Persistently increased expression of a 3-methylcholanthrene-inducible phenol uridine diphosphate-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas.

Increased UDP-glucuronosyltransferase in rat hepatocyte nodules and hepatocellular carcinomas produced by feeding 2-acetylaminofluorene or N-nitrosomorpholine was studied using isozyme-selective substrates, antibodies, and DNA probes. UDP-glucuronosyltransferase (UDP-GT) activities toward 4-methylumbelliferone, 1-naphthol, and benzo[a]pyrene-3,6-quinol were reversibly increased by short term feeding of 2-acetylaminofluorene but were persistently increased in hepatocyte nodules and differentiated hepatocellular carcinomas. Immunoblot analysis revealed that short term feeding of 2-acetylaminofluorene increased a Mr 55,000 polypeptide corresponding to the previously characterized UDP-GTI or phenol UDP-GT. However, in some hepatocyte nodules and hepatocellular carcinomas either the Mr 55,000 or a new Mr 53,000 polypeptide was preferentially increased, suggesting heterogeneous UDP-GT forms in liver nodules and carcinomas. Northern blot hybridization with a synthetic DNA probe to phenol UDP-GT demonstrated increased levels of mRNA in liver nodules. The results suggest persistently increased expression of at least two phenol UDP-GT enzyme forms in hepatocyte nodules, which may contribute to the toxin-resistance phenotype frequently observed at cancer prestages.

Alterations of antioxidant enzyme expression in response to hydrogen peroxide.

The effect of hydrogen peroxide (H2O2) on the expression of different antioxidant enzymes was investigated in primary rat hepatocytes and the rat hepatoma H4IIE cell line. Catalase mRNA expression and enzyme activity decreased during rat hepatocyte culture. Exposure of hepatocytes to H2O2 prevented this decrease in catalase mRNA expression, catalase expression was induced 2-fold. MnSOD message levels showed a peak after 12 h of culture and MnSOD enzyme activity increased similarly. MnSOD mRNA expression was also induced after exposure to H2O2. Cu/ZnSOD mRNA expression remained constant during culturing and was not affected by H2O2 treatment. In confluent hepatoma H4IIE cells catalase mRNA expression was lower than in early hepatocyte cultures and could be induced 2-fold upon treatment with H2O2. Actinomycin D alone caused the same amount of induction of catalase mRNA in rat hepatocytes as in combination with H2O2. Exposure of hepatocytes to cycloheximide did not influence the induction of catalase mRNA by H2O2. In rat hepatoma H4IIE cells the induction of catalase mRNA by H2O2 was prevented by the addition of actinomycin D or cycloheximide. Although induction of catalase mRNA by H2O2 was found in rat hepatocytes and H4IIE cells, gene expression of catalase does not appear to be regulated in both cell types in the same manner.

Site-specific hypomethylation of c-myc protooncogene in liver nodules and inhibition of DNA methylation by N-nitrosomorpholine.

The protooncogene c-myc was investigated in N-nitrosomorpholine-induced rat liver nodules to elucidate the role of altered DNA methylation in chemical carcinogenesis. Furthermore, Micrococcus luteus DNA and chicken erythrocyte DNA were modified in vitro by reactive metabolites of N-nitrosomorpholine, generated by P450-dependent monooxygenases. The modified DNAs were less methylated in vitro than control DNAs by DNA-(cytosine-5)-methyltransferase (DNA methylase). The DNA methylase assay and 32P-postlabeling analysis revealed lowered levels of DNA methylation in nodular DNA. In nodular tissue, c-myc messenger RNA levels were found to be increased compared to normal liver. DNA methylation analysis using the restriction endonucleases HpaII/MspI indicated hypomethylation in the first intron of c-myc DNA in liver nodules. The results suggest that genotoxic lesions may cause stably inherited, aberrant DNA methylation patterns which may be responsible for site-specific hypomethylation of the c-myc protooncogene in liver nodules.

Drug metabolizing enzyme activities in rat liver epithelial cell lines, hepatocytes and bile duct cells.

