Lysosomal Fragility Research Articles

MEDB-32. Reducing treatment-related toxicity for children with WNT-activated medulloblastoma

WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care (SOC) surgical resection, radiotherapy, and chemotherapy. Unfortunately, while curative, this treatment is associated with major, long-term, debilitating motor, developmental, and neuroendocrine side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. Similarities have been demonstrated between cancer cell lysosomes and those of patients with Niemann-Pick, a lysosomal storage disease characterised by lysosomal fragility and sphingomyelin accumulation. A class of drugs known as Functional Inhibitors of Acid Sphingomyelinase (FIASMAs), increase lysosomal sphingomyelin and destabilise the cancer cell’s more fragile lysosomal membrane which leads to the induction of cell-death pathways via lysosomal membrane permeabilisation. Loratadine, an antihistamine with high FIASMA activity, consistently induced lysosomal membrane permeabilisation, leading to increased cell-death, in our panel of mouse and human WNT-medulloblastoma lines. Loratadine exhibited no detrimental effect on normal mouse embryonic stem cells from the lower rhombic lip – the putative cell of origin in WNT-medulloblastoma. Luciferase-expressing mouse WNT-medulloblastoma cells were orthotopically implanted into CD1-nude mice and monitored for tumour development via bioluminescent imaging. Upon tumour engraftment, mice were subjected to reduced SOC (radiotherapy and adjuvant vincristine) plus a clinically relevant dose of loratadine. Response and survival were compared to mice treated with full SOC (radiotherapy, vincristine, cisplatin, and etoposide). Mice treated with 2mg/kg/day of loratadine following reduced SOC demonstrated increased survival when compared to those treated with full SOC (p=0.02) along with a significant reduction in weight loss during treatment (p=<0.0001). This work suggests that loratadine, or other FIASMA compounds, may be good alternative adjuvant therapies for WNT-medulloblastoma. Using less toxic adjuvants could improve long-term outcomes through reducing therapeutic related toxicities for children with this devastating disease.

Cytotoxicity and genotoxicity of methomyl, carbaryl, metalaxyl, and pendimethalin in human umbilical vein endothelial cells

AbstractPesticides have adverse effects on the cellular functionality, which may trigger myriad of health consequences. However, pesticides‐mediated toxicity in the endothelial cells (ECs) is still elusive. Hence, in this study, we have used human umbilical vein endothelial cells (HUVECs) as a model to quantify the cytotoxicity and genotoxicity of four pesticides (methomyl, carbaryl, metalaxyl, and pendimethalin). In the MTT assay, HUVECs exposed to methomyl, carbaryl, metalaxyl, and pendimethalin demonstrated significant proliferation inhibition only at higher concentrations (500 and 1000 μM). Likewise, neutral red uptake (NRU) assay also showed proliferation inhibition of HUVECs at 500 and 1000 μM by the four pesticides, confirming lysosomal fragility. HUVECs exposed to the four pesticides significantly increased the level of intracellular reactive oxygen species (ROS). Comet assay and flow cytometric data exhibited DNA damage and apoptotic cell death in HUVECs after 24 h of exposure with methomyl, metalaxyl, carbaryl, and pendimethalin. This is a first study on HUVECs signifying the cytotoxic–genotoxic and apoptotic potential of carbamate insecticides (methomyl and carbaryl), fungicide (metalaxyl), and herbicide (pendimethalin). Overall, these pesticides may affect ECs functions and angiogenesis; nonetheless, mechanistic studies are warranted from the perspective of vascular biology using in vivo test models.

Mechanisms of iron hepatotoxicity

Mechanisms of iron hepatotoxicity

Ferritin-stimulated lipid peroxidation, lysosomal leak, and macroautophagy promote lysosomal “metastability” in primary hepatocytes determining in vitro cell survival

Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal “metastability” in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.

