Dietary fats altered nephrotoxicity profile of methylmercury in rats

Research Article

Received: 20 June 2008, Revised: 1 August 2008, Accepted: 18 August 2008, Published online 29 September 2008 in Wiley Interscience

(www.interscience.wiley.com) DOI 10.1002/jat.1389

Copyright © 2008 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2009; 29: 126–140

126

John Wiley & Sons, Ltd.Dietary fats altered nephrotoxicity profile of methylmercury in ratsDietary fats altered nephrotoxicity profile of methylmercury in ratsXiaolei Jin,a* Eric Lok,a Don Caldwell,a Rudi Mueller,a Kamla Kapal,a Virginia Liston,a Stan Kubow,b Hing Man Chanc and Rekha Mehtaa

ABSTRACT: Weanling male Sprague–Dawley rats were administered semi-purified isocaloric diet containing soy oil (SO), sealoil (SE), docosahexaenoic acid (DHA), fish oil (FO) or lard (LA) for 28 days, and then gavaged with 0, 1 or 3 mg MeHg kg-1 bodyweight per day and fed the same diet for 14 days. Serum and 24 h urine samples were collected on the day of necropsy, andanalyzed for markers of kidney function and diseases. Kidney slices were analyzed for para-amino-hippurate (PAH) and tetra-ethylammonium (TEA) uptake, total mercury and MeHg content, and examined for pathological lesions. Total mercury andMeHg contents increased significantly and dose-dependently in all dietary groups. MeHg significantly increased relative kid-ney weight in all groups, serum creatinine in all except SO group, serum uric acid in the DHA and LA groups, serum Mg in allexcept the LA group, and urinary protein in the SO group. MeHg significantly decreased serum urea nitrogen in SE, FO and LAgroups, urinary creatinine in the DHA group, PAH uptake in all except the SE group, and TEA uptake in all groups. MeHgcaused nephrosis in all dietary groups. MeHg also significantly increased neutrophil counts in all except the SE group,decreased serum albumin and triglyceride in all except the DHA group, and increased serum total cholesterol in all groups,suggesting a nephrotic syndrome-like outcome. These results confirmed that kidney tubules are major targets of MeHgnephrotoxicity. Treatment with dietary fats did not prevent, but rather altered the profile of, nephrotoxicity of MeHg in rats.Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: methylmercury; nephrotoxicity; dietary fats

Introduction

Human exposure to MeHg, a widespread environmental con-taminant, through consumption of contaminated fish, continuesto pose a significant health concern. MeHg is a potent neurotoxinin humans, especially in early developmental stages (Bland andRand, 2006). In addition to the central and peripheral nervoussystem, the kidney is also a known target organ of mercuryaccumulation and toxicity in both animals (Folsom and Fishbein,1972; Ohi et al., 1976; Yasutake et al., 1997; Zalups, 2000) andhumans (Jalili and Abbasi, 1961; Kevorkian et al., 1972; Himenoet al., 1986; Barregard et al., 1988; Ohno et al., 2007; Johansenet al., 2007). The ultrastructural, histological, clinical, and bio-chemical changes associated with renal toxicity of MeHg havebeen well described (Chang et al., 1973; Ware et al., 1975; Fowlerand Woods, 1977; Fair et al., 1985; WHO, 2000). In the rodentkidney, MeHg produced ultrastructural changes in pars rectaepithelia of proximal tubules, consisting of occupational epithe-lial exfoliation, a modest increase in cellular cytoplasmic density,floccular degeneration of mitochondrial matrix material, and theloss of cristae and disruption of inner limiting membrane (Wareet al., 1975). Along with the ultrastructural damage, MeHg alsoinhibited a number of renal enzymes such as γ-glutamyl trans-peptidase, glucose-6 phosphatase, alkaline phosphatase, ATPase,succinic dehydrogenase, δ-aminolevulinic acid synthetase andmonoamine oxidase in exposed rodents (Chang et al., 1973; Fowlerand Woods, 1977). The inhibition of renal δ-aminolevulinic acidsynthetase, a rate-limiting enzyme in the heme biosyntheticpathway, occurred even prior to the onset of clinical changes inblood urea nitrogen, serum creatinine and overt neurologicalsymptoms (Fowler and Woods, 1977). Lifetime exposure to MeHg

elevated lipid peroxidation and altered activities of antioxidantenzymes such as superoxide dismutase, catalase and glutathioneperoxidase in the rat kidney, even at doses that cause no overtneurological symptoms or changes in survival rate (Yasutakeet al., 1997). In humans, MeHg caused fatty degeneration ofrenal epithelial cells (Takeuchi, 1970). The standardized mortalityratios for nephritis, nephrosis and nephrotic syndrome were sig-nificantly higher in the female patients with Minamata disease, adisease occurring mainly among fisherman and their familieswho repeatedly consume fish contaminated with MeHg, com-pared with the general population (Tamashiro et al., 1984, 1986).A very recent study conducted in Japan revealed a positivecorrelation between urinary N-acety-β-D-glucosaminidase (NAG)activity and α-microglobulin (AMG), a measure of renal tubulefunction, and daily mercury intake and mercury levels in hair,toenails and urine in women free from occupational exposures(Ohno et al., 2007). These findings suggest that MeHg-inducednephrotoxicity may occur at exposure levels even lower than

* Correspondence to: Xiaolei Jin, Toxicology Research Division, Food Directo-rate, HPFB, Health Canada, Postal Locator 2202C, 251 Sir Frederick BantingDrive Way, Ottawa, Ontario, Canada K1A 0L2. E-mail: [email protected]

a Toxicology Research Division, Food Directorate, HPFB, Health Canada,Ottawa, Ontario, Canada K1A 0L2.

b School of Dietetics and Human Nutrition, McGill University, Montreal, Quebec,Canada H9X 3V9.

c Community Health Program, University of Northern British Columbia, PrinceGeorge, British Columbia, Canada V2N 4Z9.

Dietary fats altered nephrotoxicity profile of methylmercury in rats

J. Appl. Toxicol. 2009; 29: 126–140 Copyright © 2008 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jat

127

those giving rise to central nervous system effects (Kazantzis,2002).

Despite the numerous investigation of MeHg-induced renaltoxicity, the modulating effects of dietary factors such as fats havenot been extensively explored. Højbjerg et al. (1992) reported thatmice fed a diet containing 50% energy from coconut oil retainedsignificantly greater amounts of MeHg than those fed a diet con-taining 5% energy from coconut oil. The relative dispositionof MeHg in kidney was significantly greater in mice fed a dietcontaining 20% energy from cod liver oil than those fed a dietcontaining 20% energy from soya oil. Sakamoto et al. (1988)observed that the renal levels of thiobarbituric acid reactive sub-stances (TBARS) as well as oxidative hemolysis were significantlyhigher in rats fed a vitamin E-deficient diet containing 10% codliver oil than that containing 10% beef tallow, even though therelative mercury disposition in the kidney was about the samefor both diets. This was attributed to the significantly highercontent of polyunsaturated fatty acids in the kidney of rats fedcod liver oil diet than those fed the beef tallow diet. However,polyunsaturated omega-3 fatty acids found in fish oil and flax-seed oil have been shown to be protective in experimental kidneydisease (Lu et al., 2003; Ogborn et al., 2002, 2006; Sankaran et al.,2004). Sankaran et al. (2004) reported that dietary oils with dif-ferent fatty acid compositions have different effects on the pro-gression of renal injury in pcy mice. Nephrotic syndrome inhumans is known to be associated with serum lipid disorders,such as hypercholesterolemia and altered plasma fatty acidcomposition (Fujita et al., 2006). The question remains whetherdietary fats rich in omega-3 fatty acids protect against MeHg-induced renal injury; it warrants further clarification as to howdietary fats with varying fatty acid composition may alter thenephrotoxicity of MeHg. Therefore, we undertook a study in ratsin which the effects of dietary soy oil, docosahexaenoic acid(DHA), seal oil, fish oil and lard on acute renal toxicity of MeHgwere investigated.

