Gene expression profile in oral squamous cell carcinomas and matching normal oral mucosal tissues from black Africans and white Caucasians: the case of the Sudan vs. Norway.

Expression profile of 588 known genes relating to tumour biology, was examined between oral squamous cell carcinomas (OSCCs) and matching normal oral mucosal tissues (NOMTs) obtained from Sudanese (n=11) and Norwegian (n=11) patients. cDNA probes were synthesised from total RNA and hybridised with the Atlas human cancer cDNA expression array membranes. RT-PCR and immunohistochemistry were applied to confirm the expression pattern of a subset of the 588 genes. Differences in expression of the genes examined were found between the OSCCs and the NOMTs on the Atlas membranes. Several of these genes were either up- or down-regulated 1.6-fold or higher in the OSCCs compared to the NOMTs in the cases from the two populations. We found that 181 (31%) and 195 (33%) genes were either up-regulated or down-regulated in the OSCCs from the Sudan and Norway, respectively. From the total number of genes (n=376) found expressed in the OSCCs investigated from the two countries, 53 genes (14%) showed common expression profile [35 (66%) were up-regulated and 18 (34%) were down-regulated] and 70 genes (19%) showed opposite regulation status. Results of the RT-PCR and immunohistochemistry confirmed the hybridisation data. These findings may provide an OSCCs-specific gene expression profile in patients from the two countries, suggesting that alterations of 123 genes are common in these OSCCs regardless of ethnic differences or other socio-cultural risk factors between the patients from the two countries. The findings might further suggest that specific genes are frequently involved in these OSCCs, which may provide novel clues as diagnostic, prognostic biomarkers and/or targets for therapy. The Atlas human cancer cDNA expression array technique can be useful to examine and describe the expression profile of known genes frequently involved in OSCCs from different populations.

[Nucleotide excision repair in human cells. Biochemistry and implications in diseases]. / Nukleotideksisjonsreparasjon i humane celler. Biokjemi og implikasjoner ved sykdommer.

It is of vital importance for all living systems to ensure proper functioning and propagation of their genetic information. A severe threat to the stability of DNA is posed by the ubiquitous occurrence of chemical and physical genotoxic agents that damage DNA, thus interfering with the processes of DNA metabolization and inducing mutations. This threat has necessitated the generation of an intricate network of DNA repair systems. Recently several laboratories have made great progress in the field of DNA repair, and have contributed to unravelling the molecular mechanism of the major process, the nucleotide excision repair pathway in mammals, and its biological impact and involvement in several human DNA repair disorders.

Coordinate induction of hepatic fatty acyl-CoA oxidase and P4504A1 in rat after activation of the peroxisome proliferator-activated receptor (PPAR) by sulphur-substituted fatty acid analogues.

1. In the liver of rat fed a single dose of 3-thia fatty acids, 3-dithiahexadecanedioic acid (3-thiadicarboxylic acid) and tetradecylthioacetic acid, steady-state levels of P4504A1 and fatty acyl-CoA oxidase mRNAs increased in parallel. The increases were significant 8 h after administration, reaching a maximum after 12 h and decreased from 12 to 24 h after administration. 2. The corresponding enzyme activities of P4504A1 and fatty acyl-CoA oxidase were also induced in a parallel manner by the 3-thia fatty acids. The enzyme activities were significantly increased 12 h after administration and increased further after 24 h. This may reflect a possible effect of the 3-thia fatty acids not only on mRNA levels, but also on the translation and degradation rate of the two enzymes. 3. Repeated administration of 3-thia fatty acids resulted in an increase of the specific P4504A1 protein accompanied with an increased lauric acid hydroxylase activity. The correlation between induction of P4504A1 and fatty acyl-CoA oxidase mRNAs and their enzyme activities may reflect a coordinated rather than a causative induction mechanism, and that these genes respond to a common signal. This suggests that the increased P450 activity may not be responsible or be a prerequisite for fatty acyl-CoA oxidase induction. 4. Since the peroxisome proliferator-activated receptor (PPAR) plays a role in mediating the induction of fatty acyl-CoA oxidase, we analysed the activation of PPAR by fatty acids and sulphur-substituted analogues utilizing a chimera between the N-terminal and DNA-binding domain of the glucocorticoid receptor and the putative ligand-binding domain of PPAR. Arachidonic acid activated this chimeric receptor in Chinese hamster ovary cells. Inhibitors of P450 did not affect the activation of PPAR by arachidonic acid. Furthermore, dicarboxylic acids including 1,12-dodecanedioic acid or 1,16-hexadecanedioic acid only weakly activated the chimera. 3-Thidicarboxylic acid, however, was a much more effective activator than the non-sulphur-substituted analogues. In conclusion, the data suggest that the most likely mechanism of the induction process is fatty acid-induced activation of PPAR, which then leads to a coordinated induction of P4504A1 and fatty acyl-CoA oxidase.