Hepcidin knockout mice fed with iron-rich diet develop chronic liver injury and liver fibrosis due to lysosomal iron overload

Hepcidin is the central regulator of iron homeostasis and altered hepcidin signalling results in both hereditary and acquired iron overload. While the association between iron overload and development of end-stage liver disease is well established, the underlying mechanisms are largely unknown. To improve that, we analysed hepcidin knockout (KO) mice as a model of iron overload-associated liver disease. Hepcidin wild type (WT) and KO mice fed with 3% carbonyl iron-containing diet starting at one month of age were compared to age-matched animals kept on standard chow. Liver histology and serum parameters were used to assess the extent of liver injury and fibrosis. Iron distribution was determined by subcellular fractionation and electron microscopy. Among mice kept on iron-rich diet, 6 months old hepcidin KO mice (vs. WT) displayed profound hepatic iron overload (3,186 ± 411 vs. 1,045 ± 159 μg/mg tissue, p<0.005), elevated liver enzymes (ALT: KO 128 ± 6, WT 56 ± 5 IU/L, p<0.05), mild hepatic inflammation and hepatocellular apoptosis. Twelve, but not six months old KO mice fed with iron-rich diet developed moderate liver fibrosis. The liver injury was accompanied by a marked lysosomal iron overload and lysosomal fragility with release of cathepsin B into the cytoplasm. Increased p62 levels and autofluorescent iron complexes suggested impaired protein degradation. As a mechanism leading to lysosomal iron overload, the autophagy (lysosomal influx) was increased. Hepcidin KO mice represent a novel model of iron overload-related liver diseases and implicate lysosomal injury as a crucial event in iron toxicity.

Density shift, morphological damage, lysosomal fragility and apoptosis of hemocytes of Indian molluscs exposed to pyrethroid pesticides

Bellamya bengalensis (Gastropoda: Prosobranchia) and Lamellidens marginalis (Bivalvia: Eulamellibranchiata) are the molluscs of Indian freshwater ecosystem and important biological resources. These edible species bear economical, ecological, nutritional and medicinal importance. Natural habitat of these organisms is under the ecological threat of contamination by cypermethrin and fenvalerate, the common pyrethroid pesticides of India. Hemocytes are chief immunoeffector cells of molluscs which exhibit responsiveness against environmental toxins and perform diverse immunological functions including phagocytosis, encapsulation and cytotoxicity. Experimental exposure of cypermethrin and fenvalerate resulted in significant shift in density and morphological damage in hemocytes of B. bengalensis and L. marginalis respectively. Pyrethroid induced fragility and destabilization of hemocyte lysosomal membrane was recorded and proposed as an indication of toxin induced stress in molluscs. Apoptosis is an immunologically important cellular response which is modulated by environmental toxins. Pyrethroid exposure suppressed the physiological level of apoptosis and necrosis in hemocytes of B. bengalensis and L. marginalis indicating possible impairment of apoptosis mediated immunoprotection. Differential responses of B. bengalensis and L. marginalis hemocytes may be due to species specificity, toxin specificity, nonidentical immune strategies of Gastropoda and Bivalvia, specific habitat preference and related ecological niches.

Hepcidin knockout mice develop chronic liver injury and liver fibrosis as a consequence of lysosomal iron overload

Background and Aims: Hepcidin is the central regulator of iron homeostasis. Disrupted hepcidin signaling results in hereditary hemochromatosis and iron overload seen in chronic liver disorders. While the association between iron overload and development of end-stage liver disease is well established, the understanding of the underlying mechanisms is hampered by the lack of a suitable animal model. To circumvent this problem, we analyzed hepcidin-knockout (KO) mice as a model of iron-overload associated liver disease. Methods: Hepcidin wild-type (WT) and KO animals fed 3% carbonyl iron-containing chow were compared to mice kept on standard diet. Liver histology and serum parameters were employed to assess the extent of liver injury/fibrosis. Iron distribution was determined by subcellular fractionation and electron microscopy. Results: Among mice kept on iron-rich diet, 6 month old hepcidin KOs (vs. WTs) displayed profound hepatic iron overload (2543±114 vs. 1493±136p<0,005), elevated liver enzyme (AST: KO 261±15, WT 142±34p<0,05) and serum iron levels, mild hepatocellular inflammation and apoptosis. 12, but not 6 month old KOs fed iron-rich diet developed moderate liver fibrosis as determined by Sirius red staining and increased hydroxyproline levels. The liver injury was likely due to a marked lysosomal iron overload and lysosomal fragility with release of cathepsins into the cytoplasm, while no increased lipid peroxidation or protein carbonylation was noted. As a potential mechanism, the expression of DMT1 and STEAP3, i.e. the molecules needed for lysosomal iron export, was greatly reduced. Conclusions: Hepcidin KOs represent a unique tool to study the mechanism of iron overload-related liver diseases and implicate lysosomal injury as a crucial event in iron toxicity.

