Obesity as a cause of hepatocellular carcinoma.

During recent years the incidence of obesity has increased significantly, and in some instances rapidly, in many resource-rich countries. Paralleling this increase has been an increase in the incidence of hepatocellular carcinoma. It has been estimated that as many as 90% of obese adults will develop the metabolic syndrome. The worldwide incidence of this syndrome in adults at this time ranges from 9 to 34%. Furthermore, obesity in childhood increases the risk of obesity in adulthood, and hence the development of the metabolic syndrome and hepatocellular carcinoma. Ten to 20% of patients with non-alcoholic fatty liver disease progress to non-alcoholic steatohepatitis, and 8.3% of the latter develop cirrhosis. Up to 50% of these patients with cirrhosis, and a significant proportion of those without cirrhosis, progress to hepatocellular carcinoma. Much remains to be learnt about the mechanisms by which obesity and the metabolic syndrome cause hepatocellular carcinoma, although insulin resistance, increased tissue necrosis factor activity, alterations in serum lipids, non-alcoholic fatty liver disease and non-alcoholic steatosis play important roles. There is also increasing evidence that gut microbiota play a role in the development of the metabolic syndrome and hence of hepatocellular carcinoma.

Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus-associated hepatocellular carcinoma.

BACKGROUND: The molecular mechanisms whereby hepatitis B virus (HBV) induces hepatocellular carcinoma (HCC) remain elusive. We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC. METHODS: We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes along with the intrahepatic expression of HBV. Gene expression profiling was performed on up to 17 WLT specimens obtained at various distances from the tumor center from individual livers of 11 patients with HCC and on selected LCM samples. HBV markers in liver and serum were determined by real-time polymerase chain reaction (PCR) and confocal immunofluorescence. RESULTS: Analysis of 5 areas of the liver showed a sharp change in gene expression between the immediate perilesional area and tumor periphery that correlated with a significant decrease in the intrahepatic expression of HB surface antigen (HBsAg). The tumor was characterized by a large preponderance of down-regulated genes, mostly involved in the metabolism of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of PXR/RXR and PPARα/RXRα nuclear receptors, comprising PGC-1α and FOXO1, two key regulators critically involved not only in the metabolic functions of the liver but also in the life cycle of HBV, acting as essential transcription factors for viral gene expression. These findings were confirmed by gene expression of microdissected hepatocytes. Moreover, LCM of malignant hepatocytes also revealed up-regulation of unique genes associated with cancer and signaling pathways, including two novel HCC-associated cancer testis antigen genes, NUF2 and TTK. CONCLUSIONS: Integrated gene expression profiling of whole liver tissue with that of microdissected hepatocytes demonstrated that HBV-associated HCC is characterized by a metabolism switch-off and by a significant reduction in HBsAg. LCM proved to be a critical tool to validate gene signatures associated with HCC and to identify genes that may play a role in hepatocarcinogenesis, opening new perspectives for the discovery of novel diagnostic markers and therapeutic targets.

Occult hepatitis B virus infection in chacma baboons, South Africa.

During previous studies of susceptibility to hepatitis B virus (HBV) infection, HBV DNA was detected in 2/6 wild-caught baboons. In the present study, HBV DNA was amplified from 15/69 wild-caught baboons. All animals were negative for HBV surface antigen and antibody against HBV core antigen. Liver tissue from 1 baboon was immunohistochemically negative for HBV surface antigen but positive for HBV core antigen. The complete HBV genome of an isolate from this liver clustered with subgenotype A2. Reverse transcription PCR of liver RNA amplified virus precore and surface protein genes, indicating replication of virus in baboon liver tissue. Four experimentally naive baboons were injected with serum from HBV DNA-positive baboons. These 4 baboons showed transient seroconversion, and HBV DNA was amplified from serum at various times after infection. The presence of HBV DNA at relatively low levels and in the absence of serologic markers in the baboon, a nonhuman primate, indicates an occult infection.

Epidemiology of hepatocellular carcinoma in sub-Saharan Africa.