P450-dependent mono-oxygenase and conjugating enzyme activities were studied in rat liver epithelial cells (RLEs) and compared to those in hepatocytes and bile duct cells. Various RLE cell lines were investigated since (a) they are suspected to be derived from cells in the lineage from putative pluripotent stem cells to either hepatocytes or bile duct cells, and (b) they may represent targets of chemical carcinogens. Despite considerable variation between lines, common features were recognized. P450-dependent monooxygenase activities (7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase) were undetectable in all RLEs and bile duct cells, and were uninducible by benz(a)anthracene. In contrast, glucuronosyltransferase (GT), sulfotransferase and GSH transferase activities were clearly detectable. Conjugating enzyme activities increased until confluency of the cell cultures was reached. Under the latter conditions, GT activities towards 4-methylumbelliferone or benzo(a)pyrene-3,6-quinol (substrates of a 3-methylcholanthrene-inducible phenol GT) were similar to those found in hepatocytes or bile duct cells. Using a selective cDNA probe, phenol GT mRNA was clearly detectable in RLE1. In contrast, GT activity towards 4-hydroxybiphenyl was much lower than in hepatocytes or bile duct cells (0.04- and 0.03-fold). Sulfotransferase and GSH transferase activities were also roughly comparable to those found in hepatocytes and in bile duct cells. The results suggest that RLEs and bile duct cells exhibit both high conjugating enzyme activities and a lack of P450-dependent mono-oxygenase activities, a pattern resembling the 'toxin-resistance phenotype' found in putative preneoplastic hepatocyte foci and nodules.

The influence of oxidative stress on catalase and MnSOD gene transcription in astrocytes.

The brain is particularly vulnerable to oxygen free radicals, which have been implicated in the pathology of several neurological disorders. The antioxidant enzyme (AOE) system of the brain may play an important role in the protection against such oxidative stress. We investigated the influence of oxidative stress on the transcription of catalase and MnSOD mRNA. Primary rat astroglial cell cultures were treated either with hydrogen peroxide (H2O2), as a direct mediator of oxidative stress, or with the redox cycling compound paraquat. Both substances led to an increase of catalase and MnSOD mRNA levels. To further elucidate the mechanisms residing behind this increase, transfection experiments were performed. Transient transfection of primary astroglial cells with a reporter plasmid containing the upstream region of the catalase gene showed a decrease in reporter gene activity after exposure of transfected cells to either H2O2 or paraquat. In contrast, transfection experiments done with reporter plasmids for the MnSOD upstream region resulted in an increase of reporter gene activity after H2O2 as well as after paraquat treatment of transfected cells. These results indicate transcriptional regulation of MnSOD and post-transcriptional regulation of catalase gene expression after oxidative stress in primary rat astrocytes.

Impairment of TNF-alpha expression and secretion in primary rat liver cell cultures by acetaminophen treatment.

Tumor necrosis factor-alpha (TNF-alpha) is assumed to act as a mediator in toxic liver injury, aggravating the primary damage to the parenchymal liver cell, but also stimulating liver regeneration. Reports on the effect of acetaminophen in vivo on TNF-alpha transcript concentrations and serum TNF-alpha concentrations, under different experimental, or clinical conditions have yielded controversial results. We used primary rat hepatocyte and Kupffer cell cultures to test the direct action of subtoxic and toxic concentrations of acetaminophen on TNF-alpha expression and release. We observed a dose-dependent decrease of TNF-alpha mRNA in the hepatocytes, and of TNF-alpha release into the medium of hepatocyte cultures. The data also indicate an impairment of TNF-alpha release in Kupffer cell cultures after treatment with nontoxic, as well as with toxic, acetaminophen concentrations. The results suggest that inhibition of TNF-alpha expression and release in the liver is a consequence of acetaminophen exposure. It is at present unknown how this effect modulates the course of acetaminophen intoxication.

Isolation and characterization of the human glutathione S-transferase A2 subunit gene.

We have isolated and characterized a second human liver glutathione S-transferase (GST) subunit gene. The nucleotide sequence of this gene indicates that it encodes the alpha class subunit A2, with a coding region of about 13 kb. Using reverse transcription assays it could be shown that the A2 subunit gene is expressed in human liver and HepG2 cells. The transcription initiation site has been determined by primer extension analysis. A "TATA"-sequence was found 26 nucleotides upstream from the transcription start site. A comparison of the structure of the A2 subunit gene with that of the A1 subunit gene shows significant sequence identity between the two genes. Southern blot analysis of restriction endonuclease digests of human DNA indicates that there may be several more human alpha class GST genes.

Oxidative stress in rat cortical neurons and astrocytes induced by paraquat in vitro.