Haemocyte lysosomal fragility facing an environmental reality: A toxicological perspective with atrazine and Lymnaea stagnalis (Gastropoda, Pulmonata) as a test case

The present study investigates the potential use of haemocyte lysosomal fragility as a reliable indicator of toxic injury in snails subjected to herbicide atrazine. Two cytochemical assays were used. The first one was based on microscopic observations of neutral red dye, and the second one on a fluorescence spectroscopy approach using acridine orange, which had the advantage of being more reliable in assessing minor cell damage. Two sets of experiments were carried out in controlled laboratory conditions (temperature, photoperiod) and in outdoor microcosms, which produce more realistic ecological conditions. An unequivocal relationship was shown, linking atrazine exposure to Lymnaea stagnalis haemocytes lysosomal permeability. High temperatures also had a negative effect on the lysosomal membrane. In the microcosms, the response was affected by seasonal factors, and led to an integrated cell response. It was concluded that lysosomal sensitivity reflects environmental changes. Moreover, the technique using acridine orange was shown to be sufficiently sensitive and reliable to serve as a lysosomal marker in a controlled environment.

Elevated serum β-glucuronidase reflects hepatic lysosomal fragility following toxic liver injury in rats

The level of serum beta-glucuronidase increases in various pathological conditions, including liver disorders. The aim of this investigation was to study the changes in liver lysosomal membrane stability during experimentally induced hepatic fibrosis that may result in the elevation of serum beta-glucuronidase. Liver injury was induced by intraperitoneal injections of N-nitrosodimethylamine (NDMA) in adult male albino rats over 3 weeks. The progression of fibrosis was evaluated histopathologically as well as by monitoring liver collagen content. Lipid peroxides and beta-glucuronidase levels were measured in the liver homogenate and subcellular fractions on days 0, 7, 14, and 21 after the start of NDMA administration. Serum beta-glucuronidase levels were also determined. A significant increase was observed in beta-glucuronidase levels in the serum, liver homogenate, and subcellular fractions, but not in the nuclear fraction on days 7, 14, and 21 after the start of NDMA administration. Lipid peroxides also increased in the liver homogenate and the lysosomal fraction. The measurement of lysosomal membrane stability revealed a maximum lysosomal fragility on day 21 during NDMA-induced fibrosis. In vitro studies showed that NDMA has no significant effect on liver lysosomal membrane permeability. The results of this investigation demonstrated that lysosomal fragility increases during NDMA-induced hepatic fibrosis, which could be attributed to increased lipid peroxidation of lysosomal membrane. In this study, we also elucidated the mechanism of increased beta-glucuronidase and other lysosomal glycohydrolases in the serum during hepatic fibrosis.

Metabolic basis of ethanol-induced hepatic and pancreatic injury in hepatic alcohol dehydrogenase deficient deer mice