Published incidences of hepatocellular carcinoma in the Black population of sub-Saharan Africa underestimate the true incidence of the tumor because of the many instances in which hepatocellular carcinoma is either not definitively diagnosed or is not recorded in a cancer registry. Despite this, it is manifestly evident that the tumor occurs commonly and is a major cause of cancer deaths in Black African peoples living in the sub-continent, particularly in those living in rural areas. 46,000 new cases of hepatocellular carcinoma have been recorded to be diagnosed in sub-Saharan Africa each year, and age-standardized incidences of the tumor as high as 41.2/100,000 persons/year have been documented. The highest incidence of hepatocellular carcinoma has been recorded in Mozambique. The tumor occurs at a young age in rural dwelling and, to a lesser extent, urban dwelling Black Africans. It is also more common in men than women, particularly in the younger patients. Cirrhosis co-exists with hepatocellular carcinoma in about 60% of patients and is equally common in the two sexes. The tumor is not only common in the Black African population, it also carries an especially grave prognosis, with about 93% of the patients dying within 12 months of the onset of symptoms. Caucasians living in the sub-continent have a low incidence of hepatocellular carcinoma and it occurs at an older age.

Molecular characterization of hepatitis B virus isolates from Zimbabwean blood donors.

Hepatitis B virus (HBV) is endemic in Africa, being hyperendemic in sub-Saharan Africa. Genotypes A, D, and E circulate in Africa, showing a distinct geographical distribution. The aim of the present study was to determine the HBV genotype distribution in blood donors from different geographical locations in Zimbabwe. Using a restriction fragment polymorphism assay, sequencing of the basic core promoter/precore region and of the complete S open reading frame showed that 29 HBV isolates from geographically distinct regions belong to subgenotype A1. The complete genome of two of these Zimbabwean HBV isolates was sequenced. Forty-four percent of the Zimbabwean HBV isolates (11/23) were characterized by a G1862C missense mutation, which causes a Val to Leu amino acid substitution at position 17 of the precore region. The majority of Zimbabwean HBV isolates clustered with a number of South African HBV isolates, with which they shared characteristic amino acids in the preS1, preS2, and polymerase spacer regions. The wide distribution of subgenotype in Africa, as well as the high intragroup divergence and the geographical clustering of the African and Asian subgenotype A1 HBV isolates indicate that this subgenotype has a long period of endemicity in these regions.

Hepatitis B virus x protein in the pathogenesis of hepatitis B virus-induced hepatocellular carcinoma.

Currently available evidence supports a role for the hepatitis B virus (HBV) x gene and protein in the pathogenesis of HBV-induced hepatocellular carcinoma (HCC). HBx gene is often included, and remains functionally active, in the HBV DNA that is frequently integrated into cellular DNA during hepatocellular carcinogenesis. HBx protein promotes cell cycle progression, inactivates negative growth regulators, and binds to and inhibits the expression of p53 tumour suppressor gene and other tumour suppressor genes and senescence-related factors. However, the molecular mechanisms responsible for HBx protein-induced HCC remain uncertain. Only some of the more fully documented or more recently recognised mechanisms are reviewed. During recent years evidence has accumulated that HBx protein modulates transcription of methyl transferases, causing regional hypermethylation of DNA that results in silencing of tumour suppressor genes, or global hypomethylation that results in chromosomal instability, thereby playing a role in hepatocarcinogenesis. HBx protein has both anti-apoptotic and pro-apoptotic actions, apparently contradictory effects that have yet to be explained. Particularly important among the anti-apoptotic properties is inhibition of p53. Recent experimental observations suggest that HBx protein may increase the expression of TERT and telomerase activity, prolonging the life-span of hepatocytes and contributing to malignant transformation. The protein also interferes with nucleotide excision repair through both p53-dependent and p53- independent mechanisms. Carboxy-terminal truncated HBx protein loses its inhibitory effects on cell proliferation and pro-apoptotic properties, and it may enhance the protein's ability to transform oncogenes. Dysregulation of IGF-II enhances proliferation and anti-apoptotic effects of oncogenes, resulting in uncontrolled cell growth.