Materials and Methods

Animals, Diets and Treatments

All animal care and handling procedures conformed to the guide-lines of the Canadian Council on Animal Care, and the experimentalprotocol was reviewed and approved by the Health CanadaOttawa Animal Care Committee prior to the initiation of thestudy. The dietary and MeHg treatment of the animals has beendescribed in detail elsewhere (Jin et al., 2007). Briefly, maleSprague Dawley rats at weights of 217 ± 9 g and ages of 42–45days were obtained from Charles River Canada (St Constant, Quebec,Canada). The rats were housed in pairs in disposable cages con-tained in glove box-style Isotec units (Harlan Sprague DawleyInc., Indiana, USA). All the animals were placed on the starch-basedAIN-93G basal diet upon arrival, for acclimatization. Ideally, allanimals should be acclimatized for the same length of time, say5 days. However, the animals could only be shipped to our researchfacility from Charles River once a week. In addition, only 18 animalscould be necropsied on a single day to allow sufficient time andresources to perform the required tissue processing within aspecified time following necropsy. Therefore, to allow for theselogistics in the study design, a batch of 90 animals was grouped,started on the experimental diet, and gavaged on five differentdays, resulting in an acclimatization period that ranged from aminimum of 5 days to a maximum of 10 days. The variation

between experimental groups was minimized by including in eachgroup animals with varied acclimatization periods on the basaldiet. The diet composition is shown in Table 1. After acclimatiza-tion, the rats were subjected to 28 days of treatment with starch-based AIN-93G isocaloric diets containing, as 15% lipid source,either soy oil, DHA, seal oil, fish oil or lard. All diets contained aminimal level of soy oil (1.6%) to provide essential fatty acidrequirements for rats (National Research Council, 1995) (Table 1).At the end of dietary treatment, the rats fed the same diet wererandomly assigned to three groups with six rats per group, andthen gavaged for 14 days with 0, 1 and 3 mg MeHg kg−1 bodyweight (BW) per day in 5 mM Na2CO3 (0.5 ml per 100 g BW) whilemaintained on the same diet. These doses were chosen becausea pilot study in our laboratory found that 1 mg MeHg kg−1 BW wasnon-toxic, and 3 mg MeHg kg−1 BW was effective in inducingacute toxicity with no or low mortality, in rats during two weeksof exposure. The pilot study was based on the previous work byvarious investigators (Chapman and Chan, 2000). On the day beforethe start of MeHg dosing, and on days 7 and 14 of dosing withMeHg, rats were housed individually in a metabolic cage, and a24 h urine sample was collected. On the day 43 of the study, therats were euthanized. Blood samples were collected from theabdominal aorta. Serum was obtained after centrifugation at500g for 10 min in a clinical bench centrifuge (Damon/IEC Division,Needham Heights, MA, USA). Following a full necropsy, both leftand right kidneys were removed and weighed. Slices of kidney werekept in buffer for immediate measurement of para-amino-hippurate(PAH) and tetraethylammonium (TEA) uptake, frozen in liquidnitrogen for analysis of total mercury and MeHg, or fixed in 10%buffered formalin (pH 7.0) for pathological analysis.

Analysis of Mercury Contents in Kidney

Frozen kidney tissue (0.5 g) was digested in concentrated nitricacid. Total mercury and MeHg were measured as described byJin et al. (2007). For statistical purposes, values below the limit ofdetection (0.001 μg g−1) were reported as zero.

Analysis of Urinary Parameters

Urine volume, osmolality, creatinine, protein and γ-glutamyl trans-ferase (GGT) were measured for samples collected on day 0, 7 and14 of exposure to MeHg using the methods described by Suzukiet al. (1995).

Analysis of Serum Clinical Parameters

Clinical biochemical analyses of serum were performed using aBeckman Synchron CX5 Clinical System (Beckman InstrumentCanada Inc., Mississauga, Ontario, Canada) and Beckman reagentkits according to the manufacturer. Measured serum clinical para-meters included creatinine, urea nitrogen (BUN), uric acid, calcium(Ca), magnesium (Mg) and osmolality (Osm).

Analysis of Organic Ion Transport

Transport of organic ion PAH and cation TEA into renal corticalslices was measured as described by Suzuki et al. (1995), andexpressed as a slice (S) to medium (M) ratio (S/M) calculatedfrom the concentration of ion per gram wet weight of slicedivided by the concentration of ion per milliliter of incubationmedium.

X. Jin et al.

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128

Pathological Analysis

Formalin-fixed tissues were processed and embedded in Para-plast wax, sectioned at 4 μm and stained with hematoxylin andeosin. Stained kidney sections from each treatment group wereevaluated under a microscope using a 20× objective lens. A sub-jective numeric grading scale from 0 to 5 was used to recordnephrosis. Zero indicated that nephrosis was not present. Grade1 indicated minimal focal nephrosis with lesions characterizedby occasional tubules in the outer stripe and inner cortex linedsegmentally or completely by basophilic regenerative epithelium.These basophilic epithelial cells had hyperchromatic nuclei, withincreased nuclear/cytoplasmic ratio and a prominent nucleolus.Grade 2 indicated mild multifocal nephrosis with lesions charac-terized sometimes by basophilic epithelium piling up or partiallyfilling the tubule, occasional mitoses among regenerative cells,and single-cell necrosis of proximal tubular epithelium. Grade 3indicated moderate nephrosis with a continuous band of lesionsin the outer stripe and inner cortex. Grade 4 indicated severenephrosis with a confluent and clearly formed band of lesions.Grade 5 indicated very severe nephrosis with a wide irregularband of lesions roughly occupying the outer stripe of the outermedulla and the inner 2/3 cortex. In grades 4 and 5, there werealso minimal to mild interstitial leukocytic infiltrates composedof lymphocytes, neutrophils, eosinophils and fibroblasts.

Statistical Analysis

For parameters measured at three time points, two-way repeatedmeasures analysis of variance (TW-RM-ANOVA) was performedon data from four to six animals per treatment group using Sigma-Stat Version 2.03 (Jandel Scientific, San Rafael, California, USA).For parameters measured at one time point, two-way ANOVA(TW-ANOVA) was performed using the same software. Tests for

normality and equal variance were performed on all data. TW-RM-ANOVA or TW-ANOVA was applied directly to parametricdata. Nonparametric data were transformed using log functionor ranked before running TW-RM-ANOVA or TW-ANOVA. All pairwisecomparisons were done using Tukey’s test. Pearson product momentcorrelation analysis was performed on original data or trans-formed data to determine the strength of association betweentoxicological endpoints and MeHg dose, and total mercury andMeHg content in kidney. Graphs were prepared using SigmaPlot2001 for Windows Version 7.101 (Jandel Scientific, San Rafael,CA, USA).

Results

Mercury Content in Kidney

In all dietary groups, the total mercury and MeHg content increasedsignificantly and in a linear fashion with MeHg dose (Fig. 1). Sig-nificantly more MeHg was found in the vehicle control rats fedthe lard diet than those fed soy oil diet. The reasons for thisobservation are unknown. Analysis of the lard diet indicated nodetectable mercury (Jin et al., 2007).

Relative Kidney Weight

Both diet (p = 0.002) and MeHg (p < 0.001) showed a significantmain effect on relative kidney weight (Fig. 2). The vehicle controlrats fed the DHA diet had significantly higher (p = 0.02) relativekidney weight than those fed the soy oil diet. Relative kidneyweight was significantly higher (p = 0.02) in the 3 mg MeHg kg−1-dosed rats fed the DHA than seal oil diet. As compared withvehicle control, 3 mg MeHg kg−1 BW significantly increased rela-tive kidney weight in all dietary groups, while 1 mg MeHg kg−1

BW increased this parameter only in the soy oil group.

Table 1. Diet compositiona

Component (g kg−1) Basal Fish oil Lard Soy Oil Seal Oil DHA

Corn starch 441.5 433.5 433.5 433.5 433.5 433.5Vitamin-free casein 218 200 200 200 200 200Sucrose 100 100 100 100 100 100Alphacel non-nutritive bulk 50 50 50 50 50 50Butter 28 0 0 0 0 0Corn oil 14 0 0 0 0 0Fish oil 0 150 0 0 0 0Lard 28 0 150 0 0 0Soy oil 70 16 16 166 16 16Seal oil 0 0 0 0 150 0DHAb 0 0 0 0 0 150AIN-93G mineral mix 35 35 35 35 35 35AIN-93 vitamin mix 10 10 10 10 10 10L-Cystine 3 3 3 3 3 3Choline bitartrate 2.5 2.5 2.5 2.5 2.5 2.5t-Butyl hydroquinone 0.014 0.014 0.014 0.014 0.014 0.014aThe fatty acid compositions were: 7.7% saturated fatty acids (SFA), 61.67% monounsaturated fatty acids (MUFA) and 26.23% poly-unsaturated fatty acids (PUFA) for soy oil; 100% PUFA for DHA; 15.84% SFA, 60.30% MUFA and 19.46% PUFA for seal oil; 31.79% SFA,29.3% MUFA and 33.32% PUFA for fish oil; and 39.2% SFA, 45.1% MUFA and 11.2% PUFA for lard.bDocosahexaenoic acid.



