Hepatic effects of phthalate esters.

Di(2-ethylhexyl) phthalate (DEHP), a commonly used plasticizer and microchemical environmental pollutant, produces subtle changes in hepatic function as judged by increase in liver weight and morphological and biochemical alterations. It can modify the biological response of drugs and other xenobiotics. Such interactions appear to occur at the pharmacokinetic phase, as DEHP was found to alter the activity of microsomal drug-metabolizing enzymes and ethanol metabolism. DEHP produced a time- and route-dependent effect on the hepatic cytochrome P-450 contents and activity of aminopyrine N-demethylase, aniline hydroxylase, alcohol dehydrogenase and high and low Km aldehyde dehydrogenases when given orally or intraperitoneally. Under in vitro conditions, DEHP produced no effect on the activity of aminopyrine N-demethylase or aniline hydroxylase, while mono(2-ethylhexyl) phthalate (MEHP) and 2-ethylhexanol (2-EH) significantly inhibited their activity at concentrations ranging from 2.5 to 15.0 mM. Activity of aminopyrine N-demethylase and aniline hydroxylase was also inhibited by dimethyl phthalate (DMP) and dibutyl phthalate (DBP) after a single oral administration. In view of the possibility of the human exposure to phthalates and other xenobiotics simultaneously, these observations are of great significance.


Introduction
Phthalic acid esters (PAEs) are commonly used as plasticizers to impart flexibility to plastics, particularly poly(vinyl chloride) (PVC) polymers, which have a wide variety of biomedical and other uses. Di(2-ethylhexyl) phthalate (DEHP) and other PAEs are known to leach out from finished PVC products into blood, physiological fluids, commercial solvents and food materials (1,2). Entry of these plasticizers into the biological system during tranfusions (blood or physiological fluids) or hemodialysis or through food chain and their ubiquitous presence in environment has led to numerous studies on their toxicology. In spite of their low order of toxicity, DEHP and other phthalates have been shown to exert hepatotoxic, cytotoxic, teratogenic and mutagenic effects. Certain renal, pulmonary and reproductive dysfunctions have also been reported on exposure to these plasticizers in aquatic invertebrates and mammalian species (1)(2)(3). Liver and testis appear to be the main target organs in phthalate toxicity.
It is well established that the biological response of xenobiotics can be altered significantly due to their interactions with variables in external and *Industrial Toxicology Research Centre, P. 0. Box 80, Lucknow-226 001, India. internal environmental and pharmacological agents. Unfortunately, this aspect of phthalate toxicity has not been studied in detail, though such information will be of great significance in assessing the toxicogenic potential of PAEs because of their ubiquity and hepatotoxic nature. This paper therefore reviews some of the hepatic effects of PAEs, with particular emphasis on their interaction with drugs and other xenobiotics.
Effect of Phthalates on Weight, Morphology and Chemical Constituents of Liver Several investigators have studied the hepatotoxic potential of PAEs in a variety of animal species. Acute and chronic administration of DEHP caused variable effects on liver, depending upon the species of the animal (4). In general, phthalates after oral or intraperitoneal administration caused enlargement of the liver (5-9), which declined to or below normal weight on prolonged or discontinued exposure (10) ( Table 1).
Histopathological examination ofphthalate-induced enlarged livers showed fatty vacuolation and congestion followed by a cloudy swelling, excessive fatty degeneration (4,11). Dilatation of smooth and rough endoplasmic reticulum, mitochondrial  Oral Absence of glycogen (12) swelling and increase in microbodies in rat liver have been reported on oral administration of DEHP for 21 days (7). Enlargement of cells, dilatation of smooth endoplasmic reticulum and changes in hepatic lysosomes of ferrets (12) and swollen mitochondria and increased smooth endoplasmic reticulum in liver of mice (13) receiving DEHP orally have also been reported.
Very little information is available on the hepatic effects of phthalates in primates. Jacobson (14) has reported liver necrosis and inflammatory cell inifitrations in rhesus monkeys receiving plasmasolubilized DEHP intravenously. Subtle changes in the clearance time of sulfobromophthalein were also observed ( Table 1).

