植物保護學會會刊
44：37 - 50, 2002
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Aflatoxin B1 and 2-acetylaminofluorene induced hepatic carcinogenicity and gamma-glutamyltranspeptidase expression via chronic feeding in rats Jiunn-Wang Liao1, San-Fu Tsai1, Jenn-Sheng Hwang1, Victor Fei Pang2, Chian-Ren Jeng2, and Shun-Cheng Wang1*
1
Department of Applied Toxicology, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Wufeng, Taichung, Taiwan, ROC 2 Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, ROC
(Accepted for publication: Mar. 25, 2002)
ABSTRACT Liao, J. W., Tsai, S. F., Hwang, J. S., Pang, V. F., Jeng, C. R., and Wang, S. C.* 2002. Aflatoxin B1 and 2-acetylaminofluorene induced hepatic carcinogenicity and gamma-glutamyltranspeptidase expression via chronic feeding in rats. Plant Prot. Bull. 44: 37 - 50 The hepatic carcinogenicity and gamma-glutamyltranspeptidase induction of aflatoxin B1 (AFB1) and 2-acetyl-aminofluorene (2-AAF) was investigated by a long-term feeding assay in rats. Wistar rats were fed continuously on a diet containing 1 ppm of AFB1 for 40 weeks or 200 ppm of 2-AAF for 24 weeks. In both AFB1 and 2-AAF treated groups, the total body weight and the feed consumption of rats were markedly decreased after two weeks of treatments. The liver weights and enzymatic activities of ALP, AST, ALT and GGT were significantly elevated in both treated groups. Marked liver swelling and irregular tumor masses were found in all males of both treated groups and half of the AFB1-treated females. However, no tumor masses but multiple hepatic cysts were observed grossly in the 2-AAF-treated females. Microscopically, the tumor cells were arranged in an irregular pattern with high mitotic figures of hepatocellular carcinoma (HCC) in the male rats; small neoplastic foci and multiple cholangiocysts were found in 2-AAF-treated female rats. *Corresponding author. E-mail:
[email protected]
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植物保護學會會刊
第 44 卷
第1期
2002
The pathological incidence of the hepatocellular carcinoma was 100 % in AFB1- and 2-AAF-treated male rats, and was 50 % in AFB1-treated but none in 2-AAF-treated female rats, respectively. Additionally, the positive area of GGT histochemical staining in the affected livers was correlated expression in the AFB1 and 2-AAF groups. In conclusion, 2-AAF and AFB1 exhibited a potent of hepatocarcinogenicity and increased hepatic GGT activities in rats. The GGT histochemistry staining may be used as a biological marker for the toxicological screening of potential hepatic carcinogens of pesticides. (Key words: aflatoxin B1, acetylaminofluorene, hepatocarcinogenicity, GGT, rats)
INTRODUCTION The more and more unknown chemicals were produced from natural sources or manmade. This may present a potent carcinogen by accidental exposure in the environment. Aflatoxin B1 (AFB1) is a secondary metabolite produced by Aspergillus flavus and A. parasiticus (imperfect fungi), and is one of the potent naturally occurring carcinogenic agents(19). AFB1 exerts an acute toxic and chronic carcinogenic effect on mammalian(12). Previous studies showed that low level of AFB1 inhibited the immune function and growth performance in pigs(4) and induced liver tumor in rats(1). Furthermore, AFB1 also increased the tumor-marker of -glutamyltranspeptidase (GGT) in acute toxicity study(1, 3, 13), α-fetaprotein, and phase II components, such as glutathione(7, 23). The cytochrome P450 enzymes play a prominent role in the metabolism of AFB1, both in bioactivation and detoxification(10). In the field, the contamination of AFB1 often occurs in a variety of crops in the world. Chronic low level ingestion of aflatoxin produces liver cancer and has been found to be associated with primary cancer in certain population(6). The level of aflatoxin in peanuts and peanut
products has been regulating since 1969 under an action level of 20 ppb in US FDA(6) and 15 ppb in Taiwan(5). The 2-acetylaminofluence (2-AAF) was originally synthesized as a pesticide, and also a potent carcinogen to the rat liver, where it undergoes bioactivitation to DNA-reactive ultimate carcinogen(25). 2-AAF produced toxicity, including the DNA damage, cytotoxicity, cell proliferation, hepatocellular altered foci and eventually neoplasma were formed(9, 11, 20, 22, 24). The long-term animal toxicology data are important to assay the chemical-induced carcinogenicity in vivo. This study was conducted with AFB1 or 2-AAF, a well-known carcinogen, as the positive control materials to setup an animal model of chronic feeding or carcinigenicity study. Furthermore, we also investigated the incidence of HCC and the coincidence of GGT positive foci in rat livers caused by AFB1 and 2-AAF treatment to evaluate the chronic toxicity of pesticides in the future.
