Preferential mammary carcinogenic effects of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) in human c‐Ha‐ras proto‐oncogene transgenic rats

Abstract

he Ras family is composed of three members, Harvey Ras, Kirsten Ras and N-Ras, which exhibit mutations at variable frequencies in various human and animal tumors, 1) with involvement appearing to be tissue-specific. 2, 3) For example, with N-methyl-N-nitrosourea (MNU) in experimental animals, Haras mutations are found primarily in mammary tumors, [4][5][6] while colon tumors generally have Ki-ras mutations. 7) In humans, Ki-ras mutations are preferentially found in colon, 8,9) pancreatic duct 10) and bile duct carcinomas, 11) and N-ras mutations are typically seen in myeloid leukemia. 12) We have generated a transgenic rat line, harboring copies of a human c-Ha-ras proto-oncogene (Hras128 rat), and have shown that these animals are highly susceptible to mammary, 13,14) bladder 15) and esophageal 16) carcinogens.Heterocyclic amines found in overcooked foods are both mutagenic and carcinogenic to animals, 17) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), the most abundant in our daily diet, 18) has been shown to induce primarily colon, mammary and prostate tumors in rats. [19][20][21] PhIP intake in food has also been implicated as an important risk factor in humans 22,23) although further analysis of the interactions between PhIP and relevant macromolecules is required for a full understanding of the carcinogenic processes.Although mutational analyses of tumors induced by PhIP have been conducted, 24) specific molecular targets have yet to be elucidated. One problem is that the relatively weak carcinogenic potential requires the use of long experimental periods for lesion induction. This study was conducted to analyze the susceptibility of Hras128 rats to PhIP, in the hope of overcoming the above problem. We show here that Hras128 rats are in fact highly susceptible to PhIP mammary carcinogenesis, and multiple tumors of various morphological types develop in a short period. Furthermore, they feature mutations of the transgene along with down-regulation of BRCA1. Materials and MethodsAnimals and experimental protocol. Seven-week-old Hras128 rats of both sexes were maintained on basal diet (Oriental MF, Oriental Yeast Co., Ltd. Tokyo) with tap water ad libitum under standard conditions. 13) Seven females and 18 males were treated with PhIP HCl salt (NARD Institute, Osaka), at doses of 100 mg and 80 mg/kg body weight, respectively, by gastric intubation, from 3 to 4 times a week over a 9-week period (total, 8 times in females and 10 times in males). Five female and 20 male Hras128 rats were similarly treated with vehicle (saline). Sacrifice was at week 12 for females and week 30 for males. Seven female and 20 male wild-type littermates were also treated with PhIP, and 8 female and 20 male wild-type littermates received the vehicle (saline).The experiments were conducted according to the "Guidelines for Animal Experiments in the National Cancer Center Japan" promulgated by the Committee for Ethics of Animal Experimentation.Tissue preparation. Immediately after killing of the animals, mammary tumors wer...

Synthesis of Oligonucleotides Containing the N2-Deoxyguanosine Adduct of the Dietary Carcinogen 2-Amino-3-methylimidazo[4,5-f]quinoline

“…Activated Ras oncogenes have been identified in a large number of human tumors and are particularly frequent in colorectal cancers (59). Modification of codon-12 has been observed in rats when their diet was supplemented with IQ and other HCAs (61)(62)(63)(64)(65)(66)(67). DNA adducts of polycyclic aromatic hydrocarbons (PAHs) have been wellstudied in this sequence (60).…”

Gpx2 Is an Overexpressed Gene in Rat Breast Cancers Induced by Three Different Chemical Carcinogens

“…We have previously established a strain of human c-Ha-ras proto-oncogene transgenic rats (Hras128), which are highly susceptible to these mammary carcinogens, resulting in the development of a high incidence of mammary carcinomas over a relatively short period of time (4)(5)(6). This rat model is a valuable one for investigating mammary carcinogenesis in vivo.…”

High susceptibility of human c‐Ha‐ras proto‐oncogene transgenic rats to carcinogenesis: A cancer‐prone animal model

“…For example, intestine and prostate tumor induction in male rats was not different from that in wild type littermates after treatment with PhIP. ( 32 ) Similarly, no differences were observed in the incidences of liver, lung and thyroid tumors with dihydroxy‐ di‐n ‐propyl nitrosamine. It is interesting that all of the organs that exhibit enhanced susceptibility to carcinogens are known as organs in which endogenous c‐Ha‐ras gene mutations have been found in chemically induced tumors in wild rats.…”

“…Furthermore, activated form ras protein could be detected in carcinomas induced by PhIP. ( 32 ) In contrast, no mutations whatsoever were found in the endogenous c‐Ha‐ras gene in the mammary tumors arising in the Hras128 strain. ( 23 ) Therefore, the results indicate that preferential mutation and activation of the transduced human c‐Ha‐ras proto‐oncogene play dominant roles over the endogenous c‐Ha‐ras gene.…”

“…or multiple doses (100 mg/kg, i.g., eight times in 4 week‐period) of the food contaminant carcinogen, 2‐amino‐1‐methl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). ( 23,30,32 ) Almost all the transgenic rats rapidly developed multiple mammary carcinomas within 8–12 weeks (see Fig. 1).…”