SHORT REPORT Aryl hydrocarbon receptor polymorphism modulates DMBA-induced inflammation and carcinogenesis in phenotypically selected mice Vinicius R.C. De Souza 1 , Wafa K. Cabrera 1 , Antonella Galvan 2 , Orlando G. Ribeiro 1 , Marcelo De Franco 1 , Francisca Vorraro 1 , Nancy Starobinas 1 , Solange Massa 1 , Tommaso A. Dragani 2 * and Olga M. Iba~ nez 1 * 1 Laborat  orio de Imunogen etica, Instituto Butantan, S ~ ao Paulo, SP, Brazil 2 Department of Experimental Oncology and Laboratories, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy We tested the role of aryl hydrocarbon receptor (Ahr) gene poly- morphism in the inflammatory response and in skin and lung tu- morigenesis in 2 lines of mice phenotypically selected for maxi- mum or minimum acute inflammatory reaction (AIRmax and AIRmin, respectively). Following 7,12-dimethylbenz[a]anthracene (DMBA) treatment, AIRmin but not AIRmax mice showed early skin reactions and eventually developed malignant skin tumors and lung adenocarcinomas. In skin tissue, transcript levels of IL1b, Tnf, Il6, Tgfb1 and Cyp1b1 genes were upregulated in AIR- min but not AIRmax mice, consistent with the inflammatory responses to the carcinogen. These findings appeared to be related to the homozygosity status of the Ahr functional A375V polymor- phism, which influences the binding capability of the receptor for DMBA: the 375A allele, encoding the high-affinity ligand-binding receptor (Ahr b1 ), segregated in AIRmin mice, whereas AIRmax mice carried the 375V, corresponding to the low-affinity binding receptor (Ahr d ), to DMBA. The differential segregation of Ahr functional Ahr d versus Ahr b1 alleles in AIRmax and AIRmin sug- gests a role for the Ahr gene in the control of inflammatory responsiveness and tumor development of these mouse lines. ' 2008 Wiley-Liss, Inc. Key words: aryl hydrocarbon receptor; DMBA; inflammation; carcinogenesis; selected mice Two nonisogenic mouse lines derived by bidirectional selection based on the intensity of local acute inflammatory reactions (AIR) to polyacrylamide beads (Biogel) 1 have shown striking differences in resistance/susceptibility to chemical-induced carcinogenesis. Mice with the maximum acute inflammatory reaction (AIRmax) are significantly more resistant than minimum responders (AIRmin) to two-stage skin tumorigenesis induced by 7,12-dime- thylbenz[a]anthracene (DMBA) followed by repeated doses of the promoter agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 2 and also to lung carcinogenesis induced by urethane, 3 suggesting that a subset of loci affecting the inflammatory response also act as cancer modifier loci. DMBA and other polycyclic aromatic hydrocarbon compounds (PAHs) exert carcinogenic effects following metabolic activation mediated by the intracellular aryl hydrocarbon receptor (Ahr), a ligand-activated transcriptional factor regulating the induction of mono-oxygenase enzymes that convert the parent DMBA com- pound to a diol epoxide, the actual carcinogenic moiety. 