TOXICOKINETICS AND METABOLISM

Yuki Ito Æ Hiroshi Yokota Æ Ruisheng Wang

Osamu Yamanoshita Æ Gaku Ichihara Æ Hailan Wang

Yoshimasa Kurata Æ Kenji Takagi Æ Tamie Nakajima

Species differences in the metabolism of di(2-ethylhexyl) phthalate(DEHP) in several organs of mice, rats, and marmosets

Received: 20 June 2004 / Accepted: 23 September 2004 / Published online: 11 November 2004� Springer-Verlag 2004

Abstract To clarify species differences in the metabolismof di(2-ethylhexyl) phthalate (DEHP) we measured theactivity of four DEHP-metabolizing enzymes (lipase,UDP-glucuronyltransferase (UGT), alcohol dehydroge-nase (ADH), and aldehyde dehydrogenase (ALDH)) inseveral organs (the liver, lungs, kidneys, and smallintestine) of mice (CD-1), rats (Sprague–Dawley), andmarmosets (Callithrix jacchus). Lipase activity, mea-sured by the rate of formation of mono(2-ethylhexyl)phthalate (MEHP) from DEHP, differed by 27- to 357-fold among species; the activity was highest in the smallintestines of mice and lowest in the lungs of marmosets.This might be because of the significant differencesbetween Vmax/Km values of lipase for DEHP among thespecies. UGT activity for MEHP in the liver microsomeswas highest in mice, followed by rats and marmosets.These differences, however, were only marginal com-pared with those for lipase activity. ADH and ALDHactivity also differed among species; the activity of the

former in the livers of marmosets was 1.6–3.9 timesgreater than in those of rats or mice; the activity of thelatter was higher in rats and marmosets (2–14 times)than in mice. These results were quite different fromthose for lipase or UGT activity. Because MEHP isconsidered to be the more potent ligand to peroxisomeproliferator-activated receptor a involved in differenttoxic processes, a possibly major difference in MEHP-formation capacity could be also considered on extrap-olation from rodents to humans.

Keywords Di(2-ethylhexyl) phthalate Æ Marmoset ÆMetabolism Æ Rodent Æ Species differences

Abbreviations ADH: Alcohol dehydrogenase Æ ALDH:Aldehyde dehydrogenase Æ DEHP: Di(2-ethylhexyl)phthalate Æ 2-EH: 2-Ethylhexyanol Æ 2-EHA:2-Ethylhexanoic acid Æ MEHP: Mono(2-ethylhexyl)phthalate Æ 2-POET: 2-Phenoxyethanol Æ PPARa:Peroxisome proliferator-activated receptoralpha Æ UGT: UDP-glucuronyl transferase

Introduction

Di(2-ethylhexyl) phthalate (DEHP, CAS-No. 117-81-7),a diester (molecular weight 390.56) of o-phthalic acid, iswidely used to improve the plasticity and elasticity ofpolyvinyl chloride (PVC) products. The final DEHPcontent of the resulting products is between 10 and 60%(w/w). Recent production of DEHP in Japan hasapproached 0,25 million tons per year, which accountsfor about 60% of all plasticizers used (Japan PlasticizerIndustry Association 2003). DEHP has potentiallyadverse effects on the liver, kidney, and reproductiveorgans (Huber et al. 1996). In addition, the adverse effectof DEHP as a probable endocrine disruptor has recentlybeen attracting increasing attention (Sharpe 2001).

DEHP absorbed by the body is first hydrolyzedto mono(2-ethylhexyl) phthalate (MEHP) and 2-ethyl-

Y. Ito Æ O. Yamanoshita Æ G. Ichihara Æ H. WangT. Nakajima (&)Department of Occupational and Environmental Health,Nagoya University Graduate School of Medicine,65 Tsurumai-cho, Showa-ku, 466-8550 Nagoya, JapanE-mail: tnasu23@med.nagoya-u.ac.jpTel.: +81-52-744-2122Fax: +81-52-744-2126

