acyl-CoA Oxidase Activity Research Articles

Peroxisomal enzymes in the honey bee midgut

Peroxisomes are ubiquitous organelles that contain catalase (CAT) and an array of inducible enzymes that regulate aspects of lipid, purine, xenobiotic, eicosanoid, and phospholipid metabolism. Although peroxisomes are recognized as essential components in the cellular economy of microorganisms, plants, and mammals, little is known about their specialized functions in insect metabolism. Peroxisomal acyl-CoA oxidase (ACO) is a flavin-linked, H2O2-producing enzyme that regulates the β-oxidation of long chain fatty acids. We measured ACO and CAT activity in midgut tissues from worker honey bees, Apis mellifera, of known ages from free-flying colonies. The ACO activity peaked in young worker bees that digest and assimilate nutrients from pollen and from trophallaxis. As the bees aged, ACO activity declined. Conversely, CAT activity increased as the bees aged and reached its highest level in the oldest bees that were assayed. Isolated honey bee midguts were then fractionated using sucrose and Metrizamide (MET) density gradient centrifugation. Organelle-bound CAT activity equilibrated in sucrose at densities between 1.19 and 1.22, which are typical of spherical 1 μm peroxisomes. In the MET gradients, the organelle-bound CAT separated into two distinct fractions. The heavier fraction equilibrated at 1.21 and the lighter fraction at 1.15, a density commonly associated with microperoxisomes. These results support our ultrastructural and cytochemical data and suggest that the diverse functions of regionally specialized midgut epithelial cells lead to a heterogeneous population of peroxisomal organelles. ACO activity confirms the role of midgut peroxisomes in the intermediary metabolism of lipids and the increasing CAT activity suggests that the midgut epithelium may metabolize deleterious pro-oxidants of aerobic metabolism associated with foraging and senescence. © 1996 Wiley-Liss, Inc.

Peroxisomal enzymes in the honey bee midgut

Peroxisomes are ubiquitous organelles that contain catalase (CAT) and an array of inducible enzymes that regulate aspects of lipid, purine, xenobiotic, eicosanoid, and phospholipid metabolism. Although peroxisomes are recognized as essential components in the cellular economy of microorganisms, plants, and mammals, little is known about their specialized functions in insect metabolism. Peroxisomal acyl-CoA oxidase (ACO) is a flavin-linked, H2O2-producing enzyme that regulates the β-oxidation of long chain fatty acids. We measured ACO and CAT activity in midgut tissues from worker honey bees, Apis mellifera, of known ages from free-flying colonies. The ACO activity peaked in young worker bees that digest and assimilate nutrients from pollen and from trophallaxis. As the bees aged, ACO activity declined. Conversely, CAT activity increased as the bees aged and reached its highest level in the oldest bees that were assayed. Isolated honey bee midguts were then fractionated using sucrose and Metrizamide (MET) density gradient centrifugation. Organelle-bound CAT activity equilibrated in sucrose at densities between 1.19 and 1.22, which are typical of spherical 1 μm peroxisomes. In the MET gradients, the organelle-bound CAT separated into two distinct fractions. The heavier fraction equilibrated at 1.21 and the lighter fraction at 1.15, a density commonly associated with microperoxisomes. These results support our ultrastructural and cytochemical data and suggest that the diverse functions of regionally specialized midgut epithelial cells lead to a heterogeneous population of peroxisomal organelles. ACO activity confirms the role of midgut peroxisomes in the intermediary metabolism of lipids and the increasing CAT activity suggests that the midgut epithelium may metabolize deleterious pro-oxidants of aerobic metabolism associated with foraging and senescence. © 1996 Wiley-Liss, Inc.

Peroxisomes and peroxisomal enzymes in the human fetal small intestine.

