Supplementary Material For s4

Supplementary material for:

“Effect of taurine supplementation on hepatic metabolism and alleviation of cadmium toxicity and bioaccumulation in a marine teleost, red sea bream, Pagrus major”

in Fish Physiology and Biochemistry

Takeshi Hano†*, Katsutoshi Ito†, Kumiko Kono†, Mana Ito†, Nobuyuki Okubo†, Kazuhiko Mochida†

Institutional addresses

† National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research and Education

Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima, 739-0452, Japan

*Corresponding Author

Takeshi Hano

National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research and Education

Agency, 2-17-5,Maruishi, Hatsukaichi, Hiroshima Japan, 739-0452

Tel.: +81-829-55-0666, Fax: +81-829-54-1216

E-mail: [email protected]

In this document, we present

(1) glm and determination of best-fit model (Page S2)

(2) 5 tables and 4 figures (Pages S3 - S11)

S1 (1) Details of Glm analysis and determination of best-fit model (Page S2)

Parameters applied to the model included Cd, x, and TL as numerical elements and f as a factorial element.

Akaike’s Information Criterion (AIC) and residual deviances were firstly used to determine the best-fit models among comparable models according to the various combination of explanatory variables. To select models that statistically fitted to the data, we estimated the distribution of hypothetical differences in residual deviance between models by applying a parametric bootstrap method to each model using 50,000 iterations obtained from random binomial distributions (Roff, 2006). Parameters of random binomial distributions (n, size, probability) were as follows: n was the number of samples (n = 126); size = 1; and probability was total mortality during the acute toxicity test at a concentration of 0.277 (35 dead individuals out of the 126 specimens used throughout the toxicity experiment). Models were considered significantly different if p <

0.05 when the difference in residual deviance between comparable models was greater than the 95th percentile of the distribution of the difference in hypothetical residual difference.

Reference

Roff DA. (2006) Introduction to Computer-intensive Methods of Data Analysis in Biology: Cambridge University Press.

2 Table S1. Composition of experimental diets for red sea bream (Pagrus major)

0% 0.5% 5.0% manufacturer Vitamin-free casein 51.0 51.0 51.0 Wako Pure Chemical Ltd. (Osaka, Japan) Gelatin 11.0 11.0 11.0 Wako Pure Chemical Ltd. (Osaka, Japan) α-starch 7.0 7.0 7.0 Wako Pure Chemical Ltd. (Osaka, Japan) Pollock viscera oil 5.0 5.0 5.0 Kanematsu Shintoa Foods Coporataion, Co., Ltd. (Tokyo, Japan) Cuttlefish lecithin 10.0 10.0 10.0 Tsuji Oil Mills, Co., Ltd.(Matsuzaka, Mie, Japan) Choline chloride 0.9 0.9 0.9 Wako Pure Chemical Ltd. (Osaka, Japan) VitaminE 0.1 0.1 0.1 Wako Pure Chemical Ltd. (Osaka, Japan) Ascorbic acid sodium 0.2 0.2 0.2 Wako Pure Chemical Ltd. (Osaka, Japan) Cellulose 7.3 6.8 2.3 Sigma Aldrich Corp. (St Louis, MO, USA) Taurine 0.0 0.5 5.0 Wako Pure Chemical Ltd. (Osaka, Japan) *1 Vitamin mix 2.0 2.0 2.0 Custom-ordered (Nippon Formula Feed Mfg Co.,Ltd. Yokohama, Japan) *2 Mineral mix 5.0 5.0 5.0 Custom-ordered (Nippon Formula Feed Mfg Co.,Ltd. Yokohama, Japan) *3 Feeding stimulant 0.5 0.5 0.5 100.0 100.0 100.0 Analytical contents(dry matter basis) Taurine (mg/100g) 5.0 505.0 5070.0 Crude protein(%) 57.9 58.4 61.1 Crude lipid(%) 12.6 12.1 12.6

