Chemico-Biological Interactions 180 (2009) 262–270
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Chemico-Biological Interactions
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Modification of dietary copper levels on the early stage of tumor-promotion
with propylthiouracil in a rat two-stage thyroid carcinogenesis model
Tomomi Shima
a
, Jihei Nishimura
a,b
, Yasuaki Dewa
a,b
, Yukie Saegusa
a,b
, Sayaka Matsumoto
a,b
,
Masaomi Kawai
a,b
, Tomoaki Harada
a
, Kunitoshi Mitsumori
a
, Makoto Shibutani
a,∗
a
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan
b
Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan
article info
Article history:
Received 2 December 2008
Received in revised form 5 February 2009
Accepted 5 February 2009
Available online 13 February 2009
Keywords:
Thyroid carcinogenesis
Copper (Cu)
Ceruloplasmin (Cp)
Metallothionein (MT)
Cell proliferation
abstract
To investigate the role of copper (Cu)-related cellular responses on thyroid carcinogenesis, the expres-
sion of ceruloplasmin (Cp) and metallothionein (MT)-1/2 were examined in relation to the activities of
cell proliferation/apoptosis in the thyroid of rats at an early stage of tumor promotion under different
dietary Cu levels. Male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine by single sub-
cutaneous injection at 2800 mg/kg body weight, and 1 week later promoted with 6-propyl-2-thiouracil at
12 ppm in the drinking water for 4 weeks. Animals were fed a diet containing Cu at 0.6, 6 or 60 ppm from
the time point of initiator-treatment to create marginally deficient, normal, or non-toxic supplementary
levels of Cu. At both 0.6 and 60 ppm, the multiplicity of preneoplastic focal follicular cell hyperplasias
(FFCHs) was decreased as compared with 6 ppm Cu, while adenomas also decreased at 0.6 ppm Cu. Both
0.6 and 60 ppm Cu levels revealed decreased Ki-67-immunoreactive proliferating cells in both FFCHs and
surrounding follicles accompanied by mRNA downregulation of Cdc2a and Ccnb1, while TUNEL-positive
apoptotic cells were unaltered with change of dietary Cu. Both Cp and MT-1/2 were immunolocalized in
FFCHs and adenomas, with higher distribution in the latter. At both 0.6 and 60 ppm, the immunoreac-
tivities and/or thyroidal mRNA levels of Cp and MT-1/2 were also decreased. Transcript levels of several
antioxidant enzymes were up- or downregulated in the same direction at both Cu levels. Serum levels
of thyroid-related hormones were unaltered at both Cu levels, except for non-significant reduction of
thyroid-stimulating hormone at 0.6 ppm. These results suggest an involvement of Cp and MT-1/2 on the
thyroid tumor promotion that can be suppressed by dietary Cu level through inhibition of cell proliferation
associated with altered redox balance.
© 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
In humans, the causative factors for thyroid carcinomas are
not well understood except for secondary occurrence after radio-
therapy [1]. In rats, on the other hand, thyroid follicular cell
tumors can be produced by administration of anti-thyroid agents,
such as 6-propyl-2-thiouracil (PTU) [2] and methimazole [3] in
an initiation–promotion model. The putative mechanism for this
carcinogenesis is believed to be a decrease in the serum levels of
thyroid hormone which causes suppression of the negative feed-
back effect at the pituitary to increase serum thyroid-stimulating
hormone (TSH). TSH then stimulates thyroid functions, including
growth and proliferation of follicular cells [4,5].
To search for differentially regulated molecules involved in
the processes of thyroid carcinogenesis through a mechanism of
∗
Corresponding author. Tel.: +81 42 367 5874; fax: +81 42 367 5771.
E-mail address: mshibuta@cc.tuat.ac.jp (M. Shibutani).
hypothyroidism, we recently performed a global gene expression
profiling in the microdissected proliferative lesions obtained after
promotion with sulfadimethoxine (SDM) in a rat two-stage car-
cinogenesis model [6]. By microarray analysis, upregulation of
ceruloplasmin (Cp) and metallothionein (MT)-1/2 was specifically
found in the thyroid proliferative lesions from the early stages. Cp
is the major copper (Cu)-carrying protein in the blood, and also
plays a role in iron metabolism, exerting a Cu-dependent activity of
ferroxidase [7]. We, in the above-mentioned study, found localized
expression of Cp in the proliferative lesions as early as focal follicu-
lar cell hyperplasias. MT is a family of cysteine-rich, low-molecular
weight proteins. MT-1 and -2 are associated with homeostasis of
essential trace elements such as Cu, metabolism/detoxification of
heavymetals and scavenging of free radicals [8]. Localized increased
expression of MT has been reported in human thyroid follicular cell
cancers [9,10].
Cu is regulated homeostatically not only with excessive Cu but
also under conditions of a Cu deficiency in the body. Cu defi-
ciency is quite critical for the normal functions of the body, as can
0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2009.02.002