Physiologically Based Pharmacokinetic Model for Inorganic and

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Environmental Science & Technology

A physiologically based pharmacokinetic model for inorganic and methylmercury in a marine fish Xun Wang† and Wen-Xiong Wang*,†

†Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong

Corresponding author: [email protected]

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ABSTRACT A physiologically based pharmacokinetic (PBPK) model was developed to simulate

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the uptake, distribution and elimination of inorganic mercury [Hg(II)] and methylmercury

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(MeHg) in a marine fish, Terapon jarbua. In this model, fish were schematized as a six-

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compartment model by assuming that blood was the medium linking the exchange between

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the different compartments. The transfer rates between blood and other compartments were

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determined during a period of 10-day dietary Hg(II) or MeHg exposure, followed by a 30-day

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depuration. For both Hg species, the exchange rates between liver and blood were high,

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indicating that liver served as a ‘transferring station’ in the distribution. Their accumulation

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in the kidney was relatively constant and low. The carcass (mainly muscle) represented a

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large sink for both Hg(II) and MeHg with the highest input rate constants and relatively lower

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output rate constants. Significant differences were observed in the rate constants between the

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two Hg species, suggesting great variations in their exchange and transportation routes.

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Modeling simulation for the first time demonstrated that the gill was the most important route

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in Hg(II) elimination in marine fish, with a rate constant of 0.90 d-1. A long timeframe is

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needed to study the exact rate of MeHg elimination in marine fish. This study showed that

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the PBPK modeling provided critical information for the uptake, distribution and elimination

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of Hg(II) and MeHg in the fish body, especially in elucidating the role of each compartment.

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Keywords: Physiologically based pharmacokinetic model; Mercury; Methylmercury;

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Transfer rate; Marine fish


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INTRODUCTION

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Mercury (Hg) contamination in marine fish has aroused a worldwide concern for public

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health. Among the different species of Hg, methylmercury (MeHg) is extremely toxic for

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humans and can be readily biomagnified along food chains.1,2 Hg can be absorbed by marine

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fish from aqueous and dietary phases, with the latter route generally considered to be the

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primary exposure pathway.3,4 Several studies have determined the kinetics of Hg

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accumulation in fish feeding on contaminated diets.5-7 However, most of these earlier studies

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only focused on the kinetics of Hg from the perspectives of whole-body5,8 or isolated organs

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(e.g. gill, intestine).9,10 Little information is available on the transfer of Hg between organs

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(or tissue groups). It is therefore important to quantify the distribution kinetics of dietary Hg

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between (among) organs.

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Previous studies have shown that inorganic Hg [Hg(II)] and MeHg differed greatly in the

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uptake and accumulation by fish, with a greater trophic transfer of MeHg over Hg(II), which

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resulted in >70% of Hg in whole wild fish presenting as MeHg.11,12 Besides that, Hg(II) was

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inclined to be accumulated in liver and kidney,13 whereas MeHg was primarily accumulated

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in the muscle.14 Compared to Hg(II), the greater uptake and accumulation of MeHg can be

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attributed to its higher assimilation efficiencies (AEs) and lower excretion rates in whole

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fish.5,8,15 However, the exact roles of different organs (or tissue groups) in the uptake and

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distribution of Hg, and their contributions to the different behaviors of Hg(II) and MeHg in

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fish are still not well understood.

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Physiologically based pharmacokinetic (PBPK) modeling has been increasingly used to

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describe the absorption, distribution and excretion of compounds among multiple tissues or

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organs of aquatic organisms.16-18 Combining experimental measurements and PBPK

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modeling allows researchers to analyze and explain the temporal changes in the

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concentrations of compounds in biological compartments of the exposed organism, taking

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into account the effects of exposure scenarios, routes, doses and species.19,20 Therefore,

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PBPK modeling can not only help develop a deeper understanding of the roles, kinetics, and

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relative importance of the organs involved in the uptake, distribution and excretion of Hg, but

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can also elucidate the underlying reasons for the different biokinetics of Hg(II) and MeHg in

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fish.

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Applications of PBPK modeling in describing the kinetics of Hg uptake, distribution and

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elimination in fish are very limited.21,22 Ribeiro et al.22 quantified the distribution kinetics of 4 ACS Paragon Plus Environment

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MeHg in arctic charr (Salvelinus alpinus) with a simple three-compartment catenary model,

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which divided the fish into well-perfused viscera and blood (VB), gut (G) and the rest of

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body (R), and found that the translocation rates between compartments were surprisingly low.

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Based on a multi-compartmental model, Leaner and Mason23 determined the transfer rates

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between organs of sheepshead minnows (Cyprinodon variegatus) after a single dose of

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MeHg-spiked food, and observed that the exchange between blood and visceral organs was

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relatively low, but the calculated transfer rate from viscera to the rest of body was higher than

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those measured for larger fish. However, these studies only described the uptake and

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distribution of MeHg within fish body without addressing the elimination process. The

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related routes and transfer rates for Hg(II) and MeHg elimination thus remain elusive. In the

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present study, we for the first time developed a PBPK model of Hg(II) and MeHg in a marine

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fish Terapon jarbua, with the aims to simulate the uptake, distribution and elimination of

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Hg(II) and MeHg and determine their kinetics between the organs (or tissue groups). We

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then explored the roles of each organ in these processes using the PBPK modeling approach.

