OBJECTIVETo investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro.

METHODSHepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy.

RESULTSCompared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm.

CONCLUSIONDHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.

Aflatoxin B1 ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Cycle ; Cell Movement ; drug effects ; Docosahexaenoic Acids ; pharmacology ; Golgi Apparatus ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Mitochondria ; Neoplasm Invasiveness

Objective: This study aimed to explore the protective effect of procyanidin B2 (PCB2) on acute liver injury induced by aflatoxin B (AFB ) in rats. Methods: Forty Sprague Dawley rats were randomly divided into control, AFB , AFB + PCB2, and PCB2 groups. The latter two groups were administrated PCB2 intragastrically (30 mg/kg body weight) for 7 d, whereas the control and AFB groups were given the same dose of double distilled water intragastrically. On the sixth day of treatment, the AFB and AFB + PCB2 groups were intraperitoneally injected with AFB (2 mg/kg). The control and PCB2 groups were intraperitoneally administered the same dose of dimethyl sulfoxide (DMSO). On the eighth day, all rats were euthanized: serum and liver tissue were isolated for further examination. Hepatic histological features were assessed by hematoxylin and eosin-stained sections. Weight, organ coefficient (liver, spleen, and kidney), liver function (serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, and direct bilirubin), oxidative index (catalase, glutathione, superoxide dismutase, malondialdehyde, and 8-hydroxy-2'-deoxyguanosine), inflammation factor [hepatic interleukin-6 (IL-6) mRNA expression and serum IL-6], and bcl-2/bax ratio were measured. Results: AFB significantly caused hepatic histopathological damage, abnormal liver function, oxidative stress, inflammation, and bcl-2/bax ratio reduction compared with DMSO-treated controls. Our results indicate that PCB2 treatment can partially reverse the adverse liver conditions induced by AFB . Conclusion Our findings indicate that PCB2 exhibits a protective effect on acute liver injury induced by AFB .
Aflatoxin B1 ; toxicity ; Animals ; Biflavonoids ; administration & dosage ; pharmacology ; Catechin ; administration & dosage ; pharmacology ; Chemical and Drug Induced Liver Injury ; drug therapy ; etiology ; Male ; Poisons ; toxicity ; Proanthocyanidins ; administration & dosage ; pharmacology ; Protective Agents ; administration & dosage ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

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.
Aflatoxin B1/pharmacology* ; Animal ; Carbon Tetrachloride/pharmacology* ; Drug Synergism ; Fibrosis/chemically induced ; Glutathione Transferase/metabolism* ; Immunohistochemistry ; Lipid Peroxidation/drug effects ; Liver Neoplasms, Experimental/chemically induced* ; Male ; Rats ; gamma-Glutamyltransferase/metabolism*

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.
Aflatoxin B1/pharmacology* ; Animal ; Carbon Tetrachloride ; Carcinogens/pharmacology* ; Gene Expression/drug effects* ; Genes, Immediate-Early/drug effects* ; Liver Diseases/metabolism* ; Liver Diseases/genetics* ; Liver Diseases/chemically induced ; Male ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor alpha/metabolism*

Effects of diets on hepatic aflatoxin B1 (AFB1)- DNA binding and AFB1-induced glutathione S- transferase placental (GST-P) form positive hepatic foci have been examined in young male Fischer rats. Animals were fed either AIN-76A or Purina Chow (PC) diet for 1 wk before AFB1- DNA binding studies in vivo and in vitro. Animals were injected i.p. with AFB1 (1 mg/kg body wt) and 3 days later were given either AIN-76A or PC diet with or without 0.1% phenobarbital (PB) in their drinking water. All animals were sacrificed 10 wks after AFB1 dosing for analysis of AFB1-induced GST-P positive hepatic foci by immunochemistry. Two h after i.p. injection of AFB1, hepatic AFB1-DNA binding in AIN-76A fed rats was twice as much as those in PC fed animals without affecting GSH levels. There was no significant effect of diet on either cytochrome P-450 content, GSH levels or microsomal cytochrome P-450 mediated AFB1-DNA binding to exogenous DNA. There was a 40% increase in cytosolic GSH S-transferase activity with 1-chloro-2,4-dinitrobenzene as a substrate in PC fed animals compared to those given AIN- 76A diet. The number and area of AFB1-induced GST-P positive hepatic foci were twice and fivefold as much in AIN-76A fed compared to those in PC fed rats. The number of AFB1-induced GST-P positive foci was increased 5-10 fold in the presence of PB in both groups. In summary, the present data indicate that feeding of PC diet compared to AIN-76A diet inhibits the initiation phase whereas AIN-76A stimulates the promotion phase of AFB1 hepatocarcinogenesis in rats by inhibiting AFB1-DNA binding and increasing AFB1-induced hepatic foci respectively.
Aflatoxin B1/metabolism/*pharmacology ; Animals ; Cell Transformation, Neoplastic ; Cytochrome P-450 Enzyme System/metabolism ; DNA/*metabolism ; *Diet ; Glutathione Transferase/analysis/*metabolism ; Hepatocytes/drug effects/*enzymology ; Liver Neoplasms/*etiology ; Microsomes, Liver/enzymology ; Rats ; Research Support, U.S. Gov't, P.H.S.