Phthalate Esters Enhance Quinolinate Production by Inhibiting {alpha}-Amino-ß-Carboxymuconate-{varepsilon}-Semialdehyde Decarboxylase (ACMSD), a Key Enzyme of the Tryptophan Pathway

Tsutomu Fukuwatari*, Seiko Ohsaki*, Shin-ichi Fukuoka{dagger}, Ryuzo Sasaki* and Katsumi Shibata*,1

* Laboratory of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures, University of Shiga Prefecture, Hikone, Japan; and {dagger} Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Japan

Received March 18, 2004; accepted June 20, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tryptophan is metabolized to {alpha}-amino-ß-carboxymuconate-{varepsilon}-semialdehyde (ACMS) via 3-hydroxyanthranilate (3-HA). ACMS decarboxylase (ACMSD) directs ACMS to acetyl CoA; otherwise ACMS is non-enzymatically converted to quinolinate (QA), leading to the formation of NAD and its degradation products. Thus, ACMSD is a critical enzyme for tryptophan metabolism. Phthalate esters have been suspected of being environmental endocrine disrupters. Because of the structural similarity of phthalate esters with tryptophan metabolites, we examined the effects of phthalate esters on tryptophan metabolism. Phthalate esters containing diets were orally given to rats and the urinary excreted tryptophan metabolites were quantified. Of the phthalate esters with different side chains tested, di(2-ethylhexyl)phthalate (DEHP) and its metabolite, mono(2-ethylhexyl)phthalate (MEHP), most strongly enhanced the production of QA and degradation products of nicotinamide, while 3-HA was unchanged. This pattern of metabolic change led us to assume that these esters lowered ACMSD protein or its activity. Although DEHP could not be tested because of its low solubility, MEHP reversibly inhibited ACMSD from rat liver and mouse kidney, and also the recombinant human enzyme. Correlation between inhibition of ACMSD by phthalate esters with different side chains and urinary excretion of QA supports the notion that phthalate esters perturb tryptophan metabolism by inhibiting ACMSD. Quinolinate is a potential endogenous toxin and has been implicated in the pathogenesis of various disorders. Although toxicity of phthalate esters through accumulation of QA remains to be investigated, they may be detrimental by acting as metabolic disrupters when intake of a tryptophan-rich diet and exposure to phthalate esters occur coincidentally.

Key Words: phthalate ester; endocrine disrupter; tryptophan metabolism; quinolinate; metabolic disrupter.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phthalate esters are used as plasticizers in the manufacture of polyvinylchloride plastics, as solvents in certain industrial processes, and as vehicles for pesticides (Giam et al., 1994Go). These esters are widely distributed in the ecosystem and have been suspected of being environmental endocrine disrupters. Of a variety of industrially important phthalate esters, di(2-ethylhexyl) phthalate (DEHP) has perhaps been most extensively used for the formation of plastics. A number of papers have reported that some phthalate esters are noxious to experimental animals (reviewed in Koizumi et al., 2001Go; Shea et al., 2003Go); administration of phthalate esters exhibits reproductive and developmental toxicity (David et al., 2000Go; Davis et al., 1994Go; Lamb et al., 1987Go; Wine et al., 1997Go). It is believed that phthalate esters taken orally are hydrolyzed in the intestine before absorption, and the resulting products, monoesters, are primarily responsible for the toxicity of phthalate esters (Lake et al., 1977Go).

The tryptophan–NAD pathway consists of the kynurenine pathway and the NAD pathway. The kynurenine pathway is the main route of tryptophan metabolism (Fig. 1). This pathway is initiated by the oxidation of tryptophan by tryptophan oxygenase (TDO) in the liver or by indoleamine dioxygenase (IDO) in other tissues including the brain. The metabolite at a branching point in the tryptophan–NAD pathway is {alpha}-amino-ß-carboxymuconate-{varepsilon}-semialdehyde (ACMS), which is converted by ACMS decarboxylase (ACMSD, EC4.1.1.45) to {alpha}-aminomuconate-{varepsilon}-semialdehyde (AMS). AMS eventually leads to acetyl-CoA through the glutarate pathway, or otherwise non-enzymatic cyclization of ACMS results in the formation of quinolinate (QA), from which NAD is synthesized through the NAD pathway. Thus, ACMSD activity plays a critical role in the tryptophan–NAD pathway. In mammals, NAD is also synthesized from niacin (nicotinate (NiA) and nicotinamide (Nam)) that can be obtained primarily from dietary sources.