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MATERIALS AND METHODS

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Fish and Diet. Juvenile thornfish T. jarbua (7-8 cm in length, ~4 g) were collected from a

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fish farm located in Sai Kung, Hong Kong, and were acclimated in natural sand-filtered Clear

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Water Bay seawater (pH = 8.0, salinity = 33 psu, DOC = 4.2 mg L-1, dissolved Hg = 0.2 ng L-

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1

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(Spectrum small fish formula, New Life International, Inc.) for 2 weeks at a daily rate of 4%

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body weight. The clean fish diet contained 0.037±0.002 µg total Hg g-1, 0.022±0.004 µg

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MeHg g-1 (n = 4), with the macronutrient being 38% crude protein, 7% fat, 5% fiber and 8%

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ash. The concentrations of Hg(II) and MeHg in the spiked food were set as 50 µg g-1 and 1.5

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µg g-1, respectively. Since the duration of exposure was within weeks, we employed a

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relatively high Hg(II) spiked concentration to ensure an observable Hg(II) trend. Briefly, 100

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grams of clean food pellets were incubated with 125 mL of freshly prepared solution of

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HgCl2 or MeHgCl in MiliQ water for 4 h, and then dried at room temperature for two days.

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The measured concentrations of Hg(II) and MeHg in spiked fish diet were 47.6±1.04 µg g-1

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and 1.56±0.04 µg g-1, respectively (n = 4).

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Fish Exposure, Depuration and Sampling. Fish were randomly divided into 10 tanks (size

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of 60 × 30 × 45 cm3, 32 fish per tank), with 4 tanks for each Hg treatment and 2 tanks for

) at 25 oC under a 14 h light:10 h dark regime. Fish were fed clean commercial fish diet



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control, and then acclimated under the same conditions as described above for another week

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before exposure. The fish were not fed for 2 d before Hg exposure in order to empty the gut

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and maximize their digestion and assimilation of Hg-spiked food. The dietary-exposed fish

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were fed the Hg-spiked fish diet for 10 d (exposure) and then were fed clean fish diet for 30 d

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(depuration), whereas the control fish were fed clean fish diet during the entire period. All the

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fish were fed twice a day at a daily rate of 4% body weight. The diet consumption time by

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fish was limited to 1 h. Fish feeding behavior was monitored and almost all food pellets

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(>95%) were found to be eaten; the remaining food pellets and feces were then siphoned off.

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For all the treatments, the seawater was cycled at a flow rate of 1.5 L/min to ensure that the

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water was kept clean.

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Sampling of the Hg-exposed fish took place at 0, 2, 4, 6, 8, 10, 11, 12, 14, 16, 20, 25, 31

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and 40 d, whereas that of the control fish took place every 5 d during the experiment

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(primarily because the change of Hg concentration in the fish body was small for the control

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fish). For each treatment at each time point, eight fish (two from each of the four tanks) were

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randomly collected. Four of the collected fish were rinsed by deionized water and narcotized

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in cold ice water, then were dissected into gill, liver, intestine, kidney and carcass (mostly

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muscle) immediately after blood was collected (100 to 150 µL) from caudal fin by capillary

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pipet. The remaining four fish were sampled as whole body. All samples were weighed for

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fresh weight, freeze-dried at -80 oC (except blood), and stored at -20 oC for further analysis.

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Chemical Analysis. THg concentrations in the tissues were analyzed followed the method

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of EPA 7474 with a few modifications. Briefly, the homogenized tissues were digested in

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aqua regia in a heating block at 80 oC overnight. In the hydrochloride/bromate/bromide

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mixture (Sigma–Aldrich), mercury was oxidized by stannous chloride (Sigma–Aldrich) and

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analyzed by cold vapor atomic fluorescence spectrometry (CVAFS, Brooks Rand Model III).

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MeHg analysis followed the method 1630.24 Tissues were digested in 25% KOH/methanol

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solution in an oven at 80 oC for 4 h, and were converted to volatile MeHg after addition of

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citrate buffer and freshly thawed 1% NaBEt4 solution. MeHg was determined with a MERX

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Automatic Methylmercury System (Brooks Rand Laboratories, Seattle, WA). Standard

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reference materials (Fish protein DORM-4, National Research Council of Canada) were

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concurrently digested and measured for total mercury and MeHg. Recoveries of the

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standards were 93-101% for THg and 96-106% for MeHg.


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Model Development. Hg(II) and MeHg taken up with food pellets were firstly received in

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the intestine (I) of the fish, and were then absorbed across the intestinal wall and transferred

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into blood (B). Since blood was directly in contact with the other organs (or tissue groups),

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metals were then distributed to the gill (G), liver (L), kidney (K) and carcass (C) via blood

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stream. The Hg previously transferred and stored in these organs was transferred back into

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blood and then redistributed around the fish body. This process was simulated by a

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pharmacokinetic model as shown in Fig. 1. Each of the heavily perfused organs (gill, liver

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and kidney) was treated as a separate compartment, since liver had been shown to play an

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important role in Hg redistribution in fish,25 while gill and kidney might serve as the

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excretion route of Hg by fish.26 The rate coefficient, k(i, j) (d-1) represents the transfer rate

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constant between the compartments, where the first subscript (i) refers to the receptor organ

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and the second (j) refers to the source. The coefficient k(0, 2) represents the elimination rate

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constant of Hg through feces (fast phase) from intestine to outside.

