Turnover of enzymes of peroxisomal β-oxidation in rat liver

Male Wistar rats were given a diet containing 2% (w/w) di-(2-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal β-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal β-oxidation was 5–7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the β-oxidation system and of catalase decreased to the control levels with a half-life of 2–3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.     

Induction of peroxisomal Lon protease in rat liver after di-(2-ethylhexyl)phthalate treatment

Previously, we identified a peroxisome-specific isoform of Lon protease using subcellular proteomics. In the present study, we investigated changes in the level of the Lon protease in peroxisomes during recovery from peroxisomal proliferation induced by di-(2-ethylhexyl)phthalate (DEHP) to elucidate the function of peroxisomal Lon protease (PSLP). Following a 2-week treatment with DEHP, the level of PSLP was monitored for 15 days. The amount of protease was greatly increased after the 2-week treatment, followed by a further increase 3 days after cessation of the treatment. Afterward, it decreased and reached the control level on day 15. On the other hand, level peroxisomal β-oxidation enzymes induced to express by DEHP started to decrease soon after discontinuation of treatment. The results suggest that PSLP functions to degrade β-oxidation enzymes induced by DEHP during recovery from perxisomal proliferation.     

Benzoapyrene-induced DNA damage in Mytilus galloprovincialis: measurement of bulky DNA adducts and DNA oxidative damage in terms of 8-oxo-7,8-dihydro-2´-deoxyguanosine formation

Bulky DNA adducts and 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) were measured in gill DNA of benzo[a]pyrene (B[a]P)-exposed mussels (50 mg kg^-1 dw day^-1), respectively by the ³²P-post-labelling technique and high performance liquid chromatography coupled to electrochemical detection assay. A time-course study was performed for both biomarkers and their potential use for marine biomonitoring discussed for the sentinel species studied. In gills, B[a]P-related DNA adducts were positively correlated with B[a     

Peroxisomal oxidases and catalase in liver and kidney homogenates of normal and di(ethylhexyl)phthalate-fed rats.

1. Activities of peroxisomal oxidases and catalase were assayed at neutral and alkaline pH in liver and kidney homogenates from male rats fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. 2. All enzyme activities were higher at alkaline than at neutral pH in both groups. 3. The effect of the DEHP-diet on the peroxisomal enzymes was different in kidney and liver. Acyl-CoA oxidase activity was raised three- and sixfold in kidney and liver homogenates, respectively. The activity of D-amino acid oxidase decrease in liver, but increased in kidney homogenates. In liver homogenates, urate oxidase activity was not affected by the DEHP diet. The catalase activity was twofold induced in liver, but not in kidney. 4. The differences suggest that the changes of peroxisomal enzyme activities by DEHP treatment are not directly related to peroxisome proliferation. 5. DEHP treatment caused a marked increase of total and peroxisomal fatty acid oxidation in rat liver homogenates. 6. In the control group the rate of peroxisomal fatty acid oxidation was higher at alkaline pH than at neutral pH. 7. This rate was equal at both pH values in the DEHP-fed group, in contrast to the acyl-CoA oxidase activity. These results indicate that after DEHP treatment other parameters than acyl-CoA oxidase activity become limiting for peroxisomal beta-oxidation.     

Cd (II) stress response during the growth of Aspergillus niger B 77

Aims:  To investigate the relationship between growth, heavy metal ions uptake and participation of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the protection of Apergillus niger B 77 against cadmium stress.
Methods and Results:  The stress response of the model fungal strain, under conditions of a wide range of Cd (II) ion concentrations, was investigated by determining the biomass formation, protein biosynthesis, SOD and CAT activities and heavy metal uptake in growing cells. Exposure to heavy metal ions induced an increase in protein content, heavy metal uptake and SOD activity, and a heavy decrease in CAT activity.
Conclusion:  The results obtained indicated that the tolerance of A. niger to Cd (II) was correlated with the heavy metal uptake, reactive oxygen species generation in the cells and the efficiency of antioxidative defence system.
Significance and Impact of the Study:  Evidence is provided for the possibility that oxidative stress plays a major role in the effect of Cd (II) ions on A. niger . These data could offer useful information when creating new strategies and methodological improvements for bioremediation with the participation of fungi.     

Lysosomal enlargement in digestive cells of mussels exposed to cadmium, benzo[a]pyrene and their combination

Digestive cell lysosomes in mussels are known to respond to individual organic chemicals and metals after experimental exposure under laboratory conditions but reports dealing with the response to mixtures of pollutants are scarce. The aim of the present investigation was to compare the lysosomal responses elicited by exposure to a model organic chemical compound (benzo(a)pyrene, B[a]P), a model toxic metal (Cd) and their combination (B[a]P+Cd) under controlled laboratory conditions. Dimethylsulfoxide (DMSO) was used as vehicle to dissolve organic chemicals into seawater. Control mussels were either kept untreated in clean seawater or treated with DMSO. Digestive glands were excised on Day 21. beta-Glucuronidase activity was demonstrated in 8 mum cryotome sections. Lysosomal volume, surface and numerical densities (Vv, Sv and Nv), and surface-to-volume ratio (S/V) were quantified by image analysis. Lysosomal enlargement was evident in digestive cells of mussels exposed to either Cd, B[a]P or B[a]P+Cd. Such enlargement was more marked after exposure to B[a]P+Cd than to B[a]P, but did not reach the levels recorded after Cd exposure. It seems therefore that the presence of B[a]P reduced to some extent the effects of Cd on digestive cell lysosomes in mussels.     

