other hand, may also cause multiple chromosome breakage that may be observed as multiple, small MN. Further studies, more specifically directed at evaluating whether aneugens or clastogens are responsible for the effect, would elucidate the origin of these multi-micronucleated cells.
The greater frequency of damage was found in Regions 1 and 2, which are close to the Atoyac river where industrial discharges occur and to the Xochiac river, where discharges from the denim laundries take place (Figure 1); the two polluted rivers form a semicircle that surrounds the communities studied. The least affected, San Francisco Tepeyacac and Sta Justina Ecatepec (Region 3), are more distant to these rivers. Hence, considering all the former results, we arrived at the conclusion that the damage found is due to pollutants carried by the rivers as they pass across the zone of study, affecting people who have an occupational exposure in the same way as they affect people that are not occupationally exposed to toxic chemicals. For this reason, we are planning to do new studies oriented at establishing the health risks for youngsters who were born when the environmental decay started, after 1991. It should be said that the total amount of MN was the only biomarker showing an increase with age with an R value of 0.3; this value refers to all the donors including those from México City.
The inclusion of the data from donors living outside this area, was useful in the sense that it allowed us to identify increased frequencies of MN and CHB in addition to the extraordinary frequency with which NPB are found in the zone; in spite of the fact that our reference donors were exposed to the urban pollution, and spend 2–4 h inside gasoline powered public transportation daily, they showed a low level of this kind of damage. These donors share a similar socio-economical status with the donors in Tlaxcala, except for dietary habits, which probably reflected on the BMI showing values in the normal range as compared to the donors of the zone where excessive weight and obesity were more frequent.
With respect to diet, a possibility exists that, in addition to the environmental pollution in the area, a factor contributing to the amount of damage found could be a deficiency in folate and vitamin B12 in the diet of the persons studied due to a low consumption of meat and of food supplements; additionally, the consumption of raw fruit and vegetables is low as was established from the questionnaire applied previous to the present biological study. A new biomonitoring study is being planned among school students in order to better characterize this factor as a health risk.
Another parameter studied was the individual susceptibility to chemical exposure due to the polymorphisms of enzymes GSTM1 and GSTT1. GSTM1-null polymorphism has been found to increase the frequency of chromosome aberrations after tobacco-specific N-nitrosamine exposure in vitro (33). Many studies have shown that this deletion increases the susceptibility conferred by the CYP1A1*2C allele for PAH exposure-associated cancer (16,19,34). It was found that GSTM1-null was present with a frequency of 45% in this study; Coughlin and Hall (35), in a review of this polymorphism, found that the GSTM1-null frequency ranges from 23 to 48% in African populations, 33 to 63% in Asian populations, and 39 to 62% among Europeans. No increased susceptibility to genotoxic damage was found in relation to this polymorphism in the present study, in spite of the fact that an oxidative effect was expected due to the exposure to volatile organic compounds (VOCs) as has been documented in relation to benzene exposure (36), which should have shown as increased damage in the null-genotyped individuals.
The activity of the GSTT1 enzyme has been found to be related with the activation of exogenous procarcinogens, such as small halogenated compounds and to contribute to the inflammatory response of oesophageal mucosa, which is a strong risk factor for adenocarcinoma in the respiratory tract, possibly through leukotriene synthesis. Furthermore, GSTT1 has been demonstrated to participate in the activation of trihalomethanes (like chloroform), constituting the via of activation of this kind of compounds (15,37). The GSTT1-null genotype showed a frequency of 9.5% among our donors, lower than the one reported in Asiatic populations (38), ranging from 20 to 53% and lower than reported in caucasian populations ranging from 16 to 22% (39); this frequency is in agreement to the one reported by Nelson et al. (40) for a group of Mexican-Americans, of 9.7%. The presence of the enzyme in the individuals studied would signify that they are susceptible to the effects of trihalomethane exposure, such as chloroform and, probably, methylene chloride; however, the GSTT1-null polymorphism significantly correlated with increased MN and NPB frequencies, even though only 10 individuals carried this genotype.
In conclusion, the amount and the kind of genotoxic damage found in the donors of the communities indicate the action of toxic agents in their cells and the evidence points to the environment as the most probable source of that exposure. Further characterization of that contamination in the air will be done in future studies. The mixture found in the rivers Atoyac and Xochiac is not normed in the Mexican law (41,42) (Leyes y Normas, http://portal.semarnat.gob.mx/semarnat/portal) which does not contemplate the presence of chloroform, methylene chloride, toluene, indigo and aniline either in discharges to national waters or in discharges to sewage; the VOCs mentioned are not contemplated either in the norm for air pollutants, where these agents might reach the most important concentration in the environment, and in the case of chloroform, its breakdown products too, such as phosgene and hydrogen chloride (43) (ATSDR-ToxFAQsTM, http://www.atsdr.cdc.gov/tfacts6.html). Although exposure to these chemicals in the occupational environment has been regulated (44) (Marco Jurídico, http://www.stps.gob.mx/) at levels of 10–100 p.p.m. (except indigo which is not included in the norm), the potential exposure in the zone is chronic, with variations according to the frequency and volumes of the discharges, but permanent, leading to a high frequency of complex genotoxic damage in populations with the characteristics of the one studied in the present investigation. Given the tendency for modifying the use of land in natural areas with water, and given the fact that México is a country with very limited resources of freshwater, the situation encountered in Tlaxcala should be a warning of what might happen in other areas where the same transformations are taking place. Determinations in air will contribute to the characterization of the regime of exposure for the inhabitants of this area in order to contribute to the control of pollutants and to the rescue of this environment.
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Acknowledgments |
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The authors thank the collaboration of Javier Belmont for technical support. This investigation was supported by a grant FANCA-Centro Fray Julián Garcés, Derechos Humanos y Desarrollo Local A.C., by collaborative agreement no. BM-137, and by the grant no. IN220506 by PAPIIT-UNAM.
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*To whom correspondence should be addressed at: Circuito Mario de la Cueva, P.O. Box 70228, C.P. 04510, México City, DF, México. Tel: +5255 5622 9175; Fax: +5255 5622 9182; Email: dorinda{at}servidor.unam.mx
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Received on June 1, 2006; revised on August 3, 2006; accepted on August 4, 2006.
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