Culture of impure human islet fractions in the presence of alpha-1 antitrypsin prevents insulin cleavage and improves islet recovery.

BACKGROUND: Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function. METHODS: Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM). RESULTS: Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 +/- 23.7% and 34 +/- 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% +/- 1.3% in impure and 78% +/- 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% +/- 9% vs 47% +/- 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% +/- 2%) and the A1AT-treated groups (99% +/- 1%). EM results revealed a reduction or absence of secretory granules after exposure to proteases (TCE). CONCLUSION: Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation.

Exposure to the fern Onychium contiguum causes increase in lipid peroxidation and alters antioxidant status in urinary bladder.

The status of lipid peroxidation, glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase, catalase, ascorbic acid, and alpha-tocopherol was studied in the urinary bladder of guinea pigs exposed to the carcinogenic fern Onychium contiguum. There was significant increase in the preformed lipid peroxides in the urinary bladders from fern exposed animals. The amount of lipid peroxides produced on incubation of urinary bladder homogenates with or without catalyst was significantly higher in the fern exposed animals. The concentrations of glutathione and alpha-tocopherol and the activities of glutathione reductase and catalase were elevated in the urinary bladders of the animals exposed to the fern. No effect was observed on the concentration of ascorbic acid and the activities of glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase. It is summarized that the fern toxins increased oxidative stress in the urinary bladder and antioxidant status was altered. However, the altered antioxidant status did not provide protection from the toxin induced injury. Histopathology of the urinary bladder in the fern exposed animals revealed oedema, haemorrhages, and congestion. This is the first study to show increase in lipid peroxidation along with altered antioxidant status in the urinary bladder of fern exposed animals.

Biochemical alterations in the blood plasma of rats associated with hepatotoxicity induced by Eupatorium adenophorum.

Eupatorium adenophorum (Crofton weed), a native of Central America. has appeared as a major weed in several areas in different parts of the world. Horses that eat this plant are poisoned on prolonged exposure. Toxicity due to consumption of this plant by other grazing animals is not clear. Administration of freeze-dried leaf powder to mice results in hepatotoxicity. Earlier attempts to produce toxicity in rats using the leaves of this plant were not successful. In the present study, administration of oven-dried E. adenophorum leaves collected at the flowering stage elicited hepatotoxicity in rats. The affected animals had a marked increase in the concentration of plasma bilirubin and in the activities of 5'-nucleotidase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase. There were no significant differences in plasma creatinine, urea or total protein values in the affected animals compared to controls. The livers of the affected animals had focal areas of necrosis throughout the parenchyma and hepatocytes showed megalocytosis. The bile ducts were dilated and the epithelium showed degenerative to necrotic changes. The alterations in bilirubin, enzymes and histopathological changes imply cholestasis and liver injury.

A preliminary study on the carcinogenicity of the common fern Onychium contiguum.

Onychium contiguum (Family Cryptogrammaceae) is a common terrestrial fern in the Himalayas and in many other parts of the world. It is also present on the pastures in areas where grazing animals suffer from bovine urinary bladder cancer. This fern is occasionally grazed by animals and in some areas it is present as a contaminant in grasses stored for winter feeding. Certain species of the genus Onychium are used in folk medicine. Long-term exposure of experimental animals to O. contiguum appeared to cause tumours of the ileum. urinary bladder and mammary glands.

Hepatotoxicity and cholestasis in rats induced by the sesquiterpene, 9-oxo-10,11-dehydroageraphorone, isolated from Eupatorium adenophorum.

Eupatorium adenophorum leaves cause hepatotoxicity and cholestasis in rats. The hepatotoxicant has been characterized as 9-oxo-10,11-dehydroageraphorone (ODA), a cadinene sesquiterpene. Oral administration of ODA, mixed in feed to rats, caused jaundice in 24 h. The liver of the intoxicated animals had focal areas of hepatocellular necrosis, proliferation, and dilation of bile ducts with degenerative changes in the lining epithelium. There was marked increase in the conjugated form of plasma bilirubin and in the activities of the enzymes glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltranspeptidase, glutamate dehydrogenase, and 5'-nucleotidase. The histopathological lesions in liver and biochemical profile of marker enzymes show that ODA induced hepatotoxicity and cholestasis in rats. This is the first report on the toxicity of a cadinene sesquiterpene in rats.