129

Urine Parameters

Urine Volume. A significant interaction between diet and MeHgwas observed on day 7 of dosing with MeHg (data not shown). At1 mg MeHg kg−1 BW, the fish oil group had a significantly greaterurine volume than the seal oil group. Diet expressed significantmain effect on urine volume on day 14 of exposure to MeHg. Thefish oil group had overall significantly higher urine volume thanthe soy oil group (data not shown). Within the soy oil and fish oilgroups, the urine volume increased significantly with days ofexposure to MeHg for both 1 and 3 mg MeHg kg−1 BW dosed rats(data not shown).

Urine Osmolality. A significant main effect on osmolality wasfound for diet on day 0 of exposure to MeHg with the highestmain value for seal oil group and the lowest for fish oil group(Table 2). A significant interaction between diet and MeHg onurinary osmolality was found on day 7 of exposure to MeHg. On

day 7 of exposure, urinary osmolality was significantly lower in3 than 0 mg MeHg kg−1 BW-dosed rats fed the fish oil diet, andalso in 3 than 1 mg MeHg kg−1 BW-dosed rats fed the DHA or theseal oil diet. On the day 14 of exposure, MeHg expressed a sig-nificant main effect on urinary osmolality. A lower urinary osmo-lality was found in 3 than 0 mg MeHg kg−1 BW-dosed rats in alldietary groups except the lard group, and the difference reacheda statistically significant level in the seal oil and fish oil groups. Inthese two dietary groups, urinary osmolality also decreased sig-nificantly from day 0 to day 14 of exposure in 3 mg MeHg kg−1-dosed rats.

Urinary Protein. On the day 7 of exposure to MeHg, a significantmain effect on urinary protein was detected for diet (Table 2).Urinary protein level was significantly higher 1 than 0 mg MeHgkg−1 BW-treated rats fed fish oil diet, and in 3 than 1 mg MeHgkg−1 BW-treated rats fed seal oil diet. On day 14 of exposure toMeHg, both diet and MeHg imposed significant main effects on

Figure 1. Total mercury and MeHg content (mg g−1 tissue) in kidney of rats fed soy oil, DHA, seal oil, fish oil or lard diet and exposed to 0 (blue bars),1 (pink bars) or 3 (green bars) mg MeHg kg−1 BW for 14 days. The value represents the mean of four to six rats in the same treatment group. The errorbar stands for standard error of the mean. All comparisons between MeHg dose groups were significantly different at p < 0.001.

Figure 2. Relative kidney weight (kidney weight/body weight) of rats fed soy oil, DHA, seal oil, fish oil or lard diet and exposed to 0 (empty bar), 1(right striped bars) or 3 (left striped bars) mg MeHg kg−1 BW for 14 days. The value represents the mean of four to six rats in the same treatment group.The error bar stands for standard error of the mean. a, aa: Significant difference at p < 0.05 and p < 0.001, respectively, when compared with the 0 mgMeHg kg−1 BW dose group. b, bb: Significant difference at p < 0.05 and p < 0.001, respectively, when compared with 1 mg MeHg kg−1 BW dose group.

X. Jin et al.


130

Tabl

e 2.

Effe

cts

of d

iet a

nd M

eHg

on s

ome

urin

ary

para

met

ers

of k

idne

y fu

nctio

n m

easu

red

on d

iffer

ent d

ays

of d

osin

g

Urin

ary

para

met

ers

Day

s of

ex

posu

reto

MeH

g

Mai

n ef

fect

(p-v

alue

)M

eHg

dose

grou

p

Die

tary

gro

up1

Refe

renc

eva

lue

Die

tM

eHg

Inte

ract

ion

Soy

oil

DH

ASe

al o

ilFi

sh o

ilLa

rd

Osm

olal

ity (m

Osm

)02

0.03

2N

/AN

/A0

1240

±10

311

97±

156

1641

±15

211

09±

130

1575

±16

294

3±

3273

70.

754

0.10

20.

021

012

39±

9911

22±

134

1269

±19

517

21±

283

1071

±79

110

04±

9616

73±

274

1794

±22

910

34±

235

1501

±30

33

1285

±27

296

2±

166b

960±

202b

941±

142a

1565

±24

914

0.07

30.

025

0.05

40

1175

±15

081

9±

132

1333

±17

212

17±

295

1114

±97

168

6±

7110

00±

118

1253

±18

072

5±

117

1008

±11

73A

749±

114

968±

292

751±

50aπ

500±

96axπ

1264

±27

9y

Prot

ein

(mg

per 2

4h)

00.

117

N/A

N/A

016

.16±

1.60

20.9

6±

2.26

19.3

7±

1.39

18.6

8±

4.82

19.3

0±

1.77

5–20

4

70.

005

0.09

40.

099

013

.54±

1.47

21.3

0±

2.91

16.6

0±

3.01

18.3

1±

3.74

21.3

6±

1.71

115

.61±

1.31

20.4

5±

3.48

11.5

5±

0.66

34.2

6±

6.13

a 16

.04±

3.24

314

.75±

2.10

25.6

2±

4.28

24.6

3±

4.68

b31

.55±

8.8

24.0

8±

3.06

140.

034

0.00

70.

354

011

.86±

1.76

19.5

8±

2.12

15.6

5±

1.44

22.6

1±

3.64

π17

.37±

0.99

115

.65±

1.78

18.3

4±

2.97

14.4

4±

0.96

26.7

0±

1.83

13.9

7±

3.74

3AB

21.5

1±

2.25

aπ21

.33±

5.14

24.3

0±

3.49

b25

.20±

3.80

21.5

1±

4.27

Crea

tinin

e (m

g pe

r 24

h)0

0.36

1N

/AN

/A0

14.0

5±

0.94

12.9

4±

0.70

12.5

6±

0.51

12.5

6±

0.63

12.0

5±

0.73

19.4±

5.85

70.

375

0.17

60.

587

014

.23±

0.92

13.5

3±

1.10

13.5

9±

1.15

12.2

6±

1.35

12.7

2±

0.62

113

.53±

0.95

12.7

0±

0.56

13.1

4±

0.83

13.2

9±

0.76

11.6

3±

0.71

311

.08±

0.56

13.2

5±

1.34

13.3

9±

0.82

10.9

6±

1.32

12.0

1±

0.96

140.

170

<0.0

010.

459

015

.06±

0.61

14.7

8±

0.75

15.5

2±

0.69

15.5

2±

1.39

13.4

5±

0.87

114

.62±

0.60

15.7±

0.94

14.3

1±

0.92

16.1

8±

1.41

15.0

6±

0.86

3 AA

BB13

.56±

0.80

10.4

0±

1.21

ab12

.95±

1.11

14.0

0±

0.57

11.4

6±

1.60

b

Crea

tinin

e cl

eara

nce

(ml m

in−1

kg−1

)14

0.71

5<0

.001

0.83

50

4.54

±0.

264.

90±

0.38

4.97

±0.

185.

19±

0.75

4.41

±0.

477.

24

14.

77±

0.30

4.72

±0.

364.

70±

0.40

5.00

±0.

575.

27±

0.50

3 AA

BB3.

89±

0.28

3.13

±0.

49ab

3.91

±0.

364.

07±

0.40

3.55

±0.

62b

GG

T (IU

per

24

h)0

0.01

2N

/AN

/A0

10.4

7±

0.97

9.95

±1.

009.

39±

0.87

13.8

9±

0.77

10.4

7±

1.33

3.29

±1.

285

70.

321

<0.0

010.

246

08.

25±

0.84

9.15

±1.

028.

24±

0.85

8.31

±0.

94π

10.3

6±

0.53

1A7.

27±

1.35

6.92

±0.

566.

17±

0.56

x10

.15±

1.05

y6.

84±

0.70

a

3AA

BB5.

02±

0.71

aπ6.

43±

1.42

π5.

12±

0.48

a 5.

66±

0.87

bππ

5.11

±0.

59aaππ

14<0

.001

<0.0

010.

049

06.

88±

0.58

π7.

11±

1.03

8.07

±0.

2711

.37±

1.64

9.32

±0.

751

7.90

±0.

637.

59±

0.63

5.24

±0.

57aπ

10.4

1±

1.43

7.04

±0.

553A

ABB

5.01

±0.

66bπ

3.24

±0.

34aa

bbππϕ

4.46

±0.

