Human biofluid concentrations of mono(2-ethylhexyl)phthalate extrapolated from pharmacokinetics in chimeric mice with humanized liver administered with di(2-ethylhexyl)phthalate and physiologically based pharmacokinetic modeling

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Human biofluid concentrations of mono(2-ethylhexyl)phthalate extrapolated from pharmacokinetics in chimeric mice with humanized liver administered with di(2-ethylhexyl)phthalate and physiologically based pharmacokinetic modeling Koichiro Adachi a , Hiroshi Suemizu b , Norie Murayama a , Makiko Shimizu a , Hiroshi Yamazaki a,∗ a b

Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan Central Institute for Experimental Animals, Kawasaki-ku, Kawasaki 210-0821, Japan

a r t i c l e

i n f o

a b s t r a c t

Article history:

Di(2-ethylhexyl)phthalate (DEHP) is a reproductive toxicant in male rodents. The aim of

Received 31 December 2014

the current study was to extrapolate the pharmacokinetics and toxicokinetics of mono(2-

Received in revised form

ethylhexyl)phthalate (MEHP, a primary metabolite of DEHP) in humans by using data from

11 February 2015

oral administration of DEHP to chimeric mice transplanted with human hepatocytes. MEHP

Accepted 14 February 2015

and its glucuronide were detected in plasma from control mice and chimeric mice after sin-

Available online 20 March 2015

gle oral doses of 250 mg DEHP/kg body weight. Biphasic plasma concentration–time curves

Keywords:

extensively excreted in urine within 24 h in mice with humanized liver. In contrast, fecal

of MEHP and its glucuronide were seen only in control mice. MEHP and its glucuronide were PBPK modeling

excretion levels of MEHP glucuronide were high in control mice compared with those with

Allometric scaling

humanized liver. Adjusted animal biomonitoring equivalents from chimeric mice studies

Species difference

were scaled to human biomonitoring equivalents using known species allometric scaling

Urine

factors and in vitro metabolic clearance data with a simple physiologically based pharma-

Phthalate

cokinetic (PBPK) model. Estimated urine MEHP concentrations in humans were consistent with reported concentrations. This research illustrates how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of pharmacokinetics or toxicokinetics of the primary or secondary metabolites of DEHP. © 2015 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165 Higashitamagawa Gakuen, Machida, Tokyo 194-8543, Japan. Tel.: +81 42 721 1406; fax: +81 42 721 1406. E-mail address: [email protected] (H. Yamazaki).

http://dx.doi.org/10.1016/j.etap.2015.02.011 1382-6689/© 2015 Elsevier B.V. All rights reserved.

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1.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 1067–1073

Introduction

Recent biomonitoring techniques for determining various manmade and naturally occurring chemicals have become valuable tools for quantitatively evaluating human exposure from environmental and/or incidental sources. The phthalate di(2-ethylhexyl)phthalate (DEHP, Fig. 1) is used in the production of polyvinyl chloride as a plasticizer and exhibits low toxicity from both acute and chronic exposures (Koch et al., 2006). Primary and secondary phthalate monoester metabolites of DEHP (Fig. 1) have been detected in human urine (Herr et al., 2009). DEHP is reportedly rapidly hydrolyzed to mono(2-ethylhexyl)phthalate (MEHP) in microsomal/cytosolic fractions of selected human organs (Choi et al., 2012). Studies have shown that absorbed monoester metabolites are usually oxidized in human bodies and excreted in urine, largely as glucuronide conjugates (Albro et al., 1982). Although no information is available on the chronic, reproductive, developmental, or carcinogenic effects of DEHP in humans, animal studies have reported that oral exposure has resulted in developmental, reproductive, or carcinogenic effects in rats and mice (Kluwe et al., 1983). A study by the United State National Toxicology Program showed that DEHP administered orally increased the incidence of liver tumors in rats and mice: DEHP has been classified as a Group B2, probable human carcinogen (Rusyn and Corton, 2012), as its hazard summary shows (US EPA, 1998). Comparisons of metabolic profiles of MEHP, a primary metabolite of DEHP, between rats, marmosets, and/or humans have been recently reported (Rhodes et al., 1986; Kurata et al., 2012a,b) without significant toxicological effects on animals in the range of 100–2000 mg DEHP/kg body weight. In a German study conducted in order to assess infant exposure to DEHP in infants (Volkel et al., 2014), the 95-percentile daily intake values of DEHP calculated from biomonitoring data have been 0.0054 mg/kg for infants and 0.0233 mg/kg for their mothers. There has been important understanding that a wide range of assumed linearity from the non-toxicological levels in animal toxicokinetic experiments to actual human daily exposure doses of chemicals is generally accepted. A lot of work has been reported on the in vitro–in vivo extrapolations of hepatic clearance, volume of distribution, and on the estimation of unbound microsomal fraction (Poulin and Haddad, 2013). In the present study, the pharmacokinetics of DEHP in chimeric mice transplanted with human hepatocytes were investigated. Our observations showed that transplanted human hepatocytes were able to effect the excretion of primary and secondary metabolites of DEHP into urine in chimeric mice. A simplified physiologically based pharmacokinetic (PBPK) model was able to estimate human plasma