Nuclear and mitochondrial forms of human uracil-DNA glycosylase are encoded by the same gene.

Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800. This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting. To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration. An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.

The hypolipidemic peroxisome-proliferating drug, bis(carboxymethylthio)-1.10 decane, a dicarboxylic metabolite of tiadenol, is activated to an acylcoenzyme A thioester.

Bis(carboxymethylthio)-1.10 decane (BCMTD), a thiodicarboxylic acid, was shown to be a hypolipidemic peroxisome-proliferating drug as it: (a) decreased the total serum triacylglycerols and cholesterol; (b) induced hepatomegaly; (c) increased the peroxisomal beta-oxidation and catalase activity and the activities of the multiorganelle localized enzymes: palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, glycerophosphate acyltransferase; (d) decreased the carnitine palmitoyltransferase and urate oxidase activities; and (e) induced the bifunctional eonyl-CoA hydratase in peroxisomes. The present study has confirmed the effect of tiadenol administration on the activities of key enzymes involved in hepatic fatty acid metabolism in male rats. However, the hepatic pleiotropic response was more marked with the dicarboxylic acid than with its alcohol. In a separate dose-response study BCMTD was found to be a more potent inducer of peroxisomal beta-oxidation compared to tiadenol. BCMTD can be activated in vitro to its coenzyme A thioester by a dicarboxyl-CoA synthetase. In control and BCMTD-treated animals, the synthetase activity was found in all cellular fractions except the cytosolic. Whether the acyl-CoA thioesters of peroxisome-proliferating drugs may be mediators of peroxisomal proliferation should be considered.

Effects of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate on peroxisomal activities and enzyme activities involved in lipid metabolism in rat liver.

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on hepatic lipids and key enzymes involved in esterification, hydrolysis and oxidation of long-chain fatty acids at increasing doses were investigated in rats. TPA administration tended to decrease the mitochondrial activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase. The microsomal palmitoyl-CoA synthetase activity was increased. TPA administration was also associated with a dose-dependent increase of glycerophosphate acyltransferase activity both in the mitochondrial and microsomal fractions in particular. The data are consistent with a decreased catabolism of long-chain fatty acids at the mitochondrial level, and an increased capacity for esterification of fatty acids in the microsomal fraction. Peroxisomal beta-oxidation was increased about 2-fold in the peroxisome-enriched fraction of TPA-treated rats while the catalase and urate oxidase activities were only marginally affected. TPA administration revealed elevated capacity for hydrolysis of palmitoyl-CoA and palmitoyl-L-carnitine in the microsomal fraction. Neither increased cytosolic palmitoyl-CoA hydrolase activity nor increased hydroxylation of lauric acid nor changes of the hepatic content of cytochrome P-450 isoenzymic forms were observed in the TPA-treated animals. There was no induction of the protein content of the bifunctional enoyl-CoA hydratase. Thus, TPA behaves more like choline-deficient diet and ethionine treatment than well-known peroxisome proliferators. It seems possible that TPA selectively stimulated the peroxisomal activities, i.e., peroxisomal beta-oxidation rather than evoking a peroxisome proliferation capacity.

