J Toxicol Pathol 2009; 22: 199–203 Case Report Spontaneous Iron Accumulation in Hepatocytes of a 7-Week-Old Female Rat Kaori Miyata1, Tokuo Sukata1, Masahiko Kushida1, Keiko Ogata1, Manabu Suzuki2, Masakazu Ozaki2, Keisuke Ozaki1, and Satoshi Uwagawa1 1Environmental Health Science Lab., Sumitomo Chemical Co., Ltd., 1–98, Kasugadenaka 3-chome, Konohana-ku, Osaka 554-8558, Japan 2Animal Clinic Support Center, Sumika Technoservice Corporation, 1–98, Kasugadenaka 3-chome, Konohana-ku, Osaka, 554-8558 Japan Abstract: Spontaneous iron accumulation in hepatocytes was observed in a 7-week-old female Han Wistar GALAS rat. Very fine yellowish brown pigments, which showed a positive reaction with Berlin Blue stain, were apparent in the cytoplasm close to the bile canaliculi, with a diminishing periportal-to-centrilobular gradient. There were also differences in distribution between and within lobes. Transmission electron microscopy revealed cytosolic ferritin and pericanalicular siderosomes in hepatocytes. No degeneration or necrotic changes were observed, and non-hepatocyte cells did not demonstrate any obvious accumulation of iron. There were no abnormalities in the animal other than this finding in the liver. (J Toxicol Pathol 2009; 22: 199–203) Key words: iron overload, iron accumulation, hepatocyte, spontaneous, rats Iron can accumulate in the liver under a variety of conditions, including systemic iron-loading, some types of hepatitis and alcoholic liver disease1. Hemochromatosis is a well-known congenital metabolic disorder disease in which iron accumulates predominantly in liver parenchymal cells in the early phase and subsequently in liver Kupffer cells, the pancreas, the heart, endocrine glands and so on2. It is clear that the fundamental abnormality existing in this condition is excessive absorption of iron in the duodenum. In a toxicological study, we recently encountered a female rat at 7 weeks of age with iron overloading in the liver. Because this animal was a control fed a normal diet, the abnormality was presumed to be spontaneous. There has only been one previous article documenting spontaneous iron loading in rats3. According to that report, iron deposition began to be observed in animals around 9 weeks of age at very low incidences (0.07% in males and 0.56% in females). Here, we describe the characteristics of the changes seen in a young animal. The 4-week-old female BrlHan: WIST@Jcl(GALAS) (SPF) rat was purchased from Charles River Japan Inc. as one batch of breeding to be utilized for a 2-week feeding Received: 13 April 2009, Accepted: 8 June 2009 Mailing address: Kaori Miyata, Environmental Health Science Lab., Sumitomo Chemical Co., Ltd., 1–98, Kasugadenaka 3-chome, Konohana-ku, Osaka 554-8558, Japan TEL: 81-6-6466-5346 FAX: 81-6-6466-5354 E-mail: miyatak1@sc.sumitomo-chem.co.jp toxicological study. The animal was housed with one cage mate in a suspended aluminum cage with a stainless-steel wire-mesh front and floor. The room temperature and relative humidity were controlled at 22–26°C and 40–70%, respectively. The room air was changed 10 or more times per hour. Fluorescent tube lighting was employed to provide a 12-hr light/12-hr dark cycle (lighting period, 0800–2000). After a 7-day period of quarantine and acclimation, this animal was assigned to the control group and fed the control diet, CRF-1 (Oriental Yeast, Co. Ltd., Japan). After the 2- week treatment period, the animal was killed under ether anesthesia along with the other animals and subjected to a complete necropsy. The organs removed were fixed in 10% neutral buffered formalin solution. Routinely prepared paraffin embedded sections were stained with haematoxylin and eosin for light microscopic examination. A liver section was also stained with Berlin Blue for hemosiderin. Pieces of liver, originally fixed in 10% neutral buffered formalin solution, were refixed in 2.5% glutaraldehyde solution, post- fixed in 2% osmium tetroxide, dehydrated and embedded in epoxy resin for transmission electron microscopic examination. Ultrathin sections were stained with uranyl acetate and lead citrate. This experiment was performed in accordance with The Guide for Animal Care and Use of Sumitomo Chemical Co., Ltd. There was no evidence of any abnormal condition in this animal in regard to clinical observations, body weight, f o o d c o n s u m p t i o n , h e m a t o l o g y ( T a b l e 1 ) , b l o o d biochemistry (Table 2) and necropsy. The serum ferritin,