Alcoholic liver disease (ALD) and alcoholic pancreatitis (AP) are major diseases causing high mortality and morbidity among chronic alcohol abusers. Neutral lipid accumulation (steatosis) is an early stage of ALD or AP and progresses to inflammation and other advanced stages of diseases in a subset of chronic alcohol abusers. However, the mechanisms of alcoholic steatosis leading to ALD and AP are not well understood. Chronic alcohol abuse impairs hepatic alcohol dehydrogenase (ADH, a major enzyme involved in ethanol oxidative metabolism) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol). These esters are implicated in the pathogenesis of various alcoholic diseases and shown to cause hepatocellular and pancreatitis-like injury. Ethanol exposure is known to increase synthesis of FAEEs by several-fold in the livers and pancreata of rats pretreated with hepatic ADH inhibitor. Therefore, studies were undertaken to evaluate hepatocellular and pancreatic injury in hepatic ADH-deficient (ADH −) deer mice versus ADH-normal (ADH +) deer mice fed ethanol (4% wt/vol) via Lieber-DeCarli liquid diet for 60 days. A significant mortality was found in ethanol-fed ADH − deer mice (11 out of 18) versus ADH + deer mice (1 out of 16); most of the deaths occurred during the first 2 weeks of ethanol exposure. The surviving animals, sacrificed at the end of 60th day, showed distinct changes in hepatic and pancreatic histology and several-fold increases in nonoxidative metabolism of ethanol in ethanol-fed ADH − versus ADH + deer mice. Extensive vacuolization with displacement or absence of nucleus in some hepatocytes, and significant increase in hepatic neutral lipids were found in ethanol-fed ADH − versus ADH + deer mice. Ultrastructural changes showed perinuclear space, edema, presence of apoptotic bodies and disintegration, and/or dilatation of endoplasmic reticulum (ER) in the pancreata of ethanol-fed ADH − deer mice. FAEE levels were significantly higher in ADH − versus ADH + deer mice, approximately four-fold increases in the livers and seven-fold increases in the pancreata. Ethyl esters of oleic, linoleic, and arachidonic acids were the major FAEEs detected in ethanol-fed groups. The role of FAEEs in pancreatic lysosomal fragility is reflected by higher activity of cathepsin B (five-fold) in ethanol-fed ADH − versus ADH + deer mice. Although the present studies clearly indicate a metabolic basis of ethanol-induced hepatic and pancreatic injury, detailed dose- and time-dependent toxicity studies in this ADH − deer mouse model could reveal further a better understanding of mechanism(s) of ethanol-induced hepatic and pancreatic injuries.

Metal‐induced oxidative damage in cultured hepatocytes and hepatic lysosomal fraction: beneficial effect of a curcumin/absinthium compound

Metals undergo redox cycling and there is increasing evidence of free radical generation and oxidative injury in the pathogenesis of liver injury and fibrosis in metal storage diseases. The aim of the present study was to test a natural hepatoprotective compound in metal-induced liver injury. Hepatocytes were isolated from Wistar rats by collagenase perfusion method and cultured as such and also with alpha-linolenic acid (LNA)-bovine serum albumin (BSA). Hepatocytes were then cultured with a graded dilution of PN-M001 (100 microg/mL and 200 microg/mL), which is a curcuma/absinthium-containing compound, or sylibin (100 microg/mL) dissolved in dimethyl sulfoxide for 10 min before the addition of metallic salts (iron, copper and vanadium). Lysosomal fractions were prepared for lysosome fragility tests in which beta-galactosidase activity and lactate dehydrogenase (LDH) leakage were measured, as well as oxidative damage tests in the presence of hydrophilic and lipophilic free radical generators. Quenching activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) was also assessed. Malonildialdehyde accumulation in the medium showed a direct time-course increase with incubation time. Both PN-M001 and sylibin showed a significant protective effect against all challenge metal ions, as expressed by the half inhibition concentration (IC(50)) against lipid peroxidation. However, on a molar ratio, sylibin seemed to be more effective than PN-M001 in Fe-induced peroxidative damage (P < 0.05). Both test compounds, irrespective of the concentration, significantly reduced the LDH and beta-galactosidase concentration in the lysosomal fractions. As compared with untreated lysosomal fractions challenged with the two peroxide radicals generators, either PN-M001 or sylibin exerted significant protection However, PN-M001 was significantly better than sylibin in suppressing acid phosphatase enzyme activity. Both compounds showed comparable and significant DPPH radical-scavenging activity. These data support the potential clinical application of curcumin-containing compounds.

Differential susceptibilities of murine hepatoma 1c1c7 and Tao cells to the lysosomal photosensitizer NPe6: influence of aryl hydrocarbon receptor on lysosomal fragility and protease contents.

