RESUMEN

The karyotypes of four species of freshwater triclads of the genus Girardia (Platyhelminthes), i.e. G. schubarti, G. tigrina, G. anderlani, and G. biapertura, from populations of different localities of the Rio Grande do Sul State, in southern Brazil, were analyzed. The karyotype of G. biapertura is presented for the first time. Three basic complements of 4, 8, and 9 chromosomes were found. Diploids, triploids, or mixoploids (2n/3n) specimens were frequently detected in these populations. The basic chromosomal complement of n = 9 was verified in two different species (G. biapertura and G. anderlani), presenting a large acrocentric chromosome which is rare in the family Dugesiidae. An intra and interspecific chromosomal variability was also detected and its evolutionary implications are discussed.

Os cariótipos de quatro espécies de planárias de água doce do gênero Girardia (Platyhelminthes), a saber, G. schubarti, G. tigrina, G. anderlani e G. biapertura, de populações ocorrentes em diferentes locais do estado do Rio Grande do Sul, na região sul do Brasil, foram analisados. O cariótipo de G. biapertura é apresentado pela primeira vez. Foram observados três complementos básicos, de 4, 8 e 9 cromossomos. Espécimes diplóides, triplóides e mixoplóides (2n/3n) foram observados freqüentemente nessas populações. O complemento cromossômico básico de n = 9 foi verificado em duas espécies (G. biapertura e G. anderlani), apresentando um grande cromossomo acrocêntrico que é raro na família Dugesiidae. Também foi observada certa variabilidade cromossômica, tanto intra- como interespecífica, cujas implicações evolutivas são discutidas.

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RESUMEN

The karyotypes of four species of freshwater triclads of the genus Girardia (Platyhelminthes), i.e. G. schubarti, G. tigrina, G. anderlani, and G. biapertura, from populations of different localities of the Rio Grande do Sul State, in southern Brazil, were analyzed. The karyotype of G. biapertura is presented for the first time. Three basic complements of 4, 8, and 9 chromosomes were found. Diploids, triploids, or mixoploids (2n/3n) specimens were frequently detected in these populations. The basic chromosomal complement of n = 9 was verified in two different species (G. biapertura and G. anderlani), presenting a large acrocentric chromosome which is rare in the family Dugesiidae. An intra and interspecific chromosomal variability was also detected and its evolutionary implications are discussed.

Os cariótipos de quatro espécies de planárias de água doce do gênero Girardia (Platyhelminthes), a saber, G. schubarti, G. tigrina, G. anderlani e G. biapertura, de populações ocorrentes em diferentes locais do estado do Rio Grande do Sul, na região sul do Brasil, foram analisados. O cariótipo de G. biapertura é apresentado pela primeira vez. Foram observados três complementos básicos, de 4, 8 e 9 cromossomos. Espécimes diplóides, triplóides e mixoplóides (2n/3n) foram observados freqüentemente nessas populações. O complemento cromossômico básico de n = 9 foi verificado em duas espécies (G. biapertura e G. anderlani), apresentando um grande cromossomo acrocêntrico que é raro na família Dugesiidae. Também foi observada certa variabilidade cromossômica, tanto intra- como interespecífica, cujas implicações evolutivas são discutidas.

RESUMEN

The karyotypes of four species of freshwater triclads of the genus Girardia (Platyhelminthes), i.e. G. schubarti, G. tigrina, G. anderlani, and G. biapertura, from populations of different localities of the Rio Grande do Sul State, in southern Brazil, were analyzed. The karyotype of G. biapertura is presented for the first time. Three basic complements of 4, 8, and 9 chromosomes were found. Diploids, triploids, or mixoploids (2n/3n) specimens were frequently detected in these populations. The basic chromosomal complement of n=9 was verified in two different species (G. biapertura and G. anderlani), presenting a large acrocentric chromosome which is rare in the family Dugesiidae. An intra and interspecific chromosomal variability was also detected and its evolutionary implications are discussed.