Oxidative stress has been discussed as crucial mechanism of neuronal cell death in the adult brain. However, it was not clear until now whether neurons are more sensitive to oxidative stress than the other cells in the brain, e.g. astrocytes. Therefore both cell types were exposed to oxidative stress provoked by the redox-cycling compound paraquat. Cortical neurons were found to be more sensitive towards paraquat toxicity than astrocytes as shown by MTT and Neutral Red assay, two different cytotoxicity assays. Mitochondrial functions were determined by the mitochondrial membrane potential and intracellular ATP concentrations. Again cortical neurons were more severely impaired (by paraquat than astrocytes). The production of reactive oxygen species after paraquat exposure was much higher in cortical neurons than in astrocytes and correlated with a higher depletion of GSH (intracellular glutathion). Lipid peroxidation could be shown in astrocytes via the breakdown product malondialdehyde (MDA) whereas in cortical neurons 4-hydroxynonenal (4-HNE) was detected as this endpoint. If and how oxidative stress influences the antioxidant defense was determined via changes in the expression of antioxidant enzymes. Paraquat exposure lead to a 2-3 fold increase of catalase, MnSOD and CuZnSOD mRNA expression in astrocytes. In contrast to astrocytes, in cortical neurons catalase and MnSOD mRNA levels were only marginally elevated above 1.5-fold by treatment with paraquat. Expression levels of glutathione peroxidase (GPx) mRNA were the only one that were not changed in both cell types after paraquat exposure. It is concluded that the more marked increase in expression levels of antioxidant enzymes may render astrocytes more resistant to oxidative stress than neuronal cells.

Changes in antioxidant enzyme expression in response to hydrogen peroxide in rat astroglial cells.

Oxidative stress has been causally linked to a variety of neurodegenerative diseases. To clarify the role of the antioxidant enzyme (AOE) system in oxidative brain damage primary cultures of rat astroglial cells were exposed to hydrogen peroxide (H2O2). Expression of AOEs and several parameters for cell viability and functionality were measured. In our experiments astrocytes responded to low concentrations of H2O2 exposure with a pronounced generation of ROS which ran parallel with induction of lipid peroxidation. This distinct oxidative stress was not reflected in cell viability or functionality parameters measured. Cytotoxicity, a decrease in glutathione content of astrocytes, and impairment of mitochondrial functions became obvious only for higher concentrations of H2O2. After H2O2 exposure catalase, manganese superoxide dismutase, and glutathione peroxidase expression levels were found to be increased, whereas copper/zinc superoxide dismutase mRNA expression was not affected. These data indicate that the AOE system of astrocytes can be directly regulated by oxidative stress and may thus contribute to protection of cells against oxidative insults.

Influence of Adriamycin and paraquat on antioxidant enzyme expression in primary rat hepatocytes.

The cytostatic Adriamycin and the herbicide paraquat form reactive oxygen species during enzymatic activation. Adriamycin, but not paraquat, is also able to intercalate into DNA and to interfere with DNA synthesis and transcription. We investigated the influence of both substances on antioxidant enzyme expression in primary rat hepatocytes. Treatment of hepatocytes with Adriamycin led to an increase in catalase and a decrease in MnSOD mRNA expression. In contrast, exposure of hepatocytes to paraquat resulted in an increase in both catalase and MnSOD message levels. CuZnSOD mRNA was not responsive to either treatment. Adriamycin almost completely inhibited RNA synthesis, but paraquat did not change either RNA or protein synthesis. Both substances induced lipid peroxidation as measured by the accumulation of malondialdehyde in the medium. These findings indicate that catalase and MnSOD are not regulated coordinately in hepatocytes and that ROS-producing agents can differentially influence expression of antioxidant enzymes depending on their capacity to inhibit transcription.

Influence of tumor necrosis factor-alpha and silibin on the cytotoxic action of alpha-amanitin in rat hepatocyte culture.

Tumor necrosis factor-alpha is assumed to play a role in toxic liver damage. We examined whether exogenous tumor necrosis factor-alpha must be present for alpha-amanitin cytotoxicity in rat hepatocyte culture. alpha-Amanitin at a concentration of 0.1 microM, which is close to that found in intoxicated patients, inhibits RNA and protein synthesis within 12 h but cytotoxicity only occurs after a latency period and is pronounced at 36 h after the start of treatment. Tumor necrosis factor-alpha is not indispensable for the development of cytotoxicity but aggravates it and leads to a time shift towards earlier times. Lipid peroxidation is low with alpha-amanitin alone even at 36 h but markedly increased by cotreatment with tumor necrosis factor-alpha. The antioxidant silibin prevents the effect of tumor necrosis factor-alpha, indicating an involvement of reactive oxygen species. alpha-Amanitin alone does not increase but dose-dependently inhibits the expression of the antioxidant enzyme manganous superoxide dismutase and decreases the inducing effect of TNF-alpha on the expression of this enzyme. The gene expression of endogenous tumor necrosis factor-alpha in the hepatocytes is not increased but rather inhibited by alpha-amanitin treatment. The results suggest that alpha-amanitin causes delayed cytotoxicity following rapid inhibition of RNA and protein synthesis and that tumor necrosis factor-alpha shortens the latency period and aggravates the cytotoxicity by a mechanism which may involve reactive oxygen species.