*1 Vitamin mix ingredients(%) *2 Mineral mix ingredients(%) Vitamin B1 0.900 NaCl 1.000 Vitamin B2 1.500 MgSO4 7H20 15.000 Vitamin B6 0.600 KH2PO4 32.000 Vitamin B12 0.002 Ca(H2PO4)2 H20 32.000 Niacin 6.000 Fe citrate 2.500 Ca-pantotenate 1.500 Ca lactate 3.500 Inositol 30.000 Cellulose 13.422 Biotin 0.090 ZnSO4 7H20 0.353 Folic acid 0.225 MnSO4 4H2O 0.175 p-aminobenzoic acid 0.750 CuSO4 5H2O 0.031 Vitamin K3 0.750 AlCl3 6H2O 0.015 VitaminA (600000IU) 0.198 KIO3 0.003 Vitamin D3(600000IU) 0.015 CoCl2 6H20 0.001 Cellulose 57.500

*3 Feeding stimulant ingredients(%) Proline 35.400 Wako Pure Chemical Ltd. (Osaka, Japan) Alanine 23.200 Wako Pure Chemical Ltd. (Osaka, Japan) Inosin 5'-monophosphate 41.400 Wako Pure Chemical Ltd. (Osaka, Japan)

Table S2. Summary of statistical outcomes (residual deviance, AIC, and odds ratio) after glm analysis of factors influencing the survival of red sea bream (Pagrus major) exposed to Cd in an acute toxicity experiment

S3 Odd`s ratio 2

Taurine content in Feed 3 Model 1 Residual AIC Cd TL deviance 0.5% 5%

(a) Y ~ Cd 85.8 89.8 2.7 (2.0, 4.0)

(b) Y ~ x 149 148 0.93 (0.86, 0.99)

(c) Y ~ f 144 150 0.33 (0.11, 0.88) 0.65 (0.26, 1.6)

(d) Y ~ Cd + x 81.2 87.2 2.8 (2.0, 4.2) 0.91 (0.83, 0.99)

(e) Y ~ Cd + f 76.2 84.2 3.1 (2.2, 4.9) 0.10 (0.019, 0.45) 0.38 (0.089, 1.5)

(f) Y ~ x + f 142 150 0.94 (0.86, 1.0) 0.56 (0.15, 2.0) 1.1 (0.33, 3.6)

(g) Y ~ Cd + x + f 76.0 86.0 3.1 (2.2, 4.9) 0.98 (0.86, 1.1) 0.13 (0.014, 0.96) 0.49 (0.065, 3.3)

1Y, mortality; Cd, cadmium concentration; x, total length and f, taurine content of feed 2Odds ratios are expressed as median odds ratios with 95% confidence intervals in parentheses. 3Odds ratios were calculated relative to taurine-free (0%) treatment.

4 Table S3. Metallothionein concentrations (ng/mg) in the liver of red sea bream (Pagrus major) during a Cd acute toxicity test 0h 2 24h 2 96h 2 Treatment 1 (day55) (day56) (day59)

TAU0% Control 4.6 ± 3.3 6.0 ± 1.7 3.1 ± 1.3 L group 6.3 ± 2.5 3.1 ± 0.8 H group 6.4 ± 2.3 3.6 ± 1.3

TAU0.5% Control 5.1 ± 2.2 9.5 ± 3.2 3.8 ± 1.9 L group 9.7 ± 3.0 3.6 ± 0.9 H group 11.0 ± 4.0 4.9 ± 2.6

TAU5.0% Control 6.2 ± 2.8 9.5 ± 3.1 3.7 ± 1.4 L group 9.4 ± 2.2 3.6 ± 1.1 H group 6.9 ± 2.3 3.9 ± 1.3

1 L group, 0.27 mg/L; and H group, 2.7 mg/L of Cd. 2 Values are expressed as average ± standard deviation.

S5 Table S4. Total length, body weight, and hepatosomatic index (HSI) of red sea bream (Pagrus major) during a Cd bioaccumulation test

