Steady-State Human Pharmacokinetics of Monobutyl Phthalate

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Steady-State Human Pharmacokinetics of Monobutyl Phthalate Predicted by Physiologically Based Pharmacokinetic Modeling Using Single-Dose Data from Humanized-Liver Mice Orally Administered with Dibutyl Phthalate Tomonori Miura, Shotaro Uehara, Sawa Mizuno, Manae Yoshizawa, Norie Murayama, Yusuke Kamiya, Makiko Shimizu, Hiroshi Suemizu, and Hiroshi Yamazaki Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00361 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 18, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Chemical Research in Toxicology

Steady-State Human Pharmacokinetics of Monobutyl Phthalate Predicted by Physiologically Based Pharmacokinetic Modeling Using Single-Dose Data from Humanized-Liver Mice Orally Administered with Dibutyl Phthalate

Tomonori Miura,† Shotaro Uehara,‡ Sawa Mizuno,† Manae Yoshizawa,† Norie Murayama,† Yusuke Kamiya,† Makiko Shimizu,† Hiroshi Suemizu,‡ and Hiroshi Yamazaki*,†

†

Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan

‡

Laboratory Animal Research Department, Central Institute for Experimental Animals, Kawasaki-ku, Kawasaki 210-0821, Japan

*Corresponding

author:

Hiroshi Yamazaki, Ph.D. Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165

Higashi-tamagawa

Gakuen,

Machida,

Tokyo

194-8543,

Japan.

Phone:

+81-42-721-1406. Fax: +81-42-721-1406. E-mail address: [email protected]

1 ACS Paragon Plus Environment


Table of Contents Graphic

[MBP] and [MBP-O-glucuronide], g/mL in plasma

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

100 MBPglucuronide

10

1

Humanized-liver mouse

0.1

Control mouse MBP

0.01 0

8

16

24

Time after oral administration of dibutyl phthalate (DBP), h


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Abstract: Dibutylphthalate (DBP) was widely used as a plasticizer, but it has been recently replaced with other kinds of phthalates such as di(2-ethylhexyl)phthalate and diisononylphthalate because of its toxicity. To evaluate the human risk of DBP, forward and reverse dosimetry was conducted using in silico simplified physiologically based pharmacokinetic (PBPK) modeling based on in vivo experimental pharmacokinetic data in humanized-liver mice (HL-mice) obtained after an oral dose of 100 mg/kg. Absorbed DBP was converted to monobutylphthalate (MBP) and its glucuronide extensively in vivo. HL-mice had higher concentrations of MBP glucuronide in plasma than the control mice did. Concentrations of MBP glucuronide in 0–7 h accumulated urine samples from HL-mice were significantly higher than those in control mice. Similarly, in vitro MBP glucuronidation rates mediated by pooled microsomes from rat or mouse livers were lower than those mediated by human liver microsomes. Liver damage by MBP to humanized liver was detected by measuring human albumin mRNA in HL-mouse plasma. By simple PBPK modeling, in silico concentration curves in plasma, liver or urine following virtual oral administration of DBP were created for rats, control mice, and HL-mice. A human PBPK model for MBP was established based on the HL-mouse PBPK model using allometric scaling without consideration of interspecies factors in terms of liver metabolism. Human PBPK models were used to estimate urinary and plasma concentrations of MBP and its glucuronide throughout 14 days of oral DBP administration (1.2 and 13 µg/kg/day). Reverse dosimetry PBPK modeling found that reported 50th and 95th percentile MBP urine and plasma concentrations of the general population could potentially imply exposures similar to or exceeding tolerable daily intake levels (5–10 μg/kg/day) recommended by the European and Japanese authorities. Further in-depth assessment of DBP is needed to assess the validity of assumptions made based on human biomonitoring data.


Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Keywords: Glucuronide; chimeric mouse with humanized liver; Species difference; urinary excretion; reverse dosimetry.

Introduction Rodents (most commonly mice and rats) are extensively used to assess the toxicity of chemicals. However, species differences in terms of drug metabolism and disposition often make it difficult to predict human toxicity based on rodent data. Recent biomonitoring techniques for determining internal concentrations in human fluids of various naturally occurring and human-made chemicals have proven useful to evaluate human exposures to external doses of environmental or incidental sources. Dibutylphthalate (DBP),1,2 which was widely used as a plasticizer for interior and outdoor polymer applications and the primary packaging of medicinal products, has been recently replaced by other phthalates with longer carbon chains such as di(2-ethylhexyl)phthalate (DEHP) and diisononylphthalate (DINP). Monobutylphthalate (MBP), the primary phthalate monoester derived from DBP, and its secondary metabolites have been detected in human milk3 and urine.4,5 Studies have shown that dialkylphthalates are rapidly and extensively converted to monoalkylphthalates by intestinal lipases in vivo, and that the absorbed primary monoester phthalate metabolites are generally oxidized in human livers and excreted into urine, mostly as glucuronidated metabolites.1,6,7 Animal studies have reported that oral exposure to DBP can result in reproductive effects in rats2 and mice8, but not in hamsters.9 However, no consistent results of the effects of DBP have been obtained in human epidemiological studies. In animals treated with DBP or MBP, the concentrations of unconjugated MBP in the urine are reportedly three- to four-fold higher in rats than in hamsters.10 It may be possible that these differences in 4 ACS Paragon Plus Environment