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FIG. 1. Schematic diagram of the tryptophan–NAD pathway. Enzymes are underlined. 3-HA: 3-hydroxyanthranilate; ACMS: {alpha}-amino-ß-carboxymuconate-{varepsilon}-semialdehyde; AMS: {alpha}-aminomuconate-{varepsilon}-semialdehyde; QA: quinolinate; NaMN: nicotinic acid mononucleotide; NiA: nicotinate; NMN: nicotinamide mononucleotide; Nam: nicotinamide; MNA: N1-methylnicotinamide; 2-Py: N1-methyl-2-pyridone-5-carboxamide; 4-Py: N1-methyl-4-pyridone-3-carboxamide; TDO: tryptophan 2,3-dioxygenase; 3-HAO: 3-hydroxyanthranilic acid 2,3-dioxygenase; ACMSD: {alpha}-amino-ß-carboxymuconate-{varepsilon}-semialdehyde decarboxylase; QPRT: quinolinate phosphoribosyltransferase.

 
Quinolinate is a potential endogenous toxin; QA is neurotoxic by acting as an agonist at the N-methyl-D-aspartate (NMDA)–sensitive glutamate receptors. Schwarcz et al. (1983)Go and more recently Pawlak et al. (2003)Go have shown that QA can be a uremic toxin responsible for anemia associated with renal failure by reducing production of erythropoietin, a glycoprotein that promotes erythrocyte formation. Elevation of QA concentration has been implicated in the pathogenesis of various diseases including cerebral ischemia, spinal cord injury, Huntington's disease, and multiple sclerosis (see review by Stone and Darlington, 2002Go).

The structural similarity of phthalates with tryptophan metabolites prompted us to examine the effects of phthalate esters on the pathway of tryptophan metabolism. NAD can be supplied from tryptophan in the dietary protein. Therefore, administration of a niacin-deficient diet containing phthalate esters to rats and measurement of the tryptophan metabolites excreted in the urine make it possible to estimate phthalate ester–induced changes in tryptophan metabolism (Fukuwatari et al., 2002aGo, 2002bGo; Shibata et al., 2001Go). We previously reported that di-n-butyly phthalate (DBP) (Shibata et al., 2001Go) and DEHP (Fukuwatari et al., 2002aGo, 2002bGo) stimulated conversion of tryptophan to NAD. In this article, we show that phthalate esters elevate QA and its downstream metabolites in the urine, whereas excretion of 3-hydroxyanthranilate (3-HA) remains unchanged. Of the phthalate esters tested, DEHP and its primary metabolite, mono(2-ethylhexyl)phthalate (MEHP), were the most potent disrupters of tryptophan metabolism. We also present results showing that direct inhibition of ACMSD by phthalate esters is primarily responsible for the phthalate ester–induced change in tryptophan metabolism.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. The materials used were obtained from the indicated sources: vitamin-free milk casein, sucrose, L-methionine, dimethyl phthalate (DMP), diethyl phthalate (DEP), DBP, di-n-octyl phthalate (DOP), DEHP, monoethyl phthalate (MEP), Nam, and QA (Wako Pure Chemical Industries, Osaka, Japan); mono-n-butyl phthalate (MBP), mono-n-hexyl phthalate (MHP), MEHP, and N1-methylnicotinamide (MNA) chloride (Tokyo Chemical Industry, Tokyo); gelatinized cornstarch (Nichiden Kagaku, Tokyo); corn oil (Ajinomoto, Tokyo); mineral and vitamin mixtures (Oriental Yeast Kogyo, Tokyo). N1-methyl-2-pyridone-5-carboxamide (2-Py) and N1-methyl-4-pyridone-3-carboxamide (4-Py) were synthesized by the method of Shibata et al. (1988)Go. All other chemicals used were of the highest purity available from commercial sources.