69aa

bππ

6.22

±0.

60abππ

4.58

±0.

77aa

bππ

1 Valu

es fo

r ea

ch p

aram

eter

are

the

mea

ns±

stan

dard

err

or f

rom

fiv

e or

six

ani

mal

s of

the

sam

e tr

eatm

ent

grou

p. T

he s

ingl

e-un

derli

ned

diet

ary

grou

p is

sig

nific

antly

diff

eren

t(p<

0.05

) fro

m th

e do

uble

-und

erlin

ed d

ieta

ry g

roup

whe

n co

mpa

red

for t

he s

ame

urin

ary

para

met

er.

2 For d

ay 0

, the

dat

a re

pres

ents

the

mea

n of

18

anim

als

fed

the

sam

e di

et.

3 Valu

e fo

r mal

e Sp

ragu

e–D

awle

y ra

ts (K

ohn

and

Cliff

ord,

200

2).

4 Valu

e fo

r mal

e W

ista

r rat

s (W

atan

abe

et a

l., 1

980)

.5 Va

lue

for m

ale

Spra

gue–

Daw

ley

rats

(She

vock

et a

l., 1

993)

.x a

nd y : T

he v

alue

s ar

e si

gnifi

cant

ly d

iffer

ent f

rom

eac

h ot

her a

t p<

0.05

whe

n co

mpa

red

for t

he s

ame

MeH

g do

se g

roup

.a a

nd aa

: The

val

ue is

sig

nific

antly

diff

eren

t fro

m th

e va

lue

for t

he v

ehic

le c

ontr

ol ra

ts (0

mg

MeH

g kg

−1 B

W) a

t p<

0.05

and

0.0

01, r

espe

ctiv

ely,

whe

n co

mpa

red

with

in th

e sa

me

diet

ary

grou

p.b

and

bb: T

he v

alue

is s

igni

fican

tly d

iffer

ent f

rom

the

valu

e fo

r the

1m

g M

eHg

kg−1

BW

dos

ed ra

ts a

t p<

0.05

and

0.0

01, r

espe

ctiv

ely,

whe

n co

mpa

red

with

in th

e sa

me

diet

ary

grou

p.π

and ππ

: The

val

ue is

sig

nific

antly

diff

eren

t fro

m th

e va

lue

obta

ined

on

day

0 fo

r the

sam

e M

eHg

dose

gro

up a

t p<

0.05

and

0.0

01, r

espe

ctiv

ely.

ϕ : The

val

ue is

sig

nific

antly

diff

eren

t fro

m th

e va

lue

obta

ined

on

day

7 fo

r the

sam

e M

eHg

dose

gro

up a

t p<

0.05

.A a

nd A

A: T

he v

alue

s for

this

MeH

g do

se g

roup

are

sign

ifica

ntly

diff

eren

t fro

m th

e va

lues

for t

he 0

mg

MeH

g kg

−1 B

W d

ose

grou

p at

p<

0.05

and

0.0

01, r

espe

ctiv

ely,

rega

rdle

ss o

f the

die

ts.

B and

BB: T

he v

alue

s for

this

MeH

g do

se g

roup

are

sign

ifica

ntly

diff

eren

t fro

m th

e va

lues

for t

he 1

mg

MeH

g kg

−1 B

W d

ose

grou

p at

p<

0.05

and

0.0

01, r

espe

ctiv

ely,

rega

rdle

ss o

f the

die

ts.



131

urinary protein level. Urinary protein level was generally higherin 3 than 0 mg MeHg kg−1 BW-dosed rats, and the differencereached statistically significant level for the soy oil group. Urinaryprotein level in 3 mg MeHg kg−1 BW-treated rats fed soy oil dietwas significantly higher on day 14 than day 0 of exposure.

Urinary Creatinine. On day 14 of exposure to MeHg, a signifi-cant main effect on urinary creatinine level was found for MeHg(Table 2). In all dietary groups, the urinary creatinine level waslower in the 3 than 0 mg MeHg kg−1 BW-treated rats, and the dif-ference was statistically significant in the DHA group.

Creatinine Clearance. MeHg expressed a significant main effecton creatinine clearance level (Table 2). Although on the whole,creatinine clearance was significantly lower in the 3 mg MeHg kg−1

BW-dosed than vehicle control rats, this was only statistically sig-nificant in the DHA group. In the lard group, however, creatinineclearance was significantly lower in the 3 than 1 mg MeHg kg−1

BW-dosed rats.

Urinary GGT. On day 0 of exposure to MeHg, diet expressed asignificant main effect on urinary GGT activity, with the highestactivity found in the fish oil group (Table 2). On day 7 of exposure,MeHg showed a significant main effect on urinary GGT activity,which was significantly decreased by 3 mg MeHg kg−1 vs vehiclecontrol in the soy oil, seal oil and lard groups. In the lard group,1 mg MeHg kg−1 BW also significantly decreased urinary GGTactivity. In all the dietary groups, the rats treated with 3 mgMeHg kg−1 BW had significantly lower urinary GGT on days 7 and14 than on day 0 of exposure. A significant main effect and inter-action on urinary GGT activity was observed for both diet andMeHg on day 14 of exposure. Urinary GGT activity was higherin the fish oil than in other groups, and significantly decreasedby 3 mg MeHg kg−1 BW vs vehicle control in all except soy oilgroups. In all dietary groups, urinary GGT activity in 3 mg MeHgkg−1 BW-treated rats decreased on day 14 vs day 0 of exposure.

Serum Parameters of Kidney Function

Serum Creatinine. MeHg imposed a significant main effect onserum creatinine level (Table 3). The 3 mg MeHg kg−1 BW signifi-cantly increased serum creatinine level over 0 mg MeHg kg−1 BWin all dietary groups and over 1 mg MeHg kg−1 BW in all exceptthe soy oil group.

Serum BUN. A significant main effect on BUN was found for MeHg(Table 3). MeHg at 3 mg kg−1 BW significantly decreased serumBUN level as compared with vehicle control, especially in theseal oil, fish oil and lard groups. Within the fish oil group, the1 mg MeHg kg−1 BW also significantly decreased serum BUNlevel against vehicle control.

Serum Uric Acid. Both diet and MeHg expressed a significantmain effect and interaction on serum uric acid (Table 3). Serumuric acid level in the vehicle control rats was higher in the soy oil,seal oil and fish oil groups than in the lard group, and wasincreased by 3 mg MeHg kg−1 BW treatment as compared withvehicle control, especially in the DHA and lard groups.

Serum Ca. A significant main effect and interaction on serumCa level was detected for both diet and MeHg (Table 3). Thevehicle control rats fed the soy oil diet had significantly lower

serum Ca level than those fed the seal oil, fish oil or lard diet. Sig-nificantly higher serum Ca level was found in the 1 than 0 or3 mg MeHg kg−1 BW-dosed rats fed the seal oil diet.

Serum Mg. Both diet and MeHg showed a significant maineffect and interaction on serum Mg level (Table 3). The vehiclecontrol rats fed the lard diet had significantly higher serum Mglevel than those fed the seal oil or fish oil diet. Rats fed the soyoil diet had overall greater serum Mg level than those fed theseal oil diet. In all dietary groups except the lard group, 3 mgMeHg kg−1 BW significantly increased serum Mg level over theirvehicle controls, and in the seal oil and fish oil groups, also overthe 1 mg MeHg kg−1 BW.

Serum Parameters Known to be Associated with Kidney Disease

Neutrophil Counts. Both diet and MeHg expressed a signifi-cant main effect on neutrophil counts (Table 4). Overall, the ratsfed DHA or fish oil diet had higher neutrophil counts than thosefed other diets. Within all groups except the seal oil group, theneutrophil counts were significantly increased by 3 mg MeHgkg−1 BW over the vehicle control, and also over the 1 mg MeHgkg−1 BW dose within the soy oil, fish oil or lard group.

Serum Albumin. MeHg showed a significant main effect onserum albumin level (Table 4). There was a general trend of adecrease in serum albumin with increasing MeHg dose. In allexcept DHA groups, 3 mg MeHg kg−1 BW significantly decreasedserum albumin level over vehicle control. In the fish oil group,1 mg MeHg kg−1 BW also significantly decreased serum albuminlevel.

Serum Cholesterol. Both diet and MeHg imposed a significantmain effect on serum total cholesterol level (Table 4). The ratsfed DHA diet had significantly lower serum total cholesterollevel than those fed soy oil, seal oil or lard. In all dietary groups,serum total cholesterol increased with MeHg dose, and was sig-nificantly higher in 3 mg MeHg kg−1 BW-treated than vehiclecontrol rats.