Asunto(s)

RESUMEN

The karyotypes of four species of freshwater triclads of the genus Girardia (Platyhelminthes), i.e. G. schubarti, G. tigrina, G. anderlani, and G. biapertura, from populations of different localities of the Rio Grande do Sul State, in southern Brazil, were analyzed. The karyotype of G. biapertura is presented for the first time. Three basic complements of 4, 8, and 9 chromosomes were found. Diploids, triploids, or mixoploids (2n/3n) specimens were frequently detected in these populations. The basic chromosomal complement of n = 9 was verified in two different species (G. biapertura and G. anderlani), presenting a large acrocentric chromosome which is rare in the family Dugesiidae. An intra and interspecific chromosomal variability was also detected and its evolutionary implications are discussed.

Os cariótipos de quatro espécies de planárias de água doce do gênero Girardia (Platyhelminthes), a saber, G. schubarti, G. tigrina, G. anderlani e G. biapertura, de populações ocorrentes em diferentes locais do estado do Rio Grande do Sul, na região sul do Brasil, foram analisados. O cariótipo de G. biapertura é apresentado pela primeira vez. Foram observados três complementos básicos, de 4, 8 e 9 cromossomos. Espécimes diplóides, triplóides e mixoplóides (2n/3n) foram observados freqüentemente nessas populações. O complemento cromossômico básico de n = 9 foi verificado em duas espécies (G. biapertura e G. anderlani), apresentando um grande cromossomo acrocêntrico que é raro na família Dugesiidae. Também foi observada certa variabilidade cromossômica, tanto intra- como interespecífica, cujas implicações evolutivas são discutidas.

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In this study, the micronuclei test (MNT) was applied in exfoliated cells of buccal mucosa, in order to evaluate the genotoxic risk associated with occupational exposure of mechanics, storage battery renovation workers, and car painters. For each individual, 3000 exfoliated buccal cells were analyzed. There was a significantly higher frequency of micronucleated cells (MNC) in the exposed workers than in controls. Smoking and drinking habits, age, and working time did not represent significant factors in terms of increasing the production of micronuclei (MN), when the control and the exposed groups were compared. These results allowed to conclude that the studied individuals belong to a risk group and should periodically undergo biological monitoring and proper care

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Flavonoids are phenolic compounds, naturally found in vegetables, tea and red wines. A recent study has demonstrated that the flavonoids rutin and quercetin show a protective role against the deleterious effects of free radicals in cirrhotic rats. Considering this finding and the controversial results concerning the mutagenicity of rutin and quercetin recorded in the literature, the capacity of these flavonoids to cause damage to the DNA was evaluated using the alkaline single-cell gel electrophoresis (SCG) and micronucleus test in the bone marrow of mice. The doses for both compounds were 2 x 2500, 2 x 1250 and 2 x 625 mg/kg. Micronucleus test showed that rutin caused no damage to the DNA of the mice bone marrow cells, and the SCG assay demonstrated an increase of damage only at the dose of 2 x 1250 mg/kg. But when the mice cells of the three quercetin doses were compared with the negative control, significantly higher damage was observed by SCG assay, although not proportional to the dose. The micronucleus test also demonstrated a significant increase of damage, but only at the 2 x 1250 mg/kg dose. Considering the results obtained in this study with very high doses, it is unlikely that the consumption of rutin and quercetin produces any clastogenic effects. Our results also indicated that SCG could profitably be used in drug genotoxicity evaluation protocols.

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The alkaline single-cell gel electrophoresis, or comet assay, was used to evaluate the genotoxic potential of copper sulphate in planarians. Concentration-related increase in DNA damage was induced after 2h and 7 days exposure to CuSO4 dissolved in culture water. To study the influence of copper ions on the persistence of mutagen-induced DNA lesions, planarians were treated with methyl methanesulphonate (MMS), and further incubated in the absence (post-incubation) or presence (post-treatment) of CuSO4. After 2h of post-treatment enhanced persistence of DNA effects in relation to the corresponding post-incubation value was detected, which indicate inhibition of DNA repair by CuSO4. At 4h an increase of DNA migration in relation to the 2h value was observed, which is significant for the post-incubation group. After 24h, DNA damage decreased but was still significantly elevated in relation to the control. From our results, we conclude that planarians are suitable organisms for in vivo detection of copper genotoxicity in the comet assay, and can be used to assess both acute and chronic exposure to this chemical in aquatic ecosystems. The inhibition effect of copper ions on repair of MMS-induced DNA damage suggests that copper could modulate the genotoxic effects associated with complex mixture exposure in the environment.

