Primary cultured hepatocytes of the bony fish, Oreochromis mossambicus, the tilapia: a valid tool for physiological studies on IGF-I expression in liver.
(9/7533)
In spite of the importance of IGF-I for growth and development, knowledge about regulation of its production in submammalian species is rather limited. In order to create a tool for investigation of direct regulatory effects on the expression of IGF-I in bony fish liver, a primary cell culture of hepatocytes from Oreochromis mossambicus, the tilapia, was established. The cells were viable for up to 3 days and IGF-I mRNA synthesis was detected by northern blot and semiquantitative reverse transcriptase (RT)-PCR. Northern blot analysis of the primary cultured hepatocytes revealed four different IGF-I transcripts, 0.5, 1.9, 3.9 and 6.0 kb in size, which were identical to those in liver tissue. However, the expression rate was weaker than that in liver. The direct effects of recombinant tilapia (rt) growth hormone (GH) and salmon (s) IGF-I on the expression of IGF-I in primary cultured hepatocytes were investigated in time-course and dose-response experiments. In untreated cultures, IGF-I mRNA decreased with time. Hepatocytes treated with 100 nM rtGH resulted in a pronounced stimulation of IGF-I mRNA expression throughout the experiment. Treatment with rtGH in concentrations ranging from 0.1 nM to 1 microM caused a clear dose-dependent increase in the amount of IGF-I mRNA. Significant stimulation was obtained even with 0.1 nM, reaching a plateau with 10 nM. Neither significant inhibitory nor stimulatory effects were detected by adding sIGF-I from 0.1 nM to 1 microM to the hepataocytes. Our results indicate that the established primary cell culture of tilapia hepatocytes is a useful system in which to study direct effects of potential regulators of bony fish liver cell function. (+info)
Hormonal regulation of glutathione S-transferase expression in co-cultured adult rat hepatocytes.
(10/7533)
Glutathione S-transferases (GSTs) are subject to regulation by thyroid and sex hormones and by GH. We have used an in vitro experimental system comprising adult rat hepatocytes co-cultured with rat liver epithelial cells of primitive biliary origin, to distinguish between direct and indirect effects of various hormones on GSTs; to identify the GST subunits affected by individual hormones; and to investigate the level at which the hormones act. Tri-iodothyronine (T3), thyroxine (T4) and 17beta-oestradiol (OE2) reduced GST activities, whereas testosterone, dihydrotestosterone, and human growth hormone (hGH) had little effect on total GST activity. HPLC separation of the various GST subunits revealed that T3 and T4 reduced total GST content, in particular the abundance of subunits M1 and M2. The amount of the Pi-class subunit P1 was reduced by OE2. Treatment of the co-cultured cells with this hormone altered the GST subunit profile to one that is more similar to that observed in freshly isolated hepatocytes. Analysis of mRNAs demonstrated that some of the hormones act at a pre-translational level, whereas others act at a translational or post-translational level to regulate the expression of various GST subunits. (+info)
The nongenotoxic hepatocarcinogens diethylhexylphthalate and methylclofenapate induce DNA synthesis preferentially in octoploid rat hepatocytes.
(11/7533)
Diethylhexylphthalate (DEHP), a rodent carcinogen, and 1,4-dichlorobenzene (DCB), a noncarcinogen in rat liver, are potent hepatomitogens. We have reported previously that 7-day dosing with DEHP induced a higher bromodeoxyuridine labeling index (LI) in binuclear octoploid (2x4N) rat hepatocytes than did DCB, suggesting that induction of DNA synthesis in 2x4N hepatocytes might represent a more substantial carcinogenic risk. We compared 2 additional rodent hepatocarcinogens, methylclofenapate (MCP) and phenobarbitone, with ethylene thiourea (ETU), a noncarcinogenic hepatomitogen in rat. All 3 chemicals increased hepatic LI; the 8N population had the highest LI, but only the carcinogens increased LI in the 2x4N and 4N populations. To identify the target population for induction of DNA synthesis, we used a 1-hour pulse label at the peak of induction. The results were consistent with the 7-day data, and again the highest LI was in the 8N population. The nongenotoxic rodent carcinogens MCP and DEHP induced a significant increase in the LI in the 2x4N population, whereas ETU and DCB did not. These data support the hypothesis that increased DNA synthesis within the minority 2x4N population may be more significant for subsequent hepatocarcinogenesis. (+info)
Carcinogenicity of chloral hydrate administered in drinking water to the male F344/N rat and male B6C3F1 mouse.
