In cultured hepatocytes, as in vivo, mono-2-ethylhexyl phthalate (MEHP) and its straight chain analogues mono-n-hexyl phthalate (MnHP) and mono-n-octyl phthalate (MnOP) each cause accumulation of lipid but only MEHP produces significant induction of peroxisomal fatty acid oxidizing enzymes. To elucidate the mechanisms underlying this lipid accumulation we investigated the effects of these phthalates and the drug clofibric acid on fatty acid metabolism in suspensions of isolated hepatocytes. The effects were found to be markedly dependent on the nutritional state of the animals from which the hepatocytes were isolated. In hepatocytes isolated from animals fasted overnight, or animals fed ab libitum but killed at approximately 2.30 p.m., MEHP, MnHP, MnOP and clofibric acid each caused a marked rapid stimulation of fatty acid oxidation and the synthesis of triglycerides in hepatocytes when incubated in Hanks saline. Export of very low density lipoprotein (VLDL) from the cells was either unchanged or somewhat reduced. In contrast, in hepatocytes isolated from rats fed ad libitum but killed at approximately 9.30 a.m. MEHP and clofibric acid did not alter fatty acid oxidation or triglyceride synthesis, while MnOP and MnHP increased triglyceride synthesis but decreased fatty acid oxidation. The effects of fasting were largely abolished by incubations of the cells in a complete tissue culture medium (Liebowitz L-15). The results suggest that MEHP and its straight chain analogues can, either as the free acid or the CoA ester, mimic the action of fatty acids in the allosteric regulation of fatty acid metabolism.

Groups of male Wistar albino rats were administered diets containing sufficient fenofibrate to ensure intakes of either 200, 60 or 13 mg/kg/day or sufficient clofibrate to ensure an intake of 400 mg/kg/day. Four rats from each experimental group and 6 control rats were killed, 3, 7, 14 and 28 days, 8, 12 and 20 weeks and 6, 9, 12 and 18 months after commencement of treatment. At all time points livers were subjected to histological, electron microscopic and biochemical examination, the other major abdominal organs were removed for histological examination. A more extensive necropsy was carried out on rats killed after 12 and 18 months. The major alterations were observed in the liver, although there were also morphological changes in the thyroid, pancreas and kidney after prolonged treatment. The hepatic changes followed a distinct time course. Within 24 h of offering diets containing the compounds to the rats there was accumulation of small droplets of lipid, induction of peroxisomal enzymes and of the specific cytochrome P-450 catalysing ω-hydroxylation of fatty acids and an increase in the number of mitotic figures. More slowly developing changes were loss from the centrilobular zone of fat, glycogen and of glucose 6-phosphatase activity. Here maximal changes were observed after 14 days of treatment. A still more slowly developing change was accumulation of enlarged lipid-loaded lysosomes, which was maximal at 26 weeks, accompanied by the development of lipofuscin bodies. Finally, in animals treated for 12 months or more there was evidence for increasing cell turnover as indicated by an increased number of mitotic figures, more dark cells and induction of serum alanine transaminase. The last 2 groups of changes were not observed in rats treated with 13 mg/kg/day of fenofibrate. In general the degree of change in rats treated with 400 mg/kg/day of clofibrate was similar to those found in rats treated with 60 mg/kg/day of fenofibrate.

Conference Article| June 01 1986 Comparison of the effects of hypolipidaemic agents on isolated hepatocytes SHIRLEY C. PRICE; SHIRLEY C. PRICE 1Roberts Institute of Industrial and Environmental Health and Safety, University of Surrey, Guildford GU2 5XH, Surrey, U.K. Search for other works by this author on: This Site PubMed Google Scholar FIONA E. MITCHELL; FIONA E. MITCHELL * Search for other works by this author on: This Site PubMed Google Scholar RICHARD H. HINTON RICHARD H. HINTON 1Roberts Institute of Industrial and Environmental Health and Safety, University of Surrey, Guildford GU2 5XH, Surrey, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1986) 14 (3): 636–637. https://doi.org/10.1042/bst0140636 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation SHIRLEY C. PRICE, FIONA E. MITCHELL, RICHARD H. HINTON; Comparison of the effects of hypolipidaemic agents on isolated hepatocytes. Biochem Soc Trans 1 June 1986; 14 (3): 636–637. doi: https://doi.org/10.1042/bst0140636 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search © 1986 Biochemical Society1986 Article PDF first page preview Close Modal You do not currently have access to this content.

The effects, over periods from 3 days to 9 months of administration, of diets containing di-2-ethylhexyl phthalate are very similar to those observed in rats administered diets containing hypolipidemic drugs such as clofibrate. Changes occur in a characteristic order commencing with alterations in the distribution of lipid within the liver, quickly followed by proliferation of hepatic peroxisomes and induction of the specialized P-450 isoenzyme(s) catalyzing omega oxidation of fatty acids. There follows a phase of mild liver damage indicated by induction of glucose-6-phosphatase activity and a loss of glycogen, eventually leading to the formation of enlarged lysosomes through autophagy and the accumulation of lipofuscin. Associated changes are found in the kidney and thyroid. The renal changes are limited to the proximal convoluted tubules and are generally similar to changes found in the liver. The effects on the thyroid are more marked. Although the levels of thyroxine in plasma fail to about half normal values, serum triiodothyronine remains close to normal values while the appearance of the thyroid varies, very marked hyperactivity being noted 7 days after commencement of treatment, this is less marked at 14 days, but even after 9 months treatment there is clear cut evidence for hyperactivity with colloid changes which indicate this has persisted for some time. Straight chain analogs of di-2-ethylhexyl phthalate, di-n-hexyl phthalate and di-n-oxtyl phthalate differ entirely in their short-term effects on the liver and kidney but have similar effects on the thyroid. The short-term in vivo hepatic effects of the three phthalate esters can be reproduced in hepatocytes in tissue culture. All three phthalate esters, as well as clofibrate, have early marked effects on the metabolism of fatty acids in isolated hepatocytes. The nature of these changes is such as to increase storage of lipid in the liver. A hypothesis is presented to explain the progress from these initial metabolic effects to the final formation of liver tumors.

