Tamotsu Takizawa

Inhibitory effects of cinnamaldehyde on 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone-induced lung carcinogenesis in rasH2 mice

Previously we reported a lack of modification by cinnamaldehyde (CNMA) of development of lung proliferative lesions induced by urethane in CB6F1-TgHras2 (rasH2) mice. In the present study, we re-evaluated CNMA effects using the same rasH2 strain and non-transgenic littermates initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Sixteen mice/strain/sex received intraperitoneal NNK injections at a dose of 3 mg/mouse once a week for 2 weeks followed by free feeding of commercial diet containing 5000 ppm CNMA for 26 weeks. Additional groups were maintained without NNK injection and/or CNMA feeding for 28 weeks. Lung tumors were induced by NNK in both rasH2 and non-Tg males and females at incidence ranging from 63 to 100%. CNMA treatment significantly reduced the combined incidence of adenomas and carcinomas from 86 to 31% in rasH2 males (P<0.05), but no significant influence was evident in females. The multiplicity of NNK-induced lung tumors was also significantly reduced in rasH2 males given CNMA (P<0.01). Similar effects were also observed in non-Tg females given CNMA after NNK initiation. The results of our study strongly indicate that CNMA is capable of inhibiting development of NNK-initiated pulmonary tumorigenesis in rasH2 and non-Tg mice.

Possible enhancing effects of atrazine on growth of 7,12-dimethylbenz(a) anthracene-induced mammary tumors in ovariectomized Sprague-Dawley rats

Atrazine, one of the most commonly used herbicides in the world, has been reported to have endocrine disrupting effects in vivo. In the present experiment, influence of dietary atrazine on the late promotion/progression stage of mammary carcinogenesis in ovariectomized female Sprague–Dawley rats was examined after a single intragastric administration of 7,12‐dimethylbenz(a)anthracene (DMBA). When the incidence of palpable mammary tumors reached about 50%, the animals were subjected to ovariectomy and divided into tumor bearing [DMBA‐Tumor(+)] and non‐tumor bearing [DMBA‐Tumor(–)] groups, with subgroups of each fed a soybean‐free diet containing 0, 5, 50, or 500 p.p.m. atrazine for 34 weeks. At the completion of the study, the tumor volume in the 50 and 500 p.p.m. treatment Tumor(+) subgroups was greater than in the 0 p.p.m. control case. In the DMBA‐Tumor(–) group, higher incidences and volumes of the mammary tumors, with or without statistical significance (P < 0.05), were observed in the 50 and 500 p.p.m. subgroups. Atrazine treatment tended to increase proportion of estrogen receptor α‐positive tumors and stimulated cell proliferation in the DMBA‐Tumor(+) group, but with no clear effects on serum hormone levels. The present study indicates that atrazine has a potential for enhancing the growth of mammary tumors, partly through increasing cell proliferation in the promotion/progression stage in female rats under ovarian hormone‐free conditions. (Cancer Sci 2005; 96: 19 –25)

Sequential analysis of development of invasive thyroid follicular cell carcinomas in inflamed capsular regions of rats treated with sulfadimethoxine after N-bis (2-hydroxypropyl) nitrosamine-initiation

A 2-stage thyroid follicular carcinogenesis model in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN) is widely used to detect modifying effects of chemicals on thyroid carcinogenesis. A number of goitrogens are known to strongly promote carcinogenesis, and the carcinomas often originate adjacent to the thyroid capsule and show invasive growth into the capsule or adjacent tissues. To clarify mechanisms of progression to invasive carcinomas, we sequentially evaluated histopathological and immunohistochemical characteristics of thyroids in male F344 rats treated with sulfadimethoxine (SDM, 0.1% in drinking water) for 0—10 weeks beginning 1 week after DHPN initiation (2800 mg/kg body weight, single s.c. injection). In DHPN-SDM-treated rats, multiple focal hyperplasias and adenomas developed in thyroid follicular parenchyma at weeks 4 to 6. Apart from the proliferative lesions, capsular thickening with inflammatory cell infiltration, mainly consisting of macrophages, and migration of follicular epithelium into the capsule were also observed. Focal hyperplasias/adenomas adjacent to the capsule progressively developed to invasive carcinomas at weeks 6 to 10. In thyroid parenchyma, malignant lesions were seldom observed. With SDM-treatment alone, although no neoplastic lesions were observed, capsular thickening with inflammation and epithelial migration resulted in intracapsular residual follicles. Intracapsular residual follicular cells as well as invasive and intrathyroidal carcinoma cells generally showed increased cell proliferative activity, coincidental with cytoplasmic/nuclear positivity for β-catenin. These results suggested that β -catenin activation related to capsular inflammation may play a role in development of invasive carcinomas but is insufficient for tumor formation by itself. Whether this is associated with mutations in the β-catenin gene remains to be clarified.

