Trace Nutrients Research 25 : 91­95 (2008)

Original Article

Effect of Dietary Oyster Extract on the p-aminophenol-induced
Nephrotoxicity in Rats
Ryota HOSOMI1）, Yoshikazu MATSUDA2）, Yoshinori WATANABE1）
Kenji FUKUNAGA1）and Munehiro YOSHIDA1）,
1）
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University ＊,2）
Central Research Institute, Japan Clinic Co., Ltd. ＊＊Summary
Recently, chronic kidney disease (CKD) has been estimated to affect about 20 million people in Japan. CKD is associated with a several fold increased risk of arteriosclerosis related disease. Oysters (Crassostrea gigas) contain many nutrients and are used widely in Japan. Oyster extract contains major components including minerals, carbohydrates, protein,and amino acids. Various effects on health maintenance and life-style related diseases of oyster extracts have been reported so far. The present study evaluated the preventive effects of oyster extract on the formation of chemically induced nephrotoxicity in rats. Rats fed an AIN-93G modified diet containing oyster extract (1.0 %) After 6 weeks, the rats
were injected intraperitoneally with p-aminophenol (PAP, 0.5 mmol/kg body weight). Urine was collected before and after injection, and the kidney was removed at 48 h after injection.
Dietary oyster extract caused a decrease in blood urea nitrogen (BUN), return to the normal levels of urine volume, urinary N-acetyl-β-D-glucosaminidase (NAG) activity, and creatinine concentration, and reduction in the severity of tubular
basophilic change and nuclear division in tubular epithelium compared with control diets. However, the severity of tubular necrosis and tubular basophilic change in the midzone area and vacuolar degeneration in renal and arcuate arteries
were not significantly different. Dietary oyster extract recovered proximal tubular epithelial cell function in PAPinduced nephrotoxicity in rats.

Introduction

affecting the proximal straight tubule and causes elevatedblood urea nitrogen (BUN) concentration within 24 h of ad-

Recently in Japan, chronic kidney disease (CKD) has
been estimated to affect about 20 million people. CKD is

ministration in rats 9）. Certain urinary enzyme activities
can be used as an index of renal damage 10）.

associated with a several fold increased risk of arterioscle-Oyster (Crassostrea gigas) extract contains many nutri-rosis related disease such as cardiovascular diseaseents and is used widely in Japan. The major components of(CVD)1）. In addition, kidney injury in diabetes is mediatedoyster extract include minerals, carbohydrates, protein,by several growth factors that are synthesized in higher

and amino acids. In particular, many trace nutrients suchamounts, e.g., angiotensin II (Ang II), transforming growth

as zinc are present at high levels. Various nutrient avail-factor beta, vascular endothelial growth factor, and con-abilities for health maintenance and life-style related dis-

nective tissue growth factor2, 3）. Therefore, it is important

eases from oyster extract have been reported 11­13）.

to consider strategies to reduce CKD associated with

The present study examined the effect of oyster extract

metabolic syndrome because CKD is also a risk factor foron the biochemical parameters of urine and serum and re-the development of CVD, and consequently for the in-nal histopathology on PAP-induced nephrotoxicity in rats.

creased morbidity and mortality associated with this disease 4）.

Material and Methods

p-aminophenol (PAP) is a well-known metabolite ofacetaminophen and phenacetin and a derivative of anilines

1. Materials

in industrial use 5­8）. PAP induces acute tubular necrosis

Oyster extract was obtained from Japan Clinic Co., Ltd.

―――――――――――――――――――――――――――――――――――――――――――――――――――――――――――――＊Address : Yamate-cho 3­3­35, Suita, Osaka, 564­8680, Japan＊＊Address : Kai-nichi 10­1, Uzumasa Ukyo-ku, Kyoto, 616­8555, Japan− 91 −

(Kyoto, Japan). p-aminophenol hydrochloride (PAP) was

3. Biochemical measurements of urine and serum

purchased from Nacalai Tesque (Kyoto, Japan). All of theUrine level of urea creatinine (U-Cre), γ-glutamyl-diet components were products of Oriental Yeast (Tokyo,transferase(GTP),andN-acetyl-β-D-glucosaminidase

Japan).

