Carcinogenesis vol 26 no 2 pp 503 509 2005 doi 10 1093 carcin bgh330 Gene expression profiling of chemically induced rat mammary gland cancer Liang Shan Minshu Yu and Elizabeth G Snyderwine1 Chemical Carcinogenesis Section Laboratory of Experimental Carcinogenesis Center for Cancer Research National Cancer Institute NIH Bethesda MD 20892 4262 USA 1 To whom correspondence should be addressed Tel 1 301 496 5688 Fax 1 301 496 0734 Email elizabeth snyderwine nih gov Exposure to carcinogens through diet the atmosphere and other means is generally regarded as influencing human cancer risk but the impact of specific environmental carcinogens on human breast cancer incidence is still unknown We examined whether distinct chemical carcinogens induce a unique transcriptional profile in mammary gland cancer that is characteristic of the etiologic agent Rat mammary gland cancers n 34 were generated by various carcinogens including the food derived heterocyclic amines 2 amino 1 methyl 6 phenylimidazo 4 5 b pyridine and 2 amino 3 8 dimethylimidazo 4 5 f quinoxaline 7 12 dimethylbenz a anthracene N nitrosomethylurea and 4 aminobiphenyl The histopathology of the carcinomas was graded using a modified ScarffBloom Richardson scheme and the gene expression profiles in the carcinomas were evaluated on a 10K cDNA microarray Unsupervised hierarchical clustering analysis revealed two major clusters of carcinomas irrespective of the carcinogenic agent that distinguished two groups with different histopathological parameters degree of differentiation nuclear grade mitotic activity epithelial cell growth pattern and necrosis Using class comparison analysis and hierarchical clustering of all carcinomas irrespective of histopathology gene expression profiles were further shown to be statistically differentially expressed according to the carcinogenic agent These findings indicate that the transcriptional program in carcinomas is unique to the etiologic agent and can be observed among a diverse set of carcinogens despite variations in carcinoma histopathology The ability to use microarray analysis to discern an etiology specific profile among a pathologically heterogeneous group of breast carcinomas may ultimately be valuable in determining the role of environmental chemical carcinogens in human breast cancer risk breast cancer remains a challenge in cancer research 1 4 The rat has served as a valuable model for understanding the development of human breast cancer because of similarities in pathology cell of origin and hormone dependency 5 6 Furthermore the rat model provides a means to study specific etiological factors in breast carcinogenesis in a way not feasible in humans Recent cDNA microarray analysis of rat mammary gland cancer has suggested that the profile of gene expression may be characteristic of the etiological agent 7 8 Comparison of the gene expression profiles in histologically similar rat mammary gland cancers revealed carcinogen specific gene expression profiles in carcinomas induced by 2 amino 1methyl 6 phenylimidazo 4 5 b pyridine PhIP a heterocyclic amine in the human diet and 7 12 dimethylbenz a anthracene DMBA an experimental carcinogen belonging to the class of polycyclic aromatic hydrocarbons PhIP and DMBA induced carcinomas could be distinguished by array tools including hierarchical clustering and multidimensional scaling analysis Although pioneering these prior studies used just two carcinogens and only examined carcinomas of similar histopathology These studies therefore begged the question of whether microarray analysis could distinguish breast cancers according to etiology when carcinomas were induced by multiple chemical carcinogens and were histologically varied In the light of the diversity in human breast cancers and wide variations in environmental exposures among individuals the feasibility of using microarray analysis to define the etiology of human breast cancers would likely depend on the ability to categorize specific carcinomas from a heterogeneous group of breast cancers Herein we have analyzed the gene expression profiles of 34 rat mammary gland carcinomas induced by carcinogens including PhIP DMBA N nitrosomethylurea NMU 2 amino 3 8 dimethylimidazo 4 5 f quinoxaline MelQx and 4 aminobiphenyl 4ABP These carcinogens represent major classes of environmental carcinogens that have been linked to various human cancers and include the cooked meat derived heterocyclic amines aromatic amines polycyclic aromatic hydrocarbons and nitrosoureas 1 9 10 The current study extends earlier work to determine whether carcinogens induce characteristic and etiology specific gene expression profiles in mammary gland cancers that are resolvable by microarray analysis Introduction Human breast cancer is a heterogeneous disease with respect to pathology biochemistry and etiology While environmental carcinogen exposure is often regarded as contributing to human cancer risk identifying specific etiological factors in Abbreviations 4ABP 4 aminobiphenyl DMBA 7 12 dimethylbenz a anthracene MDS multidimensional scaling MelQx 2 amino 3 8 dimethylimidazo 4 5 f quinoxaline NMU N nitrosomethylurea PhIP 2 amino 1 methyl 6 phenylimidazo 4 5 b pyridine Carcinogenesis vol 26 no 2 Oxford University Press 2005 all rights reserved Materials and methods Chemical carcinogens and rat mammary gland tumor induction Female Sprague Dawley rats 43 days old were obtained from the NIH animal supply Animal Production Area Frederick MD All animals were provided NIH Lab Chow and water ad libitum and housed in a NIH animal facility on a 12 h light 12 h dark cycle Mammary gland carcinomas were induced by PhIPHCl Toronto Research Chemicals North York Canada DMBA Sigma St Louis MO NMU Sigma MelQx Toronto Research Chemicals Toronto Canada and 4ABP provided by F F Kadlubar NCTR Jefferson AR PhIP HCl MelQx and 4ABP were all given at 75 mg kg p o once per day 503 L Shan M Yu and E G Snyderwine Clone selection and statistical analysis Data were analyzed by median normalization total intensity normalization 50th percentile using BRB ArrayTools software version 3 2 This is an integrated software package for the visualization and statistical analysis of gene expression data developed by the Biometric Research Branch of the National Cancer Institute http linus nci nih gov brb The spots of the array were excluded from analysis if any of the following were observed the spot diameter was 510 mm both red Cy5 and green Cy3 intensities