# Aflatoxin Mixture-Driven Intrahepatic Cholangiocarcinoma in Rats Involving G1/S Checkpoint Dysregulation

## TL;DR

Exposure to an aflatoxin mixture in rats led to intrahepatic cholangiocarcinoma with disrupted cell cycle control and high proliferation.

## Contribution

This study demonstrates aflatoxin-induced iCCA in rats involving G1/S checkpoint dysregulation and p53 accumulation.

## Key findings

- iCCA tumors showed high cell proliferation (PCNA ≈ 69%) and p53 nuclear accumulation in 83% of cases.
- G1/S checkpoint disruption was marked by cyclin D1 overexpression (67%) and Rb positivity in 58% of tumors.
- Aberrant Wnt activation was rare, with nuclear β-catenin in only 8% of tumors.

## Abstract

Aflatoxins (AFs) are potent hepatotropic mycotoxins—AFB1 being the best-characterized—yet their ability to induce intrahepatic cholangiocarcinoma (iCCA) remains underexplored. Male Wistar rats received vehicle (controls; n = 5) or an AFB1-dominant AF mixture (AFB1 39.46 μg/mL; AFB2 1.13 μg/mL; AFG1 17.44 μg/mL; AFG2 0.59 μg/mL— n = 10) by daily gavage for 90 days, at a dose equivalent to 400 μg AFB1 per kg of diet. After 12 months, twelve iCCA tumors were resected and analyzed by histology (H&E) and tissue-microarray-based immunohistochemistry (Cytokeratin-19, Hep Par-1, p53, Cyclin D1, Rb, β-catenin, and PCNA). Lesions predominantly showed glandular/tubular architecture consistent with iCCA and were cytokeratin-19-positive and Hep Par-1-negative. Cell proliferation was high (PCNA ≈ 69%). p53 displayed nuclear accumulation in 83% of tumors. G1/S control was perturbed, with cyclin D1 overexpression (67%), and Rb was positive in 58% of iCCA. Aberrant Wnt activation was rare (nuclear β-catenin in 8%). Subchronic exposure to an AFB1-dominant AF mixture in rats was associated with iCCA characterized by high proliferative activity, p53 accumulation, and disruption of G1/S checkpoint components. These findings broaden the oncogenic spectrum of AFs and warrant genomic confirmation of AF mutational signatures.