Lysosomal abnormalities during benzo(a)pyrene-induced experimental lung carcinogenesis – defensive role of capsaicin

The objective of the present study was to investigate whether lysosome is a target in benzo(a)pyrene-induced, oxidative stress-mediated lung cancer in Swiss albino mice and the plausible role of the phytochemical substance capsaicin in mitigating lysosomal damage. Oxidative stress was assessed based on the level of carbonyl content. The activities of lysosomal proteases like cathepsin-D, cathepsin-B, β- d -glucosidase, β- d -galactosidase, β- d -glucuronidase, β- d - N -acetylglucosaminidase and acid phosphatase were assessed to evaluate lysosomal function. Administration of benzo(a)pyrene (50 mg/kg body weight) to mice induced a increase in the activities of lysosomal enzymes and oxidative stress was evident by the increase in carbonyl content. Treatment with capsaicin (10 mg/kg body weight) decreased carbonyl content and restored the activities of lysosomal enzymes to near normalcy. Transmission electron microscopic study of lysosomes further showed the defensive action of capsaicin against the lysosomal damage caused in benzo(a)pyrene-induced lung cancer. From the present study, it can be concluded that lysosomal damage is an indispensable event in benzo(a)pyrene-induced lung cancer, and capsaicin was able to effectively prevent it, which proves the chemoprotective effect of capsaicin against benzo(a)pyrene-induced experimental lung carcinogenesis.     

Role of bioantioxidants depression and deficiency of protease inhibitor alpha 1-antitrypsin in mechanisms activating free radical oxidation and proteolysis in chronic pancreatitis

AIM: To elucidate the reason and mechanisms of neutralization of protease inhibitors and antioxidants by proteolytic enzymes and oxidants. MATERIALS AND METHODS: The trial included 92 patients with exacerbation of chronic pancreatitis. 47 of them had chronic recurrent pancreatitis (CRP), 45 patients had chronic fibrozing pancreatitis (CFP). Measurements were made of blood catalase and ceruloplasmin (according to P. Hubl, R. Breschneider and O. Houchin, respectively), alpha1-antitrypsin (by Reiderman), schiff bases (by B. Fletcher et al.), dienic conjugates (by Z. Placer), serum acid phosphatase (by Bodansky), acid phosphatase of polynuclear cells (by R. Nartsissiv), NBT-test was made according to B. Park. RESULTS: Exacerbation of CRP was associated with enhancement of free radical lipid peroxidation (FPOL), release of proteolytic and lysosomal enzymes from acinar cells, a fall in catalase level. Catalase depression depends on the level of blood lysosomal enzymes and partially on FPOL activity. In CFP moderate activity of FPOL and trypsin is associated with normal levels of lysosomal enzymes and catalase. In both pancreatitis forms, alpha1-antitrypsin levels are low. This lowering is primary and unrelated with inflammatory process in the pancreas. A trypsin rise in both forms depends on lowering of alpha1-antitrypsin which via trypsin inhibits formation of lysosomal enzymes in polynuclear cells. Inability of protease inhibitor to block proteolytic (lysosomal) enzymes manifests in initial intraacinar activation of trypsin from trypsinogen, in inflammatory focus under polynuclear cells release of lysosomal enzymes and in proteolytic enzymes release from the affected acinar structures. CONCLUSION: Lack of alpha1-antitrypsin--protease inhibitor--and depression of antioxidant catalase are main and intermediate elements in activation of mechanisms of proteolytic aggression and FPOL.     

A Comparison of the β-Glycosidase Excretion during Kidney Damage Induced by 4-Nitrophenylarsonic Acid and by Rabbit Anti-Rat Kidney Antibodies

