Investigation of the adjuvant and immuno-suppressive effects of benzyl butyl phthalate, phthalic acid and benzyl alcohol in a murine injection model.

In a recent study, di-(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono-2-ethylhexyl phthalate, were shown to possess adjuvant effect [Toxicology 169 (2001) 37; Toxicology Letters 125 (2001) 11]. The present study investigates the adjuvant effect of another important commercial phthalate plasticizer, benzyl butyl phthalate (BBP) as well as its degradation products, phthalic acid and benzyl alcohol (BA) in a murine model. The model antigen, ovalbumin (OA), was injected either alone (OA control group), together with one of the test substances (test group) or together with aluminium hydroxide, which served as the positive adjuvant control. The mice were boosted either once or twice with OA before blood was collected and assayed for the content of OA-specific IgE, IgG1 and IgG2a antibodies by ELISA methods. Adjuvant effect was defined as a statistically significant increased antibody level in the test groups compared with the OA control group. Conversely, if the antibody production in a test group was significantly lower than the OA control group, it was deemed to be immunosuppression. This study demonstrated that BBP, in contrast to DEHP, did not possess adjuvant effect. Furthermore, immunosuppression was apparent in the case of BA. The study also demonstrated that if the injections give rise to formation of wounds, it may cause false positive results.     

The adjuvant effect of di-(2-ethylhexyl) phthalate is mediated through a PPARalpha-independent mechanism

Previous studies in BALB/c mice revealed an adjuvant effect of di-(2-ethylhexyl) phthalate (DEHP) to simultaneously administered ovalbumin. DEHP is the most commonly used phthalate plasticizer. In vivo formed metabolites of DEHP are peroxisome-proliferator-activated receptor (PPAR) ligands, a group of chemicals that may have immunomodulatory properties. To study whether the PPARalpha receptor was involved in the adjuvant effect of DEHP, PPARalpha-deficient 129/Sv mice were exposed intraperitoneally to a mixture of OVA and DEHP, and the OVA-specific IgE, IgG1 and IgG2a responses were compared to the corresponding responses in the wild-type strain. The study showed that the adjuvant mechanism of DEHP is mediated through a PPARalpha-independent mechanism. Compared to mice only given OVA, DEHP induced highly increased levels of OVA-specific IgG1 and IgG2a, both in the wild-type and in the PPARalpha knock-out strains, indicating that DEHP is a mixed Th1/Th2 adjuvant.     

Airway inflammation and adjuvant effect after repeated airborne exposures to di-(2-ethylhexyl)phthalate and ovalbumin in BALB/c mice

BACKGROUND: Epidemiological studies have suggested an association between exposure to phthalate plasticizers, including di-(2-ethylhexyl)phthalate (DEHP), and increased prevalence of asthma, rhinitis or wheezing. Furthermore, studies in mice have demonstrated an adjuvant effect from DEHP after parenteral administration with the model allergen ovalbumin (OVA). OBJECTIVE: Exposures to DEHP were investigated for adjuvant effects and airway inflammation in a mouse inhalation model. METHODS: BALB/cJ mice were exposed to aerosols of 0.022-13 mg/m(3) DEHP and 0.14 mg/m(3) OVA 5 days/week for 2 weeks and thereafter weekly for 12 weeks. Mice exposed to OVA alone or OVA+Al(OH)(3) served as control groups. Finally, all groups were exposed to a nebulized 1% OVA solution on three consecutive days. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. RESULTS: In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. DEHP affected OVA-specific IgG1 production in a concentration-dependent manner, whereas little effect was seen on IgE and IgG2a. Dose-dependent increases in inflammatory cells were observed in BAL fluids, leading to significantly higher lymphocyte, neutrophil and eosinophil numbers in the OVA+13 mg/m(3) DEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested that DEHP has a mixed Th1/Th2 cytokine profile. CONCLUSION: Airborne DEHP is able to increase serum IgG1 and lung inflammatory cell levels, but only at very high concentrations. Realistic DEHP levels do not have an adjuvant effect or induce allergic lung inflammation in the present mouse model.     

