Steatosis is increasingly recognized as a cofactor influencing the progression of fibrosis in chronic hepatitis C; however, the mechanisms by which it contributes to liver injury remain uncertain. We studied 125 patients with chronic hepatitis C to assess the effect of steatosis on liver cell apoptosis and the expression of Bcl-2, Bcl-xL, Bax, and tumor necrosis factor alpha (TNF-α) and the relationship between liver cell apoptosis and disease severity. A significant increase in liver cell apoptosis was seen in liver sections with increasing grade of steatosis (r = 0.42; P < .0001). Hepatic steatosis and previous heavy alcohol consumption were the only two variables independently associated with the apoptotic index. Increasing steatosis was associated with decreased Bcl-2 mRNA levels and an increase in the proapoptotic Bax/Bcl-2 ratio (r = −0.32, P = .007; and r = 0.27, P = .02, respectively). In the absence of steatosis, increased liver cell apoptosis was not associated with stellate cell activation or fibrosis (r = 0.26, P = .11; r = 0.06, P = .71, respectively). In contrast, in the presence of steatosis, increasing apoptosis was associated with activation of stellate cells and increased stage of fibrosis (r = 0.35, P = .047; r = 0.33, P = .03, respectively), supporting the premise that the steatotic liver is more vulnerable to liver injury. In patients with hepatitis C virus genotype 3, there was a significant correlation between TNF-α mRNA levels and active caspase-3 (r = 0.54, P = .007). In conclusion, these observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C. Further investigation will be required to determine the molecular pathways responsible for the proapoptotic effect of steatosis and whether this increase in apoptosis contributes directly to fibrogenesis. (HEPATOLOGY 2004.39:1230-1238.)
Hepatic steatosis is increasingly recognized as a cofactor influencing the presence and progression of fibrosis in chronic hepatitis C.1–6 Higher grades of steatosis are associated with more advanced stages of fibrosis,2, 3 and worsening of steatosis is an independent factor associated with progression of fibrosis in paired liver biopsies.6 Both host and viral factors contribute to the development of steatosis. Patients infected with hepatitis C virus (HCV) genotype 3 have a higher prevalence and more severe steatosis than those infected with other genotypes.7 In addition, steatosis is associated with increased body mass index (BMI), and overweight patients have significantly more steatosis than lean subjects, regardless of viral genotype.8 The mechanisms by which steatosis contributes to progressive fibrosis in chronic hepatitis C remain uncertain.
Hepatocyte apoptosis has been identified as an important feature of liver injury in chronic hepatitis C. The amount of liver cell apoptosis as assessed by DNA fragmentation or the extent of caspase activation has been shown to be elevated compared with healthy liver, and a significant correlation has been seen with the grade of necroinflammatory activity.9, 10 The cellular and molecular processes responsible for the increase in apoptosis remain unclear. The CD95 pathway, the tumor necrosis factor (TNF) system, TNF-related apoptosis-inducing ligand (TRAIL) receptors, and the perforin/granzyme B pathway have all been implicated in HCV-mediated liver injury.11–13 In hepatocytes, these mechanisms seem to require a mitochondrial amplification pathway.14 HCV proteins may modulate the apoptotic process, but their effects have been difficult to evaluate. In different experimental conditions, the HCV core protein has been reported either to sensitize cells to death receptor-mediated apoptosis or to exert inhibitory effects through the activation of nuclear factor (NF)-κB and induction of antiapoptotic gene products, including Bcl-2 and Bcl-xL.15–18
It has not been determined whether steatosis or host factors such as BMI influence the apoptotic process in chronic hepatitis C. Interestingly, hepatocyte apoptosis recently was shown to be a prominent feature of nonalcoholic fatty liver disease.19 In that study, a positive correlation was observed between hepatocyte apoptosis and both hepatic inflammatory activity and fibrosis, implicating a role for the apoptotic process in the progression of fatty liver disease.
We hypothesized that in chronic hepatitis C, steatosis results in altered expression of proapoptotic and antiapoptotic factors and an increase in liver cell apoptosis. To examine this, the extent of apoptosis and the expression of Bcl-2, Bcl-xL, Bax, TNF-α, and the Bax/Bcl-2 ratio was determined in liver biopsy specimens from patients with chronic hepatitis C and varying grades of steatosis. The relationship between liver cell apoptosis and stellate cell activation and disease severity also was determined.
The study assessed 125 white patients with chronic hepatitis C who had undergone a liver biopsy at the Princess Alexandra Hospital, Brisbane. Informed consent was obtained from each patient and the protocol was approved by the Princess Alexandra Hospital Research Ethics Committee. Diagnosis of chronic hepatitis C was based on standard serological assay results and abnormal serum aminotransferase levels for at least 6 months. All patients were positive for HCV antibody by the third-generation enzyme-linked immunosorbent assay (Abbott Laboratories, North Chicago, IL), with infection confirmed by detection of circulating HCV RNA by polymerase chain reaction using the Amplicor HCV assay (Roche, Branchburg, NJ). Viral genotyping was performed using the Inno-Lipa HCV II assay (Innogenetics, Zwijnaarde, Belgium). Patients with other forms of chronic liver disease or antibodies to human immunodeficiency virus were not considered for the analysis.
Details about weight and height and average alcohol intake (grams per day) during the preceding 12 months were obtained from all patients at the time of liver biopsy. Information regarding average alcohol intake (grams per day) before the last 12 months also was obtained. Alcohol consumption was assessed retrospectively by interview on at least three occasions. The number and types of alcoholic drinks consumed each day were recorded, and the alcohol content of each drink was calculated. The alcohol intake during a weekly period was averaged and recorded in grams per day.