(NAG) activity were assayed using commercial kits ; for
U-Cre, Creatinine Test Wako (Wako Pure Chemical Industries) ; γ-GTP, γ-GTP c-Test Wako (Wako Pure Chemical

2. Animals careAll experimental protocols were reviewed and approved by the Animal Ethics Committee of Kansai Medi-Industries) ; and for NAG, NAG-Test SHIONOGI (Shionogi
Pharmaceuticals Ltd., Osaka).

cal University and followed the “Guide for the Care andSerum levels of total protein (TP), albumin (Alb), glu-Use of Experimental Animals” of the Ministry of Educa-

tamic oxaloacetic transaminase (GOT), glutamic pyruvic

tion, Culture, Sports, Science and Technology of Japan.

transaminase (GPT) were assayed using an Olympus AU

Three-week-old male Fischer 344 rats were purchased

5431 automatic analyzer. Blood urea nitrogen (BUN), cre-from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan).

atinine, and uric acid (UA) were assayed using commercial

The rats were housed in plastic cages in a temperature-kits ; for BUN, BUN B-Test Wako (Wako Pure Chemical

controlled room (22­24℃) under a 12 h light/dark cycle.

Industries) ; for Cre, Creatinine Test Wako (Wako Pure

Rats were assigned to groups (12 rats each) that were fed

Chemical Industries) ; and for UA, Uric Acid c-Test Wako

either of two diets (Control group, Oyster Group) prepared(Wako Pure Chemical Industries).

according to the AIN-93G (Control group) and modified
diet containing 1 % (w/w) oyster extract (Oyster group)(Table 1)

4. Renal histopathology14）. After 42 days, the rats were divided into

Kidneys were removed from rats immediately after sac-groups of six (Control, Control+PAP, Oyster, and Oyster+rifice. Kidneys were sectioned longitudinally through thehilum, and blocks were fixed in 10 % neutral-buffered for-Table 1 Composition of experimental diets (g/kg)
Component
α-corn starchβ-corn starch
CaseinSucroseCellulose powder
Mineral mixture*
Vitamin mixture*
L-cystine
Choline bitartrate
Soybean oil
Oyster extract

malin solution. Tissues were embedded in paraffin, sec-Control
Oyster(g/kg diet)
132.0
130.7
397.5
393.5
200.0
198.0
100.0
99.0
50.0
49.5
35.0
34.7
10.0
9.9
3.0
3.0
2.5
2.5
70.0
69.3
10.0
-tioned 5 µm thick, and stained with hematoxylin and eosin.
Tubular necrosis, tubular basophilic changes, nuclear division, and vacuolar degeneration were observed using alight microscope.
5. Statistical analysis
Data are expressed as means

SEM of six rats. The sig-nificance of differences between means was determinedusing Tukey-Kramer test. Differences with P < 0.05 wereconsidered significant.

Diets were prepared based on the AIN-93G composition.
*AIN-93G formula.

Results and Discussion

PAP). Control+PAP and Oyster+PAP groups were in-

1. Biochemical alternations in serum of rats fed

jected intraperitoneally with 0.5 mmol/kg body weight ofoyster extract

PAP in 0.9 % NaCl. Urine was collected every 24 h for two

Table 2 shows the serum biochemical parameters. BUN

days prior to and for two days following the injection,

and Cre were the most significant parameters indicative ofurine volume was measured by graduated cylinder before

renal damage. The PAP injection groups (Control+PAP

being stored at ­80℃ until analysis.

and Oyster+PAP) showed higher levels of BUN and Cre

After injection of PAP for 48 h, under ether anesthesia,

compared with the non-injection groups. The concentra-

blood was collected, and serum was harvested by centrifu-tion of BUN tended to be lower in the Oyster+PAP groupgation at 3,000 g for 15 min before being stored at ­80℃

compared with the Control+PAP group. In contrast, theuntil analysis. The kidney was rapidly removed in theirconcentrations of Cre were similar between the Control+

entirety and were rinsed, frozen in liquid nitrogen, andPAP and Oyster+PAP groups. The concentration of TP,

stored at ­80℃.