Adjuvant effects of inhaled mono-2-ethylhexyl phthalate in BALB/cJ mice

Phthalates, including di(2-ethylhexyl) phthalate (DEHP), are widely used and have been linked with the development of wheezing and asthma. The main metabolite of DEHP, mono-2-ethylhexyl phthalate (MEHP), was investigated for adjuvant effects in a mouse inhalation model. BALB/cJ mice were exposed to aerosols of 0.03 or 0.4 mg/m(3) MEHP 5 days/week for 2 weeks and thereafter weekly for 12 weeks together with a low dose of ovalbumin (OVA) as a model allergen. Mice exposed to OVA alone or OVA+Al(OH)(3) served as negative and positive controls, respectively. Finally, all groups were exposed to a nebulized 1% OVA solution on 3 consecutive days to investigate the development of an inflammatory response. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. OVA-specific IgG1 production in both MEHP groups was significantly increased. OVA-specific IgE and IgG2a were not increased significantly. A dose-dependent increase in inflammatory cells was observed in BAL fluid, leading to significantly higher lymphocyte and eosinophil numbers in the OVA+0.4 mg/m(3) MEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested a T(H)2 profile of MEHP. In conclusion, MEHP acted as a T(H)2 adjuvant after inhalation. However, it is suggested that the inflammation in the MEHP groups was primarily mediated by an IgG1-dependent mechanism. To address implications for humans, a margin-of-exposure was estimated based on the lack of significant effects on IgE production and inflammation after exposures to 0.03 mg/m(3) MEHP observed in the present study and estimated human exposure levels.     

Adjuvant and immuno-suppressive effect of six monophthalates in a subcutaneous injection model with BALB/c mice.

The prevalence of allergic airway diseases is rapidly increasing in Western Europe and North America. This increase in disease prevalence may be associated with environmental pollutants. The present study investigated the adjuvant and immuno-suppressive effect of a series of monophthalates which are considered to be important metabolites of commonly used phthalate plasticizers. The effects were studied in a screening model. Ovalbumin (OA), used as the model antigen, was injected subcutaneously in the neck region of BALB/cJ mice with or without one of the test substances, mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBnP), mono-n-octyl phthalate (MnOP), mono-2-ethylhexyl phthalate (MEHP), mono-iso-nonyl phthalate (MiNP) or mono-iso-decyl phthalate (MiDP). The levels of OA-specific IgE, IgG1 and IgG2a in sera were measured by ELISA. Immuno-suppressive effect, defined as a statistically significant reduction in IgE or IgG1 antibody production, was observed with MEHP (1000 microg/ml, IgE and IgG1), MnOP (1000 microg/ml, IgE and IgG1), MiNP (1000 microg/ml, IgE and 10 microg/ml, IgG1) and MiDP (100 microg/ml, IgE and IgG1). Adjuvant effect, defined as a statistically significant increase in IgE or IgG1 antibody level, occurred with MEHP (10 microg/ml, IgE), MnOP (100 microg/ml, and 10 microg/ml, IgG1) and MiNP (100 microg/ml, IgE). No statistically significant immune modulating effect was seen with MBnP and MnBP.     

Interaction of di-(2-ethylhexyl) phthalate with the pharmacological response and metabolic aspects of ethanol in mice

The interactions of di-(2-ethylhexyl) phthalate (DEHP) with the pharmacological response and metabolic aspects of ethanol in mice were investigated at oral doses of DEHP of 1.5, 3.0 and 7.5 g/kg or intraperitoneal doses of 3.7, 7.5 and 18.9 g/kg, administered once or daily for 7 days. A single oral or intraperitoneal administration of DEHP resulted in a significant increase in the ethanol-induced sleeping time, associated with an inhibition of alcohol dehydrogenase activity in liver; the effect of intraperitoneal administration was significant only at the highest dose. The activities of high and low Km aldehyde dehydrogenases in mouse liver were not affected by a single dose of DEHP by either route. Repeated oral doses of DEHP produced significant reductions in the ethanol-induced sleeping time and increases in the activities of alcohol and aldehyde dehydrogenases, whereas repeated intraperitoneal doses of DEHP significantly increased the sleeping time and decreased the activity of alcohol dehydrogenase, without any perceptible effect on the activities of aldehyde dehydrogenases. In vitro studies with mouse liver preparations revealed significant inhibition of alcohol dehydrogenase activity by mono-(2-ethylhexyl) phthalate and 2-ethylhexanol and of high and low Km aldehyde dehydrogenase activities by DEHP and mono-(2-ethylhexyl) phthalate at concentrations ranging from 0.03 to 1.00 mM. In all cases, in vitro enzyme inhibition by mono-(2-ethylhexyl) phthalate was most pronounced.     

