A comparative study of proliferation and hepatic differentiation of human adipose-derived stem cells.

Human adipose-derived stem cells possess a lot of stem cell characteristics, so they may be considered a source of stem cell population. On the basis of that, we have investigated the hepatic potential of adipose-derived stem cells, obtained from liposuction, following two differentiation protocols. In the first procedure, medium was supplemented with epidermal growth factor (EGF), basic fibroblast growth factor, hepatocyte growth factor (HGF) and nicotinamide; the second involved the addition of factors such as dexametasone, EGF, insulin-transferrin-sodium selenite, HGF, dimethyl sulfoxide and oncostatin. In parallel, we carried out our study in the Hep G2 cell line, as human hepatic differentiated in vitro model. Immunocytochemical analysis and RT-PCR were performed using hepatic markers to evaluate cell differentiation. DNA content, MTT test and carboxyl fluorescein succinimidyl ester staining were carried out to evaluate cell proliferation. We reported the evidence of basal hepatic marker in undifferentiated adipose-derived stem cells, which confirmed their multipotency. A strong expression of albumin and alpha-fetoprotein was observed in hepatic-induced adipose-derived stem cells following both differentiation procedures. Morphological aspects of the two types of hepatic adipose-derived stem cells were alike. Proliferation index suggested that the first differentiation procedure promoted better growth than the second. These preliminary findings suggest adipose-derived stem cells may be induced into hepatic lineage, and the most significant difference between the two standard differentiation procedures concerns proliferation rate. This aspect is to be considered when adipose-derived stem cells are employed in research and clinical studies.

Salvia somalensis essential oil as a potential cosmetic ingredient: solvent-free microwave extraction, hydrodistillation, GC-MS analysis, odour evaluation and in vitro cytotoxicity assays.

Salvia somalensis Vatke, a wild sage native of Somalia, has been studied with the aim of assessing the potential cosmetic application of its essential oil, recovered from fresh aerial parts by solvent-free microwave extraction - SFME. To evaluate the efficiency and reliability of this eco-friendly procedure, the recovery of the essential oil was also processed by conventional hydrodistillation (HD) and the results compared. The essential oils obtained by both SFME and HD were analysed by gas chromatography-mass spectrometry using apolar and polar capillary columns. The essential oil recovered by SFME was submitted to an odour evaluation that revealed peculiar olfactive characteristics interesting in alcoholic male perfumery and body detergents.In vitro cytotoxicity assays were carried out using NCTC 2544 human keratinocytes as target cells. The oil displayed slight cytotoxic effects, which were three orders of magnitude lower than those found for sodium dodecyl sulphate positive control. The promising results in terms of chemical composition, scent and safety seem to indicate this essential oil as an interesting potential functional ingredient useful in a cosmetic context.

3,3,5-Trimethylcyclohexanols and derived esters: green synthetic procedures, odour evaluation and in vitro skin cytotoxicity assays.

The alcohols 3,3,5-trimethylcyclohexanols (cis, trans epimers, cosmetic fragrance) and some derived esters, potential and well-known actives in the cosmetic field, such as Homosalate, were synthesized using fast solvent-free methodologies with the aim of renewing and simplifying the conventional procedures. The alcohols were prepared by reduction of 3,3,5-trimethylcyclohexanone (dihydroisophorone) with sodium borohydride/alumina in solid state. The esters from propanoic, butanoic, octanoic, 10-undecenoic, cyclopropanecarboxylic, mandelic and salicylic acids were synthesized with microwave-mediated solvent-free procedures under acidic and basic catalysis. Several experiments were carried out to study advantages and limits of the selected methodologies and the results are reported. Microwave irradiation was carried out using a scientific monomode reactor. In order to evaluate the cosmetic interest of the studied compounds, the sweet-scented substances were submitted to an odour evaluation test; the most promising fragrances and the ester from 10-undecenoic acid, as an example of lipophilic derivatives, were tested to assess their in vitro skin toxicity. Résumé

Chitosan-clodronate nanoparticles loaded in poloxamer gel for intra-articular administration.