Animals and diets. The care and treatment of the experimental animals conformed to The University of Shiga Prefecture guidelines for the ethical treatment of laboratory animals. Rats and mice were obtained from Clea Japan (Tokyo), and housed in a room maintained at 22 ± 1°C with 60% humidity and a 12 h light/12 h dark cycle (light onset at 6:00 A.M.). Mice were used for preparation of ACMSD as described later. Body weight and food intake were measured daily at 10:00 A.M., and food and water were renewed daily. Male Wistar rats at 5 weeks old were placed in individual metabolic cages (CT-10; Clea Japan) and acclimated for 1 week. They were fed the control diet containing no phthalate esters. Experiments (five animals per group) were started by using rats at 6 weeks of age. The control diet consisted of 20% casein, 0.2% L-methionine, 45.9% gelatinized cornstarch, 22.9% sucrose, 5% corn oil, 5% mineral mixture (AIN-93 mineral mixture), and 1% vitamin mixture (niacin-free AIN-93 vitamin mixture). The phthalate esters tested were DMP, DEP, DBP, DOP, DEHP, MBP, MHP, or MEHP. Rats were fed with a diet containing 2.6 mmol phthalate ester/kg diet ad libitum for 21 days, and controls were fed without phthalate ester. The weight percent of individual phthalate esters in the diet ranges from 0.05% (500 ppm) of DMP to 0.1% (1000 ppm) of DEHP depending on their molecular weight values. Urine samples on the last day (10:00 A.M.–10:00 A.M.; 24-h urine) were collected in amber bottles containing 1 ml of 1 mol/l HCl, and stored at –25°C until use.

Determination of tryptophan metabolites in the urine. Tryptophan metabolites were determined by high-performance liquid chromatography (HPLC). To determine 3-HA (Shibata and Onodera, 1992Go), urine samples were filtered through a 0.45-µm microfilter, and 20 µl of the filtrates was injected into a STR ODS II column (4.6 x 250 mm I.D., particle size 7 µm) (Shinwa Chemical, Kyoto, Japan). The mobile phase was 50 mmol/l KH2PO4 (pH 3.0)-acetonitrile (100:10 v/v) containing 3 mg/l ethylene diamine tetraacetic acid (EDTA)-2Na, the flow rate was 1 ml/min, the column temperature was maintained at 40°C, and 3-HA was detected at +500 mV electrochemical detection (ECD).

To determine QA (Mawatari et al., 1995Go), urine samples were filtered through a 0.45-µm microfilter, and 20 µl of the filtrates was injected into a Unisil Q C18 column (4.6 x 250 mm I.D., particle size 5 µm) (GL Sciences, Tokyo). The mobile phase was 20 mmol/l KH2PO4, pH 3.8, containing 0.00045% tetramethylammonium hydroxide and 1.2% hydrogen peroxide, the flow rate was 0.6 ml/min, and the column temperature was maintained at 40°C. The fluorescence intensity at 380 nm was measured upon excitation at 326 nm.

Nam, 2-Py, and 4-Py in the urine samples were measured simultaneously (Shibata, 1987aGo). Briefly, 1 ml of urine samples was mixed with 10 µl of 1 mg/ml isonicotinamide as an internal standard, 1.2 g of potassium carbonate, and 10 ml of diethylether. The mixtures were shaken vigorously for 5 min, and centrifuged at 800 x g for 5 min. The organic layers were evaporated, and dissolved in 0.5 ml of water. Aliquots of each sample were filtered through a 0.45-µm microfilter, and 20 µl of the filtrates was injected into a CHEMCOSORB 7-ODS-L column (4.6 x 250 mm I.D., particle size 7 µm) (Chemco Scientific, Osaka, Japan). The mobile phase was 10 mmol/l KH2PO4 (pH 3.0)–acetonitrile (96:4 v/v), the flow rate was 1 ml/min, the column temperature was maintained at 40°C, and the detection wavelength was 260 nm.

To determine MNA (Shibata, 1987bGo), urine samples (0.1 ml each) were mixed with 0.7 ml of water, 0.2 ml of 1 mmol/l isonicotinamide, 0.5 ml of 0.1 mmol/l acetophenone, and 1 ml of 6 mol/l sodium hydroxide. After the mixtures were cooled on ice for 10 min, 0.5 ml of 99% formic acid was added, followed by boiling in a water bath for 5 min. The mixtures were cooled on ice, filtered through a 0.45-µm microfilter, and 20 µl of the filtrates was injected into a Tosoh 80Ts column (4.6 x 250 mm I.D., particle size 7 µm) (Tosoh, Tokyo). The mobile phase was a mixture of 20 mmol/l KH2PO4, pH 3.0-acetonitrile (97:3 v/v) containing 1 g/l sodium hepansulfonate and 1 mmol/l EDTA-2Na, the flow rate was 1 ml/min, and the column temperature was maintained at 40°C. The fluorescence intensity at 440 nm was measured upon excitation at 382 nm.

