Paulo Cezar de Freitas Mathias

Metformin's performance in in vitro and in vivo genetic toxicology studies.

Metformin is a hypoglycemiant drug prescribed for the treatment and control of the type 2 diabetes mellitus. Recently, the potential efficacy of this antidiabetic drug as an anticancer agent has been demonstrated in various mammalian cancer cells. This report evaluates the mutagenic as well as the recombinogenic potentials of the metformin drug in therapeutically relevant plasma concentrations (12.5 µM, 25.0 µM or 50.0 µM). Since the loss of heterozygosity is a process associated with carcinogenesis, the recombinogenic potential of such a drug was evaluated by the homozygotization assay using a heterozygous diploid strain of Aspergillus nidulans. The homozigotization indices (HI) for the genetic markers from the metformin-treated diploids were not statistically different from the negative control (non-treated diploids). For the first time, this indicated a lack of recombinogenic activity of the antidiabetic drug. The mutagenic potential of the metformin drug was evaluated by the chromosome aberrations and the micronuclei tests in human lymphocytes cultures. The metformin drug did not show any significant increase either in the numerical or in the structural chromosome aberrations and did not affect significantly the mitotic index when compared to the negative control. In the in vitro micronucleus test, the drug did not increase the number of micronuclei or nuclear buds when compared with the negative control. The data in this study suggest that the metformin drug is not a secondary cancer inducer, since it has neither showed recombinogenic nor mutagenic activities when used in pharmacological concentrations.

Monosodium L‐glutamate‐induced obesity impaired the adrenal medullae activity

Impairment of sympathoadrenomedullar system has been suggested to have a role in obesity. The activity of adrenal medullae (AM) isolated from 90-day-old obese-induced L-monosodium glutamate (MSG) male mice was studied. MSG treatment induced obesity without hyperphagia, while MSG-animals were shorter and lighter than the controls. Catecholamines (CA) gland content decreased in MSG-mice, while secretory response to carbachol was similar in isolated AM of both groups. However, high extracellular potassium did not stimulate CA secretion on AM isolated from obese animals as occurred in controls. Cholinesterase activity of adrenal glands homogenate was impaired in MSG-mice. Results suggest that MSG-hypothalamus lesions could cause impairment of sympathetic transmission in AM, which impose less CA accumulation and alter secretion response in AM. These defects might be involved at the onset of MSG obesity.

A High Fat Diet during Adolescence in Male Rats Negatively Programs Reproductive and Metabolic Function Which Is Partially Ameliorated by Exercise

An interaction between obesity, impaired glucose metabolism and sperm function in adults has been observed but it is not known whether exposure to a diet high in fat during the peri-pubertal period can have longstanding programmed effects on reproductive function and gonadal structure. This study examined metabolic and reproductive function in obese rats programmed by exposure to a high fat (HF) diet during adolescence. The effect of physical training (Ex) in ameliorating this phenotype was also assessed. Thirty-day-old male Wistar rats were fed a HF diet (35% lard w/w) for 30 days then subsequently fed a normal fat diet (NF) for a 40-day recovery period. Control animals were fed a NF diet throughout life. At 70 days of life, animals started a low frequency moderate exercise training that lasted 30 days. Control animals remained sedentary (Se). At 100 days of life, biometric, metabolic and reproductive parameters were evaluated. Animals exposed to HF diet showed greater body weight, glucose intolerance, increased fat tissue deposition, reduced VO2max and reduced energy expenditure. Consumption of the HF diet led to an increase in the number of abnormal seminiferous tubule and a reduction in seminiferous epithelium height and seminiferous tubular diameter, which was reversed by moderate exercise. Compared with the NF-Se group, a high fat diet decreased the number of seminiferous tubules in stages VII-VIII and the NF-Ex group showed an increase in stages XI-XIII. HF-Se and NF-Ex animals showed a decreased number of spermatozoa in the cauda epididymis compared with animals from the NF-Se group. Animals exposed to both treatments (HF and Ex) were similar to all the other groups, thus these alterations induced by HF or Ex alone were partially prevented. Physical training reduced fat pad deposition and restored altered reproductive parameters. HF diet consumption during the peri-pubertal period induces long-term changes on metabolism and the reproductive system, but moderate and low frequency physical training is able to recover adipose tissue deposition and reproductive system alterations induced by high fat diet. This study highlights the importance of a balanced diet and continued physical activity during adolescence, with regard to metabolic and reproductive health.