Abstract. In a study of the value of urinary enzyme activities as an indication of glomerular or tubular damage, the effects of two nephrotoxic agents (4-nitrophenylarsonic acid and rabbit anti-rat kidney serum) on the urinary excretion of four β-glycosidases were compared using fluorimetric assays with 4-methylumbelliferyl substrates. The electrophoretic mobilities on starch gel of urinary β-galactosidase, β-glucosidase, β-glucosaminidase and β-glucuronidase, at various times up to 26 days after the injection of the nephrotoxins into the rat, were compared with those of the corresponding enzymes present in normal rat urine, kidney and serum. 4-Nitrophenylarsonic acid causes tubular damage characterised by an immediate rise in the rates of excretion of the four β-glycosidases, followed by a return to normal values. In Masugi glomerulonephritis the immediate rise in enzyme excretion is much less marked but greater increases occur after 10 to 12 days. The increases in excretion rate correlate roughly with the stages of the disease. Both glomerular and tubular damage produce characteristic changes in the excretion of all of the enzymes studied, but β-glucosidase appears to be the most useful indicator of kidney tubular damage, as it is the least widely distributed of these enzymes and is not normally found in urine or serum at significant levels of activity.     

Lysosomal enzyme activities in normals and in patients with chronic liver diseases

The maximal activities of liver lysosomal enzymes (acid phosphatase and cathepsin D) were found to be increased in patients with chronic active hepatitis, cirrhosis and primary hepatocellular carcinoma. The ratio between maximal and basal activity (an expression of the degree of retention of the enzymes to lysosome) of acid phosphatase was significantly decreased in patients with chronic active hepatitis and cirrhosis whereas that of cathepsin D did not show any significant changes between normal and various liver disorders. Serum levels of both the enzymes were elevated significantly in patients with cirrhosis and primary hepatocellular carcinoma.     

Insulin-like growth factor-I,interleukin-1 α and β in pancreatic cancer: role in tumor invasiveness and associated diabetes

Summary We evaluated levels of insulin-like growth factor-I and interleukin-1 α and β in patients with pancreatic cancer; the role of these substances in tumor spread and in hyperglycemia was also investigated. Thirty pancreatic cancer patients (21 with hyperglycemia) were compared with others with diseases causing hyperglycemia [liver cirrhosis (14 cases, 12 with hyperglycemia), chronic pancreatitis (20 cases, 12 with hyperglycemia), type I diabetes mellitus (13 cases, all hyperglycemic)]. Insulin-like growth factor-I was significantly reduced in patients with liver cirrhosis, probably due to a reduced hepatic capacity for synthesis. It was increased in 6 of 30 pancreatic cancer patients; in these subjects it was correlated with alanine aminotransferase and C-peptide, but not with tumor diameter or the presence of metastases. Interleukin-1α and β were both elevated in pancreatic cancer patients. The former was high, while the latter was low when liver metastases were present. Neither was related to glucose or C-peptide levels. In summary, insulin-like growth factor-I levels are increased in some pancreatic cancer patients but this does not seem to favor tumor spread; however IGF-I could be involved influencing glucose homeostasis. Interleukin-1 α increased, while interleukin-1β decreased in pancreatic cancer patients with metastases, suggesting a different involvement of these two substances in pancreatic cancer spread.     

Enzyme activities and properties of lysosomes and brush borders in jejunal biopsies from control subjects and patients with coeliac disease.

1. Reduced activities of four enzymes from brush borders were found in intestianl biopsies from patients with untreated coeliac disease. The activities returned towards control values after treatment by gluten withdrawal. Parallel changes were noted for the cytosol enzyme lactate dehydrogenase. 2. Measurement of brush-border integrity by differential centrifugation of biopsy extracts indicated increased fragility of the brush borders in biopsies from untreated patients. Normal values were obtained for biopsies from treated patients. 3. Increased activites of six acid hydrolases (lysosomal enzymes) were found in biopsies from untreated coeliac patients. Normal values were obtained for biopsies from treated patients. 4. Assement of lysosomal integrity by assay of latent N-acetyl-beta-glucosaminidase and of sedimentable activity of four acid hydrolases demonstrated increased lysosomal fragility in untreated coeliac mucosa. These lysosomal changes return to within the normal range after treatment by gluten withdrawal. 5. The lysosomal changes are consistent with their having a pathogenic role in the enterocyte damage of coeliac disease. Possible mechanisms for the lysosomal changes are discussed.     

