Claudia Sartori

Chromatin sphingomyelin changes in cell proliferation and/or apoptosis induced by ciprofibrate

It has been shown that neutral‐sphingomyelinase and sphingomyelin‐synthase activities are present in chromatin and they modify the sphingomyelin (SM) content. The activity of the first enzyme is stimulated and the second inhibited, when the hepatocytes enter into the S‐phase after partial hepatectomy, thus suggesting that ceramide may have a pivotal role in cell proliferation. An opposite function was attributed to ceramide in hepatocytes which undergo apoptosis after lobular ligature. In order to clarify this point, a model was developed in which the same liver cells undergo proliferation followed by induced apoptosis. To this purpose, the rats were treated for 7 days with ciprofibrate and then left without treatment for 4 days. During the treatment, the peroxisome enzyme markers increase their activity and the number of proliferating cells increases, reaching a maximum after 3 days of treatment, as shown by the number of cells positive for the proliferating cell nuclear antigen. At the same time, the chromatin sphingomyelinase activity reaches the maximum, while a similar increase is not found in the cytoplasm or in the isolated nuclei. On the contrary, SM‐synthase activity is depressed in chromatin, but not in the nuclei in which a peak is shown after 3 days of ciprofibrate treatment. After drug withdrawal, the hepatocytes undergo apoptosis as confirmed by the increase of Bax and tissue transglutaminase (tTGase) expression; the chromatin SM increases as a consequence of an increase of SM‐synthase activity. It can be hypothesised that chromatin SM may have a role in cell duplication by influencing the chromatin structure stability. J. Cell. Physiol. 196: 354–361, 2003.

Plasmalogens in rat liver chromatin: new molecules involved in cell proliferation.

A minor component of chromatin, the phospholipid fraction, changes during cell cycle as result of the activation of intranuclear lipid metabolism enzymes including phosphatidylcholine‐dependent phospholipase C activity. It is known that this enzyme may be activated by phosphatidylcholine plasmalogen (Plg). Until now, there has been little evidences for the presence of Plgs inside the nucleus. The aim of our study is to ascertain if they are present in the nucleus and are responsible of the activation of phosphatidylcholine‐dependent phospholipase C during cell proliferation and apoptosis. Therefore, we have analysed the Plg composition of the whole homogenate, cytosol, nuclei and chromatin of hepatocytes. The phosphatidylcholine‐dependent phospholipase C activity was assayed using both phosphatidylcholine and plasmalogenyl‐phosphatidylcholine as substrates. Our results show, for the first time, that Plgs are present in chromatin and the plasmalogenyl‐phosphatidylcholine stimulates the phosphatidylcholine‐dependent phospholipase C activity more than phosphatidylcholine. Finally, in order to verify the possible role of these molecules during cell proliferation and apoptosis, we used liver of rats fed with ciprofibrate which stimulates hepatocytes proliferation during the treatment and, after withdrawal, apoptosis. After 3 days of ciprofibrate treatment, the chromatin plasmalogenyl‐phosphatidylcholine increases as well as the phosphatidylcholine‐dependent phospholipase C activity. After drug withdrawal, when the hepatocytes undergo to apoptosis, the plasmalogenyl‐phosphatidylcholine content together with phosphatidylcholine‐dependent phospholipase C activity decreases. Therefore, it can be concluded that plamalogens are present in the chromatin, and probably may have a function both in regulating phosphatidylcholine dependent phospholipase C and cell cycle.