The appearance and development of peroxisomes and the expression of their enzymes in the human fetal intestine have been investigated between 11 and 22 weeks of gestation. In the youngest samples (11-16 weeks of age), cytochemistry at the ultrastructural level revealed the presence of rare, mostly circular peroxisomes. From 16 weeks of gestation onwards, an increase was noted in the number of peroxisomes. Two peroxisomal types were distinguished: round to oval forms and elongated and/or tailed organelles. Biochemical assays revealed that total and specific intestinal catalase activities increased gradually between 11 and 20 weeks of gestation. The activity of fatty acylCoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was detectable as early as 11 weeks of gestation. Thereafter, total and specific activities of the enzyme increased steadily. Activities of other peroxisomal oxidases (D-amino acid oxidase, L-alpha-hydroxyacid oxidase) appeared more slowly in the fetal intestine during the period studied. This investigation establishes the presence and the morphological changes that occur in intestinal peroxisomes during human fetal development as well as the developmental patterns of associated enzymes.

Hydrogen peroxide metabolism during peroxisome proliferation by fenofibrate

Fenofibrate, the hypolipidemic drug and peroxisome proliferator, was given to mice (0.23% w/w in the diet) during 1–3 weeks and enzyme activities, H 2O 2 concentration, and H 2O 2 production rate were determined. A maximal increase of 150% in liver/body weight ratio was observed after 3 weeks of treatment. Acyl-CoA oxidase, catalase and uricase activities were increased by 712%, 506% and 41% respectively by treatment with fenofibrate. Se- and non Se-glutathione peroxidase and Mn-superoxide dismutase activities were increased by 331 %, 188% and 130% respectively in the liver of 2 weeks-treated mice. CuZn superoxide dismutase activity was not affected by fenofibrate treatment. H 2O 2 steady-state concentration showed an increase of 89% after 2 weeks of treatment. H 2O 2 production rates, and the steady-state concentrations of the intermediates HO , R and ROO , calculated using experimental data, were higher in the liver of fenofibrate-treated mice than in control animals. According to our findings, the imbalance between H 2O 2 production and its degradation by its metabolizing enzymes during peroxisome proliferation, would result in an increased level of H 2O 2 steady-state concentration, with the resulting oxidative stress which may lead to the generation of oxidative damage and to the induction of liver carcinogenesis.

Gemfibrozil-induced peroxisome proliferation and hepatomegaly in male F344 rats.

Gemfibrozil is a widely used hypolipidemic drug in humans that causes peroxisome proliferation and hepatocarcinogenesis in rodents. The induction of hepatomegaly and hepatic peroxisome proliferation (measured as peroxisomal acyl CoA oxidase activity), was determined and compared to another peroxisome proliferator, WY-14,643 (0.1% in the diet) in male F344 rats. In a 21-day study, dietary no-observable-effect and lowest-observable-effect levels of gemfibrozil for both hepatomegaly and peroxisome proliferation were 0.002% and 0.005%, respectively. In a 42-day study, dietary concentrations of 0.9-2.0% gemfibrozil induced a similar magnitude of hepatomegaly to WY-14,643 (2.3-fold) but a higher level of peroxisome proliferation (16-18-fold) than the maximum induction for WY-14,643 (13-fold). The plateau in magnitude of gemfibrozil-induced peroxisome proliferation across the 0.9-2.0% dietary concentrations was associated with a plateau in serum concentration of gemfibrozil (approximately 20 micrograms/ml), similar to concentrations reported in human subjects receiving oral gemfibrozil. These results indicate that maximal induction of peroxisome proliferation by gemfibrozil can exceed that of a more potent compound such as WY-14,643, and further suggest that maximal induction of peroxisome proliferation can be limited by steady-state serum concentrations. Moreover, the reported lack of hepatic responses to gemfibrozil in humans is unlikely to be the result of inefficacy or unavailability of this drug, compared to other peroxisome proliferators, in rodents.

