Different mechanisms of DEHP-induced hepatocellular adenoma tumorigenesis in wild-type and Ppar alpha-null mice

Abstract

Di (2-ethylhexyl) phthalate (DEHP) exposure is thought to lead to hepatocellular hypertrophy and hyperplasia in rodents mediated via peroxisome proliferator-activated receptor alpha (PPAR alpha). A recent study revealed that long-term exposure to relatively low-dose DEHP (0.05%) caused liver tumors including hepatocellular carcinomas, hepatocellular adenomas, and chologiocellular carcinomas at a higher incidence in Ppar alpha-null mice (25.8%) than in wild-type mice (10.0%). Using tissues with hepatocellular adenoma, microarray (Affymetrix MOE430A) as well as, in part, real-time quantitative PCR analysis was conducted to elucidate the mechanisms of the adenoma formation resulting from DEHP exposure in both genotyped mice. The microarray profiles showed that the up- or down-regulated genes were quite different between hepatocellular adenoma tissues of wild-type and Ppar alpha-null mice exposed to DEHP. The gene expressions of apoptotic peptidase activating factor 1 (Apaf1) and DNA-damage-inducible 45 alpha (Gadd45a) were increased in the hepatocellular adenoma tissues of wild-type mice exposed to DEHP, whereas they were unchanged in corresponding tissues of Ppar alpha-null mice. On the other hand, the expressions of cyclin B2 and myeloid cell leukemia sequence 1 were increased only in the hepatocellular adenoma tissues of Ppar alpha-null mice. Taken together, DEHP may induce hepatocellular adenomas, in part, via suppression of G2/M arrest regulated by Gadd45a and caspase 3-dependent apoptosis in Ppar alpha-null mice, but these genes may not be involved in tumorigenesis in the wild-type mice. In contrast, the expression level of Met was notably increased in the liver adenoma tissue of wild-type mice, which may suggest the involvement of Met in DEHP-induced tumorigenesis in wild-type mice.

Fig. 1.

mRNA levels of cyclin B2, Gadd45a, Mcl1, Apaf1 and Bcl2l1 in wild-type mice livers with adenoma. mRNA levels of GAPDH mRNA, cyclin B2, Gadd45a, Mcl1, Apaf1 and Bcl2l1 were measured by real-time quantitative PCR method in normal livers (n=7) of control and hepatocellular adenoma tissues (n=3) of wild-type or Pparα-null mice exposed to 0, 0.01 or 0.05% DEHP. mRNA levels of each gene were normalized to those of GAPDH mRNA, and were expressed as an n-fold differences. Open and closed rectangles, normal liver and hepatocellular adenoma tissues of wild-type mice exposed to 0 and DEHP, respectively; open and closed circles, normal liver and hepatocellular adenoma tissues of Pparα-null mice exposed to 0 and DEHP. *Significant difference between normal and tumor tissues, p<0.05. **Significant difference between normal and tumor tissues, p<0.01.

Fig. 2.

G2/M arrest regulated by Gadd45. Gadd45 protein interacts with Cdc2-cyclin B complexes and promotes G2/M arrest. Since Gadd45 in the hepatocellular adenoma tissues of wild-type mice was induced by DEHP exposure, but not in those of Pparα-null mice, the promotion of the arrest might not have occurred in the Pparα-null mice, but may have been in the wild-type mice.

Fig. 3.

Apoptosis pathway diagram via caspase 3. Since expression of Mcl1 was increased only in the hepatocellular adenoma tissues of Pparα-null mice exposed to DEHP, while expression of Apaf1 was induced only in those of wild-type mice, apoptosis via caspase 3 might be inhibited in the Pparα-null mice, but not in the wild-type mice.