Metformin induces ultrastructural alterations in hepatocytes of spontaneously hypertensive rats / La metformina induce alteraciones ultraestructurales en hepatocitos de ratas espontáneamente hipertensas

Metformin, an oral biguanide approved for the treatment of type II diabetes is widely prescribed for other clinical conditions. Currently, metformin is being investigated as potential anti-tumor agent. However, there have been recent concerns about hepatotoxicity associated with the use of metformin. This study, by means of high resolution transmission electron microscopy (TEM) and morphometry, investigated potential ultrastructural changes induced by metformin treatment on the hepatocytes of spontaneously hypertensive rats (SHR). Morphometric analysis was carried out on images of randomly selected cells from sectioned gluteraldehyde-osmium-fixed, Epon embedded liver tissue. One-way analysis of variance (ANOVA) on morphometric data showed statistically significant differences in the mean volume density (MVD) of lipid bodies (F=136.48, P<0.0001)and mean surface density (MSD) of endoplasmic reticulum (ER) (F=12.45, P<0.003) between hepatocytes of control (n=8) and metformin-treated (MT) (n=8) animals. MVD for control group was 5.42% (±0.36 SEM) but decreased significantly in the MT group (1.13%, ±0.04 SEM). Similarly, MSD of ER for control was 24.7 µm2/µm3 (±1.64 SEM) but decreased for MT animals (18.90 µm2/µm3, ±0.28 SEM). These data are most likely consistent with the effects of metformin on lipid metabolism, and may not reflect on hepatotoxicity induced by the drug, in SHRs.

La metformina, una biguanida oral aprobada para el tratamiento de la diabetes tipo II, es también ampliamente prescrita para otros cuadros clínicos. Actualmente, la metformina está siendo investigada como posible agente anti-tumoral. Sin embargo, ha habido recientes preocupaciones acerca de la hepatotoxicidad asociada con el uso de metformina. En este estudio, por medio de la microscopía electrónica de transmisión (MET) de alta resolución y morfometría, se investigaron los posibles cambios ultraestructurales, inducidos por el tratamiento con metformina, en los hepatocitos de ratas espontáneamente hipertensas (REH). El análisis morfométrico se llevó a cabo en imágenes de células seleccionadas al azar a partir de tejido hepático seccionado, fijado con glutaraldehído-osmio e inmerso en Epon. El análisis de la varianza (ANOVA) de los datos morfométricos mostró diferencias significativas en la densidad de volumen medio (DVM) de cuerpos lipídicos (F=136,48, P<0,0001) y la densidad de superficie media (DSM) del retículo endoplasmático (RE) (F=12,45, P<0,003) entre los hepatocitos control (n=8) y los animales tratados con metformina (MT) (n=8). La DVM para el grupo control fue de 5,42% (±0,36 EEM), pero disminuyó significativamente en el grupo MT (1,13%, ±0,04 EEM). Del mismo modo, la DSM del RE para el grupo control fue de 24,7 µm2/µm3 (±1,64 EEM), pero disminuyó para los animales MT (18,90 µm2/µm3, ±0,28 EEM). Estos datos están probablemente más relacionados con los efectos de la metformina sobre el metabolismo de los lípidos, y no se relacionarían con la hepatotoxicidad por inducción de la droga, en REH.

Endoplasmic reticulum stress (ER-stress) by 2-deoxy-D-glucose (2DG) reduces cyclooxygenase-2 (COX-2) expression and N-glycosylation and induces a loss of COX-2 activity via a Src kinase-dependent pathway in rabbit articular chondrocytes

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG-modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.

2-Deoxy-D-glucose regulates dedifferentiation through beta-catenin pathway in rabbit articular chondrocytes

2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the beta-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and beta-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of beta-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of beta-catenin, as the 2DG stimulated accumulation of beta-catenin, which is characterized by translocation of beta-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of beta-catenin degradation by inhibition of glycogen synthase kinase 3-beta with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of beta-catenin whereas the transcriptional level of beta-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via beta-catenin pathway in rabbit articular chondrocytes.

Baicalein protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis through inhibition of reactive oxygen species production and CHOP induction

Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi and possesses various effects, including cytoprotection and anti-inflammation. Because endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, we investigated the effects of baicalein on apoptotic death of HT22 mouse hippocampal neuronal cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were measured by flow cytometry. Expression level and phosphorylation status of ER stress-associated proteins and activation and cleavage of apoptosis-associated proteins were analyzed by Western blot. Baicalein reduced TG- and BFA-induced apoptosis of HT22 cells and activation and cleavage of apoptosis-associated proteins, such as caspase-12 and -3 and poly(ADP-ribose) polymerase. Baicalein also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78, the cleavage of X-box binding protein-1 and activating transcription factor 6alpha, and the phosphorylation of eukaryotic initiation factor-2alpha and mitogen-activated protein kinases, such as p38, JNK, and ERK. Knock-down of CHOP expression by siRNA transfection and specific inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) as well as anti-oxidant (N-acetylcysteine) reduced TG- or BFA-induced cell death. Baicalein also reduced TG- and BFA-induced ROS accumulation and MMP reduction. Taken together, these results suggest that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.