Serum lysosomal hydrolases in cystic fibrosis

The activities of a number of lysosomal hydrolases were determined in sera from 100 patients with cystic fibrosis (CF), age 2-35 yr, and age-matched controls: beta-hexosaminidase activity was significantly elevated (p less than 0.005) in CF patients from all age groups. alpha-Mannosidase activity was increased only in the older CF patients (greater than 13 yr). The following enzyme activities were not altered in CF serum: alpha-fucosidase, beta-glucuronidase and acid phosphatase. The abnormal patterns of serum alpha-mannosidase and beta-hexosaminidase in CF cannot be explained by pancreatic disease or undernutrition, since serum values of these hydrolases in patients with anorexia nervosa or acute pancreatitis were not altered. However, the altered activities of the alpha-mannosidase and beta-hexosaminidase were proportional to the degree of pulmonary insufficiency in the CF group, indicating that these changes are probably a secondary consequence of the primary disease process. Except for beta-hexosaminidase, because differences in the serum hydrolases in CF do not become apparent until the second decade of life, determinations of lysosomal enzyme activities in serum will probably be of little diagnostic value.     

Serum phospholipase A2 activity in chronic pancreatic diseases.

This study was performed to investigate the phospholipase A2 (PLA2) serum activity in patients with chronic pancreatic disease. PLA2, elastase-1, total, and pancreatic isoamylase were evaluated in 40 control subjects, 28 patients with pancreatic cancer, 51 with chronic pancreatitis, and 36 with extrapancreatic diseases, mainly of gastrointestinal origin. Elastase-1, PLA2, and pancreatic isoamylase were increased in 56%, 25%, and 15% of patients with pancreatic cancer, and in 40%, 31%, and 41% of subjects with chronic pancreatitis. All four enzymes gave pathological values in a number of patients with extrapancreatic diseases. We conclude that the diagnostic efficacy of phospholipase A2 in chronic pancreatic disease is similar to that of other well known pancreatic enzymes, with an unsatisfactory sensitivity and specificity.     

Morphofunctional elements of the pathogenesis of acute pancreatitis]

Nineteen patients with acute pancreatitis were examined for the activity of LDH, NADH-tetrazolium oxidoreductase, acid phosphatase, the content of calcium salts, cAMP and cGMP in biopsy tissue of the pancreas; pancreatic enzymes and bicarbonates in the duodenal contents and pancreatic juice. The activity of enzymes participating in oxidative metabolism in epithelial cells of the intact pancreas appeared elevated. During the development of destructive changes in the pancreatic parenchyma, the processes of intracellular oxidation get inhibited, the enzymes go out into the intercellular space, calcium transport gets impaired, and acid phosphatase is activated. It has been found that in acute destructive pancreatitis, primarily impaired are epithelial cells of the islets, followed by the impairment of the epithelium of the acini and at the last moment of that of the excretory ducts. The data obtained enable one to regard cyclonucleotides, calcium, pancreatic enzymes and lysosomal hydrolases as pathogenetic elements of acute pancreatitis.     

Apical secretion of lysosomal enzymes in rabbit pancreas occurs via a secretagogue regulated pathway and is increased after pancreatic duct obstruction.

Lysosomal hydrolases such as cathepsin B are apically secreted from rabbit pancreatic acinar cells via a regulated as opposed to a constitutive pathway. Intravenous infusion of the cholecystokinin analogue caerulein results in highly correlated apical secretion of digestive and lysosomal enzymes, suggesting that they are discharged from the same presecretory compartment (zymogen granules). Lysosomal enzymes appear to enter that compartment as a result of missorting. After 7 h of duct obstruction is relieved, caerulein-stimulated apical secretion of cathepsin B and amylase is increased, but the ratio of cathepsin B to amylase secretion is not different than that following caerulein stimulation of animals never obstructed. These findings indicate that duct obstruction causes an increased amount of both lysosomal and digestive enzymes to accumulate within the secretagogue releasable compartment but that duct obstruction does not increase the degree of lysosomal enzyme missorting into that compartment. Pancreatic duct obstruction causes lysosomal hydrolases to become colocalized with digestive enzymes in organelles that, in size and distribution, resemble zymogen granules but that are not subject to secretion in response to secretagogue stimulation. These organelles may be of importance in the development of pancreatitis.     

