Maria E. Biocca

New Data on Kinetics of Lipid Peroxidation in Experimental Hepatomas and Preneoplastic Nodules

Lipid peroxidation has been found decreased in several hepatomas. The decline has been shown already at the level of preneoplastic nodules obtained after DEN treatment of rats. A substantial exception is represented by the hepatoma cell line MH1C1, deriving from a slightly deviated Morris tumor. Most of the described experiments estimated lipid peroxidation levels in terms of malonaldehyde production by the thiobarbituric acid test. It is now clear that this test does not account for several other aldehydes produced during lipid peroxidation. We now investigated by high performance liquid chromatography (HPLC) the whole range of non-polar aldehydes produced by tumor homogenates and by preneoplastic nodules both in basal conditions and after stimulation with ADP-iron or ascorbate. It was reduced in the preneoplastic nodules as well as in the DEN-induced hepatoma. The susceptibility to the prooxidant effect of ADP-iron or ascorbate was strongly decreased in all hepatomas as well as in preneoplastic nodules. It has been recently published that hepatoma cells are more susceptible than normal liver to the toxic action of aldehydes. This was attributed at least in part to the decreased activity of aldehyde dehydrogenases, as well as to their different distribution in tumor cells. A deeper study on aldehyde metabolism in hepatomas has shown that alcohol dehydrogenase and NADPH-aldehyde reductase also are markedly decreased in Yoshida hepatoma cells and the MH1C1 cell line. However, glutathione transferase, that can use hydroxynonenal as a substrate, is strongly decreased in Yoshida hepatoma cells but not in MH1C1 cells.

Biochemical studies on bile duct epithelial cells isolated from rat liver.

In the present paper we provide a basic enzymatic characterization of biliary epithelial cells (BEC) that have been isolated from normal rat liver. When compared with liver parenchymal cells, BEC display the following major features: (a) a very high specific activity of gamma-glutamyltranspeptidase (approx. 200-times higher than the value usually found in hepatocytes); (b) a lack of enzymes that are usually associated with the endoplasmic reticulum in hepatocytes such as cytochrome P-450, aminopyrine demethylase, glucose 6-phosphatase and NADPH cytochrome-c reductase; (c) the presence of enzymes related to the glutathione redox cycle (e.g., GSH-peroxidase, GSSG-reductase and different isozymes of GSH-transferase), but accompanied by a very low content in reduced glutathione. The enzyme pattern of BEC correlates well with histochemical and immunohistochemical studies, as well as with biochemical studies on bile ductular cells isolated from rat liver during cholestasis.

Isolation and characterization of biliary epithelial cells from normal rat liver

A cell fraction enriched in biliary epithelial cells (BEC) has been isolated from the liver of normal rats. The procedure involved proteolytic digestion by trypsin and mild mechanical disruption of biliary ductular and connective tissue that remained undigested after collagenase-hyaluronidase perfusion. An adherence procedure removed the large majority of contaminating Kupffer cells. The majority (87.4 +/- 3.5%) of the cells were positive to an indirect immunofluorescence staining that used an antiserum against bovine hoof prekeratin that specifically recognizes intermediate filaments of biliary epithelium. Similar results were obtained by histochemical staining for gamma-glutamyltranspeptidase activity. The contamination of the BEC fraction with Kupffer cells and hepatocytes was approximately 7% and 2%, respectively. The viability of the BEC population was always more than 90%. The BEC and hepatocytes were analysed for their lipid composition; the BEC were found to have a cholesterol content approximately 6-times higher than hepatic parenchymal cells, with a cholesterol/phospholipid molar ratio of 0.53 in comparison to a value of 0.11 for hepatocytes. No detectable evidence of cytochrome P-450 or cytochrome P-450-related enzymatic activities was found in the BEC.

The subcellular distribution and properties of aldehyde dehydrogenase of hepatoma AH-130☆

Aldehyde dehydrogenase subcellular distribution and activity were studied in the Yoshida hepatoma AH-130 and rat liver. NAD+- and NADP+-dependent dehydrogenase activities were lower in all hepatoma subfractions (except the cytosol) than in liver subfractions. In the presence of 0.025 mM substrate 78-80% of the liver NAD+- or NADP+-dependent aldehyde dehydrogenase was found in the mitochondria. With 10 mM substrate the enzyme activity was primarily in the mitochondria and microsomes. In the hepatoma a sharp increase of the soluble aldehyde dehydrogenase (either NAD+- or NADP+ dependent) was observed at all substrate concentrations. The Km of the different isoenzymes (either identified by their localization or coenzyme dependency) were of the same order for liver and hepatoma. However, a high Km enzyme was present in liver mitochondria outer membranes but not in hepatoma. Hepatoma acetaldehyde dehydrogenase was inhibited, as was the liver enzyme, by diethyldithiocarbamate. The return of activity was slower for the hepatoma and neonatal liver than for the adult liver enzyme.