and urine concentrations of MEHP after ingestion of DEHP and was capable of both forward and reverse dosimetry.

2.

Materials and methods

2.1.

Chemicals, animals, and enzyme preparations

DEHP, MEHP, and ␤-glucuronidase (2000 units/mg protein, Ampullaria source) were purchased from Wako Pure Chemicals (Osaka, Japan). Uridine diphosphate glucuronic acid (UDPGA) was obtained from Sigma–Aldrich (St. Louis, MO, USA) and pooled human liver microsomes (H150) from Corning (Woburn, MA, USA). Liver microsomes from mice were prepared as described previously (Tsukada et al., 2013). Recently developed TK-NOG mice (Hasegawa et al., 2011; Yamazaki et al., 2012; Higuchi et al., 2014) are treated to express a herpes simplex virus type 1 thymidine kinase within the livers of severely immunodeficient NOG (non-obese diabetic/severe combined immunodeficiency/interleukin-2 receptor gamma chain-deficient) mice. TK-NOG mice were induced by a nontoxic dose of ganciclovir and then human liver cells were transplanted without the need for ongoing drug treatment. Control mice (TK-NOG mice with no transplanted human hepatocytes) and humanized TK-NOG mice (∼20–30 g body weight) (Hasegawa et al., 2011) were used in this study. In the chimeric mice, more than 70% of liver cells were estimated to have been replaced with human hepatocytes, as judged by measurements of human albumin concentrations in plasma (Hasegawa et al., 2011; Yamazaki et al., 2012). Hereafter, the terms “mouse” or “mice” refer to control TK-NOG mice. The use of animals for this study was approved by the Ethics Committees of the Central Institute for Experimental Animals and Showa Pharmaceutical University. Other reagents used in this study were obtained from sources described previously or were of the highest quality commercially available (Suemizu et al., 2014).

In vitro and in vivo metabolic studies of DEHP 2.2. and MEHP Elimination rates of MEHP for liver microsomes from control mice, humanized mice, and humans were measured using a liquid chromatography (LC) system. Briefly, a typical incubation mixture consisted of 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system, 10 mM MgCl2 , 3 mM UDPGA, liver microsomes (0.10 mg protein/mL) with pretreatment with 50 ␮g/mL alamethicin, and DEHP or MEHP (100 ␮M) in a final volume of 0.25 mL. Incubations were carried out at 37 ◦ C for 15–30 min. Reactions were terminated by adding 0.5 mL of acetonitrile. Supernatant samples (50 ␮L)

Fig. 1 – Metabolic pathway of di(2-ethylhexyl)phthalate to mono(2-ethylhexyl)phthalate and its glucuronide.