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The SAAM II modeling software version 2.3.1 (SAAM Institute, University of

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Washington, Seattle, WA, USA)27 was used for model development and parameter

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calculation for each Hg species. This software has been widely used to simulate the kinetic

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processes in multiple compartments,16,28,29 due to its convenience in the production of

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differential equations of the model structures and association of these equations to the

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experimental data. Assuming that the transfer of Hg between the compartments follows first-

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order kinetics, the flux between compartments can be expressed by the following equations:

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F(j, i) = k(i, j)•Qj

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where F(j, i) is the mass flux (µg d-1) of Hg from the source compartment (j) to the receptor

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compartment (i) and Qj refers to the mass (µg) of Hg located in the source compartment (j) at

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the time point t. The equations for the different compartments can be expressed by the

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following equations:

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Blood

dQB/dt = k(1,2)•QI + k(1,3)•QL + k(1,4)•QG + k(1,5)•QK + k(1,6)•Qc - [k(2,1) + k(3,1) + k(4,1) + k(5,1) + k(6,1)]•QB

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Intestine dQI/dt = D + k(2,1)•QB - k(1,2)•QI - k(0,2)•QI

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Liver

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Gill

dQL/dt = k(3,1)•QB - k(1,3)•QL dQG/dt = k(4,1)•QB - k(1,4)•QG - k(0,4)•QG 7 ACS Paragon Plus Environment


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Kidney

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Carcass dQC/dt = k(6,1)•QB - k(1,6)•QC

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where parameter D refers to the amount of Hg assimilated in the fish body daily (µg d-1). The

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coefficients k(0,2) and k(0,4) refer to the elimination rates of Hg through feces and gills to

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outside environments, respectively. Qj equals to the concentration of Hg in the compartment

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(Cj) multiplied by the fresh weight (wj). The total weight of blood in fish body is calculated

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by assuming 40 ml blood/kg tissue in teleosts and 1 mL blood ≈ 1 g.30 The fish weights

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parameters used in the calibration are listed in Table S1.

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dQK/dt = k(5,1)•QB - k(1,5)•QK

When the Hg was taken up along with food pellets into the intestine lumen, some would be

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absorbed by intestine cells and transported into the blood, whereas the residuals would be

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eliminated through feces. In this study, the Heaviside function was introduced to simulate the

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elimination process of Hg(II) and MeHg through feces and to calculate their residence time in

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the lumen. The Heaviside function is a discontinuous step function whose value is zero for

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negative argument and one for positive argument. It can be used in operational calculus for

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the solution of differential equations, and represents a signal that switches on at a specified

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time and stays switched on indefinitely. The Heaviside function is expressed by the

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following equation:

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heaviside = 0.5•(1.0 + atan[lamba•(t - tlag)]•2/π)

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where atan is the arctangent function which is used to obtain a smooth approximation of the

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step function, t is the time, and tlag is the value at which the Heaviside function changes

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between 0 and 1. For values of t less than tlag, the value of the Heaviside function equals to

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0; for values of t greater than tlag, the value equals to 1. The parameter, lambda, controls the

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sharpness of the function, i.e. how fast the values change between zero and 1. In this study,

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tlag refers to the residence time of Hg(II) and MeHg in the intestine, and the elimination rate

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is expressed by the following equation:

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k(0,2) = (1-heaviside)*loss1

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Thus, Hg was eliminated through feces at a constant rate of loss1 during the residence period

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(t < tlag, k(0,2) = loss1). This process would be terminated after that period (t ≥ tlag, k(0,2) = 0).


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The set of different model equations was solved using the Runge-Kutta integrator. The

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SAAM II software is very functional since it can evaluate the standard deviations of the

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parameter estimates, the parameter correlation matrixes, the weighted sums of squares of the

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residuals, and the appearance of the data-model plots. To evaluate the adequacy of the

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parameter estimates, we chose a correlation coefficient threshold of 0.9, as advocated by

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Miller et al.31

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RESULTS AND DISCUSSION Uptake and Distribution Kinetics of Hg(II) and MeHg. In the present study, fish (T.

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jarbua) were schematized as a six-compartment pharmacokinetic model based on the

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biological properties, containing blood, intestine, gill, liver, kidney and carcass. The

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concentrations of Hg(II) and MeHg within the body of control fish did not show significant

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change during the experiment period (Fig. S1), indicating that the clean fish diet had no

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impact on the accumulation and distribution of Hg in fish. By contrast, both concentrations

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of Hg(II) and MeHg in different compartments increased dramatically during the exposure

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(0-10 d) (Fig. 2). During depuration (10-40 d), Hg(II) decreased continuously and resulted in

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this trend at the end of experiment: kidney > liver ~ intestine > gill ~ carcass > blood. MeHg

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in liver kept declining during depuration, whereas that in carcass kept relatively stable. At

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the end, the levels of MeHg followed carcass ~ kidney > liver ~ intestine > gill > blood.

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Similarly, previous studies have shown that kidney and liver were the target organs for Hg(II)

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accumulation,13 whereas MeHg was primarily accumulated in the muscle.32 For whole-body

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concentrations (Fig. 3), Hg(II) showed a similar trend to the compartments, whereas MeHg

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increased up to day 12, decreased to day 16, and did not show obvious change towards the

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end of experiment. The estimated rate constants of Hg(II) and MeHg are listed in Table 1,

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and the simulation curves obtained by PBPK models are shown in Fig. 4 and Fig. 5. The

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correlation coefficients were highly correlated (correlation coefficient > 0.9) in each of the

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Hg treatment. Dietary metals were taken up from and eliminated into the environment through the

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intestine. The coefficient from intestine to blood (k(1,2)) reflects the transfer of metals across

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the inner membrane of the intestinal epithelium. The estimated k(1,2) of MeHg (1.31±0.156 d-

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1

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much more easily absorbed by the intestine. Previous study has reported that the assimilation

) was significantly higher than that of Hg(II) (0.04±0.004 d-1), indicating that MeHg was



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efficiency (AE) of MeHg (90.0±1.8%) was significantly higher than that of Hg(II)

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(41.4±9.7%) in the same fish species.8 The different efficiencies in the absorption also

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suggest that they were taken up through different routes. For MeHg, the uptake by gut tissues

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was suggested to be comprised of about 40% active and 60% passive mechanisms, and the

