electron microscopic study of the effects of aflatoxins on skeletal muscles ultra structures and mitochondrial succinat dehydrogenase activity

The problem of using contaminated food with toxigenic fungi is still one of the most important stigmas in the field of nourishment of human and animals. Aflatoxins are one of the toxigenic fungi that draw attention for researcher. This study was designed to determine how Aflatoxins contaminated food and feeding regimen might effect and induce specific changes in the muscle. Aflatoxins producing fungi were isolated from seed samples according to method of Shotwell et al. Spore suspension were prepared according to Faraj method. Rats were fed daily with diet contaminated with the spore. The Extensor digitorum longus muscle were removed and cut into small pieces incubated in Tetrazolium solution [NBT] incubation solution. The specimens were prepared and examined by electron microscope. Animals treated with AFB[1] have shown a marked increase in body weight Haziness and loss of the banding pattern of the sarcomers was a marked feature Reaction product of succinat-dehydrogenase showed increase reaction in treated animals at the site if mitochondria. Skeletal muscle nuclei showed a pronounced effect of Aflatoxins on the nuclei, they have become pyknotic and there number were less than normal. It has been concluded that AFB[1] have a marked effects on the structure of the sarcomere and on the activity of succinic dehydrogenase enzyme of the mitochondria

Effect of aflatoxin B1 on phosphoinositide signal transduction pathway during regeneration of liver cells following partial hepatectomy.

Aflatoxin B1 (AFB1) when administered to partially hepatectomised rats 4 hr prior to sacrifice, activated the signalling pathway in regenerating rat liver. The activity of phosphatidylinositol (PI) kinase was found decreased at 30 min but increased at 24 hr and returned to normal at 48 hr. At 30 min, inositol-1,4,5-triphosphate (IP3) level increased significantly whereas diacylglycerol (DAG) level dropped. However, at 24 hr and 48 hr, DAG and IP3 showed the same trend i.e. an increase in their levels. Phosphatidylinositol-4-phosphate levels were found to increase at 24 hr. Protein kinase C (PKC), activity from the particulate fraction was significantly inhibited at 30 min, followed by increase in activity at 24 hr and return to normal at 48 hr. Cytosolic PKC showed a decrease at 24 hr and a significant increase at 48 hr. At the peak of DNA synthesis (24 hr) following partial hepatectomy, all these signalling steps had earlier been found to be inhibited, but the present study shows that aflatoxin B1 administration 4 hr prior to sacrifice reverses the action. Activation of PKC by aflatoxin B1, during regeneration of liver cells when PKC in normally inhibited, may possibly create conditions conducive to carcinogenesis.

Enhancement of aflatoxin B1-induced enzyme altered hepatic foci in rats by treatment with carbon tetrachloride

The effect of carbon tetrachloride (CCl4) on aflatoxin B1 (AFB1)-induced enzyme altered hepatic foci has been examined in young male Fischer rats given AIN-76A diet. A single i.p. dose of AFB1 (0.2 mg/kg body wt) was given to rats 24 h after partial hepatectomy. Two weeks later, CCl4 (0.8 ml/kg body wt) was injected i.p. once a week for 9 weeks. Animals were sacrificed 24 h after the last dose of CCl4 and glutathione S-transferase placental form (GST-P) and gamma-glutamyl transpeptidase (GGT) positive hepatic foci were analyzed by immunohistochemical and histochemical methods, respectively. Ten weeks after AFB1 dosing, treatment with CCl4 increased the number of AFB1-induced enzyme altered foci several fold and produced a ten to twenty-fold increase in area and volume. GST-P was more sensitive than GGT in detecting AFB1-induced enzyme altered foci. Treatment with AFB1 or CCl4 produced mild hepatic fibrosis in zones 1 and 3 respectively, whereas both treatments produced severe fibrosis in zones 1 to 3 areas. Treatment with CCl4 after AFB1 dosing lowered hepatic GSH levels by 20% and increased lipid peroxidation by 40%. It appears that CCl4, by being an effective enhancer of AFB1-induced enzyme altered hepatic foci in the rat, may mimic cirrhosis observed in human hepatocellular carcinoma.

Effect of aflatoxin B in vitro on rat liver mitochondrial respiratory functions.

Isolated rat liver mitochondria were incubated with various concentrations of aflatoxin B (AFB) for different periods of time. Respiration rates were then measured with two substrates (succinate and glutamate). State 3 respiration rate (with added ADP) declined with increase in preincubation concentrations of AFB1 (0.15-0.50 mM). On the other hand, state 4 respiration rate (after depletion of added ADP) was found to increase with increased pretreatment concentration of AFB. As a consequence, respiratory control index was reduced attaining minimum value with 0.25 mM AFB and preincubation time of 10 min. The induced anomaly in mitochondrial respiratory functions appear to be due to membrane damage caused by interaction of reactive AFB1 metabolite generated by mitochondrial cytochrome P-450 enzymes with mitochondrial components.