Alkylthio acetic acids (3-thia fatty acids)--a new group of non-beta-oxidizable peroxisome-inducing fatty acid analogues--II. Dose-response studies on hepatic peroxisomal- and mitochondrial changes and long-chain fatty acid metabolizing enzymes in rats.

The activity of key enzymes involved in oxidation and esterification of long-chain fatty acids was investigated after male Wistar rats were treated with different doses of sulfur substituted fatty acid analogues, 1,10-bis(carboxymethylthiodecane) (BCMTD, non-beta-oxidizable and non-omega-oxidizable), 1-mono(carboxymethylthiotetradecane) (CMTTD, trivial name, alkylthio acetic acid, non-beta-oxidizable) and 1-mono(carboxyethylthiotetradecane) (CETTD trivial name, alkylthio propionic acid, beta-oxidizable). The sulfur substituted dicarboxylic acid and the alkylthio acetic acid induced in a dose-dependent manner the mitochondrial, microsomal and especially the peroxisomal palmitoyl-CoA synthetase activity, the mitochondrial and cytosolic palmitoyl-CoA hydrolase activity, the mitochondrial and especially the microsomal glycerophosphate acyltransferase activity and the peroxisomal beta-oxidation, especially revealed in the microsomal fraction. Morphometric analysis of randomly selected hepatocytes revealed that BCMTD and CMTTD treatment increased the number, size and volume fraction of peroxisomes and mitochondria. Thus, the observed changes in the specific activity of fatty acid metabolizing enzymes with multiple subcellular localization can partly be explained as an effect of changes in the s-values of the organelles as proliferation of mitochondria and peroxisomes occurred. The most striking effect of the alkylthio propionic acid was the formation of numerous fat droplets in the liver cells and enhancement of the hepatic triglyceride level. This was in contrast to BCMTD treatment which decreased the hepatic triglyceride content. In conclusion, the results provide evidence that administration of non-beta-oxidizable fatty acid analogues had much higher in vivo potency in inducing hepatomegaly and key enzymes involved in fatty acid metabolism, including proliferation of peroxisomes and mitochondria than is exhibited in the beta-oxidizable, alkylthio propionic acid. Moreover, the dicarboxylic acid was apparently three to six times more potent than the alkylthio acetic acid in inducing peroxisomal beta-oxidation and peroxisome proliferation when considered on a mumol/day basis. As palmitic acid and hexadecanedioic acid only marginally affected these hepatic responses, it is conceivable that the potency of the selected compounds as proliferators of peroxisomes and inducers of the associated enzymes depends on their accessibility for beta-oxidation.

Alkylthioacetic acids (3-thia fatty acids) as non-beta-oxidizable fatty acid analogues: a new group of hypolipidemic drugs. III. Dissociation of cholesterol- and triglyceride-lowering effects and the induction of peroxisomal beta-oxidation.

Previous work in this laboratory indicated that sulfur-substituted fatty acid analogues, 1.10-bis(carboxymethylthio)decane and alkylthioacetic acid, both non-beta-oxidizable compounds, and the beta-oxidizable alkylthiopropionic acid (1) caused, to different extents, dose-related hepatomegaly and proliferation of peroxisomes and enhanced peroxisomal fatty acid beta-oxidation. In the present study, treatment of normolipidemic rats with alkylthioacetic acid resulted in a dose- and time-dependent decrease in serum cholesterol and serum and liver triglycerides to an extent comparable to that of the 3-thiadicarboxylic acid. At hypolipidemic doses, alkylthioacetic acid caused no hepatomegaly, did not significantly alter peroxisome morphology, and only marginally affected peroxisomal beta-oxidation activity. Only at the highest, nonpharmacological doses of alkylthioacetic acid were these hepatic parameters increased, although to a lesser extent than by the 3-thiadicarboxylic acid. Hence, on the basis of dose- and time-related studies of the two compounds, data indicate that the hypotriglyceridemia and hypocholesterolemia were dissociated from induction of peroxisomal beta-oxidation and peroxisome proliferation. Palmitic acid and hexadecanedioic acid, both beta-oxidizable fatty acids, only marginally affected the serum and liver parameters. The beta-oxidizable fatty acid analogue, alkylthiopropionic acid lowered the serum triglycerides in normolipidemic rats. In contrast to the 3-thiadicarboxylic acid and alkylthioacetic acid, alkylthiopropionic acid treatment at hypolipidemic doses caused accumulation of triglycerides in the liver.