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active uptake was predominantly mediated by neutral amino-acid carriers, which targeted

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MeHg-L-cysteine complexes, thus the uptake was dependent on the reaction between MeHg

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and thiolates in the gastric and intestinal fluids.33 On the other hand, the major routes for

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inorganic Hg entering into intestine cells were diffusion of electroneutral complexes10 and

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active uptake through mucosal Na+ channels or divalent cation transporter.34,35 However, the

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absorption can be blocked by the gut mucosal membrane due to the high affinity of mucus for

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Hg(II) ions,36 and this may contribute to the relatively lower transfer rate of Hg(II). It is

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worth noting that the simulated curve of Hg(II) concentrations in the intestine was lower than

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the determined values during the uptake phase (Fig. 4). When the fish were sampled, the

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residual feces in the intestine were inevitable. During the exposure period, the concentration

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of Hg(II) in the intestine would be elevated by the residues containing some unabsorbed Hg.

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Therefore, the actual concentrations of Hg(II) in the intestine might be lower than the

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determined values. This also suggested the accuracy of the PBPK models.

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Since the intestine was highly vascularized and kept in contact with blood, the exchange of

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mercury was expected to be bi-directional. The rate constants of Hg(II) and MeHg from

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blood to intestine were estimated to be 0.07 ± 0.402 d-1 and 7.93 ± 1.51 d-1, respectively.

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Similarly, it has been reported that the rate constants of MeHg from blood to intestine in

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sheepshead minnow were 7.34 ± 2.84 and 7.11 ± 2.68 d-1, respectively, after algae or flake-

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food dosage.23 It has been reported that approximately 90% of MeHg was quickly taken up

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into erythrocytes and bound to hemoglobin thiol groups within the cells after being

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transferred into the blood.22,37 This binding was reversible and the loss of MeHg from

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erythrocytes to the surrounding medium was determined by the relative proportion of thiol

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groups inside and outside the cells.37 Thus, MeHg is more likely drawn out from the red

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blood cells when the bloodstream is passing by the intestine, since there is large amount of

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available amino acids with thiol groups existed in digestive fluids or derived from tissue

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digestion.33 MeHg can then be transferred into intestinal cells through the b0,+ and B0,+ amino

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acid transporters, which are involved in the transport of MeHg-Cys in the intestinal cells.38

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However, little is known about how the Hg(II) in blood is exchanged with other organs,

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although it has been suggested that Hg(II) was initially associated almost exclusively (91%) 10 ACS Paragon Plus Environment

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with plasma following exposure,39 and was associated dominantly with erythrocytes over

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long time (>150 d).40 In this study, the relatively lower rate constants of Hg(II) from blood to

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intestine suggest that the Hg(II) transfer across membranes is rather difficult.

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Following the exchange across the intestinal epithelium to the blood, metals are distributed

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to other visceral organs where they are either stored, detoxified, or further redistributed to

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other parts of the body. The concentrations of Hg(II) and MeHg in the kidney were relatively

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higher and stable, compared to other viscera organs (Fig. 2), whereas the proportions

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transferred to kidney were lower and could only reach up to approximately 3% and 1% of the

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body burden, respectively (Fig. S2). It appeared that kidney played a role as ‘binding site’,

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where the binding and release of Hg were simultaneously processed. When the sites for Hg-

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binding were saturated, the rates of binding and release would be balanced and the levels of

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Hg in kidney were stable. Previous studies have shown that most Hg(II) was restricted to

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lysosomes in the kidney cells,41 whereas MeHg was associated with numerous organelles as

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well as dispersed throughout the cytoplasm.42 Thus, MeHg in the cytoplasm of kidney cells

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was probably more easily released and transferred back to the blood, which might contribute

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to a higher transfer rate from kidney to blood (1.42 ± 0.697 d-1) than that of Hg(II) (0.26 ±

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0.053 d-1). Comparatively, larger proportions of both Hg(II) and MeHg were transported

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from the blood to liver and reached a maximum of approximately 10% and 8% of the body

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burden, respectively (Fig. S2). Besides, the exchange rates between liver and blood were

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high (Table 1). All of these evidences indicated that the liver served as a ‘transfer site’ in the

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distribution of Hg. This was similar to the previous reports for freshwater fish,22 in which the

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liver was considered to be an important site for storage and possibly detoxification during the

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first few days of exposure. Sequestration of Hg(II) and MeHg in the lysosomes of the liver

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functioned as a storage mechanism to remove them from cellular process.41,42 GSH was

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involved in Hg(II) and MeHg detoxification,43,44 but the related mechanisms remained

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unclear. After the initial handling, Hg in the liver was transferred back to the blood and

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redistributed into other parts of the body (especially to the muscle), thus the concentrations of

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Hg kept decreasing during the depuration (Fig. 2). During the experiment, little Hg(II) or

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MeHg was observed to be methylated or demethylated (Fig. S3), possibly due to the

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extremely low rate of methylation25 and lack of selenium to promote the demethylation.45

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The transfer rates of Hg(II) and MeHg from blood to carcass (muscle) were much higher compared to the other compartments (Table 1), indicating that metals were distributed 11 ACS Paragon Plus Environment


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effectively to this compartment, where >60% of Hg(II) and >90% of MeHg in the body were

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located at the end of experiment (Fig. S2). The carcass was dominantly comprised of muscle

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tissue, which contributed to the bulk of the body mass of fish, thus this compartment

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represented a large sink for Hg. The storage of Hg in the muscle helped to remove it from

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circulation and cellular function, thus protecting other tissues from the threat. The estimated

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rate constants of Hg(II) (11.9 ± 1.98 d-1) and MeHg (10.8 ± 0.753 d-1) from blood to carcass

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in our study were even higher than those of MeHg determined in a freshwater fish (sheeshead

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minnow),23 with values of 8.39 ± 1.57 and 7.26 ± 1.37 d-1, respectively, after algae or flake-

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food dosage. This suggested that the transfer of Hg into muscle tissue by marine fish was

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more efficient than freshwater fish. It is worthy to note that the transfer rate of MeHg from

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carcass to blood (0.04 ± 0.004 d-1) was much lower than that of Hg(II) (0.23 ± 0.040 d-1).