Di-(2-ethylhexyl) phthalate enhances skin sensitization to isocyanate haptens in mice

Toxicological and epidemiological studies have suggested the involvement of di-(2-ethylhexyl) phthalate (DEHP) in increases in allergies. The effects of DEHP have been implicated in the deflection to T helper 2 (Th2)-biased immune responses. However, we could not observe an adjuvant effect of DEHP in a Th2-dominant fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model when applied epicutaneously together with a hapten. In the present study, we focused whether there is a hapten to which DEHP exhibits an adjuvant effect through the epicutaneous route. BALB/c mice were epicutaneously sensitized with 2-methoxy-4-nitrophenyl isocyanate (MNICN) or phenethyl isocyanate (PEICN) in the presence or absence of phthalate esters of various kinds including DEHP. After challenge with a respective hapten, the ear-swelling response was measured to determine the level of hapten sensitization. Sensitization to MNICN and PEICN was enhanced when DEHP was present in the vehicle for the hapten preparation. The enhancement of sensitization to MNICN was accompanied by an elevated production of interferon-γ and reduced production of interleukin-4 (IL-4), suggesting polarization to a T helper 1 (Th1)-dominant response. The results suggested that the combination of a hapten and a phthalate ester significantly affects the outcome of CHS.     

Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds

Male rats were fed the plasticizers di-(2-ethylhexyl) phthalate (DEHP), di-(2-ethylhexyl) adipate (DEHA), di-(2-ethylhexyl) sebacate (DEHS), adipic acid, and diethyl phthalate at a dietary concentration of 2% for 3 weeks. Hepatic peroxisome proliferation in association with an increase in liver size, increase of hepatic activities of the peroxisome-associated enzymes catalase and carnitine acetyltransferase, and hypolipidemia were observed in animals treated with DEHP, DEHA, and DEHS but not in animals fed adipic acid and diethyl phthalate. To relate structure to biological activity, additional groups of rats were fed 2-ethylhexyl alcohol (a metabolite of DEHP), hexyl alcohol. 2-ethylhexanoic acid, hexanoic acid, 2-ethylhexyl aldehyde, hexylaldehyde, and 2-ethylhexyl amine at a 2% dose level. The changes induced by 2-ethylhexyl alcohol and 2-ethylhexanoic acid were comparable to those induced by DEHP, DEHA, and DEHS, suggesting that 2-ethylhexyl alcohol is the active part of the molecule responsible for peroxisome proliferation. 2-Ethylhexyl aldehyde induced a moderate increase in peroxisome population. No effect on hepatic peroxisomes or their associated enzymes was induced by the straight-chained analogs hexyl alcohol, hexanoic acid, and hexyl aldehyde. The hepatic effects of plasticizers capable of inducing peroxisome proliferation are similar to those resulting from treatment with clofibrate and other hypolipidemic drugs.     

Phthalate esters detected in various water samples and biodegradation of the phthalates by microbes isolated from river water

Phthalate esters (PEs), especially di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) were detected in various water samples such as river water, well water and tap water. On degradation tests of PEs, Tempaku River water degraded almost 100% of diethyl phthalate (DEP), di-isobutyl phthalate and DBP, and approximately 70% of DEHP. All eight isolates from Tempaku River water (R1-R7, D1) did not degrade dimethyl phthalate (DMP), but showed biodegrading ability for the other PEs. The DBP-degrading ability was particularly high for the isolates R1-R3 and D1 of Acinetobacter iwoffii. Crude enzyme solutions prepared from bacterial cells of these isolates showed a higher degrading activity for DEHP compared with that for microbially-degradable DBP. Particularly high DEHP-degrading activity was found for crude enzyme solutions of the isolate D1. As metabolites from the river water and bacterial isolates, DMP and an unknown diester were produced from DEP. DMP, DEP, monomethyl phthalate, monobutyl phthalate (MBP) and an unknown diester were produced from DBP. DBP, DEP, DMP and an unknown diester were produced from DEHP. As metabolites by the crude enzyme solutions, DMP, MBP and an unknown diester derivative were produced from DBP. DBP, mono-(2-ethylhexyl) phthalate and an unknown diester derivative were produced from DEHP. Diesters with shortened alkyl carbon chains were also found as metabolites by the isolates and their crude enzyme solutions. The results suggest that the alkyl chains in the diesters are also decomposed in addition to monoester formation from DBP or DEHP at the first step reported for animals and some types of bacteria.     