Hepatotoxicity in rat induced by partially purified toxins from Eupatorium adenophorum (Ageratina adenophora).

A group of rats were administered a methanolic extract of Eupatorium adenophorum (Ageratina adenophora) oven-dried (60 degrees C) leaf powder and a partially purified fraction from the methanolic extract. Administration of the methanolic extract and the partially purified fraction elicited a significant increase in total and conjugated bilirubin, alkaline phosphatase, 5'-nucleotidase and transaminases. Histopathology of the livers from these animals revealed dilated bile ducts and proliferative changes. Hepatocytes around the bile ducts showed necrotic changes. Biochemical and histopathological changes resembled those observed in response to administration of whole leaf powder. The hepatotoxin present in E. adenophorum leaves can be extracted with methanol and partially purified further using the procedure described.

A spectrophotometric method for assay of tannase using rhodanine.

A method for assay of microbial tannase (tannin acyl hydrolase) based on the formation of chromogen between gallic acid and rhodanine is reported. Unlike the previous protocols, this method is sensitive up to gallic acid concentration of 5 nmol and has a precision of 1.7% (relative standard deviation). The assay is complete in a short time, very convenient, and reproducible.

Hepatotoxicity of Eupatorium adenophorum to rats.

Freeze dried Eupatorium adenophorum leaf powder mixed in rat feed at a level of 25% elicited hepatotoxicity. The affected animals were jaundiced and had marked increase in plasma bilirubin levels and activities of alkaline phosphatase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase. The liver of intoxicated animals had focal areas of necrosis and bile duct proliferation. Elevation in plasma bilirubin concomitant with alterations in enzyme profile and histopathological lesions are consistent with liver injury and cholestasis. This is the first report of the toxicity of E. adenophorum to rats.

Disposition of lantadene A, the pentacyclic triterpenoid hepatotoxin, orally administered to guinea pigs.

Lantadene A (LA) administered orally to guinea pigs elicited cholestasis. LA could not be detected in liver, bile, gall bladder, blood and urine. LA and its biotransformation product reduced lantadene A (RLA) could be detected in caecum, large intestine, and faeces. In vitro incubation of LA with liver homogenates under aerobic and anaerobic conditions did not elicit its biotransformation to RLA. On the other hand, in vitro incubation of LA with guinea pig caecal and large intestinal contents under anaerobic conditions elicited conversion of LA to RLA. This is the first report of the biotransformation of LA in the animal system.

Biotransformation of lantadene A (22 beta-angeloyloxy-3-oxoolean-12-en-28-oic acid), the pentacyclic triterpenoid, by Alcaligenes faecalis.

A bacterial strain capable of biotransformation of lantadene A (22 beta-angeloyloxy-3-oxo-olean-12-en-28-oic acid), the pentacyclic hepatotoxin of lantana (Lantana camara var. aculeata) has been isolated from soil using lantadene A as the sole carbon source. The organism is Gram negative, rod shaped, motile, catalase positive and has been identified as Alcaligenes faecalis. The isolate has been found to be specific for lantadene A and did not utilize lantadene B. In studies using sucrose as an additional carbon source, A. faecalis elicited biotransformation of lantadene A to its trans isomer 22 beta-tigloyloxy-3-oxoolean-12-en-28-oic acid, designated as lantadene X and two other minor metabolites which could not be isolated in pure state.

A review of the toxicosis and biological properties of the genus Eupatorium.

Eupatorium genus grows wild in many parts of the world. A number of species of Eupatorium are toxic to grazing animals. Milk sickness in humans is caused by ingestion of milk of the animals reared on the pastures infested with Eupatorium rugosum (white snakeroot). While some information is available on the toxins in various species of Eupatorium, ambiguities still persist in extrapolation of the data to field incidence of toxicosis. Eupatorium genus has been used for its medicinal properties for many decades. A number of bioactive natural products have been reported in the extracts of Eupatorium spp. and the genus is a promising bioresource for preparation of drugs and value-added products.