0d 1,2 7d 1,2 14d 1,2 28d 1,2 35d (eli 7d) 1,2 49d (eli 21d) 1,2 (day 40) (day 47) (day 54) (day 68) (day 75) (day 89)

a a a a a,c a 0% Control 7.4 ±0.4 6.7 ± 0.6 7.7 ± 0.5 8.4 ± 0.6 8.9 ± 0.7 8.0 ± 0.4 b,c a,c a,c c a Cd 7.9 ± 0.4 7.9 ± 0.4 8.7 ± 0.6 8.0 ± 0.6 8.1 ± 0.5

Total ab a,c b,c b a,d b ± ± ± ± ± ± length 0.5% Control 7.6 0.2 6.8 0.6 b,c 8.9 0.4 a,c,d 10.0 0.3 a,c 10.0 0.4a,d 11.0 0.6 b (cm) Cd 8.0 ± 0.7 8.6 ± 0.6 8.8 ± 0.3 9.6 ± 1.0 10.4 ± 0.4 b a,c a,c,d b b,d b 5.0% Control 8.0 ±0.3 7.1 ± 0.7 8.6 ± 0.4 9.7 ± 0.3 10.4 ± 0.6 11.2 ± 0.2 b b,d b b,d b Cd 8.5 ± 0.5 9.1 ± 0.5 10.0 ± 0.4 10.3 ± 0.2 10.5 ± 0.7 0% Control 6.2 ±1.3 6.9 ± 1.5 6.7 ± 1.6 a 8.1 ± 2.3 a 9.8 ± 2.3 a 6.8 ± 1.1 a Cd 7.2 ± 0.9 7.5 ± 0.8 a,d 9.6 ± 2.6 a 7.0 ± 1.9 a 7.5 ± 1.6 a Body 0.5% Control 6.6 ±1.2 7.7 ± 1.6 10.6 ± 1.6 b,c 15.1 ± 1.2 b 15.7 ± 1.6b 22.0 ± 3.2 b weight Cd 7.9 ± 2.1 10.0 ± 1.8 b,c,d 11.4 ± 1.4 a,c 15.3 ± 4.0b 20.0 ± 1.8 b (g) 5.0% Control 7.4 ±0.7 8.4 ± 1.9 9.3 ± 1.2 a,c 14.6 ± 1.0 b,c 18.8 ± 3.3b 24.4 ± 1.5 b Cd 9.1 ± 1.4 11.9 ±1.2 b,c 17.0 ± 1.8 b 19.7 ± 1.5b 21.6 ± 3.7 b a,c 0% Control 0.8 ± 0.2 0.8 ± 0.2 0.7 ± 0.2 1.0 ± 0.4 1.2 ± 0.4a 0.9 ± 0.2 Cd 0.9 ± 0.2 0.9 ± 0.3 1.3 ± 0.6 0.9 ± 0.2 1.1 ± 0.5

a HSI 0.5% Control 0.8 ± 0.1 0.8 ± 0.2 0.8 ± 0.2 0.8 ± 0.1 0.9 ± 0.2 1.0 ± 0.2 b,c (%) Cd 0.9 ± 0.2 0.9 ± 0.2 0.9 ± 0.2 1.3 ± 0.2 1.0 ± 0.2

5.0% Control 0.9 ± 0.2 0.8 ± 0.2 0.7 ± 0.2 1.0 ± 0.1 1.1 ± 0.2a,c 1.2 ± 0.1 Cd 0.8 ± 0.2 0.7 ± 0.3 1.1 ± 0.2 1.4 ± 0.1b,c 1.3 ± 0.3

1 Values are expressed as average ± standard deviation. 2 For each parameter, values with different superscript letters (a,b,c,d) in a column differ significantly ( p < 0.05)

6 Table S5 Metallothionein concentration (ng/mg) in the liver of red sea bream (Pagrus major) during a Cd bioaccumulation test

