Chromosome polymorphism and complements in populations of Girardia species (Platyhelminthes, Tricladida, Paludicola) from southern Brazil.

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.

Differentiation between malignant and benign solitary pulmonary nodules with proton density weighted and ECG-gated magnetic resonance imaging.

OBJECTIVE: To estimate performance of MRI for differentiating malignant from benign solitary pulmonary nodules (SPN) using morphological characteristics. MATERIAL AND METHODS: MRI in 46 patients with SPN (mean diameter: 19 mm) was carried out on 1.0 Tesla scanner using ECG-gated, gradient echo sequence. Morphological signs of SPN were determined and compared with previously performed helical-CT, where final diagnosis served as reference with 52% frequency of malignancy. Furthermore, three observers evaluated all images. RESULTS: Significant differences between the two groups were found for nodules shape, margin, inhomogeneity and the vessel-sign in MRI, nodules shape, margin, the vessel-sign, and presence of spicules in CT. Using these signs, AUC were 0.746 for MRI and 0.765 for CT. The mean sensitivity, specificity, and accuracy of observers for MRI/CT were 89%/95%, 42%/41%, 66%/68%, respectively. CONCLUSIONS: Despite discrepancies in morphologic appearance, no significant difference of accuracy between MRI and CT was determined. Further investigations are necessary to demonstrate the clinical use in combination with functional parameters, establishing MRI as a comprehensive diagnostic modality for SPN.

[Detection of pulmonary nodules with breath-hold magnetic resonance imaging in comparison with computed tomography]. / Detektion von Lungenrundherden mit der Magnetresonanztomographie in Atemanhaltetechnik im Vergleich zur Spiral-Computertomographie.

PURPOSE: Evaluation of sensitivity and false positive findings of two fast MRI sequences for the detection of pulmonary nodules in comparison with spiral CT by two independent observers. MATERIALS AND METHODS: All 30 enrolled patients had a spiral CT or MSCT as base line study. MRI was performed with a 1.5 T MR scanner (Sonata, Siemens) using a transverse 3D gradient echo sequence (3D-GRE: TR/TE/flip = 2.9 ms/1.1 ms/5 degrees ) and a half-Fourier single-shot fast spin-echo sequence (HASTE: TR/TE/flip = 800/25/150 degrees ) acquired in three planes. A separate analysis for both sequences was carried out prospectively by two independent readers (A and B) with different experience regarding pulmonary MRI. Additionally, a retrospective reading with knowledge of the CT scans was done. Results were calculated for all lesions and for lesions larger than 4 mm. RESULTS: The sensitivities were 73 %, 70 % and 84 % for the 3D-GRE sequence (reader A, reader B, retrospective reading) and 65 %, 68 % and 81 % for the HASTE sequence. For lesions larger than 4 mm, the sensitivities were 93 %, 89 %, 96 % for the 3D-GRE sequence and 85 %, 85 %, 96 % for the HASTE sequence. The rate of false positive findings depended on the reader's experience, but was generally lower for the 3D-GRE sequence with 2 and 16 (reader A and B) false positive nodules compared to 4 and 40 false positive findings for the HASTE sequence. The 3D-GRE sequence was more accurate for both readers (reader A: p = 0.08, reader B: p = 0.00003). CONCLUSION: The sensitivity of MRI for the detection of lung nodules was only acceptable for lesions larger than 4 mm. The 3D-GRE sequence is superior to the HASTE sequence due to the reduced amount of false positive findings with comparable sensitivity.

Epidemiologic studies among Amerindian populations of Amazonia. II. Prepvalence of Mansonella ozzardi.

The prevalence of microfilaremia among Indians in 13 Amazon Indian villages was determined by examining Giemsa-stained peripheral blood smears and preparations from peripheral blood lymphocyte cultures. Mansonella ozzardi was the only species found in the 601 persons tested. Prevalence was highly village-specific, ranging from 0% in four villages to as high as 93% among persons aged 10 years and older in others. Comparisons of the two methods showed that the concentration effect of the peripheral blood lymphocyte culture preparations allowed the detection of a greater number of microfilaria-positive persons, especially women and children with lower levels of parasitemia.

Genomic instability in Down syndrome and Fanconi anemia assessed by micronucleus analysis and single-cell gel electrophoresis.

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.

Densitometric measurements of C bands of chromosomes 1, 9, 16, and Y in leukemic and preleukemic disorders.

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.

Genotoxicity biomonitoring in coal regions using wild rodent Ctenomys torquatus by Comet assay and micronucleus test.

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.

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

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.

Cytogenetic evaluation of aquatic genotoxicity in human cultured lymphocytes.

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.

Follow-up study of the genetic damage in lymphocytes of pharmacists and nurses handling antineoplastic drugs evaluated by cytokinesis-block micronuclei analysis and single cell gel electrophoresis assay.