Irradiation of murine hepatoma 1c1c7 cultures presensitized with N-aspartyl chlorin e6 (NPe6) caused lysosomal disruption and apoptosis. Tao cells, a variant of the 1c1c7 line having lower aryl hydrocarbon receptor (AhR) contents, were resistant to the pro-apoptotic effects of NPe6 in the same photodynamic therapy protocol. Colony-forming assays were used to establish light dose-dependent and NPe6 concentration-dependent cytotoxicity curves. Lysosomal breakage and cell survival paralleled one another in both cell types. When analyzed at comparable lethal dose conditions, the onset of apoptosis was delayed, and the magnitude of the apoptotic response was muted in Tao cells, as assessed by morphology, annexin V binding, caspase-3 activities, and analyses of Bid, procaspase-9, and pro-caspase-3 cleavage. In contrast, the kinetics/magnitude of pro-caspase-3 activation in the two cell lines were identical after exposure to HA14 -1 or Jo2 antibody, inducers of the intrinsic and extrinsic apoptotic pathways, respectively. Tao endosomal/lysosomal extracts contained approximately 50%, 35%, and 55% of the Bid cleavage and cathepsin B and D activities of 1c1c7 endosomes/lysosomes, respectively. Western blot analyses confirmed reduced cathepsin B/D contents in Tao cells. Analyses of 1c1c7/Tao variants engineered to express antisense/sense AhR constructs suggested that endosomal/lysosomal cathepsin B and D content, but not whole cell content, correlated with AhR expression. These studies provide a mechanism for the resistance of Tao cultures to the proapoptotic effects of a protocol causing targeted disruption of lysosomes. They also suggest that the AhR, in the absence of exogenous ligand, may affect the trafficking/processing of proteases normally found in endosomes/lysosomes.

Lysosomal Fragility in Earthworms (Eisenia Veneta) Exposed to Heavy Metal Contaminated Soils from Two Abandoned Pyrite Ore Mines in Southern Norway

In this study we have investigated the toxicity of soils from thetwo abandoned pyrite mines Ertelien and Konnerud in the southernpart of Norway. Soil samples were collected close to the pitheadof the mines, and earthworms were exposed to different concentrations of the contaminated mine soil mixed with commercial plant soil for a period of 14 days. Life-cycle responses such as growth and survival, as well as the biomarkerresponse lysosomal fragility were measured. The body burdens ofthe four heavy metals Cu, Cd, Zn and Pb were detected in the worms, and the body concentration of the metals generally increased with increasing heavy metal concentration in the soil.The growth and mortality of the earthworms were not influenced when exposed to any of the mine soil concentrations used. The lysosomal fragility of the earthworm coelomocytes measured by the neutral red retention time (NRR-time) was found to be markedly reduced in all the exposed earthworms. This assay seemsto be a sensitive and dose dependent endpoint for the toxicity assessment of heavy metal mixtures in pyrite mine soils.

Iron toxicity and chelation therapy.

Iron is an essential mineral for normal cellular physiology, but an excess can result in cell injury. Iron in low-molecular-weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates; the result is wide-ranging impairment in cellular function and integrity. The rate of free radical production must overwhelm the cytoprotective defenses of cells before injury occurs. There is substantial evidence that iron overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of iron exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria and lysosomes. Iron overload impairs hepatic mitochondrial respiration primarily through a decrease in cytochrome C oxidase activity, and hepatocellular calcium homeostasis may be compromised through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of iron-induced damage, and this may have consequences in regard to malignant transformation. Mitochondrial respiratory enzymes and plasma membrane enzymes such as sodium-potassium-adenosine triphosphatase (Na(+) + K(+)-ATPase) may be key targets of damage by non-transferrin-bound iron in cardiac myocytes. Levels of some antioxidants are decreased during iron overload, a finding suggestive of ongoing oxidative stress. Reduced cellular levels of ATP, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA all may contribute to cellular injury in iron overload. Evidence is accumulating that free-radical production is increased in patients with iron overload. Iron-loaded patients have elevated plasma levels of thiobarbituric acid reactants and increased hepatic levels of aldehyde-protein adducts, indicating lipid peroxidation. Hepatic DNA of iron-loaded patients shows evidence of damage, including mutations of the tumor suppressor gene p53. Although phlebotomy therapy is effective in removing excess iron in hereditary hemochromatosis, chelation therapy is required in the treatment of many patients who have combined secondary and transfusional iron overload due to disorders in erythropoiesis. In patients with beta-thalassemia who undergo regular transfusions, deferoxamine treatment has been shown to be effective in preventing iron-induced tissue injury and in prolonging life expectancy. The use of the oral chelator deferiprone remains controversial, and work is continuing on the development of new orally effective iron chelators.