0d 1 7d 1,2 14d 1 28d 1 35d (eli7d) 1 49d (eli21d) 1,2 (day 40) (day 47) (day 54) (day 68) (day 75) (day89)

a,b a ± ± ± ± ± ± TAU0% Control 5.3 3.8 5.4 1.4a 4.6 0.6 5.8 1.6 5.4 1.0 3.5 1.0a,b Cd 3.6 ± 1.7 4.9 ± 0.9 4.4 ± 0.6 6.2 ± 3.4 3.9 ± 1.1 a,b a,b TAU0.5% Control 7.0 ± 2.3 6.3 ± 2.0 6.6 ± 1.3 6.3 ± 1.6 6.7 ± 3.0 6.3 ± 2.3 a,b a,b Cd 5.6 ± 1.0 5.8 ± 0.7 6.3 ± 1.9 5.4 ± 1.7 5.7 ± 2.8

a,b b TAU5.0% Control 5.6 ± 1.8 6.5 ± 1.8 6.2 ± 0.7 7.2 ± 2.4 6.5 ± 1.9 8.0 ± 3.8 b a,b Cd 7.4 ± 2.9 5.9 ± 1.8 5.6 ± 1.5 6.1 ± 1.5 4.4 ± 1.2

1 Values are expressed as average ± standard deviation. 2 For each parameter, values with different superscript letters (a,b) in a column differ significantly (p < 0.05)

S7 0h (day 55) 1 TAU 0% TAU 5.0%

% TAU0% 8 1

( 0 2 TAU0.5% C -1 0 1 2 3 P -0.5 TAU5.0%

-1 PC1(71%)

24h (day56) TAU 0% TAU 5.0% 2 TAU0%_Cont TAU0.5%_Cont ) 1 % TAU5.0%_Cont 2 1 (

2 TAU0%_Cd_L C

P 0 -3 -2 -1 0 1 2 3 TAU0.5%_Cd_L TAU5.0%_Cd_L -1 PC1(66%) TAU0%_Cd_H

96h (day59) TAU 0% TAU 5.0% 2 TAU0%_Cont 1 TAU0.5%_Cont )

% TAU5.0%_Cont 3 1

2 TAU0%_Cd_L C -4 -2 0 2 4 P -1 TAU0.5%_Cd_L TAU5.0%_Cd_L -2 PC1(76%) TAU0%_Cd_H

Fig. S1. PCA score plots of the 55 hepatic metabolites at 0, 24h and 96h during the Cd acute toxicity experiment

8 0d (day40) TAU 0% TAU 5.0% 1

0.5 ) %

9 TAU0% . 5

2 TAU0.5% C -4 -2 0 2 4 P -0.5 TAU5.0%

-1 PC1(89%)

07d (day47) 28d (day68) TAU 0% TAU 5.0% TAU 0% TAU 5.0% 1 1 TAU0%_Cont TAU0%_Cont 0.5 0.5 ) ) TAU0.5%_Cont TAU0.5%_Cont % % 6 1 . 1

TAU5.0%_Cont 8 TAU5.0%_Cont ( 0

( 0 2 2 C -4 -2 0 2 4 C -4 -2 0 2 4 P

TAU0%_Cd P TAU0%_Cd -0.5 -0.5 TAU0.5%_Cd TAU0.5%_Cd -1 -1 PC1(80%) TAU5.0%_Cd PC1(85%) TAU5.0%_Cd

35d (day75) 49d (day89) TAU 0% TAU 5.0% TAU 0% TAU 5.0% 1.5 1.5 TAU0%_Cont TAU0%_Cont 1 1 ) ) TAU0.5%_Cont TAU0.5%_Cont % %

4 0.5 3 0.5 1 1

TAU5.0%_Cont ( TAU5.0%_Cont ( 2 2 C C 0 0

P TAU0%_Cd P TAU0%_Cd -2 -1 0 1 2 -2 -1 0 1 2 3 -0.5 -0.5 TAU0.5%_Cd TAU0.5%_Cd -1 -1 PC1(71%) TAU5.0%_Cd PC1(79%) TAU5.0%_Cd

Fig. S2. PCA score plots of the 55 hepatic metabolites at 0, 7, 28, 35, 49d in the Cd bioaccumulation experiment

S9 (A) (B) (C) Serine Aspartate ß-alanine 0.15 0h 24h 96h 0.2 0.2 0h 24h 96h 0h 24h 96h