Serum Triglycerides. MeHg and diet both showed a significantmain effect and interaction on serum triglyceride level (Table 4).The lard group had significantly higher serum triglyceride levelthan all the other groups. MeHg at 3 mg kg−1 BW significantlydecreased serum triglyceride level over vehicle control in the soyoil, fish oil and lard groups, and at 1 mg kg−1 BW in the soy oil andlard groups.

Organic Ion Transport

PAH Transport. Only MeHg expressed a significant main effect(p < 0.001) on PAH transport into the kidney slices (Fig. 3). In alldietary groups except the seal oil group, 3 mg MeHg kg−1 BWsignificantly decreased PAH transport into kidney slice over thevehicle control. In the soy oil and lard groups, the PAH transportwas also significantly lower in the 3 than 1 mg MeHg kg−1 BW-treated rats.

TEA Transport. Both diet (p < 0.001) and MeHg (p < 0.001)expressed significant main effect on transport of TEA by kidneyslices (Fig. 4). The DHA group had greater TEA transport into the

X. Jin et al.


132

Tabl

e 3.

Effe

cts

of d

iet a

nd M

eHg

on s

ome

seru

m p

aram

eter

s of

kid

ney

func

tion

Seru

m p

aram

eter

sM

ain

effe

ct (p

-val

ue)

MeH

gdo

segr

oup

Die

tary

gro

up1

Refe

renc

eva

lue

Die

tM

eHg

Inte

ract

ion

Soy

oil

DH

ASe

al o

ilFi

sh o

ilLa

rd

Crea

tinin

e (μ

mol

l−1 )

0.79

2<0

.001

0.31

30

45.6

7±

1.05

40.1

7±

1.58

44.1

7±

0.98

42.6

0±

2.32

42.2

9±

2.15

47.6±

7.42

140

.67±

1.31

44.0

0±

1.77

42.6

7±

2.03

44.0

0±

1.73

42.5

0±

2.50

3AA

BB51

.00±

0.86

b50

.83±

2.12

aab

51.0

0±

1.37

ab50

.33±

1.54

ab48

.33±

1.87

ab

BUN

(mg

dl−1

)0.

871

<0.0

010.

081

017

.20±

1.56

14.5

0±

0.56

18.0

0±

0.68

18.2

0±

1.32

17.7

1±

0.99

19±

2.23

113

.83±

0.87

15.5

0±

1.34

15.8

3±

0.31

14.5

0±

0.56

a14

.67±

0.92

3AA

B14

.00±

0.89

14.5

0±

0.88

12.5

0±

0.76

aa11

.83±

1.14

aa14

.17±

1.82

a

Uric

aci

d (μ

mol

l−1 )

0.00

10.

001

0.02

80

83.0±

4.4x

75.7±

9.4

83.7±

4.7x

94.0±

16.4

x59

.0±

4.3y

69.2±

33.8

2

110

5.3±

9.5y

70.3±

5.6x

73.8±

2.0x

81.0±

7.1

75.3±

8.0x

3AB

103.

7±

6.4

108.

8±

10.1

ab87

.0±

6.4

84.8±

4.2

85.8±

6.3a

Ca (m

mol

l−1 )

<0.0

010.

012

0.00

80

2.59

±0.

02x

2.67

±0.

032.

74±

0.03

y2.

74±

0.02

y2.

79±

0.03

y2.

62±

0.07

2

12.

65±

0.03

yy2.

69±

0.04

y2.

85±

0.04

ax2.

73±

0.03

2.72

±0.

02y

3B2.

66±

0.03

2.69

±0.

022.

67±

0.05

bb2.

63±

0.03

2.68

±0.

03M

g (m

g dl

−1)

0.00

8<0

.001

0.02

20

2.18

±0.

052.

03±

0.05

1.98

±0.

04y

1.98

±0.

07y

2.27

±0.

07x

2.53

±0.

152

12.

25±

0.06

2.08

±0.

052.

07±

0.04

2.02

±0.

052.

23±

0.07

3AA

BB2.

42±

0.05

a2.

38±

0.09

aa2.

32±

0.04

ab2.

43±

0.09

aabb

2.25

±0.

101 : V

alue

s fo

r ea

ch p

aram

eter

are

the

mea

ns±

stan

dard

err

or fr

om fi

ve o

r si

x an

imal

s of

the

sam

e tr

eatm

ent

grou

p. T

he s

ingl

e-un

derli

ned

diet

ary

grou

p is

sig

nific

antly

diff

eren

t(p

<0.

05) f

rom

the

doub

le-u

nder

lined

die

tary

gro

up w

hen

com

pare

d fo

r the

sam

e se

rum

par

amet

er.

2 : Val

ue fo

r you

ng m

ale

Spra

gue–

Daw

ley

rats

(Lill

ie e

t al.,

199

6).

3 : Val

ue fo

r adu

lt m

ale

Spra

gue–

Daw

ley

rats

(Koh

n an

d Cl

iffor

d, 2

002)

.x a

nd y

or y

y : The

val

ues

are

sign

ifica

ntly

diff

eren

t fro

m e

ach

othe

r at p

<0.

05 o

r p<

0.00

1 w

hen

com

pare

d fo

r the

sam

e M

eHg

dose

gro

up.

a and

aa: T

he v

alue

is s

igni

fican

tly d

iffer

ent

from

the

val

ue fo

r th

e ve

hicl

e co

ntro

l rat

s (0

mg

MeH

g kg

−1 B

W) a

t p

<0.

05 a

nd 0

.001

, res

pect

ivel

y, w

hen

com

pare

d w

ithin

the

sam

edi

etar

y gr

oup.

b an

d bb

: The

val

ue is

sig

nific

antly

diff

eren

t fro

m th

e va

lue

for t

he 1

mg

MeH

g kg

−1 B

W d

osed

rats

at p

<0.

05 a

nd 0

.001

, res

pect

ivel

y, w

hen

com

pare

d w

ithin

the

sam

e di

etar

y gr

oup.

A a

nd A

A: T

he v

alue

s fo

r thi

s M

eHg

dose

gro

up a

re s

igni

fican

tly d

iffer

ent f

rom

the

valu

es fo

r the

0m

g M

eHg

kg−1

BW

dos

e gr

oup

at p

<0.

05 a

nd 0

.001

, res

pect

ivel

y, re

gard

less

of t

hedi

ets.

B and

BB: T

he v

alue

s fo

r thi

s M

eHg

dose

gro

up a

re s

igni

fican

tly d

iffer

ent f

rom

the

valu

es fo

r the

1m

g M

eHg

kg−1

BW

dos

e gr

oup

at p

<0.

05 a

nd 0

.001

, res

pect

ivel

y, re

gard

less

of t

hedi

ets.



133

Tabl

e 4.

Effe

cts

of d

iet a

nd M

eHg

on s

ome

seru

m p

aram

eter

s kn

own

to b

e as

soci

ated

with

rena

l inj

ury

Bloo

d/se

rum

par

amet

ers

Mai

n ef

fect

(p-v

alue

)M

eHg

dose

gr

oup

Die

tary

gro

up1

Refe

renc

eva

lue2

Die

tM

eHg

Inte

ract

ion

Soy

oil

DH

ASe

al o

ilFi

sh o

ilLa

rd

Neu

trop

hil C

ount

s (×

109 l−

1 )<0

.001

<0.0

010.

266

00.

86±

0.12

1.62

±0.

621.

27±

0.27

1.40

±0.

311.

11±

0.13

0.87

±0.

661

1.19

±0.

25y

2.73

±0.

36x

1.21

±0.

23y

1.36

±0.

191.

10±

0.15

y

3AA

BB2.

62±

0.79

ab4.

42±

1.11

ay1.

58±

0.35

x4.

96±

0.61

aabb

y2.

67±

0.57

ab

Alb

umin

(g l−

1 )0.

189

<0.0

010.

184

015

.8±

0.3

16.0±

0.5

16.8±

0.4

16.6±

0.3

17.6±

0.5

30.8±

1.1

1A15

.7±

0.2

15.3±

0.7

16.3±

0.3

14.5±

0.2a

16.2±

0.4

3AA

BB13

.8±

1.1a

15.2±

0.4

14.7±

0.5a

13.7±

0.3a

13.5±

0.9aa

b

Tota

l cho

lest

erol

(mm

ol l−

1 )<0

.001

<0.0

010.

910

01.

11±

0.06

0.86

±0.

