Peroxisome proliferators induce apoptosis in hepatoma cells.

In the AH-130 hepatoma, a poorly differentiated tumor, maintained by weekly transplantations in rats, a low percentage of cells spontaneously underwent apoptosis, mainly during the transition from logarithmic- to stationary-growth phase. It was possible to induce massive apoptosis of cells by treating them with clofibrate, a peroxisome proliferator and hypolipidemic drug. Similar results were obtained with HepG2 cells. With 1 mM clofibrate, apoptosis began to manifest itself after 1 h of treatment in vitro, and was assessed by morphological analysis, by DNA fragmentation carried out with agarose gel electrophoresis, and with flow cytometric determination of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. The mechanisms whereby clofibrate induces apoptosis are still unclear. Since the peroxisome proliferator-activated receptor was expressed at a very low level and was not stimulated by clofibrate in the AH-130 hepatoma cells, its involvement seems unlikely. Moreover, lipid peroxidation was not increased after clofibrate treatment. Phospholipids and cholesterol were significantly decreased. The decreased cholesterol content might suggest an inhibition of the mevalonate pathway and, therefore, of isoprenylation of proteins involved in cell proliferation.

Exposure of beta L-crystallin to oxidizing free radicals enhances its susceptibility to transglutaminase activity.

1. The effects of free radicals on the capacity of beta L-crystallin to act as a substrate for the enzyme transglutaminase were investigated. 2. beta L-Crystallin was exposed to defined radical species that were generated radiolytically, and transglutaminase activity, using the modified protein as substrate, was subsequently measured by monitoring the incorporation of [14C]putrescine. 3. Exposure of beta L-crystallin to hydroxyl radicals, thymine peroxyl radicals and acetone peroxyl radicals at concentrations of up to 135 microM increased the capacity of the protein to incorporate putrescine. With higher concentrations of these radicals this capacity of beta L-crystallin to act as a transglutaminase substrate declined to control levels or lower. 4. Superoxide radicals were inactive in this regard; hydroperoxyl radicals were active only at high concentrations. 5. It has previously been suggested that changes in the crystallins that occur during aging and with cataract may be due to oxidative reactions and to transglutaminase activity. This study suggests that these phenomena may be considered together rather than separately.

Effects of 4-hydroxynonenal, a product of lipid peroxidation, on cell proliferation and ornithine decarboxylase activity.

4-hydroxynonenal (HNE) is one of the major breakdown products of cellular lipid peroxidation. Its effects on proliferation, ornithine decarboxylase (ODC) activity and DNA synthesis have been investigated in leukemic cell lines. The cells were incubated for 1 hour with different aldehyde concentrations, then washed and resuspended in medium with fresh foetal calf serum. HNE concentrations ranging from 10(-5) to 10(-6) M significantly inhibited ODC activity when induced by addition of fresh foetal calf serum both in K562 and HL-60 cells. 3H-Thymidine incorporation in K562 cells was also inhibited from 6 to 12 hours after the treatment. The same HNE concentrations did not inhibit ODC activity when added to cytosol, thus a direct action on the enzyme can be excluded. Moreover, HNE did not affect the half-life of ODC, so that a specific effect on ODC synthesis may be supposed. These data indicate a reduction of proliferative capacity of the cells and are consistent with the possibility that HNE, at concentrations close to those found in normal cells, plays a role in the control of cell proliferation.

Effect of calvatic acid and its analogs on ornithine decarboxylase activity in tumour cells.

Calvatic acid (p-carboxyphenylazoxycyanide) is an antibiotic containing an azoxycyano group that displays carcinostatic activity. In the present work it has been shown that in AH-130 hepatoma and K562 leukemia cells the antibiotic, at low concentration, decreases ornithine decarboxylase (ODC) levels. The change depends on two summative effects of the drug, impairment of overall protein synthesis and inhibition of enzyme activity. Some analogs of calvatic acid have been tested in order to gain more insight into the structure-activity relationship. The decarboxylated derivative phenylazoxycyanide proved to be more effective in reducing protein synthesis and ODC activity in the whole tumor cells. The rapidly growing K562 cells displayed high sensitivity to this compound. Calvatic acid analogs devoid of the cyano group were less effective on the same parameters.

Increased susceptibility to transglutaminase of eye lens proteins exposed to activated oxygen species produced in the glucose-glucose oxidase reaction.

In order to test whether a mild oxidative stress could promote the transglutaminase damaging effect on eye lens proteins, total lens soluble proteins and purified beta L-crystallin have been exposed to H2O2 slowly produced by the glucose-glucose oxidase reaction. Soon after the pretreatment, the substrate capacity of the lens proteins for an exogenous transglutaminase has been evaluated. Exposure to the oxidative stress increased the susceptibility of the lens proteins to transglutaminase. When ferrous ions were added to the preincubation medium, in order to convert the H2O2 into the hydroxyl radical, the increase was more evident.

Ornithine decarboxylase properties: is there a role for a microsome-bound inactivating activity?

