Innovative Journal of Medical and Health Sciences

Purpose: Hepatitis B virus (HBV) infection causes epigenetic changes through DNA hypermethylation. HBV integrates into the human genome thereby facilitating viral persistence. Infected hepatocytes express DNA methyltransferases that hypermethylate and inactivate integrated HBV genome inserts. This defence mechanism will methylate adjacent host DNA; this may result in the methylation of tumour suppressor genes and cell-cycle regulators leading to hepatocyte injury and malignancy. The aim of this study was to investigate for the presence of genome-wide promoter region cytosine-phosphate-guanine islands hypermethylation in HBV infected patients.

Materials and methods: Affymetrix Human Promoter 1.0R microarray chip was used for microarray analysis of DNA obtained from liver tissue of 17 HBV-infected patients, 2 normal liver histopathology controls, 7 hepatocellular carcinoma (HCC), 2 from liver tissue surrounding HCC lesions and 4 from patients with autoimmune hepatitis. Microarray data was analysed using Partek Genomic Suite focusing on the core promoter region within 100bp from transcription start site. Partek Pathway was used for biological pathway analysis. The discovered hypermethylation patterns were validated using bisulfite DNA sequencing in an additional set of 59 samples.

Results: HBV infected patients had significant hypermethylation compared to other study groups. Genes controlling hepatic inflammation, tissue regeneration and carcinogenesis were significantly affected (P<0.01). Age over 40 years, cirrhosis, hepatic inflammation and HBV e antigen status correlated with hypermethylation (P<0.01). On bisulfite DNA sequencing, Cyclin D3gene promoter was particularly highly methylated. This was associated with HBV e antigen positive status, high viral load and genotype D (P=0.01).

Conclusions: HBV-induced epigenetic changes were widespread, affecting genes controlling cell growth, differentiation, proliferation and apoptosis. These changes may explain the development of hepatic inflammation, fibrosis and malignancy observed in patients with chronic HBV infection.

A dose-response study has been undertaken to clarify our proposed mechanistic axiom relating manifest UV-induced nephrotoxicity to protective effects of gradated T supplementation. Study was for 20days (d) at exposure rate of 50min/d for 5d in 50 acclimatized pre-pubertal, unsexed rabbits (PR) randomly assigned to 5 groups of 10PR each, fed concentrate feed and forage (Tridax procumbens) – basal diet (BD) and BD supplemented with 0% (Control), 2%, 4%, 6% and 12% pulverized crude T respectively. Feed and water were available ad libitum. Blood was collected by marginal venopuncture on 21d from 0900h for assessment of renal nephrotoxicity indexed as changes in plasma/serum concentrations of urea and creatinine (CR) determined by standard titrimetric/colorimetric methods. Data were analyzed by ANOVA.  Dose dependent increases in secretion of both indices were observed. Highest amount of secreted urea occurred in rabbits fed 2% T followed by 4% T and were significantly (p<0.05) higher than control and 6% T rabbits. At 12% T amount of measured urea dropped very significantly (p<0.05) below control value with a reversal to negative value. Similar increasing trend occurred in CR secretion, exhibiting initial non-significant (p>0.05) splay at 2% T followed by continuous significant increases (p<0.05). Unlike urea, highest CR secretion occurred at 6% T and without a reversal to negative value. These results strongly demonstrated potent dose dependent nephroprotection by gradated T supplementation.