061.

19±

0.10

1.02

±0.

101.

23±

0.06

1.01

±0.

221A

1.42

±0.

041.

11±

0.09

1.52

±0.

111.

23±

0.11

1.47

±0.

063A

ABB

1.66

±0.

09ay

1.55

±0.

08aa

bbx

1.85

±0.

14aa

y1.

66±

0.07

aab

1.87

±0.

11aa

by

Trig

lyce

rides

(mm

ol l−

1 )<0

.001

<0.0

01<0

.001

01.

48±

0.21

yyz

0.91

±0.

18yy

0.72

±0.

10yy

ww

0.88

±0.

12yy

3.56

±0.

45x

2.16

±0.

811A

0.82

±0.

08 y

ya0.

87±

0.15

yy0.

89±

0.04

yy0.

69±

0.06

yy2.

05±

0.38

xa

3AA

BB0.

61±

0.06

aa0.

61±

0.03

0.51

±0.

02b

0.48

±0.

06a

0.72

±0.

11aa

bb

1 : Val

ues

for e

ach

para

met

er a

re th

e m

eans

±st

anda

rd e

rror

from

5–6

ani

mal

s of

the

sam

e tr

eatm

ent g

roup

. The

sin

gle-

unde

rline

d di

etar

y gr

oup

is s

igni

fican

tly d

iffer

ent (

p<

0.05

)fr

om th

e do

uble

-und

erlin

ed d

ieta

ry g

roup

whe

n co

mpa

red

for t

he s

ame

seru

m p

aram

eter

.2 : V

alue

for y

oung

mal

e Sp

ragu

e–D

awle

y ra

ts (L

illie

et a

l., 1

996)

.x a

nd y

or y

y : The

val

ues

are

sign

ifica

ntly

diff

eren

t fro

m e

ach

othe

r at p

<0.

05 o

r p<

0.00

1 w

hen

com

pare

d fo

r the

sam

e M

eHg

dose

gro

up.

z and

ww

: The

val

ues

are

sign

ifica

ntly

diff

eren

t fro

m e

ach

othe

r at p

<0.

001

whe

n co

mpa

red

for t

he s

ame

MeH

g do

se g

roup

.a a

nd aa

: The

val

ue is

sig

nific

antly

diff

eren

t fr

om t

he v

alue

for

the

vehi

cle

cont

rol r

ats

(0m

g M

eHg

kg−1

BW

) at

p<

0.05

and

0.0

01, r

espe

ctiv

ely,

whe

n co

mpa

red

with

in t

he s

ame

diet

ary

grou

p.b

and

bb: T

he v

alue

is s

igni

fican

tly d

iffer

ent f

rom

the

valu

e fo

r the

1m

g M

eHg

kg−1

BW

dos

ed ra

ts a

t p<

0.05

and

0.0

01, r

espe

ctiv

ely,

whe

n co

mpa

red

with

in th

e sa

me

diet

ary

grou

p.A a

nd A

A: T

he v

alue

s fo

r thi

s M

eHg

dose

gro

up a

re s

igni

fican

tly d

iffer

ent f

rom

the

valu

es fo

r the

0m

g M

eHg

kg−1

BW

dos

e gr

oup

at p

<0.

05 a

nd 0

.001

, res

pect

ivel

y, re

gard

less

of t

hedi

ets.

B and

BB: T

he v

alue

s fo

r thi

s M

eHg

dose

gro

up a

re s

igni

fican

tly d

iffer

ent f

rom

the

valu

es fo

r the

1m

g M

eHg

kg−1

BW

dos

e gr

oup

at p

<0.

05 a

nd 0

.001

, res

pect

ivel

y, re

gard

less

of t

hedi

ets.

X. Jin et al.


134kidney slice than the soy oil, fish oil and lard groups. MeHg causeda general dose-dependent decrease in TEA transport by kidneyslice. In all dietary groups, 3 mg MeHg kg−1 BW significantlydecreased TEA transport over 0 and 1 mg MeHg kg−1 BW.

Pathology of Kidney

Very mild focal interstitial nephritis (grade 1) was rarely observedor absent in the vehicle control rats [Fig. 5(A)]. Varying degreesof nephrosis were observed in all rats treated with MeHg[Fig. 5(B, C)]. The nephrosis rating ranged from grade 1 to 3 forrats treated with 1 mg MeHg kg−1 BW [Fig. 5(B)] and increased to4 and/or 5 with 3 mg MeHg kg−1 BW [Fig. 5(C)], regardless of thedietary fats. There was a striking uniformity of the renal lesionsin the rats treated with 3 mg MeHg kg−1 BW. With this MeHg

dose, minimal or mild interstitial leukocytic infiltrates composedof lymphocytes, neutrophils, eosinophils and fibroblasts werealso observed [Fig. 5(C)]. TW-ANOVA on ranked grading datarevealed a significant main effect of MeHg on nephrosis. Withineach dietary group, both 1 and 3 mg MeHg kg−1 BW significantlyincreased the level of nephrosis over the vehicle control, and3 mg MeHg kg−1 BW over 1 mg MeHg kg−1 BW (Fig. 6). The ratsdosed with 1 mg MeHg kg−1 BW in the soy oil group had a signi-ficantly higher level of nephrosis than those in the seal oil andDHA groups.

Correlation Analysis

Relative kidney weight, serum total cholesterol and TEA uptakeby kidney slices significantly correlated, either negatively or

Figure 3. Uptake of PAH (S/M) by kidney slice from rats fed soy oil, DHA, seal oil, fish oil or lard diet and exposed to 0 (empty bar), 1 (right stripedbars) or 3 (left striped bars) mg MeHg kg−1 BW for 14 days. The value represents the mean of four to six rats in the same treatment group. The error barstands for standard error of the mean. a: The value is significantly different from that of 0 mg MeHg group at p < 0.05. b: The value is significantly differ-ent from that of 1 mg MeHg group at p < 0.05.

Figure 4. Uptake of TEA (S/M) by kidney slice from rats fed soy oil, DHA, seal oil, fish oil or lard diet and exposed to 0 (empty bar), 1 (right stripedbars) or 3 (left striped bars) mg MeHg kg−1 BW for 14 days. The value represents the mean of four to six rats in the same treatment group. The error barstands for standard error of the mean. aa: The value is significantly different from that of 0 mg MeHg group at p < 0.001. b: The value is significantly dif-ferent from that of 1 mg MeHg group at p < 0.05.



135

positively, with MeHg dose, kidney total mercury content orkidney MeHg content in all dietary groups (Table 5). For someparameters such as urinary GGT, serum Mg, triglyceride, neu-trophil counts and PAH uptake, significant correlations werefound in four dietary groups, while for other parameters such as

urinary osmolality, protein, creatinine, serum creatinine, BUN,uric acid, Ca and albumin, significant correlations were identifiedonly in two or three dietary groups.

DiscussionOur data revealed that, regardless of the dietary fats used, thekidney is an important organ of mercury accumulation in exposedrats, confirming the findings of others (Folsom and Fishbein,1972; Ohi et al., 1976; Mangos et al., 1981; Yasutake et al., 1997).Similar content of either total mercury or MeHg in kidney wasfound among all dietary fat groups, suggesting that dietary fatsdid not affect the transportation and transformation of MeHg inthe kidney of the dosed rats. This agrees with the finding ofSakamoto et al. (1988), who reported no significant differencesin total or inorganic mercury content in the kidney of rats fed acod-liver oil diet compared with those fed a beef tallow dietafter 5 weeks of exposure to MeHg.

MeHg at 3 mg kg−1 BW increased relative kidney weight regard-less of the diets used, supporting the reports of others (Kleinet al., 1973; Slotkin et al., 1985; Verschuuren et al., 1976a,b,c).Although the exact mechanism of MeHg-induced increase inrelative kidney weight is not fully understood, it was shown thatin neonatal rats, MeHg altered adrenergic receptor binding andcaused pronounced and persistent renal hypertrophy and func-tion impairment at doses that are associated primarily with neu-rotoxicity (Slotkin et al., 1985, 1986a). Chemical sympathectomycompletely inhibited the initial phase of MeHg-induced renalhypertrophy, suggesting that the sympathetic system may playa partial role in some of the actions of MeHg on kidney growth(Bartolome et al., 1985).