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Previous studies have also shown that the elimination of MeHg from muscle tissue was a

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slow process,14,15 whereas Hg(II) did not present a similarly high affinity to muscle tissue.46

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Inside the muscle cells, MeHg was bound tightly to cysteine-rich proteins47 or peptide

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glutathione in heat-stable protein, whereas Hg(II) was bound preferentially to heat-denatured

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protein.8 Thus, the variations in the binding sites of Hg(II) and MeHg in muscle cells may be

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responsible for the differences in transfer rates.

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Hg transport across membranes can occur through channels or mediated by Hg binding to

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proteins and low molecular weight thiols such as GSH and cysteine.48,49 Carrier-mediated

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transport and protein binding are commonly described by Michaelis-Menten kinetics due to

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the saturation of carriers- and binding sites at high concentrations. However, the

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concentrations of Hg(II) and MeHg in our experiments were not high enough to induce the

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saturation of carriers- and binding sites. Taking the blood as an example, the concentration

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of Hg(II) and MeHg increased continuously following a linear-like trend during the exposure

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period (0-10 d) (Fig. 2), suggesting that the transport process of Hg into blood was not

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saturated. Similar trends were also observed in other organs such as gill, liver, kidney and

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carcass. Based on these observations, the first-order kinetics was appropriate for describing

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the uptake and distribution of Hg in our experiments. The application of Michaelis-Menten

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kinetics may be needed under higher Hg concentrations or longer exposure period.

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Elimination Kinetics of Hg(II) and MeHg. Generally, the elimination of Hg in fish body

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occurred in two phases, with a fast phase associated with feces from intestine and a slow

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phase associated with other organs.5 The results showed that the elimination of Hg(II) 12 ACS Paragon Plus Environment

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through feces (4.32 ± 0.324 d-1) was faster than that of MeHg (2.84 ± 0.141 d-1), consistent

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with the finding that the whole-body elimination rate of Hg(II) (0.0957 ± 0.0179 d-1) in

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Plectorhinchus gibbosus was much higher than that of MeHg (0.0116 ± 0.0059 d-1) during

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the first 7 d in depuration.5 Based on the observations of whole-body levels (Fig. 2), the

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elimination rate constant (ke, d-1) for slow phase Hg(II) could be estimated as the slope of the

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least-squares regression equation relating Ln Ct/C0 to the number of the days,8 where Ct

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referred to the concentrations of Hg(II) at the time points after day 14, and C0 referred to the

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initial concentrations of Hg(II) at day 10. The estimated ke value for Hg(II) was 0.0258 ±

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0.0017 d-1, which was lower than those measured in P. gibbosus (0.0547 ± 0.0093 d-1)5 and

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Acanthopagrus schlegeli (0.042 ± 0.023 d-1),50 but comparable to those in Gambusia affinis

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(0.021-0.042 d-1) and Lepomis microlophus (0.003-0.035 d-1).6 The present study also

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simulated the elimination process of slow phase Hg(II) through gills, and the value of k(0,4)

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was estimated to be 0.90 ± 0.148 d-1. Although high loss rates were observed in the gills

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following aqueous and dietary exposures to Hg(II),26,51 the contributions of gills to the whole-

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body elimination of Hg(II) have not been thoroughly investigated previously. Our modeling

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strongly suggested that gills played the most important role in the excretion of Hg(II) in

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marine fish. We further calculated the efflux rates (µg d-1) of the gills during the last 25 days

324

in the experiment (Fig. 6). Based on these data, the simulated elimination rate constant of

325

Hg(II) was 0.0303 ± 0.0025 d-1, which was very close to the actual measured ke (0.0258 ±

326

0.0017 d-1). Such comparison indicated that our simulation of the Hg(II) excretion was

327

satisfactory and confirmed the role of gills in Hg elimination. On the other hand, the

328

excretion of Hg(II) through urine or bile has been suggested by previous studies,40,52 although

329

the relative importance of these routes is still unknown. In this study, these two pathways

330

were also considered and the related rate constants were added in the model. However, the

331

simulation results showed that the related rate constants were infinitely close to zero,

332

suggesting that the excretion through urine or bile was minimal, thus these two routes could

333

be excluded in the present study. As mentioned above, Hg(II) entered into cells through

334

mucosal Na+ channels, as well as possibly by passive diffusion. It can be speculated that

335

Hg(II) was transported to the gills and excreted into the environment through similar

336

pathways, since the gills were the important sites for ion exchange in fish.53 Further studies

337

are needed to test this notion.

338 339

In this study, the value of k(0,4) for MeHg was set to be zero since the elimination of slow phase MeHg was limited and undetectable. This might be caused by the following two 13 ACS Paragon Plus Environment


340

reasons. First, the whole-body ke of MeHg in fish was very low, as shown by many previous

341

studies, such as in Gadus morhua (0.0018 ± 0.0008 d-1),14 Perca flavescens (approximately

342

0.0013 d-1)15 and A. schlegeli (0.0058 ± 0.0034 d-1).50 The elimination process may exceed

343

well beyond the time-frame of this study (30 d depuration). Second, the ke was usually

344

measured based on the radioisotope method, which is sufficiently enough to detect the minor

345

changes of MeHg radioactivity even within a relatively short time scale,54 whereas the

346

determination of MeHg concentrations in this study could not achieve such a high sensitivity.

