Abstract: Cytochrome P4501A1 (CYP1A1), an enzyme known to metabolize polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR). The involvement of protein kinase C (PKC) in the regulation of AhR signal transduction pathway, has been widely studied but the role of specific PKC isoform(s) involved in this process it is not well clarified. To study which PKC isoform(s) is implicated in the regulation of CYP1A1, in the poorly tumorigenic MH1C1 rat hepatoma cells, we examined the effects of some PKC pharmacological inhibitors, Calphostin C (CAL), Staurosporine (STA) and H7, and of 12-0-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, on basal and 3-methylcholanthrene (MC)-induced CYP1A1 protein expression and mediated ethoxyresorufin O-deethylation (EROD) activity.
In parallel, the activities of PKC-alpha, -beta I, -delta and -epsilon isoforms, the most expressed in MH1C1 cells, were monitored.
After pre-treatment with CAL, STA and H7, the MC-induced CYP1A1 protein and EROD activity were rapidly reduced with temporal profile similar to the profile of the activity of alpha and beta1 PKC isoforms. Moreover, TPA pre-treatment induced a biphasic effect on EROD activity, and a decline of PKC -beta I and -alpha, in first instance, and -delta and -epsilon activities later on. These findings clearly show that, in MH1C1 cells, PKC is involved in CYP1A1 regulation and that alpha and beta I classic PKC isoforms play an active role in modulating this process.
Abstract: 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.
Abstract: 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.
Abstract: A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimer's disease and motor neuron disease). A stem cell population (i.e. pre-adipocytes) is present in the adipose stromal compartment. Pre-adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre-adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre-adipocytes, in parallel with neural morphology. The passage of the choline-acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic-like phenotype from pre-adipocytes.
Abstract: 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.
Abstract: Indian frankincense is a gum resin from Boswellia serrata of Burseraceae used in Ayurveda and Western medicine for the antinflammatory effects of boswellic acids, particularly 3-O-acetyl-11-keto-beta-boswellic acid (AKBA). We evaluated in vitro cytotoxicities of B. serrata extract and AKBA on differentiated and undifferentiated keratinocytes (HaCaT and NCTC 2544), and foetal dermal fibroblasts (HFFF2), using neutral red uptake (NRU), MTT, and DNA assays. Comparison between NRU and MTT, and between the extract and AKBA, suggested a relatively higher toxicity of both substances on lysosomes respect to mitochondria. Extract cytotoxicity on lysosomes was higher in NCTC and HFFF2 than on the more differentiated HaCaT. DNA assay showed low extract inhibition on HFFF2 proliferation, possibly due to lower growth rate, and a stronger effect on NCTC than on HaCaT, possibly related to higher proapoptotic effect on the less differentiated NCTC, as also suggested by higher AKBA toxicity on NCTC than on HaCaT. In general, gum resin and AKBA toxicities were slightly lower or higher than that of the reference compound SDS. Our in vitro model allowed to compare the sensitivities of different human skin cells to B. serrata, and indicated that the gum resin and AKBA exert moderate to low toxicity on the skin.
Abstract: BACKGROUND/AIMS: In thioacetamide-induced liver injury a modification of isoprenoid content and an increase of reactive oxygen species has been described. We have examined how reactive oxygen species influence the 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate limiting enzyme of the isoprenoid biosynthetic pathway, to verify if changes of that enzyme activity are involved in the changed lipid composition of the liver. METHODS: In chronic and acute thioacetamide-treated rat liver we measured the reactive oxygen species content, the activation state and K(M), the level and degradation rate of the hepatic reductase, its short term regulatory enzymes and the liver lipid profile. RESULTS: In thioacetamide-treated rat liver, the reactive oxygen species content is high and the reductase is fully activated with no modifications in its K(M) and its short term regulatory enzymes. The reductase level is reduced in chronic thioacetamide treated rats and its degradation rate is altered. CONCLUSIONS: The data show a relationship between reactive oxygen species production and altered 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. It is suggested that reducing the levels of reactive oxygen species may improve the altered lipid profile found in liver injury.