Stabilization of lysosomal membrane and cell membrane glycoprotein profile bySemecarpus anacardium Linn. nut milk extract in experimental hepatocellular carcinoma

Semecarpus anacardium Linn. nut milk extract administered orally at a dose of 200 mg/kg/day for 14 days exerted an in vivo stabilizing effect on lysosomal membrane and glycoprotein content in rat hepatocellular carcinoma. This was demonstrated in normal rats and in animals whose biomembranes were rendered fragile by induction of hepatocellular carcinoma with aflatoxin B(1) and subsequent treatment with Semecarpus anacardium nut extract. In this condition, the discharge of lysosomal enzymes increased significantly with a subsequent increase in glycoprotein components. The nut extract administration reversed these adverse changes to near normal in treated animals. The possible reason for this reversal is discussed. Such stabilization of biomembranes by Semecarpus anacardium nut extract may have a beneficial effect in the treatment of hepatocellular carcinoma and other cancers involving abnormal fragility of lysosomes and glycoprotein content providing the extract demonstrates safety in a full toxicity study.

Cytoprotective Effect of Ulinastatin, a Kunitz-type Protease Inhibitor, on Hypoxic Injury in L2 Cells Treated with Antimycin A via Stabilization of Lysosomal Fragility

The cytoprotective effect of ulinastatin, a Kunitz-type protease inhibitor, was studied in L2 cells treated with antimycin A, which induces depletion of cellular adenosine triphosphate (ATP), so-called «chemical hypoxia». Antimycin A treatment with 2 μM significantly elevated the release of N-acetyl-β-D-glucosaminidase (NAG), a lysosomal enzyme, to 51.99±7.36%. In this condition, ulinastatin tended to inhibit the release of NAG at a concentration of 1000 U/ml (39.74±3.80%) and significantly ameliorated it at a concentration of 3000 U/ml (32.35±4.17%). Furthermore, ulinastatin at 10 U/ml showed a suppression on the fragility of lysosomal membrane isolated from L2 cells. These results indicate that ulinastatin has a prominent protective effect on hypoxic injury in L2 cells, and the lysosomal stabilizing effect is possibly involved as a mechanism of this action.

Effect of high fiber intake on pancreatic lysosomal stability in ethanol-fed rats

Chronic ethanol consumption increases the fragility of pancreatic lysosomes, but the effect of a high fiber intake, alone or combined with alcohol abuse, on the lysosomal stability has not been studied. Furthermore, it is not yet known whether these treatments could predispose the exocrine pancreas to a greater damage after cerulein-induced acute pancreatitis. Cytosolic specific activity of three lysosomal enzymes, N-acetyl-β- d-glucosaminidase (NAG), cathepsin B, and β-glucuronidase were measured, as an index of lysosomal stability in pancreas from control rats and rats under chronic alcohol and/or high fiber intake. Cathepsin B is the only enzyme with significantly increased specific activity after chronic ethanol consumption and, moreover, its specific activity undergoes the highest increase after cerulein-induced acute pancreatitis, in all the groups of rats, when compared with the remainder enzymes. When pancreatitis was induced by cerulein, the combination of chronic alcohol and high fiber intake produces a significant decrease in the cytosolic specific activity of N-acetyl-β- d-glucosaminidase and β-glucuronidase when compared with chronic alcohol alone. Our results suggest that fiber partially avoids the damage of ethanol on pancreatic lysosomes, reducing the effects of pancreatitis.