O tratamento com isoflavonas mimetiza a ação do estradiol no acúmulo de gordura em ratas ovariectomizadas

OBJECTIVE Isoflavones (ISO) present in soybean are named phytoestrogens because they show estrogen effect. The use of isoflavones has beneficial effect in disturbance of post-menopause, which is characterized by ovarian function suppression. Decreasing of estrogen secretion and consequent morphologic and metabolic disarrangements are observed in female hormonal decline. The aim of present work was to investigate the effect of ISO on the fat accretion of uterine endometric tissue, and HDL and glucose blood concentration from ovariectomized rats (OVX). METHODS Female Wistar rats with 60 days-old were submitted a surgery to remove bilaterally the ovarium. After 8-day recovery period the animals were distributed into three groups: sham operate (GC); OVX ISO untreated (GI) and OVX supplemented with ISO (G II). Total uterus mass, uterus fat and retroperitoneal fat pad, were removed, washed and weighted. Samples of uterus were histological processed to measure endometrium thickness. Blood samples were also collected to analyze the concentration of HDL and glucose. The OVX caused endometric atrophy, decrease of uterus weight and HDL reduction. The treatment with ISO provoked decrease of uterine and retroperitoneal fat pad. HDL increase and glycemia reduction were also observed. However, there was no uterotrophic effect. CONCLUSIONS ISO treatment causes decrease in tissue fat accretion from ovariectomized rats.

Assessment of in vivo and in vitro genotoxicity of glibenclamide in eukaryotic cells.

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.

Genotoxic investigation of a thiazolidinedione PPARγ agonist using the in vitro micronucleus test and the in vivo homozygotization assay

Pioglitazone (PTZ) is an oral antidiabetic agent whose anti-cancer properties have been described recently. Since PTZ increases the production of reactive oxygen species in mammalian cells, the aim of current study was to evaluate the cytotoxic, mutagenic and recombinogenic effects of PTZ using respectively the in vitro mitotic index assay and the in vitro mammalian cell micronucleus test in human peripheral lymphocytes, and the in vivo homozygotization assay in Aspergillus nidulans, which detects the loss of heterozygosity due to somatic recombination. Although the lowest PTZ concentrations (4-36 μM) did not show any significant rise in the micronucleus production, the higher PTZ concentration (108 μM) produced a statistically higher number of micronuclei than the negative control and significantly altered the cell-proliferation kinetics, demonstrating the mutagenic and antiproliferative effects of PTZ, respectively. The recombinogenic activity of PTZ, demonstrated here for the first time, was observed at the highest tested concentration (400 μM) through the homozygotization index rates significantly different from the negative control. Taken together, our results show that PTZ is genotoxic at a concentration higher than the therapeutic plasma concentration. This PTZ genotoxicity may be a potential benefit to its previously described antitumour activity.

Atividade colinesterásica em tireóide de ratos: resposta a uma sobrecarga de iodo

A utilizacao do iodo antes de cirurgias de tireoide visa a reducao do fluxo sanguineo para a glândula e melhora da hemostasia. Para tentar elucidar o mecanismo pelo qual a vasoconstriccao se instala, foi determinada a atividade colinesterasica em tireoides de ratos Wistar machos (240-250g de peso corporal), submetidos a uma sobrecarga oral de iodo pela oferta de solucao aquosa de iodato de potassio (KIO3 3mg/ml) como agua de beber, por 3 e 7 dias; o grupo controle recebeu agua durante o mesmo periodo. Ao final do tratamento os animais foram sacrificados e suas tireoides retiradas, limpas e pesadas; o rim direito e o lobo direito do figado foram usados como controles da atividade colinesterasica. Foram preparados homogeneizados em tampao fosfato (pH=8,0), contendo 0,32M de sacarose, na concentracao de 40mg de tecido/ml. Os ratos que receberam KIO3 por 7 dias tiveram diminuicao no peso da tireoide quando comparados aos controles (p<0,05), o mesmo nao acontecendo aos 3 dias de tratamento. Aos 3 dias de tratamento, a atividade colinesterasica tireoidea foi de 5,38±0,36 nmol/min.mg para o grupo controle (n=12) e de 5,43±0,98 nmol/min.mg para o grupo tratado (n=11) (NS); valores bastante similares foram tambem encontrados com o tratamento de 7 dias (controles: 5,42±0,27 nmol/min.mg (n=8) e tratados: 5,63±0,88 nmol/min.mg (n=8); NS). Nossos resultados sugerem que, a despeito da vasoconstriccao, nao ha alteracao da atividade colinesterasica na tireoide em resposta a sobrecarga oral de iodo.