The glomerular membrane normally serves to prevent thepassage of high molecular weight protein into the urine. Pro-teinuria is primarily an indicator of glomerular damage, but mayalso occur with renal tubule damage (Ragan and Weller, 1999).MeHg induced proteinuria has been reported previously (Vers-chuuren et al., 1976a,b,c). In the present study, MeHg imposeda significant main effect on urinary protein level, but did notinduce severe proteinuria in any of the dietary groups after 14days of exposure. However, a severe proteinuria may occur withprolonged, or at a specific stage of, exposure to MeHg. Omega-3fatty acids have been shown to improve the condition of pro-teinuria in human subjects (De Caterina et al., 1993; Lenzi et al.,1996; Sulikowska et al., 2004). Ohtake et al. (2002) found thatdietary cod liver oil was protective against the renal toxicity ofdaunomycin in mice when compared with dietary soy oil. In thisstudy, however, the highest urinary protein level was found inthe rats fed a fish oil diet, which is rich in omega-3 fatty acids,with or without MeHg treatment. A significant decrease in urinarycreatinine and creatinine clearance level was found in MeHg-treated rats fed the DHA diet, but not other diets, suggestingthat DHA, also a rich source of omega-3 fatty acids, did not pro-tect, and may even have aggravated, the renal toxicity of MeHg.

GGT is a brush border membrane enzyme of the proximalconvoluted tubule, and known to play an important role in thedisposition and secretion of mercury (Ballatori et al., 1998; DeCeaurriz et al., 1994). It was shown that MeHg excretion into theurine was more rapid in the GGT-deficient mice than in wild-typeand heterozygous mice (Ballatori et al., 1998), and that the inhibi-tion of GGT activity using avicin dramatically increased urinaryexcretion of MeHg (Gregus et al., 1987). Increased enzymuria hasbeen considered as a very early sign of reversible tubular damage,

Figure 5. H&E staining of kidney sections from rats fed the lard dietand exposed to 0 (A), 1 (B) or 3 (C) mg MeHg kg−1 BW for 14 days.Medium size arrow heads point to basophilic epithelial cells of the proxi-mal or distal convoluted tubules. Small arrows point to the proteinaciouscell debris or dead cells in the tubule lumen. Large arrow heads point tomitotic figure in the tubule epithelial cells.

X. Jin et al.


136

Tabl

e 5.

Corr

elat

ion

(coe

ffici

ents

) bet

wee

n to

xico

logi

cal e

ndpo

ints

and

MeH

g do

se, t

otal

mer

cury

and

MeH

g co

nten

t in

kidn

ey

Die

tM

ercu

ry

dose

or c

onte

nt

in k

idne

y

Toxi

colo

gica

l end

poin

ts

Org

anic

Ion

Tran

spor

tRe

lativ

e ki

dney

w

eigh

t

Urin

e bi

oche

mis

try

Seru

m b

ioch

emis

try

Osm

olal

ityG

GTa

Prot

ein

Crea

tinin

eCr

eatin

ine

BUN

bU

ricac

idCa

Mg

Neu

trop

hil

coun

tsA

lbum

inTo

tal

chol

este

rol

Trig

lyce

rides

PAH

cTE

Ad

Soy

oil

Dos

e0.

807*

**e

−0.5

13*f

NSg

0.67

0**h

NS

NS

NS

NS

NS

0.63

5**

0.57

1*N

S0.

804*

**−0

.773

***

−0.6

63**

−0.7

04**

*To

tal

0.88

3***

NS

NS

0.66

6***

−0.5

07*

NS

NS

NS

NS

0.61

9*0.

480*

NS

0.80

9***

−0.6

25**

−0.5

70*

−0.6

34**

MeH

g 0.

733*

**−0

.641

**N

S0.

712*

*N

SN

SN

SN

SN

S0.

536*

0.55

0*N

S0.

695*

*−0

.689

**−0

.588

*−0

.584

*D

HA

Dos

e0.

716*

*N

S−0

.624

**N

S−0

.561

*0.

722*

**N

S0.

474*

NS

0.68

0***

686*

*N

S0.

665*

*N

S−0

.696

**−0

.897

***

Tota

l 0.

640*

*N

S−0

.610

**N

S−0

.589

*0.

633*

*N

S0.

559*

NS

0.64

9**

0.59

1**

NS

0.52

9*−0

.482

*−0

.680

**−0

.866

***

MeH

g 0.

663*

*N

S−0

.572

*N

S−0

.520

*0.

585*

NS

0.51

3*N

S0.

674*

*0.

574*

NS

0.57

3*N

S−0

.669

**−0

.865

***

Seal

oil

Dos

e0.

786*

**−0

.570

*−0

.751

***

0.52

9*N

S0.

561*

−0.8

47**

*N

SN

S0.

808*

**N

S−0

.654

**0.

679*

*N

SN

S−0

.903

***

Tota

l 0.

783*

**−0

.529

*−0

.718

***

NS

−0.5

00*

NS

−0.8

89**

*N

SN

S0.

763*

**N

S−0

.637

**0.

616*

*N

SN

S−0

.864

***

MeH

g0.

717*

**N

S−0

.770

***

NS

NS

0.50

5*−0

.780

***

NS

NS

0.66

4**

NS

−0.6

26**

0.56

2*N

SN

S−0

.894

***

Fish

oil

Dos

e0.

590*

−0.5

65*

−0.5

89*

NS

NS

0.55

6*−0

.744

***

NS

−0.5

47*

0.71

5**

0.73

6**

−0.8

88**

*0.

747*

**−0

.584

**−0

.595

*−0

.793

***

Tota

l 0.

558*

NS

−0.6

13*

NS

NS

0.59

7*−0

.747

***

NS

−0.6

03*

0.65

0**

0.69

1**

−0.8

24**

*0.

649*

*−0

.691

**−0

.519

*−0

.790

***

MeH

gN

SN

S−0

.563

*N

SN

SN

S−0

.841

***

NS

−0.5

37*

0.61

2*0.

612*

−0.8

81**

*0.

675*

*−0

.529

**N

S−0

.710

**La

rdD

ose

0.57

6**

NS

−0.7

68**

*N

SN

SN

SN

S0.

612*

*−0

.555

*N

S0.

615*

*−0

.715

***

0.79

8***

−0.7

93**

*−0

.488

*−0

.665

**To

tal

0.50

8*N

S−0

.735

***

NS

NS

NS

−0.4

76*

0.55

0*−0

.495

*N

S0.

549*

−0.5

76**

0.76

5***

−0.6

31**

NS

−0.5

91**

MeH

g0.

471*

NS

−0.7

64**

*N

SN

S0.

480*

NS

0.46

2*−0

.520

*N

S0.

613*

*−0

.592

**0.

734*

**−0

.758

***

−0.4

78*

−0.6

07**

a Gam

ma-

glut

amyl

tran

sfer

ase.

b Ure

a ni

trog

en.

c para

-Am

ino-

hipp

urat

e.d Te

trae

thyl

amm

oniu

m.

e The

corr

elat

ion

was

sig

nific

ant a

t p<

0.00

1.f Th

e co

rrel

atio

n w

as s

igni

fican

t at p

<0.

05.

g Not

sig

nific

ant o

r p>

0.05

.h Th

e co

rrel

atio

n w

as s

igni

fican

t at p

<0.

01.



137

which often correlates directly with a rise in serum creatinine,and a fall in creatinine clearance (Trof et al., 2006). Increased urinaryGGT has been associated with exposure to inorganic mercury inexperimental animals (Sener et al., 1979). However, in this study,MeHg significantly decreased urinary GGT in four dietary groups.This may indicate a more advanced stage or a higher degree ofbrush border membrane damage by MeHg.

Although serum creatinine level is influenced by multiplefactors such as diet, muscle mass and metabolism, and tubularsecretion and reabsorption, it has been most widely used as ameasure of the glomerular filtration rate (GFR) and a commonlyaccepted measure of renal function in clinical medicine (Perroneet al., 1992). In this study, MeHg increased serum creatinine levelsover the vehicle control in all dietary groups, which concurs withthe finding of Yasutake et al. (1990, 1997). This could be an indica-tion of a reduced GFR by MeHg, especially in the DHA groupwhere it was also associated with decreased creatinine clearance.It was unexpected that the increased serum creatinine by MeHgin the seal oil, fish oil and lard groups was not accompanied byincreased, but rather decreased, BUN. This is concordant withthe report of Fair et al. (1985). In contrast, Verschuuren et al.(1976a), Kempson et al. (1977), and Slotkin et al. (1985) revealeda significant increase in serum urea in MeHg-exposed rats, whileKlein et al. (1973) and Stroo and Hook (1977) detected nochanges in serum urea in dosed rats. This discrepancy reflects ahigh degree of complexity of MeHg nephrotoxicity. The decreasedserum BUN observed in this study could be a result of suppressedprotein catabolism, and/or an increased urinary excretion of urea(Slotkin et al. 1986b). It has been shown that, after chronic expo-sure, MeHg accumulated in the cortical lysosomes, which is acenter of protein catabolism, in the proximal tubule of the kidney,and altered lysosomal profiles in male monkeys (Chen et al., 1983).In mice, MeHg altered the lysosomal profile after only 7 days ofi.p. injection, which was also accompanied by decreased GGTactivity and serum urea nitrogen (Fair et al., 1985). In fact, bothMeHg and inorganic mercury have been shown to inhibit theactivity of a number of lysosomal enzymes in experimental ani-mals (Fowler et al., 1975; Zalme et al., 1976; Stroo and Hook, 1977;

Nakano and Itoh, 1983), and both have been found to inhibitprotein degradation by lysosomal enzymes in kidney proximaltubule (Madsen and Christensen, 1978).