Alkylthioacetic acid (3-thia fatty acids)--a new group of non-beta-oxidizable, peroxisome-inducing fatty acid analogues. I. A study on the structural requirements for proliferation of peroxisomes and mitochondria in rat liver.

The induction of peroxisome proliferation was examined in rat liver after administration of equal concentrations (1 mmol/kg body weight) of 1,10-bis(carboxymethylthiodecane) (BCMTD), 1-mono(carboxymethylthiotetradecane) (CMTTD), 1-mono(carboxymethylthiooctane) (CMTO), 1-mono(carboxyethylthiotetradecane) (CETTD), palmitic acid and hexadecanedioic acid (HDDA). BCMTD, a non-beta-oxidizable and non-omega-oxidizable sulphur-substituted fatty acid analogue was considerably more potent than CMTTD (only non-beta-oxidizable) in inducing enlargement of the liver and increasing peroxisomal activities (monitored by peroxisomal beta-oxidation, palmitoyl-CoA hydrolase and catalase activities). Morphometric analysis of randomly selected hepatocytes revealed that BCMTD and CMTTD treatment increased the number and size of peroxisomes and the relative volume fraction of the peroxisomes. All these cellular responses were more marked with BCMTD than compared with CMTTD. CMTO, a non-beta-oxidizable fatty acid analogue containing a lower hydrophobic alkyl-end than CMTTD and CETTD (a beta-oxidizable fatty acid analogue), showed a slight increase (1.4-1.8-fold) of peroxisomal beta-oxidation and caused marginally morphological changes of peroxisomes compared with CMTTD and BCMTD. The most striking effect of the alkylthiopropionic acid (CETTD) was an enhancement of the hepatic triacylglycerol level. Palmitic acid and hexadecanedioic acid only marginally affected the peroxisomal activities, but no morphological changes of peroxisomes and fat droplets were observed. The presented data strongly suggest that a minimal structural requirement for a peroxisome proliferator may be (1) a carboxylic acid group linked to (2) a hydrophobic backbone which (3) cannot be beta-oxidized i.e., the fatty acid analogues have a sulphur atom in the beta-position. It is also conceivable that blockage for omega-oxidation may potentiate the peroxisome-proliferating activities in as much as BCMTD was more potent than CMTTD. Two mitochondrial marker enzymes, carnitine palmitoyltransferase and succinate phenazine methosulphate oxidoreductase were differently affected after administration of the investigated compounds. Furthermore, BCMTD and CMTTD as well as HDDA treatments increased the number of mitochondria, but the mitochondria tended to be smaller. The overall results presented here indicate that the structural requirements for proliferation of mitochondria are not identical to those for proliferation of peroxisomes.

Changes in peroxisomes and mitochondria in liver of ethionine exposed rats: a biochemical and morphological investigation.