MATERIALS AND METHODS Sixty male and female adult Wistar rats (Rattus norvegicus), 5 week-old, were bought from the National Laboratory Animal
Aflatoxin B 1 and 2-AAF induced hepatic carcinogenicity and GGT expression in rats
Production and Research Center in Taipei. 2-AAF and AFB1 of 95 % purity were purchased from Sigma Co., (Louis, MO, USA). The stock feed of 2 % 2-AAF powder and 0.02 % AFB1 (0.0526 g diluted with 250 ml acetone) were prepared as a stock feed. Weighing 100 g of stock feed was directly added into 4.9 kg of powder feed (Rodent Laboratory Chow 5001, Purina, MO, USA) and blended with stainless mixing machine (V-type, No. 40-564, RKJ, Japan) for 30 min. Five kilogram feed containing test chemical was mixed weekly. Rats were randomly assigned to the 2-AAF, AFB1 and control groups with 10 males and 10 females in each. They were fed continuously on a diet containing 200 ppm 2-AAF for 24 weeks (2-AAF group), 1 ppm AFB1 for 40 weeks (AFB1 group) or an equal amount of acetone (control group) for 40 weeks. Health condition and behavior abnormalities were observed daily. Feed consumption and body weight gains were measured weekly. All animals received humane care in accordance with the guideline by A Guidebook for the Care and Use of Laboratory Animals(26). At the end of experiment, all rats were anesthetized with ether, and the fresh blood was collected with EDTA and plasma separated tubes (Vacationer, Franklin Lakes, NJ, USA) from abdominal artery, and prepared for the hematological and biochemical examination. The brain, liver, kidney and spleen were individually weighed. Red blood cell (RBC), hematocrit (Hct), mean corpuscular volume (MCV), white blood cell (WBC), and hemoglobin (Hb) of whole blood were determined by Microcell Counter (Sysmex, CC-150, Japan). Serum was obtained from clotted whole blood and
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centrifuged at 775xg for 15 min (Kubota 2010, Japan). Serum biochemistry of total protein, albumin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were detected by DMA kits (DMA, Inc., Arlingto, Texas, USA), and gamma glutamyltransferase (GGT) was analyzed by Ciba-Corning kit (Ciba-Corning, Diagnostics Corporation, Irvine, CA, USA), and determined individually by semiautomatic spectrophotometer (VitaLab100, German). At necropsy, the left medial lobe of liver was frozen at -80 ℃ for 10 mm cryo-section. The histochemical stain for GGT was performed with the GGT reagent (G-0141, Sigma Chemical Co., Louis, MO, USA) according to the method described by Rutenburg et al.(18); the positive neoplastic areas were assayed by an image analyzer (Q-500, Lieca, German). Representative tissues from all organs were fixed in 10 % neutral phosphate buffered formalin, and sectioned at 5-7 m stained with H&E or Masson trichrome stain(13). Data were analyzed by Microsoft Excel method in AVERAGE formula for mean and by STDEVP method for standard deviation. The significant differences between treated and control groups were analyzed by t-test method at p
