Evidence Of Mitochondrial Damage Research Articles

The Mechanism of Mitochondrial Injury in Alpha-1 Antitrypsin Deficiency Mediated Liver Disease.

Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.

Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology.

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (Pabpn1+/A17) that contains one alanine-expanded Pabpn1 allele under the control of the native promoter and one wild-type Pabpn1 allele. This mouse is the closest available genocopy of OPMD patients. We show that Pabpn1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous Pabpn1 knock-out mouse model (Pabpn1+/Δ). Like the Pabpn1+/A17 mice, Pabpn1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in Pabpn1+/Δ mice only partially overlap with those detected in Pabpn1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in Pabpn1+/A17 mice.

The role of the c-Jun N-terminal kinases 1/2 and receptor-interacting protein kinase 3 in furosemide-induced liver injury

1. The mechanisms of furosemide (FS) hepatotoxicity were explored in mice. Specifically, C57Bl/6 J mice were treated with 500 mg FS/kg bodyweight, and c-Jun N-terminal kinase (JNK) activation and receptor-interacting protein kinase 3 (RIP3) expression were measured by western blotting. Co-treatment with FS and the JNK inhibitor SP600125 was also performed, and FS-induced liver injury was compared in wild-type and RIP3 knockout (KO) mice.2. JNK phosphorylation and RIP3 expression were increased in livers from the FS-treated mice as early as 6 h after treatment and persisted until at least 24 h. JNK phosphorylation was also observed in primary mouse hepatocytes and human HepaRG cells treated with FS.3. Phosphorylated JNK translocated into mitochondria in livers, but no evidence of mitochondrial damage was observed.4. SP600125-treated mice, SP600125 co-treated primary mouse hepatocytes and RIP3 KO mice were not protected against FS hepatotoxicity. These data show that, although JNK activation and RIP3 expression are induced by FS, neither contributes to the liver injury.

Liver ultrastructural morphology and mitochondrial DNA levels in HIV/hepatitis C virus coinfection: no evidence of mitochondrial damage with highly active antiretroviral therapy

Liver mitochondrial toxicity is a concern, particularly in HIV/hepatitis C virus (HCV) coinfection. Liver biopsies from HIV/HCV co-infected patients, 14 ON-highly active antiretroviral therapy (HAART) and nine OFF-HAART, were assessed by electron microscopy quantitative morphometric analyses. Hepatocytes tended to be larger ON-HAART than OFF-HAART (P = 0.05), but mitochondrial volume, cristae density, lipid volume, mitochondrial DNA and RNA levels were similar. We found no evidence of increased mitochondrial toxicity in individuals currently on HAART, suggesting that concomitant HAART should not delay HCV therapy.

Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells

Stem cells are a key target of environmental toxicants, but little is known about their toxicological responses. We aimed at developing an in-vitro model based on adult human stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI]) and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were exposed for 48 h to 0.1 μM and 10 μM Cr(VI) or Cd. Cultures treated with 10 μM Cr(VI) or Cd showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not show the morphologic hallmarks of apoptosis. Treatment with 0.1 μM Cr(VI) or Cd did not result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum and evidence of mitochondrial damage. We conclude that autophagy is implicated in the response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd. Autophagy, which mediates cell survival and death under stress, deserves further evaluation to be established as biomarker of metal exposure.

Effect of pH on uptake and photodynamic action of chlorin p6 on human colon and breast adenocarcinoma cell lines.

The effect of reducing the extracellular pH from 7.4 to 6.0 on the uptake and photosensitivity of chlorin p6, a potential photosensitising drug, has been investigated using two mammalian cell lines, human colon (Colo-205) and breast (MCF-7) adenocarcinoma cells. In Colo-205 cells, the uptake and phototoxicity of chlorin p6 was observed to increase as the pH of the incubation medium decreased. For light doses of up to [similar]6 kJ m(-2), although there was no evidence of mitochondrial damage, a significant reduction in Neutral Red uptake was observed, signifying damage to lysosomes. At higher light doses, significant mitochondrial damage was observed, accompanied by saturation of the lysosomal damage. This suggests light-induced relocalization of the photosensitizer from lysosomes to mitochondria. Furthermore, it was found that for a given light dose, lysosomes exhibit greater photosensitivity at lower pH. Since chlorin p6 is known to aggregate at pH 6.0, this observation suggests that the dye accumulation in these cells mainly takes place through endocytosis. In contrast, no significant variation in uptake, photosensitivity, and sites of photodamage was observed for MCF-7 cells at different extracellular pH. Additionally, the lower photosensitivity of lysosomes as compared to mitochondria in these cells suggests chlorin p6 is taken up through diffusion rather than endocytosis.

Non-invasive, in-vivo electrical impedance of EMT-6 tumours during hyperthermia: Correlation with morphology and tumour-growth-delay

The electrical impedance at frequencies from 100 Hz to 40 MHz of EMT-6 tumours was measured non-invasively, in vivo, during hyperthermia using an apparatus constructed for this purpose. Histology and morphometry were performed on tumours harvested periodically during the heating. A ratio of conductivities at two frequencies (s`10MHz/s`10kHz). which minimizes the tissue temperature-coefficient effects, was used to correlate impedance changes with the histopathological changes. The bulk of the cell population followed a necrotic cell death sequence during the heating. Initial increase of the s`-ratio correlated with cell swelling, and a reversal of the rate of this increase correlated with the appearance of small membrane breaks and evidence of mitochondrial damage. A continued, slowing s`-ratio increase to a maximum correlated with continued cell swelling accompanied by increasing membrane disruption. The subsequent decrease in u-ratio correlated with continued general cell lysing. Between the appearance of the first membrane breaks (s`-ratio rate peak) and the evidence of general lysing (s`-ratio peak), the tumour-growth-delay increased non-linearly. Because the s`-ratio consistently discerned these events, these measurements were able to predict the fate of this cell population when subjected to hyperthermia. Knowledge of temperature or time of heating was not required.