Administration of ethionine resulted in a dose- and time-dependent enhancement of the activities of peroxisomal beta-oxidation, carnitine palmitoyltransferase and omega-oxidation, especially the 12-hydroxylation of lauric acid. The mitochondrial and, especially, the microsomal palmitoyl-CoA hydrolase activities were increased, whereas the peroxisomal and cytosolic activities were decreased. Ethionine administration decreased the catalase and urate oxidase activities in both a dose- and time-related manner. The liver cells and the volume fraction of cytoplasma decreased 40% in ethionine-exposed animals, whereas the average nuclei volume fraction increased approximately 50%. The volume fraction and the total number of mitochondria increased 1.5-fold after ethionine exposure and an accumulation of lipid in large droplets of the hepatocytes was observed. No proliferation of peroxisomes was observed after treatment; the volume fraction and the number of peroxisomes decreased. However, the size of peroxisomes in livers of ethionine-exposed rats tended to be greater than controls; a 1.5-fold increase in average size was observed. As there was no induction of the protein content of the bifunctional enoyl-CoA hydratase, an enzyme involved in peroxisomal beta-oxidation, it is considered that ethionine selectively stimulates the peroxisomal beta-oxidation due to increased peroxisome surface area rather than evoked a peroxisome proliferation capacity. Increased peroxisomal beta-oxidation was also observed in the kidney of ethionine-exposed rats at a dose of 750 mg/day/kg body weight. At that dose the amount of reduced glutathione (GSH) was significantly increased in kidney. The amount of GSH and the level of peroxisomal beta-oxidation were significantly increased in liver at an ethionine dose of 100 mg/day/kg body weight. These responses in liver were evident within 2 days of ethionine exposure and then leveled off whereas a significant increase in GSH and peroxisomal beta-oxidation in kidney was observed within 12 days. Whether the acute H2O2-generating peroxisomal oxidation of long-chain fatty acids in the liver may also make this organ susceptible to the long-term effects of low-dose ethionine and be an important step in the chain of events which eventually results in tumour development should be considered.

Ethionine-induced alterations of enzymes involved in lipid metabolism and their possible relationship to induction of fatty liver.

Changes of enzymes involved in the hepatic metabolism of long-chain fatty acids (palmitoyl-CoA synthetase (EC 6.2.1.3), carnitine palmitoyltransferase (EC 6.2.1.3), glycerophosphate acyltransferase (EC 2.3.1.15)) in the liver of male rats were examined after ethionine exposure. Ethionine administration resulted in a dose- and time-dependent enhancement of the palmitoyl-CoA synthetase activity both in the mitochondrial, peroxisomal and microsomal fractions. The total carnitine palmitoyltransferase activity in the mitochondrial fraction was enhanced. Ethionine administration was also associated with dose- and time-dependent changes of the microsomal glycerophosphate acyltransferase activity, whereas the mitochondrial enzyme activity was marginally affected. The hepatic triacylglycerol content of the ethionine-treated animals was increased. Hepatic lipids were accumulated in large droplets. Serum triacylglycerol and cholesterol were decreased. In particular, the serum HDL-cholesterol level was lowered. The concentration of ATP in the liver decreased. Accumulation of the metabolic product S-adenosylethionine (AdoEth) was observed for the first 2 days of exposure followed by a fall in S-adenosylmethionine (Ado-Met) during the next 10 days. Linear regression analysis of ATP content versus AdoEth and AdoMet showed highly significant correlations. A significant correlation between the hepatic triacylglycerol and AdoEth content was also observed upon ethionine treatment. The data show that ethionine perturbs the hepatic lipid metabolism. Enhanced esterification of long-chain fatty acids, but not a simple reduction of their oxidation, might contribute to ethionine-induced fatty liver in addition to a block in secretion of lipoproteins and decreased protein synthesis.

Effect of choline-deficiency and methotrexate administration on peroxisomal beta-oxidation, palmitoyl-CoA hydrolase activity and the glutathione content in rat liver.