H. YokotaDepartment of Biochemistry,Rakuno Gakuen University Schoolof Veterinary Medicine, 069-8501 Ebetsu, Japan

R. WangNational Institute of Industrial Health,214-8585 Kawasaki, Japan

Y. KurataDepartment of Safety Science Research,Mitsubishi Research Institute Company Limited,314-0255 Kashima, Japan

K. TakagiDepartment of Medical Technology,Nagoya University School of Health Sciences,461-8673 Nagoya, Japan

Arch Toxicol (2005) 79: 147–154DOI 10.1007/s00204-004-0615-7

hexyanol (2-EH) by the catalytic action of lipase. SomeMEHP is conjugated with UDP-glucuronide by thecatalytic action of UDP-glucuronyl transferase (UGT),and excreted in the urine (Albro and Lavenhar 1989).The remaining MEHP is excreted directly in the urine oris oxidized by cytochrome P450 4A, and further oxidizedby alcohol dehydorogenase (ADH) or aldehyde dehy-drogenase (ALDH) to dicarboxylic acid or ketones(Albro and Lavenhar 1989). 2-EH is metabolized mainlyto carboxylic acid (mainly 2-ethylhexanoic acid, 2-EHA)via 2-ethylhexanal by the catalytic action of ADH andALDH (Albro and Lavenhar 1989). The 2-EHA thusformed is further oxidized to a dicarboxylic acid similarto the oxidation of MEHP.

Peroxisome proliferator-activated receptor alpha(PPARa) might be involved in the processes of bothphysiological and toxicological response to differentendogenous or exogenous substances (Gonzalez 1997).Mono- and dicarboxylic acid metabolites of DEHP actas ligands for PPARa (Maloney and Waxman 1999),with a resulting effect on expression of several targetgenes, while also having pleiotropic effects on severalorgans (Huber et al. 1996). This indicates that the con-centrations of these mono- and dicarboxylic metabolitesin target organs are of primary importance in the tox-icity of DEHP, although differences in the constitutivelevels of PPARa or in its function cannot be denied(Maloney and Waxman 1999). Therefore, understandingof the kinetic behavior of these metabolites is needed toclarify the mechanisms underlying the toxicity of DEHP.

Although specific differences between metabolism ofDEHP (Lake et al. 1977; Albro et al. 1982; Rhodes et al.1986; Albro 1986; Albro et al. 1983) and organ speci-ficity (Albro and Thomas 1973; Pollack et al. 1985) havebeen investigated in several studies, the focus has beenon lipase activity only. Even so, no information hasemerged concerning kinetic data for lipase, for exampleKm and Vmax.

This study investigated species differences (mice, rats,and marmosets) and differences among organs withregard to the activity of four kinds of enzyme (lipase,UGT, ADH, ALDH) involved in the metabolism ofDEHP in several organs (liver, kidneys, lungs, and smallintestine). We selected these organs because they areimportant in the absorption (lungs and small intestine),metabolism (liver), and excretion (kidney) of DEHP.Our results show that species differences in lipaseactivity might be critical to our understanding of therespective DEHP kinetics.

Materials and methods

Experimental animals

This study was conducted according to the Guidelinesfor Animal Experiments of The Shinshu UniversityAnimal Center. Animals were housed in a cage in a cleanroom under controlled temperature, relative humidity,and light (12 h light/dark cycle). Eight-week-old malemice (CD-1) and rats (Sprague–Dawley) were purchasedfrom Charles River (Atsugi, Japan). Marmosets (com-mon marmoset, Callithrix jacchus) were purchased fromClea Japan (Tokyo, Japan) at 2 months of age. On theday the marmosets reached 18 months of age they wereall sacrificed by exsanguination from the abdominalaorta under pentobarbital anesthesia and subjected tonecropsy. Mice and rats at 11 weeks were killed by CO2

asphyxiation. The liver, kidneys, lungs, and a smallportion of the small intestines were removed and storedat �85�C until used. The body and organ weights areshown in Table 1.