Stevia Nonsweetener Fraction Displays an Insulinotropic Effect Involving Neurotransmission in Pancreatic Islets

Stevia rebaudiana (Bert.) Bertoni besides being a source of noncaloric sweeteners is also an important source of bioactive molecules. Many plant extracts, mostly obtained with ethyl acetate solvent, are rich in polyphenol compounds that present insulinotropic effects. To investigate whether the nonsweetener fraction, which is rich in phenolic compounds isolated from Stevia rebaudiana with the solvent ethyl acetate (EAF), has an insulinotropic effect, including interference at the terminals of the autonomic nervous system of the pancreatic islets of rats. Pancreatic islets were isolated from Wistar rats and incubated with EAF and inhibitory or stimulatory substances of insulin secretion, including cholinergic and adrenergic agonists and antagonists. EAF potentiates glucose-stimulated insulin secretion (GSIS) only in the presence of high glucose and calcium-dependent concentrations. EAF increased muscarinic insulinotropic effects in pancreatic islets, interfering with the muscarinic receptor subfamily M3. Adrenergic inhibitory effects on GSIS were attenuated in the presence of EAF, which interfered with the adrenergic α 2 receptor. Results suggest that EAF isolated from stevia leaves is a potential therapy for treating type 2 diabetes mellitus by stimulating insulin secretion only in high glucose concentrations, enhancing parasympathetic signal transduction and inhibiting sympathetic signal transduction in beta cells.

Assessment of bendamustine-induced genotoxicity in eukaryotic cells

Abstract Bendamustine, an anticancer drug with alkylating properties, is widely used to treat hematological malignancies. Since the nitrogen mustard family alkylators induce DNA damages and have been associated with an elevated risk of second malignancy, current study evaluates the cytotoxic, mutagenic, and recombinogenic effects of bendamustine by using, respectively the mitotic index assay, the in vitro mammalian cell micronucleus test (Mnvit) and the chromosome aberration (CA) test in human peripheral lymphocytes, and the in vivo homozygotization assay in Aspergillus nidulans, which detects the loss of heterozygosity (LOH) due to somatic recombination. Bendamustine (6.0 µg/ml, 9.0 µg/ml, and 12.0 µg/ml) induced a statistically significant concentration-related increase in the frequencies of micronuclei and a significant reduction in the cytokinesis block proliferation index (CBPI) rates when compared to negative control. In the CA test, bendamustine significantly increased the frequencies of structural aberrations at the three tested concentrations when compared to the negative control. Aspergillus nidulans diploids, obtained after bendamustine treatment (6.0 µg/ml, 12.0 µg/ml, and 24.0 µg/ml), produced, after haploidization, homozygotization index (HI) rates higher than 2.0 and significantly different from the negative control. Since bendamustine showed genotoxic effects in all tested concentrations, two of them corresponding to the peak plasma concentrations observed in cancer patients treated with bendamustine, data provided in the current research work may be useful to identify the most appropriate dosage regimen to achieve the efficacy and safety of this anticancer medication.

Efeito da redução de ninhada sobre as respostas autonômicas e metabólicas de ratos Wistar

OBJECTIVE: This study investigated the lipid profile and electric activity of the parasympathetic (vagus nerve) and sympathetic (located in the splanchnic region) nerves of obese rats from small litters. METHODS: Two distinct groups were studied, each with 12 animals: normal litter with nine pups per litter and small litter, with three pups per litter. Chow intake and body weight were monitored from weaning until the end of the experimental protocol. At age 90 days, the animals were anesthetized with Thiopental® for investigation of the electric activity of the sympathetic and parasympathetic nerves. They were then sacrificed for removal and weighing of the retroperitoneal and epididymal fat pads. Blood samples were collected for determination of blood glucose, insulin, total cholesterol, triglycerides and high-density lipoprotein cholesterol. RESULTS: The small litters rats had high food intake, body weight, white fat tissue, blood glucose, blood insulin, total cholesterol and triglycerides, and low high-density lipoprotein cholesterol levels. CONCLUSION: The vagus nerve of the small litters rats was significantly more active than that of the normal litter rats. Sympathetic activity did not differ between the groups but the small litters model effectively promoted obesity, dyslipidemia, hypercholesterolemia, hyperinsulinemia, hyperglycemia and autonomic imbalance in rats.