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Fig. 2 – PBPK model established in this study for mice with humanized liver and humans.

after centrifugation at 13,000 × g for 10 min were injected to the LC system with an auto-sampler. Plasma samples were collected from mice 1, 2, 4, 7 and 24 h after non-oxic single oral doses of DEHP (250 mg/kg). Accumulated urine and feces samples (0–24 h) from the mice were collected. Plasma and urine samples (10 ␮L) were deproteinized by adding 40 and 90 ␮L, respectively, of acetonitrile for metabolite analyses. Feces (50 mg) were suspended in 0.5 mL acetonitrile by vortex mixing. The supernatant after centrifugation at 13,000 × g for 20 min (400 ␮L) was evaporated to dryness under N2 . The residue was dissolved in 150 ␮L of 100 mM potassium phosphate buffer (pH 7.4) and used as the feces analyte (60 ␮L). To hydrolyze the ␤-glucuronides, plasma, urine, and feces analytes (10 ␮L) were treated with ␤-glucuronidase (200 units) at 52 ◦ C for 4 h in 10 mM potassium phosphate buffer (pH 7.4) in total volumes of 20 ␮L; 80 ␮L of acetonitrile was added after termination. After centrifugation at 13,000 × g for 10 min, supernatant samples (50 ␮L) were injected into the LC with an auto-sampler. The recovery of DEHP and MEHP was >90% in the deproteinized plasma and urine samples. The LC system consisted of a pump and a multi-wavelength UV detector (Shimadzu, Kyoto, Japan) using an analytical C18 reversed-phase column (5 ␮m, 4.6 mm × 250 mm, Mightysil RP-18 GP 2, Kanto Chemicals, Tokyo, Japan). DEHP, MEHP, and their metabolites were eluted with 65% (v/v) CH3 CN in 0.1% (v/v) acetic acid at a flow rate of 1.0 mL/min at 40 ◦ C and monitored at a wavelength of 240 nm. The substrate and metabolite were quantified on the basis of the standard curve peak areas of DEHP or MEHP with retention times of 5.6 and 14.7 min, respectively. Relative standard deviations for data in this study were within <15% in the range of 1–1000 ␮g/mL of DEHP or MEHP.

2.3. Estimation of plasma concentrations using a simplified PBPK model with suitable parameters The simplified PBPK model (Fig. 2) consisted of a chemical receptor gut compartment, a metabolizing liver compartment, and a central compartment and was set up as described previously (Takano et al., 2010; Yamazaki et al., 2010; Tsukada et al., 2013; Yamashita et al., 2014). Values of the plasma unbound fraction (fu,p ) and the octanol–water partition coefficient (log P) were obtained by in silico estimation using Simcyp

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and Chemdraw software (Emoto et al., 2009) and are shown in Table 1; the liver-to-plasma concentration ratio (Kp,h ) and the blood-to-plasma concentration ratio (Rb ) were estimated from fu,p and log P (Poulin and Theil, 2002). Parameters that represent physiological properties such as hepatic blood flow rates in mice (0.160 L/h) and humans (96.6 L/h) were taken from the literature (Kato et al., 2008; Gargas et al., 1995). Values of the absorption rate constant (ka ), hepatic clearance (CLh ), hepatic intrinsic clearance (CLh,int ), and the volume of the systemic circulation (V1 ) were also calculated (Tsukada et al., 2013; Yamashita et al., 2014; Suemizu et al., 2014). Subsequently, final parameter values for the chimeric mouse PBPK models were calculated using the initial values mentioned above by the user model in WinNonlin so as to give the best fit to measured Cb values; these final parameters including standard deviation values for ka , CLh,int , and V1 are shown in Table 2. Finally, a system of differential equations was solved to conduct the modeling: dXg(t) = −ka · Xg(t) when at t = 0, Xg(0) = dose dt Vh

dCh Q ·C ·R C = Qh · Cb − h h b + ka · Xg − CLh,int · h · fu,p dt Kp,h Kp,h

V1

Q ·C ·R dCb = −Qh · Cb + h h b − CLr · Cb dt Kp,h

where Xg is the substrate amount in the gut, Vh is the volume of liver, Ch is the hepatic substrate concentration, Qh is the hepatic blood flow rate of systemic circulation to the tissue compartment, and Cb is the blood substrate concentration (Fig. 2). For the respective substrate metabolites: V1