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Hexavalent chromium (Cr [VI]) genotoxicity was studied using fish micronucleus analysis in peripheral blood erythrocytes from Pimephales promelas, the fathead minnow. Forty-five- to 60-d-old fish were used to assess the spontaneous level of genotoxic damage. The genotoxic effect of Cr (VI) obtained from potassium dichromate (K2Cr2O7) in tests performed for 7-, 14-, and 21-d exposure periods was estimated. Significant micronucleated erythrocyte (MNE) induction was detected in fish exposed for 7 d to 2.5 mg/L of Cr (VI), and induction decreased after 21 d of exposure. The results suggest a handling effect in fish manipulated compared to those not manipulated, thus demonstrating the importance of including parallel negative controls in experimental design. Basal levels of MNE are reported, providing laboratory values for future assay quality control. The importance of determining the period with the highest expression of the genotoxic effects in this assay system was also confirmed.

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Cytokinesis-block micronucleus (CB-MN) assay and single-cell gel electrophoresis (SCGE) were employed to analyze leukocytes from 14 Fanconi anemia (FA) patients, 30 Down syndrome (DS) patients, and 30 control individuals, to examine the sensitivity of these techniques to detect genomic instability in these 2 diseases. The DS patients presented increased DNA damage as measured by SCGE in relation to controls. The frequencies of micronuclei and dicentric bridges were similar to those of controls. Micronucleus frequency, dicentric bridge frequencies, and DNA damage were higher in FA patients than in controls. The high frequency of micronuclei observed in FA patients seems to be due to clastogenic events, because an increase in the frequency of dicentric bridges was also observed. Micronuclei are expressed mutations and need cell division to appear. The damage detected by SCGE is repairable, and does not require cell division. Under alkaline conditions, SCGE assesses double- and single-strand breaks and alkali-labile sites. The 2 methods are efficient for monitoring mutagenic events in exposed populations or in individuals with genetic instability. While the damage measured by micronucleus analysis is accumulated over a long period of time, DNA damage measured by SCGE reflects recent, unrepaired events.

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Harman and harmine are beta-carboline alkaloids which are present in plants widely used in medical practice, in beverages used for religious purposes in Brazil, as well as in tobacco smoke and over cooked food. In view of the controversial results observed in the literature about the mutagenic effects of these alkaloids, we studied their cytotoxic and genotoxic effects in V79 Chinese hamster lung fibroblasts in vitro using single-cell gel assay, Comet assay, either in the presence or in absence of an exogenous metabolic activation system (S9-mix), and by the chromosome aberration test without S9-mix. Harmine was more cytotoxic than harman. Both harman and harmine increased aberrant cell frequency and induced DNA damage by the Comet assay. These results suggest that harman and harmine are genotoxic in V79 cells, probably as a consequence of their ability to induce DNA strand breaks.

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The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.

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A follow-up study was carried out 4 years after an initial evaluation of the micronucleus frequency in 10 healthy individuals who had been occupationally exposed to antineoplastic drugs in a Brazilian hospital. Upon the first evaluation, these 10 exposed individuals were compared with 10 non-exposed individuals matched for age, sex and smoking habits; the results revealed that the frequency of micronucleated lymphocytes in individuals exposed to antineoplastic drugs was significantly higher (P=0.038) than in controls. The frequency of dicentric bridges was also increased, although not significantly (P=0.0545). After the first analysis, the workers handling antineoplastic drugs were advised to modify their work schedule to limit exposure, and the number of workers in the group was increased from 10 to 12 individuals. In the follow-up study, 12 individuals from the same work area were assessed. In addition to micronucleus frequency, alkaline single cell gel electrophoresis was also used to monitor genetic hazard. This exposed group was compared to 12 non-exposed workers from the same hospital, matched for age, sex and smoking habits. In the follow-up study, no statistical difference was found between exposed workers and controls in terms of micronucleus and dicentric bridge frequency with the Mann--Whitney U-test (P=0.129 and 0.373, respectively). However, the mean value of SCGE analysis was significantly higher in the exposed group than in the controls (P=0.0006). Although the micronucleus analysis seems to be less sensitive to assess DNA damage, it detects chromosome aberrations and not just repairable DNA breakage and alkali-labile sites. Combination of the alkaline single cell gel electrophoresis and cytokinesis blocked micronucleus assay appears to be commendable to monitor populations chronically exposed to genotoxic agents.