Differential induction of peroxisomal populations in subcellular fractions of rat liver

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of 'small peroxisomes', containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.     

Overexpression of peroxisome proliferator-activated receptor-alpha (PPARalpha)-regulated genes in liver in the absence of peroxisome proliferation in mice deficient in both L- and D-forms of enoyl-CoA hydratase/dehydrogenase enzymes of peroxisomal beta-oxidation system

Peroxisomal beta-oxidation system consists of peroxisome proliferator-activated receptor alpha (PPARalpha)-inducible pathway capable of catalyzing straight-chain acyl-CoAs and a second noninducible pathway catalyzing the oxidation of 2-methyl-branched fatty acyl-CoAs. Disruption of the inducible beta-oxidation pathway in mice at the level of fatty acyl-CoA oxidase (AOX), the first and rate-limiting enzyme, results in spontaneous peroxisome proliferation and sustained activation of PPARalpha, leading to the development of liver tumors, whereas disruptions at the level of the second enzyme of this classical pathway or of the noninducible system had no such discernible effects. We now show that mice with complete inactivation of peroxisomal beta-oxidation at the level of the second enzyme, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-PBE) of the inducible pathway and D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase (D-PBE) of the noninducible pathway (L-PBE-/-D-PBE-/-), exhibit severe growth retardation and postnatal mortality with none surviving beyond weaning. L-PBE-/-D-PBE-/- mice that survived exceptionally beyond the age of 3 weeks exhibited overexpression of PPARalpha-regulated genes in liver, despite the absence of morphological evidence of hepatic peroxisome proliferation. These studies establish that peroxisome proliferation in rodent liver is highly correlatable with the induction mostly of the L- and D-PBE genes. We conclude that disruption of peroxisomal fatty acid beta-oxidation at the level of second enzyme in mice leads to the induction of many of the PPARalpha target genes independently of peroxisome proliferation in hepatocytes, raising the possibility that intermediate metabolites of very long-chain fatty acids and peroxisomal beta-oxidation act as ligands for PPARalpha.     

Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.     

The 70-kDa peroxisomal membrane protein (PMP70) an ATP-binding cassette transporter

The 70-kDa peroxisomal membrane protein (PMP70) is one of major components of peroxisomal membranes. In rodents, PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and the induction of peroxisomal fatty acid β-oxidation enzymes. PMP70 is an ATP-binding cassette transporter, identified for the first time in intracellular membranes of eukaryotic cells. The authors' recent studies suggest that PMP70 is synthesized on free polysomes and posttranslationally inserted into peroxisomal membranes, and assembles as dimeric or oligomeric forms on peroxisomal membranes. PMP70 is suggested to be involved in metabolic transport of long-chain acyl-CoA across peroxisomal membranes.     

邻苯二甲酸二乙基己酯对翡翠贻贝(Perna viridis)生化指标的影响

实验条件下,研究邻苯二甲酸二乙基己酯(DEHP)5个浓度组(0、0.38、1.92、9.60和48.00mg.L-1)长时间胁迫下翡翠贻贝(Perna viridis)内脏团和外套膜中抗氧化酶(SOD和CAT)活性和丙二醛(MDA)含量的变化,以及胁迫解除后这些指标的恢复情况。结果表明:在胁迫过程中,翡翠贻贝内脏团SOD活性表现为先显著升高,随后受抑制而逐渐降低(P<0.05),CAT活性则表现为先被抑制后受诱导,15d后恢复到对照组水平,MDA含量呈显著增加的趋势(P<0.05);外套膜中的SOD活性在胁迫初期在低浓度组被抑制,而在高浓度组则被诱导(P<0.05),4d后SOD活性逐渐恢复到对照组水平,各浓度组MDA含量均出现明显的增加(P<0.05);净化阶段,低浓度组(0.38mg.L-1)内脏团SOD活性和CAT活性逐渐恢复到对照组水平,但MDA含量升高;净化7d后,除高浓度组(48.00mg.L-1)外,其余浓度组外套膜中SOD活性均已经恢复到对照组水平,MDA含量也没有出现明显升高的现象。研究表明,DEHP对翡翠贻贝内脏团和外套膜抗氧化防御系统酶具有明显的影响,DEHP诱导引起2种组织内脂质过氧化损伤,并且短期内这种损伤无法消除。     

Peroxisome proliferator-activated receptor alpha-responsive genes induced in the newborn but not prenatal liver of peroxisomal fatty acyl-CoA oxidase null mice.

