Changes to the integral membrane protein composition of mouse liver peroxisomes in response to the peroxisome proliferators clofibrate,Wy-14,643 and Di(2-ethyl-hexyl)phthalate

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.     

Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

BACKGROUND: Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. METHODS: Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP), 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls).The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. RESULTS: Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. CONCLUSION: The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.     

Polar metabolites of di-(2-ethylhexyl)phthalate in the rat

Di-(2-ethylhexyl)phthalate (DEHP) is an important industrial chemical widely used as a plasticizer for vinyl and other plastics. DEHP is extensively metabolized by mammals, different species showing dramatic differences in metabolite distributions. Previous studies of the metabolism in rats led to the suggestion that the enzymatic processes normally associated with omega-, omega-1, alpha-, and beta-oxidation of fatty acids could account for the known metabolites of DEHP found in the urine. Several additional metabolites of DEHP have been identified in the present study. Their formation requires that the initial hydroxylation process be less specific than fatty acid omega- and omega-1 oxidation are thought to be. Furthermore, it is necessary to postulate either that the aliphatic chain of mono-(2-ethylhexyl)phthalate can be oxidized at two sites simultaneously, or that oxidation products can be recycled for a second hydroxylation prior to excretion.     

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.     

The different effects of the peroxisome proliferators clofibric acid and bis(2-ethylhexyl)phthalate on the activities of peroxisomal oxidases in rat liver

Treatment with peroxisome proliferators induces increased numbers and alterations in the shape of peroxisomes in liver. It ultimately leads to hepatocellular carcinomas induced by the persistent production of high amounts of H2O2 as a result of a dramatical increase in acyl-CoA oxidase activity. The effects of peroxisome proliferators on other peroxisomal oxidase activities are less well documented. In the present study, the distribution patterns of the activity of SdD-amino acid oxidase, SlD-alpha-hydroxy acid oxidase, polyamine oxidase, urate oxidase and catalase activities were investigated in unfixed cryostat sections of liver, kidney and duodenum of rats treated with either clofibrate or bis(2-ethylhexyl)phthalate. The activities of xanthine oxidoreductase, which produces urate, a potent anti-oxidant, and xanthine oxidase, which produces oxygen radicals, were studied as well. The liver was the only organ that was affected by treatment. The number of peroxisomes increased considerably. SdD-Amino acid oxidase and polyamine oxidase activities were completely abolished by the treatment, whereas SlD-alpha-hydroxy acid oxidase activity decreased and urate oxidase activity increased periportally and decreased pericentrally. Total catalase activity increased because of the larger numbers of peroxisomes, but it decreased per individual peroxisome. Xanthine oxidoreductase activity decreased, whereas the percentage of xanthine oxidase remained constant. We conclude that oxidases in rat liver are affected differentially, indicating that the expression of activity of each oxidase is regulated individually. © 1998 Chapman & Hall     

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.     

Induction of rat liver mitochondrial fatty acid elongation by the administration of peroxisome proliferator di-(2-ethylhexyl)phthalate: absence of elongation activity in peroxisomes

The administration of di-(2-ethylhexyl)phthalate (DEHP)3 to male Sprague-Dawley rats resulted in more than a threefold increase in activity of acetyl CoA-dependent hepatic mitochondrial fatty acid elongation. Peroxisomes obtained either from control or DEHP-treated rats were not capable of elongating any of the fatty acyl CoAs tested. Furthermore, the peroxisomes possessed no trans-2-enoyl CoA reductase activity. Therefore, the elongation activity in the 7500g fraction from both control and DEHP-fed animals can be attributed totally to the mitochondria. Maximal incorporation of acetyl CoA occurred in the presence of both NADH and NADPH, and octanoyl CoA (8:0) and decanoyl CoA (10:0) were found to be optimal primers for fatty acid elongation in both control and DEHP-treated animals. The apparent Km for 8:0 CoA was 17 microM in both animal groups while the Vmax was increased from 4.5 to 12.5 nmol/min/mg following treatment. The apparent Km for 10:0 CoA was 10 microM in both control and DEHP-treated groups while the apparent Vmax increased from 2.5 to 10 nmol/min/mg; palmitoyl-CoA (16:0) was a very poor primer for chain elongation. Although the acetyl CoA-dependent fatty acid elongation was stimulated by DEHP treatment, the mitochondrial trans-2-enoyl CoA reductase activity was unaffected. The mitochondrial total elongation activity following DEHP-treatment using 8:0 CoA as primer was about two times higher than enoyl CoA reductase activity using trans-2-decenoyl CoA (10:1). This was the result of accumulation of intermediates, which were identified as trans-2-10:1 (35%), beta-hydroxy 10:0 (25%), unidentified (15%), and elongated saturated product 10:0 (24%). Elongation by one acetate unit was found in both the control and DEHP-treated animals. The results are discussed in terms of physiological significance.     