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Rio Grande do Sul (RS) coal is low quality and typically obtained by strip mining. In a recent study concerning 2 years of biomonitoring in coal regions, we demonstrated the genotoxicity of coal and related products on blood cells of native rodents, from RS, Brazil. With the goal of studying the variations in the effects of RS coal on different tissues of the same rodent, we utilized, besides the single cell gel (SCG) and micronucleus (MN) assay on blood, histological analyses and SCG assay of bone marrow, spleen, kidney, liver and lung cells, and MN assay of bone marrow and spleen cells. In addition, to identify agents that can potentially influence the results, concentrations of several heavy metals were analyzed in livers and in soil, and the total concentration of hydrocarbons in the soil was determined. Rodents exposed to coal were captured at two different sites, Butiá and Candiota, in RS. Reference animals were obtained from Pelotas, where there is no coal mining. This report provides chemical and biological data from coal regions, indicating the possible association between Zn, Ni, Pb and hydrocarbons in the induction of DNA damage (e.g. single strand-breaks and alkali-labile sites) determined by the alkaline SCG assay in cells from Ctenomys torquatus. The results of the present SCG study indicate that coal and by-products not only induce DNA damage in blood cells, but also in other tissue cells, mainly liver, kidney and lung. Neither the MN assay nor histopathological observations showed significant differences; these analyses may be useful under circumstances where genotoxicity is higher. In conclusion we believe that the in vivo genotoxicity of coal can be biomonitored by the SCG assay, and our studies suggest that wild rodents, such as C. torquatus are useful for monitoring genotoxic damage by both methods, the SCG assay and the MN test.

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Coal is a mixture of a variety of chemicals, especially hydrocarbons, which may give rise to polycyclic aromatic hydrocarbons (PAH). Many PAH compounds produce mutagenic and carcinogenic effects. The quality of mineral coal in Rio Grande do Sul (RS) is low and it is typically obtained by stripping operations; it represents approximately 87% of the Brazil reserves. This report concerns the application of the Comet assay to Ctenomys torquatus to detect the effects of coal, comparing the results with a micronucleus (MN) assay, both using peripheral blood. This study was performed over a 2-year period in an attempt to evaluate seasonal patterns. The wild rodent is fossorial, and its geographic distribution in RS coincides with the distribution of coal reserves. Three localitions were studied: two coal fields, Butiá (in a strip coal mine region) and Candiota (near a strip coal mine), and one control region, Pelotas (no coal). At the end of 2 years, 240 rodents had been analyzed. Our results showed that coal and derivatives induced DNA and chromosomal lesions in rodent cells that were demonstrated by Comet and MN assays. These tests also demonstrated quantitative differences between field exposures (Candiota > Butiá). The Comet assay was more sensitive and also showed a direct relationship between age and damage, and an inverse relationship between temperature and damage index.

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This paper presents the results of a cytogenetic study to evaluate the quality of Caí river water at the area under the influence of the Petrochemical Complex of the State of Rio Grande do Sul. Cytokinesis-block micronucleus assay (CBMN) was performed on cultured human lymphocytes exposed to stream water samples. The Caí River is an important tributary to the Guaíba basin that includes Porto Alegre, the capital of Rio Grande do Sul. The Caí river water is the source of drinking water after conventional treatment, and is also used for irrigation and primary contact recreation according to Federal regulations. Water samples were collected in the Caí River and some tributaries at four sites for 20 months with a bimonthly frequency, in a total of 11 samplings, from November 1993 to July 1995. It was possible to detect the presence of substances with clastogenic and/or aneugenic potential in vitro human lymphocytes at the different sites analyzed. As to the four sites studied, site B presents nearly half of the total positive results (44%), followed by sites A (28%), D (17%) and C (11%). The CBMN assay in human lymphocytes was a sensitive cytogenetic approach for aquatic environmental studies, and should be better exploited to monitor industrial areas.

