Treatment Of Rat Hepatocytes Research Articles

Effects of indole-3-carbinol on biotransformation enzymes in the rat: in vivo changes in liver and small intestinal mucosa in comparison with primary hepatocyte cultures

Groups of male Wistar rats were fed semi-synthetic diets containing 0, 200 or 500 mg indole-3-carbinol (I3C)/kg for 2, 7, 14 or 28 days. After 2 days, P-450 activities were already induced, but the isoenzyme pattern induced was different in the liver and the small intestine. Hepatic P4501 A1, P4501 A2 and P4502 B1 apoprotein levels were dose-relatedly enhanced, whereas in the small intestine induced levels of P4502 B1 and P4501 A1 were detected but P4501 A2 was not induced. Pentoxy- and ethoxyresorufin dealkylation (PROD and EROD) were dose-relatedly enhanced in the liver (5- and 7-fold, respectively, in the higher dose group) as well as in the small intestine (8- and 13-fold, respectively, at 500 mg I3C/kg diet). Testosterone 16α- and 16β-hydroxylation in the small intestine were enhanced (6-9-fold) from day 2 onwards, but in the liver these activities were only slightly enhanced from day 7 onwards. Thus, the major forms induced in the liver appear to be P4501 A1, P4501 A2, P4502 B1 and, to a lesser extent, P4503 A, whereas in the small intestine all of the effects that were found are associated with only one cytochrome P-450, P4502 B1. After 2 days I3C (500 mg/kg) induced glutathione S-transferase in the liver (1.3-fold) and small intestine (1.5-fold). Hepatic glucuronyl transferase (GT1) was induced (about 1.6-fold) after 7, 14 and 28 days. DT-diaphorase was induced in the liver (2.7-fold) and small intestine (1.5-fold) after 14 days of exposure to 500 mg I3C/kg diet. Treatment of rat hepatocytes with indole-3-acetonitrile and 3,3′-diindolylmethane, but not I3C and indole-3-carboxaldehyde, enhanced EROD activity and halved testosterone 16α- and 2α-hydroxylation. All four indoles slightly induced glutathione S-transferase in cultured hepatocytes. Thus, the in vitro studies suggest that the in vivo effects of I3C have to be attributed to indole-condensation products, such as 3,3′-diindolylmethane, but not to I3C itself.

Hyperphosphorylation of cytokeratins 8 and 18 by microcystin-LR, a new liver tumor promoter, in primary cultured rat hepatocytes.

Microcystin-LR (MC-LR), an inhibitor of protein phosphatases 1 and 2A, is a potent tumor promoter in rat liver initiated with diethylnitrosamine. To understand its biochemical process in hepatocytes, primary cultured rat hepatocytes were treated with MC-LR. MC-LR (1 microM) induced phosphorylation of various proteins. Two 55 and 49 kDa proteins were phosphorylated at a 3-fold higher rate than other proteins, and these proteins were identified to be cytokeratins 8 and 18 respectively, by immunoprecipitation and Western blot analysis using monoclonal anti-cytokeratin 8 and 18 antibodies. The basic cytokeratins 8 and 18 showed pI 6.4 and 5.4 respectively, in two-dimensional gel electrophoresis. MC-LR dose dependently increased phosphorylation of cytokeratins 8 and 18 in a cell-free system by incubation with a cytosolic fraction of rat liver containing both protein kinases and protein phosphatases 1 and 2A, and with [gamma-32P]ATP. Cytokeratins 8 and 18 were target proteins for phosphorylation induced by inhibition of protein phosphatases 1 and 2A, in vitro and in rat hepatocytes. Thus, the treatment of rat hepatocytes with MC-LR induced hyperphosphorylation of cytokeratins 8 and 18 associated with morphological changes, indicating that intermediate filament networks were rearranged in the cytoplasm. The hyperphosphorylation of cytokeratins is a significant biochemical process associated with liver tumor promotion.

Pertussis toxin-sensitive Gi protein involvement in epidermal growth factor-induced activation of phospholipase C-gamma in rat hepatocytes.

Treatment of rat hepatocytes with epidermal growth factor (EGF) produced an enhanced tyrosine phosphorylation of the EGF receptor and phospholipase C-gamma (PLC-gamma) in conjunction with the mobilization of Ca2+. Approximately 30% of the total PLC-gamma was tyrosine-phosphorylated with a maximum being reached after 30 s of incubation with EGF. Pretreatment of the rats with pertussis toxin prior to isolation of the hepatocytes blocked EGF-induced tyrosine phosphorylation of PLC-gamma and Ca2+ mobilization but had no effect on autophosphorylation of the EGF receptor or Ca2+ responses elicited by angiotensin II or phenylephrine. Under these conditions Gi protein alpha subunits were fully ADP-ribosylated. A 41-kDa Gi protein alpha subunit was found to be present in the anti-PLC-gamma immune complex after EGF stimulation as shown by in vitro ADP-ribosylation using [32P]NAD+ and activated pertussis toxin. The kinetics of association between PLC-gamma with Gi alpha protein reached a maximum after 1 min of incubation with EGF. Antibodies specific for the EGF receptor also coimmunoprecipitated a Gi protein alpha subunit. Treatment of hepatocytes with EGF caused first an increase and then a decrease in the amount of Gi protein alpha subunit associated with the EGF receptor. In contrast, studies with cultured rat liver (WB) cells, a cell line in which EGF stimulation of phosphoinositide hydrolysis is not inhibited by pertussis toxin, showed that a stable complex of Gi alpha was not formed with either PLC-gamma or EGF receptor immunoprecipitates. These results indicate that a pertussis toxin-sensitive Gi protein is uniquely involved in the signal transduction pathway mediating EGF-induced activation of PLC-gamma and Ca2+ mobilization in hepatocytes.