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concentrations of free form of MBP, which is known to produce testicular damage in rats in vitro,9 may explain the absence of damage in hamsters seen after oral administrations of either MBP or DBP. Urinary concentrations of DBP metabolites in adult European subjects in the general population were used to estimate the daily intake of DBP. In 2003, the estimated daily intake of DBP was 1.9 μg/kg body weight/day, and this figure had decreased continuously since 1996.11 In 2005, the tolerable daily intake (TDI) of DBP was reduced to 10 μg/kg body weight/day by the European Food Safety Authority.12 A 2005 study in the United States used modeling to calculate the 95th percentile of daily exposure of DBP in the general population as 2.68 μg/kg/day.5 It has also been shown that the DBP (3.6 mg) used in the formulation of enteric coatings for medicinal capsules has the potential to cause exposures higher than the European TDI value of 10 μg/kg/day.12 The Food Safety Commission of Japan13 applied an uncertainty factor of 500 to the smallest lowest-observed-adverse-effect level for DBP of 2.5 mg/kg body weight/day (established in a dietary reproductive–developmental toxicity study in rats) and specified the TDI of DBP as 5 μg/kg/day. Further in-depth assessment of DBP is needed, especially to evaluate the appropriateness of the assumptions made based on reported biomonitoring data in humans. The pharmacokinetics of DEHP14 and DINP15 in immunodeficient mice transplanted with commercially available hepatocytes from human donors [humanized-liver mice (HL-mice)] were previously investigated. Our observations indicated that the transplanted human hepatocytes could mediate the rapid excretion of primary monophthalate esters and their glucuronidated metabolites into urine in HL-mice orally treated with DEHP14 or DINP.15 In the present study, a combination of simplified physiologically based pharmacokinetic (PBPK) modeling of DBP



(which

has

a

shorter

carbon

chain

than

DEHP

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or

DINP)

and

experimental

pharmaco/toxicokinetics was conducted. To assess the risk to humans of phthalate esters, we report herein plasma, liver, and urine concentrations of MBP after ingestion of DBP estimated using a simple PBPK model that can perform both forward and reverse dosimetry. The current results indicated that the glucuronidation rates of the primary metabolite of DBP, MBP, and its excretion routes to the urine/feces were major determinant factors in the metabolism and disposition of diallyl phthalates such as DBP in vivo.

Materials and Methods Chemicals and animals Dibutylphthalate (CAS 84-74-2) and monobutylphthalate (131-70-4) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Uridine diphosphate glucuronic acid (UDPGA) and β-glucuronidase (2000 units/mg protein, Ampullaria source) were purchased from Sigma-Aldrich (St. Louis, MO) and Fujifilm Wako Pure Chemical (Osaka, Japan), respectively. Pooled liver microsomes from Sprague–Dawley rats (3 males, aged ~8 weeks), CD-1 mice (8 males and females, aged 11 weeks), Beagle dogs (10 males, aged >12 months), Gottingen minipigs (2 males, aged 7 months), and humans (74 men and 76 women, aged 18–82 years) were obtained from Corning Life Sciences (Woburn, MA). Pooled liver microsomes from cynomolgus monkeys (5 males, sexually mature) and hamsters (Syrian, 100 males, aged ~8 weeks) were purchased

from

Xenotech

(Kansas

City,

KS).

Male

control

and

humanized-liver

immunodeficient TK-NOG mice (20–30 g body weight)16,17 were used as in vivo models. The


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plasma concentrations of human albumin were determined in HL-mice, and it was estimated that >90% of the TK-NOG mouse liver had been replaced with human hepatocytes. The use of control mice and HL-mice for this research received approval from the Ethics Committees of the Japan Central Institute for Experimental Animals. Other chemicals used in the current study were supplied by previously described sources14,15 or were of the highest quality commercially available. In vitro and in vivo metabolic studies of MBP Typical incubation mixtures contained MBP (100 µM), 3.0 mM UDPGA, 10 mM MgCl2, liver microsomes (0.10 mg protein/mL) pretreated with 50 µg/mL alamethicin, and 50 mM Tris-HCl buffer (pH 6.5) in a final volume of 0.20 mL. Incubations were carried out at 37 °C for 10 min; termination of reactions was achieved by adding 0.20 mL of acetonitrile. Blood samples (~ 30 µL) were collected at times in the range 0.5–24 h after single oral doses of DBP administered by gavage (100 mg/kg) to four animals in each of two groups. For metabolite analyses, plasma and urine samples (10 µL) were deproteinized by adding 40 and 90 µL, respectively, of acetonitrile and centrifuged at 2  103  g for 10 min at 4 °C. To hydrolyze the MBP β-glucuronides, plasma and urine samples (10 µL) were treated with β-glucuronidase (200 units) at 52 °C for 4 h in 10 mM potassium phosphate buffer (pH 7.4) in total volumes of 20 µL; after termination, acetonitrile (80 µL) was added. Centrifugation at 13,000 × g was carried out for 10 min, and 30-µL samples of supernatant were injected into the liquid chromatography (LC) system. Hereafter, MBP



glucuronide concentrations in in vivo samples indicated values calculated from total MBP concentrations minus free MBP concentrations. The LC system had an analytical C18 reversed-phase column (5 µm, 4.6 × 250 mm, Mightysil RP-18 GP 2, Kanto Chemicals, Tokyo, Japan) and consisted of a multiwavelength UV detector and pump (Shimadzu, Kyoto, Japan). MBP and its glucuronide were eluted with 60% (v/v) CH3CN in 0.1% (v/v) acetic acid at 40 °C and monitored at a wavelength of 240 nm at a flow rate of 1.0 mL/min, with retention times of 2.4 and 5.1 min, respectively. The substrate and metabolite were quantified using the standard curve peak areas of MBP. Relative standard deviations for data in this study were within 90% in the deproteinized plasma and urine samples. Statistical analysis for urinary concentrations from control and HL-mice was performed by two-way analysis of variance using Prism software (GraphPad Software, La Jolla, CA). Detection of human albumin mRNA in HL-mice plasma