Morphometric analysis of liver and kidney peroxisomes in lactating rats and their pups after treatment with the peroxisomal proliferator di‐(2‐ethylexyl)phthalate

Summary— Di‐(2‐ethylexyl)phthalate (DEHP) administered to adult lactating rats from delivery to weaning induces age‐ and organ‐specific modifications of the peroxisomal morphometric parameters (VV, NA and D) in the liver and kidney of both rats and their pups. In both tissues, peroxisomal relative volume and catalase biochemical activity show a similar pattern during the development, as well as under DEHP treatment. Morphometric results suggest that two modalities of peroxisomal proliferation exist, involving: a) increases in both number and mean diameter of the organelles; b) a purely numerical increase of the organelles, accompanied by a remarkable decrement in their mean diameter. A peroxisomal population proliferated through the latter model appears unable to return to normal conditions, following treatment withdrawal. These two proliferation systems, the first implying a swelling and the latter a fragmentation of pre‐existing peroxisomal profiles, are supposed to be tissue‐specific in the adult animal. In particular, in the liver the ‘swelling’ model appears more suitable to explain peroxisome proliferation, while in the kidney this process would follow the ‘fragmentation’ model. Immature animals might instead show in both organs intermediate features of peroxisomal proliferation modalities.

Liver peroxisomes in newborns from clofibrate-treated rats. II. A biochemical study of the recovery period.

Summary— The fatty‐acyl‐CoAβ‐oxidation (FAO) and catalase activities, as well as membrane fluidity of liver peroxisomes of newborns from normal and clofibrate‐treated rats were studied during the recovery period, ie, throughout the first week of postnatal life. In the test animals the enzyme activities, which are significantly higher than controls at birth return to normal levels showing a somewhat different time course with FAO rapidly decreasing to control values within three days but with catalase still higher than controls at day 6. The half‐life and degradation rate (Kd) of FAO are identical to those calculated by us for the whole organelles and to those reported by others for total catalase in normal or clofibrate‐treated adult animals in the presence of catalase inhibitors. Soluble catalase shows turnover values which are similar though not identical to those of FAO, while total catalase has a very long half‐life and a low Kd. Peroxisomal membrane fluidity, as determined by fluorescence anisotropy of 1‐anilinonaphthalene‐8‐sulfonate (ANS) bound to purified peroxisomal fractions is higher in tests than in controls, recovering normal values within 6 days. Our results demonstrate that liver peroxisomes of rats prenatally exposed to clofibrate return to control conditions within about 1 week. The turnover parameters of enzymes and the membrane fluidity values are discussed in terms of disposal mechanism(s) for the excess of induced peroxisomes.

A new method for qualitative and quantitative determination of di- and polyamines in animal tissues by gas-liquid chromatography

Abstract A gas chromatography method for qualitative and quantitative determination of di- and polyamines from animal tissues was developed. The polyamines were extracted either from tissue homogenates or from standard solutions with the butanol method, opportunely modified in order to improve both the recoveries and the reproducibility of the results. Chromatography was performed on a GLC column prepared with Corning glass beads coated with 1% KOH and 4‰ Carbowax 20 M, with d -amphetamine and benzoamphetamine as internal standards. This column permits a good separation and a nearly complete recovery of di- and polyamines.

Liver peroxisomes in newborns from clofibrate‐treated rats. I. A morphometric study of the recovery period

Summary— Morphological and morphometric parameters (volume density (Vv), numerical density (NA) and mean diameter (D¯)) of newborn liver peroxisomes were measured throughout the first week of life in rats born to mothers treated with clofibrate (ethyl 2 p‐clorophenoxy isobutyrate) during the last five days of pregnancy. In control studies the same analyses were carried out in newborns from untreated rats. At birth (day 0), treated animals exhibited a proliferated, pleiomorphic peroxisomal population (higher Vv NA and D¯, and a spread distribution of profile diameter with respect to the controls). In the subsequent two days, many peroxisomes disappeared (decrease of Vv and NA to values even lower than controls), with a persisting high pleiomorphism (no change of D¯ and diameter distribution) in residual ones. Starting from day 3, and up today 6, larger peroxisomes were no longer detectable in test animals, and a significant, not pleiomorphic proliferation took place (D¯ and diameter distributions strictly comparable to the controls and progressively increasing Vv and NA). The correlation analysis validated these morphological results, from which it can be surmised that the postnatal peroxisome recovery period consists of a destructive phase followed by a proliferative one. The possible mechanism(s) of disposal of the excess of drug‐induced peroxisomes are discussed.