Enzymes and assays. Because the dietary protein has been shown to induce ACMSD in the rat liver (Fukuoka et al., 1998Go), male Wistar rats (10 weeks old) were fed a high-protein diet (40% casein) for 4 weeks. Male ICR mice (9 weeks old) were fed the control diet (20% casein) for 1 week. Animals were sacrificed by decapitation, and the liver and kidneys were removed from rats and mice, respectively. The organs were immediately homogenized with a polytetrafluoroethylene (PTFE)-glass homogenizer in 5 volumes of cold 50 mmol/l potassium phosphate buffer, pH 7.0. The homogenate was centrifuged at 55,000 x g for 20 min, and the supernatant was used as an enzyme source. Four or five animals per group were used and the enzyme activities were assayed with the supernatant prepared from each organ.

Human ACMSD (Fukuoka et al., 2002Go) or human quinolinate phosphoribosyltransferase (QPRT, EC 2. 4. 2. 19) (Fukuoka et al., 1998Go) transiently expressed in COS-7 cells was prepared from cells cultured for 72 h after transfection. Cells were harvested and lysed with 50 mmol/l Tris-HCl buffer, pH 7.6, containing 137 mmol/l sodium chloride, 1% Triton X-100, 5 mmol/l EDTA, 100 µmol/l leupeptin, and 20 µg/ml FOY-305. The homogenates were centrifuged at 100,000 x g for 15 min, and the supernatants were used for assaying enzyme activity.

The activity of ACMSD was measured as described (Ichiyama et al., 1965Go). The reaction mixture containing 10 µl of 3.3 mmol/l 3-HA (in 50 mmol/l Tris-acetate buffer, pH 8.0); 0.5 ml of 0.2 mol/l Tris-acetate buffer, pH 8.0; and 0.8 ml of water was incubated in a cuvette for 5 min at 25°C. ACMS was produced by the addition of an excess quantity of the purified 3-HA oxygenase (50 µl containing 0.4 mg protein). After the formation of ACMS was complete, as judged by its absorbance at 360 nm, 0.1 ml of the ACMSD preparation was added. The decrease in absorbance at 360 nm was followed for 5 min against a control incubation that contained all the ingredients except 3-HA. When the effects of phthalate monoesters were examined, 50 µl of the esters dissolved in ethanol was added before the addition of the enzyme. The control incubation contained 50 µl of ethanol. The effects of phthalate diesters could not be tested because of their low solubility in the enzyme assay mixture.

QPRT was assayed as described (Shibata et al., 2000Go). The incubation medium contained 50 µl of 500 mmol/l potassium phosphate buffer, pH 7.0, 50 µl of 10 mmol/l QA, 50 µl of 10 mmol/l phosphoribosylpyrophosphate, 10 µl of 100 mmol/l MgCl2, 20 µl of phthalate monoester dissolved in ethanol, 270 µl of water, and 50 µl of the enzyme preparation. The control incubation contained 20 µl of ethanol. The reaction was started by addition of the enzyme, and the incubation was carried out at 37°C for 1 h. The reaction tube was placed in a boiling water bath for 5 min to stop the reaction, cooled on ice for 5 min, and centrifuged at 10,000 x g for 5 min. The supernatant was filtered through a 0.45-µm microfilter, and 20 µl of the filtrate was injected into a HPLC column, Tosoh 80Ts (4.6 x 250 mm I.D., particle size 7 µm) (Tosoh, Tokyo). The mobile phase was 10 mmol/l potassium phosphate buffer, pH 7.8, containing 1.48 g/l tetra-n-butylammonium bromide-acetonitrile (90:10 v/v), the flow rate was 1.0 ml/min, and the column temperature was maintained at 40°C. The product was detected at 265 nm.

