Cesar Koppe Grisolia

A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides

A comparative analysis between mouse and fish erythrocyte micronuclei (MN) assays was carried out using cyclophosphamide, mitomycin C and various pesticides such as alliete, brestanid, decis 25 CE (deltamethrin), kelthane 480 CE (dicofol), roundup (glyphosate), imazapyr and thiram. The aim of this study was to evaluate the fish species Tilapia rendalli as a suitable organism for the detection of genotoxicants in water. The clastogens cyclophosphamide and mitomycin C induced MN in both test-systems. Insecticides: decis 25 CE increased T. rendalli MN frequencies at doses of 1.0 and 5.0mg/kg, but not at the highest dose, and in mice there was no MN induction. Kelthane 480 CE also induced a significant MN frequency in T. rendalli, but not in mice. Fungicides: alliete and brestanid induced MN only in T. rendalli, while thiram was negative in both assays. Herbicides: imazapyr induced MN in T. rendalli at the maximum tolerated dose only, while roundup induced MN at three dosed levels. In mice both herbicides presented negative results. This study revealed that fish MN assay can be used as a genotoxicological test-system since some methodological particularities were observed.

Variability in micronucleus induction with different mutagens applied to several species of fish

Fish are often used for screening genotoxicity of water. For such programs, a knowledge of the sensitivity to clastogens, spontaneous micronucleus frequency and cell cycle kinetics of the target tissue is necessary. To investigate the pattern of inter-specific sensitivity to micronucleus induction three species of fish, Tilapia rendalli, Oreochromis niloticus and Cyprinus carpio, were exposed to the clastogens bleomycin (BLM), cyclophosphamide (CP), 5-fluorouracil (5-FU), and mitomycin C (MMC). The binucleate/mononucleate ratio in peripheral erythrocytes exposed to cytochalasin B was also used to evaluate the time-dependent response of micronucleus formation during hematopoesis in the kidney and the micronucleus peak in peripheral erythrocytes. Micronucleus frequencies induced by CP were significantly greater than their respective controls for the three fish species throughout all treatment periods. During the whole evaluation period (30 days) CP was also the most effective clastogen. In general, until the 14th day of evaluation period T. rendalii was the most sensitive species to clastogens. No difference in micronucleus frequencies among species was observed in the 4th evaluation (at the 30th day). A micronucleus peak was observed at the 7th day after treatment. After the 14th day the frequencies were stabilized. The cytochalasin B experiment was carried out to demonstrate that micronuclei induced in the young kidney erythrocyte cells were detected in the circulating blood 2-4 days later.

Micronuclei monitoring of fishes from Lake Paranoá, under influence of sewage treatment plant discharges

Fish micronuclei tests (MN) were used to evaluate the ability of wastewater from two municipal sewage treatment plants that empty into Lake Paranoá to cause genetic damage. There were no significant differences in the frequencies of micronuclei between the control and hypertrophic areas. In contrast, cyclophosphamide and mitomycin C, used to test the sensitivity of the biological assay, significantly increased the micronuclei counts in Tilapia rendalli, Oreochromis niloticus and Cyprinus carpio, T. rendalli was the most sensitive specie to both clastogens and C. carpio, the most resistant.

Comparison between the micronucleus frequencies of kidney and gill erythrocytes in tilapia fish, following mitomycin C treatment

The frequencies of mitomycin C and cyclophosphamide-induced kidney and gill erythrocyte micronuclei were compared in Tilapia rendalli and Oreochromis niloticus. Cyclophosphamide (CP) and mitomycin C (MMC) were previously used to investigate fish sensitivity to micronuclei induction in kidney erythrocytes. Fish were treated with a single dose of cyclophosphamide (40 mg/kg) or mitomycin C (2 mg/kg). Peripheral blood samples and kidney blood samples were obtained, 1, 2, 3, 5, 7, and 15 days after the intra-abdominal injection of MMC. T. rendalli showed to be more sensitive than O. niloticus to micronuclei production. Both compounds were used at the maximum tolerated dose (MTD), and mitomycin C proved to be more toxic than cyclophosphamide. The frequency of micronucleated erythrocytes observed varied significantly among the treated individuals. The difference between the kidney and gill micronuclei frequencies was not significant. Higher levels of micronucleated erythrocytes were observed in Tilapia rendalli than in O. niloticus. T. rendalli produced erythrocytes with deformed nuclei, which O. niloticus did not.

Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults

The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC50=145.7 mg/L) than adults (96 h-LC50=39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant.

Profile of micronucleus frequencies and DNA damage in different species of fish in a eutrophic tropical lake

Lake Paranoá is a tropical reservoir for the City of Brasilia, which became eutrophic due to inadequate sewage treatment associated with intensive population growth. At present, two wastewater treatment plants are capable of processing up to 95% of the domestic sewage, thereby successfully reducing eutrophization. We evaluated both genotoxic and cytotoxic parameters in several fish species (Geophagus brasiliensis, Cichla temensis, Hoplias malabaricus, Astyanax bimaculatus lacustres, Oreochromis niloticus, Cyprinus carpio and Steindachnerina insculpita) by using the micronucleus (MN) test, the comet assay and nuclear abnormality assessment in peripheral erythrocytes. The highest frequencies of MN were found in Cichla temensis and Hoplias malabaricus, which were statistically significant when compared to the other species. However, Steindachnerina insculpita (a detritivorous and lake-floor feeder species) showed the highest index of DNA damage in the comet assay, followed by C. temensis (piscivorous). Nuclear abnormalities, such as binucleated, blebbed, lobed and notched cells, were used as evidence of cytotoxicity. Oreochromis niloticus followed by Hoplias malaricus, ominivorous/detritivotous and piscivorous species, respectively, presented the highest frequency of nuclear abnormalities, especially notched cells, while the herbivorous Astyanax bimaculatus lacustres showed the lowest frequency compared to the other species studied. Thus, for biomonitoring aquatic genotoxins under field conditions, the food web should also be considered.

Dietary carotenoid-rich pequi oil reduces plasma lipid peroxidation and DNA damage in runners and evidence for an association with MnSOD genetic variant -Val9Ala.

Physical training induces beneficial adaptations; however, exhausting exercise increases reactive oxygen species generation, resulting in damage to DNA and tissues. Pequi (Caryocar brasiliense), a fruit of the Brazilian Cerrado, contains a carotenoid-rich oil. We investigated whether pequi oil had antioxidant effects in runners. Evaluations were made after outdoor races before and after ingestion of 400 mg pequi-oil capsules for 14 days. Blood samples were taken after races and submitted to comet and TBARS assays and biochemical analyses of creatine kinase (CK), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). To determine if the protective effects of pequi-oil were influenced by antioxidant enzyme genotypes, MnSOD (-Val9Ala), CAT (-21A/T) and GPX1 (Pro198Leu) gene polymorphisms were also investigated. Pequi oil was efficient in reducing tissue injuries evaluated for AST and ALT, particularly in women, and in reducing DNA damages in both sexes. Except for CK levels, the results were influenced by MnSOD genotypes; heterozygous excess was related to less DNA damage, tissue injury and lipid peroxidation, besides presenting a better response to pequi oil against exercise-induced damage.

Pequi fruit (Caryocar brasiliense Camb.) pulp oil reduces exercise-induced inflammatory markers and blood pressure of male and female runners

The objective of this study was to investigate the anti-inflammatory properties of pequi (Caryocar brasiliense) fruit oil and its effects on the postprandial lipidemia and arterial blood pressure of male and female athletes. These athletes were evaluated after races in the same environment and under the same type, intensity, and length of weekly training conditions, both before and after ingestion of 400 mg pequi oil capsules for 14 days. Pequi fruit contains several antioxidants, and its oil has been associated with anti-inflammatory properties in other pequi species. Because the oil of pequi is mostly composed of oleic and palmitic fatty acids, the oil may alter the ratio of triglyceride to cholesterol in postprandial lipidemia. Epidemiologic studies suggest that an increased intake of monounsaturated fatty acids (such as oleic acid) is inversely related to blood pressure. Thus, we hypothesize that pequi oil could reduce exercise-induced inflammation and blood pressure, and modulate postprandial lipidemia in runners. To test this hypothesis, arterial blood pressures were checked before races; blood samples were taken after the races and submitted for analysis of leukocytes and platelets analysis, high-sensitivity C-reactive protein values, and postprandial lipids. Pequi oil resulted in anti-inflammatory effects and reduced the total cholesterol and low-density lipoprotein in the age group older than 45 years, mainly for men. The results showed a general trend for reduced arterial pressure, suggesting that pequi oil may have a hypotensive effect. However, this finding needs additional investigation. Thus, pequi oil, besides possessing many nutritional properties, may be a good candidate supplement for athletes.