347

This study demonstrated that PBPK models are helpful to describe the uptake, distribution

348

and elimination kinetics of Hg(II) and MeHg within the body of T. jarbua as well as to

349

elucidate the exact role of each compartment. For both Hg species, the intestine was the

350

major site for the uptake and fast phase elimination, and the liver served as a ‘transfer site’,

351

whereas the kidney was the ‘binding site’ in the distribution. The carcass represented a large

352

sink where most Hg was stored. Compared to Hg(II), MeHg exerted higher exchange rates

353

between blood and viscera organs, but much lower transfer rates from carcass to blood,

354

indicating the great differences in the exchange and transportation routes between these two

355

species. Our study using modeling approach showed that gills played the most important role

356

in the excretion of Hg(II) during the slow phase in marine fish. Since the excretion of MeHg

357

in marine fish is usually a slow process, a longer timeframe study may be required to

358

investigate the exact rate of MeHg elimination.

359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374

REFERENCES

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(7) Dang, F.; Wang, W.-X. Antagonistic interaction of mercury and selenium in a marine fish is dependent on their chemical species. Environ Sci Technol. 2011, 45 (7), 3116-3122. (8) Dang, F.; Wang, W.-X. Subcellular controls of mercury trophic transfer to a marine fish. Aquat Toxicol. 2010, 99 (4), 500-506. (9) Pedersen, T. V.; Block, M.; Part, P. Effect of selenium on the uptake of methyl mercury across perfused gills of rainbow trout Oncorhynchus mykiss. Aquat Toxicol. 1998, 40 (4), 361-373. (10) Hoyle, I.; Handy, R. D. Dose-dependent inorganic mercury absorption by isolated perfused intestine of rainbow trout, Oncorhynchus mykiss, involves both amiloride-sensitive and energydependent pathways. Aquat Toxicol. 2005, 72 (1-2), 147-159. (11) Bank, M. S.; Chesney, E.; Shine, J. P.; Maage, A.; Senn, D. B. Mercury bioaccumulation and trophic transfer in sympatric snapper species from the Gulf of Mexico. Ecol Appl. 2007, 17 (7), 21002110. (12) Wyn, B.; Kidd, K. A.; Burgess, N. M.; Curry, R. A. Mercury biomagnification in the food webs of acidic lakes in Kejimkujik National Park and National Historic Site, Nova Scotia. Can J Fish Aquat Sci. 2009, 66 (9), 1532-1545. (13) Elia, A. C.; Galarini, R.; Taticchi, M. I.; Dorr, A. J. M.; Mantilacci, L. Antioxidant responses and bioaccumulation in Ictalurus melas under mercury exposure. Ecotox Environ Safe. 2003, 55 (2), 162-167. (14) Amlund, H.; Lundebye, A. K.; Berntssen, M. H. G. Accumulation and elimination of methylmercury in Atlantic cod (Gadus morhua L.) following dietary exposure. Aquat Toxicol. 2007, 83 (4), 323-330. (15) Van Walleghem, J. L. A.; Blanchfield, P. J.; Hintelmann, H. Elimination of mercury by yellow perch in the wild. Environ Sci Technol. 2007, 41 (16), 5895-5901. (16) Van Campenhout, K.; Bervoets, L.; Redeker, E. S.; Blust, R. A kinetic model for the relative contribution of waterborne and dietary cadmium and zinc in the common carp (Cyprinus Carpio). Environ Toxicol Chem. 2009, 28 (1), 209-219. (17) Weijs, L.; Yang, R. S. H.; Covaci, A.; Das, K.; Blust, R. Physiologically based pharmacokinetic (PBPK) models for lifetime exposure to PCB 153 in male and female harbor porpoises (Phocoena phocoena): model development and evaluation. Environ Sci Technol. 2010, 44 (18), 7023-7030. (18) Parhizgari, Z.; Li, J. A physiologically-based pharmacokinetic model for disposition of 2,3,7,8-Tcdd in fathead minnow and medaka. Environ Toxicol Chem. 2014, 33 (5), 1064-1071. (19) Thomann, R. V.; Shkreli, F.; Harrison, S. A pharmacokinetic model of cadmium in rainbow trout. Environ Toxicol Chem. 1997, 16 (11), 2268-2274. (20) Krishnan, K.; Peyret, T., Physiologically based toxicokinetic (PBTK) modeling in ecotoxicology. In Ecotoxicology modeling, Springer: 2009; pp 145-175. (21) Rouleau, C.; Gobeil, C.; Tjalve, H. Pharmacokinetics and distribution of dietary tributyltin compared to those of methylmercury in the American plaice Hippoglossoides platessoides. Mar Ecol Prog Ser. 1998, 171, 275-284. (22) Ribeiro, C. A. O.; Rouleau, C.; Pelletier, E.; Audet, C.; Tjalve, H. Distribution kinetics of dietary methylmercury in the arctic charr (Salvelinus alpinus). Environ Sci Technol. 1999, 33 (6), 902-907.