Q ·C ·R dCb = −Qh · Cb + h h b − CLr · Cb dt Kp,h

Vh

dCh Q ·C ·R C = Qh · Cb − h h b − CLh,int · h · fu,p dt Kp,h Kp,h + CLh,int,metabolite ·

Ch,metabolite ·f Kp,h,metabolite u,p,metabolite

To define a simplified human PBPK model based on the chimeric mouse PBPK model, we used relevant liver microsomes and physiological parameters (CL, ka , and V1 ) derived from the literature and applied the systems approach to fit them into the traditional parallelogram for risk assessment (Edwards and Preston, 2008). The values of CLh,int , ka , and V1 in the human PBPK model were estimated using a scale-up strategy from mice to humans as described previously (Tsukada et al., 2013). The human absorption rate constant (ka ) was estimated as reported (Amidon et al., 1988). The human hepatic (or renal) clearance CLhuman and the systemic circulation volume (V1,human ) were estimated using Vh,human , volume of blood, Vb,rodent , and Vb,human values of 1.50 L, 0.00160 L, and 4.90 L, respectively (Tsukada et al., 2013). Physicochemical parameters such as Kp,h , Rb , and fu,p (Tsukada et al., 2013) were assumed to be the same for humans and for mice. The in vivo hepatic intrinsic clearance (CLh,int ) in humans was estimated by multiplying the calculated initial parameters for in vitro hepatic intrinsic clearance values in humans by the

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Table 1 – Chemical properties of MEHP and MEHP-O-glucuronide. Parameter

Symbol

Molecular weight Octanol–water partition coefficient Plasma unbound fraction Fraction unbound in microsomes Blood-to-plasma concentration ratio Liver-to-plasma concentration ratio

MW log P fu,p fu,mic Rb Kp,h

MEHP

MEHP-O-glucuronide

278 4.71 0.00700 0.786 0.718 2.90

454 3.06 0.113 0.840 2.73

Table 2 – Physiological, experimental, and calculated parameters for humanized mouse and human PBPK models for MEHP. Parameter Absorption rate constant Volume of systemic circulation for MEHP Hepatic clearance for MEHP Hepatic intrinsic clearance for MEHP Metabolic ratio to MEHP-O-glucuronide Renal clearance for MEHP Volume of systemic circulation for MEHP-O-glucuronide Hepatic clearance for MEHP-O-glucuronide Hepatic intrinsic clearance for MEHP-O-glucuronide Renal clearance for MEHP-O-glucuronide

Symbol (unit) ka (1/h) V1 MEHP (L) CLh,MEHP (L/h) CLh,int MEHP (L/h) CLr MEHP (L/h) V1 MEHP-o-glucuronide (L) CLh,MEHP-o-glucuronide (L/h) CLh,int MEHP-o-glucuronide (L/h) CLr MEHP-o-glucuronide (L/h)

Humanized mouse

Human

9.44 (±3.30) 0.0362 (±0.0125) 0.00147 1.66 (±0.35) 1.00 0.00161 0.000100 (±0.00040) 0 0 0.00184

7.02 112 27.0 3840 1.00 0.319 16.0 0 0 1.00

Values in parentheses are standard deviations.

ratio of in vivo to in vitro hepatic intrinsic clearance in mice, as mentioned above for modeling. Then, the final parameters for the human PBPK model were calculated using these initial values by the user model in WinNonlin. As was done for the mouse and chimeric mouse models, systems of differential equations were solved to determine the concentrations in each compartment in humans.

3.

