Bernardo Erdtmann

DNA damage in bipolar disorder

Bipolar disorder (BD) is a prevalent, chronic, severe, and highly disabling psychiatric disorder that is associated with increased morbidity and mortality due to general medical conditions. There is an emerging body of evidence correlating chronic medical conditions with DNA damage. The present study was designed to assess DNA damage in BD patients using the comet assay (CA). Thirty-two bipolar-I outpatients diagnosed using the Structured Clinical Interview for DSM-IV were matched with 32 healthy volunteers. Manic and depressive symptoms were assessed using the Young Mania Rating Scale and the Hamilton Depression Rating Scale, respectively. Peripheral blood samples were collected and a standard protocol for CA preparation and analysis was performed. The present study showed that BD outpatients present an increased frequency of DNA damage relative to controls. The frequency of DNA damage correlated with the severity of symptoms of depression and mania.

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.

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.

An alkaline single-cell gel electrophoresis (comet) assay for environmental biomonitoring with native rodents

The main advantages of single-cell gel electrophoresis (SCG) are its applicability to any eukaryotic organism and cell type, it s low cost and the short time required to obtain results. These properties make the SCG assay particularly useful in screening for environ mental genotoxicity. The present study describes a modified version of this technique for use in field work with native rodents and ex amines some factors which influence the outcome of the assay. Wild rodents ( Ctenomys torquatus, “tuco-tuco”) from a region close to a strip coal mine and from a region with no coal mines were used. Animals from the coal mining region had significantly more DNA damage than those from the control area. The use of this SCG technique for direct sampling in the field should facilitate environmental genotoxic ity studies with natural populations, without the need to remove the animals from their habitat or to sacrifice them.

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.

Evaluation of genetic damage in a Brazilian population occupationally exposed to pesticides and its correlation with polymorphisms in metabolizing genes.

Cytogenetic damage in individuals occupationally exposed to pesticides has received the attention of investigators in several countries, but no definitive conclusions can yet be made. The present study aimed at assessing if prolonged exposure to complex mixtures of pesticides leads to an increase in cytogenetic damage. Vineyard workers exposed to pesticides in Caxias do Sul (Brazil) were evaluated using the micronucleus (MN) test in binucleated lymphocytes and the comet assay in peripheral leukocytes. In order to evaluate if genetically determined individual variations in xenobiotic metabolizing capacity could modify individual susceptibility to the possible genotoxic effects of pesticides, the subjects were genotyped for several genes: GSTT1, GSTM1, GSTP1, CYP1A1, CYP2E1 and PON. The study involved a total number of 173 men: 108 were agricultural workers exposed to pesticides and 65 were controls. The present study showed a high rate of MN and DNA damage in pesticide-exposed individuals (P <or= 0.001; Mann-Whitney U-test). In addition, some effects of genetic polymorphisms in PON in the modulation of MN results were observed in the exposed group, and an association between GSTM1, GSTT1 and CYP2E1 polymorphisms was suggested.

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.

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. Environ. Mol. Mutagen. 35:270–278, 2000

Chronic hyperhomocysteinemia alters antioxidant defenses and increases DNA damage in brain and blood of rats: protective effect of folic acid.

We have previously demonstrated that acute hyperhomocysteinemia induces oxidative stress in rat brain. In the present study, we initially investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative damage, namely total radical-trapping antioxidant potential and activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), as well as on DNA damage in parietal cortex and blood of rats. We also evaluated the effect of folic acid on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of Hcy (0.3-0.6 micromol/g body weight), and/or folic acid (0.011 micromol/g body weight) from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, parietal cortex and total blood was collected. Results showed that chronic homocysteine administration increased DNA damage, evaluated by comet assay, and disrupted antioxidant defenses (enzymatic and non-enzymatic) in parietal cortex and blood/plasma. Folic acid concurrent administration prevented homocysteine effects, possibly by its antioxidant and DNA stability maintenance properties. If confirmed in human beings, our results could propose that the supplementation of folic acid can be used as an adjuvant therapy in disorders that accumulate homocysteine.

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.