Biomedical subjects

S F Muakkassah-Kelly

Publications and source records attributed to S F Muakkassah-Kelly.

9 recordsLinked to original sources

PubMed1991

Studies on the mechanism of induction of microsomal cytochrome P452 and peroxisomal bifunctional enzyme mRNAs by nafenopin in primary cultures of adult rat hepatocytes.

The amount of the two mRNAs although lower in cultured hepatocytes than in freshly isolated cells was found to be rapidly inducible upon the addition of 32 microM nafenopin. The induction of cyt.P452 mRNA always preceded the induction of PBE mRNA, but for both, the maximal induction (10-20-fold over control) was obtained within 24 hr and was achieved by transcriptional activation. At early time points (1 and 2 hr after the addition of nafenopin), in the absence of on-going protein synthesis, the amount of cyt.P452 mRNA (and not of PBE mRNA) was transiently higher in the presence of cycloheximide and nafenopin than in the presence of nafenopin alone.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1991

The effect of short term feeding of the antioxidant triethyleneglycol-bis-3(3-tert-butyl-4-hydroxy-5-methyl)propionate on serum thyrotropin and thyroid hormones in the male rat.

Male rats were fed triethyleneglycol-bis-3(3-tert-butyl-4-hydroxy-5-methyl)propionate (TK 12627) admixed with the food at a concentration of 1000 ppm for 3, 6, 13, and 20 days. Treatment resulted in time-dependent and marked increases in serum levels of thyrotropin (TSH) and reverse triiodothyronine (rT3). Serum levels of thyroxine (T4) were slightly and transiently decreased, whereas triiodothyronine (T3) levels decreased by 35-50% at all time periods. Treatment with 50, 150, 500, and 1000 ppm for 2 weeks resulted in dose-related increases in thyroid and liver weights, follicular hypertrophy of the thyroid, morphological changes of the pituitaries, liver hypertrophy, and similar changes in the serum parameters described above. At 50 ppm, no alterations in the weights and morphology of the liver, thyroid, or pituitary nor in the serum levels of TSH or T4 were observed. The effects of TK 12627 observed at a dose of 1000 ppm for 2 weeks were reversible after cessation of treatment. Decreases in T3, increases in rT3, and no change in T4 serum levels were also obtained when thyroidectomized T4-substituted rats were treated with 1000 ppm TK 12627 for 28 days, indicating that the effects of TK 12627 are probably due to inhibition of the 5' monodeiodination of T4 to T3 and rT3 to diiodothyronine with compensatory increases in thyroid hormone conjugation at extrathyroidal sites.

Animals↗

PubMed1990

Nafenopin, a hypolipidemic and non-genotoxic hepatocarcinogen increases intracellular calcium and transiently decreases intracellular pH in hepatocytes without generation of inositol phosphates.

Addition of nafenopin (30-300 microM to 45Ca2+ preloaded cultured hepatocytes caused a rapid and concentration-dependent increase in 45Ca2+ efflux in a manner similar to vasopressin, as evidenced by the loss of radioactivity from the cells. In contrast to vasopressin, addition of nafenopin to [3H]inositol prelabelled hepatocytes in culture did not increase [3H]inositol phosphate production. When added simultaneously with vasopressin, nafenopin inhibited the vasopressin-stimulated [3H]inositol phosphate production. In hepatocyte suspensions isolated from rats treated for 1 week with a carcinogenic dose of nafenopin (1000 ppm in their daily food) the incorporation of [3H]inositol into the phosphoinositide fraction, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, was much less than that in hepatocytes isolated from untreated rats. The vasopressin-stimulated [3H]inositol phosphate production was also decreased. Experiments with hepatocyte suspensions preloaded with Ca2+ or pH sensitive fluorescent indicators demonstrated that addition of nafenopin caused an increase in intracellular free Ca2+ and transient acidification of the cells. The increase in [Ca2+]i was decreased by only about 25% when extracellular calcium was removed indicating that nafenopin mainly mobilizes Ca2+ from intracellular stores. The recovery to basal pH was amiloride-sensitive indicating the importance of Na+/H+ exchange in pH recovery after intracellular acidification. Amiloride also inhibited DNA synthesis induced by nafenopin and by epidermal growth factor in cultured hepatocytes; but this effect occurred concomitantly with inhibition of basal DNA synthesis. We suggest that hepatic Ca2+ mobilization induced by nafenopin may play an important role in the mechanism by which nafenopin exerts its physiological as well as its tumour promotive activity upon chronic treatment with carcinogenic doses.