(12/7533)
Male B6C3F1 mice and male F344/N rats were exposed to chloral hydrate (chloral) in the drinking water for 2 years. Rats: Measured chloral hydrate drinking water concentrations for the study were 0.12 g/L, 0.58 g/L, and 2.51 g/L chloral hydrate that yielded time-weighted mean daily doses (MDDs) of 7.4, 37.4, and 162.6 mg/kg per day. Water consumptions, survival, body weights, and organ weights were not altered in any of the chloral hydrate treatments. Life-time exposures to chloral hydrate failed to increase the prevalence (percentage of animals with a tumor) or the multiplicity (tumors/animal) of hepatocellular neoplasia. Chloral hydrate did not increase the prevalence of neoplasia at any other organ site. Mice: Measured chloral hydrate drinking water concentrations for the study were 0.12 g/L, 0.58 g/L, and 1.28 g/L that gave MDDs of 13.5, 65.0, and 146.6 mg/kg per day. Water consumptions, survival, body and organ weights, were not altered from the control values by any of the chloral hydrate treatments. Enhanced neoplasia was observed only in the liver. Prevalence and multiplicity of hepatocellular carcinoma (HC) were increased only for the high-dose group (84.4%; 0.72 HC/animal; p < or = 0.05). Values of 54.3%; 0.72 HC/animal and 59%; 1.03 HC/animal were observed for the 13.5- and 65.0-mg/kg per day treatment groups. Prevalence and multiplicity for the control group were 54.8%; 0.74 HC/animal. Hepatoadenoma (HA) prevalence and multiplicity were significantly increased (p < or = 0.05) at all chloral hydrate concentrations: 43.5%; 0.65 HA/animal, 51.3%; 0.95 HA/animal and 50%; 0.72 HA/animal at 13.5, 65.0, and 146.6 mg/kg per day chloral hydrate compared to 21.4%; 0.21 HA/animal in the untreated group. Altered foci of cells were evident in all doses tested in the mouse, but no significant differences were observed over the control values. Hepatocellular necrosis was minimal and did not exceed that seen in untreated rats and mice. Chloral hydrate exposure did not alter serum chemistry and hepatocyte proliferation in rats and mice or increase hepatic palmitoyl CoA oxidase in mice at any of the time periods monitored. It was concluded that chloral hydrate was carcinogenic (hepatocellular neoplasia) in the male mouse, but not in the rat, following a lifetime exposure in the drinking water. Based upon the increased HA and combined tumors at all chloral hydrate doses tested, a no observed adverse effect level was not determined. (+info)
Insulin effects on sterol regulatory-element-binding protein-1c (SREBP-1c) transcriptional activity in rat hepatocytes.
(13/7533)
The transcription factor sterol regulatory-element-binding protein-1c (SREBP-1c) plays a major role in the effect of insulin on the transcription of hepatic genes such as glucokinase and fatty acid synthase. We show here in cultured rat hepatocytes that insulin, through activation of the phosphatidylinositol 3-kinase pathway increases the abundance of the precursor form of SREBP-1c in endoplasmic reticulum. This precursor form is then rapidly cleaved, possibly irrespective of the continuous presence of insulin, leading to an increased content of the nuclear mature form of SREBP-1c. Nevertheless, the increased amount of the mature form of SREBP-1c in the nucleus is not a prerequisite for the rapid effect of insulin on the transcription of genes such as glucokinase, suggesting that additional actions of the hormone are involved, such as the activation of the nuclear form of SREBP-1c or of an unidentified SREBP-1c partner. (+info)
Physiological oxygen tensions modulate expression of the mdr1b multidrug-resistance gene in primary rat hepatocyte cultures.