This work was based on the study of an intra-articular delivery system constituted by a poloxamer gel vehiculating clodronate in chitosan nanoparticles. This system has been conceived to obtain a specific and controlled release of clodronate in the joints to reduce the arthritis rheumatoid degenerative effect. Clodronate (CLO) is a first-generation bisphosphonate with anti-inflammatory properties, inhibiting the cytokine and NO secretion from macrophages, therefore causing apoptosis in these cells. This is related to its ability to be metabolized by cells and converted into a cytotoxic intermediate as a non-hydrolysable analogue of ATP. Chitosan (CHI) was used to develop nanosystems, by ionotropic gelation induced by clodronate itself. A fractional factorial experimental design allowed us to obtain nanoparticles, the diameter of which ranged from 200 to 300nm. Glutaraldehyde was used to increase nanoparticle stability and modify the drug release profile. The zeta potential value of crosslinked nanopaparticles was 21.0mV±1.3, while drug loading was 31.0%±5.4 w/w; nanoparticle yield was 18.2%±1.8 w/w, the encapsulation efficiency was 48.8%±9.9 w/w. Nanoparticles were homogenously loaded in a poloxamer sol, and the drug delivery system is produced in-situ after local administration, when sol become gel at physiological temperature. The properties of poloxamer gels containing CHI-CLO nanoparticles, such as viscosity, gelation temperature and drug release properties, were evaluated. In vitro studies were conducted to evaluate the effects of these nanoparticles on a human monocytic cell line (THP1). The results showed that this drug delivery system is more efficient, with respect to the free drug, to counteract the inflammatory process characteristic of several degenerative diseases.

Thioacetamide impairs retinol storage and dolichol content in rat liver cells in vivo.

The aim of this paper was to ascertain whether chronic pretreatment with thioacetamide (TAA) might alter the uptake of a load of retinol and dolichol distribution in hepatocytes (HC), hepatic stellate cells (HSC) (Ito-1 and Ito-2 subfractions), Kupffer (KC) and sinusoidal endothelial cells (SEC). The reason why retinol and dolichol content was studied is that their metabolism and transport might be interrelated and that the two isoprenoids might exert different functions in the cells of the hepatic sinusoid. Rats were treated for 2 and 4 months with TAA, a known fibrogenic hepatotoxin, at a low dosage, to produce an early stage of damage. Three days before sacrifice, the rats were given a load of vitamin A, and cells were isolated to investigate its uptake. In HC, the load of retinol was taken up and accumulated, while a decrease in dolichol preceded retinol increase. In HSC, much less of the retinol load was stored than in controls, and dolichol content also decreased. Various minor modifications were seen in KC and SEC.Collectively, the results show that the distribution of these two isoprenoids, which play important roles in cellular differentiation and proliferation, is differently altered in the multiple cell types that line the hepatic sinusoid, and that both isoprenoids seem to participate in the first steps of liver damage.

Changes of CYP1A1, GST, and ALDH3 enzymes in hepatoma cell lines undergoing enhanced lipid peroxidation.

Hepatoma cells show alterations in the response to oxidative stress (decreased lipid peroxidation) and in xenobiotic metabolism enzymes (decreased P450, increased GST and ALDH3). This study examined the effect of lipid peroxidation on the expression of the above enzymes in two rat hepatoma cell lines (MH(1)C(1) and 7777). To induce oxidative stress, cells were exposed to arachidonic acid (to increase lipid peroxidation substrate) and/or to beta-naphthoflavone (to increase CYP450), and treated with one dose of iron/histidine. The cells, that were still viable after the challenge, were refed with the culture medium and CYP1A1, GST, and ALDH3 enzymes monitored for 1, 6, 12, and 24 h. Treatments that increased markers indicative of lipid peroxidation are associated with a decrease in enzyme activities, which was permanent for CYP1A1 and transient for the other enzymes. We speculate from these data that aldehydic byproducts of lipid peroxidation may be responsible for these effects. Thus, restoration of lipid peroxidation in hepatoma cells seems to induce a rapid adaptation to oxidative stress, which is achieved by a simultaneous decrease of reactive oxygen species production and an increase in the two main enzymes involved in the removal of the aldehydic products of lipid peroxidation.

Enrichment with arachidonic acid increases the sensitivity of hepatoma cells to the cytotoxic effects of oxidative stress.