Effects of ethylene glycol on the ultrastructure of hepatocytes.

The ultrastructure of rat hepatocytes after acute experimental ethylene glycol poisoning was examined. On the 1st, 5th and 14th days after poisoning the material from the centrolobular zone (zone III) was collected. Proliferation and enlargement of SER at the early period of poisoning and evidence of mitochondrial damage both at the early and late time after ethylene glycol intoxication were found. In the liver extensive capillary deposits surrounded by membranes were seen, filled with flocculent material of middle electron density. The results show destruction of the cytoplasmic organelles, especially mitochondria, on the 1st and 5th days after ethylene glycol intoxication, and symptoms of damage removing together with regeneration on the 15th day of the experiment.

Mitochondrial damage as a mechanism of cell injury in the killing of cultured hepatocytes by tert-butyl hydroperoxide

The killing of cultured hepatocytes by tert-butyl hydroperoxide (TBHP) occurs by different mechanisms depending on the presence or absence of the antioxidant N,N - ́ diphenylphenylenediamine (DPPD). In either situation there is evidence of mitochondrial damage. The mitochondrial inner membrane potential is lost, a result determined by the release from the cells of the lipophilic cation [ 3H]triphenylmethylphosphonium (TPMP +). Deenergization of the mitochondria is accompanied by a loss of ATP. Oligomycin reduced ATP stores without release of TPMP + or without effect on the viability of the hepatocytes over the same time course that TBHP killed the majority of the cells. Monensin, a H + Na + ionophore, potentiated the toxicity of tert-butyl hydroperoxide in the presence or absence of DPPD. By contrast, extracellular acidosis reduced the toxicity of tert-butyl hydroperoxide in the presence or absence of DPPD. Neither monensin nor extracellular acidosis affected the metabolism of tert-butyl hydroperoxide, the release of TPMP +, or the extent of the peroxidation of cellular lipids. These data document the presence of mitochondrial damage in hepatocytes intoxicated with TBHP in both the presence and absence of DPPD. Furthermore, the potentiation by monensin is readily explained by the proposal that mitochondrial deenergization is accompanied by an intracellular acidosis. Such acidosis tends to delay the development of lethal cell injury. The protective effect of extracellular acidosis supports this interpretation.

Subcellular fractionation studies on hepatic tissue from a patient with Pompe's disease (type II glycogen-storage disease).

1. An homogenate of liver biopsy taken from a patient with Pompe's disease (type II glycogen-storage disease) was analysed by enzyme microassay and subcellular fractionation by sucrose-density-gradient centrifugation. 2. A strikingly low activity of lysosomal acid alpha-D-glucosidase was noted but there was also increased activity of neutral alpha-D-glucosidase (found in endoplasmic reticulum). 3. Activities of the acid hydrolases, acid phosphatase and beta-N-acetyl-D-glucosaminidase, were elevated compared with those of controls. Measurement of latent beta-N-acetyl-D-glucosaminidase activity and results of subcellular fractionation experiments, indicated a marked fragility of certain populations of lysosomes. Lysosomes containing predominantly acid phosphatase and beta-D-glucuronidase activities appeared to have normal integrity. 4. Assessment of organelle pathology by enzyme microassay indicated low mitochondrial and peroxisomal enzyme activities. In addition there was evidence of mitochondrial damage as reflected by increased sucrose permeability and by ultrastructural studies.

Mitochondria in Allomyces under experimental conditions

1. 1. The vegetative hyphae of aquatic fungi are described as favorable material for the study of mitochondria in the living state, particularly when observed with phase contrast optics. 2. 2. Immobilization of mitochondria without morphological change was realized by treatment of hyphal cells with 1 per cent solution of OsO 4. 3. 3. When hyphae were mounted in isotonic NaCl, the mitochondria, being as sensitive to mechanical shock as they were when the hyphae were in water, disintegrated under pressure. In hypertonic saline, cells were at first plasmolysed, but recovered. Mitochondria in plasmolysed cells changed shape, fragmented, became vesiculated, and disappeared after becoming pale and non-refractive. 4. 4. In cells treated with 0.88 M sucrose, which has been useful in isolating mitochondrial particles from animal tissues, the fungal mitochondria quickly fragmented into spheres about 1 micron in diameter. 5. 5. A solution of 10 −2 M KCN in 10 −2 M glutamic acid, at pH 7.0, caused cessation of movement of mitochondria and inhibited staining by Janus green B. No evidence of mitochondrial damage was apparent. 6. 6. Temperatures of 8 °–45 ° C. had no visible effect on mitochondria. At higher temperatures, the results were as follows: 50 ° C., mitochondria became slender, and almost invisible; at 65 ° C., all mitochondrial motion ceased; at 75 ° C., mitochondria broke up into beads which became hollow spheres and disappeared. 7. 7. Mitochondrial orientation is determined more by the direction of protoplasmic streaming than by direction of diffusion. 8. 8. Small granules, never positively identified as normal mitochondria, were “stained” red after 48 hours with triphenyl tetrazolium chloride when the salt was incorporated in the medium at a concentration of 0.005 per cent. Higher concentrations were lethal. The sites at which reduction of the tetrazolium was most active were in the fat droplets. 9. 9. Because of the diversity of behavior and function of the particles referred to as mitochondria, it is suggested that many cytoplasmic particles, even though they do accumulate Janus green, are scarcely comparable to one another.