Effects of co-administration of Di(2-ethylhexyl)phthalate and testosterone on several parameters in the testis and pharmacokinetics of its mono-de-esterified metabolite

The administration of 1 g/kg di(2-ethylhexyl) phthalate (DEHP) or 5 mg/kg testosterone for 1 week did not affect the testicular and prostatic gland weights in rats. However, co-administration of DEHP and testosterone induced severe testicular atrophy accompanied by a decrease of zinc concentration in the testis and reduction of the activity of testicular specific lactate dehydrogenase isozyme. These changes were similar to the results of high dose administration of DEHP alone. Values of biological half-life and area under the concentration-time curve (AUC) of mono(2-ethylhexyl)phthalate, the main metabolite of DEHP, in testes after a single co-administration of DEHP (p.o.) and testosterone (i.p.) were higher than those after DEHP administration alone. Results suggest that the co-administration of DEHP and testosterone enhanced the adverse effects of DEHP on testes as the result of changes in pharmacokinetic values of MEHP.     

Studies on the hepatic effects of orally administered di-)2-ethylhexyl) phthalate in the rat.

A sequential study of hepatic effects was conducted in young male Wistar albino rats following daily oral intubation of di-(2-ethylhexyl) phthalate (DEHP) at a dose of 2000 mg/kg for 21 days. The relative liver weights of the animals increased progressively with the duration of the treatment. Alcohol dehydrogenase activity and microsomal protein and cytochrome P-450 contents showed a marked initial increase followed by a reversal as the treatment progressed. In contrast to this biphasic response, the activities of microsomal glucose-6-phosphatase, aniline 4-hydroxylase, and mitochondrial succinate dehydrogenase activity were significantly depressed, as observed both biochemically and histochemically, throughout the period of exposure. Parallel electronmicroscopic studies revealed a progressive dilatation of the smooth and rough endoplasmic reticulum, mitochondrial swelling and an increase in microbodies. Investigations carried out on the metabolic fate of [¹⁴C]DEHP in animals prior to and during the course of DEHP treatment showed no significant differences in the excretion pattern of radioactivity. Furthermore, there was no evidence indicating the storage of phthalate residues in the liver. Studies on the comparative effects of phthalic acid, 2-ethylhexanol, and mono-(2-ethylhexyl)phthalate (MEHP) orally administered at dose levels equimolar to DEHP for 7 days showed that the biochemical and ultrastructural changes in the hepatic endoplasmic reticulum and mitochondria in DEHP pretreatment were substantially reproducible by the administration of MEHP. Additionally, it was found that 2-ethylhexanol treatment led to an increase in the number of microbodies. The results indicate that the partial hydrolysis of DEHP to the monoester (MEHP) is the degradative step which determines the hepatic changes produced by DEHP.     

Di-(2-ethylhexyl) phthalate is without adjuvant effect in mice on ovalbumin

It has been suggested that one possible contributor to the increasing prevalence of IgE-mediated allergic diseases in Europe and the US is exposure to chemicals that may act as adjuvants. It has been reported previously that certain commonly used phthalate plasticizers, such as di-(2-ethylhexyl) phthalate (DEHP), are able to modify immune responses induced in mice by the common hens' egg allergen ovalbumin (OVA). However, the significance of these observations for human health is unclear, not least because the relevant studies have been conducted exclusively using subcutaneous administration of phthalates. We have therefore investigated the ability of DEHP when applied topically to affect anti-OVA antibody responses induced by subcutaneous exposure to OVA in BALB/c strain mice. Doses of DEHP (50mg) were used that resulted in a marked (approximately 30%) increase in liver weight. Dose-responses were conducted in order to identify doses of OVA that were sub-optimal for both anti-OVA IgG1 and IgE antibody responses: 1microg and 0.05microg, respectively. Under these conditions of exposure, topical administration of DEHP was without impact on antibody responses, regardless of whether DEHP was applied local or distant to the site of OVA immunization. Topical application of concentrations of DEHP that provoked marked systemic effects was without effect on the induction of immune responses.     

Studies on the effects of orally administered di-(2-ethylhexyl) phthalate in the ferret

A target-organ study of the effects of the phthalate ester di-(2-ethylhexyl) phthalate (DEHP) has been conducted in mature male albino ferrets. DEHP treatment caused a loss of body weight when administered as a 1% (w/w) diet for 14 months. Additionally, marked liver enlargement with associated morphological and biochemical changes was observed. These changes consisted of liver cell enlargement, lysosomal changes, dilatation of the endoplasmic reticulum and the depression of a number of marker enzyme activities. The only other tissue observed to be affected by DEHP treatment was the testes where histological evidence of tissue damage was observed in some animals.Studies on the metabolism of [¹⁴C]DEHP in the ferret indicated that the diester was metabolised to derivatives of mono-(2-ethylhexyl) phthalate which were excreted in the urine both unconjugated and as glucuronides.The results obtained have been compared with previous studies in the rat and it is concluded that DEHP is hepatotoxic in both species.     

Studies on gamma-glutamyl transpeptidase (GGT) after di(2-ethylhexyl) phthalate (DEHP) exposure

Administration of Di(2-ethylhexyl)phthalate (DEHP) at doses of 250, 500, 1000 and 2000 mg/kg to adult rats was found to significantly increase the activity of gamma-glutamyl transpeptidase (GGT) in liver and serum in a dose-dependent manner. The data indicate that DEHP can be hepatotoxic since an increase in serum GGT levels are indicative of hepatobiliary dysfunction and liver malignancy. An assay of GGT in serum of the individuals exposed to DEHP could be helpful in early detection of liver disorders due to this widely used plasticizer.     

Effects of Di-2-ethylhexyl phthalate (DEHP) on gap-junctional intercellular communication (GJIC), DNA synthesis, and peroxisomal beta oxidation (PBOX) in rat, mouse, and hamster liver.

The present study evaluated the effect of di-2-ethylhexyl phthalate (DEHP) on gap-junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX) activity, and replicative DNA synthesis in several rodent species with differing susceptibilities to peroxisome proliferator-induced hepatic tumorigenesis. A low (non-tumorigenic) and high (tumorigenic) dietary concentration of DEHP was administered to male F344 rats for 1, 2, 4, and 6 weeks. Additionally, a previously non-tumorigenic dose (1000 ppm) and tumorigenic dose of DEHP (12,000 ppm), as determined by chronic bioassay data, were examined following 2 weeks dietary administration. Male B6C3F1 mice were fed the non-tumorigenic concentration, 500 ppm, and the tumorigenic concentration, 6000 ppm, of DEHP for two and four weeks. The hepatic effects of low and high concentrations of DEHP, 1000 and 6000 ppm, were also examined in male Syrian Golden hamsters (refractory to peroxisome proliferator-induced tumorigenicity). In rat and mouse liver, a concentration-dependent increase in the relative liver weight, PBOX activity, and replicative DNA synthesis was observed at the earliest time point examined. Concurrent to these observations was an inhibition of GJIC. In hamster liver, a slight increase in the relative liver weight, PBOX activity, and replicative DNA synthesis was observed. However, these effects were not of the same magnitude or consistency as those observed in rats or mice. Furthermore, DEHP had no effect on GJIC in hamster liver at any of the time points examined (2 and 4 weeks). HPLC analysis of DEHP and its primary metabolites, mono-2-ethylhexyl phthalate (MEHP), and phthalate acid (PA), indicated a time- and concentration-dependent increase in the hepatic concentration of MEHP. At equivalent dietary concentrations and time points, the presence of MEHP, the primary metabolite responsible for the hepatic effects of DEHP, demonstrated a species-specific response. The largest increase in the hepatic concentration of MEHP was observed in mice, which was greater than the concentration observed in rats. The hepatic concentration of MEHP was lowest in hamsters. Hepatic concentrations of DEHP and phthalic acid were minimal and did not correlate with concentration and time. Collectively, these data demonstrate the inhibition of hepatic GJIC and increased replicative DNA synthesis correlated with the observed dose- and species-specific tumorigenicity of DEHP and may be predictive indicators of the nongenotoxic carcinogenic potential of phthalate esters.     

High-throughput determination of mono- and di(2-ethylhexyl)phthalate migration from PVC tubing to drugs using liquid chromatography-tandem mass spectrometry

The risk assessment of di(2-ethylhexyl) phthalate (DEHP) that migrated from polyvinyl chloride (PVC) medical devices is an important issue for hospitalized patients. Many studies have been conducted to determine the level of DEHP migration. A recent report has indicated that DEHP in blood bags was hydrolyzed by esterase to mono(2-ethylhexyl) phthalate (MEHP). Therefore, a method for the simultaneous determination of DEHP and MEHP was developed. The migration of DEHP and MEHP from PVC tubing to drugs was examined. Although we detected MEHP in the drugs, we found no enzymatic activity involved in the migration process. Some reports have indicated that hydrolysis may have occurred during sterilization by autoclaving. However, we did not perform any heat treatment. It is speculated that the MEHP migrated directly from the PVC tubing. The simultaneous determination of DEHP and MEHP is required for risk assessment, as MEHP may be even more toxic than the parent compound.     

Identification of the proximate peroxisome proliferator(s) derived from di(2-ethylhexyl) phthalate

A primary rat hepatocyte culture system was utilized to determine the proximate peroxisome proliferator(s) derived from di(2-ethylhexyl) phthalate (DEHP). DEHP was administered to rats and the urinary metabolites were identified and isolated. The major metabolites were those resulting from initial omega- or omega - 1-carbon oxidation of the mono(2-ethylhexyl) phthalate (MEHP) moiety. These metabolites, together with MEHP and 2-ethylhexanol, were added to primary rat hepatocyte cultures and the effect on peroxisomal enzyme activity was determined. The omega-carbon oxidation products [mono(3-carboxy-2-ethylpropyl) phthalate (I) and mono(5-carboxy-2-ethylpentyl) phthalate (V)] and 2-ethylhexanol produced little or no effect on CN- -insensitive palmitoyl-CoA oxidation (a peroxisomal marker). MEHP and the omega - 1-carbon oxidation products [mono-(2-ethyl-5-oxohexyl) phthalate (VI) and mono(2-ethyl-5-hydroxyhexyl) phthalate (IX)] produced a large (7- to 11-fold) induction of peroxisomal enzyme activity. Similar structure-activity relationships were observed for the induction of cytochrome P-450-mediated lauric acid hydroxylase and increase in cellular coenzyme A content. This identification of the proximate proliferators will aid in the elucidation of the mechanism by which DEHP causes proliferation of peroxisomes in the rodent liver. Oral administration of MEHP (150 or 250 mg/kg) to male guinea pigs did not produce hepatic peroxisome proliferation. Addition of MEHP (0 to 0.5 mM) or one of the "active" proliferators in the rat (metabolite IX, 0 to 0.5 mM) to primary guinea pig hepatocyte cultures also failed to produce an induction of peroxisomal beta-oxidation. Possible reasons for this species difference are discussed.     

Phthalate esters enhance quinolinate production by inhibiting alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), a key enzyme of the tryptophan pathway.

Tryptophan is metabolized to alpha-amino-beta-carboxymuconate-epsilon-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.     

Effects of phthalic acid esters on drug metabolizing enzymes of rat liver

Di(2-ethylhexyl)phthalate (DEHP) inhibited UDP-glucuronyltransferase activity of rat liver in vitro and in vivo. Diethyl phthalate and dimethoxyethyl phthalate also inhibited this enzyme in vitro. On the other hand, DEHP did not inhibit the activity of the cytosolic enzyme N-acetyltransferase; it also did not alter the levels of rat liver microsomal cytochrome P-450 in vitro. It is suggested that DEHP may alter the composition of microsomal phospholipids.     

Role of PPARalpha in mediating the effects of phthalates and metabolites in the liver

Phthalate esters belong to a large class of compounds known as peroxisome proliferators (PP). PP include chemicals that activate different subtypes of the peroxisome proliferator-activated receptor (PPAR) family. The ability of phthalate esters and their metabolites to activate responses through different PPAR subtypes is not fully characterized. We investigated the ability of two phthalate esters di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) and selected metabolites to activate PPAR (alpha, beta/delta, gamma) using a transient transfection assay. The monoester of DEHP, mono-(2-ethylhexyl) phthalate (MEHP) activated all three subtypes of PPAR, but preferentially activated PPARalpha. A second metabolite of DEHP, 2-ethylhexanoic acid (2-EHXA) was a weaker activator of all three subtypes. DBP, but not the primary metabolite mono-n-butyl phthalate weakly activated all three PPAR subtypes. MEHP and DBP but not DEHP and MBP interacted directly with human PPARalpha and PPARgamma as determined by scintillation proximity assays. Both DEHP and DBP activated expression of PP-inducible gene products in wild-type but not PPARalpha-null mice suggesting that both of these phthalates exert their effects by activation of PPARalpha in vivo. The preferential activation of PPARalpha by phthalate ester metabolites suggests that these phthalates mediate their toxic effects in rodent liver in a manner indistinguishable from other PP.