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beta-Carboline alkaloids, found in medicinal plants, tobacco smoke and well-cooked foods, have shown a variety of actions in biological systems related to their interaction with DNA. Therefore, these alkaloids can be considered potentially mutagenic. In this work, the genotoxic, mutagenic, and cytotoxic activities of three aromatic beta-carboline alkaloids (harman, harmine, and harmol) and two dihydro-beta-carboline alkaloids (harmaline and harmalol) were evaluated by means of the Salmonella/microsome assay (Salmonella typhimurium TA98, TA97, TA100, and TA102) and SOS chromotest (Escherichia coli PQ37) with and without metabolic activation. Moreover, harman and harmine were analyzed by the micronucleus assay in vivo. It was shown that genotoxicity was inhibited by the addition of S9 mix for aromatic beta-carbolines harman and harmol in TA97. However, harmine showed signs of mutagenicity only in the presence of S9 mix in TA98 and TA97 frameshift strains. In the SOS chromotest, only harman induced SOS functions in the absence of S9 mix. Dihydro-beta-carbolines were not genotoxic in any of the microorganisms used. The negative responses obtained in the micronucleus assay indicated that harman and harmine were not able to induce chromosomal mutations.

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Studies were conducted to evaluate the clastogenic activity of drinking water from Porto Alegre and Guaíba (Rio Grande do Sul, Brazil) estuarine waters. Mouse bone marrow was the target organ. C57B1/6 male and female mice received the water samples as their only liquid supply. Bone marrow cells were collected on the 16th day after the beginning of treatment. The analysis of metaphases demonstrated that the water supplies did not increase the structural chromosome aberration frequencies compared to the control groups. Concerning numerical alterations, only one treated female group showed a significant difference (loss of one chromosome) when compared to the control group, but this result is not considered relevant.

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In an investigation of the action of integerrimine on chromosomes, the bone marrow was taken as target organ. Male and female mice of the C57Bl/6 strain received a single acute dose of this pyrrolizidine alkaloid, in 2 concentrations: 18.75 and 37.50 mg/kg. Bone marrow cells were collected 6, 12 and 24 h after treatment. The analysis of metaphasic chromosomes demonstrated that chromosomal damage occurs, correlated with drug concentration. The greatest frequency of chromosomal aberrations was detected 12 h after treatment.

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Fifty-six patients with blood disorders (23 with chronic myeloid leukemia, 14 with acute myeloblastic leukemia, seven with acute lymphoblastic leukemia, one with chronic lymphocytic leukemia, and 11 with preleukemia states) were studied. A quantitative and objective method of C band length analysis with well-matched controls was used. The C bands of chromosome pairs 1, 9, and 16 presented a normal distribution that was similar in patients and controls, whereas the Y chromosome presented an abnormal distribution. Smaller C bands in 1qh and higher indexes of intrapair heteromorphism in pairs 1 and 9 were detected in the CML group; the group of acute leukemias (myeloblastic and lymphoblastic) presented a smaller index only in pair 1qh. No other differences in length, heteromorphism, inversion frequency, or sex were detected.

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A variabilidade das regiöes heterocromática e eucromática do cromossomo Y foi estudada, pelo emprego de uma metodologia de análise quantitativa e através de medidas feitas em densitometria, em 30 caucasóides e 30 japoneses de Curitiba. Observou-se que o comprimento total do cromossomo Y é dependente tanto das variaçöes da regiäo heterocromática como da eucromática. Em ambos os casos, os coeficientes de regressäo apresentaram valores altamente significantes. O maior tamanho do cromossomo Y dos japoneses do que o dos caucasóides foi devido somente às diferenças encontradas entre as regiöes heterocromáticas. O estudo da herdabilidade dos comprimentos das regiöes heterocromáticas, eucromática e total do cromossomo Y, feito em 30 pares de pais-filhos (15 japoneses e 15 caucasóides) apresentou valores altos, estatisticamente significantes. Contudo, enquanto os valores da heterocromatina se distribuíram ao longo da linha de regressäo, conforme o esperado, a eucromatina apresentou uma distribuiçäo mais central com poucos valores fora da distribuiçäo geral

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