Influence of dietary protein levels on the fate of methylmercury and glutathione metabolism in mice

Toxwology, 72 (1992) 17-26 Elsewer Scientific Pubhshers Ireland Ltd

17

Influence of dietary protein levels on the fate of methylmercury and glutathione metabolism in mice T a t s u m i A d a c h i a, A k i r a Y a s u t a k e a a n d K l m i k o H l r a y a m a b aBtochemtstry Sectton, Department of Bastc Medtcal S~wnces, Nattonal ln~tttute Jor Mmamata Dtsease. Mmamata Ctty, Kumamoto 867 and hKumamoto Umverstty College of Medt~al Sewme. Kumamoto Ctty. Kumamoto 862 (Japan) (Recewed July 17th, 1991, accepted December 4th. 1991)

Summary We mvestlgated the influence of dietary protein levels on the fate of methylmercury (MeHg), the tissue glutathlone (GSH) levels and the efflux rates of GSH m C57BL/6N male mice One group of mice was fed a 7 5% protein diet (low protein diet, LPD) and the other was fed a 24 8% protein &et (normal protein diet, NPD) The cumulative a m o u n t of Hg in urine m LPD-fed mice was approximately 3 7-times lower than in N P D group during the 7 days after oral admm~stratlon of MeHg (20 #mol/kg), although the fecal Hg levels were identical m the two groups Hg concentration m kidney, hver and blood decreased timedependently for 7 days after the administration m both groups of mice, whereas the brain levels continued to increase during th)s period Tmsue Hg levels m the LPD group were slgmficantly h~gher than m the N P D group except for the hver Although the hepattc GSH level m LPD-fed mice was slgmficantly lower than m NPD-fed mice, the levels m the kidney, brain, blood and plasma were not different between the two groups The efflux rate (#mol/g body weight per day) of hepatic GSH m LPD-fed mice was slgmficantly lower than m the N P D group, whereas the efflux rates of renal GSH were identical m both groups. When MeHg (20 #mol/kg)-pretreated mtce were m.lected with acwlcln, a specific mhtbltor of "rglutamyltranspeptxdase, the urinary Hg levels increased by 60- and 36-fold m groups fed LPD and NPD. respectively As a result, the &fference m urinary Hg levels between the two groups &sappeared w~th acw~cm treatment. This result m&cated that LPD feeding might decrease urinary Hg excreuon by increasing the retention of MeHg metabohte(s) m renal cells Thus, our present study suggested that the dietary protein status, whach could modulate the metabohsm of thlol compounds, played an important role m determining the fate of MeHg

Key words Methylmercury, Glutathlone, D~etary protein

Introduction Through a ser/es of experiments using mice, the toxicity and the fate of methylmercury (MeHg) have been revealed to vary with strain [1,2], sex [1,3-5] and age [4]. Similar differences were also observed in other animal species [6-8]. These vanaUons were suggested to occur by vartous endogenous factors, such as glutathione Correspondence to Tatsum~ Adachl, Biochemistry Section, Department of Basic Me&cal Sciences, National Institute for M m a m a t a Disease, 4058-18 Hama, M m a m a t a C~ty, K u m a m o t o 867, Japan 0300-483X/92/$05 00 © 1992 Elsevier SclenUfic Pubhshers Ireland Ltd Printed and Pubhshed m Ireland

18 (GSH) [3], hemoglobin [2] and other thiol compounds, because MeHg has a high affintty for thiol group [9,10]. Since the levels of these factors are affected by nutrition, tt ts considered that the nutritional condttlon is one of the most important factors for the toxicity and the fate of MeHg. However, there are few reports about the effect of dietary protein levels on MeHg toxicity [1 l] and none of the reports served to elucllate the mechamsm. It has been well documented that the hepatic GSH levels are affected by various nutritional conditions such as fasting and lowered protein levels [12-18] These facts led us to speculate that dietary protein levels might contribute to the modulation of MeHg toxicity through the alteration of GSH metabolism. In the present study, in order to investigate the influence of &etary protem levels on the fate of MeHg, tissue distribution and excretion of Hg after administration of MeHg chloride were compared between two groups of C57BL/6N mice which were fed a 24.8% normal protein diet, (NPD) or a 7.5% protein diet, low protein diet, (LPD). The results were discussed from the viewpoint of GSH metabolism.

Materials and methods

Animals C57BL/6N male mice (aged 7 weeks) were obtained from C L E A Japan Co. (Tokyo). They were maintained at 24 ± I°C and 50-60% relative humidity and were exposed to a 12-h light cycle beginning at 06:00 h. They were acclimated to each of two kinds of casein-based pellet diets (CLEA Japan Co.) for 5 days before use in the experiment. The compositions of the diets are shown m Table I.

Time-dependent changes of tissue distrtbunon and excretion of Hg MeHg chloride (Tokyo Chemical Industry Co.) was dissolved in saline and administered orally to mice at a dose of 20/~mol/kg on day 0. Mice were housed m

TABLEI COMPOSITIONS OF DIETS Ingredient (g/100 g)

Casein (Protem) Cornstarch Sucrose Corn oil Cellulose Vitamin mix Mineral mix or-Starch

Diets NPD

LPD

29 0 (24 8) 41 0 10 0 60 50 1.0 7.0 10

88 (7 5) 61 2 10 0 60 50 I0 70 10

19 metabolism cages (1 mouse/cage) after MeHg administration. On days 1, 3, 5 and 7, urine and feces were collected and mice were killed under pentobarbital anesthesia. Blood was collected from the inferior caval vein in heparimzed syringes. After perfusion with sahne, the kidney, liver and brain were removed. An aliquot of blood was centrifuged to separate the plasma. Hg content in each sample was determined by the oxygen combustion-gold amalgamation method [19] using a Rigaku Mercury Analyzer SP-3 and expressed as total Hg.

Tissue GSH and half-hves Total GSH levels in the blood, plasma, kidney, liver and brain were determined by the method of Tietze [20]. GSH turnover rates in the kidney and hver were determined by the method reported previously [3] using L-buthionine-(S,R)-sulfoximine (BSO, Sigma) a specific inhibitor of 3,-glutamylcystelne synthetase [21].

Effect of acivicm After (24 h) oral administration of MeHg (20 ~mol/kg), mice were lntraperltoneally 0.P-) injected with acivicin (2 mmol/kg, Sigma), a specific inhibitor of "rglutamyltranspeptidase (7-GTP) [22,23]. After (3 h) the injection, the urinary and renal Hg levels were determined.

7-GTP actlvtty 3,-GTP activity in the kidney was determined by the method of Orlowski and Meister using L-3,-glutamyl-p-nitroanillde (Wako Pure Chemical Industries) as a substrate [24]. Protein content was determined by the method of Lowry et al. usmg bovine serum albumin (BSA) as a standard [25].

Statistical analysis Statistical analyses of variance and difference were carried out according to ANOVA and Student's t-test, respectively; the level of significance was put at P < 0.01 or 0.05. Results

After feeding on N P D or LPD for 5 days, no difference was observed in body weights between the two groups of mice, whereas liver and kidney weights in LPDfed mice were significantly lower than in NPD-fed mice (Table II). After the oral administration of MeHg (20 ~mol/kg), Hg levels in the kidney, liver and blood showed a monotonic decrease for 7 days, while the brain levels continued to increase m both groups (Figs. 1 and 2) The plasma Hg levels scarcely changed during this period. Although Hg levels m the kidney, brain, blood and plasma in LPD-fed mice were significantly higher than in NPD-fed mice, there was no marked difference in the hepatic levels. However, the percentage of dosed Hg m liver in LPD-fed mice was lower than m the NPD group and the kidney levels were identical in the two groups (data not shown), because of the differences in the tissue weights. Cumulative amounts of Hg excretion m mice fed LPD or NPD are shown in Fig. 3. NPD-fed mice excreted 12.8 and 6.0% of dosed Hg m urine and feces, respectwely,