Effect of Phthalates on Energy and Carbohydrate Metabolism
Associated with liver enlargement, changes (increases or decreases) in many hepatic enzymes have been reported in phthalate-exposed animals ( Table 2). A decrease in succinic dehydrogenase (SDH) activity after oral or intraperitoneal administration of DEHP has been observed consistently (7,15). Histochemical studies have revealed that loss ofthe enzyme occurs specifically in the periportal zones (7).
Mitochondria appear to be the target of phthalates. Adverse effects of phthalates on mitochondrial function have been reported by several investigators. In vitro studies have shown that several PAEs can produce significant inhibition of mitochondrial respiration (16)(17)(18). The studies of Ohyama (16) suggest that phthalates are electron and energy transport inhibitors, while the observations of Inouye et al. (10) indicate that phthalates can also cause uncoupling ofoxidative phosphorylation. Phthalates were found to produce aberrations in mitochondrial function, e.g., di-n-butyl phthalate enhanced K+ efflux from isolated mitochondria, usually not produced by standard uncoupling agents (18). Sensitivity of various components of electron transport chain towards phthalates was indicated under in vivo conditions as well as by decreased activities of mitochondrial cytochrome-c oxidase, malic dehydrogenase and diaphorase in liver of DEHP-treated 28 P. K. SETH HEPATOTOXICITY OF PHTHALATES rats (9). The inhibition of these mitochondrial enzymes also indicates an interaction of DEHP with mitochondrial membranes. DEHP was also found to inhibit the activity of total and Mg' -stimulated adenosine triphosphatase (ATPase) activity of rat liver (9,19). Besides liver, the activity of SDH and ATPase was also inhibited in rat heart, lung, kidney (19) and gonads (20), indicating that suppression ofenergy-linked reactions may be a generalized effect of DEHP. In vivo effects of DEHP (9,15,19) were present 11 days after the final treatment with plasticizers. This suggests that enzymic alterations were perhaps not related to the physical presence of DEHP, as by this time all of the plasticizer would have been excreted (6,21,23). Detailed studies have been conducted on the effects of DEHP on carbohydrate metabolism ( Table  2). A decrease in glycogen in livers of mice (13), rats (19) and ferrets (12) receiving DEHP has been reported. Sakurai et al. (24) showed marked depression of glucose and glycogen in livers of rat receiving a diet containing 2% or 4% DEHP. Studies on the rates of incorporation of labeled pyruvate into blood glucose and liver glycogen and alteration in the levels of intermediates of carbohydrate metabolism indicated retardation of gluconeogenesis (24). Recent studies of Mushtaq et al. (25) using cold and labeled glucose have shown that DEHP inhibits both glycogenesis and glycogenolysis in rat liver. These authors have observed a significant decrease in the activity of glucose 6-phosphate dehydrogenase, phosphorylase and glucose 6-phosphatase and no change in the activity of fructose 1,6-diphosphatase and aldolase in livers of DEHPexposed rats (25).

Effect of Phthalates on Lipid and Protein Metabolism
Since the detection of DEHP in association with triglycerides of beef mitochondria (26), its interaction with lipid metabolism has been a subject of active 29 investigation (Table 3). Stein et al. (27) showed an interaction between dietary fat and DEHP in lipid metabolism where presence of the phthalate in diet potentiated the growth-promoting effect of lipids in rats. Substantial reduction in serum cholesterol and proliferation ofperoxisomes and elevation of catalase and carnitine acetyltransferase activity in livers of animals receiving DEHP via diet was reported by Reddy et al. (28). Induction of the fatty acyl-CoA oxidizing enzyme system located in peroxisomes by DEHP has been suggested by Osumi and Hashimoto (29), and inhibition of lipid synthesis in rats has been shown by Bell et al. (30,31). Reduced serum glucose and cholesterol and elevated serum free fatty acid and ketone body have also been reported (26,32,33). The rats receiving 0.5% of DEHP in a normal protein diet showed accumulation of phospholipids, decrease in cholesterol and triglyceride contents in liver and a rise in levels of plasma fatty acids. Such observations suggest the utilization of lipids for the energy source. However, the significance of this altered state of energy metabolism is not understood at present (Table 3).
No significant studies have been conducted on the effects of phthalate on protein metabolism, though their effects on the other proximate principles, carbohydrate and fat have been studied in detail. An increase in the protein content in liver of DEHP-treated rats has been described by Yanagita et al. (32). Recently Pillai et al. (34) have shown that a DEHP-caused elevation in rat liver protein content is due to a decrease in protein breakdown ( Table 3).

