Micronuclei in diabetes: Folate supplementation diminishes micronuclei in diabetic patients but not in an animal model

Diabetes mellitus (DM) is associated with a high risk of health complications, mainly due to excessive free radical (FRs) production that could result in an increased frequency of micronuclei. The consumption of antioxidants, like folic acid (FA), may mitigate the effects of the FRs. In the present study, micronucleated polychromatic erythrocyte (MNPCE) frequencies were determined in blood sampled weekly from the tails of pregnant female Wistar rats and pregnant Wistar rats with experimental diabetes that were given unsupplemented diets and diets supplemented with FA. At birth, the pups were sampled to analyze micronucleated erythrocyte (MNE) and MNPCE frequencies. Moreover micronucleated cells (MNCs) were evaluated in buccal mucosa samples taken from 81 healthy adult subjects, 48 patients with DM, and 30 DM patients who were sampled before and after FA treatment. Increases in MNPCE frequencies were significant only at the first sampling (P < 0.01 and P < 0.03) in pregnant rats with experimental diabetes. In addition, the pups from the diabetic group and from diabetic group treated with FA had higher frequencies of MNEs (P < 0.03 and P < 0.001, respectively) and MNPCEs (P < 0.009 and P < 0.05, respectively) than the controls. No differences were found in diabetic rats and newborn rats born to diabetic mothers treated with FA compared with untreated animals. Patients with DM had a higher frequency of MNCs compared with healthy subjects (P < 0.001). Also FA reduced the frequency of MNCs in DM patients (P < 0.001). The results of this study indicate that diabetes results in elevated frequencies of micronuclei, and that, at least in humans, FA can protect against the elevation.     

Induction of micronuclei in mouse bone-marrow erythrocytes in association with hypothermia after administration of the sigma receptor ligand E-5842

Oral administration of E-5842, a new sigma1 receptor ligand being developed as an antipsychotic drug, to male mice at single doses of 50, 100, 200 and 400 mg/kg produced marked and sustained decreases in rectal temperature. Both the intensity and the duration of the hypothermic effect increased with dose. Maximum decreases from the mean pre-administration temperature (36.2 degrees C) ranged from 7.5 to 12.9 degrees C for animals receiving 50 and 400 mg/kg doses, respectively. Examination of bone-marrow smears obtained 24, 48 and 72 h after administration revealed a slight but statistically significant (p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) at the 48 h sampling for animals receiving the 200 mg/kg dose. These animals showed decreases from pre-administration temperature of approximately 12 degrees C, with recovery being observed 24 h after administration. When the hypothermic effect of E-5842 administration was avoided by housing treated animals under conditions of increased environmental temperature (30 degrees C) for 24 h, MNPCE frequency reverted to vehicle control values. Further, in E-5842-treated animals with an increased MNPCE frequency there was a shift in the distribution of the relative areas of micronuclei in MNPCE towards higher values. In addition, there was a statistically significant increase (p < 0.001) in the number of relatively large micronuclei (micronucleus diameter > or = 1/4 cytoplasm diameter) similar to that produced by administration of the mitotic spindle inhibitor colchicine (1 mg/kg), suggesting disturbance of mitotic apparatus as the possible underlying mechanism. The results suggest that the slight increase in MNPCE frequency observed 48 h after administration of a 200 mg/kg dose of E-5842 is due to a hypothermic effect and not to a direct effect of E-5842 on DNA.     

Protective effects of vitamin E against atrazine-induced genotoxicity in rats

Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) is one of the most commonly used herbicides to control grasses and weeds. The widespread contamination and persistence of atrazine residues in the environment has resulted in human exposure. Vitamin E is a primary antioxidant that plays an important role in protecting cells against toxicity by inactivating free radicals generated following pesticides exposure. The present study was undertaken to investigate the protective effect of vitamin E against atrazine-induced genotoxicity. Three different methods: gel electrophoresis, comet assay and micronucleus test were used to assess the atrazine-induced genotoxicity and to evaluate the protective effects of vitamin E. Atrazine was administered to male rats at a dose of 300 mg/kg body weight for a period of 7, 14 and 21 days. There was a significant increase (P<0.001) in tail length of comets from blood and liver cells treated with atrazine as compared to controls. Co-administration of vitamin E (100 mg/kg body weight) along with atrazine resulted in decrease in tail length of comets as compared to the group treated with atrazine alone. Micronucleus assay revealed a significant increase (P<0.001) in the frequency of micronucleated cells (MNCs) following atrazine administration. In the animals administrated vitamin E along with atrazine there was a significant decrease in percentage of micronuclei as compared to atrazine treated rats. The increase in frequency of micronuclei in liver cells and tail length of comets confirm genotoxicity induced by atrazine in blood and liver cells. In addition, the findings clearly demonstrate protective effect of vitamin E in attenuating atrazine-induced DNA damage.     