Synergistic induction of acyl-CoA oxidase activity, an indicator of peroxisome proliferation, by arachidonic acid and retinoic acid in Morris hepatoma 7800C1 cells

Morris hepatoma 7800C1 cells (a Wistar rat cell line) were exposed to 100 μM arachidonic acid in the medium for seven days. This treatment resulted in 150% and 60% increases (above control activities) in acyl-CoA oxidase (which catalyzes the first step in peroxisomal β-oxidation) and catalase activities, respectively. Arachidonic acid (C20:4) can be metabolized to 20- and 19-hydroxyarachidonic acid by cytochrome P-450IVA and it was shown that our cells are capable of forming 20-hydroxyarachidonic acid. However, 20-hydroxyarachidonic acid (0.l–0.8 μM, 4 days) had no effects on lauroyl-CoA oxidase and catalase activities in Morris hepatoma cells. Treatment of 7800C1 cells with 100 μM all- trans-retinoic acid resulted in inductions of catalase (160% above the control activity) and carnitine acetyltransferase (140% above the control activity) activities. The activity of lauroyl-CoA oxidase was often, but not always, slightly induced by treatment with all- trans-retinoic acid. When all- trans-retinoic acid was administered together with arachidonic acid, these two compounds had a synergistic effect on the induction of acyl-CoA oxidase activity (almost 700% above the control activity). However, treatment of Morris hepatoma cells with the man-made peroxisome proliferator, perfluorooctanoic acid, together with all- trans-retinoic acid did not result in any synergistic effect on this same enzyme activity. In summary, this study (1) corroborates findings from transfection experiments indicating that the heterodimer PPAR-RXR α activates transcription of the acyl-CoA oxidase gene using the Morris hepatoma cell line; (2) shows that arachidonic acid induces the activity of lauroyl-CoA oxidase; (3) suggests that transcription of the catalase gene is not regulated by a PPAR-RXR α heterodimer in this system; and (4) demonstrates that peroxisome proliferation in Morris hepatoma cells by perfluorooctanoic acid is not as dependent on the level of retinoic caused by arachidonic acid.

Regulation of acyl-CoA oxidases in maize seedlings

Because of the importance of acyl-CoA oxidases in controlling peroxisomal β-oxidation in higher plants, it is necessary to determine their activities and the factors regulating their expression. Acyl-CoA oxidase activities were determined in extracts from different tissues of dark-grown maize plantlets, and in peroxisomes isolated from normal, carbohydrate-starved and carbohydrate-fed maize root tips. Acyl-CoA oxidases exhibited differential and coordinated expression depending on tissue type, tissue development and cellular carbohydrate status.

Peroxisome Proliferation and Microsomal Enzyme Induction by the Hypolipidemic CI-924 in Rats and Mice: Relationship to Tumorgenicity

Peroxisome Proliferation and Microsomal Enzyme Induction by the Hypolipidemic CI-924 in Rats and Mice: Relationship to Tumorgenicity. Hofstra, A. H., King, L. M., and Walker, R. M. (1995). Fundam. Appl. Toxicol. 27, 277-286. CI-924 (5,5′-[[1,1′-biphenyl]-2,5-diylbis(oxy)]bis[2,2-dimethylpentanoic acid]), a lipid lowering agent, was previously shown to be hepatotumorigenic in male and female B6C3F1 mice but not in male and female albino Wistar rats. To determine if the difference between the species in tumorigenic response correlated with the extent of peroxisome proliferation or microsomal changes the effects of CI-924 on liver were characterized in rats and mice. CI-924 doses of 0, 5, 25, 75, and 150 mg/kg were administered in the diet for 4 weeks to B6C3F1 mice and albino Wistar rats. Peroxisomal β-oxidation activity was significantly increased in all groups at doses of 25 mg/kg or higher and was induced up to 25 times in male rats. Peroxisomal carnitine acyltransferase and acyl-CoA oxidase activities were also increased, with the greatest induction observed in male rats. Catalase activity quadrupled in rats and doubled in mice. Serum liver enzyme activities were unchanged with the exception of 5′-nucleotidase which was elevated in mice and decreased in male, but not female, rats. Glutathione S-transferase decreased in the males of both species and glutathione peroxidase increased in the mice. Cytochrome P450 4A1 increased in both species at doses of 25 mg/kg or greater and correlated with increased lauric acid hydroxylation. The high degree of peroxisome proliferation in male rats was unexpected in light of the lack of tumorgenicity demonstrated in a previous 2-year study and these results indicate that early peroxisome proliferation alone is not always a good predictor of hepatocarcinogenicity.