Initiation Site of Ca2+ Entry Evoked by Endoplasmic Reticulum Ca2+ Depletion in Mouse Parotid and Pancreatic Acinar Cells

PURPOSE: In non-excitable cells, which include parotid and pancreatic acinar cells, Ca(2+) entry is triggered via a mechanism known as capacitative Ca(2+) entry, or store-operated Ca(2+) entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca(2+) stores, which acts as an important factor triggering Ca(2+) entry. However, both the mechanism of store-mediated Ca(2+) entry and the molecular identity of store-operated Ca(2+) channel (SOCC) remain uncertain. MATERIALS AND METHODS: In the present study we investigated the Ca(2+) entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system. RESULTS: Treatment with thapsigargin (Tg), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase, in an extracellular Ca(2+) free state, and subsequent exposure to a high external calcium state evoked Ca(2+) entry, while treatment with lanthanum, a non-specific blocker of plasma Ca(2+) channel, completely blocked Tg-induced Ca(2+) entry. Microfluorometric imaging showed that Tg-induced Ca(2+) entry started at a basal membrane, not a apical membrane. CONCLUSION: These results suggest that Ca2+ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.

Experimental pathological study of acute intoxication by Chloranthus serratus Roem. Et Schalt / 法医学杂志

OBJECTIVE@#To find out the pathological change and the toxic mechanism of Chloranthus serratus Roem. et Schalt in mice.@*METHODS@#Mice were intoxicated by oral administration with extracts of Chloranthus serratus Roem. et Schalt followed by pathological, serum biochemical, and coagulation mechanism examination.@*RESULTS@#The LD50 in mice was 41.12 g/kg; All poisoned mice serum BUN and ALT increased markedly; Thrombocyte decreased and coagulation time increased; The organ index of liver, spleen and kidneys increased significantly; The cells of liver, kidney and heart were degeneration and necrosis, There were extensive hyperemia and hemorrhage in many organs.@*CONCLUSION@#The experiment suggests that the target organs were liver, kidney, heart and blood vessels; The toxic mechanism was the damage on the mitochondrional, endoplasmic reticulum and coagulation system.

Ultrastructural changes in cauda epididymidal epithelial cell types of Azadirachta indica leaf treated rats.

To assess if cauda epididymis is a target for the effect of A. indica leaves, Wistar strain male albino rats were administered (po) A. indica leaves (100 mg/rat/day for 24 days). Transmission electron microscopic analysis revealed that in the cauda epididymal epithelium the nuclei of principal cells were enlarged and the number of coated micropinocytotic vesicles of the apical cytoplasm decreased. Microvilli were missing and mitochondrial cristae and Golgi complex were highly disrupted. The cytoplasm was abounding with lysosomal bodies. The clear cells increased in perimeter and their nuclei increased in size and contained lesser chromatin. The nuclear membrane bulged out. The cytoplasm was vacuolized. Further, there was decrease in size of the lipid droplets, mitochondria, Golgi complex, endoplasmic reticulum and there was accumulation of lysosomal bodies. The changes in the principal and clear cells appear to be due to the effect of the hypoandrogen status caused by treatment with A. indica leaves and a direct action on the epididymal epithelium.

Tissue alterations in the Guinea pig lateral prostate following antiandrogen flutamide therapy

The flutamide antiandrogenic effects on the Guinea pig male prostate morphology in puberal, post-puberal and adult ages were evaluated in the present study. Daily-treated group animals received flutamide subcutaneous injection at a dose of 10 mg/Kg body weight for 10 days. The control group animals received a pharmacological vehicle under the same conditions. The lateral prostate was removed, fixed and processed for light and transmission electron microscopy. The results revealed an increase of the acinus diameter in the treated puberal animals and straitness in the stromal compartment around the acini. The epithelial cells exhibited cubic phenotype. In the post-puberal and adult animals, a decrease of the acinus diameter was observed, as well as an increase of the smooth muscle layer and presence of the folds at epithelium. The ultrastructural evaluation of the secretory cells in the treated group demonstrated endomembrane enlargement, mainly in the rough endoplasmic reticulum and Golgi apparatus. In addition, a decrease of the microvilli and alterations in the distribution patterns and density of the stromal fibrillar components were observed. In conclusion, the flutamide treatment exerts tissue effects on the lateral prostate, promoting stroma/epithelium alterations.