In vitro studies of the inhibition of protein kinase C from rat brain by di-(2-ethylhexyl)phthalate

The environmental contaminant di(2-ethylhexyl)phthalate (DEHP) has been shown to inhibit the phosphorylation of histone by purified protein kinase C (PK-C) from rat brain in a concentration-dependent manner. The inhibition does not involve making the substrate unavailable, although DEHP does bind to some extent to histone. DEHP displaces phorbol dibutyrate from PK-C, indicating that DEHP binds to the regulatory domain of the enzyme. Since DEHP does not affect the PK-C dependent phosphorylation of protamine, DEHP probably does not bind at the catalytic site. DEHP non-competitively blocked activation of PK-C by either phosphatidyl serine or calcium ion. Inhibition of histone phosphorylation by DEHP was enhanced if diglyceride was present, and the enhancement was stereoselective for the isomeric form of the diglyceride. The mechanism of the inhibition is thought to involve interference with the interaction between calcium ion and the regulatory domain of PK-C, and would have significance only for those PK-C substrates that require calcium activation of the enzyme. Thus the presence of DEHP in the high nanomolar concentration range alters the effective substrate specificity of PK-C.     

In utero exposure to the antiandrogen di-(2-ethylhexyl) phthalate decreases adrenal aldosterone production in the adult rat

We previously reported that in utero exposure of the male fetus to the plasticizer di-(2-ethylhexyl) phthalate (DEHP) resulted in decreased circulating levels of testosterone in the adult without affecting Leydig cell numbers, luteinizing hormone levels, or steroidogenic enzyme expression. Fetal exposure to DEHP resulted in reduced mineralocorticoid receptor (MR; NR3C2) expression in adult Leydig cells. In the present studies, treatment of pregnant Sprague-Dawley dams from Gestational Day 14 until birth with 20, 50, 100, 300, or 750 mg kg(-1) day(-1) of DEHP resulted in significant sex-specific decreases in serum aldosterone but not corticosterone levels at Postnatal Day 60 (PND60) but not at PND21. There was no effect on circulating levels of potassium, angiotensin II or adrenocorticotropin hormone (ACTH). However, there was reduced expression of AT receptor Agtr1a, Agtr1b, and Agtr2 mRNAs. The mRNA levels of proteins and enzymes implicated in aldosterone biosynthesis were not affected by in utero DEHP treatment except for Cyp11b2, which was decreased at high (≥ 500 mg kg(-1) day(-1)) doses. The data presented herein, together with our previous observation that aldosterone stimulates testosterone production via an MR-mediated mechanism, suggest that in utero exposure to DEHP causes reduction in both adrenal aldosterone synthesis and MR expression in Leydig cells, leading to reduced testosterone production in the adult. Moreover, these results suggest the existence of a DEHP-sensitive adrenal-testis axis regulating androgen formation.     

Peroxisomes in the rat brain and the effects of di-(2-ethylhexyl) phthalate during postnatal development. An electron-microscopic study

Peroxisomes were identified in the neurons of cerebral cortex of 12-day-old suckling rats from normal and plasticizer di-(2-ethylhexyl) phthalate (DEHP)-fed mothers. The study suggests that DEHP passed to the pups through the mother's milk during nursing can induce peroxisomal proliferation in neurons during postnatal development.     