20 TABLE II INFLUENCE OF DIETARY PROTEIN LEVELS ON BODY A N D TISSUE WEIGHTS IN MALE MICE

Body weight (g) Tissue weight (g)

Liver Kidney Brain

NPD group

LPD group

2233 ± 086 I 22 ± 0 09

21 92 ± 095 0 92 ± 0 10"

0 30 ± 0 02 043 ± 001

0 26 ± 0 02* 044 + 001

The values represent the mean ± S D obtained from 4 m~ce *P < 0 01, slgmficantly different from NPD group

during the 7 days following MeHg admimstratlon It should be noted that urinary excreuon of Hg m LPD-fed mice was approximately 3.7-times lower than in NPDfed mice, whereas the fecal Hg level in the LPD-fed group was similar to that in the N P D group.

80 50

~..~'-~ -""" - .-~-------,~ "6 . . . . . .

Kidney o • . . . . . .

LPD

•

NPD

¢-

10

9

I' . . . . .

.~ _ __

Ltver

.. L P D

E

NPD

O O E O o

Brain

LPD

'1" .......

1

NPD

A~

05 I

1

I

I

1

3

5

7

Days

after

MeHg

administration

Fig 1 Time-dependent change of tissue Hg levels m LPD- or NPD-fed male mice Mice were orally administered MeHg (20 ~tmol/kg) on day 0 The values represent the mean ± S D obtained from 3 ~ mice ANOVA days I, 3, 5, 7, NPD and LPD Brain diets P < 0 01, days P < 0 01, &ets and days interaction P < 0 01 Lwer d]ets NS, days P < 0 05, &ets and days interact]on NS Kidney &ets P < 0 01, days P < 0 01, &ets and days mteracuon NS

21

E

5

o') -i tO

~

LPD

--" . . . . .

I ' ~ NPD

E o o

c: 0 o GI)

3-

I

~

LPD

. . . . 'I'. . . . .

"I' "- NPD

0.5 |

I

I

I

1

3

5

7

Days after MeHg administration Fig 2 Time-dependent change of blood and plasma Hg levels m LPD- or NPD-fed male mice. M~ce were orally administered MeHg (20 #mol/kg) on day 0 The values represent the mean ± S D obtained from 3 ~ 4 m l c e . ANOVA days 1, 3, 5, 7, N P D a n d LPD Blood dJetsP < 005, d a y s P < 001, dletsand days interaction NS Plasma diets P < 0 01, days NS, diets and days interaction NS

L

15

_......- NPD

1 /

-

( Urine )

,/ !

.~

10

"6 a~ . LPD ~Feces o~

5

////

z/, j

/

)

( Feces ) ~

"~

/

LPD ( Urine )

t

0

1

3

5

7

Days after MeHg admm)strat)on Fig 3 Cumulative amount of Hg excretion m LPD- or NPD-fed male mice Mice were orally admlmstered MeHg (20/zmol/kg) on day 0 The values represent the mean + S D obtained from 3 4 mice The values on day 1, 3, 5 and 7 were slgmficantly &fferent from the NPD group, *P < 0 01

