ATT-Myc Transgenic Mouse Model and Gene Expression Identify Genotoxic and Non-Genotoxic Chemicals That Accelerating Liver Tumor Growth in Short-Term Toxicity

Abstract

Introduction: Diethyl nitrosamine (DEN), a known carcinogen, has been used for validating the RasH2 and P53 transgenic models in chemical testing and has been shown to enhance primary liver tumor growth in the ATT-Myc transgenic mouse model of liver cancer. Material and Methods: to better understand the mechanism of hepatocellular carcinoma acceleration following DEN, BHT and vehicles treatments in ATT-Myc, transgenic and non-transgenic, mice. We employed an exon array, RT-PCR, Western blotting, and IHC to investigate the complex interplay between the c-Myc transgene and other growth factors in treated mice versus control transgenic and non-transgenic mice. Results: Notably, DEN treatment induced a 12-fold increase in c-Myc expression compared to non-transgenic mice. Furthermore, tumor growth in the DEN group was strongly associated with increased proliferation of transformed or carcinogenic hepatocytes, as evidenced by proliferative cell nuclear antigen and bromodeoxyuridine expression. Internally, the loss of c-Met signaling, enriched transcription factors, and the diminished expression of antioxidants, such as superoxide dismutase (SOD1) and NRF2, further enhanced c-Myc-induced liver tumor growth as early as four months post-DEN treatment. Discussion: Extensive tumor growth was observed at 8.5 months, coinciding with the downregulation of tumor suppressors such as p53. In contrast, at these time points, ATT-Myc transgenic mice exhibited only dysplastic hepatocytes without tumor formation. Additionally, the antioxidant butylated hydroxytoluene maintained c-Met expression and did not promote liver tumor formation. Conclusions: the persistent upregulation of c-Myc in the ATT-Myc liver cancer model, at both the gene and protein levels following DEN treatment inhibited the ETS1 transcription factor, further exacerbating the decline of c-Met signaling, SOD1, and NRF2. These changes led to increased reactive oxygen species production and promoted rapid liver tumor growth.

Keywords: ATT-Myc transgenic mouse model of liver cancer; DEN; HCC; NRF2; SOD1; c-Met/HGF signaling.

Figure 1

(A1,A2) ATT-Myc transgene construction and the positive band for transgenic ATT-Myc transgenes were confirmed by PCR tail DNA testing. (B1) The increase in liver weight to body weight ratio in DEN-treated transgenic mice at 8.5 months was due to massive liver tumor growth, as shown in the corresponding gross images. Moreover, these images illustrate a large liver in DEN-treated transgenic mice, small nodular structures in BHT-treated transgenic animals, and normal liver architecture in both transgenic and non-transgenic controls (B2). (C1) Histopathological analysis revealed that the normal liver parenchyma in non-transgenic mice remained unaltered. (C2) In BHT-treated transgenic animals, macrocytic dysplastic nodules were observed. (C3) illustrates liver cirrhosis and nodular regeneration in DEN-treated transgenic mice, large trabecular HCC, and pseudoglandular HCC (C4). Necrosis (C5) and fat changes (C6) were also detected. * p < 0.05.

Figure 2

(A) shows HCA and PCA of gene expression changes in the livers of AAT-Myc transgenic mice induced by BHT and DEN at 8.5 months compared to transgenic control mice treated with a vehicle. FCs of 666 SRGs were analyzed. (B) presents the HCA and PCA of gene expression changes in the livers of AAT-Myc transgenic mice induced by BHT and DEN at 8.5 months compared to non-transgenic control mice treated with a vehicle, where FCs of 533 SRGs were analyzed. (C) illustrates PCA of the whole dataset of gene expression changes in the livers of AAT-Myc transgenic mice induced by BHT and DEN at 8.5 months compared to transgenic control mice treated with vehicles, with FCs of 666 SRGs included. The figure also depicts the volume differential (VD) analysis of genes significantly regulated in the liver of AAT-Myc transgenic mice treated with BHT and DEN versus transgenic control mice treated with vehicle (NaCl) at 8.5 months.

Figure 3

Group (A) represents most of the strongly varying gene expressions in the GOMolFn classification groups. Group (B) of the GOProcess classification exhibits the most significant gene expression. The GOCellLoc classification’s group with significant gene expression is represented in (C), while group (D) shows significant gene expression within the Pathway classification group.

Figure 4

RT-PCR products were analyzed using gel electrophoresis and visualized using Kodak Image Station 440CF under UV light to validate exon array expression. All samples were normalized to β-actin levels. The densometrical scan of the gene expression analysis showed elevated levels of RPL23, RFC4, MMP12, and BZWZ after treatment with DEN in comparison to the non-transgenic control. In contrast, C9 displayed a decrease, while no significant variations were detected in DYNL1, SLC10A, GAS6, or the housekeeping gene β-actin. * p < 0.05.

Figure 5

(A) This shows the loss of c-Met and its transcriptional factor ETS1 in liver tumors after DEN treatment at 8.5 months. (B) Loss of c-Met is time-dependent in liver tumors induced by DEN at different time points, while BHT maintains c-Met expression. additionally, Liver protein extracts from DEN-treated transgenic mice showing increased expressions of c-Myc, TGFα, PCNA, and CDK4 contributing to accelerated tumor growth. (C) DEN treatment enhances the loss of antioxidants, such as SOD1, NRF2, Neuropilin-1 (NP-1), and PPARγ, compared to control non-transgenic animals at 8.5 months. (D) DEN treatment reduces enriched transcriptional factors, such as HNF4α and HNFγ, while increasing the expression of C/EBPα and CD44 compared to control transgenic and non-transgenic mice.

Figure 6

Immunohistochemistry staining demonstrated increased c-Myc expression in transgenic mice (A), while the distribution of c-Met expression was uneven (B). The unstained control group provided a reference for comparison (C). Increased PCNA expression was noted in DEN-treated mice (D) compared to the BHT-treated group (E), along with increased BrdU in the BHT-treated group (F). (G) shows control unstained sections.