Antigenotoxic activity and antioxidant properties of organic and aqueous extracts of pequi fruit (Caryocar brasiliense Camb.) pulp

The daily consumption of natural antioxidants protects against oxidative damage caused by reactive oxygen species (ROS), including DNA damage, and can reduce the risk of cancer, atherosclerosis and other degenerative diseases. The pulp of pequi (Caryocar brasiliense Camb.) fruit, a tree native to the Brazilian savannah, contains several compounds with antioxidant properties, including carotenoids, vitamin C, phenolic compounds such as flavonoids, saponins and tannins, and essential oils. In this work, we examined the ability of organic and aqueous extracts of pequi fruit pulp to protect against the genotoxicity induced by two antineoplastic drugs, cyclophosphamide (CP) and bleomycin (BLM). Micronucleus tests with mouse bone marrow cells and single-cell gel electrophoresis (comet assay) with peripheral blood leukocytes were used to examine the effects of CP and BLM, respectively. The antioxidant activity of the extracts was assessed by measuring lipid peroxidation with the TBARS method in mouse plasma. The fruit pulp extracts had no clastogenic or genotoxic effects in the cells studied, but both extracts protected against oxidative DNA damage caused by BLM or CP, indicating an ability to inhibit chemical mutagenesis in vivo. However, the protective effect against oxidative DNA damage depended on the dose of extract used. At the doses tested, the aqueous extract enhanced lipid peroxidation in mice of both sexes, especially in males. In contrast, the organic extract enhanced lipid peroxidation only in male mice, with no significant effect in females. These results suggest that, with adequate adjustment of the dose, an organic extract of pequi fruit pulp could be a useful dietary supplement with natural antioxidant activity, at least in females.

Gene polymorphisms against DNA damage induced by hydrogen peroxide in leukocytes of healthy humans through comet assay: a quasi-experimental study

BackgroundNormal cellular metabolism is well established as the source of endogenous reactive oxygen species which account for the background levels of oxidative DNA damage detected in normal tissue. Hydrogen peroxide imposes an oxidative stress condition on cells that can result in DNA damage, leading to mutagenesis and cell death. Several potentially significant genetic variants related to oxidative stress have already been identified, and angiotensin I-converting enzyme (ACE) inhibitors have been reported as possible antioxidant agents that can reduce vascular oxidative stress in cardiovascular events.MethodsWe investigate the influences of haptoglobin, manganese superoxide dismutase (MnSOD Val9Ala), catalase (CAT -21A/T), glutathione peroxidase 1 (GPx-1 Pro198Leu), ACE (I/D) and gluthatione S-transferases GSTM1 and GSTT1 gene polymorphisms against DNA damage and oxidative stress. These were induced by exposing leukocytes from peripheral blood of healthy humans (N = 135) to hydrogen peroxide (H2O2), and the effects were tested by comet assay. Blood samples were submitted to genotyping and comet assay (before and after treatment with H2O2 at 250 μM and 1 mM).ResultsAfter treatment with H2O2 at 250 μM, the GPx-1 polymorphism significantly influenced results of comet assay and a possible association of the Pro/Leu genotype with higher DNA damage was found. The highest or lowest DNA damage also depended on interaction between GPX-1/ACE and Hp/GSTM1T1 polymorphisms when hydrogen peroxide treatment increased oxidative stress.ConclusionsThe GPx-1 polymorphism and the interactions between GPX-1/ACE and Hp/GSTM1T1 can be determining factors for DNA oxidation provoked by hydrogen peroxide, and thus for higher susceptibility to or protection against oxidative stress suffered by healthy individuals.