Abstract: In skin, vitamin E acts as the predominant lipophilic antioxidant with a protective function against irradiation and oxidative stress. In addition to that, vitamin E can also modulate signal transduction and gene expression. To study whether the four natural tocopherol analogues (alpha-, beta-, gamma-, delta-tocopherol) can influence transcriptional activity by modulating the activity of nuclear receptors, a human keratinocytes cell line (NCTC 2544) was transfected with plasmids containing the luciferase reporter gene under control by direct repeat elements (DR1-DR4), representing binding sites for four different classes of nuclear receptors. In this model, the tocopherols positively modulated only the reporter construct containing a consensus element for peroxisome proliferator-activated receptors (PPARs). The induction was strongest with gamma-tocopherol and was most likely the direct consequence of stimulation of PPARgamma protein expression in keratinocytes. Vitamin E treatment also led to increased expression of a known PPARgamma target gene involved in terminal keratinocytes differentiation, the transglutaminase-1.
Abstract: Advanced glycation end-products (AGEs) are linked to aging and correlated diseases. The aim of present study was to evaluate oxidative stress related parameters in J774A.1 murine macrophage cells during chronic exposure to a subtoxic concentration of AGE (5% ribose-glycated serum (GS)) and subsequently for 48 h to a higher dose (10% GS). No effects on cell viability were evident in either experimental condition. During chronic treatment, glycative markers (free and bound pentosidine) increased significantly in intra- and extracellular environments, but the production and release of thiobarbituric acid reactive substances (TBARs), as an index of lipid peroxidation, underwent a time-dependent decrease. Exposure to 10% GS evidenced that glycative markers rose further, while TBARs elicited a cellular defence against oxidative stress. Nonadapted cultures showed an accumulation of AGEs, a marked oxidative stress, and a loss of viability. During 10% GS exposure, reduced glutathione levels in adapted cultures remained constant, as did the oxidized glutathione to reduced glutathione ratio, while nonadapted cells showed a markedly increased redox ratio. A constant increase of heat shock protein 70 (HSP70) mRNA was observed in all experimental conditions. On the contrary, HSP70 expression became undetectable for a longer exposure time; this could be due to the direct involvement of HSP70 in the refolding of damaged proteins. Our findings suggest an adaptive response of macrophages to subtoxic doses of AGE, which could constitute an important factor in the spread of damage to other cellular types during aging.
Abstract: We analysed the action, in rats in vivo, of the protein isoprenylation inhibitor perillyl alcohol (POH) and that of vitamin A, alone or in association, on m-RNA and protein expression of farnesyltransferases (FTases alpha and beta subunits) and their protein substrates RhoA and RhoB, in isolated hepatocytes. Combined administration of POH and vitamin A induced a sharp decrease in FTase alpha protein after 96 h, suggesting an involvement not only of farnesyltransferases but also of geranylgeranyltransferases, which share the FTase alpha protein. FTase beta protein did not decrease. POH plus vitamin A, in contrast with POH or vitamin A alone, induced a decrease in RhoB protein, probably because of different cleavages. No modification was observed in RhoA protein. Vitamin A alone increased RhoB m-RNA and protein expression. As one of the functions of RhoB is cell polarisation, these data support our previous hypothesis of a polarised transport of vitamin A from hepatocytes to hepatic stellate cells. As the behaviours of m-RNAs and proteins in this study were often different, cytoplasmic metabolic pathways must be considered for the parameters studied. The behaviour of Rho B, which is thought to have an antioncogene function, is discussed in view of its isoprenylated forms in the membranes. These preliminary findings stress the need, when studying the association of two isoprenoids in cancer therapy, to consider normal as well as tumour-bearing animals.