Lysosomal enzyme pattern in lung lymph and blood during E. coli sepsis in sheep

Systemic release of lysosomal enzymes and local release in the pulmonary microcirculation from sequestrated and activated leucocytes could be an important factor in the development of the lung microvascular injury seen after septicaemia. The maximal activities of 11 lysosomal acid hydrolases (acid phosphatase, alpha- and beta-glucosidase, alpha- and beta-galactosidase, alpha-mannosidase, beta-acetylglucosaminidase, beta-glucuronidase, arylamidase and cathepsins B and C) were measured in serum and lung lymph from seven sheep before and after infusion of live E. coli bacteria. In the early phase of septicaemia (the first hour) the activities of eight enzymes were increased in serum and/or lung lymph (1.1 to 2X pre-infusion values). In the late phase, 3-4 h after sepsis, there were significantly elevated serum activities of beta-glucosidase (5.4X), alpha- and beta-galactosidases (2.7X, 1.5X), beta-acetylglucosaminidase (2.0X) arylamidase (1.2X) and cathespin B (1.7X). In lymph acid phosphatase (1.7X), alpha- and beta-glucosidases (1.6X, 6.4X), alpha- and beta-galactosidases (2.1X, 1.7X). Beta-acetylglucosaminidase (2.6X), and beta-glucuronidase (4.0X pre-infusion) were elevated. The findings of a heterogenicity of changes in serum and lymph activities, as well as the large molecular sizes of some of the enzymes with changed activities indicated to us that permeability changes were not major causes of increased lymph enzyme activities. The results could indicate a local release of enzymes either from sequestrated leucocytes or lung tissue due to local reactions in the lung or lung microvessels. The heterogenous changes in activities for the various lysosomal enzymes as found in the present study indicated that measurement of only one enzyme could be misleading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid hydrolase activities and lysosomal integrity in liver biopsies from patients with iron overload.

1. Iron, acid phosphatase and N-acetyl-beta-glucosaminidase were assayed in liver biopsies from control subjects and patients with primary and secondary haemochromatosis. 2. The activities of the lysosomal enzymes were significantly higher in liver biopsies from patients with iron overload than in those from other patient groups. 3. Lysosomes from the livers of patients with iron overload were strikingly more fragile than those of control subjects as demonstrated by assays of latent and sedimentable N-acetyl-beta-glucosaminidase. 4. Lysosomal integrity was essentially normal in biopsies from patients with a wide variety of chronic liver diseases. 5. It is suggested that iron accumulation damages lysosomal membrane, releasing acid hydrolases into the cytoplasm and thus initiating cell damage.     

The subcellular localization of peptidase activity in the human jejunum

Abstract. The subcellular localization of peptidase activity in the normal human jejunum has been investigated. Subcellular organelles were fractionated by density gradient centrifugation. The localization of peptidases was determined by comparing the distributions of peptidase activities with those of organelle 'marker' enzymes. The organelles and their markers were: cytosol—lactate dehydrogenase; brush border—neutral α-glucosidase, γ-glutamyl transferase and leucyl-2-naphthylamidase; plasma membrane—5'-nucleotidase; lysosomes—N-acetyl- β -glucosaminidase; mitochrondria—malate dehydrogenase; endoplasmic reticulum—alkaline α-glucosidase; peroxisomes—catalase.
Thirteen dipeptides, seven tripeptides, two tetrapeptides, two pentapeptides and a hexapeptide were used as substrates. The distribution of dipeptidyl peptidase IV was also determined.
Irrespective of whether the NH₂-terminal or COOH-terminal amino acid was neutral, basic or acidic, the major or exclusive locus of dipeptidase activity was cytosolic. All of the activity against a dipeptide with the amino acid proline at the NH₂-terminus was in the cytosol.
The distribution of tripeptidase activity was quite different. Although the cytosol hydrolysed all tripeptides, as much as 50% of tripeptidase activity was particulate. For both tetrapeptides, one of the pentapeptides and the hexapeptide, the major or exclusive locus of activity was the brush border membrane. Pentaphenylalanine, however, was hydrolysed by both the cytosol and the brush border. Dipeptidyl peptidase IV was localized in the brush border.     