This study compares changes in the livers of rats treated with di(2-ethylhexyl) phthalate (DEHP) and its straight-chain analogs di(n-hexyl) phthalate (DnHP) and di(n-octyl phthalate (DnOP). Groups of rats were fed diets containing 20,000 ppm of one of these compounds. Subgroups were killed after 3, 10, and 21 days, and the livers were examined by histological, cytological, and biochemical methods. The results show considerable differences between the effects of the branched-chain phthalate ester DEHP and its straight-chain analogs. The major effects on the liver following administration of diets containing DEHP were midzonal and periportal accumulation of small droplets of lipid, hepatomegaly accompanied by an initial burst of mitosis, proliferation of hepatic peroxisomes and of smooth endoplasmic reticulum accompanied by induction of peroxisomal fatty acid oxidation, damage to the peroxisomal membranes as evidenced by increased leakage of catalase to the cytosol, and centrilobular loss of glycogen and falls in glucose-6-phosphatase activity and in low-molecular-weight reducing agents. In contrast, diets containing DnHP or DnOP induced accumulation of large droplets of fat around central veins leading, by 10 days, to mild centrilobular necrosis and a very slight induction of one peroxisomal enzyme and an increase in liver weight, but no significant changes in any other parameters which were affected by DEHP.

Groups of male and groups of female Wistar albino rats were administered diets containing sufficient di(2-ethylhexyl) phthalate (DEHP) to ensure intakes of either 1000, 200, or 50 mg/kg/day. Four rats from each experimental group and six control rats of the same sex were killed 3, 7, 14, and 28 days and 9 months after commencement of treatment. At all time points the major abdominal organs were removed and subjected to histological examination. A more extensive necropsy was performed on those rats killed after 9 months of treatment. At all time points the livers of the rats were subjected to extensive histologic, electron microscopic, and biochemical examination. Changes could be grouped according to their time course. Two early and transient alterations were noticed. First, there were morphologic changes in the bile canaliculi of male rats treated with 1000 mg/kg/day of DEHP. Second, there was a burst of mitosis immediately after the start of administration of the compound. The time course of this mitotic burst varied; the increase in mitosis was greatest at 3 days in rats treated with 1000 mg/kg/day of DEHP and was smaller but more prolonged in rats treated with 200 or 50 mg/kg/day. Other changes, namely, a midzonal to periportal accumulation of fat, induction of peroxisomal enzymes, and induction of the P-450 isoenzyme also developed rapidly but were sustained throughout the study. The maximal change was usually attained within 7 days of commencement of treatment. More slowly developing changes were hypertrophy of the hepatocytes, centrilobular loss of glycogen, and a fall in glucose-6-phosphatase activity. Here maximal changes were not attained until 28 days after commencement of treatment. These three effects were clearly observed in rats treated with 200 or 1000 mg/kg/day of DEHP but were only marginally altered in rats treated with 50 mg/kg/day. Finally accumulation of lipid-loaded lysosomes assessed by light and electron microscopy and by assay of β-galactosidase activity was only apparent in rats treated with DEHP for 9 months with 200 or 1000 mg/kg/day of DEHP. Changes in female rats were qualitatively similar to those observed in male rats. The alterations were, however, less pronounced than in male rats treated with an equal dose of DEHP and the degree of liver enlargement was much less because, although the initial hyperplasia was clearly apparent, there was a much smaller degree of hypertrophy.

Conference Article| February 01 1985 Effect of phthalate esters on triglyceride synthesis and export in hepatocytes FIONA E. MITCHELL; FIONA E. MITCHELL 1Robens Institute of Industrial and Environmental Health and Safety, University of Surrey, Guildford GU2 5XH, Surrey, U.K. Search for other works by this author on: This Site PubMed Google Scholar RICHARD H. HINTON RICHARD H. HINTON 1Robens Institute of Industrial and Environmental Health and Safety, University of Surrey, Guildford GU2 5XH, Surrey, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1985) 13 (1): 154–155. https://doi.org/10.1042/bst0130154 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation FIONA E. MITCHELL, RICHARD H. HINTON; Effect of phthalate esters on triglyceride synthesis and export in hepatocytes. Biochem Soc Trans 1 February 1985; 13 (1): 154–155. doi: https://doi.org/10.1042/bst0130154 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: MEHP, mono-2-ethylhexylphthalate, MnOP, mono-n-octylphthalate, MnHP, mono-n-hexylphthalate, VLDL, very-low-density lipoprotein This content is only available as a PDF. © 1985 Biochemical Society1985 Article PDF first page preview Close Modal You do not currently have access to this content.

1 Treatment of rats with hypolipidaemic drugs or with the plasticizer di-2-ethyl hexyl phthalate caused significant alterations in the concentration of certain plasma proteins. 2 Certain proteins showed dose-dependent increases, in other cases the plasma concentrations fell in treated animals. 3 The changes were quite distinct from the changes in plasma proteins which occur during the acute-phase response to inflammatory agents. 4 Some changes appeared specific to agents which produce peroxisome proliferation in liver, other alterations appeared associated with mild, but sustained, liver injury.