The concentrations of human albumin mRNA in plasma from HL-mice were analyzed semi-quantitatively using reverse transcription-polymerase chain reaction (PCR), as described previously.18 Briefly, using an miRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany), total RNA fractions were extracted from 15 µL plasma taken from blood samples collected from HL-mice with EDTA-coated tubes. Total RNA (9.0 µg) was reverse transcribed using a High-Capacity RNA-to-cDNA Kit (ThermoFisher Scientific, Waltham, MA). Thirty-three cycles of PCR were performed with the primers for human albumin described previously18 and DNA polymerase TaKaRa La-Taq (Takara, Kusatsu, Japan) using an Applied Biosystems 2720 thermocycler (Thermo Fisher Scientific, Waltham, MA). The semi-quantitative analysis of PCR 8 ACS Paragon Plus Environment

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products (146 bp) was carried out by electrophoresis in 4% agarose gels containing ethidium bromide. Gel images were detected and normalized using units in comparison with those of human albumin transcripts (3.0 fmol) under the present conditions. Utilizing simplified pharmacokinetic models to estimate plasma, liver, and urinary concentrations of phthalates The simplified PBPK models (Figure 1) were composed of a chemical receptor (gut) compartment, a metabolizing (liver) compartment, a central compartment, and a perirenal compartment. The models were established as described previously19 for an orally administered substance and its primary metabolite. The chemical properties of MBP and its glucuronide (Table 1) were calculated as described elsewhere.20-22 The fraction absorbed (Fa) × intestinal availability (Fg) was estimated to be 1. This figure was obtained by applying curve fitting to several plasma concentrations in rats taken from the literature1 and to plasma concentrations in control mice and HL-mice measured in the current experiments. Subsequently, as described elsewhere,23 the final parameter values (with standard deviations) for the simple PBPK models were calculated (Table 2). The important parameters [the absorption rate constant (ka), the hepatic intrinsic clearance (CLh,int), and the systemic circulation volume (V1)] and the rate constants for transfer of the drug from/to the central (first) compartment and to/from the peripheral (second) compartment (k12/k21)19 for the rat, mouse, and HL-mouse models were calculated by the user model in WinNonlin, ver. 5 (Certara, Princeton, NJ), using simplex and modified Marquardt methods, so as to fit reported or determined plasma substrate concentrations. Renal clearance (CLr) was also calculated by fitting curves to several plasma concentrations.19



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The following system of differential equations15,24 were used to model the concentrations of the substrate (MBP) and its primary glucuronidated metabolite (indicated with subscript m): 𝑑𝑋𝑔(𝑡) 𝑑𝑡

= ― 𝑘𝑎 ⋅ 𝑋𝑔(𝑡) when at 𝑡 = 0, 𝑋𝑔(0) = 𝑑𝑜𝑠𝑒

𝑑𝑉ℎ

𝑉ℎ 𝑑𝑡 = 𝑄ℎ ⋅ 𝐶𝑏 ―

𝑄ℎ ⋅ 𝐶ℎ ⋅ 𝑅𝑏 𝐾𝑝,ℎ

𝐶ℎ

+ 𝑘𝑎 ⋅ 𝑋𝑔 ― 𝐶𝐿ℎ,𝑖𝑛𝑡 ⋅ 𝐾𝑝,ℎ ⋅ 𝑓𝑢,𝑝

𝑑𝐶𝑏 𝑄ℎ ⋅ 𝐶ℎ ⋅ 𝑅𝑏 𝑉1 = ― 𝑄ℎ ⋅ 𝐶𝑏 + ― 𝑘12 ⋅ 𝑉1 ⋅ 𝐶𝑏 + 𝑘21 ⋅ 𝑋𝑝𝑒𝑟𝑖𝑝ℎ𝑒𝑟𝑎𝑙 ― 𝐶𝐿𝑟 ⋅ 𝐶𝑏 𝑑𝑡 𝐾𝑝,ℎ 𝑑𝐶𝑢 𝑉𝑢 = 𝐶𝐿𝑟 ⋅ 𝐶𝑏 𝑑𝑡 𝑑𝑋𝑝𝑒𝑟𝑖𝑝ℎ𝑒𝑟𝑎𝑙 𝑑𝑡 𝑉ℎ,𝑚