Statistical analysis. The values are expressed as the mean ± SEM. The statistical significance was determined by ANOVA followed by Tukey's multiple comparison test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body Weight, Food Intake, and Liver Weight
Body weight of rats (6 weeks of age) at starting point of experiments was 140 ± 3 g and their weight increased almost linearly with gains of 6.2 ± 0.4 g per day. There was no significant difference in growth between groups fed phthalate esters and the control group. Food intake (g/day) of rats was 10 ± 0.5 at 6 weeks of age and increased to 17 ± 0.5, 20 ± 0.7, and 22 ± 0.8 at 7, 8, and 9 weeks, respectively. Phthalate esters showed no significant effect on food intake. Food intake/kg body weight/day varies depending on age (66 g at 6 weeks and 77 g at 9 weeks). When we used an average value of food intake (70 g/kg body weight/day), intake of phthalate esters was calculated to be 0.182 mmol/kg body weight/day, and therefore the weight values of phthalate esters ingested ranged from 35 mg/kg body weight/day of DMP to 70 mg of DEHP, depending on their molecular weight values.

DEHP causes hepatomegaly in rodents by proliferating peroxisome (Elcombe and Mitchell, 1986Go; Ward et al., 1986Go). However, the liver weights of phthalate ester–fed groups measured at the end point of experiments (9 weeks of age) did not differ from those of the control groups, indicating that DHEP at the dose level given in this experiment does not cause significant peroxisome proliferation.

Effects of Phthalate Diesters on the Urinary Excretion of the Tryptophan Metabolites
To assess the effects of various phthalate diesters on the tryptophan–NAD pathway, rats at 6 weeks of age were fed with a diet containing DMP, DEP, DBP, DOP, or DEHP for 21 days, and the urinary contents of tryptophan metabolites such as 3-HA, QA, Nam, MNA, 2-Py, and 4-Py were measured. The sum of Nam, MNA, 2-Py, and 4-Py was expressed as Nam metabolites. As shown in Figure 2A, the urinary excretion of 3-HA was not changed by any of the phthalate diesters used. In contrast, QA (Fig. 2B) and its downstream metabolites (Nam metabolites in Fig. 2C) were markedly elevated by DEHP. Both DBP and DOP also increased the urinary excretion of QA but to a lesser extent; DMP and DEP, however, had no effect (Fig. 2B). DME, DEP, DBP, and DOP did not affect the excretion of Nam metabolites (Fig. 2C). Thus the length and structure of side chains in the esters appear to be crucial for the urinary excretion of tryptophan metabolites. DEHP that has long and branched side chains was the most powerful disruptor of tryptophan metabolism.



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FIG. 2. Effects of phthalate diesters on the urinary excretion of 3-HA (A), QA (B), and Nam metabolites (C) in rats. Male Wistar rats at 6 weeks old were fed with a diet containing 2.6 mmol phthalate ester/kg diet ad libitum for 21 days. The phthalate esters used were DMP, DEP, DBP, DOP, or DEHP. Urine samples on the last day (10:00 A.M.–10:00 A.M.; 24-h urine) were collected in amber bottles containing 1 ml of 1 mol/L HCl. Values are means ± SEM; n = 5. *P < 0.05 versus the control.

 
Effects of Phthalate Monoesters on the Urinary Excretion of the Tryptophan Metabolites
Because the phthalate diester–induced effects may be due to the monoesters that are produced in the digestive organs (Lake et al., 1977Go), we also examined phthalate monoesters. Rats at 6 weeks of age were fed with a diet containing MBP, MHP, or MEHP for 21 days, and the urinary excretion of the tryptophan metabolites was assayed. The experiments with DEHP were performed again for comparison with MEHP. As shown in Figure 3, the results were very similar to those when the diesters were used. The urinary excretion of 3-HA was unchanged after administration of the monoesters (Fig. 3A). Large increases in QA and Nam metabolites were found when MEHP was given, and those increases were similar to the ones found with DEHP (Fig. 3B and 3C). When MBP and MHP were given, there was an increase in the mean values of urinary QA and Nam metabolites, but the increase was not statistically significant.



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FIG. 3. Effects of phthalate monoesters on the urinary excretion of 3-HA (A), QA (B), and Nam metabolites (C) in rats. Male Wistar rats at 6 weeks old were fed with a diet containing 2.6 mmol phthalate ester/kg diet ad libitum for 21 days. The phthalate esters used were MBP, MHP, MEHP, or DEHP. Urine samples on the last day (10:00 A.M.–10:00 A.M.; 24-h urine) were collected in amber bottles containing 1 ml of 1 mol/L HCl. Values are means ± SEM, n = 5. *P < 0.05 versus the control.