The effect of aflatoxin B1 on the expression of early response genes and transforming growth factor-alpha in CCl4 induced rat liver injury

Aflatoxin B1 (AFB1), a fungal toxin produced by Aspergillus flavus, is known to be a possible hepatocarcinogen. But the molecular biologic changes which may occur following exposure to AFB1 are not known and thus the carcinogenesis is not yet understood. This study was performed to examine the expressions of c-myc, c-fos and TGF-alpha genes and to investigate the possible role of those molecular biologic changes in hepatic regeneration and in the development of hepatocellular carcinoma (HCC). Sprague-Dawley rats were divided into 3 groups: Carbon tetrachloride (CCl4) only was administered to group I, AFB1 only was administered to group II and a combination of AFB1 and CCl4 was administered to group III. The animals were sacrificed at 0.5, 1, 2, 6, 12, 24, 48, and 72 hours after treatment. In addition to the examination of the hematoxylin-eosin stained sections, hepatic regeneration and apoptosis were analyzed quantitatively by bromodeoxyuridine (BrdU)-anti-BrdU immunohistochemistry and TUNEL assay utilizing apoptosis kit, respectively. The hepatic expressions of c-myc, c-fos and transforming growth factor-alpha (TGF-alpha) were examined by immunohistochemistry and studied by Western blot. The number of BrdU labelled cells and the degree of necrosis/apoptosis were comparable among the different groups. Livers of the group II rats showed nearly normal histology without regeneration and necrosis/apoptosis. In groups I and III, the number of BrdU- labelled cells showed an increase at 48 hours after treatment, and the increment was significantly higher in group I than in group III. Most BrdU-labelled cells were mature hepatocytes in group I, whereas in group III they appeared to be less mature. In group I, apoptosis showed an increase at around 24 hours, but appeared in group III as early as 12 hours after treatment and persisted through 48 hours. The expression of c-myc and c-fos were also different between the experimental groups. The expression intensity of c-myc in group I was highest at 1 hour and decreased thereafter. In groups II and III, the expressions were much more intense than in group I, except at 1 hour, and the increased intensity persisted throughout the experiment. Group II in particular showed a peak intensity at 30 minutes and at 6 hours after treatment. In group I, c-fos was strongly expressed only at 24 hours, but in group III, there was progressively increased expression with peak intensity at 24 hours. TGF-alpha was expressed in similar intensities in all groups throughout the experiment. These results suggest that AFB1 may evoke an intense and protracted expression of c-myc, provocating the CCl4-induced necrosis of hepatocytes, and a prolonged expression of c-fos, including persistent signals for regeneration which in turn may activate the replication of immature cells. These findings will aid further investigation of molecular biologic and histologic characteristics of the hepatotoxic and hepatocarcinogenic mechanism of AFB1 in rats. And these results in rats, together with clinico-epidemiologic and molecular biologic investigations in humans and other animals, suggest that AFB1 may supply hepatocarcinogenic background in early exposure time in AFB1-contaminated areas of China and Korea.

Acute effect of aflatoxin B_ on inbred strains of mice-I

O efeito agudo de aflatoxina AFB1 foi avaliado em camundongos C57B1/6, machos, com 30 dias de idade e peso médio de 20g inoculados com dose única de AFB1 (60mg/Kg peso corpóreo). Os animais foram divididos em 3 grupos intoxicado (30 animais) e grupo controle II (30 animais). O grupo intoxicado foi inoculado por via intraperitoneal com AFB1 (60mg/Kg peso corpóreo) dissolvida em óleo de milho (0,0l mL/g) e o grupo controle II recebeu pela mesma via somente óleo de milho (0,01mL/g). Lotes de 10 animais de ambos os grupos foram sacrificados após 24,72 e 168 horas da inoculaçäo. O grupo controle II näo foi inoculado e serviu como padräo de normalidade para as análises bioquímicas (funçäo hepática e renal e hematológica. A AFB1 foi detectada no fígado do grupo intoxicado após 24 (1,46ng/g),72 horas 2,30ng/g) e 168 horas (2,18ng/g) da inoculaçäo. As lesöes histológicas mais evidentes ocorreram após 168 horas da inoculaçäo com AFB1 , com vacuolizaçäo dos hepátocitos e desarronjo da arquitetura do parênquina hepático. Os níveis sérios de fosfatase alcalina estavam elevados após 24 e72 horas da inaculaçäo no grupo intoxicado. Esse estudo indicou queo fígado foi o órgäo alvo da AFB1 nos camundongos C57B1/6,provocando lesöes histopatológicas e alteraçöes bioquímicas. Provavelmente essas alteraçöes estäo diretamente relacionadas ao processo mais lento de biotransformaçäo e eliminaçäo dos camundongos para os efeitos dessa micotoxina