Genomic and Metabolic Disposition of Non-Obese Type 2 Diabetic Rats to Increased Myocardial Fatty Acid Metabolism

Lipotoxicity of the heart has been implicated as a leading cause of morbidity in Type 2 Diabetes Mellitus (T2DM). While numerous reports have demonstrated increased myocardial fatty acid (FA) utilization in obese T2DM animal models, this diabetic phenotype has yet to be demonstrated in non-obese animal models of T2DM. Therefore, the present study investigates functional, metabolic, and genomic differences in myocardial FA metabolism in non-obese type 2 diabetic rats. The study utilized Goto-Kakizaki (GK) rats at the age of 24 weeks. Each rat was imaged with small animal positron emission tomography (PET) to estimate myocardial blood flow (MBF) and myocardial FA metabolism. Echocardiograms (ECHOs) were performed to assess cardiac function. Levels of triglycerides (TG) and non-esterified fatty acids (NEFA) were measured in both plasma and cardiac tissues. Finally, expression profiles for 168 genes that have been implicated in diabetes and FA metabolism were measured using quantitative PCR (qPCR) arrays. GK rats exhibited increased NEFA and TG in both plasma and cardiac tissue. Quantitative PET imaging suggests that GK rats have increased FA metabolism. ECHO data indicates that GK rats have a significant increase in left ventricle mass index (LVMI) and decrease in peak early diastolic mitral annular velocity (E’) compared to Wistar rats, suggesting structural remodeling and impaired diastolic function. Of the 84 genes in each the diabetes and FA metabolism arrays, 17 genes in the diabetes array and 41 genes in the FA metabolism array were significantly up-regulated in GK rats. Our data suggest that GK rats’ exhibit increased genomic disposition to FA and TG metabolism independent of obesity.     

Clastogenic activity of urethane in mice

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 × DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6–8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p < 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p < 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

Clastogenic activity of urethane in mice.

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 x DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6-8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p less than 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p less than 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.     

Formation of micronucleated erythrocytes in mouse bone-marrow under conditions of hypothermia is not associated with stimulation of erythropoiesis

Conditions of marked and long-lasting hypothermia have been shown to increase the formation of micronucleated polychromatic erythrocyte (MNPCE) in mouse bone-marrow. Stimulation of erythropoiesis as a consequence of anoxic conditions associated with decreased body temperature has been suggested as a possible mechanism for hypothermia-induced micronucleus formation. We examined whether chemically induced hypothermic conditions that produced increased MNPCE formation were associated with stimulation of erythropoiesis by measuring erythropoietin (EPO) concentrations in blood. Marked and long-lasting hypothermia was induced in male mice by oral administration of the antipsychotic compounds E-5842 (200 mg/kg) or chlorpromazine (100 mg/kg). Maximum decreases from the basal temperature, achieved 8 h after treatment, were 14.8 and 12.8 degrees C, respectively. A statistically significant increase in bone-marrow MNPCE frequency was observed 48 h after administration of E-5842 (p<0.01) or chlorpromazine (p<0.05). Mice made anaemic by retro-orbital bleeding (0.5 ml), which acted as positive control for stimulation of erythropoiesis, showed no relevant variation in mean rectal temperature and a slight non-statistically significant increase in MNPCE frequency after 48 h. Blood samples for determination of EPO levels were obtained 4 (bleed-control animals only), 8, 16 and 24 h after treatment. In spite of the induced hypothermia, no significant variation in EPO blood levels was observed after administration of E-5842 or chlorpromazine. Bleed-induced anaemic mice showed a clear increase in EPO blood levels at all sampled time points, differences from baseline values being statistically significant (p<0.001) at the 8-h samplings and beyond. These results indicate that induction of MNPCE secondary to chemically induced hypothermia is not mediated by stimulation of erythropoiesis.     