Prevention of hepatocellular carcinoma.

Because of its frequency and grave prognosis, preventing hepatocellular carcinoma is an urgent priority. Prevention should be possible because environmental carcinogens-chronic hepatitis B and C virus infections, dietary exposure to aflatoxins, and iron overload-cause the great majority of these tumors. Chronic hepatitis B virus infection accounts for 55% of global hepatocellular carcinomas and 80% of those in the high-incidence Asia Pacific and sub-Saharan African regions. In these regions the infection that becomes chronic is predominantly acquired very early in life. A safe and effective vaccine against this virus is available and its universal inclusion in the immunization of infants has already resulted in a marked reduction of chronic infection and a 70% decrease in the occurrence of hepatocellular carcinoma in those immunized. Chronic hepatitis C virus infection is the major cause of hepatocellular carcinoma in industrialized countries. The infection is mainly acquired in adulthood and, until a vaccine becomes available, prevention will consist mainly of identifying, counselling, and treating chronically infected individuals, preventing spread of the virus by the use of safe injection practices (particularly in intravenous drug abusers), and screening all donated blood for the presence of the virus. 4.5 billion of the world.s population are exposed to dietary aflatoxins. Prevention involves treating susceptible crops to prevent fungal contamination, and handling the foodstuffs in such a way as to prevent contamination during storage. Iron overload in hereditary hemochromatosis can be prevented by repeated venesection and in African dietary iron overload by fermenting the home-brewed beer in iron-free containers.

Possible adverse effect of high delta-alpha-tocopherol intake on hepatic iron overload: enhanced production of vitamin C and the genotoxin, 8-hydroxy-2'- deoxyguanosine.

Excess hepatic iron generates reactive oxygen species that result in oxidative stress and oxidative damage to the liver. Vitamins have hitherto been considered to be a possible remedy. The aim of this study was to determine if high doses of delta-alpha-tocopherol supplementation in iron overload would ameliorate the oxidative stress. Four groups of 20 male Wistar albino rats were studied: group 1 (control) was fed normal diet, group 2 (Fe) 0.75% Ferrocene iron, group 3 (FV gp) 0.75% Ferrocene/delta-alpha-tocopherol (10x RDA), group 4 (V gp) normal diet/delta-alpha-tocopherol. After 12 months, serum iron, reduced glutathione, catalase, vitamin C, Oxygen Radical Absorbance Capacity, lipid peroxidation, 8-hydroxy-2'-deoxyguanosine (8-OHdG), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. Vitamin C levels were: F gp = 5.04 +/- 0.09; FV gp = 5.85 +/- 0.13 (micromol/l) (p < 0.05). 8-hydroxy-2'-deoxyguanosine levels were: F gp = 143.6 +/- 6.4; FV gp = 179.2 +/- 18.2 (ng/ml) (p < 0.05). Oxidative liver damage, as determined by serum AST and ALT levels, was not attenuated by alpha-tocopherol. A positive correlation existed between vitamin C and 8-OHdG, suggesting possible delta-alpha-tocopherol toxicity.

Increased expression of ErbB-2 in liver is associated with hepatitis B x antigen and shorter survival in patients with liver cancer.

Hepatitis B x antigen, or HBxAg, contributes importantly to the pathogenesis of hepatocellular carcinoma (HCC). Given that HBxAg constitutively activates beta-catenin and that upregulated ErbB-2 promotes beta-catenin signaling in other tumor types, experiments were designed to ask whether HBxAg was associated with upregulated expression of ErbB-2. When HBxAg positive and negative HepG2 cells were subjected to proteomics analysis, ErbB-2 was shown to be upregulated in HepG2X but not control cells. ErbB-2 was also strongly upregulated in HB infected liver, and weakly in some HCC nodules, where it correlated with HBxAg expression. Among tumor bearing patients, strong ErbB-2 staining in the liver was associated with dysplasia, and a shorter survival after tumor diagnosis. This implies that elevated ErbB-2 is an early marker of HCC. Treatment of HepG2X cells with ErbB-2 specific siRNA not only reduced ErbB-2 expression, but also reduced the expression of beta-catenin, suggesting that ErbB-2 contributed to the stabilization of beta-catenin. ErbB-2 specific siRNA also partially blocked the ability of HBxAg to promote DNA synthesis and growth of HepG2 cells. These results suggest that ErbB-2/beta-catenin up-regulation contributes importantly to the mechanism of HBxAg mediated hepatocellular growth.