𝑉1,𝑚

𝑑𝑉ℎ.𝑚 𝑑𝑡

𝑑𝐶𝑏,𝑚

𝑉𝑢,𝑚

𝑑𝑡 𝑑𝐶𝑢,𝑚 𝑑𝑡

= 𝑘12 ⋅ 𝑉1 ⋅ 𝐶𝑏 + 𝑘21 ⋅ 𝑋𝑝𝑒𝑟𝑖𝑝ℎ𝑒𝑟𝑎𝑙

= 𝑄ℎ ⋅ 𝐶𝑏,𝑚 ―

= ― 𝑄ℎ ⋅ 𝐶𝑏,𝑚 +

𝐾𝑝,ℎ,𝑚

+ 𝐶𝐿ℎ,𝑖𝑛𝑡 ⋅

𝑄ℎ ⋅ 𝐶ℎ,𝑚 ⋅ 𝑅𝑏,𝑚 𝐾𝑝,ℎ,𝑚

𝐶ℎ 𝐾𝑝,ℎ

⋅ 𝑓𝑢,𝑝 ― 𝐶𝐿ℎ,𝑖𝑛𝑡,𝑚 ⋅

𝐶ℎ,𝑚 𝐾𝑝,ℎ,𝑚

⋅ 𝑓𝑢,𝑝,𝑚

― 𝑘12,𝑚 ⋅ 𝑉1,𝑚 ⋅ 𝐶𝑏,𝑚 + 𝑘21,𝑚 ⋅ 𝑋𝑝𝑒𝑟𝑖𝑝ℎ𝑒𝑟𝑎𝑙,𝑚 ― 𝐶𝐿𝑟,𝑚 ⋅ 𝐶𝑏,𝑚

= 𝐶𝐿𝑟,𝑚 ⋅ 𝐶𝑏,𝑚

𝑑𝑋𝑝𝑒𝑟𝑖𝑝ℎ𝑒𝑟𝑎𝑙,𝑚 𝑑𝑡

𝑄ℎ ⋅ 𝐶ℎ,𝑚 ⋅ 𝑅𝑏,𝑚

= 𝑘12,𝑚 ⋅ 𝑉1,𝑚 ⋅ 𝐶𝑏,𝑚 + 𝑘21,𝑚 ⋅ 𝑋𝑝𝑒𝑟𝑖𝑝ℎ𝑒𝑟𝑎𝑙,𝑚

where Xg and Xperipheral are the amounts of the chemicals in the gut and peripheral compartments respectively; Ch, Cb, and Cu are the hepatic, blood, and urinary chemical concentrations, respectively; Vh, V1, and Vu are the volumes of the liver, the central compartment, and urine, respectively; and Qh is the hepatic blood flow rate of systemic circulation to the hepatic compartment. The definitions of other parameters in the above equations are given in Tables 1 and 2. To establish a simplified human PBPK model, the human values for ka, V1, and CLh,int, as shown in Table 2, were estimated by employing a scale-up strategy from HL-mice to humans. A 10 ACS Paragon Plus Environment

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multiplicative factor of 0.744 was applied to the rodent ka value to obtain the human ka value.21-25 V1,human was derived using the reported equation25 based on data from rats, dogs, and monkeys26 and using fixed values of the human liver volume (Vh,human, 1.50 L) and blood volume (Vb,human, 4.90 L).27,28 The in vivo hepatic intrinsic clearance (CLh,int) in humans was estimated based on that of HL-mice, with no consideration for interspecies factors, as previously described18,24,29:

CLh,human =

CLh,rodent Body weightrodent

() 2 3

∙ Body weighthuman

(23)

A system of differential equations, as described above, was also solved to model the concentrations in each compartment in humans. Forward and reverse dosimetry was conducted using this human PBPK model for DBP/MBP.

Results In vitro and in vivo metabolic studies of MBP Pooled liver microsomes from a range of species mediated in vitro MBP glucuronidation, and the rates were measured (Table 3). The in vitro MBP glucuronidation rates mediated by rat liver microsomes were found to be lower than those mediated by hamster or mouse liver microsomes. Dog and minipig liver microsomes mediated MBP glucuronidations at rates higher than those for rodents (Table 3). Liver microsomes from primates (cynomolgus monkey and human) efficiently catalyzed MBP glucuronidations. In our preliminary experiments with recombinant human or monkey UDP-glucuronosyltransferases (UGTs), UGT2B forms would be mainly responsible for the MBP glucuronidations in liver microsomes from humans and monkeys (data not shown). 11 ACS Paragon Plus Environment


Male control mice and HL-mice were orally administered 100 mg/kg DBP. Because absorbed DBP was extensively metabolized in vivo to MBP and its glucuronide, DBP was not detected in plasma or urine samples under the current conditions. Figure 2 shows the mean plasma concentrations of MBP and its glucuronide for control mice and HL-mice. HL-mice had higher plasma MBP glucuronide concentrations than did the control mice (Figure 2A and 2B). The maximum MBP concentrations (Cmax) in control and HL-mice were 5.8 and 6.8 µg/mL, respectively, and the areas under the plasma–concentration curves (AUCs) were 10.4 and 19.3 µg h/mL, respectively. These values were an order of magnitude less than those reported for rats (Table 4). Excreted levels of MBP and its glucuronide in 0–7 h urine samples were, respectively, 1440 and 1550 µg/mL in control (48% of free MBP in total MBP) and 2400 and 7210 µg/mL in HL-mice (25% of free MBP). The concentrations of MBP glucuronide excreted to urine in HL-mice (Figure 2D) were significantly higher than those in control mice (Figure 2C) 7 h after oral administrations of DBP. Apparent injury to the humanized liver caused by MBP was evaluated by detecting the human albumin mRNA in mouse plasma 0.5–24 h (Figure 3) after oral administration of a relatively high dose of DBP (100 mg/kg). Under the current conditions, control plasma samples from untreated HL-mice had human albumin mRNA background levels below the detection limit (data not shown). Human plasma albumin mRNA concentrations were also estimated to be ~0.01% of the albumin mRNA levels in primary human hepatocytes (data not shown). Using simplified pharmacokinetic models to estimate plasma, liver, and urinary concentrations of phthalates Based on the reported plasma concentration data in rats30 and the present results of in vivo 12 ACS Paragon Plus Environment