Asymmetric distribution of the phosphatidylinositol‐linked phospho‐oligosaccharide that mimics insulin action in the plasma membrane

We have investigated the topography of a glycosyl-phosphatidylinositol implicated in insulin action by a combination of two complementary methods: (a) chemical labelling with a non-permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with beta-galactosidase (EC 3.2.1.23) or phosphatidylinositol-specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl-phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl-phosphatidylinositol to the exterior. Upon insulin addition (10 nM), about 60% of the total glycosyl-phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with beta-galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosyl-phosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol-specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl-phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulin-sensitive cells.

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A 2

The effect of phospholipase A 2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membranebound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A 2 (0.9 units /dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 ± 0.15 nmol/min per mg compared to 3.9 ± 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units /dish of phospholipase A 2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 ± 0.10 nmol/min per mg to 2.6 ± 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 ± 0.16 nmol/min per mg to 1.8 ± 0.20 nmol/min per mg). The effect of phospholipase A 2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepaotcytes in the presence of phospholipase A 2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 ± 0.02· 10 −5 dpm/dish to 3.1 ± 0.1 · 10 −5 dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 ± 0.05 · 10 −5 to 1.4 ± 0.03 · 10 −5 dpm) and phosphocholine fractions (from 8.5 ± 0.07 · 10 −5 to 6.9 ± 0.05 · 10 −5 dpm). We conclude that phospholipase A 2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.

Application of a hepatocyte-erythrocyte coincubation system to studies of cyanide antidotal mechanisms

A coincubation system composed of hepatocytes in primary monolayer culture and erythrocytes suspended in the culture medium was developed and used as a model for investigations of mechanisms of cyanide antidote action at the cellular level. Hepatocyte ATP was used as the cytotoxicity indicator. Treatment of rat hepatocytes in the coincubation system with KCN (1.0 m m) for 10 min at 37°C selectively reduced hepatocyte ATP levels to 33 ± 15% of control (no KCN added) levels. 4-Dimethylaminophenol (DMAP), cobalt(II) chloride, sodium nitrite, sodium thiosulfate, or a combination of the last two antidotes added to the KCN-containing medium significantly reversed ATP depression and the response was concentration dependent. The relative effectiveness, on a molar basis, was estimated to be DMAP > CoCl 2 ⪢ NaNO 2 ⋍ Na 2S 2O 3. NaNO 2 and DMAP induced methemoglobin formation in the absence of cyanide and cyanmethemoglobin formation in its presence; erythrocytes were required in the medium for effectiveness. CoCl 2 produced neither cyanmethemoglobin nor thiocyanate in appreciable quantities nor required erythrocytes for antagonism. Na 2S 2O 3 converted cyanide to thiocyanate and reversed ATP depression without erythrocytes in the medium. The addition of erythrocytes increased these rates significantly and to a greater extent than albumin. The overall results are consistent with previously proposed modes of action for these antidotes. However, the enhancement in cyanide metabolism and ATP recovery with Na 2S 2O 3 and erythrocytes in the system was unexpected and raises the possibility that erythrocytes may contribute to cyanide disposition and antagonism in vivo when this antidote is administered.

U.v.-enhanced reactivation of u.v.-irradiated herpes virus by primary cultures of rat hepatocytes.

Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. In this paper, we report the development of a primary rat hepatocyte culture system to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. We have obtained data demonstrating that enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurs in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured by direct plaque assay. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions that may have a role in the initiation of hepatocarcinogenesis.

Effect of hormones on adenine and adenosine metabolism in mammary gland in vitro.

Little is known regarding the effect of hormones on purine metabolism. It has been shown that injection of some hormones, such as insulin, glucagon, increase PRPP concentration in mouse liver1. Also integrative action of insulin in purine metabolism has been reported2. According to these authors, hepatic PRPP amidotransferase activity in diabetic rats decreased 50%, and hepatic concentrations of ATP and GTP decreased markedly. Injection of insulin restored enzyme activity and ribonucleotide level increased. Insulin treatment of rat hepatocytes also increased adenine nucleotide pool3.