Tetradecylthioacetic acid reduces the amount of lipid droplets, induces megamitochondria formation and increases the fatty acid oxidation in rat heart

The effects of prolonged administration (3 months) of a 3-thia fatty acid analogue and omega-3-fatty acids on cardiac fatty acid oxidation and the volume fraction of lipid droplets and mitochondria in cardiomyocytes were investigated. Doses were 1 g/day/kg body weight, except 150 mg/day/kg body weight for tetradecylthioacetic acid (a 3-thia fatty acid). One group served as control and did not receive any treatment. The volume fraction of lipid droplets in cardiomyocytes was significantly lower in the tetradecylthioacetic acid group compared to the other groups. Mitochondrial β-oxidation was 60% greater and fatty acyl-CoA oxidase activity was increased by 430% in the tetradecylthioacetic acid group compared to control. This was accompanied by a greater volume fraction of mitochondria in cardiomyocytes (0.514±0.032% in tetradecylthioacetic acid v 0.318±0.007% in control) which was due to an increased size of mitochondria. The volume fraction of mitochondria was also greater in eicosapentaenoic acid (EPA) treated rats compared to control, but the enzymic activities were unaffected. Docosahexaenoic acid (DHA) treatment resulted in a greater volume fraction of lipid droplets in the cardiomyocytes, but the volume fraction of mitochondria and enzymic activities were unaltered. These results indicate that EPA and DHA have different effects on the modulation of mitochondrial biogenesis. Tetradecylthioacetic acid treatment results in megamitochondria formation and increased peroxisomal and mitochondrial β-oxidation with a concomitant reduction of lipid droplets in the cardiomyocytes.

Studies on the mode of action of the herbicidal effect of 2,4,5-trichlorophenoxyacetic acid on germinating Norway spruce

A dramatic effect of 2,4,5-trichlorophenoxyacetic acid on the growth of germinating plants was observed. This effect was expressed as abnormal growth. A pronounced increase in ethylene formation (10-fold) was observed in dark-grown plants treated with 200 μM 2,4,5-trichlorophenoxyacetic acid. In a crude peroxisomal fraction prepared from cotyledons of seedlings treated with 200 μM trichlorophenoxyacetic acid, large increases in fatty acyl-CoA oxidase (6.9-fold) and catalase (2.7-fold) activities were observed. A reduced activity of photosystem 11 was observed in chloroplasts from cotyledons of seedlings treated with 2,4,5-trichlorophenoxyacetic acid. In the dark-grown seedlings, this activity decreased to 35% of control. An inhibitory effect of 2,4,5-trichlorophenoxyacetic acid on photosystem II was also found in in vitro experiments with chloroplasts isolated from cotyledons of untreated plants. An increase in lipid peroxidation in chloroplasts treated with 200 μM 2,4,5-trichlorophenoxyacetic acid was also observed. Electron microscope studies on germinating Norway spruce seedlings revealed a pronounced degenerative effect of 2,4,5-trichlorophenoxyacetic acid on chloroplasts, expressed as swollen chloroplats and disorganized orientation of thylakoid membranes. Vesicles were often present in the stroma and the thylakoid. In some chloroplasts, a complete disintegration of compartments was observed. The present study suggests that the principal mode of action of 2,4,5-trichlorophenoxyacetic acid on Norway spruce seedlings is via damage of the thylakoid membrane and inhibition of photosystem II. Additionally, our results support a possible role of increased oxidative stress as an additional mode of action of 2,45-trichlorophenoxyacetic acid.