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experiments in both types of mice as shown in Figure 2, the kinetic parameters for MBP disposition in rats, control mice, and HL-mice were calculated (Table 2). Complicated pharmacokinetics of MBP in rats taken from the literature under the enterohepatic circulation30 were fitted using a simple PBPK modeling without a perirenal compartment. The equations of the simplified PBPK models were solved to generate in silico plasma concentration curves following virtual administration of DBP in rats, control mice, and HL-mice (Figure 4). Estimated values of Cmax and AUC for MBP after virtual single doses in rats, control mice, and HL-mice were consistent with the reported/observed values in vivo (Table 4). Estimated urinary AUC0-7 h values of MBP for MBP and its glucuronide after virtual single doses were 754 and 7110 µg h/mL in control mice (9.6% of free MBP in a total MBP) and 1390 and 10,100 µg h/mL in HL-mice (14% of free MBP), respectively, following virtual administration of DBP (Figure 4). These estimated values resulted in mean urinary concentrations for 0–7 h of MBP glucuronide of 1020 and 1440 µg/mL, which are roughly consistent with the observed scales in vivo (Figure 2). Using these PBPK models, it is also possible to calculate hepatic concentrations of MBP after single or repeated doses of DBP. Hepatic concentrations and their AUC values in rats were high compared with those of the control mice and HL-mice (Table 4). A human PBPK model for MBP was set up based on the HL-mouse PBPK model by employing allometric scaling methods without consideration of interspecies factors between in vitro liver clearances (Table 2). Figure 5 shows the estimated plasma and urinary concentrations of MBP and its glucuronide generated using the human PBPK models to model virtual oral administrations of DBP (13 and 1.2 µg/kg/day) over 14 days. The levels of the repeated doses (13 and 1.2 µg/kg/day) were reverse calculated using the 50th percentiles of human plasma and urinary levels, respectively, of total MBP (combined free and glucuronidated MBP) published in 13 ACS Paragon Plus Environment


a biomonitoring report (Table 5). On modeling daily administration of DBP for 2 weeks, we found that excretions of MBP and its glucuronide would be evident in humans (Figure 5).

Discussion Phthalate esters exhibit low toxicity from common levels of exposure.31 However, species differences in toxicity and metabolism between rodents and nonhuman primates reportedly exist.32 To assess the safety of DBP, extensive toxicokinetic studies on MBP have previously been carried out.2,9,10,33 Rats were found to excrete MBP (14% and 17% of doses) and its glucuronide (38% and 48% of doses) into urine after administration of 2000 mg of DBP and 800 mg of MBP/kg body weight, respectively, whereas hamsters excreted MBP (4% and 6% of doses) and its glucuronide (53% and 67% of doses) into urine after receiving the same doses.10 After oral treatment with either DBP or MBP, the levels of unconjugated toxicant MBP9 in rodent urine were reportedly higher in the rat than in the hamster,10 which may be a causal factor for species-dependent testicular damage in rats in vitro.9 A special simulation model for rats showing enterohepatic circulation of MBP has been reported.30 In the current study, in vitro glucuronidation rates of MBP mediated by liver microsomes showed species-dependent differences (Table 3). The livers of HL-mice extensively mediated glucuronidation of MBP in vivo (Figures 2 and 4) and in vitro (Table 3). The pharmacokinetics of MBP in HL-mice (Figures. 2 and 4) pointed to a higher clearance from plasma of MBP glucuronide by transplanted human hepatocytes via the urinary pathway in HL-mice, compared with that in control mice. In the point of view of urinary excretion profile of MBP glucuronide, estimated free MBP levels of total of MBP and its glucuronide in urinary AUC0-7 h and AUC0-24 h values 14 ACS Paragon Plus Environment

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were 14% and 8.7%, respectively (Figure 4). Similarly, free MBP levels in human urine samples from 17 subjects accumulated 0-24 h after oral dose of DBP (3.6 mg) have reportedly correspond to 1–11% (median 4%) of total MBP.12 It could be noted that pharmacokinetic profile of MBP in HL-mice were similar to that in humans in term of urinary excretion profile of MBP glucuronide. After a single administration of 100 mg/kg DBP, even HL-mice exhibited liver damage, possibly as a result of accumulated MBP (as estimated by PBPK modeling). The liver damage was determined by detection of circulating human albumin mRNA in plasma. Although the mechanism by which MBP resulted in elevated human albumin mRNA (a liver-specific toxicity marker) in plasma is currently unknown, one causal factor for DBP toxicity is likely hepatic accumulation of MBP and its metabolism and disposition mediated by hepatic clearance. Using the PBPK model for control mouse, some accumulation of MBP in plasma and livers in mice were shown after repeated doses of DBP (Table 4), presumably because of different liver function between control and HL-mice in terms of their excretion routes to urine/feces. Human plasma and urinary levels of total MBP (combined free and glucuronidated) published in a biomonitoring report underwent reverse dosimetry PBPK modeling to calculate the repeated daily intake doses needed to achieve those levels (Table 5). The 50th and 95th percentile MBP concentrations in the urine and plasma could potentially imply exposures similar to or higher than the TDI levels (5–10 μg/kg/day) recommended by the European Food Safety Authority12 and the Food Safety Commission of Japan.13 In-depth forward and reverse assessment of DBP and/or MBP levels could be facilitated by adopting the current human PBPK model established in this study (Table 2) [based on the pharmacokinetics in HL-mice (Figure 3)], thereby evaluating the implications of reported biomonitoring data in humans.30



