A large series of assays of the hepatocarcinogenic potential of 112 different compounds were carried out using a rapid bioassay system developed in this laboratory based on the two-step concept of hepatocarcinogenesis. Rats were initially given a single dose (200 mg/kg) of diethylnitrosamine (DEN) i.p. and starting 2 weeks later were treated with test compounds for 6 weeks and then killed, all rats being subjected to two-thirds partial hepatectomy (PH) at week 3. Carcinogenic potential was scored by comparing the number and area per cm2 of induced glutathione S-transferase placental form-positive (GST-P+) foci in the liver with those of the corresponding control group given DEN alone. Positive was scored for a significant increase in the value of GST-P+ foci, negative for no change or a decrease. Results were compared to reported Salmonella/microsome and long-term carcinogenicity test findings. Of the liver carcinogens, 10 out of 11 (90.9%) mutagenic, and 11 out of 13 (84.6%) non-mutagenic compounds gave positive results (mean, 87.5%). Carcinogens other than the hepatocarcinogens gave less positive results (two out of 17, 11.8%). None of the compounds reported as non-carcinogenic demonstrated positivity suggesting that the assay system does not suffer from the disadvantage of false-positive results. The protocol system also provided information concerning the inhibitory potential of compounds such as anti-oxidants. It is concluded that the present experimental protocol which requires far fewer animals and shorter duration than a long-term carcinogenicity test has practical applications for the rapid and economical screening of environmental hepatocarcinogens and their inhibitory agents.

Dietary phytochemicals can inhibit the development of certain types of tumors. We here investigated the effects of nobiletin (Nob), garcinol (Gar), auraptene (Aur), β β β β-cryptoxanthin-and hesperidine-rich pulp (CHRP) and 1,1′ ′ ′ ′-acetoxychavicol acetate (ACA) on hepatocarcinogenesis in a rat medium-term liver bioassay, and also examined their influence on cell proliferation, cell cycle kinetics, apoptosis and cell invasion of rat and human hepatocellular carcinoma ( ietary phytochemicals are attractive as chemopreventive agents for cancer development and there are a number of promising candidates. For instance, it has been shown that soybean isoflavones can inhibit experimental prostate cancer development in vivo 1) and in vitro 2, 3) and intake of citrus fruits suppresses certain types of tumors. 4,5) Recent examples include nobiletin (Nob, a polymethoxyflavonoid in citrus fruits), garcinol (Gar, an antioxidant isolated from Garcinia indica fruit rind), auraptene (Aur, a Citrus antioxidant), β-cryptoxanthinand hesperidine-rich pulp (CHRP, a powder containing large amounts of β-cryptoxanthin and hesperidine, prepared from a Satsuma mandarin juice) and 1,1′-acetoxychavicol acetate (ACA, present in seeds and rhizomes of Languas galanga, used as a ginger substitute and a stomach medicine in Thailand), which inhibit the development of several types of tumors. [6][7][8][9][10][11] When given orally they were found to reduce cancers of the tongue, esophagus and colon in rat experiments.7, 12, 13) They also downregulated expression of cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) in rat colon, with a concomitant decrease in inflammatory responses and oxidative stress, suggesting a potential for colon cancer inhibition. 7) Induction of detoxification enzymes and inhibition of cell proliferation 14,15) are also possible mechanisms of their suppressive effects.Rat medium-term liver bioassays are effective to evaluate the effects of chemicals on hepatocarcinogenesis.16) In our model, rats initiated with diethylnitrosamine (DEN) are treated with test chemicals by oral administration and pre-neoplastic lesions of the liver are measured by quantitation of immunohistochemically detected glutathione S-transferase placental form (GST-P)-positive foci in the liver, 17) with the help of an image analyzer. In the present study, we focused on effects of a series of phytochemicals in this model and also investigated the influence of these compounds on the growth of a human HCC cell line, HepG2 and a rat HCC cell line, MH1C1, looking at cell proliferation and apoptosis. Moreover, we performed microarray analyses to identify changes in gene expression in HepG2 cells treated with these chemicals. We also investigated their effects on invasion of HCC cells using "Matrigel" invasion assays.

The effects of phenylethyl isothiocyanate (PEITC) on urinary bladder and liver carcinogenesis were analyzed in a rat model. Diets containing 0.1%, 0.05%, or 0.01% PEITC were administered for 32 wk to male Fischer 344 rats with and without pretreatment with an injection of diethylnitrosamine (200 mg/kg body wt i.p.) and 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water for 4 wk for initiation. In the initiated groups, PEITC administration significantly increased the incidences of papillary or nodular hyperplasia, dysplasia, and transitional cell carcinomas at higher doses of 0.01%, 0.01%, and 0.05%, respectively, compared with the control group, given initiation alone, in a dose-dependent manner. Without initiation, administration of 0.1% and 0.05% PEITC induced simple and papillary or nodular hyperplasia and dysplasia in the urinary bladder. In the liver, induction of glutathione S-transferase placental form-positive foci was dose dependently enhanced by PEITC administration, but the incidences of liver tumors were not different among the groups. From the present experiment, we can conclude that > 0.01% PEITC enhances rat urinary bladder carcinogenesis, while weakly promoting hepatocarcinogenesis. In addition, it is suggested that > 0.05% PEITC has tumorigenic potential.