Animals↗

PubMed1990

Increase in hepatocyte and nuclear volume and decrease in the population of binucleated cells in preneoplastic foci of rat liver: a stereological study using the nucleator method.

Gamma-glutamyltranspeptidase-positive hepatocyte foci were produced in female rats given a single dose of diethylnitrosamine neonatally after birth and, after weaning, a diet containing phenobarbitone for 30 wk. The nucleator method, a new stereological approach, provided an efficient, unbiased estimate of mean cell volume in focal lesions and extrafocal areas. It also provided an unbiased sample of cells to estimate hepatocyte nuclear volume and the percentage of binucleated cells. The results showed an increase in the mean volume of mononucleated cells--from 4,700 micron3 in extrafocal areas to 12,700 micron2 in foci--and of binucleated cells--from 6,900 micron3 to 25,000 micron3. This demonstrated the hypertrophic effect of the carcinogenic treatment in focal lesions. A striking reduction in the proportion of binucleated cells was also observed in the preneoplastic lesions. Nuclear volume measurements from mononucleated and binucleated hepatocytes were used to assess ploidy. An apparent increase in nuclear ploidy, with no change in cellular ploidy, was noted in focal tissue when compared with nonfocal tissue. This appeared to be caused by an increase in mononucleated tetraploid cells and a reduction in binucleated cells with two diploid nuclei, indicating an altered mitotic mechanism in focal lesions. The significance of these changes in cell volume, apparent ploidy levels and binuclearity in preneoplastic foci is discussed in relation to the hepatocarcinogenic process.

Animals↗

PubMed1990

Ultrastructural changes in chemically induced preneoplastic focal lesions in the rat liver: a stereological study.

Ultrastructural changes were investigated and quantified, using a stereological approach, in early gamma-glutamyltranspeptidase (GGT)-positive focal lesions, induced in the rat liver by treatment with a single initiating dose of diethylnitrosamine (DENA) followed by promotion with phenobarbitone (PB) for 30 weeks. Within the extra-hepatocyte environment of focal tissue, the mean volume occupied by Ito cells was markedly decreased, whilst that occupied by endothelial and Kupffer cells was increased, when compared to uninvolved tissue from the same rat livers. The bile canaliculi were dilated, but no significant differences in the mean volume occupied by the sinusoidal and Disse spaces were noted. In focal hepatocytes there was a striking overproduction of lipid droplets and proliferation of smooth endoplasmic reticulum (sER). Whorls of concentrically arranged, parallel ER membranes were found only in the hepatocytes of preneoplastic foci, in association with the proliferated sER, and never in the surrounding, uninvolved tissue. The increase in mean volume of the sER, lipid droplet and cytoplasmic matrix compartments, together with the appearance of whorls, were the major contributing factors to the marked hypertrophy seen in focal hepatocytes. The mean volume of the rough endoplasmic reticulum, mitochondrial, lysosomal, peroxisomal and nuclear compartments per hepatocyte also increased, but contributed to a lesser extent to the cellular hypertrophy. It is speculated that whorls may be structural adaptations, resulting from a possible alteration in the normal feedback control of cholesterol synthesis, for the production of sterols and the biogenesis of sER in eosinophilic-type focal cells. The significance of changes observed in focal tissue, and the high biological variation noted between foci, is discussed in relation to the hepatocarcinogenic process.

Animals↗

PubMed1988

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation.

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.

Animals↗

PubMed1988

Electroporation of cultured adult rat hepatocytes with the c-myc gene potentiates DNA synthesis in response to epidermal growth factor.