Abstract: Our line of researches follows the hypothesis that dolichol and retinol metabolism might be interrelated and involved in liver fibrosis. To this end, in this study rats were subjected to chronic treatment with thioacetamide (TAA) (300 mg/L liquid diet) for 1 and 2 months and, after liver damage had occurred, supplemented with vitamin A before sacrifice. Dolichol, dolichol isoprene units, and retinol content were determined in isolated parenchymal and sinusoidal liver cells (hepatic stellate cells; Kupffer cells; sinusoidal endothelial cells). Dolichol increased in hepatocytes after TAA treatment, with or without vitamin A. Dolichol decreased in the other cells. Retinol in general decreased. In hepatocytes, retinol decreased only on normal nutrition, while the vitamin A load was taken up normally. The percentages of dolichol isoprene units (Dol-16 to Dol-20, in rats) confirm that Dol-18, which was not modified in percentage by TAA on normal nutrition, did not increase after vitamin A, as it did in control cells (7-12%). The behavior of Dol-18 was similar in all the cells studied. Vitamin A might reveal a latent damage produced by TAA on dolichol homologues. These data support previous hypotheses that the action of TAA depends on the administration modality, the dosage, and the diet, and that Dol-18 might have different functions and compartmentalization in the cells. Furthermore, the results support the hypothesis that dolichol chain length might be interrelated with retinol metabolism, perhaps through their metabolites.
Abstract: Our aim was to study the distribution of dolichol, dolichol isoprenoids, and retinol in hepatocytes, Kupffer, sinusoidal endothelial and two subfractions of hepatic stellate cells, --Ito-1 and Ito-2--, after chronic treatment of rats for 2 and 4 months with a low dosage of thioacetamide associated with ethanol. Each type of cell responded differently to the two hepatotoxins. Overall, ethanol rarely affected the action of thioacetamide. Some new information emerges with regard to these hepatotoxins in comparison with the effects exerted by each of the drugs separately: treatment with thioacetamide plus ethanol determined an early decrease in dolichol in Kupffer cells (about 13% and 50% after 2 and 4 months, respectively). Moreover, after liver damage, a load of vitamin A evidenced altered percentages of the form of dolichol with eighteen isoprene units; these percentages were modified by all treatments in all cell types. The results confirm that dolichol is the preferred target of oxidative stress and suggest a relationship between dolichol and retinol metabolisms, and a possible new role of dolichol precursors, of prenyltransferases and of retinol metabolites in liver pathology.
Abstract: This study examines how treatment with a single dose of thioacetamide, a known experimental hepatotoxin, alters the content of dolichol, dolichol isoprene units and retinol in isolated rat parenchymal and non-parenchymal liver cells at different times and when the animals are supplemented with Vitamin A. Thioacetamide (300 mg/kg i.p.) was administered in a single injection to rats, sacrificed at intervals of 0.5, 1, 2, 3, 4, 15 and 30 days. Rats supplemented, following thioacetamide, with Vitamin A, 3 days before sacrifice showed increased mortality and cellular necrosis on the third and fourth days. Parameters indicating tissue necrosis returned to normal values in surviving animals. Dolichol and retinol content showed a variable, reversible decrease, with normal levels being restored in 15-30 days. After Vitamin A, dolichol content only in hepatic stellate cells (HSC) was lower then the controls 3 and 4 days after thioacetamide treatment, in parallel with the decrease of retinol storage. The percentage of dolichol-18 is not modified by thioacetamide alone. When supplemented with Vitamin A the percentage of dolichol-18 always decreased after thioacetamide, showing that damage was still present. Mechanisms that might be operative in liver cells are briefly discussed. This approach would provide an indication to investigate how the length of the dolichol chain is determined.
Abstract: The aim of this study was to use chronic ethanol intoxication for 2 and 4 months as a means of studying the distribution of dolichol and retinol in isolated rat liver parenchymal cells, Kupffer cells, sinusoidal endothelial cells, and two subfractions of hepatic stellate cells: Ito 1 and Ito 2. Dolichol and retinol were studied in two batches of rats: on normal nutrition and after a load of vitamin A given 3 d before sacrifice. New observations reported are: (i) on normal nutrition, after 2 months of treatment, dolichol in HC seems to be the first target of chronic ethanol, while retinol is the first target in hepatic stellate cells; (ii) the various types of liver cells are differently affected by chronic ethanol, which highlights the importance of studying each type of sinusoidal cell; (iii) a load of vitamin A given when the damage has already occurred restores dolichol content in HC while retinol decreases; and, (iv) a link between dolichol and vitamin A metabolism might be supposed after the load of vitamin A: the percentage distribution of dolichol with 18 isoprene units (Dolichol -18) increases in all the control cells but decreases after chronic ethanol treatment. A different role of this dolichol and/or a different compartmentalization within the cell need to be further investigated.