22 TABLE Ill INFLUENCE OF DIETARY PROTEIN LEVELS ON TISSUE TOTAL GSH LEVELS IN MALE MICE T~ssue

Lwera Kidneya Braina Blooda Plasmab

Total GSH levels (mM a o r p.Mb) NPD group

LPD group

8 62 4 04 I 93 I 02 319

6 12 4 45 1 92 085 249

+ 0 69 -4- 0 56 4- 003 4- 0 15 4- 72

+ 0.52* -4- 0.41 4- 0 11 4- 0 11 4- 68

The values represent the mean -4- S D obtained from 4 mice *P < 0 01, slgmficantly &fferent from NPD group Tissue total G S H levels m L P D - or N P D - f e d mice are summarized in Table III. After feeding on the diets for 5 days, the hepatic G S H level m LPD-fed mice was 29% lower than m N P D - f e d mice. However, the differences were not slgmficant m the levels of the brain, kidney, b l o o d a n d plasma. Since the t u r n o v e r rate of tissue G S H is one of the i m p o r t a n t factors to modify the fate of M e H g [3], the half-lives o f hepatic a n d renal G S H in the mice were determined using BSO (Table IV). C o n t r a r y to the expectation, no marked difference was observed in either tissue m the two groups; half-lives of G S H were 215 a n d 195 mln in the liver a n d 15.0 a n d 16.6 m i n in the kidney for L P D and N P D groups, respectively. However, the hepatic G S H effiux rate (/~mol/g body weight per day), calculated from G S H levels a n d its t u r n o v e r rates in LPD-fed mice, was only half that in the N P D group, whereas the efflux rates in the kidney were identical m the two groups (Fig. 4). W h e n the mice were treated with acivlcm 24 h after M e H g a d m i m s t r a t i o n , u r i n a r y Hg levels for the following 3 h dramatically increased in both groups with a conc o m i t a n t decrease in the renal Hg levels (Fig 5) The increased p o r t i o n in u r i n a r y Hg accounted for 73 a n d 93% of the decreased p o r t i o n of renal Hg for the L P D a n d N P D groups, respectively. It is w o r t h n o t i n g that the u r i n a r y Hg levels m acwlcmtreated mice were similar in the two dietary groups, although the level in LPD-fed mice was significantly lower t h a n m the N P D group without the inhibitor TABLE IV INFLUENCE OF DIETARY PROTEIN LEVELS ON RENAL AND HEPATIC TOTAL GSH HALFLIVES IN MALE MICE Tissue

Ktdney Liver

TI/2 (mm) NPD group

LPD group

16 6 195

15 0 215

The values were estimated from the semdoganthmlc plot of tissue total GSH levelsbefore and 10, 20, 30, 60, 120 and 180 mm after administration of BSO

23

GSH efflux (pmol/body weight (g)/day) 1.0

0

2.0

3.0

w

,

Liver

I'

NPD LPD

iii!iiiiiiiiiiiiiiiiiiiiiii ,, Kidney

NPD

I

LPD ,

|

I

Fig 4 Influence of &etary protein levels on hepato-renal efflux of total GSH m male mice The values represent the mean + S.D. obtained from 3 ~ 4 mice *P < 0.01, slgmficantly different from NPD group

Discussion

In the present study, we found that feeding LPD to C57BL/6N male mice accelerated renal Hg accumulation and depressed urinary Hg excretion. Previously, we suggested that decreased renal accumulation and urinary ehmmation of Hg observed in 15

301

(B) Kidney

(A) Urine

-8 ~0 "6

"6 v

o t-

t-

o O

O

5

o

"r"

•

Control

[ ] Acwic,n

NPD

v

LPD

NPD

LPD

Fig 5 Effect of acwlcm on urinary excretion and renal accumulation of Hg m LPD- or NPD-fed mice Mice were treated with a o v l c m (2 mmol/kg, 1 p ) or sahne 24 h after MeI4g administration (20 tzmol/kg, p o.) Urine was collected for 3 h following acwlcm or sahne treatment The mice were kdled 3 h after ac~wcm or sahne treatment The values represent the mean + S D obtained from 4 - - 6 m~ce *P < 0 01, slgmficantly &fferent from control * P < 0 05, significantly different from NPD group