Development of invasive follicular cell carcinomas in a rat thyroid carcinogenesis model : Biological impact of capsular inflammation and reduced cyclooxygenase-2 expression

We have previously reported that thyroid capsular inflammation induced by sulfadimethoxine (SDM), a goitrogen, might play a role in development of invasive follicular cell adenocarcinomas in rats initiated with N‐bis(2‐hydroxypropyl)nitrosamine (DHPN). The present study was designed to examine the role of cyclooxygenase (COX)‐2, widely known to be up‐regulated in inflammatory states, during chemically induced rat thyroid carcinogenesis. Male F344 rats received a subcutaneous DHPN (2800 mg/kg) injection, and 1 week later were allowed free access to drinking water containing antithyroidal propylthiouracil (PTU, 0.003%) or SDM (0.1%) for 4 or 10 weeks. Control groups receiving goitrogen alone and no treatment were also included. At week 4, diffuse follicular cell hyperplasia was induced in all PTU‐ and SDM‐treated groups, along with fibrous capsular thickening and capsular thickening with inflammation, respectively. Additionally, multiple focal follicular cell hyperplasias and adenomas were observed in the DHPN + PTU and DHPN + SDM cases. At week 10, adenocarcinomas invasive to the capsule and restricted to the capsular adjacent region, were frequent in the DHPN + SDM group, but not observed in the animals given DHPN + PTU. Western blots and immunohistochemistry revealed constitutive COX‐2 expression in non‐neoplastic follicular cells of the control and all of the PTU‐ and SDM‐treated rats. However, COX‐2 reactivity was significantly reduced or negative in the preneoplastic/neoplastic lesions in the DHPN‐treated groups. In fibrous or inflamed thickened capsules, only a few component cells with inflammatory elements were positive for COX‐2, and there was no significant difference in this regard between the PTU and SDM treatments. The present results suggest that capsular inflammation could play a role in development of invasive carcinomas, but COX‐2 expression does not make a major contribution. (Cancer Sci 2005; 96: 31–38)

Comparison of enhancing effects of different goitrogen treatments in combination with β‐estradiol‐3‐benzoate for establishing a rat two‐stage thyroid carcinogenesis model to detect modifying effects of estrogenic compounds

With the aim of establishing a sensitive model for the detection of weak effects of endocrine disrupting chemicals on thyroid carcinogenesis, thyrotrophic and tumor‐promoting influences of β‐estradiol‐3‐benzoate (EB) in combination with representative antithyroidal agents (goitrogens), sulfadimethoxine (SDM), propylthiouracil (PTU), potassium perchlorate (PPC), iopanoic acid (IOP) or an iodine‐deficient diet were evaluated in a short‐term (7‐day) experiment without N‐bis(2‐hydroxypropyl)nitrosamine (DHPN) initiation and a long‐term (30‐week) experiment with DHPN initiation in ovariectomized F344 rats. In the short‐term experiment, the most remarkable thyrotrophic effects were found in the PTU‐treated group, followed by the SDM and PPC cases. EB treatment alone caused slight increases in thyroidal weights but no apparent morphological changes. Concomitant treatment with EB and antithyroidal agents enhanced the changes in thyroid weights, histopathological findings and/or serum thyroid hormone levels in the SDM (30 and 100 p.p.m), PTU (5 and 30 p.p.m) and PPC (100 p.p.m), IOP (30 and 100 mg/kg) or iodine‐deficient diet groups. In the long‐term experiment after DHPN initiation, EB alone slightly increased small numbers of animals with follicular hyperplasias, adenomas and adenocarcinomas. Simultaneous treatment with antithyroidal chemicals was associated with an increase in the incidences of focal hyperplasias, adenomas and/or adenocarcinomas. The enhancement was most remarkable with PTU (5 p.p.m), followed by PTU (2 p.p.m), SDM (100 p.p.m) and PPC (100 p.p.m). The results showed that EB has only a marginal promoting effect on DHPN‐induced rat thyroid carcinogenesis and that antithyroidal chemicals, particularly PTU, are effective as copromoting agents. (Cancer Sci 2005)