Abstract: The content of dolichol, an isoprenoid present in all biological membranes, was determined in isolated sinusoidal liver cells after treatment of rats for 2 and 4 months with a low dosage of the hepatotoxin thioacetamide. The significant decrease in dolichol observed in hepatocytes after 2 months might be explained by peroxidation of the isoprenoid. At the same time point, retinol was retained, and decreased only after 4 months of treatment. After 4 months of treatment therefore both lipids decreased. In a subfraction of hepatic stellate cells, Ito-1 cells, the main storage site of vitamin A, dolichol decreased significantly only after 4 months. A remarkable difference from hepatocytes is that in Ito-1 cells retinol content significantly decreased after 2 months of treatment. In another subfraction, Ito-2 cells, the content of the two isoprenoids decreased in parallel. This heterogeneous subfraction might represent those transitional hepatic stellate cells that, while losing retinol, are in the process of differentiating into myofibroblasts secreting extracellular matrix components. In Kupffer cells and sinusoidal endothelial cells, impairment of dolichol might be observed later, only after 4 months of treatment, while retinol decreases uniformly over time. Starting after two months of treatment, the decrease of dolichol and the increase of retinol in hepatocytes, at the same time as retinol decreases in hepatic stellate cells, might be taken as an early index of incipient liver injury due to thioacetamide. This hypothesis is discussed with regard to a role of dolichol in the modulation of membrane fluidity for intracellular and intercellular retinol transport.
Abstract: 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.
Abstract: 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.
Abstract: Aldehyde dehydrogenase (ALDH) is a family of several isoenzymes important in cell defence against both exogenous and endogenous aldehydes. Compared with normal hepatocytes, in rat hepatoma cells the following changes in the expression of ALDH occur: cytosolic class 3 ALDH expression appears and mitochondrial class 2 ALDH decreases. In parallel with these changes, a decrease in the polyunsaturated fatty acid content in membrane phospholipids occurs. In the present study we demonstrated that restoring the levels of arachidonic acid in 7777 and JM2 rat hepatoma cell lines to those seen in hepatocytes decreases hepatoma cell growth, and increases class 2 ALDH activity. This latter effect appears to be due to an increased gene transcription of class 2 ALDH. To account for this increase, we examined whether peroxisome-proliferator-activated receptors (PPARs) or lipid peroxidation were involved. We demonstrated a stimulation of PPAR expression, which is different in the two hepatoma cell lines: in the 7777 cell line, there was an increase in PPAR alpha expression, whereas PPAR gamma expression increased in JM2 cells. We also found increased lipid peroxidation, but this increase became evident at a later stage when class 2 ALDH expression had already increased. In conclusion, arachidonic acid added to the culture medium of hepatoma cell lines is able to partially restore the normal phenotype of class 2 ALDH, in addition to a decrease in cell growth.
Abstract: 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.
Abstract: 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.
Abstract: The metabolism of acetaldehyde (ACA), benzaldehyde (BA), propionaldehyde (PA) and valeraldehyde (VA) has been studied in two hepatoma cell lines, the rat HTC and mouse Hepa 1c1c7 cells. The cytotoxicity of the four aldehydes to these two cell lines has been compared. The end-points for evaluating cytotoxicity were 1) total macromolecular content (TMC) of confluent cultures, and 2) colony forming ability of dividing cells. These two assay systems had different sensitivities for the toxicity of aldehydes, probably due to different numbers of target cells. The activities of aldehyde dehydrogenases (NAD- and NADP-dependent, ALDH), alcohol dehydrogenase and aldehyde reductase were markedly greater in the HTC cell line compared to the Hepa 1c1c7 cell line, especially with BA as substrate. The cytotoxicities of aldehydes were generally stronger in the HTC cell line than in the Hepa 1c1c7 cell line; with the CF test. Particularly, BA was highly toxic to the HTC cells, which possessed the highest ALDH levels. Moreover, the treatment with (diethylamino)benzaldehyde, an ALDH inhibitor, completely abolished the toxicity of BA. Taken together, all these findings suggest that several cell lines expressing different aldehyde metabolizing activities could be used especially in the pre-screening phase to distinguish the metabolism-dependent cytotoxic effects from the metabolism independent effects.