24 C57BL/6N female mice compared with male mice might be accounted for by slower effiux rates of hepatic and renal GSH [3] Thus, it is considered that GSH effiux from liver and kidney may control MeHg transfer to the kidney and urine, respectively. Since the efflux rate of the hepatic GSH m LPD-fed mice was slower than in NPD-fed mice, LPD-fed mice should have a lower renal Hg than the NPD group due to similar hepatic Hg levels in the groups Similarly, LPD-fed mice should have somewhat higher urinary Hg levels due to higher renal Hg levels compared with N P D group, since the effiux rates of renal GSH were identical However, LPD-fed mice showed significantly higher renal Hg and lower urinary Hg levels Accordingly, the reason for the difference in the fate of MeHg between the two dietary groups cannot be simply explained by hepato-renal GSH efflux Previously, we found that most low molecular weight MeHg metabolite(s) in the kidney and urine were accounted for by GSH and cystelne conjugates, respectively [26] From this result, we suggested that MeHg in the kidney was secreted as its GSH conjugate into the proximal luminal space and reabsorbed from proximal tubule cells as cystelne conjugate after degradation by 3,-GTP and peptldase and that a portion of the metabolate(s) which escaped reabsorptlon was detected m t, rlne as MeHgcystelne Since renal 3,-GTP activities were also similar m the two groups of mice (data not shown), efflux and subsequent degradation rates of MeHg metabohte(s) at the luminal space would be comparable. Accordingly, the marked difference m the urinary Hg excretion rates observed between the two dietary groups m~ght conceivably be attributed to the difference m reabsorptlon efficiency of the MeHg metabohte(s) at the renal tubules The LPD-fed mice might reabsorb the metabohte(s) more effectively than the NPD-fed group The reabsorptlon of the MeHg metabohte(s) could be prevented by the inhibition of 3,-GTP m the renal tubules. If the enzyme activity was inhibited, MeHg metabohte (GSH conjugate) secreted m the tubular lumen would rea&ly appear m the urine without reabsorpt~on [26,27] The fact that the aClVlCln treatment markedly increased urinary Hg and removed the difference in urinary Hg excretion between the two dietary groups, supported the conslderatlon described above. Although LPD feeding drastically decreased urinary Hg, fecal Hg excretion rates were completely unaffected It is known that MeHg was absorbed as its cystelne conjugate via an amino acid transport system m the intestine [28]. Since intestinal absorption of amino acids was suggested to be rarely affected by low protein diet in mice [29], the difference m the reabsorptlon rates of MeHg metabohte(s) at the intestine might not be observed between the dietary groups m the present circumstances. The living organism maintains tissue amino acid levels by controlhng ~ts influx and effiux balance Where the dietary protein level is lowered, an influx of various amino acids in the various tissues might serve to maintain their cellular levels as observed in the kidney. It is reported that MeHg was taken up by the brain as its cystexne conjugate via a transport system for neutral amino acids [30-32]. Recently, we suggested that the Hg levels m the low molecular weight fraction of blood plasma, in which MeHg-cystelne was the dominant component, were a drlwng force in brain uptake of MeHg [33] The increased brain Hg observed in LPD-fed mice might be accounted for, at least partly, by the improved uptake of MeHg-cysteme

25

Despite the marked difference in the effiux rates of hepatic GSH, the Hg levels in the laver were identical between the two groups. The influx of MeHg an the liver might be slower in LPD-fed mice probably due to accelerated uptake by other tissues such as the brain and kidney compared with the NPD-fed group. Accordingly, it might be difficult to observe the difference in hepatic Hg levels between the groups. The pronounced changes in the distribution and excretion of MeHg achieved by lowering dietary protein levels in this study m~ght be, at least partly, due to the lack of sulfur amino acids which are required for G S H biosynthesis It is well known that MeHg is slowly converted to an inorganic form m vlvo. In short-term experiments, most Hg was detected in an organic form except m feces in mice [3,5,34]. Dietary protein levels may have apprecmble effects on the blotransformation (demethylation) in long-term experiments. The present results suggest that dietary protein status plays an important role m determining the fate of MeHg and suscephbihty to the hazardous metals by modulating the metabolism of thiol compounds such as cysteine and GSH.

Acknowledgements The authors are grateful to Mrs. Tamlko Terada for technical assistance and to Dr. Yoshlhide Kmjo for help and advice on analysis of variation, respectively.

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