Analysis of lipase activity

Each tissue was homogenized with a threefold volume of10 mmol L�1 phosphate buffer (pH 7.4) containing0.25 mol L�1 sucrose. Lipase activity was determined bymeasuring the rate of formation of MEHP from DEHPin each tissue homogenate or microsomal fraction pre-pared using the method of Wang et al. (1999). Mea-surements were performed using 100 lg microsomalprotein or homogenate per 0.5 mL reaction mixturecontaining a final concentration of 50 mmol L�1

potassium–sodium phosphate buffer (pH 7.4) and1 mmol L�1 DEHP (Tokyo Kasei Kogyo, Tokyo,Japan). DEHP was dissolved in 99.5% ethanol (WakoPure Chemical Industries, Osaka, Japan) because DEHPis hardly soluble in water. The reaction was initiated byaddition of substrate and the reaction tube was placed ina thermoregulated shaking water bath at 37�C (incu-bated samples). After 10 min incubation, 120 lL of1 mol L�1 HCl solution was added to stop the reaction.The MEHP formed was then extracted twice with 1 mLethyl acetate. The ethyl acetate layer was evaporated,followed by addition of 40 lL ethyl acetate. Under theseconditions, the extraction recovery of MEHP was95.6±1.9 (n=6, mean±SD). N-Methyl-N-(tert-butyl-

Table 1 Body and organ weights

Animal n Body weight(g)

Liver weight(g)

Liver/body(%)

Kidney weight(g)

Kidney/body(%)

Mouse 6 35.6±0.9 1.99±0.12 5.57±0.34 0.54±0.07 1.53±0.21Rat 5 351±17 13.3±0.6 3.80±0.13 2.60±0.29 0.73±0.06Marmoset 5 264±20 9.9±2.0 3.93±0.55 1.52±0.31 0.61±0.11

Values represent the mean±standard deviation for each group

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dimethylsilyl)trifluoroacetamide (GL Sciences, Tokyo,Japan; 20 lL) was added to the MEHP-extractedvial and left at room temperature for 60 min. TheMEHP tert-butyldimethylsilyl derivative produced wasanalyzed by gas chromatography with mass-selectivedetection (6890 N, 5973 N; Agilent Technologies, CA,USA). The analytical conditions were: column, HP-5MS 5% phenyl methyl siloxane capillary column(30 m·0.25 mm·0.25 lm, Agilent Technologies), carriergas, helium 1 mL min�1; injection type, splitless(1 min)-split; oven temperature, initially 80�C for 2 min,elevated at 20�C min�1 to 260�C and held for 5 min;split ratio 50:1, selective ion monitoring (SIM) mode. Inthis analysis, 5 nmol trans-cinnamic acid (Wako PureChemical Industries) was used as internal standard. Twoions each, m/z 223 and 335, and m/z 205 and 131 wereused for the detection of MEHP and trans-cinnamicacid, respectively, with the first ions, m/z 223 and 205,being used for quantification. The MEHP level in a tubecontaining the same medium as the reaction vial butexcluding the incubation step at 37�C (non-incubatedsamples) was also measured. The net MEHP contentenzymatically formed from DEHP was calculated bysubtracting the MEHP in the non-incubated samplesfrom that in the incubated samples. Under these con-ditions, lipase activity increased proportionately up to100 lg protein and was linear with time for 60 minincubation. All glassware was heated at 200�C for 2 h toexclude the possibility of environmental contaminationof DEHP.

Lipase kinetics

Lipase kinetics were determined for DEHP by recordingthe activity toward a range of concentrations (10–1000 lmol L�1 for mice, 5–1000 lmol L�1 for rats, and300–5000 lmol L�1 for marmosets). We pooled hepaticmicrosomes from mice, rats, and marmosets and deter-mined lipase activity as mentioned above using severalsubstrate concentrations. Maximum velocity Vmax andMichaelis constant Km values for DEHP were deter-mined from Lineweaver–Burk plots.

Analysis of UGT activity

UGT activity for MEHP (>90%, Tokyo Kasei Kogyo,Tokyo, Japan) was determined by the high-performanceliquid chromatography (HPLC) method of Sjoberg et al.(1991) except for using cholic acid (final concentration0.01%) instead of lubrol. UGT activity was also mea-sured using 1-naphthol (analytical grade, NakaraiChemical, Kyoto, Japan) and bis-phenol A (analyticalgrade, Kanto Chemical, Tokyo, Japan) as substrates forthe UGT 1A and UGT 2B families, respectively (Yokotaet al. 1999). Each standard, such as MEHP glucuronide,was converted from the reaction mixtures with livermicrosomes in the presence of UDP-glucuronic acid,

and quantified by HPLC by calculating the differencebetween b-glucuronidase (Sigma–Aldrich, Tokyo,Japan)-treated samples and untreated samples. We useda final concentration of 0.1 mg mL�1 microsomes andchecked the linearity with time between 20 min to40 min.