Eicosapentaenoic acid causes transient accumulation of lipids in rat myocardium

Rats were given eicosapentaenoic acid (EPA) or palmitic acid (PALM) up to 15 days, and control animals were given carboxymethyl-cellulose. All suspensions which were given by gastric intubation contained tocopherol. Heart triacylglycerols, heart cholesterol and heart phospholipids significantly increased after one day of EPA treatment, but they were normalized within 15 days. Both after 2 and 10 days of treatment with palmitic acid the heart triacylglycerols were significantly greater than control. The heart cholesterol and heart phospholipids were significantly greater than control after ]0 days of treatment with palmitic acid. Total camitine palmitoyltransferase (CPT) activity in heart was significantly greater in rats treated with EPA for 15 days compared to control, but treatment with palmitic acid had no effect. The fatty acyl-CoA oxidase activity was greater in rats treated with EPA for 15 days and palmitic acid for 10 days compared to control. The fractional volume of lipid droplets in myocardial cells was calculated from electronmicrographs and was 0.112 ± 0.016% after 1 day of EPA treatment compared to 0.035 ± 0.016% in the control group. After 5 and 15 days the fractional volume was the same as control. The fractional volume of lipid droplets in rats treated with palmitic acid for 10 days was 0.120 ± 0.023%. Treatment with EPA caused an immediate accumulation of lipids and lipid droplets in the rat heart which after few days normalized in parallel with an increased activity of total CPT in the heart.

The Herbicide Dicamba (2-Methoxy-3,6-dichlorobenzoic Acid) Is a Peroxisome Proliferator in Rats

The Herbicide Dicamba (2-Methoxy-3,6-dichlorobenzoic Acid) Is a Peroxisome Proliferator in Rats. Espandiari, P., Thomas, V, A., Glauert, H. P., O'Brien, M., Noonan, D., and Robertson, L. W. (1995). Fundam. Appl. Toxicol. 26, 85-90.The widely used broad leaf herbicide, dicamba, or Banvel, is similar in structure to xenobiotics which induce hepatic drug metabolism or proliferation of hepatic peroxisomes in rodents. The ability of xenobiotics to effect these hepatic changes often portends their positive outcomes in chronic bioassays. Dicamba's ability to induce hepatomegaly and peroxisome proliferation was studied in male and female Sprague-Dawley rats. Rats were placed on feed containing 0, 0.001, 0.01, 0.1, or 1% dicamba or 0.01% ciprofibrate for 3 weeks. Dicamba had no effect on relative liver weights or feed efficiency in either female or male rats at all doses tested. Dicamba, however, caused a statistically significant increase in peroxisomal β-oxidation activity in liver homogenates from rats of both sexes fed 1% dicamba. Fatty acyl CoA-oxidase activity was increased in male rats fed 1% dicamba. A protein of Mr 80 kDa was visible when liver homogenates of female or male rats fed 1% dicamba were subjected to SDS-PAGE. Lauric acid hydroxylase activity and CYP4A-reactive protein were increased in microsomes from male rats fed the highest level of dicamba. Moreover, dicamba was observed to transcriptionally upregulate the peroxisome proliferator-activated receptor (PPAR), a peroxisome prolifexator sensitive receptor previously shown to be linked to the transcriptional regulation of a variety of peroxisome-specific enzymes. These studies show that dicamba is a peroxisome proliferator in rats. Although dicamba was not an efficacious inducer of peroxisomal enzymes in these rats, dicamba's ability to transcriptionally activate the PPAR and induce peroxisomal and related enzymes must be considered in the safety evaluation of this herbicide.

Suppression of clofibrate-induction of peroxisomal and microsomal fatty acid-oxidizing enzymes by growth hormone and thyroid hormone in primary cultures of rat hepatocytes

Using primary cultures of rat hepatocytes on a matri-gel, effects of peroxisome proliferator and ω-hydroxydodecanoic acid on cellular levels of acyl-CoA oxidase and CYP4A have been studied to determine the hormonal influence in serum-free media. Peroxisomal acyl-CoA oxidation, microsomal CYP4A content and laurate ω-hydroxylation were increased in rat hepatocytes by the addition of 100 μM clofibrate or Wy14,643 for two days. ω-Hydroxydodecanoic acid (100 μM) also increased peroxisomal acyl-CoA oxidation, but had no clear effect on microsomal CYP4A level and laurate ω-hydroxylation. CYP4A-mediated laurate ω-hydroxylation in hepatocytes was suppressed by the addition of pituitary growth hormone (0.05 mU/ml), but was not altered by the addition of triiodothyronine (30 nM). In contrast, clofibrate-mediated induction of acyl-CoA oxidase activity was decreased by the addition of either one of the hormones in hepatocytes. Suppression by those hormones was also observed with ω-hydroxydodecanoic acid-mediated induction of acyl-CoA oxidase activity. These results indicate the possibility that GH and T 3 exert the suppressive effects on peroxisomal acyl-CoA oxidation through plural mechanisms with and without the alteration of CYP4A levels in livers.