Resistance to oxidative stress by hyperplastic and neoplastic rat liver tissue monitored in terms of production of unpolar and medium polar carbonyls

The susceptibility of rat liver tissue to oxidative stress during its neoplastic transformation was analyzed by both qualitative and quantitative measurements of the carbonyl products of lipid peroxidation. Diethylnitrosamine was used as initiating agent of hepatocarcinogenesis and lipid peroxidation levels were monitored in the homogenates from normal liver, hyperplastic nodules and tumour, incubated in the presence or in the absence of ascorbate or adenosine diphosphate-iron complex. While the basal levels of lipid peroxidation in the three experimental conditions were found to be quite similar, in the presence of the pro-oxidant stimulus a remarkable reduction in aldehyde production was shown not only by the hepatoma tissue but also by the preneoplastic nodules.

Glutathione-S-transferase, alcohol dehydrogenase and aldehyde reductase activities during diethylnitrosamine-carcinogenesis in rat liver

Several enzymes metabolize the toxic aldehydes produced during lipid peroxidation, such as 4-hydroxynonenal. During carcinogenesis induced by diethylnitrosamine in rat liver, an increase in aldehyde dehydrogenase, in comparison with normal liver, has already been shown. This paper demonstrates that, although to a lesser extent than aldehyde dehydrogenase, aldehyde reductase and glutathione-S-transferase also increase during carcinogenesis. Of the latter two enzymes, aldehyde reductase increases more markedly in a progressive fashion during the months of development of nodules and hepatoma. The increase of enzymes able to metabolize 4-hydroxynonenal, as well as other aldehydes, is certainly important in protecting tumour cells against cytotoxic effect of aldehydes.

Oxidative metabolism of 4-hydroxy-2,3-nonenal during diethyl-nitrosamine-induced carcinogenesis in rat liver

In some chemically-induced hepatomas and in cultured transformed cells the aldehyde dehydrogenase activity was found increased in the presence of aromatic aldehyde as substrate. We studied this enzyme during diethyl-nitrosamine carcinogenesis in rat liver by using an aliphatic aldehyde, 4-hydroxynonenal, as substrate. 4-Hydroxynonenal is an important product of lipid peroxidation. The NAD- and NADP-dependent aldehyde dehydrogenase of the cytosolic fraction and the NADP-dependent aldehyde dehydrogenase of the microsomes show higher values in nodules and hepatoma than in normal liver. These results suggest that increased aldehyde dehydrogenase, when 4-hydroxynonenal is used, can be considered a marker of the neoplastic process, in the same way as the level of aldehyde dehydrogenase increased in presence of aromatic aldehyde.

Further Experiments on Lipid Peroxidation in Transplanted and Experimental Hepatomas

The results of experiments on the subject of lipid peroxidation in hepatomas are described. It is now clear that lipid peroxidation is strongly decreased in most highly dedifferentiated hepatomas. It seems evident that the extent of the decline is strictly related to the degree of dedifferentiation. The model of diethylnitrosamine carcinogenesis, according to the method by Soh, Medline and Farber, has been now adopted to study the stages of carcinogenesis. It was shown that a net decline in lipid peroxidation occurs as early as at the stage of reversible nodules and progresses until the development of clear hepatomas. This change is practically simultaneous with a decline in the efficiency of the enzymes of the drug metabolizing system and in the content of cytochrome P450. Glutathione content and metabolism show also important changes. In fact, a dramatic increase in gamma-glutamyl-transpeptidase takes place very early during carcinogenesis, and is responsible for large decline in total glutathione during incubation of the homogenates. Glutathione peroxidase activity, on the contrary, is decreased, whereas glutathione reductase does not show significant changes. The supernatant of highly anaplastic tumors inhibits lipid peroxidation in normal liver homogenates, suggesting the presence of substances provided with antioxidant properties. These cannot be, however, related to a higher glutathione content. Supernatants from early nodules seem to be unable to block lipid peroxidation in normal liver homogenates. Preliminary experiments done to study the aldehyde pattern produced during lipid peroxidation, both in hepatomas and in nodules, confirm the presence of very poor lipid peroxidation and possibly of different peroxidation kinetics.

Electron Spin Resonance Studies on Isolated Hepatocytes Treated with Ferrous Or Ferric Iron

Isolated rat hepatocytes incubated with iron salts in the presence of the spin trapping agent alpha-4-pyridyl-l-oxide N-tert-butyl nitrone (4-POBN) generate a clear electron spin resonance signal; this signal is not detectable in the absence of exogenous iron. The hyperfine splitting constants are identical whether ferrous or ferric iron is used. The free radical trapped does not appear to be an active oxygen species but rather a carbon-centred radical, which we here ascribe to a lipodienyl radical on the basis of its hyperfine splitting features. Support to this interpretation is lent by the fact that no such radical could be generated in hepatocytes fully protected against lipid peroxidation by pretreating the donor rats with alpha-tocopherol.

Phospholipid composition of inner and outer mitochondrial membranes isolated from Yoshida hepatoma AH-130

Outer and inner membranes isolated from hepatoma AH-130 and rat liver mitochondria were used to study phospholipid composition. The phosphatidylethanolamine content increased and that of phosphatidylcholine decreased in ;whole mitochondria and isolated membranes. The ratio between the 2 phospholipids increased 47% and 117% in the inner and outer membranes respectively. A small decrease of diphosphatidylglycerol also occurred in the hepatoma mitochondria inner membrane. In contrast to the results of previous work, no sphingomyelin was found in hepatoma mitochondria and isolated membranes.