Differential modulation of PPARα and γ target gene expression in the liver and kidney of rats treated with aspirin

Aspirin modified peroxisomal enzymatic activities both in the liver and renal cortex of rats, producing typical effects of peroxisomal proliferators (PPs). Although similar increments in beta-oxidation system and catalase activities were observed in both organs, induction of mRNA-Cyp4a10 and mRNA-FAT/CD36, target genes for peroxisome proliferator-activated receptors alpha (PPARalpha) and gamma (PPARgamma), respectively, was only present in the liver. There was no effect on liver mRNA-PPARalpha, while mRNA-PPARgamma was down-regulated, probably as a result of enzymatic inhibition of cyclooxygenases (COXs) by aspirin which has been shown to decrease the levels of PGJ2 and its metabolites, known as strong endogenous ligands for PPARgamma. Typical PP alterations in cell replication and apoptosis were not found during aspirin treatment or after withdrawal, suggesting that peroxisome proliferation occurs without inducing cell cycle alterations. Probably, the synergic action of both PPARalpha and PPARgamma receptors might reduce the impact on cell proliferation and apoptosis.

Subcellular localization of diamine oxidase in rabbit kidney cortex.

The intracellular localization of diamine oxidase (EC 1.4.3.6) in rabbit kidney cortex was studied. The distribution of diamine oxidase in the subcellular fractions, obtained by modifying the classical method of Wattiaux-De Coninck, S., Rutgeerts, M.T. and Wattiaux, R. (Biochim. Biophys. Acta (1965) 105, 446-459) demonstrated that this activity is concentrated (greater than 60%) in the microsomal fraction. Biochemical and morphological data indicate a 20-30% contamination of this fraction by plasma membrane and brush border fragments. Subfractionation of the microsomes, obtained by centrifuging in a continuous sucrose-Ficoll gradient (d 1.038-1.064) for 75 min, showed that diamine oxidase is concentrated in membrane deriving from the endoplasmic reticulum. In fact the bulk of diamine oxidase activity was recovered in a subfraction of the gradient which was shown both biochemically and morphologically to derive from the endoplasmic reticulum. The possible significance of this result is discussed.

Tyrosine aminotransferase activity of frog (Rana esculenta) liver—III. A circannual study

A circannual study of tyrosine aminotransferase and other metabolic enzymes in frog liver is reported. The subcellular distribution of all enzymatic activities under investigation was also studied. Results show significant oscillations of all enzymatic activities throughout the year; in particular tyrosine aminotransferase has a marked summer maximum. The subcellular distribution of tyrosine aminotransferase shows significant variations: the soluble activity of the enzyme presents a bimodal circannual distribution, which has its counterpart in an increased activity of heavier fractions.

Lysosomal involvement in the removal of clofibrate-induced rat liver peroxisomes. A biochemical and morphological analysis

Peroxisomal proliferators induce in rodents hepatic hyperplasia and hypertrophy; the significant increase in the peroxisomal population is accompanied by specific and reversible induction of some peroxisomal enzymes. In suckling rats born from clofibrate‐treated mothers, a massive removal of proliferated organelles occurs within 3 days of recovery. In the present paper we examined the early stages of the recovery period in liver of male rats treated with clofibrate for 5 days. The lysosomal involvement in the removal of drug‐induced peroxisomes was investigated under physiological conditions, ie in the absence of inhibitors of the autophagic process. Biochemical results indicate that peroxisomal β‐oxidation, but not catalase activity, returns to the control values within the examined period. Total acid phosphatase activity is not affected by clofibrate treatment, but following fractionation on a linear density gradient the lysosomal marker enzyme activity is shifted towards lower density values, particularly at day 1 and 2 of recovery. This class of organelles possibly represents lysosomes involved in active autophagic processes. Acid phosphatase cytochemistry shows an increase of lysosome number at day 1 of recovery. Combination of acid phosphatase cytochemistry either with catalase cytochemistry or with catalase immunogold labelling allows to reveal organelles containing both marker enzymes. These results strongly support the involvement of autophagic processes in the removal of proliferated peroxisomes.