Expression of the FOX1 gene of Saccharomyces cerevisiae is regulated by carbon source, but not by the known glucose repression genes

We have investigated the regulation of expression of the FOX1 gene of Saccharomyces cerevisiae which encodes acyl-CoA oxidase, the first enzyme in the peroxisomal beta oxidation of fatty acids. We have found that the FOX1 steady state mRNA level is repressed by glucose, partially induced by ethanol (but not by raffinose) and fully induced by oleic acid as a carbon source. Glucose repression was observed even if cultures were grown to stationary phase; however, if the glucose supply was limited initially then partial induction of FOX1 mRNA occurred upon growth to high cell density. A variety of mutants are known to affect the glucose repression of many genes, including the FOX3 gene which encodes the thiolase activity in peroxisomal beta oxidation. However, upon examination none of these mutants showed de-repression of FOX1 expression. Similarly we investigated the role of two inducers of genes encoding peroxisomal enzymes (namely SNF1 and ADR1). No evidence was found to suggest that either of these plays a significant role in the induction of FOX1 mRNA levels. These observations indicate that the regulation of FOX1 is under the control of as yet unidentified genes involved in catabolite repression and suggest that the regulatory circuit influencing acyl CoA oxidase activity, and hence beta oxidation and peroxisome function, is significantly different than that which might have been assumed from other studies.

Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to mitochondrial alterations.

Recent studies have indicated a lack of correlation between hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and the carcinogenicity of peroxisome proliferators (PP) and suggested that DNA in intact hepatic nuclei may be insensitive to increases in 8-OHdG resulting from PP exposure. The possibility that PP-induced elevations in acyl CoA oxidase (ACO) activity might result in oxidative damage to mitochondrial DNA (mtDNA) was therefore investigated by feeding male F344 rats the hepatocarcinogenic PP Wy-14,643 (Wy, 0.1% in the diet) for 3, 6, 11, or 22 weeks, or clofibric acid (CA, 0.5% in the diet) for 22 weeks. Following the respective PP exposures, hepatic peroxisomal acyl CoA oxidase activity was determined and DNA isolated from either mitochondria or unfractionated liver homogenates and analysed for the presence of 8-OHdG. PP treatment caused an increase in ACO activity (10- to 15-fold) at all time points examined and an increase of 8-OHdG (1.5- to 2-fold) in DNA isolated from unfractionated liver homogenates following PP treatment for 11 or 22 weeks. No increase of 8-OHdG in mtDNA was detected. However, quantitation of a PCR amplified region from the D-loop of mtDNA demonstrated a 2- to 3-fold increase in the relative amount of mtDNA in DNA isolated from unfractionated liver homogenates following 3, 11, and 22 weeks exposure to Wy or CA (22 weeks only). In addition, a slight increase in the mitochondrial volume density (1.4-fold) was observed in electron micrographs of liver samples from rats exposed to Wy for 22 weeks. These results (i) demonstrate that PP treatment, at levels which cause an increase in ACO activity, does not cause oxidative damage to mtDNA, and (ii) suggest that one reason for the observed increase of 8-OHdG in DNA from unfractionated liver homogenates may be an increase in the amount of mtDNA present in these samples. Furthermore, these studies provide additional evidence against a role of oxidative DNA damage, measured as 8-OHdG, in PP-induced rodent hepatocarcinogenesis and suggest that alterations in mitochondria or other effects may be more pertinent to PP-related carcinogenesis.

Fatty acyl-CoA oxidase activity is induced before long-chain acyl-CoA hydrolase activity and acyl-CoA binding protein in liver of rat treated with peroxisome proliferating 3-thia fatty acids.