0.1 0.1

0 0 0 % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % 5 5 0 5 0 5 0 0 5 0 0 5 0 0 0 0 0 5 0 0 0 5 0 5 0 0 0 0 0 5 0 0 0 0 5 0 0 0 0 0 5 5 0 0 0 0 0 0 0 5 0 5 0 5 5 0 5 0 5 0 5 0 5 ...... ______0 5 0 5 0 5 0 5 0 5 0 5 0 5 5 0 5 0 5 0 5 0 5 0 5 5 0 5 0 5 0 0 5 0 5 0 5 0 5 0 0 5 L L L L L L C C C C C C C C C H H H H H H ______L L L L L L L L L L L L C C C C C C C C C C C C C C C C C C H H H H H H H H H H H H (D) (E) (F) Urea 0.25 Myo-inositol Threitol

0.4 0h 24h 96h 0.03 0h 24h 96h 0h 24h 96h 0.2

0.15 0.02

0.2 0.1 0.01 0.05

0 0 0 % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % 5 0 5 0 5 0 5 0 5 0 0 5 0 0 5 0 0 5 0 0 5 0 0 0 0 5 0 0 0 5 0 0 0 5 0 0 0 0 5 0 5 0 0 0 0 5 0 5 0 0 0 5 0 0 5 0 5 0 5 0 0 5 0 ...... ______0 5 0 5 0 5 5 0 5 0 5 0 5 5 0 5 0 5 0 0 5 0 5 0 5 0 0 5 L L L L 0 5 0 5 0 5 0 5 0 5 0 5 0 5 C C C C C C H H H H L L ______C C C ______H H ______L L L L L L L L C C C C C C C C C C C C H H H H H H H H L L L L C C C C C C H H H H

(G) Ethanolamine (H) Threonine (I) Glutathione

0.08 0h 24h 96h 0.1 0h 24h 96h 0.015 0h 24h 96h

0.04 0.05

0 0 0 % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % 5 5 5 0 0 5 0 0 0 5 0 0 0 5 0 0 5 0 5 0 0 5 0 0 0 0 0 0 0 0 0 5 0 0 5 0 0 5 0 5 0 0 5 0 0 0 5 0 5 5 0 0 0 5 0 0 0 5 0 0 5 0 5 ...... ______0 5 0 5 0 0 5 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 0 5 L L L L L L C C C C C C C C C H H H H H H ______L L L L L L L L L L L L C C C C C C C C C C C C C C C C C C H H H H H H H H H H H H Fig. S3. Remarkably altered metabolites in the liver of the acute toxicity test: metabolites that commonly altered by taurine administration in both acute and bioconcentration experiments ((A) – (H)) and that induced by Cd exposure (I). 0 h, 24 h, and 96 h indicated the exposure duration after the initiation of exposure on day 55. The vertical axes of graphs are the intensities (mean ± standard deviation) of each metabolite peak relative to an internal standard (ribitol). C, control; L, 0.27 mg/L; and H, 2.7 mg/L of Cd. Statistical significances are shown in Tables 2 and 3.

10 (A) Serine (B) Asparate (C) ß-alanine 0.12 0.2 0d 7d 28d 35d 49d 0d 7d 28d 35d 49d 0.2 0d 7d 28d 35d 49d 0.1

0.06 0.1 0.1 0.04 0.02 Myo-inositol Myo-inositol 0 0 0 % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 5 0 5 0 0 5 0 5 0 5 0 0 5 0 0 0 0 0 0 0.5 0 5 5 0 ...... 0.5 ...... ______0 5 0 5 0 5 5 0 5 0 5 0 5 0 5 0 5 5 0 5 0 5 0 5 0 0 5 0 0 5 0 0 5 0 5 5 x x x x x x x x 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 C C C C C C C C C C x x x x ______C C C C C E E E E E E E E ______x x x x x x x x x x x x x x x x E E E E C C C C C C C C C C C C C C C C C C C C x x x x x x x x E E E E E E E E E E E E E E E E C C C C C C C C C C E E E E E E E E 0.4 0.4