Hepatitis B viral loads in southern African Blacks with hepatocellular carcinoma.

UNLABELLED: Although viral loads are known to influence the development of hepatitis B virus-induced hepatocellular carcinoma in a number of populations, little information is available in the Black African population. Black African patients with hepatocellular carcinoma differ from those in other populations in having a lower frequency of e antigen-positivity and in other respects that might affect viral loads. Hepatitis B viral loads were measured using real-time polymerase chain reaction assay in 124 Black Africans with hepatocellular carcinoma and compared with those in 125 Black adult asymptomatic viral carriers. The geometric mean viral load in the cancer patients was 553,618 copies/ml (95% CI 301,953-1,015,033 copies/ml), with 62.1% having loads >1 x 10(5) copies/ml and 87.1% >1 x 10(4) copies/ml, whereas that in the carriers was 16,084 copies/ml (95% CI 9,184-28,168 copies/ml), with only 15.2% having values >1 x 10(5) copies/ml and 49.6% >1 x 10(4) copies/ml (P < 0.001 in each instance). Mean viral load was significantly higher in e antigen-positive than e antigen-negative cancer patients (5,905,357 copies/ml [1,362,847-25,588,520] cf 238,173 copies/ml [97,200-685,730]: P < 0.001) after adjusting for age and sex. No statistically significant difference existed between patients in different age groups, in men and women, or in patients infected with genotype A or D after adjusting for the other variables. CONCLUSION: Black Africans with hepatocellular carcinoma have high hepatitis B viral loads in spite of the relative infrequency of e antigen-positivity.

First report of genotype e of hepatitis B virus in an Indian population.

Twenty-one hepatitis B virus (HBV) isolates from the state of Haryana (North India) were studied for genotype, subgenotype, serotype distribution and precore mutations. Assays of alanine aminotransminase (ALT) and HBeAg were performed on all samples. Genotypes, subgenotypes and serotypes were determined by amplification of pre-S1/S2 regions followed by RFLP and also by phylogenetic analysis of amplified products. Mutations were studied by amplification and sequencing of the precore region. Twenty-four percent of the samples had high ALT levels and 90% were HBeAg negative. It was observed that 90% of the samples were HBV D genotype, (subgenotype D1, serotype ayw2), 5% HBV A genotype (subgenotype A1, serotype adw2), and the remaining 5% were HBV E genotype (serotype ayw4). The subgenotype A1 was quite similar to the South African isolates. Phylogenetic analysis of the HBV isolates, based on the pre-S1/S2 gene sequences, confirmed genotype E. Amplification and sequencing of the precore region showed 1762(A-T) and 1764(G-A) mutations in 38 and 15% of the samples, respectively. 1809(T) was observed in 5% of the cases under study. This is the first report of the genotype E of hepatitis B virus in the Indian population. Efforts are underway to amplify and sequence the full length of this genotype E isolate.

Effects of exogenous antioxidants on dietary iron overload.