Analysis of ADH and ALDH

ADH (Boehringer Mannheim, Tokyo, Japan) activity inthe cytosol fraction was assayed by the method ofBurton et al. (1988). ALDH (Boehringer Mannheim,Tokyo, Japan) activity in the mitochondrial and post-mitochondrial fractions was measured by the method ofWang et al. (1999). Because there were no authenticalcohol or acetaldehyde metabolites of MEHP byCYP4A, we used the structurally analogous 2-phen-oxyethanol (2-POET) (Wako Pure Chemical Industries)as substrate for ADH and 3-phenylpropionaldehyde(Wako Pure Chemical Industries) as substrate forALDH. 2-EH and 2-ethylhexanal were also used assubstrates for ADH and ALDH, respectively.

Analysis of protein concentrations

Protein concentrations in the samples were measuredusing a Protein Assay Kit (Bio-Rad, Tokyo, Japan).

Statistical analysis

Comparisons were made by using analysis of varianceand the Tukey–Kramer HSD post-hoc test. Values ofP<0.05 were considered to indicate statistical sig-nificance. A logarithmic transformation was applied tolipase activities in microsome and homogenate samplesfrom the small intestine and kidneys before Tukey–Kramer analysis.

Results

Lipase

Lipase activity in the liver, small intestine, kidneys, andlungs from mice, rats, and marmosets (Table 2) werefound to be highest in all the organs from mice andlowest in those from marmosets; differences between theresults for these species ranged from 22- to 148-fold.Lipase activity was also compared among organs withinspecies; in mice, the activity was highest in the smallintestine, whereas in rats and marmosets, it was highestin the liver. The activity in the lungs was below thedetectable limit for marmosets, but was equal in miceand rats. Lipase activity was found in the microsomalfraction from all tissues except the lungs from marmo-sets; the pattern was quite similar to those in the

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homogenates, but the range of inter-species differenceswas slightly greater (27- to 357-fold).

Enzyme parameters

Species differences in lipase Vmax and Km values forDEHP calculated from Lineweaver–Burk plots (Fig. 1)are shown in Table 3. Vmax of lipase activity for DEHPwas highest for mice, and lowest for marmosets. Incontrast, Km for DEHP was much lower for rats andmice than that for marmosets. This resulted in majorspecies differences in the Vmax/Km ratio (often referredto as an index of intrinsic clearance) among mice, rats,and marmosets. These findings suggest that significantdifferences in lipase activity among these species resultfrom the different affinity of DEHP for lipase and thedifferent levels of the enzyme.

UDP-glucuronyl transferase

Although UGT activity for MEHP was detected inhepatic microsomes from all species investigated, none

could be detected in microsomes of other tissues(Table 4). Although the activity was greater in mice andrats than in marmosets, the differences among specieswere not as large as those for lipase activity. UGTactivity was also measured using 1-naphthol and bis-phenol A as a substrate for the UGT 1A and UGT 2Bfamily, respectively. The pattern of species differencesfor the activity of MEHP was not similar to that ofeither 1-naphthol or bis-phenol A. In addition, UGTactivity for MEHP was less than one-tenth that for 1-naphthol or bis-phenol A in all organs, suggesting thatUGT is relatively less active for MEHP.

Table 2 Species differences between lipase activity (pmol mg�1 protein in homogenates or microsomal fragment min�1)

n Liver Small intestine Kidney LungHomogenates

Mouse 6 1339±261 5764±1147 956±87 53±20Rat 5 718±152a 428±146a 105±31a 57±19Marmoset 5 62±11b,c 39±17b#,c 21±7b,c NDb,c

MicrosomesMouse 6 4964±1040 11790±2740 1054±254 82±47Rat 5 2129±333a 400±146a 171±35a 104±23Marmoset 5 186±111b,c 33±33b#,c 27±11b#,c NDb,c

Table 3 Species differences between Kmand Vmax values of lipaseactivity for DEHP in hepatic microsomes from mouse, rat, andmarmoset

Mouse Rat Marmoset

Km (mmol L�1) 0.012 0.006 1.357Vmax(nmol mg�1 protein min�1)

3.91 1.32 0.49

Vmax/Km 333 227 1.38

Values represent the mean of triplicate analyses for each group

Fig. 1 Lineweaver–Burk plot oflipase activity using hepaticmicrosomes from mouse, rat,and marmoset. Each pointrepresents results from triplicatedetermination using pooledmicrosomes

The substrate concentration (DEHP) used was 1.0 mmol L�1

ND, not detected (<1 pmol mg�1 protein min�1)Values represent the mean±standard deviation for each groupaSignificant difference between mice and rats (P<0.05)

bSignificant difference between rats and marmosets (P<0.05)cSignificant difference between mice and marmosets (P<0.05)#Significant difference using logarithmic transformation

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ADH

Whereas ADH activity against 2-POET was not de-tected in mouse or rat livers, nor in the lungs or kidneysof any of the species examined (Table 5), it was detectedin the livers from marmosets and in the small intestinefrom all species. ADH activity against 2-EH was, how-ever, detected in all organs, most prominently in thelivers of all the species, followed in order by the smallintestine and lungs. It should be noted that this activitywas highest for marmosets, which was quite differentfrom the results for lipase or UGT activity.

ALDH

ALDH activity against 3-phenylpropionaldehyde and 2-ethylhexanal was detected in all species (Table 6). In

general, activity against these two substrates was lowestin all the mice tissues examined. When 3-phenylpropi-onaldehyde was used as substrate, activity was mostprominent in either the mitochondrial or post-mito-chondrial fractions of all marmoset organs examined,with particularly high activity observed in the smallintestine and kidneys. ALDH activity against 2-ethyl-hexanal was highest in the mitochondria of rat livers andmarmoset kidneys, however. No species differencesamong ALDH activity were seen in the small intestine orlungs. These results are very similar to those for ADHactivity, suggesting that ADH and ALDH activity arehigher in primates than in small experimental animals.

Discussion

Clear-cut differences were seen among the activity of thefour enzymes involved in DEHP metabolism of mice,rats, and marmosets. The most prominent difference wasobserved in the first step of DEHP metabolism (hydro-lysis to MEHP and 2-EH by lipase) with an almost 150-to 350- fold difference between the highest activity inmice and the lowest in marmosets. These differenceswere comparable with those between the kinetic dataVmax and Km, i.e. the affinity of DEHP for lipase inmarmosets was much lower than in rats or mice, andconstitutive expression of the enzyme was markedlylower in the former than in the latter. Species differenceswere also found for the activity of the other enzyme, butthey were not as great as those detected in lipase activity.Taken together, these results suggest that lipase mightplay a major role in interspecies differences in DEHPmetabolism.

Table 5 Species differences between ADH activity (nmol mg�1

protein min�1) for 2-ethylhexanol and 2-phenoxyethanol

Mouse(n=6)

Rat(n=5)

Marmoset(n=5)

2-EH (10 lmol L�1)Liver 32.7±4.4 26.3±6.2 43.3±6.2b,c

Small intestine 3.55±2.37 10.72±1.36a 13.78±7.26c

Kidney 0.43±0.85 0.65±0.38 NDLung 2.21±2.48 3.47±1.47 4.72±0.29

2-POET (10 lmol L�1)Liver ND ND 11.25±2.5Small intestine 1.80±1.97 5.69±2.47 1.72±2.55Kidney ND ND NDLung ND ND ND

ND, not detected (<0.16 nmol min�1)Values represent the mean±standard deviation for each groupaSignificant difference between mice and rats (P<0.05)bSignificant difference between rats and marmosets (P<0.05)cSignificant difference between mice and marmosets (P<0.05)

Table 6 Species differences between ALDH activity (nmol mg�1

protein min�1) for 3-phenylpropionaldehyde and 2-ethylhexanal

Mouse(n=6)

Rat(n=5)

Marmoset(n=5)

2-Ethylhexanal (10 lmol L�1)Post-mitochondrial fraction

Liver 7.7±2.9 14.0±2.4a 12.9±2.7Mitochondrial fraction

Liver 5.4±1.9 32.8±10.8a 14.4±7.6b

Small intestine 2.56±1.38 6.68±2.77 7.86±4.89Kidney ND 4.74±0.81 22.11±1.71b,c

Lung 2.68±0.93 5.14±1.60a 4.64±0.723-Phenyl propionaldehyde (10 lmol L�1)Post-mitochondrial fraction

Liver 9.9±1.8 15.9±2.4a 25.8±0.6b,c

Mitochondrial fractionLiver 6.1±1.3 21.0±5.2a 27.3±4.5c

Small intestine 2.77±2.40 9.95±0.98a 38.65±5.10b,c

Kidney ND 3.79±0.92 39.30±1.98b

Lung 2.62±1.24 4.07±1.93 8.15±2.70b,c

ND, not detected (<0.16 nmol min�1)Values represent the mean±standard deviation for each groupaSignificant difference between mice and rats (P<0.05)bSignificant difference between rats and marmosets (P<0.05)cSignificant difference between mice and marmosets (P<0.05)

Table 4 Species differences between hepatic UGT activity (nmolmg�1 protein min�1)

Substrate Mouse(n=6)

Rat(n=5)

Marmoset(n=5)

MEHP (1 mmol L�1)Liver 0.66±0.10 0.51±0.12 0.25±0.04b,c

Bis-phenol A (1 mmol L�1)Liver 21.63±2.10 10.80±2.82 79.22±10.33b,c

Small intestine 4.21±1.01 1.84±1.11 15.34±4.02Kidney 2.43±0.23 0.93±0.25 NDLung ND ND ND1-Naphthol (1 mmol L�1)Liver 5.91±1.41 8.53±1.23 6.30±2.18Small intestine 1.55±0.52 2.80±2.03 1.23±0.90Kidney 1.19±0.67 11.38±4.35a 3.11±0.74Lung 9.05±7.41 1.67±0.11a ND

ND, not detected (bis-phenol A, <0.005 nmol min�1 mg�1;1-naphthol, <0.01 nmol min�1 mg�1)Values represent the mean±standard deviation for each groupaSignificant difference between mice and rats (P<0.05)bSignificant difference between rats and marmosets (P<0.05)cSignificant difference between mice and marmosets (P<0.05)

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The toxicity of DEHP is thought to be due to MEHPor its metabolites, rather than to DEHP itself (Daviset al. 1994; Albro et al. 1989). A plausible mechanism ofthe toxic action of MEHP might be that it arises via thenuclear receptor PPARa—MEHP acts on the nuclearreceptor PPARa, which regulates several kinds of per-oxisomal and mitochondrial enzymes (Moody et al.1991). Thus, the pleiotropic toxic actions of DEHPoccur in several organs (Huber et al. 1996). Our resultssuggest that the significant species differences in themetabolism of DEHP lie primarily in the first step, thehydrolysis of DEHP by lipase, which might result indifferent levels of MEHP in the tissues. The internaldoses of MEHP in organs might be greatest in mice,followed by those in rats and marmosets, whenever theexternal exposure is similar. In fact, we recently con-firmed this hypothesis (unpublished data), and this resultagreed with that of Kessler et al. (2004): the maximumconcentration of MEHP in blood from DEHP-treatedrats averaged 3.2 times higher (range 1.3–7.5) than inmarmosets exposed to the same concentration of DEHP.This information should be very valuable for assessmentof the risk of DEHP, where external doses are usuallyused. Species differences might also exist in the func-tional activation of PPARa by peroxisomal proliferatorssuch as DEHP (Hasmall et al. 2000; Tugwood et al.1996; Palmer et al. 1998; Kurata et al. 1998). It is gen-erally accepted that the activation in rats or mice occurson exposure to DEHP, though not to any appreciableextent in primates (Roberts 1999). Our results suggestthat the species difference in the kinetics of DEHP, i.e.with regard to its ability to form MEHP, contribute tothe species difference in the activation of PPARa,although it is also possible that there are constitutivevariations in the expression or functional differences ofPPARa among species (Sher et al. 1993; Tugwood et al.1996; Tugwood et al. 1998; Palmer et al. 1998; Robertset al. 1998).

