Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines

AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7 B mutations in cell culture. METHODS: The most common Wilson disease(WD) mutations p.H1069 Q, p.R778 L and p.C271*, found in the ATP7 B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7 B was used to stably express WD mutants. m RNA and protein expression of mutant ATP7 B, survival of cells, apoptosis, and protein trafficking were determined.RESULTS: Low temperature increased ATP7 B protein expression in several mutants. Intracellular ATP7 B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069 Q and to a lesser extent p.C271* improved by D-penicillamine(DPA) treatment, while mutant p.R778 L showed a pronounced response to zinc(Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability. CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines.

Dietary copper triggers onset of fulminant hepatitis in the Long-Evans cinnamon rat model

AIM： To investigate the impact of dietary copper given at different time points on the onset of fulminant hepatitis. METHODS： The Long-Evans cinnamon （LEC） rat mod- el of Wilson＇s disease （WD） was used to study the im- pact of high dietary copper （hCu） on the induction of fulminant hepatitis at early or late time points of life. High Cu diet was started in rat pups or in adults （month 5） for three months. Animals that received reduced di- etary copper （rCu） throughout their lifetime served as a control. Hepatitis-associated serum markers （alanine aminotransferase, aspartate transaminase, bilirubin） were analyzed in animal groups receiving hCu or rCu. Liver copper content and liver histology were revealed at sacrifice. A set of 5 marker genes previously found to be affected in injured liver and which are related to angiogenesis （Vegfa）, fat metabolism （Srebf1）, ex- tracellular matrix （Timp1）, oxidative stress （Hmox1）, and the cell cycle （Cdknla） were analyzed by real-time polymerase chain reaction. RESULTS： Regardless of the time point when hCu was started, LEC rats （35/36） developed fulminant hepati- tis and died. Animals receiving rCu （36/36） remained healthy, did not develop hepatitis, and survived long term without symptoms of overt disease, although liver copper accumulated in adult animals （477 ± 75 μg/g）. With regard to start of hCu, onset of fulminant hepatitis was significantly （P 〈 0.001） earlier in adults （35±9 d） that showed pre-accumulation of liver copper as com- pared to the pup group （77±15 d）. Hepatitis-associ- ated serum markers, liver copper and liver histology, as well as gene expression, were affected in LEC rats re- ceiving hCu. However, except for early and rapid onset of hepatitis, biochemical and molecular markers were similar at the early and late time points of disease. CONCLUSION： Rapid onset of fulminant hepatitis in asymptomatic LEC rats with elevated liver copper sug- gests that there is a critical threshold of liver copper which is important to trigger the course of WD.