Gene expression during tumor enhancement by 3,3’-diindolylmethane in rainbow trout: HCC tumor samples

Indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM), a primary I3C derivative in vivo, are known dietary chemopreventive agents also available as dietary supplements. However, I3C has been found to act as a tumor promoter in rat (multi-organ) and trout (liver) models. I3C and DIM were previously found to be estrogenic in trout liver based on toxicogenomic profiles. In this study, we compare the post-initiation effects of DIM and 17β-estradiol (E2) on aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in trout. Trout were initiated as embryos with 50 ppb AFB1, fed control diet for three months followed by diets containing 0, 120 or 400 ppm DIM or 5 ppm E2 for 18 weeks before returning all groups to control diet. Tumor incidence was determined 13 months later and found to be significantly elevated in AFB1-initiated trout fed either 400 ppm DIM or 5 ppm E2 compared to control animals. To evaluate the mechanism of tumor enhancement, hepatic gene expression profiles were examined in animals fed promotional diets during the course of tumorigenesis and in hepatocellular carcinomas (HCCs) of initiated animals using a rainbow trout 70-mer custom oligonucleotide array. We demonstrate that DIM alters gene expression profiles similar to E2 in liver samples during tumorigenesis and in HCC tumors. Further, HCCs from animals on DIM and E2 promotional diets had a transcriptional signature indicating decreased invasive or metastatic potential compared to HCCs from control animals. Overall, these findings are the first to demonstrate tumor promotion by DIM. They confirm the importance of estrogenic signaling in the mechanism of promotion by dietary indoles in trout liver and indicate a possible dual effect that enhances tumor incidence and decreases potential for metastasis. Keywords: treatment effect Trout embryos were initiated with aqueous 50 ppb aflatoxin B1 in 0.01% ethanol for 30 min. At 3 months of age, fish were fed 0.1% DMSO vehicle, 400 ppm DIM or 5 ppm E2 (n=3 replicates per treatment) in the diet 5 days/week for 18 weeks. At 13 months, trout were sampled for tumors. Only HCC tumors (determined by histopathology) were used for microarray analysis. Hybridizations were performed using standard reference design with dye-swapping. cDNA from two of the three biological replicates were dye-swapped and hybridized to two slides as technical replicates (5 arrays per treatment).