Alb, GOT, GPT, and UA were not significantly different
among the all groups.

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Table 2 Biochemical parameters in serum of rats fed the experimental dietsCreAlbControl
Control+PAP

Data are means SEM (n = 6). Values not sharing a common letter are significantly different at P < 0.05.
TP, total protein ; Alb, albumin ; GOT, glutamic oxaloacetic transaminase ; GPT, glutamic pyruvic transaminase ; BUN, Blood urea nitrogen ; Cre, Creatinine ; UA, Uric Acid.

Urine volume

2. Urine volume and biochemical alternations inurine of rats fed oyster extract
mL/dayFigure 1 shows the changes in urine volume before andafter the injection of PAP. Injection of PAP caused a remarkable elevation in the urine volume. The urine volumes in the 2 days after the PAP injection tend to belower in the Oyster+PAP group compared with the Control+PAP group.
Figure 2 shows the alterations in urinary NAG activity,
U-Cre concentration and γ-GTP activity. Administration ofPAP caused a remarkable reduction in the U-Cre concen-Fig. 1 Effect of oyster extract on urine volume in Control+PAP(●) and Oyster+PAP (■) rats.
Male Fischer 344 rats were injected intraperitoneallywith PAP (0.5 mmol/kg body weight) on day 3 at 9 a.m.
Urine was collected every 24 h (9 a.m. to 9 a.m.) for 2 days
prior to and for 2 days following the injection.
Data are means SEM (n = 6). Asterisks shows significant difference at P < 0.05.

tration and elevation of NAG and γ-GTP activities. Therats in the Oyster+PAP group showed a significant returnto the normal level of U-Cre and NAG activities comparedwith rats in the Control+PAP group.
Because serum GOT and GPT of liver origin did notchange, elevation of γ-GTP activity in the urine was con-NAG

U/L

mg/dL

U-Cre

U/mL

ǫ-GTPFig. 2 Alteration of U-Cre concentration, urinary NAG activitiesand γ-GTP activity in Control+PAP (●) and Oyster+PAP(■) rats.
Experimental conditions are the same as described in Figure. 1.
Data are means ± SEM (n = 6). Asterisk shows significant
difference at P < 0.05.

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sidered to be of kidney origin. The γ-GTP level in the urine

crosis/changes, and Figure 3-e, f shows renal sections of

did not change in the Control+PAP and Oyster+PAP

Oyster+PAP rats in overview and of tubular necrosis.

groups. γ-GTP localizes to the plasma membrane of tubu-Table 3 shows a summary of the histopathologicallar epithelial cells in the kidney, and NAG is abundant inchanges seen in renal sections from rats fed the experi-

lysosomes in proximal tubular epithelial cells. Hence, rats

mental diets and injected with PAP. When the rats werein the Oyster+PAP group showed quicker recovery of

not injected with PAP, the severity of each pathologicalproximal tubular epithelial cell function compared withchange was not significantly different between the two

rats in the Control+PAP group, but no significant recov-groups. When the rats were injected with PAP, the sever-

ery of the plasma membrane of tubular epithelial cells in

ity of nuclear division and vacuolar degeneration in the tu-the kidney.

bular epithelium were significant lower in rats fed oyster

Cre was filtrated in glomerulus of kidney. Therefore, theextract containing diet compared with the control diet.

rats of the Oyster+PAP group showed quicker recovery

The severity of tubular necrosis and tubular basophilicof glomerulus function resulting in the rapid recovery ofchanges in the midzone area and vacuolar degeneration in

U-Cre concentration to the normal level compared withthe renal and arcuate artery were not significantly differ-

the rats in the Control+PAP group.

ent between the Control+PAP and Oyster+PAP groups.
Therefore, dietary oyster extract results in a reduction in
the severity of pathology changes in the tubular epithe-

