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Expression patterns of cell cycle proteins in the livers of rats treated with hepatocarcinogens for 28 days

  • Genotoxicity and Carcinogenicity
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Abstract

Some hepatocarcinogens induce cytomegaly, which reflects aberrant cell cycling and increased ploidy, from the early stages of administration to animals. To clarify the regulatory molecular mechanisms behind cell cycle aberrations related to the early stages of hepatocarcinogenesis, we performed gene expression analysis using microarrays and real-time reverse transcription polymerase chain reaction followed by immunohistochemical analysis in the livers of rats treated with the cytomegaly inducing hepatocarcinogens thioacetamide (TAA), fenbendazole, and methyleugenol, the cytomegaly non-inducing hepatocarcinogen piperonyl butoxide (PBO), or the non-carcinogenic hepatotoxicants acetaminophen and α-naphthyl isothiocyanate, for 28 days. Gene expression profiling showed that cell cycle-related genes, especially those of G2/M phase, were mostly upregulated after TAA treatment. Immunohistochemical analysis was performed on cell cycle proteins that were upregulated by TAA treatment and on related proteins. All hepatocarcinogens, irrespective of their cytomegaly inducing potential, increased liver cells immunoreactive for p21Cip1, which acts on cells arrested in G1 phase, and for Aurora B or Incenp, which is suggestive of an increase in a cell population with chromosomal instability caused by overexpression. PBO did not induce cell proliferation after 28-day treatment. Hepatocarcinogens that induced cell proliferation after 28-day treatment also caused an increase in p53+ cells in parallel with increased apoptotic cells, as well as increased population of cells expressing M phase-related proteins nuclear Cdc2, phospho-Histone H3, and HP1α. These results suggest that hepatocarcinogens may increase cellular populations arrested in G1 phase or showing chromosomal instability after 28-day treatment. Hepatocarcinogens that induce cell cycle facilitation may cause M phase arrest accompanied by apoptosis.

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Abbreviations

ANIT:

α-Naphthyl isothiocyanate

APAP:

Acetaminophen

Cdc2:

Cell division cycle 2

Cdk:

Cyclin-dependent kinase

FB:

Fenbendazole

HP1α :

Heterochromatin protein 1α

Klf6:

Kruppel-like factor 6

MEG:

Methyleugenol

NDRG1:

N-myc downstream regulated gene 1

p-Histone H3:

Phosphorylated-Histone H3

PBO:

Piperonyl butoxide

TAA:

Thioacetamide

Topo IIα :

Topoisomerase IIα

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Acknowledgments

The authors thank Mrs. Shigeko Suzuki for her technical assistance in preparing the histological specimens. This work was supported by Health and Labour Sciences Research Grants (Research on Food Safety) from the Ministry of Health, Labour and Welfare of Japan.

Conflict of interest

The authors disclose that there are no competing financial interests that could inappropriately influence the outcome of this study.

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Authors and Affiliations

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

    Atsunori Yafune, Eriko Taniai, Reiko Morita, Fumiyuki Nakane, Kazuhiko Suzuki, Kunitoshi Mitsumori & Makoto Shibutani

  2. Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu, 501-1193, Japan

    Atsunori Yafune, Eriko Taniai & Reiko Morita

  3. Gotemba Laboratory, Bozo Research Center Inc., 1284 Kamado, Gotemba, Shizuoka, 412-0039, Japan

    Atsunori Yafune

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  1. Atsunori Yafune
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  2. Eriko Taniai
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  3. Reiko Morita
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  4. Fumiyuki Nakane
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Correspondence to Makoto Shibutani.

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Yafune, A., Taniai, E., Morita, R. et al. Expression patterns of cell cycle proteins in the livers of rats treated with hepatocarcinogens for 28 days. Arch Toxicol 87, 1141–1153 (2013). https://doi.org/10.1007/s00204-013-1011-y

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