Enhancing Effects of β‐Estradiol 3–Benzoate but Not Methoxychlor on the Promotion/Progression Stage of Chemically‐induced Mammary Carcinogenesis in Ovariectomized Rats

Modifying effects of β‐estradiol 3–benzoate (EB) and methoxychlor (MXC), a pesticide which possesses weak estrogenic activity, on 7,12–dimethylbenz(a)anthracene (DMBA)‐induced mammary carcinogenesis were investigated in ovariectomized or intact female Sprague‐Dawley rats. Twenty‐eight weeks after a single DMBA (100 mg/kg body weight) initiation, when the incidence of mammary tumor‐bearing rats had reached 75%, a number of the animals were subjected to ovariectomy in order to obtain 3 groups: i) tumor‐bearing, ovariectomized group; ii) tumor‐bearing, intact group; iii) no‐tumor, ovariectomized group. Subsequently animals of each group were subjected to subcutaneous implantation of 0.5 mg EB or given diet containing 1000 ppm MXC for 13 weeks. Although the incidences, multiplicities and volumes of the palpable tumors gradually decreased after ovariectomy, EB treatment stimulated tumor growth in the tumor‐bearing, ovariectomized group thereafter. A similar effect of EB treatment was also observed in the no‐tumor, ovariectomized group. However, MXC did not show any effect in the tumor‐bearing, or no‐tumor ovariectomized groups, except that the multiplicity of tumors was significantly decreased by MXC treatment in the tumor‐bearing, intact group. The results of our study suggest that MXC has no promotion/progression effect, but rather possesses a weak inhibitory effect, whereas the strongly estrogenic substance EB clearly enhanced DMBA‐induced mammary tumorigenesis.

Inhibition by Ethinylestradiol of N-Ethyl-N-Nitrosourea-Initiated Uterine Carcinogenesis in Transgenic Mice Carrying a Human Prototype C-Ha-ras Gene (rasH2 Mice)

In order to demonstrate the tumor promoting effect of ethinylestradiol (EE) in our uterine carcinogenesis model, rasH2 or ICR mice given an intraperitoneal injection of 120 mg/kg body weight of N-ethyl-N-nitrosourea (ENU) or an intra-uterine injection of 50 mg/kg body weight of ENU, respectively, followed by 2.5 or 0 ppm EE in the diet for 24 weeks in experiment 1 and 6 weeks in experiment 2. In experiment 1, in ICR mice, the incidences of adenocarcinomas in the ENU alone and the ENU+EE groups were 0% and 37.5%, respectively, the difference being statistically significant. The incidences of atypical hyperplasias and endometrial hyperplasias in the ENU+EE group were also significantly higher than those in the ENU alone group. In rasH2 mice, on the other hand, no endometrial proliferative lesions were induced in the uterus of the ENU+EE group, although uterine adenocarcinomas (55.6%), atypical hyperplasias (33.3%), and endometrial hyperplasias (22.2%) were observed in the ENU alone group. Proliferating cell nuclear antigen (PCNA) positive indices for uterine adenocarcinomas and atypical hyperplasias in ICR mice treated with ENU+EE showed high values, but those in rasH2 mice given ENU alone were comparable to data for intact epithelium. In experiment 2, the immunohistochemical expression of estrogen receptor α (ERα) in the uterine luminal and glandular epithelium in the ENU+EE group of ICR mice was moderate to marked, but that in the ENU alone group was slight. There was no consistent difference in ER α expression in the uterine luminal and glandular epithelium between ENU+EE and ENU alone groups of rasH2 mice. These results suggest that 2.5 ppm EE paradoxically inhibits the uterine carcinogenesis in rasH2 mice initiated with ENU.