Responses of serum calcium and inorganic phosphate levels, parathyroid gland and C cells of Rattus norvegicus to heptachlor administration.

Intramuscular administration of two doses: 0.50 LD50 (14.70 mg/kg b w) and 0.75 LD50 (22.30 mg/kg b w) of heptachlor in Rattus norvegicus for 14 days induced significant hypocalcemia without altering serum inorganic phosphate value. Parathyroid chief cells of the experimental rats exhibited degranulation, vacuolation, loss of secretory granules and lipid droplets, reduction in chromatin, and degenerative changes in endoplasmic reticulum and cristae of the mitochondria. Not much of histological and ultrastructural changes could be seen in C cells of the heptachlor treated rats.

Inhibition of membrane Na(+)-K+ Atpase of the brain, liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plasticizer used in polyvinyl chloride (PVC) blood storage bags.

Significant amounts of di(2-ethylhexyl) phthalate (DEHP) leach out into blood stored in DEHP plasticized polyvinyl chloride (PVC) bags resulting in the exposure of recipients of blood transfusion to this compound. The aim of this study was to find out whether DEHP at these low levels has any effect on the activity of membrane Na(+)-K+ ATPase, since a decrease in this enzyme activity has been reported to take place in a number of disorders like neurodegenerative and psychiatric disorders, coronary artery disease and stroke, syndrome-X, tumours etc. DEHP was administered (ip) at a low dose of 750 microg/100 g body weight to rats and the activity of membrane Na(+)-K+ ATPase in liver, brain and RBC was estimated. Histopathology of brain, activity of HMG CoA reductase (a major rate limiting enzyme in the isoprenoid pathway of which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is a product), intracellular concentration of Ca2+ and Mg2+ in RBC (which is altered as a result of inhibition of Na(+)-K+ ATPase) were also studied. (In the light of the observation of increase of intracellular Ca2+ load and intracellular depletion of Mg2+ when Na(+)-K+ ATPase is inhibited). Histopathology of brain revealed areas of degeneration in the rats administered DEHP. There was significant inhibition of membrane Na(+)-K+ ATPase in brain, liver and RBC. Intracellular Ca2+ increased in the RBC while intracellular Mg2+ decreased. However activity of hepatic HMG CoA reductase decreased. Activity of Na(+)-K+ ATPase and HMG CoA reductase, however returned to normal levels within 7 days of stopping administration of DEHP. The inhibition of membrane Na(+)-K+ ATPase activity by DEHP may indicate the possibility of predisposing recipients of transfusion of blood or hemodialysis to the various disorders mentioned above. However since this effect is reversed when DEHP administration is stopped, it may not be a serious problem in the case of a few transfusion; but in patients receiving repeated blood transfusion as in thalassemia patients or patients undergoing hemodialysis, possibility of this risk has to be considered. This inhibition is a direct effect of DEHP or its metabolites, since activity of HMG CoA reductase, (an enzyme which catalyses a major rate limiting step in the isoprenoid pathway by which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is synthesized) showed a decrease.

Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species.

Effect of propriety herbal formulation against chronic carbon tetrachloride induced hepatotoxicity.

Efficacy of propriety herbal formulation (PHF) against carbon tetrachloride (CCl4) induced liver damage was investigated in adult rats. Administration of CCl4 (0.2 ml/kg; i.p.) twice a week for 12 weeks resulted in significant elevation in serum transaminases activity. Level of reduced glutathione was significantly decreased. On the contrary, significant elevation was found in the hepatic lipid peroxidation level. Proliferation of fibroblast replaced the hepatic parenchyma cells in focal areas. Cell organelles like mitochondria, endoplasmic reticulum and nucleus showed severe degeneration after CCl4 exposure. PHF was effective in restoring the CCl4 induced biochemical and histological ultrastructural changes.

Morphological changes in the liver on chronic administration of small doses of paracetamol in albino rats.

30 male albino rats of Wistar strain were used to study the effect of repeated administration of small doses of paracetamol over a period of 24 weeks. The animals were administered 0.3 g of paracetamol orally admixed in the diet, after an overnight fast. Three animals from the test group and one control were sacrificed at the end of 4, 8, 12, 16, 20 and 24 weeks, under ether anaesthesia. Liver cell necrosis was not seen at any stage of the experiment. Fatty change was focal at 4 to 8 weeks and diffuse at 12 to 24 weeks. Chronic administration of paracetamol admixed in the diet showed less extensive changes comprising of focal ballooning degeneration. Ultrastructural changes involved mitochondria and RER. Fibrosis was not seen.