Biodegradation of lantadene A, the pentacyclic triterpenoid hepatotoxin by Pseudomonas pickettii.

A bacterial strain capable of biodegradation of lantadene A (22 beta-angeloyloxy-3-oxoolean-12-en-28-oic acid) has been isolated from soil using lantadene A as the sole carbon source. The organism is rod shaped, Gram negative, motile and has been identified as Pseudomonas pickettii. This is the first report of the biodegradation of a pentacyclic triterpenoid.

Inositol trisphosphate-independent agonist-stimulated calcium influx in rat pancreatic acinar cells.

CCK-JMV-180 is a cholecystokinin analog that stimulates digestive enzyme secretion from pancreatic acinar cells but does not cause either generation of inositol 1,4,5-trisphosphate or depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ storage pool. We report that CCK-JMV-180 can accelerate Ca2+ influx into fura-2-loaded dispersed rat pancreatic acini and single acinar cells. Furthermore, CCK-JMV-180 accelerates Ca2+ influx into cells microinjected with the inositol 1,4,5-trisphosphate receptor antagonist heparin and into acini loaded with the Ca(2+)-chelating agent BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). These results indicate that agonist-stimulated Ca2+ influx can occur (a) without depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ storage pool, (b) without a rise in cytoplasmic free Ca2+ concentrations, and (c) after blockade of inositol 1,4,5-trisphosphate receptors. They suggest that depletion of an inositol 1,4,5-trisphosphate-independent intracellular Ca2+ storage pool and/or generation of a non-inositol 1,4,5-trisphosphate second messenger by CCK-JMV-180 may be a sufficient signal for acceleration of Ca2+ influx into rat pancreatic acinar cells.

CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini.

In pancreatic acinar cells cholecystokinin and its analogs, caerulein and CCK-JMV-180, stimulate an increase in intracellular free [Ca2+] by releasing Ca2+ from non-mitochondrial intracellular pools. It is generally believed that the caerulein-induced release of Ca2+ is mediated by phospholipase C-catalyzed production of 1,4,5-inositol triphosphate (IP3). In this study we have investigated the source and mechanism of Ca2+ release induced by CCK-JMV-180 using streptolysin O-permeabilized pancreatic acinar cells. Caerulein-stimulated release of Ca2+ was completely blocked by either neomycin, an inhibitor of phospholipase C, or by heparin, an IP3 receptor antagonist. These observations are compatible with the conclusion that caerulein releases Ca2+ from an IP3-sensitive pool. In contrast to caerulein, however, CCK-JMV-180-stimulated release of Ca2+ was not blocked by either neomycin or by heparin, indicating that CCK-JMV-180 releases Ca2+ by mechanisms which do not involve the generation or action of IP3. CCK-JMV-180 stimulated the release of Ca2+ even after the IP3-sensitive pool had been completely emptied by prior exposure to a supramaximally stimulating concentration of IP3 (40 microM). Prestimulation of permeabilized acini with 20 mM caffeine did not abolish the CCK-JMV-180-induced Ca2+ release. These results indicate that CCK-JMV-180 stimulates release of Ca2+ from a hitherto uncharacterized intracellular storage pool which is insensitive to either IP3 or caffeine.

An outbreak of aflatoxicosis in Angora rabbits.

An outbreak of aflatoxicosis in Angora rabbits involving a large number of rabbitries was investigated. Mortality was more in weaners than in adults. Affected animals showed anorexia, dullness and weight loss followed by jaundice in terminal stages. Death occurred within 3-4 d of the appearance of clinical signs. Livers were moderately to severely congested, icteric and were hard to cut. Gall bladders were distended and had inspissated bile. Liver sections showed degenerative changes of hepatic cells along with dilatation and engorgement of sinusoids. Bile ducts had mild to severe periportal fibrosis. Focal areas of pseudolobulation and regenerative foci were also predominant. The level of aflatoxin B1 in feed samples from various farms submitted at the time of the investigation varied from 90 to 540 ug aflatoxin B1/kg of feed. Withdrawal of feed and supplementary therapy resulted in gradual disappearance of signs and mortality.