Elevated serum uric acid as a result of both increased genera-tion and decreased excretion is a well known characteristic ofacute renal failure (Ejaz et al., 2007). Recently, Kang and Nakagawa(2005) demonstrated that hyperuricemia increased systemicblood pressure, proteinuria, renal dysfunction, vascular diseaseand progressive renal scarring in rats, suggesting a role of uricacid in the progression of renal disease. A number of epidemio-logical studies have linked hyperuricemia to cardiovascular andcerebrovascular disease, hypertension, diabetes and metabolicdisease (Hikita et al., 2007). Although the mechanisms by whichuric acid may play a pathogenic role in these diseases are unclear,hyperuricemia has been shown to impose deleterious effects onendothelial function, oxidative metabolism, platelet adhesive-ness, and aggregation (Alderman and Redfern, 2004). In thepresent study, we observed a significant increase in serum uricacid in MeHg-treated animals fed DHA or lard diet, indicatingthat, with these two diets, MeHg exposure may contribute notonly to renal injury, but also the risk of other diseases associatedwith hyperuricemia.

Dyslipidemia is one of the characteristics of nephrotic syndromewith elevated low density lipoprotein cholesterol, triglyceride,and lipoprotein(a) (Kronenberg, 2005; Akyol et al., 2007). In addi-tion, proteinuria, hypoalbuminemia, hyperoxidative stress andinflammation are also known to be associated with nephroticsyndrome (Dogra et al., 2002; Camici, 2007). Nephrotic syndromeassociated with exposure to inorganic mercury has been observedrepeatedly in humans (Barr et al., 1972; Kibukamusoke et al.,1974; Gerstner and Huff, 1977; Agner and Jans, 1978; Tubbset al., 1982; Meeks et al., 1990; Yo et al., 2003; Tang et al., 2006)and animals (Shull et al., 1981; Michaud et al., 1984). However, itsassociation with MeHg exposure has rarely been reported,although patients with Minimata disease in Japan showed sig-nificantly increased standardized mortality ratio for nephritis–nephrotic syndrome in both females and males (Tamashiro et al.,1985). In the present study, MeHg did not cause severe proteinuria,

Figure 6. Qualitative grading of nephrosis observed in rats fed soy oil, DHA, seal oil, fish oil or lard diet and exposed to 0 (empty bar), 1 (right stripedbars) or 3 (left striped bars) mg MeHg kg−1 BW for 14 days. The value represents the mean of four to six rats in the same treatment group. The error barstands for standard error of the mean. aa: The value is significantly different from that of 0 mg MeHg dose group at p < 0.001. bb: The value is signifi-cantly different from that of 1 mg MeHg dose group at p < 0.001.

X. Jin et al.


138

but significantly decreased serum albumin, caused neutrophilinfiltration in the kidney and increased circulating neutrophil,total cholesterol and systemic oxidative stress (Jin et al., 2008),which are all typical characteristics of nephrotic syndrome.However, it decreased rather than increased serum triglyceridelevel. This could be a result of decreased transport from theintestine and/or increased hydrolysis by circulating lipase oftriglyceride by MeHg. In fact, in a different study, we did find anincreased serum lipase activity in rats treated with the samedose of MeHg (unpublished data). Although the effects ofMeHg on serum cholesterol and triglyceride were universalamong the five dietary groups, the effects on serum albuminwere nearly absent in the DHA group, and the effects on neutro-phil counts were hardly seen in the seal oil group. This suggeststhat the dietary fat can influence the expression of the nephroticsyndrome induced by MeHg.

Although it has been shown that MeHg and its amino acidconjugates are transportable substrates of organic anion trans-porter in kidney (Tanaka et al., 1992; Koh et al., 2002; Zalups andAhmad, 2005), little is known about the effects of MeHg on thetransport of organic anion. Hirtsch (1971) noted a significantdecrease in the ability of kidney slices to accumulate PAH or N-methylnicotinamide (NMN) by MeHg, while Stroo and Hook (1977)only found a decrease in the accumulation of NMN by kidneyslice exposed to MeHg. In the present study, MeHg dramaticallydecreased uptake of an organic anion, PAH, by kidney slice in allgroups except the seal oil group. This could be caused byMeHg-induced generation of reactive oxygen species whichcould down-regulate organic anion transporter in the kidney(Takeda et al., 2000), or a decreased ATP production in the mito-chondria by MeHg (Stroo and Hook, 1977). However, the protec-tive role of seal oil cannot be explained at present. In addition toPAH, MeHg also markedly decreased uptake of an organiccation, TEA, by kidney slices in all dietary groups. Although theexact mechanism behind this is not clear, inorganic mercury ionhas been shown to interact with cysteine residues of humanorganic cation transporter 2 (hOCT2), and reduce hOCT2-mediated transport of TEA into Chinese hamster ovary cellsstably expressing hOCT2 (Pelis et al. 2007).

We observed a dose-dependent focal nephrosis in the kidneyof MeHg-treated rats, confirming the findings by others (Kleinet al., 1973; Folsom and Fishbein, 1972; Verschuuren et al., 1976a;Munro et al., 1980; Eto et al., 1997). The interstitial leukocyticinfiltration of renal tubules found with 3 mg MeHg kg−1 treatmentexplained the increased neutrophil counts by the same dose ofMeHg in all dietary groups except the seal oil group. Along withhistological observations, ultrastructural changes were reported,including increased lysosomal profile, cytoplasmic degeneration,floccular degeneration of mitochondrial matrix material of themitochondria and extrusion of cellular masses into the tubularlumen (Klein et al., 1973; Ware et al., 1975; Chen et al., 1983; Fairet al., 1985). These morphological observations are in line with theaforementioned biochemical findings from urine and serum analysis.

Significant dose correlations were found for nearly all endpointsexamined with three endpoints in all dietary groups, and otherendpoints in some dietary groups, suggesting a modifying effectof dietary fats on the sensitivity of these parameters to MeHgdose changes.

In summary, regardless of the diets used, MeHg at 3 mg kg−1

BW induced numerous significant renal and systemic changesafter two weeks of exposure, pointing to a nephrotic syndrome-like renal toxicity. With the exception of relative kidney weight,

serum cholesterol and TEA transport as the parameters affectedby MeHg uniformly among all dietary groups, all other endpointsexamined were altered specifically in one or some of the dietarygroups. Decreased serum albumin level has been proposedto be the underlying cause of increased serum cholesterol innephrotic syndrome. This seemed to be true for all dietarygroups except the DHA group. In this study, some of the para-meters measured expressed different sensitivity to MeHg impactunder different dietary treatments. For example, serum albuminwas significantly decreased in the fish oil group by 1 mg MeHgkg−1, but not changed by even 3 mg MeHg kg−1 in the DHAgroup. While formulation of diets with a variety of fats did notprotect against MeHg nephrotoxicity, the specific clinical pro-files significantly differed. The observed modulating effects ofdietary fats on MeHg-induced renal toxicity are dependent onthe types of toxicological endpoints used; therefore, multipleparameters should be examined to compare MeHg toxicityunder different dietary backgrounds.

Acknowledgements

This project was supported by a research contract from the North-ern Contaminants Program, Indian and Northern Affair Canadaawarded to H.M. Chan and R. Mehta. The authors thank G. Laver,M. Barker, J. Clauseen, I. Greer and P. Smyth from the ToxicologyResearch Division, Food Directorate, HPFB, Health Canada, fortheir active participation in and contribution to this project. Wealso greatly appreciate the support of the animal care staff fromthe Animal Resources Division of Health Canada.

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