(14/7533)
P-Glycoprotein transporters encoded by mdr1 (multidrug resistance) genes mediate extrusion of an array of lipophilic xenobiotics from the cell. In rat liver, mdr transcripts have been shown to be expressed mainly in hepatocytes of the periportal region. Since gradients in oxygen tension (pO(2)) may contribute towards zonated gene expression, the influence of arterial and venous pO(2) on mRNA expression of the mdr1b isoform was examined in primary rat hepatocytes cultured for up to 3 days. Maximal mdr1b mRNA levels (100%) were observed under arterial pO(2) after 72 h, whereas less than half-maximal mRNA levels (40%) were attained under venous pO(2). Accordingly, expression of mdr protein and extrusion of the mdr1 substrate rhodamine 123 were maximal under arterial pO(2) and reduced under venous pO(2). Oxygen-dependent modulation of mdr1b mRNA expression was prevented by actinomycin D, indicating transcriptional regulation. Inhibition of haem synthesis by 25 microM CoCl(2) blocked mdr1b mRNA expression under both oxygen tensions, whereas 80 microM desferrioxamine abolished modulation by O(2). Haem (10 microM) increased mdr1b mRNA levels under arterial and venous pO(2). In hepatocytes treated with 50 microM H(2)O(2), mdr1b mRNA expression was elevated by about 1.6-fold at venous pO(2) and 1.5-fold at arterial pO(2). These results support the conclusion that haem proteins are crucial for modulation of mdr1b mRNA expression by O(2) in hepatocyte cultures and that reactive oxygen species may participate in O(2)-dependent signal transduction. Furthermore, the present study suggests that oxygen might be a critical modulator for zonated secretion of mdr1 substrates into the bile. (+info)
Clear cell carcinoma of the liver: a comparative immunohistochemical study with renal clear cell carcinoma.
(15/7533)
Morphologic differentiation of clear cell hepatocellular carcinoma (HCC-CC) from clear cell renal carcinoma (RCC-CC) may not be possible without the aid of immunohistochemical stains. We performed a battery of immunohistochemical stains on 10 previously diagnosed HCC-CCs, and 10 RCC-CCs, in order to determine which single or combination of immunostains would be most useful in diagnosis. We concluded that a positive Hepatocyte immunostain (DAKO) is sufficient for a diagnosis of HCC-CC if enough tissue is available. This immunostain distinguishes HCC-CC from other clear cell malignancies with sensitivity of 90% and specificity of 100%, when biopsy material is adequate. Other tests were much less sensitive, although several had specificity of 100%. A negative immunostain does not exclude the diagnosis of HCC-CC (negative predictive value 91%, especially in small biopsy material) and should be followed by additional immunostains such as pCEA for demonstration of tumor canaliculi, ubiquitin for Mallory bodies, and several epithelial cell markers that are typically positive in RCC-CC (epithelial membrane antigen, Leu M-1, pancytokeratin) and negative in HCC-CC. (+info)
Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes.
(16/7533)
CD14 presents as a glycosylphosphatidylinositol-linked membrane protein on the surface of monocytes/macrophages and as a soluble protein in the serum. Our previous studies have shown that an 80-kilobase pair (kb) genomic DNA fragment containing the human CD14 gene is sufficient to direct CD14 expression in a monocyte-specific manner in transgenic mice. In addition, we discovered that human CD14 is highly expressed in hepatocytes. Here, we report the generation of transgenic mice with either a 24- or 33-kb human CD14 genomic DNA fragment. Data from multiple transgenic lines show that neither the 24- nor the 33-kb transgenic mice express human CD14 in monocytes/macrophages. However, human CD14 is highly expressed in the liver of the 33-kb transgenic mice. These results demonstrate that human CD14 expression is regulated differently in monocytes and hepatocytes. Furthermore, we identified an upstream regulatory element beyond the 24-kb region, but within the 33-kb region of the human CD14 gene, which is critical for CD14 expression in hepatocytes, but not in monocytes/macrophages. Most importantly, the data demonstrate that the liver is one of the major organs for the production of soluble CD14. These transgenic mice provide an excellent system to further explore the functions of soluble CD14. (+info)