Hepatoma cells are, at most, moderately sensitive to oxidative stress. An important cause of this lack of sensitivity is the decreased content of polyunsaturated fatty acids in comparison with normal cells. These fatty acids are one cellular target of oxygen radicals, by which they are broken down into several toxic carbonyl compounds. If the membrane phospholipids of tumor cells are enriched with polyunsaturated fatty acids, such as arachidonic acid, they become able to undergo lipid peroxidation in the presence of prooxidants. This effect is studied in the highly deviated Yoshida AH-130 ascites hepatoma and in two rat hepatoma cell lines. In parallel to their increased lipid peroxidation, cells enriched with arachidonic acid and exposed to ascorbic acid/FeSO4 showed lower viability and growth than unenriched ones.

Inhibition of class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines.

Hepatoma cells have a below-normal content of polyunsaturated fatty acids; this reduces lipid peroxidation and the production of cytotoxic and cytostatic aldehydes within the cells. In proportion to the degree of deviation, hepatoma cells also show an increase in the activity of Class-3 aldehyde dehydrogenase, an enzyme important in the metabolism of lipid peroxidation products and also in that of several drugs. When hepatoma cells with different degrees of deviation were enriched with arachidonic acid and stimulated to peroxidize by ascorbate/iron sulphate, their growth rate was reduced in proportion to the quantity of aldehydes produced and to the activity of aldehyde dehydrogenase. Therefore, 7777 cells, less deviated and with low Class-3 aldehyde dehydrogenase activity, were more susceptible to lipid peroxidation products than JM2 cells. It is noteworthy that repeated treatments with prooxidant also caused a decrease in mRNA and activity of Class-3 aldehyde dehydrogenase, contributing to the decreased growth and viability. Thus, Class-3 aldehyde dehydrogenase could be considered relevant for the growth of hepatoma cells, since it defends them against cell growth inhibiting aldehydes derived from lipid peroxidation.

Carbon tetrachloride-induced inhibition of hepatocyte lipoprotein secretion: functional impairment of Golgi apparatus in the early phases of such injury.

Functional change of liver Golgi apparatus during carbon tetrachloride (CCl4) poisoning was demonstrated both in rat isolated hepatocytes and in the whole animal. The "in vitro" experimental model provided evidence of Golgi derangement early after giving the haloalkane. The "in vivo" analyses also showed that such an alteration involves both formative and secretory sides of the subcellular structure.

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by the biotic ketoaldehyde methylglyoxal.

The number of sister-chromatid exchanges (SCEs) per metaphase was determined in Chinese hamster ovary cells after 16 h exposure to methylglyoxal (MG) concentrations ranging from 0.1 to 0.75 mM. MG produced an increase of SCE frequency that proved to be dose-dependent, and to reach a maximum of 2 X baseline at the highest nontoxic concentration (0.5 mM).

Damaging effects of advanced glycation end-products in the murine macrophage cell line J774A.1.

The interaction of reducing sugars, such as aldose, with proteins and the subsequent molecular rearrangements, produces irreversible advanced glycation end-products (AGEs), a heterogeneous class of non-enzymatic glycated proteins or lipids. AGEs form cross-links, trap macromolecules and release reactive oxygen intermediates. AGEs are linked to aging, and increase in several related diseases. The aim of this study was to assess, in a murine macrophage cell line, J774A.1, the effects of 48 h of exposure to glycated serum containing a known amount of pentosidine, a well-known AGE found in the plasma and tissues of diabetic and uremic subjects. Fetal bovine serum was incubated with ribose (50 mM) for 7 days at 37 degrees C to obtain about 10 nmol/ml of pentosidine. The cytotoxic parameters studied were cell morphology and viability by neutral red uptake, lactate dehydrogenase release and tetrazolium salt test. In the medium and in the intracellular compartment, bound and free pentosidine were evaluated by HPLC, as sensitive and specific glycative markers, and thiobarbituric acid reactive substances (TBARs), as index of the extent of lipid peroxidation. Our results confirm that macrophages are able to take up pentosidine. It is conceivable that bound pentosidine is degraded and free pentosidine is released inside the cell and then into the medium. The AGE increase in the medium was combined with an increase in TBARs, meaning that an oxidative stress occurred; marked cytotoxic effects were observed, and were followed by the release of free pentosidine and TBARs into the culture medium.

Evaluation of the xenobiotic biotransformation capability of six rodent hepatoma cell lines in comparison with rat hepatocytes.