Interaction of Phthalates with Other Xenobiotics
Alterations in barbiturate sleeping time on exposure to phthalates have been reported by several investigators. A single intraperitoneal or intravenous injection of DEHP was found to increase the hexobarbital or pentobarbital sleeping time in rats or mice (6,(35)(36)(37). Repeated intraperitoneal ad- ministration of a number of phthalates caused significant and dose-dependent increases in pentobarbital sleeping time in mice (38), while repeated oral administration of DEHP resulted in reduced hexobarbital sleeping time in rats (6). DEHP has also been shown to modify the biological response of parathion, an organophosphorus insecticide (39). Parathion exerts its toxic effect through its metabolite, paraxon, which is a potent inhibitor of acetylcholine esterase enzyme. A lower inhibitory activity of parathion in DEHP-treated rats suggested an effect of the phthalate on biotransformation of the pesticide to its active metabolite (39). Alterations in the biological response of carbon tetrachloride (40) and ethanol (41) have also been described.
The mechanism by which phthalates modify the biological response ofthe above-mentioned xenobiotics is not clear, although some investigators have suggested alterations in the biodistribution and biotransformation of the test xenobiotics or an increase in sensitivity of the central nervous system towards them in the presence of phthalates (6,(36)(37)(38)(39). Estimation of concentration of barbiturates in blood during sleep and at awakening have shown that sleeping time alterations caused by DEHP were related to retarded disposition of the test drugs and not to the increased sensitivity of the central nervous system (36,38). An increase in hepatic cytochrome P-450 content, alcohol dehydrogenase activity and persistent inhibition ofaniline hydroxylase activity in livers of rats receiving DEHP for prolonged periods indicates its interaction with biotransformation mechanism.
In view of the significance of this aspect of phthalate toxicity and variable effects of phthalates on barbiturate and ethanol-induced sleeping time, the effect of DEHP on some enzymes involved in their biotransformation will be discussed in detail.

Effect of DEHP on Enzymes Involved in Biotransformation of Ethanol and Other Xenobiotics
The effect of DEHP on the activity of aminopyrine N-demethylase or aniline hydroxylase is shown in Figure 1. A single oral or intraperitoneal exposure to DEHP caused a significant dose-dependent decrease in aminopyrine N-demethylase and aniline hydroxylase activity and no change in cytochrome P-450 content (data not shown). Exposure to DEHP for 7 days produced a differential effect. Activity of these two microsomal enzymes was significantly increased upon oral administration but remained decreased upon intraperitoneal administration. Repeated oral administration also increased the levels P. K. SETH These doses are known to cause teratogenic (42), pharmacologic (38) and biochemical (7-9, 15, 19, 20) effects. of cytochrome P-450 which were not altered upon single oral administration (data not shown). Cytochrome P-450 levels were also not affected by repeated intraperitoneal administration of DEHP.
In order to see whether the alterations in activities of aminopyrine N-demethylase and aniline hydroxylase are caused by DEHP or its two active metabolites, their effects on these two enzymes were investigated in vitro at varying concentrations.
DEHP had no effect on aminopyrine N-demethylase (Fig. 2) or aniline hydroxylase activity (Fig. 3), while mono(2-ethylhexyl) phthalate (MEHP) and 2-ethylhexanol (2-EH) produced significant inhibition at concentrations ranging from 2.5 to 15mM. The effects of DEHP on activity of alcohol dehydrogenase and high and low Km aldehyde dehydrogenases are shown in Figures 4 and 5. A single oral administration of DEHP significantly inhibited the activity of alcohol dehydrogenase in a dosedependent manner while intraperitoneal administration produced the effect only at the highest dose. No effect of DEHP was observed on the activity of high or low Km aldehyde dehydrogenase after a single oral or intraperitoneal administration ( on the rat liver aniline hydroxylase activity. Conditions of assay as described in Fig. 2. 31  4). The repeated oral administration of DEHP produced a different effect than that seen after a single administration ofthe phthalate. The activities of alcohol and aldehyde dehydrogenases were significantly increased. However, repeated intraperitoneal administration of DEHP caused no significant change in the activity of these enzymes (Fig. 5).

