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Threshold dose of piperonyl butoxide that induces reactive oxygen species-mediated hepatocarcinogenesis in rats

  • Genotoxicity and Carcinogenicity
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Abstract

To determine the threshold dose of piperonyl butoxide (PBO) that induces hepatocellular tumor-promoting effects, reactive oxygen species (ROS) generation, and drug-metabolizing enzymes that protect against ROS generation, partial hepatectomized rats were fed diets containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.

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Abbreviations

ABC:

Streptavidin–biotin peroxidase complex

Akr7a3:

Aflatoxin B1-aldehyde reductase

BSA:

Bovie serum albumin

CYP1A1:

Cytochrome P4501A1

DAB:

3, 3′-Diaminobenzidine

DCFH-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

DCF:

2′,7′-Dichlorofluorescein

DEN:

N-Diethylnitrosamine

GPX-2:

Glutathione peroxidase 2

GRx:

Glutaredoxin

GST-P:

Glutathione S-transferase placental form

H&E:

Hematoxylin and eosin

HPLC:

High performance liquid chromatography

i.p.:

Intraperitoneal

Nqo1:

NAD(P):quinine oxidoreductase

Mrp3:

Multidrug resistance protein3

PBO:

α-[2-(2-Butoxyethoxy)ethoxy]-4, 5-methylenedioxy-2-propyltoluene

PBS:

Phosphate-buffered saline

ROS:

Reactive oxygen species

UDPGTr-2:

UDP-glucronosyltransferase

RT-PCR:

Reverse transcriptase polymerase chain reaction

8-OHdG:

8-Hydroxydeoxyguanosine

Slc7a5:

Solute carrier family 7 (cationic amino acid transporter, y+ system), member 5

TCDD:

2,3,7,8-Tetrachlorodibenzo-p-dioxin

PCNA:

Proliferating cell nuclear antigen

EDTA-2Na:

Ethylene diamine tetraacetic acid disodium

DTT:

Dithiothreitol

PMSF:

Phenylmethylsulfonyl fluoride

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Acknowledgments

This research was supported by a part of Grant-in-Aid 564 for the Research Program for Risk Assessment Study on 565 Food Safety from the Food Safety Commission, Japan 0501.

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Authors and Affiliations

  1. Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan

    Masako Muguruma, Masaomi Kawai, Yasuaki Dewa, Jihei Nishimura, Yukie Saegusa, Hironobu Yasuno, Meilan Jin, Sayaka Matsumoto, Masayoshi Takabatake & Kunitoshi Mitsumori

  2. Department of Tissue Physiology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan

    Katsuhiko Arai

  3. United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagida, Gifu, 501-1193, Japan

    Masaomi Kawai, Yasuaki Dewa, Jihei Nishimura & Yukie Saegusa

  4. Department of Applied Biological Science, United Graduate School of Agricultural Sciences, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan

    Meilan Jin

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  1. Masako Muguruma
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  2. Masaomi Kawai
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  9. Masayoshi Takabatake
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  11. Kunitoshi Mitsumori
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Correspondence to Masako Muguruma.

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Muguruma, M., Kawai, M., Dewa, Y. et al. Threshold dose of piperonyl butoxide that induces reactive oxygen species-mediated hepatocarcinogenesis in rats. Arch Toxicol 83, 183–193 (2009). https://doi.org/10.1007/s00204-008-0340-8

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  • DOI: https://doi.org/10.1007/s00204-008-0340-8

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