3. Renal histopathology
Figure 3-a, b shows normal renal architecture. Figure 3-lium.

c, d shows a typical section from Control+PAP rats at 48 hIn addition, the dietary oyster extract group showed

after injection with an overview and basophilic tubular ne-quicker recovery of proximal tubular epithelial cell andFig. 3 Microphotographs of rat kidneys following PAP induced kidney damage. Hematoxylin and eosin stained sections of renal cortex from Control (a) and Oyster extract fed (b) rats. A typicalsection from Control+PAP rats 48 h after injection showing renal sections of overview (c) andtubular necrosis/change basophilic (d). A typical section from Oyster+PAP rats 48 h after injection showing renal sections in overview (e) and tubular necrosis (f).
Table 3 Histopathological changes in renal of rats fed the experimental diets or PAP-induced nephrotoxicity

Rats were injected intraperitoneally with PAP at 0.5 mmol/kg body weight.
Kidneys were removed 48 h after the injection.
The severity of pathological change was scored on an arbitrary scale of 0 to 4.
0, normal ; 1, slight ; 2, mild ; 3, moderate ; 4, severe.
Data are means ± SEM (n = 6). Values not sharing a common letter are significantly different at P < 0.05.

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glomerulus function resulting in return of NAG activity,

Med 351 : 1296­1305.

urinary volume serum BUN levels and U-Cre concentra-5）Green CR, Ham KN, Tange JD (1969) Kidney lesions

tion to the normal level compared with dietary control di-induced in rats by p-aminophenol. Br Med J 1 : 162­

ets. Hence, with the dietary oyster extract there was rapid

164.

renal tubular epithelial cell regeneration following nephro-6）Newton JF, Yoshimoto M, Bernstein J, Rush GF, Hooktoxicity due to the injection of PAP compared with theJB (1983) Acetaminophen nephrotoxicity in the rat. II.

control diet.

Strain differences in nephrotoxicity and metabolism

The major components of the oyster extract were min-of p-aminophenol, a metabolite of acetaminophen.

erals, carbohydrates, protein, and amino acids. In present

Toxicol Appl Pharmacol 69 : 307­318.

study, it was not determined which components of the7）McCarthy DJ, Waud WR, Struck RF, Hill DL (1985)

oyster extract affected the quick recovered of proximal tu-Disposition and metabolism of aniline in Fischer 344

bular epithelial cell function in PAP-induced nephrotoxic-rats and C57BL/6 X C3H F1 mice. Cancer Res 45 :ity in rats. Therefore, future studies will determine which174­180.

components of the oyster extract are responsible for the8）Kanbak G, Inal M, Bayçu C (1996) The role of freerecovery of renal function.

radicals in p-aminophenol-induced nephrotoxicity :does reduced glutathione have a protective effect?

Conclusion

Clin Chim Acta 252 : 61­71.
9）Shao R, Tarloff JB (1996) Lack of correlation between

The present study evaluated the effect of oyster extract

para-aminophenol toxicity in vivo and in vitro in fe-on nephrotoxicity in rats injected with PAP using several

male Sprague-Dawley rats. Fundam Appl Toxicol 31 :indices ; BUN and Cre in serum, urine volume, urinary268­278.

NAG activity, and Cre concentration and renal histopa-10）Piperno E (1981) Detection of drug induced nephro-thological changes. Dietary oyster extract led to decreased

toxicity with urinalysis and enzymuria assessment.

serum BUN and induced a return to the normal levels ofIn : Hook JB, eds. Toxicology of the Kidney. Raven

urine volume, urinary NAG activity, and U-Cre concentra-Press, New York, pp. 31­55.

tion, and the extract reduced the severity of pathological11）Matsuda Y, Takaya E, Yamaguchi M, Ohta T, Man-changes in tubular epithelium. Therefore, the oyster ex-abe M, Kawamura Y, Hiroishi S, Shibata Y (1998) The

tract prevented CKD.

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