Mice deficient in fatty acyl-CoA oxidase (AOX(-/-)), the first enzyme of the peroxisomal beta-oxidation system, develop specific morphological and molecular changes in the liver characterized by microvesicular fatty change, increased mitosis, spontaneous peroxisome proliferation, increased mRNA and protein levels of genes regulated by peroxisome proliferator-activated receptor alpha (PPARalpha), and hepatocellular carcinoma. Based on these findings it is proposed that substrates for AOX function as ligands for PPARalpha. In this study we examined the sequential changes in morphology and gene expression in the liver of wild-type and AOX(-/-) mice at Embryonic Day 17.5, and during postnatal development up to 2 months of age. In AOX(-/-) mice high levels of expression of PPARalpha-responsive genes in the liver commenced on the day of birth and persisted throughout the postnatal period. We found no indication of PPARalpha activation in the livers of AOX(-/-) mice at embryonic age E17.5. In AOX(-/-) mice microvesicular fatty change in liver cells was evident at 7 days. At 2 months of age livers showed extensive steatosis and the presence in the periportal areas of clusters of hepatocytes with abundant granular eosinophilic cytoplasm rich in peroxisomes. These results suggest that the biological ligands for PPARalpha vis a vis substrates for AOX either are not functional in fetal liver or do not cross the placental barrier during the fetal development and that postnatally they are likely derived from milk and diet.     

Specific changes in the protein composition of rat liver in response to the peroxisome proliferators ciprofibrate, Wy-14,643 and di-(2-ethylhexyl)phthalate.

The hypolipidaemic agents ciprofibrate and Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) and the phthalate-ester plasticizer di-(2-ethylhexyl)-phthalate (DEHP), like other peroxisome proliferators, produce a significant hepatomegaly and induce the peroxisomal fatty acid beta-oxidation enzyme system together with profound proliferation of peroxisomes in hepatic parenchymal cells. Changes in the profile of liver proteins in rats following induction of peroxisome proliferation by ciprofibrate, Wy-14,643 and DEHP have been analysed by high-resolution two-dimensional gel electrophoresis. The proteins of whole liver homogenates from normal and peroxisome-proliferator-treated rats were separated by two-dimensional gel electrophoresis using isoelectric focusing for acidic proteins and nonequilibrium pH gradient electrophoresis for basic proteins. In the whole liver homogenates, the quantities of six proteins in acidic gels and six proteins in the basic gels increased following induction of peroxisome proliferation. Peroxisome proliferator administration caused a repression of three acidic proteins in the liver homogenates. By the immunoblot method using polyspecific antiserum against soluble peroxisomal proteins and monospecific antiserum against peroxisome proliferation associated Mr 80000 polypeptide (polypeptide PPA-80), the majority of basic proteins induced by these peroxisome proliferators appeared to be peroxisomal proteins. Polypeptide PPA-80 becomes the most abundant protein in the total liver homogenates of peroxisome-proliferator-treated rats. These results indicate that ciprofibrate, DEHP and Wy-14,643 induce marked changes in the profile of specific hepatic proteins and that some of these changes should serve as a baseline to identify a set of gene products that may assist in defining the specific 'peroxisome proliferator domain'.     

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase in rat liver. The effects of di-(2-ethylhexyl)phthalate, a peroxisome proliferator

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase (isomerase) was investigated in rat liver. Livers of di-(2-ethylhexyl)phthalate (DEHP)-treated or untreated rats were perfusion-fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus-lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus-lining cells decreased. The isomerase staining pattern was quite similar to that of long-chain acyl-CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus-lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2-fold whereas the mitochondrial isomerase was enhanced about 5-fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.     

Assessment of atypical DNA intercalating agents in biological and in silico systems

The carcinogenic polycyclic aromatic hydrocarbon (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are widespread environmental pollutants, however their toxicological effects within a mixture is not established. We investigated the influence of diesel exhaust (DE) on B[a]P and DB[a,l]P-induced PAH-DNA adduct formation, metabolic activation, gene expression and 8-oxo-dG adduct levels in human breast epithelial cells (MCF-10A) in culture. Following 24 and 48 h, cells co-exposed to DE plus B[a]P exhibited a significant decrease in PAH-DNA adduct levels, compared with B[a]P alone, as determined by ³³P-postlabeling combined with reversed-phase high performance liquid chromatography (HPLC). Cytochrome P450 (CYP) enzyme activity, as measured by the ethoxyresorufin O-deethylase (EROD) assay and CYP1B1 expression, significantly increased with co-exposure of DE plus DB[a,l]P, compared with DB[a,l]P alone. Aldo keto-reductase (AKR)1C1, AKR1C2, and AKR1C3 expression also significantly increased in cells exposed to DE plus PAH, compared with PAH exposure alone. Cell populations exhibiting 8-oxo-dG adducts significantly increased in response to exposure to B[a]P or DE plus B[a]P for 24 h, compared with vehicle control, as quantified by flow cytometry. These results suggest that complex mixtures may modify the carcinogenic potency of PAH by shifting the metabolic activation pathway from the production of PAH diol-epoxides to AKR pathway-derived metabolites.