1. In this study we explored the relationship between specific acyl-CoA esters and induction of acyl-CoA binding protein (ACBP) and enzymes related to the proliferation of peroxisomes. Male Wistar rats were administered a single dose (150 mg/day/kg) of sulphur-substituted fatty acid analogues, and the effects of tetradecylthioacetic acid and 3-thiadicarboxylic acid, which both act as peroxisome proliferators, were compared with the effects of tetradecylthiopropionic acid and palmitic acid which do not induce peroxisome proliferation. 2. The hepatic level of total long-chain acyl-CoA was significantly increased within 12 h of feeding these fatty acids, except in rat fed tetradecylthioacetic acid. Hplc chromatograms of liver extracts prepared from rat fed tetradecylthioacetic acid showed that tetradecylthioacetyl-CoA ester accumulated in the liver 4 h after feeding and had disappeared after 24 h. In liver extracts of the tetradecylthiopropionic acid-treated rat tetradecylthiopropionyl-CoA was not observed, but the appearance of a new long-chain acyl-CoA ester, probably a metabolite of tetradecylthiopropionic acid, was detected. This new peak reached a maximum 4h after feeding. In rat fed tetradecylthioacetic acid and 3-thiadicarboxylic acid the hepatic level of fatty acyl-CoA oxidase mRNA increased 8 h after feeding, while the acyl-CoA oxidase activity had increased after 12 h. 3. The early accumulation of specific tetradecylthioacetyl-CoA suggests that this ester may be a possible mediator of the induction of fatty acyl-CoA oxidase. The level of hepatic acyl-CoA binding protein, long-chain acyl-CoA hydrolase activity and long-chain acyl-CoA synthetase activity did not change after a single dose of all four fatty acids. Prolonged administration of 3-thia fatty acids resulted, however, in a dose- and time-dependent increase in hepatic ACBP content and ACBP mRNA level. The amount of ACBP increased in parallel to the long-chain acyl-CoA hydrolase activity. The correlated induction of fatty acyl-CoA binding protein and long-chain acyl-CoA hydrolase seems to be dependent on a sustained accumulation of total long-chain acyl-CoA esters.

The peroxisomal β-oxidation enzyme system of rat heart. Basal level and effect of the peroxisome proliferator clofibrate

Peroxisomes, isolated from homogenates of rat hearts (myocard), contain a β-oxidation enzyme system which is indistinguishable from that found in liver, but the total capacity of β-oxidation is only 0.8% of the liver value (expressed per g of tissue). Fatty acyl-CoA oxidase was assayed by an H 2O 2 based fluorescent assay avoiding important interfering side reactions. The presence of polypeptides with electrophoretic and immunological properties similar to the β-oxidation enzymes of liver peroxisomes, was demonstrated by immuno-blotting using polyclonal antibodies. The level of 72 and 52 kDa subunits of fatty acyl-CoA oxidase (FAO), quantitated by an anti-FAO 1–16 peptide antibody, was only 1% of the level in liver (expressed per g of tissue). Immunoblots of one-dimensional (1-D) SDS-PAGE of rat heart and liver peroxisomal fractions revealed a 60 kDa subunit of the fatty acyl-CoA oxidase in addition to the known 72 and 52 kDa subunits. Immunoblots of two-dimensional (2-D) IEF/SDS-PAGE revealed that all subunits are strongly basic polypeptides, with a microheterogeneity, which probably represents deamidations of the polypeptides. The 2-D immunoblot also revealed another group of polypeptides with M r 72 kDa of less basic isoelectric point, possibly representing an isoform of fatty acyl-CoA oxidase. Substrate specificity studies revealed the highest V max values with C 10–C 12. For the very long-chain fatty acids C 20–C 24, the monoenes revealed much higher V max values than the saturated fatty acids. Administration of the classical peroxisome proliferator clofibrate resulted in a similar increase in the fatty acyl-CoA oxidase activity and the 72 and 52 kDa subunits of FAO in the heart. The response (activity) was found to change from 2.2-fold increase in young (34 days) to 11.1-fold increase in adult (76 days) rats. In contrast to liver, where the ratio of the increase in FAO mRNA to the increase in FAO activity was about 4, this ratio in heart was about 0.5.