Abstract: 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.
Abstract: 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)
Abstract: It is well established that many types of tumor cells have reduced lipid peroxidation capacity compared to their normal counterparts. Changes in the activity of enzymes metabolizing aldehydes produced by lipid peroxidation have also been reported in a variety of tumor cells. We have investigated the relationship between changes in lipid peroxidation and changes in aldehyde-metabolizing enzymes in normal hepatocytes and two representative rat hepatoma cell lines, McA-RH-7777 and JM2. Compared to hepatocytes, both 7777 and JM2 cells have significantly lower basal and prooxidant-induced levels of lipid peroxidation than normal hepatocytes. Using 4-hydroxynonenal (4-HNE) as substrate, both cell lines also have significantly reduced activities of alcohol dehydrogenase (ADH) and glutathione S-transferase (GST) compared to hepatocytes. JM2 cells have significantly increased aldehyde dehydrogenase (ALDH) and aldehyde reductase (ALRD) activities with 4-HNE. In 7777 cells the ALDH and ALRD activities are not different from hepatocytes. The changes in enzyme activity are inversely correlated with the sensitivity of cells to 4-HNE. JM2 cells, with increased ALDH and ALRD and decreased ADH and GST, are much more resistant to the toxic effects of 4-HNE than 7777 cells. Normal hepatocytes and JM2 cells are approximately equally resistant to 4-HNE even though hepatocytes rely primarily on GST-mediated aldehyde conjugation to metabolize 4-HNE. Coupled with previous results from our laboratories, the overall increased sensitivity of certain hepatoma cells to lipid aldehydes appears due to decreased ability of these hepatoma cells to remove toxic products of lipid peroxidation. Moreover, hepatoma cells with increased levels of aldehyde dehydrogenase and aldehyde reductase appear most like hepatocytes in their ability to metabolize lipid aldehydes.
Abstract: 4-Hydroxynonenal (4-HNE), produced during the oxidative lipid breakdown of biological membranes, modulates various biochemical processes in normal liver and in hepatoma cells. It is very probable that the effects of 4-HNE are related to the quantity formed in the cells and to the cells' ability to metabolize it. Aldehyde catabolism takes place within the cells through oxidative and reductive enzymes, and through conjugation with intracellular glutathione. In this paper, the various enzymatic pathways involved in the metabolism of 4-HNE were studied in normal hepatocytes and in hepatoma cells. The hepatocyte pathway undergoes a complex variety of change during neoplastic transformation. In hepatoma cells, generally, 4-HNE metabolism was due mainly to aldehyde dehydrogenases, whereas in normal hepatocytes 4-HNE metabolism was mainly due to alcohol dehydrogenase and glutathione-S-transferase. The increase in oxidative enzymes compared to normal tissue was not the same in all types of hepatoma: in HTC hepatoma cells, the enzyme levels were considerably higher; in AH-130 hepatoma cells of Yoshida, they were lower in subcellular particles and similar in the cytosol. Indeed, consumption of externally-added 4-HNE in hepatoma cells was proportional to their content of 4-HNE metabolizing enzymes.