Hepatic metabolism of long-chain fatty acids was studied in male rats fed a defined choline-deficient (CD) diet with and without choline and after methotrexate (MTX) administration. Peroxisomal beta-oxidation was increased approximately 4-fold in the peroxisome-enriched fraction of CD-fed animals, whereas the catalase activity was increased 1.3-fold. The urate oxidase activity was marginally affected. The CD-fed rats also revealed elevated capacity for hydrolysis of palmitoyl-CoA in the cytosolic fraction (2.0-fold), whereas the microsomal palmitoyl-CoA hydrolase activity was decreased. Notably, the increased peroxisomal beta-oxidation, the catalase activity and palmitoyl-CoA hydrolase activities (the membrane-bounded and cytosolic) were almost fully prevented by adding choline to the CD-diet. Thus, the change in these enzyme activities appears to be a consequence of a choline-deficiency provoked by the CD diet. MTX administration of normal fed rats (ND diet) had no effects on the peroxisomal beta-oxidation, catalase activity and urate oxidase activity. MTX treatment of the ND-fed animals, however, increased the mitochondrial palmitoyl-CoA hydrolase activity and decreased the microsomal enzyme activity. As choline-deficiency and MTX increased the hepatic lipid level, the overall results suggest that fat accumulation is not an 'induction signal' for increased peroxisomal beta-oxidation. The CD diet alone increased the reduced glutathione content in liver, whereas MTX did not significantly change this level. Whether the changes of H2O2-generating peroxisomal oxidation of long-chain fatty acids may be an important step in a chain of events, which eventually results in tumour formation by choline-deficiency, should be considered.

Differential metabolic response of rat liver, kidney and spleen to ethionine exposure. S-adenosylamino acids, homocysteine and reduced glutathione in tissues.

Intraperitoneal injection of ethionine to male rats for up to 12 days caused a pronounced fall in S-adenosylmethionine (AdoMet) in liver, but did not or only slightly affect AdoMet in kidney and spleen. Liver and to a lesser degree kidney showed a dose-dependent, massive accumulation of the metabolic product, S-adenosylethionine (AdoEth), and this metabolic response was most pronounced within the first days of exposure. Trace amounts of AdoEth was demonstrated in the spleen. Both S-adenosylhomocysteine (AdoHcy) and homocysteine (Hcy) in the liver were markedly increased in a dose- and time-dependent manner. There was a moderate increase in Hcy content in spleen and kidney, whereas the AdoHcy levels in these tissues were not affected. The amount of reduced glutathione (GSH) was significantly increased in liver and kidney. This response in liver was evident within 2 days of ethionine exposure and then leveled off whereas there was a gradual increase in GSH in kidney. The GSH content in spleen was unaltered. In addition to a massive build-up of AdoEth, the unique features of the metabolic response of the liver are a pronounced decrease in the AdoMet/AdoHcy ratio (from 15 to 2) associated with an elevated Hcy content and a rapid increase in the amount of GSH. The possibility that the metabolic response of the liver could be assigned to the existence of isozymes or metabolic pathways unique to hepatic cells is discussed.

Effect of methotrexate on homocysteine and other sulfur compounds in tissues of rats fed a normal or a defined, choline-deficient diet.

Methotrexate (MTX) affects homocysteine (Hcy) metabolism in both cultured cells and patients, and this may be explained by a lack of the 5-methyltetrahydrofolate required for salvage of Hcy to methionine. We here report the effect of MTX on Hcy in serum and Hcy, S-adenosylhomocysteine (AdoHcy), S-adenosylmethionine (AdoMet) and reduced glutathione (GSH) in tissues of rats fed either a normal or a defined, choline-deficient (CD) diet. The CD diet alone did not affect the amounts of Hcy in serum and tissues, but decreased the amount of AdoMet in most tissues and increased the GSH content in the liver. MTX increased the amount of Hcy about 2-fold in serum, liver and kidney, and decreased the amount of AdoMet in liver and kidney, whereas the AdoHcy content in these tissues was essentially unaffected. Accordingly, both choline deficiency and MTX treatment reduced the AdoMet to AdoHcy ratio. The increased GSH in the liver induced by CD diet seemed to be abolished by MTX. In the spleen MTX had only a marginal effect on the Hcy and AdoMet content and decreased the GSH content. It is concluded that the increase in serum Hcy during MTX exposure probably reflects a disturbance of the Hcy metabolism in some tissues, and especially in the liver. Altered metabolism of other sulfur-containing metabolites may only partly be related to the inhibition of Hcy salvage, and some metabolic effects of MTX may be modulated by tissue-specific metabolic pathways as well as by the diet.

Effect of methotrexate on long-chain fatty acid metabolism in liver of rats fed a standard or a defined, choline-deficient diet.

