The importance of preclinical models in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an adenocarcinoma of the hepatobiliary system with a grim prognosis. Incidence is rising globally and surgery is currently the only curative treatment, but is only available for patients who are fit and diagnosed in an early-stage of disease progression. Great importance has been placed on developing preclinical models to help further our understanding of CCA and potential treatments to improve therapeutic outcomes. Preclinical models of varying complexity and cost have been established, ranging from more simplistic in vitro 2D CCA cell lines in culture, to more complex in vivo genetically engineered mouse models. Currently there is no single model that faithfully recaptures the complexities of human CCA and the in vivo tumour microenvironment. Instead a multi-model approach should be used when designing preclinical trials to study CCA and potential therapies.

Activation of hepatic Nrf2 in vivo by acetaminophen in CD-1 mice

The transcription factor NF-E2-related factor 2 (Nrf2) plays an essential role in the mammalian response to chemical and oxidative stress through induction of hepatic phase II detoxification enzymes and regulation of glutathione (GSH). Enhanced liver damage in Nrf2-deficient mice treated with acetaminophen suggests a critical role for Nrf2; however, direct evidence for Nrf2 activation following acetaminophen exposure was previously lacking. We show that acetaminophen can initiate nuclear translocation of Nrf2 in vivo, with maximum levels reached after 1 hour, in a dose dependent manner, at doses below those causing overt liver damage. Furthermore, Nrf2 was shown to be functionally active, as assessed by the induction of epoxide hydrolase, heme oxygenase-1, and glutamate cysteine ligase gene expression. Increased nuclear Nrf2 was found to be associated with depletion of hepatic GSH. Activation of Nrf2 is considered to involve dissociation from a cytoplasmic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), through a redox-sensitive mechanism involving either GSH depletion or direct chemical interaction through Michael addition. To investigate acetaminophen-induced Nrf2 activation we compared the actions of 2 other GSH depleters, diethyl maleate (DEM) and buthionine sulphoximine (BSO), only 1 of which (DEM) can function as a Michael acceptor. For each compound, greater than 60% depletion of GSH was achieved; however, in the case of BSO, this depletion did not cause nuclear translocation of Nrf2. In conclusion, GSH depletion alone is insufficient for Nrf2 activation: a more direct interaction is required, possibly involving chemical modification of Nrf2 or Keap1, which is facilitated by the prior loss of GSH.