 
Effects of Phthalate Monoesters on ACMSD and QPRT
Feeding of MEHP or DEHP strongly increased urinary excretion of QA and its downstream metabolites in the tryptophan–NAD pathway but did not change the excretion of 3-HA, suggesting involvement of ACMSD in this phthalate ester-induced change of the tryptophan metabolism; the cellular concentration of ACMSD protein may be decreased or ACMSD activity may be directly inhibited by these esters. To test the latter possibility, rat, mouse, and human ACMSD activities were measured in vitro in the presence and absence of phthalate monoesters such as MEP, MBP, MHP, and MEHP. Extracts from rat livers, mouse kidneys, and COS-7 cells that express human recombinant ACMSD were used as enzyme sources. To show the specificity of the inhibition, QPRT from rat livers and COS-7 cells producing human enzyme were also assayed. Assays with the corresponding diesters could not be performed because the addition of the diesters caused turbidity in the mixture for the enzyme assay. Table 1 shows the results when ACMSD was assayed with and without 3 mmol/l phthalate ester. The high activity of human ACMSD compared with that from rats and mice, is probably due to the high expression of this recombinant human enzyme in COS cells. The same would be true for QPRT. MEP, the monoester with the shortest chain, was not inhibitory whereas low inhibition was found with MBP. The inhibition became more potent as the length of the side chains in esters became longer; MHP and MEHP severely blocked the ACMSD activity from all sources, and MEHP was the most potent inhibitor. In contrast, neither human nor rat QPRT was affected by any of the phthalate monoesters. Figure 4A shows a dose-dependent inhibition of human ACMSD by MEHP; the presence of MEHP at 0.3 mmol/l caused a significant inhibition, and at 3 mmol/l the activity was inhibited by more than 90%. In agreement with Table 1, MEHP was not inhibitory to human QPRT at any of the concentrations tested (Fig. 4B).


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TABLE 1 Effects of Phtalate Monoesters on the Activity of ACMSD or QPRTa

 


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FIG. 4. MEHP inhibits human ACMSD activity in a dose-dependent manner. ACMSD activity is shown as the amount of enzyme that generates µmoles of AMS per hour per milligram of protein. QPRT activity is shown as the amount of enzyme that generates µmoles of nicotinic acid mononucleotide per hour per milligram of protein. Values are means ± SEM, n = 5. *P < 0.05 versus the control.

 
To investigate whether the inhibition of ACMSD by MEHP was reversible or whether MEHP irreversibly inactivated the enzyme, human ACMSD was incubated for 10 min at 25°C with 3 mmol/l MEHP, at which the ACMSD activity would be inhibited by more than 90% (see Fig. 4). The enzyme was then diluted fivefold with 50 mmol/l Tris-HCl buffer, pH 7.6, and the diluted enzyme preparation was subjected to the enzyme assay. The final concentration of MEHP in the assay mixture was 0.06 mmol/l at which the inhibition of the enzyme should be negligible if the inhibition is reversible. The activity of the diluted ACMSD was found to be comparable to that of the control enzyme diluted without treatment with MEHP (data not shown), indicating that the inhibition is reversible. A similar result was obtained using rat ACMSD.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was undertaken to investigate effects of phthalate esters orally given to rats on the tryptophan metabolism. DEHP, which has been most widely used in the formation of plastics, and its primary metabolite, MEHP, caused the most dramatic change in tryptophan metabolism. They markedly enhanced the urinary excretion of QA and its downstream metabolites, while the excretion of 3-HA was unchanged, raising the possibility that ACMSD, a critical enzyme acting at the branching point of tryptophan metabolism, is inhibited by these phthalate esters. In fact, MEHP reversibly inhibited ACMSD from human, mouse, and rat sources in a dose-dependent manner. Some of phthalate esters with different side chains promoted urinary excretion and inhibited ACMSD but their potency was far less than that of DEHP and MEHP. Thus it is very likely that the inhibition of ACMSD by phthalate esters blocks conversion of tryptophan to acetyl CoA and directs tryptophan metabolism to the NAD pathway (see Fig. 1), and thus cellular QA and Nam metabolites are accumulated, resulting in their increased excretion into the urine.