The effect of methotrexate on lipids in serum and liver and key enzymes involved in esterification and oxidation of long-chain fatty acids were investigated in rats fed a standard diet and a defined choline-deficient diet. Hepatic metabolism of long-chain fatty acids were also studied in rats fed the defined diet with or without choline. When methotrexate was administered to the rats fed the standard diet there was a slight increase in hepatic lipids and a moderate reduction in the serum level. The palmitoyl-CoA synthetase activity and the microsomal glycerophosphate acyltransferase activity in the liver of rats were increased by methotrexate. The data are consistent with those where the liver may fail to transfer the newly formed triacylglycerols into the plasma with a resultant increase in liver triacylglycerol content and a decrease in serum lipid levels. Fatty liver of methotrexate-exposed rats can not be attributed simply to a reduction of fatty acid oxidation as the carnitine palmitoyltransferase activity was increased. The methotrexate response in the rats fed the defined choline-deficient diet was different. There was a reduction in both serum and hepatic triacylglycerol and the glycerophosphate acyltransferase and palmitoyl-CoA synthetase activities. The carnitine palmitoyltransferase activity was unchanged. Hepatomegaly and increased hepatic fat content, but decreased serum triacylglycerol, total cholesterol and HDL cholesterol were found to be related to the development of choline deficiency as the pleiotropic responses were almost fully prevented by addition of choline to the choline-deficient diet. Addition of choline to the choline-deficient diet normalized the total palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities. In contrast to methotrexate exposure, choline deficiency increased the mitochondrial glycerophosphate acyltransferase activity. The data are consistent with those of where fatty liver induction of choline deficiency may be related to an enhanced esterification of long-chain fatty acids concomitant with a reduction of their oxidation.

Induction of cytosolic clofibroyl-CoA hydrolase activity in liver of rats treated with clofibrate.

Among subcellular fractions of liver homogenates of rats, the clofibroyl-CoA hydrolase activity is found mainly in the cytosolic fraction. It is here shown that the subcellular distribution of clofibroyl-CoA hydrolase appears to be different from the distribution of palmitoyl-CoA hydrolase activity. Thus, in contrast to the case with palmitoyl-CoA, no hydrolysis of clofibroyl-CoA was catalysed by the microsomal fraction. Furthermore, the hydrolysis of palmitoyl-CoA and clofibroyl-CoA in the cytosolic fraction seemed to be catalyzed by two different enzymes. Rats treated with clofibrate (0.3%, w/w) showed a significant increased clofibroyl-CoA hydrolase activity where the cytosolic hydrolase was increased 3.5-fold. Clofibrate administration also elevated the specific clofibroyl-CoA hydrolase activity by factors of 1.7 and 1.5 in the mitochondrial and the light-mitochondrial fractions, respectively. Thus, it is possible that clofibroyl-CoA hydrolase has also a multiorganelle localization.

Cell transformation and promoter activity of insulation oils in the Syrian hamster embryo cell and in the C3H/10T1/2 mouse embryo fibroblast test systems.

The ability of mineral-oil-based and synthetic cable insulating fluids to transform and promote transformation of mammalian cells in vitro have been studied. In experiments with the Syrian hamster embryo cell transformation assay, it was found that C15-C18 alkylbenzenes were the most potent inducers of transformation, followed by low-viscosity and residual mineral oils. No activity and low cytotoxicity were found for a low-viscosity polyisobutylene-based oil. In the two-stage transformation assay of C3H/10T1/2 cells, promoter activity was obtained with all fluids tested. A blend of residual and low-viscosity mineral oils showed the highest activity. This oil possessed a low cytotoxicity and was tested at a relatively high concentration. The alkylbenzenes were more potent than the polyisobutylene-based fluid. The alkylbenzenes were also found to possess initiating activity in the two-stage assay, when 12-O-tetradecanoylphorbol 13-acetate (TPA) was used as promoter. All the fluids showed low potency compared to benzo[a]pyrene and the tumor promoter TPA.