Results

Male control mice and chimeric mice were orally administered 250 mg DEHP/kg. Absorbed DEHP in the plasma was under detection limit (<1 ␮g/mL) and was judged to be completely converted to MEHP and its glucuronide in vivo under the present conditions because a small amount of DHEP was detected in the plasma from mice 1 h after oral administration of 2500 mg DEHP/kg (one-tenth of 50% lethal dose in rats) in our preliminary experiments (data not shown). Fig. 3A shows the mean levels of MEHP and its glucuronide in plasma from control mice and chimeric mice. Chimeric mice with humanized liver had lower MEHP plasma concentrations than the control mice did (Fig. 3A). Biphasic plasma concentration–time curves of MEHP and its glucuronide were seen only in control mice. The amount of MEHP-O-glucuronide excreted in urine in chimeric mice was significantly higher than that in control mice (Fig. 3B). In contrast, the amount of MEHP-O-glucuronide excreted in feces in chimeric mice was significantly lower than that in control mice (Fig. 3C). The pharmacokinetics of MEHP in chimeric mice transplanted with human hepatocytes orally administered with DEHP suggested a higher clearance of MEHP from plasma via the urinary pathway in mice with humanized livers than in control mice (Fig. 3). Renal clearance values in Table 2 were calculated from the levels of MEHP and its glucuronide excreted in the accumulated urine samples and from the area under the plasma

concentration–time curve (AUC) values in humanized mice. In vitro MEHP elimination rates mediated by pooled human liver microsomes and mouse liver microsomes were similar for the liver enzyme sources tested (Table 3). The estimated clearance values were calculated using the following values: 40 mg liver microsomal protein per 1 g liver and 1.5 g liver weight per 0.025 kg of mouse body weight (BW) and 1.5 kg liver weight per 70 kg of human BW. Thus, the hepatic intrinsic clearance (CLh,int,in vitro ) rates were calculated based on the substrate elimination rates in liver microsomes (Table 3). The final parameters such as the hepatic intrinsic clearance (CLh,int ), the volume of the systemic circulation (V1 ), and the absorption rate constant (ka ) for chimeric mouse PBPK models (consisting essentially of a chemical receptor gut compartment, a metabolizing liver compartment, and a central compartment, shown in Fig. 2) were obtained as described in Section 2.3. Based on

Table 3 – MEHP elimination and hepatic intrinsic clearance (CLh,int,in vitro ) determined using liver microsomes from pooled human livers, control mice, and chimeric mice with humanized liver. Enzyme source

MEHP elimination (nmol/min/mg protein)

CLh,int,in vitro (L/h)

Human Control mouse Chimeric mouse

5.60 5.20 4.70

255 0.130 0.110

MEHP (100 ␮M) was incubated with liver microsomes at 37 ◦ C for 30 min. Metabolites were separated with reverse-phase LC. Estimated clearance values (CLh,int,in vitro ) were extrapolated using the fraction unbound in liver microsomes and the following values: 40 mg liver microsomal protein per 1 g liver and 1.5 g liver weight per 0.025 kg of mouse body weight, or 1.5 kg liver weight per 70 kg of human body weight.

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(A)

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(C)

(B)

Fig. 3 – Plasma concentrations (A) and urinary (B) and fecal (C) excretion of mono(2-ethylhexyl)phthalate (MEHP, circles) and its glucuronide (triangles) in mice with humanized liver (closed) and without humanized liver (open) after single oral dose of 250 mg DEHP per kg body weight. Data with bars represent means ± SDs in mice with and without humanized liver for five animals after single oral administration of DEHP (***p < 0.001 and *p < 0.05, two-way ANOVA).

these in vivo experiments, the kinetic parameters in chimeric mice were calculated and are shown in Table 2. By solving the equations that make up the simplified PBPK models, plasma concentration curves were created; the resulting estimated in silico concentration curves are shown in Fig. 4. Using the algometric scaling method and the derived values shown in Table 2, human PBPK models for MEHP and its metabolite MEHP-O-glucuronide were set up based on the PBPK models for humanized mice (Fig. 4B). In terms of the goodness of fit, chi-square values for MEHP and MEHP-Oglucuronide were 28 and 493 (p < 0.001, 5 degrees of freedom), indicating a good fitting of the experimental data in the humanized mice PBPK model. The available reported human urine data (Kurata et al., 2012b) obtained after administration of a single low dose of 0.04 mg DEHP/kg in human subjects could be reasonably estimated by the human PBPK models under the linear assumption, as shown in Fig. 4C. Fig. 4D indicates the estimated urinary concentrations after repeated oral administration of DEHP (0.04 mg/kg) for 7 days using the PBPK

(A)

(B)

models for humans. When daily administration of DEHP for 7 days was modeled, metabolite excretions were evident in humans (Fig. 4D).