Isenberg et al. (2000)Go explored the metabolism of DEHP given orally to rats. MEHP was the most prominent hepatic metabolite of DEHP, and elevation of the hepatic MEHP concentration was time-dependent and dose-dependent, whereas the levels of DEHP and phthalate were minimal and did not correlate with the dose of DEHP or the time after its administration (Isenberg et al., 2000Go). Oral administration of phthalate and 2-ethylhexanol, hydrolysis products of DEHP, did not affect the conversion rate of tryptophan to NAD (Fukuwatari et al., 2002bGo). In agreement with the proposal that phthalate monoesters are produced from the diesters in the intestine before absorption (Lake et al., 1977Go), these results indicate that MEHP is mainly responsible for the perturbation of tryptophan metabolism.

According to Isenberg et al. (2000)Go, hepatic MEHP concentration in rats fed 1000 ppm DEHP for 2 weeks, conditions similar to those used in the present experiments, was 9 µmol/g tissue. In vitro inhibition of ACMSD by MEHP was apparent (33%) at 0.3 mmol/l and greater than 90% at 3 mmol/l. These results suggest that the liver in rats fed 1000 ppm DEHP accumulates MEHP at the concentration sufficient to exhibit its inhibitory effect on ACMSD, although all of the MEHP molecules in the liver may not necessarily be available for this inhibition.

Previously we showed that ACMSD activity in the liver extracts from rats fed DEHP was similar to that of control animals (Fukuwatari et al., 2002bGo). This result is not contradictory to our present finding that ACMSD is inhibited in vitro by MEHP. The inhibition is reversible, and therefore, even if MEHP is accumulated in the liver of rats fed DEHP at concentrations sufficient to block ACMSD, MEHP would be washed out during the preparation of the enzyme; the resulting enzyme preparations would contain MEHP at levels that show little inhibition of ACMSD. Taken together, we conclude that phthalate esters perturb tryptophan metabolism through direct inhibition of ACMSD but not by reducing the ACMSD protein level. The mechanism by which ACMSD is inhibited remains to be examined. The very labile nature of ACMS, the substrate of ACMSD, hampers kinetic studies of this enzyme.

Quinolinate is a potential endogenous toxin; it is neurotoxic and has been suspected of being involved in the development of a number of brain diseases (see review by Stone and Darlington, 2002Go). Although it is believed that the liver is a major site of tryptophan metabolism, expression of ACMSD, as well as its mRNA in the brain and kidney (Fukuoka et al., 2002Go), suggests that the phthalate ester–induced metabolic alteration occurs in these organs. However, to date, there are no reports that show neurotoxicity of phthalate esters. When DEHP (0–200 mg/kg/day) was given to rats via oral gavage, few adverse effects on neurobehavioral evaluations were found (Moser et al., 2003Go). DEHP given to mice through the diet to provide levels of 0.01–0.09% did not show detrimental effects on neurobehavioral parameters (Tanaka, 2002Go). Examination of tissue distribution by the use of the radioactive DEHP did not show significant accumulation of the radioactivity in the brain of rats (Tanaka et al., 1975Go) and mice during the pre-weaning period (Eriksson and Darnerud, 1985Go). Quinolinate also can be a uremic toxin responsible for anemia with renal failure by reducing the renal production of erythropoietin, a growth factor essential for erythrocyte formation (Pawlak et al., 2003Go). When DEHP at a high dose (12,500 ppm in the diet) was given to rats, the erythrocyte count, hemoglobin, and hematocrit values were significantly lower than controls, but these effects were not found with a lower dose (2500 ppm; David et al., 2000Go). Although no data indicating that phthalate esters exhibit adverse effects through accumulation of QA are available, effects of the concurrent intake of a tryptophan-rich diet and phthalate esters are worthy of further investigation. Such a series of misfortunes may contribute to triggering and/or exacerbating various diseases.


    ACKNOWLEDGMENTS
 
This investigation was supported by a Grant-in-Aid for the Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan.


    NOTES
 

1 To whom correspondence should be addressed at Laboratories of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures, University of Shiga Prefecture, Hikone, Shiga 522-8533, Japan. E-mail: kshibata{at}shc.usp.ac.jp.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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