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.

Genotoxic effects of copper sulphate in freshwater planarian in vivo, studied with the single-cell gel test (comet assay).

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.

Analysis of clastogenic effect of Porto Alegre drinking water supplies on mouse bone marrow cells.

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.

The effect of the pyrrolizidine alkaloid integerrimine on the chromosomes of mouse bone marrow cells.

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.

Genotoxic effects of structurally related beta-carboline alkaloids.

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.

Effects of chronic exposure to coal in wild rodents (Ctenomys torquatus) evaluated by multiple methods and tissues.

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.

Evaluation of the genotoxic effect of rutin and quercetin by comet assay and micronucleus test.

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.

[CT-guided therapy of osteoid osteoma by drill trepanation of the nidus. Clinical follow-up results]. / CT-gesteuerte Therapie des Osteoidosteoms durch Nidusbohrung: Klinische Langzeitergebnisse.

PURPOSE: Evaluation of technical success and follow-up of patients with osteoid osteoma. MATERIAL AND METHODS: 9 patients (3 w, 6 m) with a mean age of 22.3 years suffering from an osteoid osteoma localized in the femur were treated with a combination therapy based on drill trepanation of the nidus and subsequent installation of 1-2 ml ethanol (96%). The treatment was done under CT-guidance in general anaesthesia. RESULTS: During the follow-up period of 20.6 months (10 to 39 months) one relapse occurred within 3 months. This was treated with the same percutaneous method. CONCLUSIONS: Percutaneous removal of intracortical osteoid osteomas under CT-guidance had success in this study, was technical easy, and a had low relapse rate. In addition the duration of hospitalisation is shorten. Meanwhile other studies have used additive ablation with radiofrequency. A second study has been started in our department using a thermoablative procedure. In a comparison between surgical and radiological methods, the rates of relapse are similar (9% vs. 23% surgically). The CT-guided removal of intracortical osteoid osteomas should be the procedure of first choice.

[High resolution multislice CT of the lung: comparison with sequential HRCT slices]. / Hochauflösende Mehrschicht-CT der Lunge: Vergleich mit HRCT-Einzelschichten.

PURPOSE: To compare the image quality of high-resolution (HR) spiral CT scans from a multislice CT scanner with sequential HRCT scans from a singleslice CT scanner. MATERIALS AND METHODS: 20 patients with diffuse lung disease received a high-resolution spiral CT on a multislice scanner (4 slices) and 5 HRCT single slices (1 mm) on a singleslice scanner. Scan parameters of the multislice scanner were: Collimation 4 x 1 mm, pitch 6, slice thickness 1 mm. 5 HRCT slices were compared to the corresponding HR spiral CT slices using a 5-point scale by 5 radiologists with regard to the image quality and the number of artifacts. The evaluation was performed with a multivariate analysis (MANOVA test). RESULTS: Overall impression of image quality, noise, central vessels and bronchi, and all pathological changes were not significantly different between the two CT methods. Sequential HRCT scans were considered to be significantly better than HR spiral CT scans for spatial resolution (p = 0.02), depiction of peripheral vessels (p = 0.02), and of small bronchi (p = 0.05), and significantly worse for depiction of interlobar septa (p < 0.001). Diagnostically relevant differences were found in only 2.2%. Breathing and heartbeat artifacts each were 3 times higher in the sequential HRCT technique than in the multislice-spiral technique (p < 0.0001). CONCLUSION: HR spiral CT scans performed on a multislice CT scanner provide significantly less artifacts and an equal diagnostic image quality compared to sequential HRCT scans performed on a singleslice CT scanner. Multislice spiral CT in HR technique may replace the common scanning technique with conventional spiral CT and additional HRCT scans for diffuse lung diseases.

Evaluation of basal micronucleus frequency and hexavalent chromium effects in fish erythrocytes.

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.

Intravascular ultrasound: value of electronic and mechanical devices for quantifying mild to moderate atherosclerosis.

To define the accuracy of electronic and mechanical ultrasound (US) devices for determining the thickness of intima and media, 32 fresh normal and atherosclerotic human femoral arteries were obtained at necropsy. The samples were imaged with a 64-element array and a mechanically rotating US transducer at 20 MHz. The mean thickness of the intimal and medial layer was measured with electronic calipers followed by histopathologic and micromorphometric analysis. Morphometric correlation for intima showed r = 0.64 for the electronic and r = 0.58 for the mechanical US device. The correlation between ultrasonic and histologic measurement of medial thickness in normal and diseased specimens was r = 0.79 for the multielement and r = 0.76 for the mechanical transducer. In conclusion, multielement array transducers are equivalent to mechanically driven probes in the quantitative assessment of peripheral arterial wall layers. Both systems lack sufficient accuracy in the determination of mild to moderate intimal thickening.