Separation and measurement of clofibroyl coenzyme A and clofibric acid in rat liver after clofibrate adminstration by reversed-phase high-performance liquid chromatography with photodiode array detection.

A method to identify and quantitate clofibric acid and clofibroyl coenzyme A (CoA) products in rat liver was developed using reversed-phase high-performance liquid chromatography. The system was developed with baseline separation of clofibroyl-CoA from clofibric acid using isocratic elution, with a mobile phase consisting of 52% methanol and 28 mM potassium phosphate buffer (pH 4.2). With this high methanol concentration, the large amount of UV-absorbing materials present in the liver extracts were eluted earlier than the investigated compounds. Clofibroyl-CoA has a characteristic absorbance spectrum with distinct peaks at 260 and 230 nm, while clofibric acid showed only a distinct peak at 230 nm. Using an on-line photodiode array detector, the spectra could be recorded during analysis without interrupting the flow of the mobile phase. This spectral analysis identification possibilities and evaluation of the purity of the chromatographic peaks. In a perchloric extract of rat liver, the recovery of clofibric acid and clofibroyl-CoA added to the liver extract ranged from 70 to 80%. A linear relationship was observed between clofibric acid and clofibroyl-CoA concentration and the area of their peaks in the chromatogram. The detection limit of the method was lower than 5 pmol for both compounds when the absorbance was recorded at 230 nm. The method could be used without modification for the estimation of clofibroyl-CoA and clofibric acid in biological extracts.

Differential effects of chlorpromazine on secretion, protein phosphorylation and phosphoinositide metabolism in stimulated platelets.

Increasing concentrations of chlorpromazine (30-500 microM) caused a progressive lysis of gel-filtered platelets, as monitored by the extracellular appearance of cytoplasmic ([14C]adenine-labelled) adenine nucleotides. The chlorpromazine-induced lysis was markedly enhanced by thrombin and phorbol ester, and complete cytolysis was found at chlorpromazine concentrations of 100 microM and above in the presence of thrombin. At non-lytic concentrations, chlorpromazine caused a dramatic increase in the thrombin- or phorbol ester-mediated incorporation of 32P into phosphatidylinositol 4-phosphate and, to a lesser extent, into phosphatidylinositol 4,5-bisphosphate in platelets pulse-labelled with [32P]Pi. Chlorpromazine alone also caused an incorporation of 32P into the phosphoinositides. Non-lytic concentrations of chlorpromazine had no effect on the phosphorylation of the 47 kDa protein (regarded as the substrate for protein kinase C), but markedly inhibited the accompanying secretion of ATP + ADP and beta-hexosaminidase when platelets were incubated with 0.17 microM-phorbol ester or 0.1-0.2 unit of thrombin/ml. At lower concentrations of thrombin, chlorpromazine did not inhibit, but slightly enhanced, secretion. A protein of 82 kDa was phosphorylated during the interaction of platelets with thrombin and phorbol ester, and this phosphorylation was enhanced by chlorpromazine (non-lytic). These results suggest that the previously reported inhibition of protein kinase C by chlorpromazine is probably non-specific and due to cytolysis. However, since non-lytic concentrations of chlorpromazine inhibit secretion, but not protein kinase C, in platelets, activation of protein kinase C is not involved in the stimulation-secretion coupling, or chlorpromazine acts at a step after kinase activation. Possible mechanisms of this inhibition by chlorpromazine are discussed in the light of its effect on phosphoinositide metabolism and protein phosphorylation.

Peroxisome proliferators show tumor-promoting but no direct transforming activity in vitro.

The chemically unrelated hypolipidemic drugs, tiadenol, niadenate, and clofibrate have been tested for carcinogenic and tumor-promoting potential in the C3H/10TI/2 C18 cell test system. None of these chemicals were carcinogenic, while both niadenate and clofibrate were active tumor promoters at micromolar concentrations. All 3 drugs induced the differentiation of C3H/10T1/2 C18 cells to adipocytes. This latter finding confirms previously observed effects of the tumor promoter TPA.