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(23) Leaner, J. J.; Mason, R. P. Methylmercury uptake and distribution kinetics in sheepshead minnows, Cyprinodon variegatus, after exposure to CH3Hg-spiked food. Environ Toxicol Chem. 2004, 23 (9), 2138-2146. (24) USEPA, Method 1630: methylmercury in water by distillation, aqueous ethylation, purge and trap, and CVAFS; EPA-821-R-01-020; Washington, DC. 2001. (25) Wang, R.; Feng, X. B.; Wang, W.-X. In vivo mercury methylation and demethylation in freshwater Tilapia quantified by mercury stable isotopes. Environ Sci Technol. 2013, 47 (14), 79497957. (26) Handy, R. D.; Penrice, W. S. The influence of high oral doses of mercuric chloride on organ toxicant concentrations and histopathology in rainbow trout, Oncorhynchus mykiss. Comp Biochem Phys C. 1993, 106 (3), 717-724. (27) Barrett, P. H. R.; Bell, B. M.; Cobelli, C.; Golde, H.; Schumitzky, A.; Vicini, P.; Foster, D. M. SAAM II: simulation, analysis, and modeling software for tracer and pharmacokinetic studies. Metabolism. 1998, 47 (4), 484-492. (28) Carneiro, M. F. H.; Souza, J. M. O.; Grotto, D.; Batista, B. L.; Souza, V. C. D.; Barbosa, F. A systematic study of the disposition and metabolism of mercury species in mice after exposure to low levels of thimerosal (ethylmercury). Environ Res. 2014, 134, 218-227. (29) Consoer, D. M.; Hoffman, A. D.; Fitzsimmons, P. N.; Kosian, P. A.; Nichols, J. W. Toxicokinetics of perfluorooctanoate (PFOA) in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol. 2014, 156, 65-73. (30) Horn, M. H., Feeding and digestion. In The physiology of ﬁshes, Evans, D. H., Ed. CRC, Boca Raton, FL: USA, 1998; pp 65-97. (31) Miller, L. V.; Krebs, N. F.; Hambidge, K. M. Development of a compartmental model of human zinc metabolism: identifiability and multiple studies analyses. Am J Physiol-Reg I. 2000, 279 (5), R1671-R1684. (32) Cizdziel, J.; Hinners, T.; Cross, C.; Pollard, J. Distribution of mercury in the tissues of five species of freshwater fish from Lake Mead, USA. J Environ Monitor. 2003, 5 (5), 802-807. (33) Leaner, J. J.; Mason, R. P. Factors controlling the bioavailability of ingested methylmercury to channel catfish and Atlantic sturgeon. Environ Sci Technol. 2002, 36 (23), 5124-5129. (34) Vazquez, M.; Velez, D.; Devesa, V.; Puig, S. Participation of divalent cation transporter DMT1 in the uptake of inorganic mercury. Toxicology. 2015, 331, 119-124. (35) Vazquez, M.; Devesa, V.; Velez, D. Characterization of the intestinal absorption of inorganic mercury in Caco-2 cells. Toxicol in Vitro. 2015, 29 (1), 93-102. (36) Vazquez, M.; Calatayud, M.; Velez, D.; Devesa, V. Intestinal transport of methylmercury and inorganic mercury in various models of Caco-2 and HT29-MTX cells. Toxicology. 2013, 311 (3), 147-153. (37) Giblin, F.; Massaro, E. J. The erythrocyte transport and transfer of methylmercury to the tissues of the rainbow trout (Salmo gairdneri). Toxicology. 1975, 5 (2), 243-254. (38) Vazquez, M.; Velez, D.; Devesa, V. Participation of b0,+ and B0,+ systems in the transport of mercury bound to cysteine in intestinal cells. Toxicol Res-Uk. 2015, 4 (4), 895-900. (39) Olson, K. R.; Fromm, P. O. Mercury uptake and ion distribution in gills of rainbow trout (Salmo gairdneri): tissue scans with an electron microprobe. J Fish Res Board Can. 1973, 30 (10), 1575-1578. 16 ACS Paragon Plus Environment

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(40) Schultz, I.; Peters, E.; Newman, M. Toxicokinetics and disposition of inorganic mercury and cadmium in channel catfish after intravascular administration. Toxicol Appl Pharm. 1996, 140 (1), 3950. (41) Baatrup, E.; Nielsen, M. G.; Danscher, G. Histochemical demonstration of two mercury pools in trout tissues: mercury in kidney and liver after mercuric chloride exposure. Ecotox Environ Safe. 1986, 12 (3), 267-282. (42) Baatrup, E.; Danscher, G. Cytochemical demonstration of mercury deposits in trout liver and kidney following methyl mercury intoxication: differentiation of two mercury pools by selenium. Ecotox Environ Safe. 1987, 14 (2), 129-141. (43) Monteiro, D. A.; Rantin, F. T.; Kalinin, A. L. Inorganic mercury exposure: toxicological effects, oxidative stress biomarkers and bioaccumulation in the tropical freshwater fish matrinx, Brycon amazonicus (Spix and Agassiz, 1829). Ecotoxicology. 2010, 19 (1), 105-123. (44) Larose, C.; Canuel, R.; Lucotte, M.; Di Giulio, R. T. Toxicological effects of methylmercury on walleye (Sander vitreus) and perch (Perca flavescens) from lakes of the boreal forest. Comp Biochem Phys C. 2008, 147 (2), 139-149. (45) Khan, M. A. K.; Wang, F. Y. Chemical demethylation of methylmercury by selenoamino acids. Chem Res Toxicol. 2010, 23 (7), 1202-1206. (46) Ribeiro, C. A. O.; Guimaraes, J. R. D.; Pfeiffer, W. C. Accumulation and distribution of inorganic mercury in a tropical fish (Trichomycterus zonatus). Ecotox Environ Safe. 1996, 34 (2), 190-195. (47) Lemes, M.; Wang, F. Y. Methylmercury speciation in fish muscle by HPLC-ICP-MS following enzymatic hydrolysis. J Anal Atom Spectrom. 2009, 24 (5), 663-668. (48) Bridges, C. C.; Zalups, R. K. Molecular and ionic mimicry and the transport of toxic metals. Toxicology and applied pharmacology. 2005, 204 (3), 274-308. (49) Onsanit, S.; Wang, W.-X. Sequestration of total and methyl mercury in different subcellular pools in marine caged fish. J Hazard Mater. 2011, 198, 113-122. (50) Dang, F.; Wang, W.-X. Why mercury concentration increases with fish size? Biokinetic explanation. Environ Pollut. 2012, 163, 192-198. (51) Weisbart, M. The distribution and tissue retention of mercury-203 in the goldfish (Carassius auratus). Can J Zool. 1973, 51 (2), 143-150. (52) Greif, R. L.; Du Vigneaud, M. Distribution of a radiomercury-labelled diuretic (Chlormerodrin) in tissues of marine fish. Biol Bull-US. 1959, 117 (2), 251-257. (53) Kidd, K.; Batchelar, K., 5-Mercury. In Fish Physiology: Homeostasis and Toxicology of NonEssential Metals, Academic Press: 2011; Vol. 31, pp 237-295. (54) Wang, W.-X.; Fisher, N. S. Assimilation efficiencies of chemical contaminants in aquatic invertebrates: a synthesis. Environ Toxicol Chem. 1999, 18 (9), 2034-2045.