Role of iron in the potentiation of anthracycline cardiotoxicity: Identification of heart cell mitochondria as a major site of iron-anthracycline interaction

The role of iron in anthracycline toxicity was studied in rats in vivo in intact animals and in vitro in heart cell cultures. In animals treated with 8 mg/kg doxorubicin, iron loading resulted in severe weight loss and a twofold increase in rate of mortality. Studies in cultured heart cells aimed at defining the subcellular target of interaction between iron and anthracycline toxicity showed no evidence of anthracycline-induced damage to sarcolemmal thiolic enzymes represented by 5′-nucleotidase and only a limited increase in lysosomal fragility as monitored by an increase in β-hexosaminidase activity in cell homogenates and its release into the culture medium. By contrast, doxorubicin treatment resulted in a marked inhibition of mitochondrial function as monitored by a decrease in carbon 14-labeled palmitate utilization, to 33% ± 4% of controls, and prior iron loading resulted in a further decrease in palmitate utilization, to 18% ± 3% of controls. Conversely, iron-chelation treatment by either deferoxamine or deferiprone (L1) eliminated the harmful effects of iron loading and resulted in a partial inhibition of doxorubicin toxicity in both normal and iron-loaded cells. Our studies represent the first demonstration in intact animals of the potentiation of anthracycline toxicity by iron overload. They also indicate that mitochondria represent an important target of combined iron-anthracycline toxicity. These observations provide new insights into the mechanism of anthracycline cardiotoxicity and may be useful in developing better strategies for tumor therapy.

Protective action of ulinastatin against cisplatin nephrotoxicity in mice and its effect on the lysosomal fragility.

The development of azotemia after cisplatin injection in mice was inhibited by ulinastatin treatment in a dose-dependent manner. Reduction in creatinine clearance and elevation in fractional excretion of sodium in mice receiving cisplatin was ameliorated by ulinastatin administration. Epithelial necrosis and hyaline cast formation in the proximal tubule were also suppressed. Ulinastatin showed no influence on the kidney platinum level after cisplatin injection. In LLC-PK1 cells, addition of ulinastatin to the incubation medium markedly reduced the release of N-acetyl-beta-D-glucosaminidase, on of the lysosomal enzymes, during hypotonic treatment only when cells were damaged with cisplatin. On the other hand, ulinastatin showed no effect on the elevation of malondialdehyde concentration in the murine kidney cortical slices after the treatment with cisplatin. These results indicate that ulinastatin has a protective effect against cisplatin nephrotoxicity, and its prevention of the increase in lysosomal fragility is a probable mechanism involved in the renal protection.

Metal-induced hepatotoxicity.

Figure 3 summarizes several proposed mechanisms of iron- or copper-induced hepatotoxicity. It has long been suspected that free radicals may play a role in iron- and copper-induced cell toxicity because of the powerful prooxidant action of iron and copper salts in vitro. In the presence of available cellular reductants, iron or copper in low molecular weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates, resulting in wide-ranging impairment in cellular function and integrity. However, cells are endowed with cytoprotective mechanisms (antioxidants, scavenging enzymes, repair processes) that act to counteract the effects of free radical production. Thus, the net effect of metal-induced free radicals on cellular function will depend on the balance between radical production and the cytoprotective systems As a result, there may be a rate of free radical production that must be exceeded before cellular injury occurs. Evidence has now accumulated that iron or copper overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of the metal exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria (oxidative metabolism) and lysosomes (membrane integrity, fluidity, pH). Although these findings do not prove causality, it seems likely that lipid peroxidation is involved, since similar functional defects are produced by metal-induced lipid peroxidation in these organelles in vitro. Both iron and copper overload impair hepatic mitochondrial respiration, primarily through a decrease in cytochrome c oxidase activity. In iron overload, hepatocellular calcium homeostasis may be impaired through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of metal-induced damage in the liver; this may have consequences as regards malignant transformation. The levels of some antioxidants in the liver are decreased in rats with iron or copper overload, which is also suggestive of ongoing oxidative stress. Reduced cellular ATP levels, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA may all contribute to hepatocellular injury in iron and copper overload. There are few data addressing the key issue of whether free radical production is increased in patients with iron or copper overload. Patients with hereditary hemochromatosis have elevated plasma levels of TBA-reactants and increased hepatic levels of MDA-protein and HNE-protein adducts, indicative of lipid peroxidation. Mitochondria isolated from the livers of Wilson disease patients have evidence of lipid peroxidation, and some patients with Wilson disease have decreased hepatic and plasma levels of vitamin E. Additional investigation will be required to fully assess oxidant stress and its potential pathophysiologic role in patients with iron or copper overload.