Biochemical mechanisms of hypoxic injury to the cerebral cortex in acute blood loss and during various stages of the postresuscitation period

After 4 h of hypotension with an arterial pressure of 40 mmHg induced by acute blood loss, the activities of acid phosphatase and acid cathepsins (lysosomal enzymes) and alkaline phosphatase in the grey matter of the cerebral cortex of dogs were sharply increased. At the same time, the total RNA of the brain decreased by 36·4% and the protein in the subcellular fractions by 37·2%. During electrophoresis of the protein fractions in polyacrylamide gel, there was a considerable loss of prealbumins, postalbumins, caeruloplasmin and transferrin, as well as an increase in β-globulins. The cerebral cortex proteins in dogs that survived hypovolaemic hypotension showed changes in their physicochemical properties. During the early postresuscitation period the total RNA in the protein fractions did not return to normal. A further loss of γ-globulins took place. A profound disturbance in the plastic metabolism persisted during the 14–21 days after resuscitation; the γ-globulins diminished to 59·8% of the control values. High activity of the acid and alkaline phosphatase continued in the grey matter of the cerebral cortex. The deficit of the plastic material and the physicochemical changes in the protein molecules together with the increased activity of the acid cathepsins reflect degenerative and necrotic disturbances in the cerebral cortex during the 14–21 days after resuscitation. Histological studies of the cerebral cortex also showed areas of necrosis in all the test animals.     

Both ethanol consumption and protein deficiency increase the fragility of pancreatic lysosomes

Both ethanol abuse and protein deficiency result in pancreatic injury. Moreover, these two variables frequently coexist. As lysosomal enzymes may play a role in the initiation of pancreatic injury, the aim of this study was to determine the effects of ethanol consumption and protein deficiency on pancreatic lysosomal stability. For 3 weeks, male Sprague-Dawley rats were match-fed (in groups of four) isocaloric amounts of one of the following liquid diets: (1) protein-sufficient diet, (2) protein-sufficient diet containing ethanol as 36% of the total energy, (3) protein-deficient diet, and (4) protein-deficient diet containing ethanol as 36% of energy. Pancreatic lysosomal stability was assessed by determining (a) latency, as indicated by the percentage increase in lysosomal enzyme activity in pancreatic homogenate induced by Triton X-100, and (b) by the percentage of lysosomal enzyme remaining in the supernatant after sedimentation of the lysosomal pellet from the pancreatic homogenate. Protein deficiency was associated with a decrease in latency and an increase in supernatant enzyme. Ethanol administration was associated with a decreased latency. Both protein-deficient and ethanol-fed animals exhibited higher pancreatic activities of cathepsin B, a lysosomal protease capable of activating trypsinogen. In addition, protein-deficient animals exhibited higher pancreatic activities of acid phosphatase, N-acetyl-glucosaminidase, and beta-glucuronidase. As lysosomal enzymes are postulated to play a role in the initiation of pancreatitis, these results suggest that ethanol consumption and protein deficiency may at least partly exert their toxic effects on the pancreas by altering pancreatic lysosomal stability and increasing the glandular content of cathepsin B.     

Pancreatic beta-cell function and interleukin-1β in plasma during the acute phase response in patients with major burn injuries

Abstract. Animal experiments demonstrate that inter-leukin-lβ (IL-1β) is beta-cell cytotoxic in vitro and inhibits insulin secretion in vivo . However, it is unknown if IL-Iβ affects beta-cell function in man. Since IL-1β and other cytokines are main mediators of the acute phase response, the objectives of the present study were to examine beta-cell function in patients with major burn injuries, and to test if changes in beta-cell function correlated to systemic levels of IL-1β and tumour necrosis factor α (TNFα). We established and validated an IL-1β assay measuring free and protein bound IL-1β; protein bound IL-1β was detached from the IL-1β specific binding protein by acidification, rendering it accessible for the employed antibody. The IL-1β specific binding protein (43–60 kDa) was found in serum and plasma from all tested patients and normal subjects. Survivors of burn injuries had a stimulated beta-cell function, whereas non-survivors had an impaired beta-cell function as indicated by an increased plasma concentration of proinsulin, and an increased proinsulin/insulin ratio. In addition, non-survivors had significantly increased plasma levels of TL-1β. However, we could not demonstrate any correlation between C-peptide, proinsulin, insulin or proinsulin/insulin ratio and plasma concentration of IL-1β. In conclusion, beta-cell function abnormalities are evident in patients with major burn injuries, and a high plasma level of IL-1β correlates with a fatal outcome. However, the present study did not provide evidence for the hypothesis that beta-cell function is influenced by circulating IL-1β or TNFα during the acute phase response.     