邻苯二甲酸酯在不同品种通菜-土壤系统中的累积效应研究

在邻苯二甲酸(2-乙基己基)酯(DEHP)不同污染程度的水稻土上盆栽不同品种通菜,应用GC／MS联机检测技术研究了通菜-土壤系统中DEHP的环境行为与归宿．结果表明,通菜中DEHP的含量为0．335～12．710mg·kg^-1,与土壤的污染程度呈正相关．不同品种通菜之间对DEHP的吸收累积效应存在显著差异,与其叶子大小存在一定的正相关关系．种植不同品种通菜后土壤中DEHP的残留量(1．424～19．834mg·kg^-1)存在显著差异．通菜对土壤中DEHP的BCFs都小于1．0,与土壤的污染程度呈负相关．不同通菜品种的BCFs之间存在显著差异,中等叶子通菜品种的BCFs相对较大。     

Biochemical alterations in rat fetal liver following in utero exposure to di(2-ethylhexyl)phthalate (DEHP)

Oral administration of DEHP, 1000 mg/kg body weight, to rats daily from 6 to 15 day of gestation resulted in retardation of fetal growth and increase in fetal liver weight which contained significant quantities of DEHP. The activities of mitochondrial succinate dehydrogenase, malate dehydrogenase, cytochrome c oxidase and adenosine triphosphatase were decreased in fetal liver. The data indicate that exposure of mothers to DEHP during pregnancy could adversely affect the fetal livers by interfering with bioenergetics of the cell.     

Effects of early pubertal exposure to di-(2-ethylhexyl) phthalate on social behavior of mice

Di-(2-ethylhexyl) phthalate (DEHP), a main member of phthalates used as plasticizer in PVC plastics, is an environmental endocrine disrupter. The present study investigated the effect of DEHP on social behavior of mice following pubertal exposure (1, 10, 50, and 200 mg/kg/d) from postnatal day 28 through postnatal day 42. The results showed that, in pubertal females, DEHP reduced the time spent in social play and social investigation and inhibited sociability, but a contrary effect was found in pubertal males, suggesting that the effect of DEHP on pubertal social behavior displays sex differences. In adults, DEHP reduced sociability in females and inhibited social play and social investigation in males, suggesting that early pubertal exposure to DEHP not only plays a significant role in puberty but also alters social behavior in adults. In addition, the present study showed that the higher dose of DEHP (50, 200 mg/kg/d) reduced the relative weight of bilateral testis and anogenital distance of pubertal or adult males, suggesting an anti-androgenic activity of DEHP. These results suggest that early pubertal exposure to DEHP sex- and age- specifically affected the social behaviors of pubertal and even adult mice.     

Zonal distribution of peroxisomal 3-oxoacyl-CoA thiolase mRNA in liver from rats treated with di-(2-ethylhexyl)phthalate

Treatment of rats with di-(2-ethylhexyl)phthalate leads to a dramatic increase in peroxisomal 3-oxoacyl-CoA thiolase RNA, the concentration being higher in the pericentral than in periportal hepatocytes. These findings indicate that the production of peroxisomal thiolase and the zonal distribution of the enzyme are regulated at a pretranslational level.     

Effect of in utero exposure to di(2-ethylhexyl)phthalate on rat testes.

In utero exposure to di(2-ethylhexyl)phthalate (DEHP; 1000 mg/kg body weight) significantly decreased activities of testicular sorbitol dehydrogenase and acid phosphatase and increased gamma-glutamyl transpeptidase, lactate dehydrogenase and beta-glucuronidase activities at early ages. A decrease in the sperm count of the epididymal spermatozoa was also observed in the sexually matured animals of DEHP exposed group. The data suggest that in utero exposure to DEHP may affect the normal development of testes.     

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。