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499

AUTHOR INFORMATION

500

Corresponding Author

501

*Phone: (852) 23587346. E-mail: [email protected].

502 503

ACKNOWLEDGEMENTS

504

We are grateful to the anonymous reviewers for their comments. This study was supported

505

by the National Key Basic Research Program of China (2013CB430004) and the General

506

Research Fund from the Hong Kong Research Grants Council (663112).

507 508

ASSOCIATED CONTENT

509

Supporting Information Available

510

Figure. S1. Concentrations of Hg(II) (a) and MeHg (b) in the different compartments (blood,

511

gill, carcass, liver, intestine and kidney) in the controlled fish during the experiment. Data are

512

mean + SD; Figure. S2. Proportions of Hg(II) (a) and MeHg (b) in different compartments

513

(blood, gill, carcass, liver, intestine and kidney) of Terapon jarbua during dietary exposure

514

(10 d) and depuration (30 d); Figure. S3. Ratio of Hg(II) and MeHg in the whole-body of

515

Terapon jarbua during dietary exposure (10 d) and depuration (30 d); Table S1. Fish weights

516

parameters used for calibration. This information is available free of charge via the Internet

517

at http://pubs.acs.org.


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Figure. 1 Schematic representation of Hg(II) and MeHg uptake and distribution in the different compartments of Terapon jarbua used in the PBPK model. Values of k are the model’s intercompartmental rate constant (d-1)



Figure. 2 Concentrations of Hg(II) (a, b) and MeHg (c, d) in different compartments (blood, gill, carcass, liver, intestine and kidney) of Terapon jarbua during dietary exposure (10 d) and depuration (30 d). Data are means ± SD (n = 4).


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Figure. 3 Concentrations of Hg(II) and MeHg in the whole-body of Terapon jarbua during dietary exposure (10 d) and depuration (30 d). Data are means ± SD (n = 4).



Figure. 4 The distribution kinetics of Hg(II) in different compartments of Terapon jarbua during dietary exposure (10 d) and depuration (30 d). Data points represent means ± SD (n = 4). Fitted curves were obtained by physiologically based pharmacokinetic (PBPK) models using the SAAM II modeling software (SAAM Institute, University of Washington, Seattle, WA, USA). Compartments: (a) blood, (b) intestine, (c) liver, (d) gill, (e) kidney, and (f) carcass.


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Figure. 5 The distribution kinetics of MeHg in different compartments of Terapon jarbua during dietary exposure (10 d) and depuration (30 d). Data points represent means ± SD (n = 4). Fitted curves were obtained by physiologically based pharmacokinetic (PBPK) models using the SAAM II modeling software (SAAM Institute, University of Washington, Seattle, WA, USA). Compartments: (a) blood, (b) intestine, (c) liver, (d) gill, (e) kidney, and (f) carcass.



Figure. 6 The efflux rates (µg d-1) of whole-body (solid circles) and the gills (open circles) in Terapon jarbua during the last 25 days. The solid curve (whole-body) was calculated to be Y = 0.056e-0.0258t, and the dashed curve (the gills) was calculated to be Y = 0.062e-0.0303t.


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Table 1. Estimated rate constants for Hg(II) and MeHg in Terapon jarbua under dietary exposure (n = 4). Rate constant

Definition

(d-1)

Value ± SD

MeHg a

Value ± SD

k(2,1)

Blood to intestine

0.07 ± 0.402

7.93 ± 1.51

k(3,1)

Blood to liver

3.06 ± 0.830

6.10 ± 2.67

k(4,1)

Blood to gill

2.64 ± 1.10

1.16 ± 0.219

k(5,1)

Blood to kidney

0.58 ± 0.097

0.95 ± 0.466

k(6,1)

Blood to carcass

11.9 ± 1.98

10.8 ± 0.753

k(1,2)

Intestine to blood

0.04 ± 0.004

1.31 ± 0.156

k(1,3)

Liver to blood

0.41 ± 0.124

1.27 ± 0.587

k(1,4)

Gill to blood

0.11 ± 0.453

0.43 ± 0.096

k(1,5)

Kidney to blood

0.26 ± 0.053

1.42 ± 0.697

k(1,6)

Carcass to blood

0.23 ± 0.040

0.04 ± 0.004

k(0,2)

Intestine to feces

4.32 ± 0.324

2.84 ± 0.141

k(0,4)

Gill to water

0.90 ± 0.148

/

14.4 ± 0.191

11.4 ± 0.088

tlag (d)

a

Hg(II)

Loss time by intestine

SD = Standard deviation.


Physiologically Based Pharmacokinetic Model for Inorganic and

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