Abstract: MH1C1, HTC and HEPA 1c1c7 hepatoma cell lines were selected in this study as the bioindicators of the cytotoxicity induced by six chemicals: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), cycloheximide (CHE), cyclophosphamide (CPA), potassium dichromate (Cr VI) and 2,4-dinitrophenol (DNP). The concentrations used were in the range from 10(-6) mol/l to 10(-2) mol/l, and the exposure time was 24 h. Two end-points were measured to evaluate cytotoxicity: the detachment of dead cells from the monolayer (CS), as evaluated by detection of the total macromolecules present in the cell monolayers solubilized in alkali and the loss of colony-forming efficiency (CF). The dose-response curves were different from one compound to another, but generally similar with the two assays, the colony formation being the most sensitive test. Some technical problems like the toxicity of the solvents at the highest concentrations, the different sizes of colonies, the unspecific cellular detachment due to overgrowth during the experimental time, can be overcome by accurate standardization of the protocols used for each cell line. The sensitivity of the three cell lines was very similar, with some differences in the case of compounds exerting intermediate toxic effects, like CHE and DNP. The most toxic compound was Cr (VI), the least toxic one was CPA. The low cytotoxic effects displayed by CPA could be due to a lack of bioactivation and/or an increase of the inactivating enzymes, which are typical of hepatoma cell lines.
Abstract: The HTC hepatoma cell line was used as an "in vitro" model to detect the cytotoxicity of eighteen chemicals, chosen on the basis of different biological activities and physicochemical characteristics. Two different cytotoxicity assays measuring cell lethality (CS) or inhibition of cell growth (CF) were applicated to confluent cell monolayers or to colony-forming cells, respectively. Cells were exposed to the chemicals at doses ranging from 10(-6) M to 10(-2) M for 24 h. The results indicated a wide range of IC 50 (the concentration resulting in 50% inhibition of toxicity parameters) from as low as 1 microM (Potassium dichromate) to as high as 407.5 mM (Ethanol), the sensitivity of the CF test being greater than that of the CS test. A battery of cytotoxicity tests could be established in order to offer simple, rapid and economic methods which can be complementary and, in part, alternative to the use of laboratory animals.
Abstract: The NAD- and NADP-dependent aldehyde dehydrogenase (ALDH) activities were evaluated in two rat hepatoma cell lines, namely the well-differentiated MH1C1 line and the less differentiated HTC line. Each activity was determined in parallel in isolated rat hepatocytes, for comparison. The aliphatic aldehyde acetaldehyde (ACA) and the aromatic aldehyde benzaldehyde (BA) were used as substrates. With the first substrate the ALDH activities found in the crude cytoplasmic extracts were lower in hepatoma cells than in normal hepatocytes, especially when measured with NADP as coenzyme (ACA/NADP). Otherwise, with benzaldehyde as substrate the NAD-dependent enzyme activity (BA/NAD) was increased about 9-fold in HTC cells over hepatocytes and decreased in MH1C1 cells, while the NADP-dependent (BA/NADP) activity was increased 38- and 2.5-fold in HTC and MH1C1 cell lines, respectively. Studies on the subcellular distribution of these enzyme activities showed that the activity measured with acetaldehyde and NAD (ACA/NAD) was almost equally distributed between the cytosol and the subcellular particles in the three cell populations, but the ACA/NADP activity was shifted towards the cytosolic compartment in hepatomas, especially in HTC cells. The BA/NAD and BA/NADP ALDH activities found in the organelles of hepatoma cells were markedly reduced in comparison with hepatocytes, in favour of the cytosol. The most striking difference between the normal and the transformed cells was the 94-fold increase over hepatocytes of the BA/NADP activity, found in the cytosolic fractions of HTC cells. MH1C1 cells showed a less pronounced (7.5-fold) enhancement of this tumour-associated specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Carcinoma of the prostate is a tumor with a variable clinical course and a high incidence of local progression and/or metastasis. This study was undertaken to evaluate tissue prostate specific antigen (PSA) in patients with carcinoma of the prostate, its correlation with Gleason's grading and its value in predicting the clinical course of these patients. We studied 28 transurethral biopsies of patients with prostatic carcinoma utilizing HE and peroxidase-antiperoxidase staining techniques. These were given a score of 2 to 10 using Gleason's grading. PSA was determined according to percent positivity. The clinical course was considered favourable (F) when the lesion remained