In dietary iron overload, excess hepatic iron promotes liver damage. The aim was to attenuate free radical-induced liver damage using vitamins. Four groups of 60 Wistar rats were studied: group 1 (control) was fed normal diet, group 2 (Fe) 2.5% pentacarbonyl iron (CI) followed by 0.5% Ferrocene, group 3 (Fe + V gp) CI, Ferrocene, plus vitamins A and E (42x and 10x RDA, respectively), group 4 (Fe - V gp) CI, Ferrocene diet, minus vitamins A and E. At 20 months, glutathione peroxidase (GPx), superoxide dismutase (SOD), Oxygen Radical Absorbance Capacity (ORAC), Ames mutagenicity test, AST, ALT and 4-hydroxynonenal (4-HNE) immunohistochemistry were measured. 8OHdG levels of the Fe + V and Fe - V groups were 346 +/- 117 and 455 +/- 151, ng/g w.wt, respectively. Fe + V and Fe - V differences were significant (p<0.005). A positive correlation between DNA damage and mutagenesis existed (p<0.005) within the iron-fed gps. AST levels for Fe + V and Fe - V groups were 134.6 +/- 48.6 IU and 202.2 +/- 50.5 IU, respectively. Similarly, ALT levels were 234.6 +/- 48.3 IU and 329.0 +/- 48.6 IU, respectively. However, Fe - V and Fe + V groups transaminases were statistically insignificant. 4-HNE was detected in Fe + V and Fe - V gp livers. Vitamins A and E could not prevent hepatic damage.

Synergistic interaction between excess hepatic iron and alcohol ingestion in hepatic mutagenesis.

BACKGROUND/AIM: Hereditary hemochromatosis (HH) and dietary iron overload are the main iron-loading diseases. Fibrosis, cirrhosis and hepatocellular carcinoma (HCC) are complications to HH and dietary iron overload possibly influenced by co-factors. Alcohol may be one such factor. The aim therefore was to determine the extent of synergistic interaction between free hepatic iron and alcohol, complicating dietary iron overload in HCC pathogenesis. METHODS: Four groups of 20 Wistar albino rats were used: group 1 (C) was fed the chow diet; group 2 (Fe) was supplemented with 0.75% ferrocene iron; group 3 (Fe+Al), 0.75% iron and 7% ethanol; and group 4, 7% ethanol (Al) for 12 months. Iron profile, superoxide/nitrite free radicals, lipid peroxidation (LPO)/8-isoprostane (8-IP), 8-hydroxydeoxyguanosine (8-OHdG), oxidative lipid/DNA damage immunohistochemistry, transaminases (AST/ALT) and Ames mutagenesis tests were performed. RESULTS: Significant differences were observed in the Fe+Al group for LPO, 8-IP, AST and ALT (p<0.001, 0.001, 0.001 and 0.001, respectively) compared to other groups. A three-fold synergistic interaction was observed for the same parameters. Furthermore, significant differences of p<0.05 and 0.001 were observed for 8-OHdG and mutagenesis, respectively, with an additive synergy in the Fe+Al group. ALT/8-OHdG and ALT/mutagenesis correlated positively (p<0.04 and 0.008, respectively). The immunohistochemistry revealed iron/alcohol multiplicative synergism with hydroxyl radical involvement. CONCLUSION: Mutagenic effects of iron and alcohol are synergistically multiplicative implicating hydroxyl free radicals in hepatocarcingenesis.

A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe-antigen.

AIM: Hepatitis B virus (HBV) e antigen (HBeAg) is translated from precore mRNA as a precore/core protein, which is post-translationally modified to give rise to the protein that is secreted into the serum. The G1862T mutation in HBV occurs in the bulge of the encapsidation signal within the pregenomic RNA. When the precore mRNA is translated, this mutation results in a valine to phenylalanine substitution at the -3 position to the signal peptide cleavage site at the amino end of the precursor protein. The aim of this study was to determine whether this mutation could affect HBV replication and/or HBeAg expression. METHODS: Following transfection of Huh 7 cells, HBV replication was followed using real time polymerase reaction (PCR) and expression of HBeAg expression was monitored using confocal microscopy. RESULTS: HBV replication was reduced when this mutation was introduced into genotype D but not into genotype A replication-competent constructs. Using mutant HBeAg-expressing plasmids, we demonstrated a 54% reduction in HBeAg secretion relative to the wild type. Confocal microscopy demonstrated that the mutant HBeAg accumulated in the endoplasmic reticulum, endoplasmic reticulum intermediate compartment and Golgi. These aggregates of mutant protein increased in size following treatment of the cells with a proteasome inhibitor, MG132, and had the hallmark features of aggresomes. They attracted ubiquitin, heat shock proteins and proteasomes and were isolated from the cytosol by the intermediate filaments, vimentin and cytokeratin. CONCLUSION: The formation of aggresomes, as a result of the G1862T mutation, may play a contributory role in HBV-induced liver disease.