Liver microsomes have a strong ornithine decarboxylase (ODC) inactivating capacity in vitro. The present results suggest that this may be involved in regulation of ODC activity in vivo: (1) the ODC inactivating capacity of microsomes appears susceptible to in vivo modulation: a single administration of thioacetamide, which induces ODC, also causes a significant increase in the inactivating capacity of the microsomes; (2) under conditions leading to increased microsome-bound ODC-inactivating capacity (e.g. liver from thioacetamide-treated rates versus regenerating liver) ODC displays a greater thermal lability and inactivability in vitro. A possible involvement of this microsomal activity in an autoregulatory pathway of ODC is suggested by the fact that it is induced by the administration of polyamines. However, inhibition of ODC activity by alpha-difluoromethylornithine does not prevent the increase of the microsomal activity caused by thioacetamide. Thus, polyamine biosynthesis does not appear to be an absolute requirement for induction of the microsomal ODC-inactivating capacity. The apparent half-life of ODC in vivo, as evaluated after cycloheximide administration, does not appear to correlate with the microsomal ODC-inactivating capacity content and the stability properties of ODC in vitro.

Studies on ornithine decarboxylase from liver, kidney and tumoral tissues during activity decay following cycloheximide administration.

We have studied the activity, thiol-dependency and Km of ornithine decarboxylase (ODC) from the following sources: liver of rats subjected to partial hepatectomy or administered thioacetamide, the rat 3924A Morris hepatoma, the rat AH130 Yoshida ascites hepatoma, a mouse transplantable mammary carcinoma and kidney of rats administered testosterone propionate. In order to detect possible changes occurring during in vivo ageing of this enzyme we inhibited protein synthesis with cycloheximide. A gradual decrease of Km during ageing was observed in ODC from liver.

Hepato- and neuro-toxicity by ethylenthiourea.

Thyrotoxicity of Ethylenthiourea (2-imidazolidinethione) (ETU) is well known: this paper examines the extent to which ETU alone may damage certain non-thyroid tissues, and the possible enhancement of its effects through synergism when it is taken with substances employed for medical or alimentary reasons. It was found that the lethality of ETU is augmented by accumulation of fractionated doses, particularly in male as opposed to female rats. When accompanied by ethanol, its harmfulness is increased, whereas phenobarbital provides a certain degree of protection. Liver secretion of triglycerides is greatly impaired (delta % = -31) by acute administration, which results in steatosis. This is not observed during subacute administration. Liver microsomal cytochrome P450 is significantly reduced (delta % = -32) after a long term administration. The 30 week treatment leads to a form of "selection". Some animals die (28.6%), whereas others survive despite: i) prolonged failure to put on weight, ii) alopecia (80% loss of hair), iii) severe conjuntivitis, iv) blepharitis, v) peripheral nervous system distress. These findings underscore the importance of investigating the relation between hazardous compounds and possible potentiating factors, on the one hand, and inclusion of organs not yet recognized as targets, on the other hand, when setting tolerance limits for ambient pollution.

Impairment of erythropoiesis in rats exposed to environmental pollutants.

The extent to which halogen compounds interfere with erythropoiesis is still unclear. This paper reports an evaluation of the effect of repeated exposure to carbon tetrachloride (CCl4) in air on red blood cell (RBC) creatine concentration. Creatine is neither synthetised nor metabolised in circulating RBC and decreases over the lifespan of red cells. It can thus be taken as a reliable indicator of mean RBC age, and hence of cell viability and bone marrow efficiency. Following inducement of hemolysis with phenylhydrazine (PHH) to stimulate erythropoiesis, creatine levels rose in the controls. This increase was significantly less in the CCl4-treated animals. It is not yet certain whether this inhibition reflects impaired marrow efficiency, enhanced RBC destruction in the marrow, or block of the release of mature RBC. The fact that such inhibition takes place, however, is of importance as a predictive factor in environmental toxicology, since it appears before changes in other blood parameters or signs of liver toxicity are observed.

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria.

The phospholipid depletion of rat liver mitochondria, induced by acetoneextraction or by digestion with phospholipase A2 or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A2. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipid-deficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes. These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.

Phenobarbital stimulation of cytochrome P-450 and aminopyrine N-demethylase in hyperplastic liver nodules during LD-ethionine carcinogenesis.

Microsomes isolated from hyperplastic liver nodules and hepatomas, induced by DL-ethionine, exhibited a reduced cytochrome P-450 content and aminopyrine N-demethylase activity when compared to the organelles of control and surrounding non-nodular liver. Phenobarbital administration to rats caused an increase of microsomal protein, cytochrome P-450 and aminopyrine N-demethylase in all tissue tested. In the hepatoma the rise of cytochrome P-450 and aminopyrine N-demethylase/g of tissue was very low and it is compensated by a slight increase of microsomal protein. In hyperplastic nodules as well as in control and surrounding livers, cytochrome P-450 and aminopyrine N-demethylase increased more than microsomal protein. However, the phenobarbital-induced stimulation was significantly lower in hyperplastic nodules than in control and surrounding livers.