stable and unfavourable (U) when peri-prostatic spread was evidenced, metastasis and/or death from the disease. Statistical analysis was performed with the linear discriminatory test. PSA percentages ranged from 0 to 95 and the Gleason score from 3 to 11. There was an indirect correlation between these methods (r = 0.74): high Gleason scores corresponded to low PSA values and viceversa. PSA was highly positive in patients with F and U clinical courses whereas low positive values (less than 40%) were observed only in patients with U clinical course. High Gleason (8 to 10) and low (less than 5) scores were observed only in patients with a clinical course of U or F, respectively, while intermediate values (5 to 8) were not predictive of the clinical course. Discriminatory analysis gave Z values of -2.446 (P = 0.014) for PSA, -2.90 (P = 0.004) for the Gleason score in predicting prognosis, conferring a greater value overall to the latter.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: We have previously demonstrated the inducibility of both cytochrome P-448- and P-450-dependent monooxygenases in the differentiated rat hepatoma cell line MH1C1. Further experiments with these cells on the expression of different forms of cytochrome P-450, inducible not only by phenobarbital (PB) and 3-methylcholanthrene (MC), but also by metyrapone (MP), ethanol (E), and beta-naphthoflavone (BNF) are reported here. The effects of the in vitro addition of the inhibitors alpha-naphthoflavone and beta-naphthoflavone on the aryl hydroxylase activity (AHH) and the influence of protein synthesis on the induction of cytochrome P-450 were also assessed. Cultures were exposed to the inducers PB, MC, BNF, and MP during the last 6 days of culture and to E for 10 days. The inhibition of protein synthesis was obtained by adding cycloheximide (CY) to the cultured cells during the last 24 hr. The exposure of MH1C1 cells to various concentrations of MP resulted in a dose-dependent increase in AHH activity. The treatment of MH1C1 cells with different concentrations of ethanol produced a significant dose-dependent increase of monooxygenases. AHH activity, induced by the various treatments, was inhibited in a dose-dependent way by alpha-naphthoflavone and beta-naphthoflavone. Cy reduced the concentration of cytochrome P-450 and the AHH activity induced by the various treatments, thus indicating an implication of the protein synthesis in the mechanism(s) of induction.
Abstract: 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.
Abstract: The technique of alkaline elution was applied to study the capacity of methylglyoxal to induce DNA damage and repair in Chinese hamster ovary cells. DNA cross-linking was observed after a 90-min exposure to a subtoxic dose (1.5 mM), and the cross-links were fully repaired by 24 h. The cross-linking appeared to be DNA-protein in nature, since proteinase treatment removed the effect. When the same cells were exposed to methylglyoxal in the presence of a rat liver metabolic system, both cytotoxicity and cross-linking frequency were significantly reduced.
Abstract: 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).
Abstract: A cell line derived from a Morris hepatoma, MH1C1, was examined for its in vitro expression of monooxygenases. These cells were found to contain different forms of cytochrome P450, as shown by the response to inducers, namely phenobarbital (PB), 3-methylcholanthrene (MC) and metyrapone (MP). MH1C1 cell monolayers exposed to PB or MC showed an increase in the concentration of two spectrally distinct forms of cytochrome P450. The PB and MC treatments elicited enzyme activities towards the substrates aminopyrine and benzo(a)pyrene, respectively. The cell treatment with metyrapone led to a simultaneous stimulation of aminopyrine demethylase and benzo(a)pyrene hydroxylase activities, so underlining the peculiar features of this inducer.
Abstract: 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 Solt, 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.
Abstract: Alkaline elution was employed to study DNA damage in CHO-Kl cells treated with a series of biotic and xenobiotic aldehydes. DNA cross-linking was measured in terms of the reduction in the effect of methyl methanesulphonate on the kinetics of DNA elution and was observed in cells treated with formaldehyde, acetaldehyde, methylglyoxal and malonaldehyde. Propionaldehyde, valeraldehyde, hexanal and 4-hydroxynonenal produced DNA single-strand breaks, or lesions which were converted to breaks in alkali. Both types of DNA damage occurred in cells exposed to malealdehyde. These findings support the hypothesis of a carcinogenic effect of the aldehydic products (malonaldehyde, methylglyoxal, propionaldehyde, hexanal, 4-hydroxynonenal) released in biomembranes during lipid peroxidation.
Abstract: 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.