Human internal concentrations of chemicals can be assessed by determining exposure markers, mainly compounds or their primary metabolites, in human fluids based on assumed linearity for doses. In this study, simplified PBPK models and pharmacokinetic data from HL-mice were employed to develop a biomonitoring strategy for human exposures to DBP that was similar to that previously applied to DEHP14 and DINP.15 Our findings supported the assertion that MBP and its glucuronide, the main primary and secondary phthalate metabolites of DBP, can be utilized in human surveys to estimate the biological effects of DBP on livers. In conclusion, the current results suggested that species-dependent glucuronidation rates of the primary metabolite of DBP and their excretion routes to the urine/feces were major determinant factors in the metabolism and disposition of diallyl phthalates such as DBP in vivo. The data presented here suggest that human PBPK modeling, in combination with data from HL-mice, could effectively evaluative the toxicological potential in humans of phthalates (e.g., DBP, DEHP, and DINP) with short or long chains.

ORCID Hiroshi Yamazaki: 0000-0002-1068-4261 Funding This work was supported in part by the METI Artificial Intelligence-based Substance Hazard Integrated Prediction System project, Japan.


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Notes The authors report no conflicts of interest. Acknowledgments The authors thank Megumi Nishiwaki and Nao Yoneda for their technical help. The authors also thank David Smallbones, who copyedited an early draft of this manuscript. References (1) Silva, M. J., Barr, D. B., Reidy, J. A., Kato, K., Malek, N. A., Hodge, C. C., Hurtz, D., III, Calafat, A. M., Needham, L. L., and Brock, J. W. (2003) Glucuronidation patterns of common urinary and serum monoester phthalate metabolites. Arch. Toxicol. 77(10), 561–567. (2) Wine, R. N., Li, L. H., Barnes, L. H., Gulati, D. K., and Chapin, R. E. (1997) Reproductive toxicity of di-n-butylphthalate in a continuous breeding protocol in Sprague-Dawley rats. Environ. Health Persp. 105, 102–107. (3) Latini, G., Wittassek, M., Del Vecchio, A., Presta, G., De Felice, C., and Angerer, J. (2009) Lactational exposure to phthalates in Southern Italy. Environ. Int. 35(2), 236–239. (4) Koch, H. M., Becker, K., Wittassek, M., Seiwert, M., Angerer, J., and Kolossa-Gehring, M. (2007) Di-n-butylphthalate and butylbenzylphthalate – urinary metabolite levels and estimated daily intakes: pilot study for the German Environmental Survey on children. J. Expo. Sci. Environ. Epidemiol. 17, 378–387. (5) Marsee, K., Woodruff, T. J., Axelrad, D. A., Calafat, A. M., and Swan, S. H. (2006) Estimated daily phthalate exposures in a population of mothers of male infants exhibiting reduced anogenital distance. Environ. Health Persp. 114, 805–809. (6) Albro, P. W., Corbett, J. T., Schroeder, J. L., Jordan, S., and Matthews, H. B. (1982) Pharmacokinetics, interactions with macromolecules and species differences in metabolism of DEHP. Environ. Health Persp. 45, 19–25. (7) Ito, Y., Yokota, H., Wang, R., Yamanoshita, O., Ichihara, G., Wang, H., Kurata, Y., Takagi, K., and Nakajima, T. (2005) Species differences in the metabolism of di(2-ethylhexyl) phthalate (DEHP) in several organs of mice, rats, and marmosets. Arch. Toxicol. 79, 147–154. (8) Oishi, S., and Hiraga, K. (1980) Effects of phthalic acid monoesters on mouse testes. Toxicol. Lett. 6, 239–242. 17 ACS Paragon Plus Environment