Phase I and II activities were examined in six rodent hepatoma cell lines and compared with those of cultured rat hepatocytes both in basal conditions and after exposure to 5 microM methylcholanthrene, 2 mM phenobarbital, and 15 microM beta-naphtoflavone. The metabolic profile of testosterone was also studied. The highest aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities were found in MH1C1 cells. Comparable values for 7-ethoxyresorufin O-deethylase activity, ranging from 21.6 to 42.9 pmol/mg x min, were observed in the hepatocytes and hepatoma cells, except the HTC cells. In contrast, only Fao cells showed 7-pentoxyresorufin O-depentylase activity at levels similar to those of hepatocytes (6.2 +/- 1.0 and 7.4 +/- 1.2 pmol/mg x min, respectively). Rat hepatocytes actively hydroxylated p-nitrophenol, but this activity was not measurable in hepatoma cells. Glutathione transferase activity was maintained in all the hepatoma cell lines at similar levels to those found in hepatocytes (684 +/- 56 nmol/mg x min). The seven hydroxylated metabolites of testosterone produced by cultured hepatocytes were negligible in hepatoma cells. Exposure of cells to inducers revealed that aryl hydrocarbon hydroxylase activity was mainly increased after treatment with 3-methylcholanthrene and beta-naphtoflavone, and the highest values were found in rat hepatocytes followed by MH1C1 and Fao cells. 3-Methylcholanthrene and naphtoflavone treatment also resulted in a marked increase in 7-ethoxyresorufin O-deethylase activity in hepatocytes as well as in H4IIC3, McA-Rh7777, MH1C1, and Fao cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of proteins, phospholipids and cholesterol of rat bile during chronic drainage.

Glycerophospholipids, cholesterol and proteins of rat bile were analyzed at different time-intervals during the bile duct cannulation over a period of 24 hr, to study a possible association between the secretion of these bile components. Glycerophospholipids and cholesterol decreased between the 16th-24th hrs, then rose again, showing a bile acid synthesis dependence. In contrast, during the entire collection period, protein concentration was normal. On gel electrophoresis bile proteins give a spectrum of fifteen discrete bands, three of them being Sudan black positive. Some, but not all bile protein bands, show a pattern similar to serum proteins both by means of SDS disc electrophoresis and of immunological techniques. According to the bile composition in GPL and cholesterol and the presence in bile of lipoproteins with SDS electrophoresis migration superimposable to apo-A-IV of serum high density lipoproteins, the following hypothesis is suggested to explain the origin and pathway that some fractions of bile can follow to reach biliary canalicula: some serum or membrane components (that is GPL, cholesterol and possibly apo-A-IV) might insert themselves into the outer leaflet of hepatocyte plasmamembranes at the sinusoidal side; from here they may slip over the inner plasmamembrane monolayer, through the junctions, to the canalicular region of the membrane to give rise, by the action of bile salts, to micelles of bile, together with components coming from other subcellular compartments, following different pathways.

Antioxidant activity of timolol on endothelial cells and its relevance for glaucoma course.

PURPOSE: A growing evidence in the scientific literature suggests that oxidative damage plays a pathogenic role in primary open-angle glaucoma. Therefore, it is of interest to test whether drugs effective against glaucoma display antioxidant activity. We test the hypothesis that the classic beta-blocker therapy for glaucoma with timolol involves the activation of antioxidant protective mechanisms towards endothelial cells. METHODS: Oxidative stress was induced in cultured human endothelial cells by iron/ascorbate with or without timolol pretreatment. Analysed parameters included cell viability (neutral red uptake and tetrazolium salt tests), lipid peroxidation (thiobarbituric reactive substances), and occurrence of molecular oxidative damage to DNA (8-hydroxy-2'-deoxyguanosine). RESULTS: Oxidative stress decreased 1.8-fold cell viability, increased 3.0-fold lipid peroxidation and 64-fold oxidative damage to DNA. In the presence of timolol, oxidative stress did not modify cell viability, whereas lipid peroxidation was increased 1.3-fold, and DNA oxidative damage 3.6-fold only. CONCLUSIONS: The obtained results indicate that timolol exerts a direct antioxidant activity protecting human endothelial cells from oxidative stress. These cells employ mechanisms similar to those observed in the vascular endothelium. It is hypothesized that this antioxidant activity is involved in the therapeutic effect of this drug against glaucoma.