SUPPLEMENTAL MATERIAL

EFFECTS OF DIETARY ANTIOXIDANTS (SELENIUM & NACETYL-CYSTEINE) ON PARAOXONASE 1 (PON1) IN PCB126-EXPOSED RATS

Hua Shen2, Miao Li2, Bingxuan Wang2, Ian K. Lai2, Larry W. Robertson1,2, Gabriele Ludewig1,2

1Department of Occupational & Environmental Health and 1Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, Iowa

Corresponding author

Gabriele Ludewig, PhD

Dept of Occupational & Environmental Health

The University of Iowa College of Public Health

100 Oakdale Campus 214 IREH

Iowa City, IA 52242-5000

Phone: (319) 335-4353

Supplemental Table 1A. AIN-93 Se modified diet composition

Constituent / 0.02 ppm Se / 0.2 ppm Se / 2 ppm Se
g/kg / g/kg / g/kg
Torula Yeast / 300 / 300 / 300
DL-Methionine / 3.0 / 3.0 / 3.0
Corn Starch / 350 / 350 / 345
Dextrose, monohydrate / 200 / 200 / 200
Soybean Oil / 40 / 40 / 40
Cellulose / 50 / 50 / 50
Mineral Mix ( Se excluded) / 35 / 35 / 35
Calcium Carbonate / 9.0 / 9.0 / 9.0
Sodium Selenate (0.1% in sucrose) / 0.048 / 0.48 / 4.8
Vitamin Mix, AIN-93-VX / 10 / 10 / 10
Choline Bitartrate / 2.5 / 2.5 / 2.5
Constituent
(g/kg) / AIN93-G / AIN-93G (1%NAC)
Casein, low Cu & Fe / 200 / 200
L-Cystine / 3 / 3
Corn Starch / 397 / 387
Maltodextrin / 132 / 132
Sucrose / 100 / 100
Soybean Oil / 70 / 70
Cellulose / 50 / 50
Mineral Mix, AIN-93G-MX / 35 / 35
Vitamin Mix, AIN-93-VX / 10 / 10
Choline Bitartrate / 2.5 / 2.5
THBQ, antioxidant / 0.014 / 0.014
N-acetylcysteine / 0 / 10

Supplemental Table 1B. AIN-93 NAC modified diet composition

Supplemental Table 2: Primer pair information:

rPON1: forward 5’ TGCTGGCTCACAAGATTCAC3’;

reverse 5’ TTCCTTTGTACACAGCAGCG3’ (Varatharajalu et al. 2009)

rPON3: forward 5’CTCTCGTCCACCTGAAAACC 3’;

reverse 5’ GAAGTCCAGTGAGGGTCCAA3’ (Romani et al. 2009)

rRPL13a: forward 5’ CCCTCCACCCTATGACAAGA3’;

reverse 5’ CCTTTTCCTTCCGTTTCTCC3’ (Gaub et al. 2010)

rCYP1A1: forward 5’ ATGTCCAGCTCTCAGATGATAAGGTC’3;

reverse 5’ ATCCCTGCCAATCACTGTGTCTAAC’3 (Vondracek et al. 2006)

rApoA1: forward 5’ CCTGGATGAATTCCAGGAGA’3;

reverse 5’ TCGCTGTAGAGCCCAAACTT’3 (Boesch-Saadatmandi et al. 2009)

rAhR: forward 5’ GGGCCAAGAGCTTCTTTGATG’3;

reverse 5’ GCAAGTCCTGCCAGTCTCTGA’3 (Shipley and Waxman 2006)

Boesch-Saadatmandi C, Pospissil RT, Graeser AC, Canali R, Boomgaarden I, Doering F, et al. 2009. Effect of quercetin on paraoxonase 2 levels in raw264.7 macrophages and in human monocytes--role of quercetin metabolism. Int J Mol Sci 10:4168-4177.

Gaub P, Tedeschi A, Puttagunta R, Nguyen T, Schmandke A, Di Giovanni S. 2010. Hdac inhibition promotes neuronal outgrowth and counteracts growth cone collapse through cbp/p300 and p/caf-dependent p53 acetylation. Cell Death Differ 17:1392-1408.

Romani R, De Medio GE, di Tullio S, Lapalombella R, Pirisinu I, Margonato V, et al. 2009. Modulation of paraoxonase 1 and 3 expression after moderate exercise training in the rat. J Lipid Res 50:2036-2045.

Shipley JM, Waxman DJ. 2006. Aryl hydrocarbon receptor-independent activation of estrogen receptor-dependent transcription by 3-methylcholanthrene. Toxicol Appl Pharmacol 213:87-97.

Varatharajalu R, Garige M, Leckey LC, Gong M, Lakshman MR. 2009. Betaine protects chronic alcohol and omega-3 pufa-mediated down-regulations of pon1 gene, serum pon1 and homocysteine thiolactonase activities with restoration of liver gsh. Alcohol Clin Exp Res 34:424-431.

Vondracek J, Svihalkova-Sindlerova L, Pencikova K, Krcmar P, Andrysik Z, Chramostova K, et al. 2006. 7h-dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial 'stem-like' cells. Mutat Res 596:43-56.

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Supplemental Table 3: Ratio between HDL-C and TC and HDL-C and LDL-C in animals on different Se diets treated with PCB 126

PCB 126
Treatment / HDL-C/TC / HDL-C/LDL-C
Low
(0.02 ppm Se) / Adequate
(0.2 ppm Se) / Supplemental
(2 ppm Se) / Low
(0.02 ppm Se) / Adequate
(0.2 ppm Se) / Supplemental
(2 ppm Se)
Corn Oil
0.2 mmol/kg
1 mmol/kg
5 mmol/kg / 0.66 ± 0.04
0.56 ± 0.08
0.83 ± 0.11
0.69 ± 0.15 / 0.66 ± 0.12
0.56 ± 0.13
0.70 ± 0.06
0.52 ± 0.06 / 0.73 ± 0.20
0.57 ± 0.04
0.56 ± 0.02
0.62 ± 0.06 / 4.72 ± 0.64b
3.72 ± 0.31
5.81 ± 0.73
5.07 ± 0.91 / 2.66 ± 0.27
4.02 ± 0.76
5.14 ± 0.61a
4.63 ± 0.63 / 2.69 ± 0.59
4.76 ± 0.39a
4.69 ± 0.19a
5.18 ± 0.69a

Results are expressed as mean ± SEM. Each group contained 6 animals. Significant differences (p < 0.005) were observed between (a) a PCB 126 level and the corn oil treatment, (b) Se 0.02 or 2 ppm Se diet and 0.2ppm

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Supplemental Figure 1: Effects of PCB 126 and Dietary Se Levels on PON1 Protein Level (Western blot). Liver proteins were separated by SDS gel electrophoresis. PON1 and GRPDH (loading control) proteins were identified by antibody staining. The data of the densitometry analysis are depicted in Figure 2, bottom left.


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Supplemental Figure 2. Direct effect of NAC on rat serum PON1 activity

Rat serum was incubated with the indicated amounts of NAC for 0-30 minutes and serum PON1 activity was determined using paraoxon (top) and phenylacetate (bottom) as substrates. No statistically significant differences were observed.

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