The human c-myc gene was introduced and transiently expressed in adult rat hepatocyte cultures by the technique of electroporation and its effect on DNA synthesis was examined. Epidermal growth factor (EGF) has been found to stimulate a wave of DNA synthesis in electroporated rat hepatocytes. Hepatocyte cultures electroporated with the c-myc gene showed a potentiation of this EGF effect exhibiting rates of DNA synthesis up to 50% greater than those of control electroporated cultures, as determined by [3H]thymidine labeling of cell nuclei. This potentiation was dependent on the amount of c-myc DNA transfected. The potentiation was due neither to an alteration in the dose-response of the stimulatory effect of EGF nor to a change in the time course of the DNA synthesis wave.

Animals↗

PubMed1987

Long-term maintenance of hepatocytes in primary culture in the presence of DMSO: further characterization and effect of nafenopin, a peroxisome proliferator.

The addition of 2% dimethyl sulfoxide to adult rat hepatocytes cultured in a chemically defined medium at Day 1 after cell plating resulted in maintenance of the cytochrome P-450 content and the cyanide-insensitive palmitoyl-CoA beta-oxidation activity at 66 and 70% of the initial Day 1 values. The addition of phenobarbital, 3-methylcholanthrene, or nafenopin from Day 3 to Day 6 increased the contents of cytochrome P-450 to 128, 239, and 251%, respectively, compared to untreated controls at Day 3. In addition, nafenopin also caused a pronounced and time-dependent increase in palmitoyl-CoA beta-oxidation activity but was found to have only a weak stimulating effect on replicative DNA synthesis (2-fold) when compared to that of epidermal growth factor (6.5-fold). In the presence of dimethyl sulfoxide the hepatocyte cultures could be kept alive for more than 1 month. Exposure of such cultures to nafenopin from Day 1 do Day 37 resulted in survival which was even better than that of their untreated counterparts. This effect was accompanied by the appearance of abundant endoplasmic reticulum membranes and an increased number of peroxisomes.

Animals↗

PubMed1986

Uric acid-iron ion complexes. A new aspect of the antioxidant functions of uric acid.

In order to survive in an oxygen environment, aerobic organisms have developed numerous mechanisms to protect against oxygen radicals and singlet oxygen. One such mechanism, which appears to have attained particular significance during primate evolution, is the direct scavenging of oxygen radicals, singlet oxygen, oxo-haem oxidants and hydroperoxyl radicals by uric acid. In the present paper we demonstrate that another important 'antioxidant' property of uric acid is the ability to form stable co-ordination complexes with iron ions. Formation of urate-Fe3+ complexes dramatically inhibits Fe3+-catalysed ascorbate oxidation, as well as lipid peroxidation in liposomes and rat liver microsomal fraction. In contrast with antioxidant scavenger reactions, the inhibition of ascorbate oxidation and lipid peroxidation provided by urate's ability to bind iron ions does not involve urate oxidation. Association constants (Ka) for urate-iron ion complexes were determined by fluorescence-quenching techniques. The Ka for a 1:1 urate-Fe3+ complex was found to be 2.4 X 10(5), whereas the Ka for a 1:1 urate-Fe2+ complex was determined to be 1.9 X 10(4). Our experiments also revealed that urate can form a 2:1 complex with Fe3+ with an association constant for the second urate molecule (K'a) of approx. 4.5 X 10(5). From these data we estimate an overall stability constant (Ks approximately equal to Ka X K'a) for urate-Fe3+ complexes of approx. 1.1 X 10(11). Polarographic measurements revealed that (upon binding) urate decreases the reduction potential for the Fe2+/Fe3+ half-reaction from -0.77 V to -0.67 V. Thus urate slightly diminishes the oxidizing potential of Fe3+. The present results provide a mechanistic explanation for our previous report that urate protects ascorbate from oxidation in human blood. The almost saturating concentration of urate normally found in human plasma (up to 0.6 mM) represents 5-10 times the plasma ascorbate concentration, and is orders of magnitude higher than the 'free' iron ion concentration. These considerations point to the physiological significance of our findings.

Animals↗