Sex and type 2 diabetes: obesity-independent effects on left ventricular substrate metabolism and relaxation in humans

Patients with type 2 diabetes (T2DM), particularly women, are at risk for heart failure. Myocardial substrate metabolism derangements contribute to cardiac dysfunction in diabetic animal models. The purpose of this study was to determine the effects of diabetes and sex on myocardial metabolism and diastolic function in humans, separate from those of obesity. Thirty-six diabetic subjects (22 women) and 36 nondiabetic, BMI-matched subjects (21 women) underwent positron emission tomography (myocardial metabolism) and echocardiography (structure, function). Myocardial blood flow and oxygen consumption (MVO(2)) were higher in women than men (P = 0.003 and <0.0001, respectively). Plasma fatty acid (FA) levels were higher in diabetics (vs. obese, P < 0.003) and sex and diabetes status interacted in its prediction (P = 0.03). Myocardial FA utilization, oxidation, and esterification were higher and percent FA oxidation lower in diabetics (vs. obese, P = 0.0004, P = 0.007, P = 0.002, P = 0.02). FA utilization and esterification were higher and percent FA oxidation lower in women (vs. men, P = 0.03, P = 0.01, P = 0.03). Diabetes and sex did not affect myocardial glucose utilization, but myocardial glucose uptake/plasma insulin was lower in the diabetics (P = 0.04). Left ventricular relaxation was lower in diabetics (P < 0.0001) and in men (P = 0.001), and diabetes and sex interacted in its prediction (P = 0.03). Sex, T2DM, or their interaction affect myocardial blood flow, MVO(2), FA metabolism, and relaxation separate from obesity's effects. Sexually dimorphic myocardial metabolic and relaxation responses to diabetes may play a role in the known cardiovascular differences between men and women with diabetes.     

Micronuclei in blood lymphocytes and genetic polymorphism for GSTM1, GSTT1 and NAT2 in pesticide-exposed greenhouse workers.

The frequency of micronuclei (MN) in cultured peripheral lymphocytes was used as a biomarker of genotoxic effects in 34 Italian pesticide-exposed greenhouse workers and 33 unexposed referents matched with the exposed workers for age and smoking habits. The possible influence of the genetic polymorphisms of xenobiotic metabolizing enzymes glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and N-acetyltransferase 2 (NAT2) was also evaluated. To restrict the analysis primarily to cells that have divided once in vitro, MN were scored only in cells showing label after a 42-h incubation with bromodeoxyuridine (BrdU), as detected by immunofluorescence (anti-BrdU technique). Two different concentrations of BrdU (0.5 and 1 microg/ml) were compared. Individual frequencies of micronucleated cells (MNCs) obtained with the two concentrations of BrdU significantly correlated with each other (r=0.55, P<0.001). Higher mean MNCs frequencies (per 1000 cells) were detected among exposed smokers (9.0 at 0.5 microg/ml BrdU and 7.8 at 1 microg/ml BrdU) than in smoking referents (6.3 and 5.9, respectively). In multiple regression analysis controlling for age, sex, smoking and genotypes, a significant elevation of MNC frequency (P=0.004 at 1 microg/ml BrdU; P=0.052 at 0.5 microg/ml BrdU) was observed in greenhouse workers with a work history of extensive pesticide spraying (n=17). Increased MNC frequencies were also associated with ageing at 0.5 microg/ml BrdU, with the GSTM1-positive genotype at both 1 (P=0.028) and 0.5 (P=0.056) microg/ml BrdU in all subjects, and with the NAT2 fast acetylator genotype in smokers at 0.5 microg/ml BrdU (P=0.043). The results indicate that MN rates are increased in greenhouse workers, especially in those involved in pesticide spraying. The GSTM1 positive and NAT2 fast genotypes appear to be associated with elevated MNC frequencies, which contradicts with earlier results on elevated chromosomal aberration rates in GSTM1 null smokers and NAT2 slow subjects.     

In vivo cytogenetic effects of a commercially formulated mixture of cypermethrin and quinalphos in mice

In vivo cytogenetic effects of commercially formulated cypermethrin (CYP, synthetic pyrethroid insecticide) and/or quinalphos (QUI, organophosphate insecticide), generally used in combination, were examined through chromosomal aberrations (CA) and micronucleus test (MT) in mice. Male mice were orally gavaged to a single dose of CYP/QUI commercial mixture (22, 44 or 67 mg/kg b.wt.) for 24h (CA) or 48 h (MT). Based on the concentrations of active ingredients of CYP and QUI present in the test doses of CYP/QUI mixture, mice were orally exposed to 0.66, 1.32 and 2 mg/kg of CYP or 4.4, 8.8 and 13.4 mg/kg of QUI. For reference, a group of five mice was intraperitoneally administered to cyclophosphamide (20 or 50 mg/kg) or orally gavaged to peanut oil for vehicle control. Exposure of CYP/QUI mixture inhibited the mitotic index (MI) and induced CA in a dose-dependent manner at 24 h; however, significant (p<0.01 or 0.001) frequencies of CA were observed at 44 mg/kg onwards, whereas inhibition of MI at 67 mg/kg. Independent exposure of QUI at 8.8 mg/kg onwards also significantly (p<0.01 or 0.001) inhibited MI and induced CA, whereas CYP at 2 mg/kg (highest concentration in CYP/QUI mixture) inhibited MI significantly but failed to induce CA. Chromatid breaks and fragments found to be frequent aberrations in all the test groups. Treatment of CYP/QUI mixture also induced micronucleus formation dose-dependently at 48 h, yet statistically significant (p<0.001) frequencies of micronucleated polychromatic erythrocytes (MNPCE) were observed at 44 mg/kg onwards. QUI (8.8 and 13.4 mg/kg) alone also induced significant frequencies of MNPCE, whereas frequencies of MNPCE observed with the CYP even at 2 mg/kg were comparable to that of vehicle control. Present findings indicate the genotoxicity potential of CYP/QUI mixture and suggest that the simultaneous presence of the toxic doses of CYP and QUI can lead to synergistic genotoxicity in mice and may pose mutagenic risk in human beings.     

Municipal sludge leachate-induced genotoxicity in mice--a subacute study

Inappropriate disposal of municipal sludge (MS) results in the leaching of toxic metals and organic chemicals, which can contaminate the surface and ground water leading to the serious health hazards. In this study, the genotoxic potential of the leachate prepared from MS sample was examined in mouse bone marrow cells through chromosomal aberrations (CA), micronucleus test (MT) and comet assay. Analysis of metals and physicochemical parameters of the leachate was also carried out to correlate the genotoxic results. The dried sludge showed high concentrations of heavy metals, viz. Cr, Cu, Pb and Ni. However, in 10% leachate, concentrations of these metals were manifold lower than that of obtained in dried sludge. Male mice orally gavaged to leachates (0.1-0.4 ml/mouse/day) for 15 days revealed significant (P<0.01, P<0.001) inhibition of mitotic index (MI) and induction of chromatid/chromosome fragments and breaks in all the treatment groups. The effect was observed to be dose-dependent. Treatment of mice with leachates also induced significant (P<0.001) frequencies of micronucleated polychromatic erythrocytes (MNPCE). The results of comet assay revealed a statistically significant (P<0.05 and <0.01) DNA damage in bone marrow cells exposed to 0.2-0.4 ml/mouse/day. Findings of the present study indicate that the constant exposure of MS leachate can cause genotoxic effects in mammals and suggest risks in human population.     

GABA in the endocrine pancreas: cellular localization and function in normal and diabetic rats

Gamma amino butyric acid (GABA) and its related enzymes have been demonstrated in pancreatic beta cells of normal rat. Antibodies against GABA-synthesizing enzymes have been implicated in the pathogenesis of Type I diabetes. In spite of the importance of GABA in the aetiology of diabetes mellitus, detailed morphological data on the pattern of distribution of GABA in the pancreas of normal and diabetic rats are lacking. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (60 mg kg body weight(-1)). Four weeks after the induction of DM, normal (n = 6) and diabetic (n = 6) rats were anesthetized with chloral hydrate and their pancreata were removed and processed for the localization and effect of GABA on insulin secretion using immunohistochemistry and radioimmunoassay techniques. The number of GABA-like immunoreactive (GABA-LIR) cells in the pancreatic islets of STZ-diabetic rats decreased significantly (P<0.0001) when compared to non-diabetic control rats. The pattern and percentage distribution of GABA in the islet of Langerhans of normal and diabetic rat was similar to that of insulin. GABA induced a significant (P<0.0007) increase in insulin secretion from the pancreas of normal rats. In diabetic pancreas, GABA evoked a higher but not significant (P<0.1) increase in insulin secretion. These findings showed that the number of GABA-LIR cells is reduced significantly in diabetes. Moreover, GABA is a strong secretagogue of insulin from the pancreas of normal rat.     

Computerized automated morphometric assay including frequency estimation of pentachlorophenol induced nuclear anomalies (micronucleus) in catfish Heteropneustes fossilis

An in vivo study of the effects of pentachlorophenol was carried out with a pre-acclimatized fish species, Heteropneustes fossilis, using four sub-lethal concentrations, 0.1, 0.2, 0.3 and 0.4 ppm, and three sampling times, 48, 72 and 96 h. Cytogenetic preparations were stained by the haematoxylin-eosin technique. The incidence of micronuclei was scored by a manual and an automated method. Small-sized micronuclei appeared in the cytoplasm in addition to the main nucleus. The frequency of micronucleated erythrocytes peaked at 4 days (96 h) exposure. The percentage of single micronuclei increased with longer exposures. The Mann-Whitney U test showed all micronuclei frequencies were significantly different from control (P<0.05). No statistical difference was observed between scores obtained by the manual and automated methods. A linear relationship between the percentage of micronucleated erythrocytes and dose was confirmed at all levels. Computer image analysis of morphological variations of erythrocytes indicated a 1:5 ratio of micronuclei and main nucleus accompanied by a reduction in cell volume by 600 dot units. Pentachlorophenol-mediated genotoxicity was confirmed in this fish for the first time. Possible consequences of genotoxicity and cytotoxicity are discussed.     

Deterioration of Bone Quality by Streptozotocin (STZ)-Induced Type 2 Diabetes Mellitus in Rats

Patients with diabetes mellitus (DM) have various skeletal disorders and bone quality can be impaired in DM leading to fractures. Wistar albino male rats (270?C300?g; n?=?16) were assigned randomly to nondiabetic and diabetic rats (single dose intravenous injection of 45?mg/kg streptozotocin). All rats in each group were perpetuated for 8?weeks, and blood glucose levels as well as body weights were measured once weekly. Biomechanical measurements were performed at the mid-diaphysis of the left femur with tensile test. Extrinsic and intrinsic properties were measured or calculated. Bone mineral density (BMD) was also evaluated and measured by dual-energy X-ray absorptiometry. Cross-sectional area of the femoral shaft was evaluated by computerized tomography. Blood glucose levels in diabetic rats were significantly increased compared to that of the nondiabetic rats, while the body and femur weights were decreased (P?<?0.05). In respect to the BMD, cross-sectional area and femur length, there were no statistically significant differences between the nondiabetic and diabetic rats (P?>?0.05). The maximum load, ultimate stress, and toughness endpoints in diabetic rats were significantly decreased compared to that of the nondiabetics (P?<?0.05). There were no statistically significant differences between the nondiabetic and diabetic rats with regard to the displacement and stiffness (P?>?0.05). Femurs of diabetic rats had less absorbed energy than that in nondiabetics (P?<?0.05). Ultimate strain was lower in diabetic rats than that in nondiabetics, while the elastic modulus was higher (P?>?0.05). The bone quality of rats is decreased by streptozotocin-induced type 2 diabetes mellitus.     

The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: implications for cytogenetic screening