Occult hepatitis B virus infection in Southern African blacks with hepatocellular carcinoma.

BACKGROUND AND AIM: To ascertain the prevalence of occult hepatitis B virus (HBV) infection in southern African blacks with hepatocellular carcinoma. METHODS: Sera from 118 patients negative for HBV surface antigen but positive for HBV antibodies were studied. HBV-DNA was detected using a nested polymerase chain reaction (PCR) assay and confirmed by nucleotide sequencing of the surface and precore/core genes. RESULTS: Surface gene HBV-DNA was detected in a single PCR assay in 48.4% of the patients. Positive results increased to 57.7% after two PCR assays (not significant) and 75.7% after four assays (P < 0.001). No false positive results were obtained in these assays or in the 15 control samples for which PCR assays were performed four times. Significant differences in positivity rates were not observed between patients positive for HBV core antibody alone and those positive for core and surface antibodies. The sensitivity of the PCR amplification of the precore/core gene was significantly less than that of the surface open reading frame: the yield of positive results was 23.7% after one assay, 32.2% after two assays (not significant), and 52% after four assays (P < 0.001). Combining the results of the assays of the two genes increased the yield of positive results for the first assay (by 11.9%, P = 0.015), but not the second (6.1%) or fourth assays (4.6%). CONCLUSION: Occult HBV infection is present in the serum of the majority of hepatocellular carcinomas in southern African blacks whose serum is negative for hepatitis B surface antigen but positive for anti-HBV core antigen. The yield of positive results increases if more than one PCR assay is performed.

Silencing of O6-methylguanine DNA methyltransferase in the absence of promoter hypermethylation in hepatocellular carcinomas from Australia and South Africa.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is involved in cellular defences against alkylating agents. Alterations in the MGMT gene may result in an increase in the mutation rate and risk of malignant transformation. We have previously shown that MGMT is implicated in colorectal carcinogenesis particularly in cancers which display microsatellite instability, a marker of impaired DNA repair. The aims of the current study were to assess the roles of MGMT and microsatellite instability in hepatocellular carcinomas (HCCs) from Australia and South Africa. DNA was extracted from malignant and non-malignant liver tissue from 37 Australian and 24 South African patients, and histologically normal liver from 20 transplant donors. MGMT promoter hypermethylation and MGMT protein expression were assessed using methylation specific PCR and immunohistochemistry. Microsatellite instability was examined using a panel of 23 microsatellite markers previously used to detect allelic imbalance and two specific markers for the detection of low levels of microsatellite instability. Methylation specific PCR did not detect any methylation of the MGMT promoter in Australian and South African HCCs. Similarly, no hypermethylation of MGMT was observed in the adjacent non-malignant liver or histologically normal liver. MGMT staining was predominantly nuclear with some cytoplasmic staining. Overexpression of MGMT protein was detected in 14 (39%) HCCs, while a reduction in protein expression was evident in 14 (39%) HCCs. In the remaining 8 cases the expression of MGMT was comparable in HCCs and adjacent non-malignant tissue. Interestingly, MGMT expression decreased relative to adjacent non-malignant liver tissue in patients who had aetiologies other than viral hepatitis for their underlying liver diseases (p<0.02). No microsatellite instability was detected in this series of 61 HCCs. This suggests that epigenetic silencing of MGMT and microsatellite instability does not play an important role in this series of HCCs derived from different populations.

Interactions between aflatoxin B1 and dietary iron overload in hepatic mutagenesis.