Studies on peroxisomes of colonic mucosa in Crohn's disease

The etiology and pathogenesis of Crohn's disease, a chronic inflammatory bowel pathology, have not been elucidated yet. In particular, the behavior of peroxisomes in inflamed colonic mucosa has not been investigated despite their important role in cellular oxidative metabolism. Using cytochemistry at the ultrastructural level, we have observed these catalase-positive organelles. In addition, biochemical analyses have revealed the specific activities of catalase and cyanide-insensitive acyl-CoA oxidase. Mucosal biopsy specimens from inflamed and noninflamed areas of Crohn's patients were compared to control biopsies. We found that Crohn's disease was marked by an important diminution in the peroxisomal frequency per cell unit area. If catalase activity was not affected by this pathology, cyanide-insensitive acyl-CoA oxidase, an enzyme of the peroxisomal beta-oxidation system, was found diminished in inflamed and in noninflamed areas. In conclusion, our results showed that Crohn's disease is accompanied by peroxisomal modifications but the number and the enzyme activities of colonic peroxisomes are less deeply altered in Crohn's disease than during neoplasia. This fact suggests that a relation may exist between the degree of peroxisomal deficiency and the clinical severity of colonic disease.

Evaluation of a rodent peroxisome proliferator in two species of freshwater fish: Rainbow trout ( Onchorynchus mykiss) and Japanese medaka ( Oryzias latipes)

Rainbow trout ( Onchorynchus mykiss) and Japanese medaka ( Oryzias latipes) were exposed to the hypolipidemic drug gemfibrozil, a known rodent peroxisome proliferator. Trout were injected (i.p.) daily for 2 weeks at doses of 0, 46, 87, or 152 mg/kg/day. Medaka were exposed to the nominal concentrations of 0, 1.25, 2.5, or 5 ppm in water for 2 weeks in a static-renewal system. Peroxisome proliferation was assessed by measuring fatty acyl-CoA oxidase (FAO) activity, peroxisomal bifunctional enzyme (PBE) quantity, and changes in liver-to-body weight ratios (LWR). Results indicate that a mild peroxisome proliferative response was observed in rainbow trout (significant increases in FAO activity at all dose levels and in LWR at the highest dose level). Medaka demonstrated a significant increase in PBE at the highest dose level, while nonsignificant increases in FAO activity were observed at the mid- and high-dose levels.

Mitochondrial 2,4-dienoyl-CoA reductases in the rat: differential responses to clofibrate treatment

The effect of a peroxisome proliferator, clofibrate, on mitochondrial 2,4-dienoyl-CoA reductases was studied in rat liver. The specific activity of reductase measured with 2,4-hexadienoyl-CoA as the substrate increased 2.9-fold in the liver homogenate and 2.5-fold in the mitochondrial extract, whereas acyl-CoA oxidase activity increased 13-fold and delta 3, delta 2-enoyl-CoA isomerase activity, 25-fold in the homogenate. Chromatography of the rat liver homogenate on hydroxylapatite, which separates the mitochondrial isoforms (M(r) 120,000 and M(r) 60,000) showed that the M(r) 60,000 isoform increased 3.5-fold and the M(r) 120,000 isoform 6-fold. When the isoforms were assayed with 2,4-hexadienoyl-CoA and trans-2,cis-4,7,10,13,16,19-docosaheptaenoyl-CoA, the activity ratios of C6 to C22 were 1.5-2.1 for the both isoforms isolated from livers of either control or clofibrate-treated rats. A quantitative immunological experiment with the antibody for the 120,000 reductase in the mitochondrial extracts showed a 6.9-fold increase in the signal, confirming the observation that this isoform is induced more than the other. The mRNA levels of reductase, isomerase, and peroxisomal multifunctional enzyme (MFE) were found to rise in a parallel manner when analyzed by in situ or slot hybridizations, which suggests that the increase was mediated by the same mechanism. Peroxisome proliferators have been shown to increase the mRNA levels of MFE by inducing peroxisome proliferator-activated receptor (PPAR)-mediated expression of the corresponding gene. The short-chain delta 3, delta 2-enoyl-CoA isomerase and the M(r) 120,000 reductase are exceptions among the mitochondrial beta-oxidation proteins, which usually show only a minor response to peroxisome proliferators.