Micronucleated erythrocytes are selectively removed from the peripheral circulation of normal rats. Splenectomy prevents this selective removal. In normal rats treated daily for 20 days with 0.2 mg/kg triethylenemelamine (TEM), micronucleated normochromatic (mature) erythrocytes did not accumulate in peripheral blood. In these same animals, the frequencies of micronucleated cells among polychromatic (newly formed) erythrocytes increased from 0.21 to 5.25 per thousand in peripheral blood and from 1.75 to 31.5 per thousand in bone marrow. Since both control and induced frequencies in peripheral blood were approximately 15% of those in bone marrow, the removal appears to be equally efficient for cells containing either spontaneously occurring or clastogen-induced micronuclei. In splenectomized rats treated daily for 11 days with 0.2 mg/kg TEM, the frequency of micronucleated normochromatic erythrocytes (NCEs) in the peripheral blood rose rapidly to 9 times the control value and remained elevated for 50-55 days, indicating a life span approximately equivalent to that of normal erythrocytes. Among splenectomized rats exposed to either 0.15 mg/kg triethylenemelamine, 6.5 mg/kg cyclophosphamide, or 300 mg/kg urethane for periods exceeding the erythrocyte life span, the incidences of micronucleated NCEs in the peripheral blood rose steadily from a control value of 1.0 per thousand to maximum values of 15.0, 12.7 and 8.9 per thousand, respectively. During these extended exposures, the mean frequencies of micronucleated polychromatic erythrocytes (PCEs) in peripheral blood increased from a spontaneous value of 0.9 per thousand to 23.0, 13.0 and 6.6 per thousand, respectively, reflecting the frequencies among PCEs in the bone marrow and approximating the maximum values among NCEs in the peripheral blood. Thus, the frequency of micronucleated erythrocytes in the peripheral blood of splenectomized rats can be used as an index of both acute and cumulative chromosomal damage, while in normal rats the use of peripheral blood for cytogenetic monitoring is restricted by the selective removal of these micronucleated cells.     

Type 2 diabetes alters mesenchymal stem cell secretome composition and angiogenic properties

This study aimed at characterizing the impact of type 2 diabetes mellitus (T2DM) on the bone marrow mesenchymal stem cell (BMMSC) secretome and angiogenic properties. BMMSCs from Zucker diabetic fatty rats (ZDF) (a T2DM model) and Zucker LEAN littermates (control) were cultured. The supernatant conditioned media (CM) from BMMSCs of diabetic and control rats were collected and analysed. Compared to results obtained using CM from LEAN‐BMMSCs, the bioactive content of ZDF‐BMMSC CM (i) differently affects endothelial cell (HUVEC) functions in vitro by inducing increased (3.5‐fold; P < 0.01) formation of tubule‐like structures and migration of these cells (3‐fold; P < 0.001), as well as promotes improved vascular formation in vivo, and (ii) contains different levels of angiogenic factors (e.g. IGF1) and mediators, such as OSTP, CATD, FMOD LTBP1 and LTBP2, which are involved in angiogenesis and/or extracellular matrix composition. Addition of neutralizing antibodies against IGF‐1, LTBP1 or LTBP2 in the CM of BMMSCs from diabetic rats decreased its stimulatory effect on HUVEC migration by approximately 60%, 40% or 40%, respectively. These results demonstrate that BMMSCs from T2DM rats have a unique secretome with distinct angiogenic properties and provide new insights into the role of BMMSCs in aberrant angiogenesis in the diabetic milieu.     

Expression of GLUT4 glucose transporter protein in adipose tissue and skeletal muscle from streptozotocin-induced diabetic pregnant rats.

Depletion of GLUT4, the primary glucose transporter protein in adipose tissue and skeletal muscle, is reported to contribute to insulin resistance in pregnancy or diabetes. To examine this phenomenon, the expression of GLUT4 protein was assessed by Western blotting in streptozotocin-induced diabetic pregnant rats. In adipose tissue, relative to control, it was decreased by 30% in the normal pregnant group (p<0.001), by 37% in the diabetic nonpregnant group (p<0.01) and by 65% in the diabetic pregnant group (p<0.001). On the other hand, no significant variation was evident among the groups in skeletal muscle. To assess the mechanisms responsible for depletion of GLUT4 protein in adipose tissue, we quantitated levels of GLUT4 mRNA with a RNase protection assay. It was decreased by 44% in the normal pregnant group (p<0.05) and by 55% in the diabetic pregnant group (p<0.05), but not altered in the diabetic nonpregnant group. These results suggest that the depletion of GLUT4 protein in adipose tissue is a factor contributing to insulin resistance in pregnancy or diabetes, especially when the two states exist in combination.     

