Nathan Subramaniam

A novel mouse model of veno-occlusive disease provides strategies to prevent thioguanine-induced hepatic toxicity

Objective The anti-leukemic drugs, azathioprine and 6-mercaptopurine (6MP), are important in the treatment of inflammatory bowel disease but an alternative faster-acting, less-allergenic thiopurine, 6-thioguanine (6TG), can cause hepatic veno-occlusive disease/sinusoidal obstructive syndrome (SOS). Understanding of SOS has been hindered by inability to ethically perform serial liver biopsies on patients and the lack of an animal model. Design Normal and C57Bl/6 mice with specific genes altered to elucidate mechanisms responsible for 6TG-SOS, were gavaged daily for upto 28d with 6TG, 6MP or methylated metabolites. Animal survival was monitored and at sacrifice a histological score of SOS, haematology and liver biochemistry were measured. Results Only 6TG caused SOS, which was dose related. 6TG and to a lesser extent 6MP but not methylated metabolites were associated with dose-dependent haematopoietic toxicity. SOS was not detected with non-lethal doses of 6TG. SOS did not occur in hypoxanthine-phosphoribosyl transferase-deficient C57Bl/6 mice, demonstrating that 6TG-SOS requires thioguanine nucleotides. Hepatic inflammation was characteristic of SOS, and C57Bl/6 mice deficient in P- and E-selectins on the surface of vascular endothelial cells showed markedly reduced SOS, demonstrating a major role for leukocytes recruited from blood. Split dosing of 6TG markedly attenuated SOS but still effected immunosuppression and prevented spontaneous colitis in Winnie mice, which have a single nucleotide polymorphism mutation in Muc2. Conclusion This novel model provides clinically relevant insights into how 6TG induces SOS, and how this dangerous adverse drug reaction may be avoided by either inhibition of endothelial activation or simple changes to dosing regimens of 6TG, while still being effective treatment for colitis.

Heterozygous Deletion of the Hfe Gene Contributes to Hepatic Iron Loading But Not Steatosis in Mice Fed a High Fat Diet

Introduction: The liver is central to the metabolism of both iron and cholesterol. Cholesterol is synthesised and further metabolised to bile acids in the liver and the liver plays an important role in regulation of iron metabolism. It is also the organ in which excess iron is stored. Clinically, links have been noted between lipid and iron metabolism, with approximately one - third of patients with non - alcoholic fatty liver disease exhibiting altered iron parameters. On a molecular level, we have previously reported that wild - type mice fed iron - deficient, normal or iron - loaded diets exhibited increased hepatic cholesterol and increased hepatic gene expression of enzymes in the cholesterol biosynthesis pathway with increasing hepatic iron burden. In the genetic disorder, haemochromatosis, the liver can become overloaded with iron; however, clinical studies have suggested that lipid metabolism may not be perturbed in haemochromatosis. Methods and Materials: We investigated hepatic cholesterol metabolis m in three mouse models of hereditary haemochromatosis: Hfe - / - , Tfr2 Y245X single mutant and Hfe - / - x Tfr2 Y245X double mutant animals as well as wild - type controls. Mice were fed normal mouse chow and sacrificed at 10 weeks of age. Hepatic gene expression, total cholesterol and non – haem iron were measured. Liver non - haem iron was similar in Hfe - / - and Tfr2 Y245X mice (16.6±0.8 and 17±1 μmol Fe /g liver, respectively) and significantly higher in the double mutant animals (22.4±0.7 μmol Fe /g liver ; P<0.004) than either of the single mutant mice. Results: Only one group of genes increased significantly with increasing hepatic iron: those involved in cholesterol transport. Gene expression of apolipoproteins A4, C1, C2, C3 and E increased significantly with increasing hepatic iron as did expression of VLDL receptor. In contrast to our findings in wild - type mice, gene expression of cholesterol biosynthetic enzymes did not increase significantly with liver iron burden and there were no differences in hepatic cholesterol between the groups of mutant mice. We also measured expression of genes involved in cholesterol regulation, which similarly, did not increase with increasing hepatic iron. Approximately 50% of cholesterol synthesised in the liver is directed to bile acid synthesis; however, gene expression of bile acid pathway enzymes did not change with respect to hepatic iron burden. Conclusion: These results suggest that iron - associated cholesterol regulation may be ameliorated by the genetic changes which occur in haemochromatosis.Poster presented at Fifth Congress of the International BioIron Society that took place in University College London (London, United Kingdom) during 14-18th April 2013.