HEPATOTOXICITY OF PHTHALATES
The above results suggest that DEHP produces a timeand route-dependent effect on the activities of aminopyrine N-demethylase, aniline hydroxylase, cytochrome P450 content and alcohol dehydrogenase and high and low Km aldehyde dehydrogenases. A similar timeand route-dependent effect of DEHP was seen on the pentobarbital (43) and ethanol (44) sleeping time in rats and mice, respectively. An increase in hexobarbital sleeping time after intraperitoneal or intravenous administration and decrease in the same after repeated oral administration have also been observed by Daniel and Bratt (6). However, no suitable explanation has been provided by these investigators. DEHP is hydrolyzed to MEHP at markedly different rates by intestine and liver lipases P. K. SETH (21,45,46) and metabolized to 2-EH. These two major metabolites of DEHP have been reported to mimic some of the biological effects of plasticizer (7,16,33). Thus, the above observed route-dependent effects of DEHP may perhaps be related to the amounts of MEHP and 2-EH formed in the body, as oral administration of the phthalate may result in higher concentrations of these metabolites. A significant inhibition of aminopyrine N-demethylase and aniline hydroxylase by MEHP and 2-EH without any effect of DEHP on these enzymes under in vitro conditions suggests that these two metabolites are responsible for their in vivo effects. The DEHP-induced alterations in ethanol metabolism may also be related to MEHP and 2-EH, as these metabolites can be generated both by mitochondria and microsomes (45). Such observations are of great significance in view of a recent report on the metabolism of DEHP and accumulation of its metabolites in human blood and red cell concentrates stored in PVC blood bags (47). In order to compare the effects of DMP with DEHP on the activity of aminopyrine N-demethylase and aniline hydroxylase, male rats were exposed to these phthalates at a concentration corresponding to 1/10 of their LD50 dose by intraperitoneal route. The activity of these enzymes was examined 18 hr after the administration of phthalates. All the three phthalates inhibited the activity of aminopyrine N-demethylase and aniline hydroxylase with DMP producing the maximum effect followed by DEHP and DBP (Fig. 6). The effect of DBP on aminopyrine N-demethylase was, however, not significant.
The above results show that besides DEHP, these two phthalates can also inhibit the activity of drug-metabolizing enzymes and hence can modify the biological response of certain xenobiotics. These phthalates failed to exhibit any structure-activity relationship in affecting the activity of aminopyrine N-demethylase and aniline hydroxylase. A similar observation on the lack of any role of molecular structure ofphthalates in prolonging the pentobarbital sleeping time (38) or inhibiting the growth of nerve cell fibroblasts (48) has been reported earlier. Conclusions DEHP at high concentrations can cause functional hepatic damage, as reflected by morphological changes, and alterations in the activity of energylinked enzymes and metabolism of lipids and carbohydrates. Phthalates can interact with other xenobiotics and such interactions occur during the pharmacokinetic phase. These observations are of significance, as man may be exposed to low levels of phthalates for prolonged periods which can cause altered drug-xenobiotic reactions. Effects of DEHP are dose-and route-dependent and are also influenced by strain and species of the test animal. At present, information on the biological effects of phthalates in higher animals is very limited.