(9) Gray, T. J., Rowland, I. R., Foster, P. M., and Gangolli, S. D. (1982) Species differences in the testicular toxicity of phthalate esters. Toxicol. Lett. 11, 141–147. (10) Foster, P. M., Cook, M. W., Thomas, L. V., Walters, D. G., and Gangolli, S. D. (1983) Differences in urinary metabolic profile from di-n-butyl phthalate-treated rats and hamsters. A possible explanation for species differences in susceptibility to testicular atrophy. Drug Metab. Dispos. 11, 59–61. (11) Wittassek, M., Wiesmuller, G. A., Koch, H. M., Eckard, R., Dobler, L., Muller, J., Angerer, J., and Schluter, C. (2007) Internal phthalate exposure over the last two decades – a retrospective human biomonitoring study. Int. J. Hyg. Environ. Health 210, 319–333. (12) Seckin, E., Fromme, H., and Volkel, W. (2009) Determination of total and free mono-n-butyl phthalate in human urine samples after medication of a di-n-butyl phthalate containing capsule. Toxicol. Lett. 188, 33–37. (13) Food Safety Commission of Japan. (2014) Dibutyl phthalate (DBP): summary. Food Safety 2, 138–139. (14) Adachi, K., Suemizu, H., Murayama, N., Shimizu, M., and Yamazaki, H. (2015) 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. Environ. Toxicol. Pharmacol. 39, 1067– 1073. (15) Miura, T., Suemizu, H., Goto, M., Sakai, N., Iwata, H., Shimizu, M., Yamazaki, H. Human urinary concentrations of monoisononyl phthalate estimated using physiologically based pharmacokinetic modeling and experimental pharmacokinetics in humanized-liver mice orally administered with diisononyl phthalate. Xenobiotica, in press. (16) Hasegawa, M., Kawai, K., Mitsui, T., Taniguchi, K., Monnai, M., Wakui, M., Ito, M., Suematsu, M., Peltz, G., Nakamura, M., and Suemizu, H. (2011) The reconstituted 'humanized liver' in TK-NOG mice is mature and functional. Biochem. Biophys. Res. Commun. 405, 405–410. (17) Yamazaki, H., Suemizu, H., Kazuki, Y., Oofusa, K., Kuribayashi, S., Shimizu, M., Ninomiya, S., Horie, T., Shibata, N., and Guengerich, F. P. (2016) Assessment of protein binding of 5-hydroxythalidomide bioactivated in humanized mice with human P450 3A-chromosome or hepatocytes by two-dimensional electrophoresis/accelerator mass spectrometry. Chem. Res. Toxicol. 29, 1279–1281. (18) Murayama, N., Suemizu, H., Uehara, S., Kusama, T., Mitsui, M., Kamiya, Y., Shimizu, M., Guengerich, F. P., and Yamazaki, H. (2018) Association of pharmacokinetic profiles of lenalidomide in human plasma simulated using pharmacokinetic data in humanized-liver mice with liver toxicity detected by human serum albumin RNA. J. Toxicol. Sci. 43, 369–375. (19) Miyaguchi, T., Suemizu, H., Shimizu, M., Shida, S., Nishiyama, S., Takano, R., Murayama, N., and Yamazaki, H. (2015) Human urine and plasma concentrations of 18 ACS Paragon Plus Environment

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bisphenol A extrapolated from pharmacokinetics established in in vivo experiments with chimeric mice with humanized liver and semi-physiological pharmacokinetic modeling. Regul. Toxicol. Pharmacol. 72, 71–76. (20) Emoto, C., Murayama, N., Rostami-Hodjegan, A., and Yamazaki, H. (2009) Utilization of estimated physicochemical properties as an integrated part of predicting hepatic clearance in the early drug-discovery stage: impact of plasma and microsomal binding. Xenobiotica. 39, 227–235. (21) Takano, R., Murayama, N., Horiuchi, K., Kitajima, M., Kumamoto, M., Shono, F., and Yamazaki, H. (2010) Blood concentrations of acrylonitrile in humans after oral administration extrapolated from in vivo rat pharmacokinetics, in vitro human metabolism, and physiologically based pharmacokinetic modeling. Regul. Toxicol. Pharmacol. 58, 252–258. (22) Tsukada, A., Suemizu, H., Murayama, N., Takano, R., Shimizu, M., Nakamura, M., and Yamazaki, H. (2013) Plasma concentrations of melengestrol acetate in humans extrapolated from the pharmacokinetics established in in vivo experiments with rats and chimeric mice with humanized liver and physiologically based pharmacokinetic modeling. Regul. Toxicol. Pharmacol. 65, 316–324. (23) Yamazaki, H., Suemizu, H., Mitsui, M., Shimizu, M., and Guengerich, F. P. (2016) Combining chimeric mice with humanized liver, mass spectrometry, and physiologically-based pharmacokinetic modeling in toxicology. Chem. Res. Toxicol. 29, 1903–1911. (24) Shimizu, M., Suemizu, H., Mizuno, S., Kusama, T., Miura, T., Uehara, S., and Yamazaki, H. (2018) Human plasma concentrations of trimethylamine N-oxide extrapolated using pharmacokinetic modeling based on metabolic profiles of deuterium-labeled trimethylamine in humanized-liver mice. J. Toxicol. Sci. 43, 387–393. (25) Nishiyama, S., Suemizu, H., Shibata, N., Guengerich, F. P., and Yamazaki, H. (2015) Simulation of human plasma concentrations of thalidomide and primary 5-hydroxylated metabolites explored with pharmacokinetic data in humanized TK-NOG mice. Chem. Res. Toxicol. 28, 2088–2090. (26) Berry, L. M., Li, C., and Zhao, Z. (2011) Species differences in distribution and prediction of human V(ss) from preclinical data. Drug Metab. Dispos. 39, 2103–2116. (27) Utoh, M., Suemizu, H., Mitsui, M., Kawano, M., Toda, A., Uehara, S., Uno, Y., Shimizu, M., Sasaki, E., and Yamazaki, H. (2016) Human plasma concentrations of cytochrome P450 probe cocktails extrapolated from pharmacokinetics in mice transplanted with human hepatocytes and from pharmacokinetics in common marmosets using physiologically based pharmacokinetic modeling. Xenobiotica 46, 1049–1055. (28) Shimizu, M., Suemizu, H., Mitsui, M., Shibata, N., Guengerich, F. P., and Yamazaki, H. (2017) Metabolic profiles of pomalidomide in human plasma simulated with pharmacokinetic data in control and humanized-liver mice. Xenobiotica 47, 844–848. 19 ACS Paragon Plus Environment