Urea Myo-inositol (D) 0.3 (E) 0.3 (F) Threitol 0d 7d 28d 35d 49d 0d 7d 28d 35d 49d 0.5 0.25 0.03 0d 7d 28d 35d 49d 0.2 0.2 0.4 0.2 0.02 0.3 0.15 0.1 0.1 0.2 0.1 0 0.01 % % % % % % % % % % % % % % % % % % % % % % % 0.1 00.05 % % % % 0 5 0 5 0 5 0 5 0 5 5 0 5 0 5 0 % % % % % % % % % % 5 0 % % 0 0 % 0 0 0 % % % % % % % % % 0 0 0 0 % % % % % ...... ______5 5 0 0 5 0 0 5 5 0 5 0 5 0 5 0 5 0 0 0 0 0 0 0 0 0 0 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 ...... x x x x C C C C C ______E E E E 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 0 5 5 x x x x C C C C C x x x x x x x x C C C C C C C C C C ______E E E E

0 E E E E E E E E

0 x x x x x x x x C C C C C C C C C C 0 % % % % % % % % % % % % % % % % % % % % % % % % % % % E E E E E E E E % % % % % % % % % % % % % % % % % % % % % % % % % % % 5 0 5 0 5 0 5 0 0 5 0 5 0 0 5 0 5 0 5 0 0 0 0 0 0 0 0 % % % % % % % % % % % % % % % % % % % % % % % % % % % ...... 0 5 0 0 5 0 5 0 0 5 0 5 0 0 5 0 0 5 0 0 5 0 0 5 0 0 0 ______...... 5 0 5 0 5 0 5 0 0 5 0 0 5 0 0 0 5 0 0 0 5 0 0 0 5 0 0 0 5 0 5 0 5 0 5 0 0 5 0 5 0 5 5 0 5 ______...... x x x x 0 5 0 5 0 0 5 0 5 0 5 0 5 0 5 C C C C C 5 0 5 ______x x x x ______C C C C C 5 0 5 0 5 0 0 5 0 5 0 5 0 5 0 5 0 5 E E E E x x x x ______C C C C C x x x x x x x x E E E E C C C C C C C C C C ______x x x x x x x x E E E E C C C C C C C C C C E E E E E E E E x x x x x x x x C C C C C C C C C C E E E E E E E E E E E E E E E E

(G) Ethanolamine (H) Threonine (I) Methionine 0.05 0.08 0d 7d 28d 35d 49d 0.015 0d 7d 28d 35d 49d 0d 7d 28d 35d 49d 0.04

0.01 0.03 0.04 0.02 0.005

0 0 0 % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % 0 5 0 0 5 0 5 0 0 5 0 5 0 0 5 0 0 5 0 0 5 0 0 5 0 0 5 0 0 5 0 5 0 0 5 0 0 5 0 0 5 0 5 0 0 5 0 0 5 0 0 0 % % % % % % % % % % % % % % % % % % % % % 0 0 % % % % % % ...... ______0 5 0 0 5 0 5 0 0 5 0 0 5 0 0 5 0 0 5 0 0 5 0 5 0 _ _ 0 0 0 5 0 5 0 5 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 0 5 ...... 0 5 0 5 ...... x x x x x x x x C C C C C ______C C C C C ______0 5 0 5 0 5 0 5 0 5 5 0 5 _ _ _ _ 0 5 0 0 5 E E E E E E E E x x x x C C C C C x x x x x x x x x x x x x x x x C C C C C C C C C C ______C C C C C C C C C C _ _ _ _ _ E E E E E E E E E E E E E E E E E E E E x x x x x x x x C C C C C C C C C C E E E E E E E E Fig. S4. Remarkably altered metabolites in the liver of the bioaccumulation test: metabolites that commonly altered by taurine administration in both acute and bioconcentration experiments ((A) – (H)), and that induced by Cd exposure (I). 0, 7, 28, 35, and 49 d in each figure correspond to days 40, 47, 54, 68, 75 and 89, respectively, after the initiation of feeding trial. The vertical axes of graphs indicate the relative intensities (means ± standard deviation) of each metabolite peak relative to an internal standard (ribitol). In horizontal axes of graphs, C, control; Ex, Cd-exposed. Statistical significances are shown in Tables 5 and 6.