The enzymatic profile of urine and plasma in bovine urinary bladder cancer (enzootic bovine haematuria).

The enzymatic profile of urine and plasma in field cases of bovine bladder cancer was studied. Urinary lactate dehydrogenase activity was significantly altered along with the isoenzyme pattern. Activity of alkaline phosphatase and beta-glucuronidase was decreased in the affected animals. No significant changes were observed in acid phosphatase, or arylsulphatase A and B activity. In plasma, lactate dehydrogenase activity was elevated without any change in the isoenzyme pattern. No significant changes were observed in the other plasma enzymes studied or in the sialic acid concentration.

Molecular structure, polymorphism, and toxicity of lantadene A, the pentacyclic triterpenoid from the hepatotoxic plant Lantana camara.

Lantadene A (22 beta-angeloyloxy-3-oxo-olean-12-en-28-oic acid), a pentacyclic triterpenoid compound from lantana (Lantana camara) leaves has been obtained in two polymorphic forms I and II. Form I had white, fluffy, and rod-shaped uniform crystals. Form II particles were irregular, shining, and polyhedral. The two forms differed in melting behavior. The powder x-ray diffraction of form I showed sharp peaks whereas from II did not contain distinct peaks. From single-crystal three-dimensional x-ray structure determination, the molecular structure of form I has been established. A/B and B/C rings of the molecule are trans fused while D/E rings are cis fused. The packing of the molecule is stabilized by hydrogen bonding. Form I of lantadene A was non-toxic to guinea pigs on oral administration. Form II induced ictericity and toxicity associated with decrease in feed intake and fecal output, hepatomegaly, increase in plasma bilirubin, and acid phosphatase activity.

Erythrocyte glutathione and its metabolizing enzymes in bovine urinary bladder cancer.

Glutathione and its metabolizing enzymes were studied in erythrocytes of cattle suffering from urinary bladder cancer and associated hematuria. The concentration of glutathione (GSH) and GSH peroxidase activity were subnormal in the affected animals, whereas activities of GSH reductase and GSH S-transferase were not altered. Decreased GSH and GSH peroxidase are likely to make erythrocytes of the affected animals more susceptible to oxygen-induced injury and the radiomimetic effect of bracken fern.

Protein-phosphate complexes as an inhibitor of iron-induced lipid peroxidation.

Effect of phosphate buffer (pH 6.2) alone or in the presence of bovine serum albumin and other proteins on iron (II)-induced lipid peroxidation was studied. Phosphate buffer alone and in the presence of bovine serum albumin was found to inhibit lipid peroxidation. The inhibition was higher when bovine serum albumin was also present. Other proteins also inhibited lipid peroxidation in the presence of phosphate. Inhibition by proteins in the presence of phosphate seems to be due to binding of iron with phosphate and with protein-phosphate complexes. Reversal in inhibition was observed with an increase in iron concentration in reaction mixture. Equilibrium dialysis showed more binding of iron to protein in the presence of phosphate than in the presence of chloride ions.

Toxicity of isolated Lantana (Lantana camara L) constituents to male and female guinea pigs.

A toxin fraction was obtained from Lantana camara L (red variety) leaves by batch extraction and column chromatography on silica gel (60-120 mesh). The main constituents of the toxin preparation were lantadene A and lantadene B and it was devoid of reduced lantadene A. Oral administration (125 mg/kg bwt) of the toxin to male and female guinea pigs caused icterus and photosensitization within 48 hr. All the affected animals had hepatomegaly and significant increases in conjugated and unconjugated bilirubin in blood plasma. The intoxicated animals of either sex had marked increases in acid phosphatase activity which was inhibited 45.77% and 49.35% by 1 mM tartrate in male and female animals respectively. The corresponding inhibition of acid phosphatase activity in control male and female guinea pigs was 15.91% and 20.33% respectively.