BACKGROUND/AIM: Dietary aflatoxin B(1) (AFB(1)) exposure and iron overload are important causes of hepatocellular carcinoma in sub-Saharan Africa. The aim of this study was to investigate if the two risk factors have an interactive effect. METHODS: Four groups of Wistar albino rats were studied for 12 months. Group 1 (control) was fed the normal chow diet; group 2 (Fe) was supplemented with 0.75% ferrocene iron; group 3 (Fe+AFB(1)) was fed 0.75% ferrocene throughout and gavaged 25 microg AFB(1) for 10 days; group 4 (AFB(1)) was gavaged 25 microg AFB(1) for 10 days. Iron profile, lipid peroxidation (LPO), 8-hydroxydeoxyguanosine (8OHdG), oxidative lipid/DNA damage immunohistochemistry, superoxide/nitrite free radicals, cytokines IL6, IL-10, transaminases (ALT/AST) and Ames mutagenesis tests were performed. RESULTS: LPO and ALT showed a significant (p<0.05)/additive effect and 8OHdG a significant (p<0.05)/multiplicative effect in the Fe+AFB(1) group. IL-6 produced a negative synergy as against an additive antagonistic effect with IL-10. Massive deposits of 4-hydroxynonenal (4-HNE) and 8OHdG were observed in liver sections of the Fe+AFB(1) group, suggestive of multiplicative synergy. Significant levels of mutagenesis (p<0.001) were observed in the Fe+AFB(1) group. This multiplicative synergy was five-fold. CONCLUSION: Dietary iron overload and AFB(1) have a multiplicative effect on mutagenesis.

Molecular characterization of subgenotype A1 (subgroup Aa) of hepatitis B virus.

Subgenotypes of hepatitis B virus (HBV) were first recognized after a unique segment of genotype A was identified when sequencing the preS2/S region of southern African HBV isolates. Originally named subgroup A', subsequently called subgroup Aa (for Africa) or subgenotype A1, this subgenotype is found in South Africa, Malawi, Uganda, Tanzania, Somalia, Yemen, India, Nepal, the Philippines and Brazil. The relatively higher mean nucleotide divergence of subgenotype A1 suggests that it has been endemic and has a long evolutionary history in the populations where it prevails. Distinctive sequence characteristics could account for the high hepatitis B e-antigen (HBeAg) negativity and low HBV DNA levels in carriers of this subgenotype. Substitutions or mutations can reduce HBeAg expression at three levels: (i) 1762T1764A atthe transcriptional level; (ii) substitutions at nt 1809-1812 at the translational level; and (iii) 1862T at the post-translational level. Co-existence of 1762T1764A and nt 1809-1812 mutations reduces HBeAg expression in an additive manner. In addition, subgenotype A1 has unique sequence alterations in the transcriptional regulatory elements and the polymerase coding region. The distinct sequence characteristics of subgenotype A1 may contribute to the 4.5-fold increased risk of heptocellular carcinoma in HBV carriers infected with genotype A, which is entirely attributable to subgenotype A1.

Epidemiology of hepatitis B virus in Africa, its genotypes and clinical associations of genotypes.

Of approximately 360 million people in the world chronically infected with hepatitis B virus (HBV), 65 million reside in Africa. Thus, Africa, with 12% of the world's population, carries approximately 18% of the global burden of HBV infection, with hepatocellular carcinoma and cirrhosis accounting for 2% of the continent's annual deaths. Despite HBV being endemic or hyperendemic in Africa, there is a paucity of data on the genotypes and their distribution. Genotype A is found mainly in southern, eastern and central Africa. Most African genotype A strains belong to subgenotype A1, with subgenotype A3 found in western Africa. Genotype D prevails in northern countries and genotype E in western and central Africa. Ithas become increasingly evident that heterogeneity in the global distribution of HBV genotypes may be responsible for differences in the clinical outcomes of HBV infections and the response to antiviral treatment and vaccination. A limited number of studies have been published relating genotypes to clinical outcomes in African countries. Because observations from other regions of the world can not be extrapolated from one locale to another, the HBV strains circulating in Africa should be studied and related to clinical outcomes.