The induction of chromosomal damage in rat hepatocytes and lymphocytes I. Time-dependent changes of the clastogenic effects of diethylnitrosamine,dimethylnitrosamine and ethyl methanesulfonate

Male Wistar rats received a single injection of diethylnitrosamine (DEN), dimethylnitrosamine (DMN) or ethyl methanesulfonate (EMS). After a number of time intervals (up to 56 days) liver cells were assayed for the presence of possible preclastogenic damage by performing partial hepatectomy and subsequent analysis of chromosomal damage (micronucleus formation) in isolated hepatocytes. Peripheral blood lymphocytes from the same animals were collected, stimulated to proliferate and assayed for the frequency of sister-chromatid exchanges (SCEs). Whereas all agents significantly increased frequencies of SCEs in lymphocytes up to at least 28 days (EMS) or 56 days (DMN, DEN) after injection, only the latter 2 compounds gave rise to significantly increased incidences of micronucleated hepatocytes. DMN-induced preclastogenic damage in hepatocytes was lost between 28 and 56 days after injection. After DEN, this type of damage was persistent over the entire experimental period (56 days).When rats treated with DEN did not undergo partial hepatectomy, the frequencies of micronuclei at different time intervals after treatment were at control level. This result, together with those from hepatectomized DEN-treated rats, suggests that it is the persistent character of the preclastogenic damage that is responsible for the occurrence of micronucleated hepatocytes at later time intervals after treatment with DEN, rather than the stability of micronuclei which might eventually have been formed soon after injection.     

Association between diabetes type 1 and DQB1 alleles in a case-control study conducted in Montevideo,Uruguay

We studied HLA DQB1 allele frequencies and the relative risk (RR) of various genotypes in 72 type 1 diabetic patients and 40 control individuals in Uruguay. This is a tri-racial (Caucasian, Black and Indo-American) mixed population. The products of the polymerase chain reaction amplifications were hybridized with oligonucleotides by allele-specific oligonucleotide reverse or dot blot methods. Significant differences between these two groups were observed only for allele DQB1*0302 (35%, RR = 7.34, P<0.001). The frequency of the alleles carrying a non-aspartic acid residue at position 57 was significantly higher in the diabetic patients (85 vs 53%, P<0.001). In contrast, the frequency of Asp alleles was negatively associated with type 1 diabetes (RR = 0.20, P<0.001). The genotype DQB1*0302/DQB1*0201 (33%, RR = 5.41, P<0.05) was positively associated with this disease. The genotype frequencies associated with type 1 diabetes in our population were significantly different from what is known for Caucasian and Black populations as well as compared with another admixed population, from Chile.     

n‐3 Fatty Acids Modulate T‐Cell Calcium Signaling in Obese Macrosomic Rats

Objective: We investigated the effects of a diet containing EPAX‐7010, rich in PUFAs such as eicosapentaenoic acid [20:5(n‐3)] and docosahexaenoic acid [22:6(n‐3)], i.e., a PUFA/EPAX regimen, on T‐cell activation in diabetic pregnant rats and their obese pups. Research Methods and Procedures: Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin on Day 5 of gestation. T‐cell blastogenesis was assayed by using ³H‐thymidine, whereas intracellular free calcium concentrations ([Ca²⁺]i) were measured by using Fura‐2 in diabetic pregnant rats and their obese offspring. Results: Concavalin‐A‐stimulated T‐cell proliferation was decreased in both pregnant diabetic rats and their obese pups as compared with control animals. Feeding the PUFA/EPAX diet restored T‐cell proliferation in both groups of animals. We also employed ionomycin, which at 50 nM opens calcium channels, and thapsigargin (TG), which recruits [Ca²⁺]i from endoplasmic reticulum pool. We observed that ionomycin‐induced increases in [Ca²⁺]i in T‐cells of diabetic mothers and obese offspring were greater than in those of control rats. Furthermore, feeding PUFA/EPAX diet diminished significantly the ionomycin‐evoked rise in [Ca²⁺]i in diabetic and obese animals. TG‐induced increases in [Ca²⁺]i in T‐cells of diabetic pregnant rats and their obese offspring were greater than in those of control rats. The feeding of the experimental diet significantly curtailed the TG‐evoked increases in [Ca²⁺]i in both diabetic and obese rats. Discussion: Together, these observations provide evidence that T‐cell activation and T‐cell calcium signaling are altered during gestational diabetes and macrosomia. Hence, dietary fish oils, particularly eicosapentaenoic acid and docosahexaenoic acid, may restore these T‐cell abnormalities.