4.

Discussion

In this study, we investigated the pharmacokinetics of the phthalate DEHP, which was cleared from humanized mice mainly by liver metabolism and renal excretion. A full PBPK model for DEHP and MEHP in rats has been reported (Keys et al., 1999). To support work by regulatory and industrial researchers, simplified PBPK models for DEHP, MEHP, and their primary metabolites were developed using a combination of algorithms, in vivo experimentation with chimeric mice with humanized livers, and literature resources. According to the current simplified PBPK analysis, MEHP cleared similarly from plasma in humans, but low levels of MEHP cleared from plasma via the urine pathway in humans differently from that in control mice, i.e., fecal excretion. The

(C)

(D)

Fig. 4 – Plasma (A, B) and urinary (C, D) concentrations of MEHP (circles) and its glucuronide (triangles) in mice with humanized liver (A) and in humans (B–D) after single oral doses of 250 mg/kg (A) and 0.04 mg/kg (B, C) di(2-ethylhexyl)phthalate (DEHP), respectively, and multiple doses of 0.04 mg/kg/day for a week (D).

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reason for this difference is that humans and control mice showed enterohepatic circulation because MEHP clearance was dependent on the humanized liver function. Rates of MEHP-glucuronidation in liver microsomes were similar in humans and mice species (Table 3), but the excretion route of MEHP-O-glucuronide as evident in chimeric mice with humanized liver (Fig. 3B), may be an important determinant factor for species-dependent toxicological risk of MEHP. The same kind of differences has been reported between the MEHP pharmacokinetics of rats and marmosets (Kurata et al., 2012a). The present simplified PBPK model was able to estimate human plasma and urine concentrations of MEHP after ingestion of DEHP and was also capable of reverse dosimetry. The Fourth National Report on Human Exposure to Environmental Chemicals, Updated Tables, August 2014, provides nationally representative biomonitoring data that has become available since 2009 (US CDC, 2009; Silva et al., 2004). Geometric mean and 95th percentile values of urinary MEHP concentrations for men in the USA in 2005–2006 were 3.4 and 49.8 ␮g/L, respectively, which were the highest values in the period 1999–2010. These MEHP concentrations in urine imply exposure to 0.087 ␮g/kg/day and 1.3 ␮g/kg/day DEHP by reverse dosimetry with the current human PBPK model. These estimated DEHP exposures were less than the daily tolerable intake of DEHP (30 ␮g/kg/day, Koch et al., 2006; Rusyn and Corton, 2012; or 50 ␮g/kg/day, EU Public Health, 2008), implying little risk in humans under the present conditions. This study developed a biomonitoring strategy for evaluation of human exposure to DEHP by using chimeric mice transplanted with human hepatocytes and simple physiologically based pharmacokinetic model. The proposed method was applied to predict the urinary MEHP concentrations in humans, based on assumed linearity for chemical doses. It has been suggested that urinary phthalate metabolites are associated with decreased serum testosterone in humans (Meeker and Ferguson, 2014). To estimate this biological effects of DEHP, our findings support the assertion that the MEHP and MEHPO-glucuronide, the main primary and secondary phthalate metabolites, can be used in future surveys of human exposure to DEHP (Anderson et al., 2011). To make more precise simulation in an easily simple modeling, dust ingestion, inhalation, and dermal absorption routes of DEHP as evident by recent findings (Fromme et al., 2013) should be considered in future. The current data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential with respect to DEHP and MEHP.

Conflict of interest Drs. Suemizu and Yamazaki report grants from Ministry of Education, Science, Sports and Culture of Japan, outside the submitted work. All other authors declare that there are no conflicts of interest.

Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgments The authors thank Miyuki Kuronuma, Ryoji Takano, and Takamori Miyaguchi for their technical help. This work was supported in part by Japan Chemical Industry Association’s (JCIA) LRI program.

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