4 Ahr gene presents functional polymorphism, which accounts for differ- ences in responses of mouse strains to PAHs. 5 We investigated DMBA-induced skin contact reactions and skin and lung tumor development in AIR mice with respect to their allele status of the Ahr gene. Our results implicate Ahr in tumor susceptibility and in inflammatory response control in AIRmax and AIRmin mice. Material and methods Mice and tumor induction AIRmax and AIRmin mice from the 38th generation of selec- tive breedings were produced at Laboratory of Immunogenetics of Butantan Institute (S~ ao Paulo, Brazil). All experimental proce- dures were approved by the Animal Experimentation Ethics Com- mittee of the Institute. Doses of 50 lg DMBA (Sigma, St. Louis, MO) in 0.1 ml acetone were applied to the shaved dorsal skin of mice during 5 consecutive days. Skin tumors were scored twice a week in groups of 15 AIRmax and 15 AIRmin mice, and the results were expressed as tumor multiplicity (number of tumors/ mouse). Mice were sacrificed 250 days after treatment, for analy- sis of tumors in internal organs. Lung tumor incidence, lung tumor multiplicity and lung tumor volume (N 3 V5 total volume of tumors) were recorded. A group of 8 mice from each line was sac- rificed at 48 hr after the last dose for histology and for mRNA expression analysis in the skin. Skin fragments and lungs after per- fusion with Carnoy solution were embedded in paraffin, cut into 5-lm sections and stained with hematoxilin and eosin. DNA and RNA analyses DNAs from AIRmax and AIRmin were extracted from tails. Skin samples were powdered in liquid nitrogen and RNA was iso- lated with TRIZOL (Invitrogen, Carlsbad, CA). After DNase I treatment, 1 lg of total RNA was reverse transcribed using 50 lM poly(dT)12–18, dNTPs (10 lM) and SuperScript III RT (200 U/ll) (Invitrogen). Quantitative mRNA levels were analyzed by kinetic reverse transcriptase-PCR (kRT-PCR) using Platinum SYBR Green qPCR Supermix-UDG (Invitrogen), 1 ll cDNA template, and 5 lM specific primers for the following genes: cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1) (5 0 -TGGA- GACCTTCCGGCATTC-3 0 ,5 0 -GCCATTCAGACTTGTATCTCT TGTG-3 0 ), cytochrome P450, family 1, subfamily b, polypeptide 1 (Cyp1b1) (5 0 -TGGCTGCTCATCCTCTTTA-3 0 , 5 0 -AGGTTGG GCTGGTCACTCAT-3 0 ), interferon gamma (Ifng) (5 0 -GCT CTGAGACAATGAACGCT-3 0 , 5 0 -AAAGAGATAATCTGGCT CTGC-3 0 ), interleukin 10 (Il10) (5 0 -TCAAACAAAGGACCA GCTGGACAACATACTG-3 0 ,5 0 -CTGTCTAGGTCCTGGAGTC- CAGCAGACTCAA-3 0 ), interleukin 18 (Il18) (5 0 -GACCCAGA- GGATATTGGATC-3 0 , 5 0 -GGAGGAAGTGTGATGTGAC-3 0 ), interleukin 1 alpha (Il1a) (5 0 -CAGTTCTGCCATTGACCATC-3 0 , 5 0 -TCTCACTGAAACTCAGCCGT-3 0 ), interleukin 1 beta (Il1b) (5 0 -GAGATTGAGCTGTCTGCTCA-3 0 , 5 0 -AAGGAGAACCAA GCAACGAC-3 0 ), interleukin 6 (Il6) (5 0 -GTACTCCAGAAGAC- CAGAGG-3 0 ,5 0 -TGCTGGTGACAACCACGGCC-3 0 ), transform- ing growth factor, beta 1 (Tgfb1) (5 0 -ACCGCAACAACGCCATC- TAT-3 0 ,5 0 -GTAACGCCAGGAATTGTTGC-3 0 ), tumor necrosis factor (Tnf) (5 0 -TTGACCTCAGCGCTGAGTTG-3 0 , 5 0 -CCTG TAGCCCACGTCGTAGC-3 0 ). The mouse hydroxymethylbilane synthase (Hmbs) gene (5 0 -AAAGTGCCGTGGGGAAACCAG-3 0 , 5 0 -GAGGCGGGTGAGGTTTC-3 0 ) was used as housekeeping con- trol. The amplifications were conducted in Chromo 4 thermal cycler (MJ Research Inc., Watertown, MA, USA). Relative gene expres- sion levels were calculated by comparative Ct method. Grant sponsors: FAPESP, Fondazione Cariplo grant, CNPq. *Correspondence to: Laborat orio de Imunogen etica, Instituto Butan- tan, S~ ao Paulo, SP, Brazil. E-mail: olgaibanez@butantan.gov.br or Fonda- zione IRCCS Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy. E-mail: tommaso.dragani@istitutotumori.mi.it. Received 10 June 2008; Accepted after revision 26 September 2008 DOI 10.1002/ijc.24066 Published online 22 October 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 124, 1478–1482 (2009) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer