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المستخلص
In 1989, the hepatitis C virus was first identified. Since then, tremendous progress has been made to understand and treat HCV infection. The disease is surprisingly complex in its manifestations and complications whether hepatic or extrahepatic.
The mechanisms of liver pathogenesis are not fully understood. Innate and specific immune responses to the presence of the replicating virus play a central role in this pathogenesis. However, current treatment is poorly tolerated and achieves viral clearance in less than half of the patients. Thus, the end result of deficient knowledge about pathogenesis and lack of effective HCV treatment is hindrance of disease control and persistence of infection with increased liability to development of complications.
Many possible factors affect HCV infection consequences and fibrosis progression. Age at infection (older than 40 years), daily alcohol consumption (more than 50 g / day), male sex and interferon-α therapy, all are possible interplaying
factors.
Moreover, mechanisms by which chronic HCV infection results in HCC are not definite. During chronic active liver disease, hepatocytes death and regeneration may result in accumulation of damaged DNA and fixation of mutations during cell replication leading to hepatocytes transformation. Another possibility is free radicals mediated injury which develops as part of HCV chronic liver disease and might induce DNA damage and HCC.
HCC is one of the most common malignant tumors worldwide, accounting for about one million deaths annually. It is one of the few human cancers for which an underlying etiology can be identified.
HCC is considered as a long- term multistage disease associated with multiple genetic alterations including activation of cellular oncogenes and mutation in tumor suppressor gene P53. Moreover, infection with HCV has now been identified as a leading cause of HCC in many countries around the world. Evidence for the important role of HCV infection in HCC occurrence includes: high seroprevalence of anti-HCV among HCC patients and documented progression from chronic hepatitis to cirrhosis then to HCC among patients with chronic HCV infection.
Many of the substances synthesized and secreted by hepatocellular carcinoma are not biologically active. Nevertheless, a few are produced by a sufficiently large proportion of tumors to warrant use as serum tumor markers. The most helpful of these is alpha fetoprotein.
Although serum AFP has been associated with HCC since 1963 yet, unfortunately, it is also elevated in a wide variety of non-hepatic malignancies and benign hepatic conditions. Moreover, false-positive and -negative results are obtained when - fetoprotein is used as a serum marker for hepatocellular carcinoma. Thus, the search for an ideal marker continues. A number of alternative markers have been suggested, although none has proved to be more useful than – fetoprotein.
In addition, GGT activity increases obviously in fetal liver and HCC. Total GGT can be divided into 13 isoenzymes by using polymeracrylamide gradient gel electrophoresis. Some of these could only be detected in the serum of HCC patients. Furthermore, simultaneous determination of GGT II, dys-γ carboxy prothrombin and AFP can significantly improve the sensitivity over AFP alone.
Furthermore, ALDH2 is one of three distinct aldehyde dehydrogenases isolated from the human liver. It is a mitochondrial enzyme present primarily in adult liver, kidney, muscle and heart. Moreover, it is one of the aldehyde dehydrogenase family of proteins. Two major liver isoforms of this enzyme, cytosolic and mitochondrial, can be distinguished by their electrophoretic mobilities, kinetic properties and subcellular localizations.
A variant of this enzyme was detected. It results from a single nucleotide polymorphism (SNP) of the ALDH2 gene. Moreover, SNP is associated with phenotypic reduction of catalytic activity in both heterozygotes and homozygotes.
ALDH2 catalyzes the pyridine nucleotide – dependent oxidation of aldehydes to acids. Reduced ALDH2 activity results in higher acetaldehyde levels after alcohol intake and this is thought to contribute to the flushing and other vasomotor symptoms observed in some Asians after alcohol consumption.
In addition, acetaldehyde has mutagenic and carcinogenic effects leading to metaplasia, inhibition of DNA repair, sister chromatid ex¬changes, stimulation of apoptosis and enhanced cell injury asso¬ciated with hyper-regeneration. Moreover,
aldehydes have the potential to bind to cellular proteins and DNA, thus leading to carcinogenesis. Besides individuals possessing more susceptible ALDH2 1-2/2-2 genotypes were more likely to reveal p53 overexpression.
Furthermore, when correlating between ALDH2 and other cancers, most studies have revealed associations between the ALDH2 polymorphism on one hand and both esophageal cancer risk and multiple esophageal dysplasia on the other hand. It was also reported that the ALDH2*2 allele leads to increased susceptibility to esophageal cancer among male alcoholics.
In addition, ALDH2*2 allele is strongly associated with multiple intra-esophageal cancers and upper aero-digestive tract cancer concurrent with esophageal cancer. Both of these are frequent in alcoholics, suggesting that severe systemic acetaldehydemia caused multicentric field concretization.
Recent research has further suggested that polymorphism of the p 4502E 1 (CYP2E1) gene, which encodes an enzyme catalyzing the metabolism of N-nitrosamines and alcohol, might be associated with susceptibility to HCC. So, ALDH2 polymorphism may modify the risk of development of HCC against the background of liver cirrhosis.
Accordingly, the aim of the present work was to evaluate role of aldehyde dehydrogenase-2 in the pathogenesis of hepatocellular carcinoma in chronic HCV cirrhotics.
The present study was conducted on 40 chronic hepatitis C patients divided into 2 groups (each made of 20 patients) according to presence or absence of HCC. Moreover, 15 healthy age, sex and socioeconomic status matched volunteers were enrolled in this work as controls for comparison.
All included patients and controls were subjected to detailed history taking including history of jaundice or any manifestations of hepatic insufficiency. Patients with diabetes, obesity, HBV and/or collagenic diseases were excluded.
In addition, thorough clinical examination was done laying particular stress on the size of the liver, spleen and presence or absence of ascites. Blood samples were collected to perform complete blood picture, fasting and post prandial blood glucose, ESR, liver function tests (serum aminotransferases, serum bilirubin, total protein, serum albumin, prothrombin time and activity, GGT and alkaline phosphatase), serum cholesterol, triglycerides and α - fetoprotein.
Moreover, serological viral markers for hepatitis C virus were looked for using ELISA assay. Positive anti HCV cases were confirmed by PCR. Abdominal ultrasonography was done for all included subjects. Liver biopsies were also done whenever possible.
Furthermore, patients and controls were investigated for ALDH2 polymorphism by polymerase chain reaction- restriction fragment length polymorphism. In addition, ALDH2 activity was determined.
The present work revealed no sex or age difference among patients’ groups and controls.
On evaluating the haematological parameters of the three studied groups, it was found that, hemoglobin concentration and red blood cells’ count were significantly lower in patients with either cirrhosis or HCC than in controls. This could be due to iron sequestration in the reticuloendothelial system, reduction of red blood cells’ life span and/or insensitivity to circulating erythropoietin. Furthermore, hypersplenism, folate deficiency, inapparent gastrointestinal bleeding, HCV bone marrow suppression and autoimmune hemolytic anaemia may also contribute to the occurrence of anaemia in HCV patients.
Moreover, leukocytes’ count in cirrhotic patients was significantly lower than in controls. On the contrary, increased leucocytic count in HCC could be attributed to the occurrence of the paramalignant syndrome and /or presence of a superadded infection.
In addition, platelets’ count was significantly lower in the two herein included patients groups than in controls. This could be related with HCV stimulation of production of autoantibodies against platelet membrane protein. Other possibilities include decreased thrombopoietin level in cirrhotic HCV patients and/or hypersplenism.
Regarding erythrocyte sedimentation rate (1st hour), statistical analysis showed the presence of a significant difference between the three studied groups where HCC patients had the highest ESR value followed by HCV cirrhotic ones then controls. Cirrhosis following viral hepatitis is one of the etiological factors that could lead to extremely elevated ESR probably due to its association with chronic inflammation, anemia, high serum globulin level, decreased serum albumin, and prerenal azotemia. All these factors encourage erythrocyte aggregation and sedimentation. It is also well known that ESR increases markedly (above 100mm) in collagenic diseases, tuberculosis and metastasis.
Evaluating blood sugar showed that, although in all selected patients FBS was within normal values yet, fasting blood sugar levels were significantly higher in the two patients’ groups than in controls with rise of its values with progression of the disease severity. This could be related to defects in the formation of hormones as insulin, glucagon, growth hormone & cortisone leading to abnormal glucose homeostasis. However, post-prandial blood sugar levels showed no statistically significant changes.
In addition, serum transaminases were significantly higher in both patients groups than in controls. Altered serum transaminases’ levels seem to depend upon HCV activity and not upon disease chronicity. Moreover, chronic HCV inflammation stimulates hepatocytes’ apoptosis. Dying cells presumably synthesize less trans-aminases as they wither away. Thus, transaminases’ elevation cannot be used as a predictor of histopathological changes.
Two diseases (cirrhosis and HCC) could be incriminated in the marked increase in liver transaminases in HCC patients.
As regards serum bilirubin, it was significantly elevated in patients with cirrhosis and HCC compared to controls. Hyperbilirubinemia might be related to the extensive HCV damage to hepatocytes. Besides, decreased hepatocytes excretory ability due to the chronic inflammatory process may be another possible factor. Diminished erythrocytes’ survival, hypersplenism and autoimmune liver diseases could add hemolytic components to the causes of jaundice.
Furthermore, in the current research serum albumin and prothrombin activity were significantly lower in the two patients’ groups than in controls. Moreover, total protein was significantly lower in both cirrhotics and HCC patients than in controls. Defect in total hepatic plasma protein synthetic function occurs in advanced liver diseases and could be responsible for such changes.
Regarding serum alkaline phosphatase and serum GGT, both were significantly higher in HCC patients than in the other two groups.
In the present work, serum cholesterol was lower in both cirrhotics and HCC patients than in controls. This might be due to elevation of serum estrogen levels with cirrhosis.
Serum AFP levels were also, significantly higher in HCC patients than in the other two groups.
When evaluating the association between HCV- positive cirrhotics with HCC and inter-individual variation due to genetic ALDH2 polymorphisms, there was no significant difference between the two patients’ groups, but the control group was significantly different compared to the two patients’ groups.
As regards ALDH2 activity, there was a highly significant reduction in ALDH2 activity in the two patients’ groups in comparison to controls, with no significant difference in activity when comparing between these two groups.
Moreover, there was a significant negative correlation between ALDH2 activity and age, 1st hr ESR, alkaline phosphatase and GGT. On the other hand, there was a significant positive correlation between ALDH2 activity and hemoglobin, RBCs, platelets, total protein, serum albumin, A/G ratio, prothrombin activity and serum cholesterol. Other parameters showed no significant correlation with ALDH2 activity.
According to the above findings, it could be concluded that:
1. Study of the role of ALDH2 mutation and activity in non alcoholic HCC patients with HCV cirrhosis revealed that variations in acetaldehyde detoxification due to ALDH2 polymorphism had no role in determining occurrence of HCC in HCV cirrhosis in non-alcoholics.
2. Inter-individual variations may have relation to either cirrhosis and/or HCV infection as there was significant difference between patients’ groups and controls.
3. ALDH2 activity decreases with age progression and high values of alkaline phosphatase and GGT. Moreover, this activity increases with high values of hemoglobin, RBCs, platelets, total protein, serum albumin, A/G ratio, prothrombin activity and serum cholesterol. In addition, other parameters as FBS, PPBS, liver enzymes, serum bilirubin, triglycerides and alpha fetoprotein had no relation with ALDH2 activity.
4. Alpha fetoprotein was markedly elevated in HCC patients. This explains the fact that AFP is the most widely used tumor marker for HCC.
5. The present work also confirms previously recorded haematological findings, liver functions tests results and lipid profiles in HCV cirrhotics.
A. Hemoglobin concentration, red blood cells’ count and platelets’ count decrease in HCV cirrhotic patients with or without HCC. Moreover, leukocytes’ count also decreases in HCV cirrhotic patients but increases with HCC patients. Increased leucocytic count in HCC may be due to the occurrence of the paramalignant syndrome and /or a superadded infection.
B. Serum transaminases and serum bilirubin increase in both cirrhosis and HCC patients. Moreover, alkaline phosphatase and serum GGT also increase in these two diseases but with marked increase in HCC patients therefore, they could be used as good supplements to AFP in the diagnosis of HCC. On the contrary, serum albumin and prothrombin activity are decreased in these diseases.
C. Patients with either cirrhosis or HCC showed decreased levels of cholesterol and normal values of triglycerides. This might be due to elevation of serum estrogen levels with cirrhosis .