(29) Yamazaki-Nishioka, M., Shimizu, M., Suemizu, H., Nishiwaki, M., Mitsui, M., and Yamazaki, H. (2018) Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice. Xenobiotica 48, 117–123. (30) Keys, D. A., Wallace, D. G., Kepler, T. B., and Conolly, R. B. (2000) Quantitative evaluation of alternative mechanisms of blood disposition of di(n-butyl) phthalate and mono(n-butyl) phthalate in rats. Toxicol. Sci. 53, 173–184. (31) Hellwig, J., Freudenberger, H., and Jackh, R. (1997) Differential prenatal toxicity of branched phthalate esters in rats. Food Chem. Toxicol. 35, 501–512. (32) Kurata, Y., Kidachi, F., Yokoyama, M., Toyota, N., Tsuchitani, M., and Katoh, M. (1998) Subchronic toxicity of di(2-ethylhexyl)phthalate in common marmosets: lack of hepatic peroxisome proliferation, testicular atrophy, or pancreatic acinar cell hyperplasia. Toxicol. Sci. 42, 49–56. (33) Fennell, T. R., Krol, W. L., Sumner, S. C., and Snyder, R. W. (2004) Pharmacokinetics of dibutylphthalate in pregnant rats. Toxicol. Sci. 82, 407–418.


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Table 1. Chemical properties of MBP and its primary hydroxylated metabolite Parameter

Symbol

MBP

MBP glucuronide

Molecular weight

MW

222

398

Octanol–water partition

logP

2.72

0.563

Plasma unbound fraction

fu,p

0.0376

0.193

Blood-to-plasma concentration

Rb

0.752

0.889

Kp,h

2.17

0.495

coefficient

ratio Liver-to-plasma concentration ratio Values of fu,p and logP were obtained by in silico estimation using SimCYP and ChemDrawBioUltra software.20 The liver–plasma concentration ratio (Kp,h) and the ratio of the blood to plasma concentration (Rb) were calculated22: 𝐾p,h =

0.02289 ⋅ 𝑃 + 0.72621 1 + 𝑓u,p × 0.00396 ⋅ 𝑃 + 0.960581 2

Rb = 0.45 ∙ (Kb ∙ fu,p ― 1) +1 where log Kb = 0.617 ∙ log

(

1 ― fu,p fu,p


) +0.208

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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Table 2. Experimental and calculated parameters for PBPK models of MBP disposition Parameter

Symbol (unit)

Rat

Mouse

HL-mouse

Human (from HL-mouse) by scale-up strategy 2.35

Absorption rate constant ka (1/h) 2.52 ± 0.98 3.15 ± 1.27 3.16 ± 2.01 Transfer rate constant k12 (1/h) – 0.549 ± 0.320 0.266 ± 0.317 Transfer rate constant k21 (1/h) – 0.0238 ± 0.0342 0.129 ± 0.185 Volume of systemic circulation V1_substrate (L) 0.304 (± 0.067) 0.222 (± 0.085) 0.162 (± 0.066) 0.401 for MBP Hepatic intrinsic clearance for CLh,int_substrate 2.23 (± 0.49) 3.33 (± 1.44) 3.07 (± 0.87) 308 MBP (L/h) Hepatic clearance for MBP CLh,_substrate (L/h) 0.0763 0.0702 0.0670 10.3 Renal clearance for MBP CLr,_substrate (L/h) 0.0092 0.0050 0.0050 0.993 Transfer rate constant for k12_primary metabolite – 1.27 ± 3.41 0.773 ± 2.210 – primary metabolite (1/h) Transfer rate constant for k21_primary metabolite – 0.0858 ± 0.108 0.0991 ± 0.117 – primary metabolite (1/h) Volume of systemic circulation V1_primary metabolite – 0.0266 (± 0.0678) 0.0293 (± 0.0775) 1.29 for primary metabolite (L) Hepatic intrinsic clearance for CLh,int_primary – 0.0275 (± 0.0888) 0.0780 (± 0.0629) 7.82 primary metabolite metabolite (L/h) Hepatic clearance for primary CLh,_ primary metabolite – 0.0051 0.0138 1.48 metabolite (L/h) Renal clearance for primary CLr,_ primary metabolite – 0.0060 0.0085 1.69 metabolite (L/h) Values in parentheses are standard deviations by fitting. Accepted values for the physiological hepatic blood flow rates (Qh) in mice (0.160 L/h), rats (0.853 L/h), and humans (96.6 L/h) were used. 22 ACS Paragon Plus Environment

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Table 3. Rates of glucuronide formation from MBP determined using pooled liver microsomes from rats, hamsters, mice, dogs, minipigs, monkeys, and humans Liver microsomes

MBP glucuronide formation (nmol/min/mg protein)

Rat

0.25

Hamster

0.46

Mouse, male

0.44

Mouse, female

0.91

Minipig

1.30

Dog

2.16

Cynomolgus monkey

3.25

Human

1.83

MBP (100 μM) was incubated with liver microsomes at 37 °C for 20 min. Glucuronide metabolites were separated using reverse-phase LC. Coefficients of variation were

Steady-State Human Pharmacokinetics of Monobutyl Phthalate

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