Bile acid synthesis and excretion following release of total extrahepatic cholestasis by percutaneous transhepatic drainage

Abstract. Urinary, biliary and serum bile acids were studied in three patients before and after percutaneous transhepatic drainage for total bile duct obstruction.
Before drainage high urinary excretion often different bile acids occurred. The percentage distribution was: cholic and chenodeoxycholic acid (66–86%), hyo-cholic (3–16%), 3β 12α-dihydroxy-5-cholenoic (3–6%) and 3β-hydroxy-5-cholenoic acid (2–8%). These acids were regularly found in serum. In addition small amounts (less than 2%) of norcholic, allocholic, 3β, 7α-dihydroxy-5β-cholanoic, 3α, 7α-dihydroxy-5α-cholanoic and lithocholic acid were excreted in urine. Trace amounts of these bile acids were found in serum.
After start of drainage biliary bile acid excretion increased rapidly during the first day, dropped to a minimum during the second or third day and then slowly increased again. In spite of normal volumes of bile produced, the total serum bile acids and the urinary excretion of bile acids remained increased during a drainage period of 19 days. The bile acids were of the same type as observed during cholestasis. In serum the increase was mainly due to high concentrations of chenodeoxycholic and 3β-hydroxy-5-cholenoic acid, as sulphate esters.
Glycine and taurine conjugates of cholic, chenodeoxycholic and hyocholic acid were mainly excreted in bile. Bile acid sulphate esters were only present in trace amounts in bile and were mainly excreted in urine. This, combined with low renal clearance, explains the elevated serum levels of sulphate esters of chenodeoxycholic and 3β-hydroxy-5-cholenoic acid conjugates.     

Serum vitamin E,lipid peroxide and glutathione peroxidase in patients with chronic pancreatitis

It has been reported that lipid peroxidation increases in patients with antioxidant deficiencies, such as vitamin E and glutathione peroxidase. The relationships between serum lipid peroxide and vitamin E on the one hand and glutathione peroxidase on the other were examined in 22 patients with chronic pancreatitis, often accompanied by malabsorption of fats and fat-soluble vitamins due to the impaired exocrine pancreatic function.Both serum vitamin E concentrations and glutathione peroxidase activities were depressed, especially in patients with chronic calcifying pancreatitis. On the other hand, serum lipid peroxide levels were elevated. A significant negative correlation was found between the serum lipid peroxide levels and vitamin E concentration.These findings suggest that an elevation of the serum lipid peroxide level may be due to the lack of an antioxidative defense mechanism, such as vitamin E, against lipid peroxide.     

Diagnostic utility of a new monoclonal antibody pancreatic isoamylase assay in chronic pancreatic diseases

In order to evaluate the efficacy of a monoclonal pancreatic (P) isoamylase assay in the diagnosis of chronic pancreatic disease and to compare the behavior of this test with that of amylase and elastase 1, these three enzymes were measured in the sera of 39 healthy controls, 28 patients with pancreatic cancer, 50 with chronic pancreatitis and 60 with extra-pancreatic diseases. In patients with chronic relapsing pancreatitis, increased P-isoamylase and elastase 1 values were found in similar percentages (about 70%), whereas the percentage for elevated amylase values was lower (52%). Elastase 1 was increased in 52% of patients with pancreatic cancer, while the other two enzymes were only occasionally elevated. The levels for all three enzymes were abnormal in some patients with extra-pancreatic diseases. It may be concluded that this assay for P-isoamylase determination is sufficiently sensitive and reliable in detecting pancreatic inflammation, even though some limitations concerning its specificity should be born in mind.