Lipase activity comparable with or greater than thatin the liver was seen in the small intestine, with ten-folddifferences observed between mice and rats, and betweenrats and marmosets. Although we did not measurepancreatic lipase activity, the pancreas seems to have thehighest activity among organs (Albro and Thomas 1973;Albro 1986). These results suggest that orally-ingestedDEHP is quickly hydrolyzed to MEHP by lipase frompancreas and lipase in the small intestine, and absorbedfrom the small intestine into the system (Sjoberg et al.1985). In rats, the concentration of unhydrolyzed DEHPin the liver increased when DEHP concentration in thediet exceeded 0.43%, whereas no similar observationwas made in mice (Albro et al. 1982). In marmosets,neither induction of peroxisomes nor testicular damagewas observed after oral administration of a relativelyhigh dose of DEHP (Kurata et al. 1998; Pugh et al.2000). These results might be because lipase activityin the small intestine decreases in the ordermice>rats>marmosets.

The low lipase activity in the lungs observed in thisstudy agrees with a previous report that pancreas, liver,and intestinal mucosa contain the bulk of DEHPhydrolase activity in rats, although less activity wasfound in kidney and lung (Albro and Thomas. 1973).Because inhaled DEHP is hardly hydrolyzed in thelungs, the absorption of MEHP from the lungs might bemuch lower than that from the small intestine. Indeed,Pollack et al. (1985) reported that the rate of hydrolysisof DEHP was only 1% when the chemical was inhaled.Although there are many reports of DEHP havingreproductive or developmental toxicity when adminis-tered orally, such toxicity was not seen when DEHP wasadministered alveolarly (Klimisch et al. 1992). InhaledDEHP thus seems to have a very low toxicity comparedwith orally administered DEHP.

Daily exposure to DEHP in a medical setting mayexceed that in the general population by up to threeorders of magnitude (Tickner et al. 2001). High levels ofdistributed DEHP could be metabolized to MEHP inboth the liver and small intestine of mice or rats, butonly a small amount was metabolized in both the liverand small intestine of marmosets (and possibly in mostprimates). If humans have a metabolic capacity similarto that of marmosets, DEHP might not be readilymetabolized to MEHP in either the human liver or thesmall intestine. Although lipase activity in the smallintestine of humans was reported to be lower than thatfor the primate baboon (Lake et al. 1977), our resultscannot exclude the possibility that DEHP has adverseeffects on humans, because there is insufficient datarelating to the kinetics of DEHP in humans and thequestion remains as to whether all toxic effects could beattributed solely to MEHP levels in the target organs.ADH and ALDH activity was generally lower in miceand rats than in marmosets, suggesting that x- or x-1oxidized metabolites of MEHP by CYP4A are moredifficult to metabolize further in the former than in thelatter. In marmosets, however, formation of MEHPitself is so very low that formation of subsequentmetabolites might not be as great as that predicted byuse of these enzyme activity values. In contrast, althoughthere are species differences between the activity of ADHand ALDH for 2-EH, the metabolites 2-EHA and thedicarboxylic acids might be formed in all animals. Thesemono- and dicarboxylic acids could also act on PPARa,and might thereby be involved in adverse effects byactivation of PPARa.

Although we could not measure CYP4A activityagainst MEHP, immunoblot analysis clearly showed theconstitutive expression of CYP4A was greater in ratsthan in mice and marmosets if there was no speciesdifference for the affinity of anti-CYP4A (unpublisheddata). This finding might provide evidence of speciesdifferences in the urinary metabolites of DEHP: primarymetabolites in rats are MEHP oxidative metabolites andthose in mice and marmosets include many MEHP andMEHP-glucuronides (Astill et al. 1986).

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In conclusion, clear-cut species differences wereobserved for DEHP-metabolizing enzymes. Among theenzymes examined species differences were most prom-inent for lipase, which is involved in the first step ofDEHP metabolism. This results in higher internal con-centrations of PPARa-binding MEHP, and also suggeststhe presence of higher concentrations of 2-EHA andother dicarboxylic acids in mice or rats than in mar-mosets. If kinetic data for the action of lipase on DEHPin humans are similar to those in marmosets, we shouldnote a possible species difference on extrapolation fromrodents to humans. Internal doses of mono- and dicar-boxylic metabolites of DEHP may be more useful thanexternal doses for risk evaluation.

Acknowledgements This study was supported by a research grantfrom the Japan Ministry of Environment (2001) and a Grant-in-Aid for JSPS fellows from the Japan Ministry of Education, Cul-ture, Sports, Science and Technology (14,2531).

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