At the time of biopsy, liver tissue (2–3 mm) was immediately frozen in liquid nitrogen and stored at −80°C until the extraction of RNA was performed. The remaining biopsy core was fixed in buffered formalin and was embedded in paraffin. The sections were analyzed by an experienced hepatopathologist (AC) who was blinded to the laboratory parameters and clinical data. The degree of inflammation was graded according to the method of Knodell et al.,20 and fibrosis was staged according to the method of Scheuer.21 Steatosis was graded as follows: 0 (<5% hepatocytes affected), 1 (5%–29% of hepatocytes affected), 2 (30%–70% of hepatocytes affected), or 3 (>70% of hepatocytes affected). Perls stain was available for all patients and was graded from 0 to 4. For statistical analyses, hepatic iron was scored as present or absent.
Determination of Apoptotic Index.
Apoptosis was quantified in liver biopsies (n = 109) by in situ labeling of fragmented DNA using a modified terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay.22, 23 In short, antigen retrieval was facilitated by microwave heating (3 × 5 minutes on medium setting of 340 watts) in 0.01 M citrate buffer (pH 3) with 0.1% Tween 20 followed by incubation on ice, in a freshly prepared permeabilization solution containing 0.1% Triton X-100 in 0.1% sodium citrate (Merck, Darmstadt, Germany). Sections were incubated with phosphate-buffered saline (pH 7.4) containing 3% bovine serum albumin and 20% fetal calf serum for 30 minutes, before application of the TUNEL mixture. The in situ cell death detection kit, AP (Roche Molecular Biochemicals, West End, Queensland), was used according to the manufacturer's instructions. Positive controls received DNase 1 (grade 1, 1500 u/mL; Roche Molecular Biochemicals) and negative controls received no TUNEL enzyme solution. The converter-alkaline phosphatase solution was diluted by half with 1 × TBS. The development of blue–black nuclear staining was monitored carefully under the microscope for each slide individually to avoid background staining. Immunohistochemical staining for CD3 and CD68 was performed as previously described.24
Apoptosis was quantified by counting the number of TUNEL-positive nuclei in at least 15 random high-power fields (magnification, ×400). Cells were classified as apoptotic only if they were TUNEL positive and showed the morphological characteristics of apoptosis. The results were expressed as a percentage of the total number of hepatocyte nuclei per high-power field and defined as an apoptotic index (AI).
Immunohistochemistry (IH) for Activated Caspase-3, Bcl-2, Bax, and α-Smooth Muscle Actin (α-SMA).
Formalin-fixed paraffin-embedded liver biopsy samples (n = 115) were used for immunohistochemical studies as previously described.22, 24, 25 The primary antibodies used in the study were: activation-specific anti-caspase-3 (BD Pharmingen, North Ryde, New South Wales, Australia; dilution, 1:600), anti-Bcl-2 (DakoCytomation, Botany, New South Wales, Australia; dilution, 1:50), anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA; dilution, 1:400), and anti-α-SMA (DakoCytomation; dilution, 1:400). Tissue sections were photographed using a PixeLink Colour Digital Camera (Total Turnkey Solutions, Mona Vale, New South Wales, Australia) mounted on an Olympus BX-40 microscope (Olympus Australia Pty. Ltd., Mt. Waverley, Victoria, Australia).
Image analysis was used to quantify the immunoreactivity of active caspase-3, Bax, and α-SMA. For quantification of α-SMA, 10 consecutive and nonoverlapping fields at a magnification ×100 and for active capase-3 and Bax, 20 nonoverlapping lobular fields at a magnification of ×200 were photographed. Image analysis software (Image Pro Plus 4.5; SciTech Pty Ltd, Preston, Victoria, Australia) was used to assess the mean immunoreactive area per biopsy. The percent positive area was defined as the ratio of pixels set above the segmentation threshold to the total number of pixels within a defined area of interest. For each section, the percent positive area for replicate fields was averaged.
Determination of mRNA levels of Bcl-2, Bax, Bcl-xL, and TNF-α.
The steady-state mRNA levels of Bax, Bcl-xL, Bcl-2, and TNF-α were assessed by semiquantitative real-time polymerase chain reaction (PCR) assays using glyceraldehyde-3-phosphate dehydrogenase as a housekeeping gene. Total RNA was extracted from liver biopsy tissue (n = 76) in the presence of guanidinium thiocyanate.26 Contaminating DNA was removed by treatment of each sample with DNAse I, according to the manufacturer's instructions (Invitrogen, Melbourne, Victoria, Australia). cDNA was prepared by reverse transcription at 50°C for 60 minutes in a 20-μl reaction mixture containing up to 2 μg of total RNA, 1 μg oligo(dT), 2 mM 2′-deoxy-nucleotide-5′-triphosphates, 4 μl of First-Strand Buffer (Invitrogen), 5 mM dithiothreitol (DTT), 25 U RNasin (Promega, Sydney, New South Wales, Australia), and 200 U SuperScript III (Invitrogen).
The probe and primer sequences for the glyceraldehyde-3-phosphate dehydrogenase gene were as described previously.27 The primer sequences for the other genes of interest were designed using online software Primer 3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3www.cgi; Table 1) and were purchased from Proligo Australia Pty. Ltd. (Lismore, New South Wales, Australia). Probes were labeled at the 5′ end with the reporter dye FAM and at the 3′ end with the quencher dye TAMRA.
Table 1. Primer and Probe Sequences for RT-PCR
Sequence 5′– 3′
Five microliters of diluted (1:20) cDNA were added to a PCR mix containing 6.45 μL sterile water, 12.5 μL 2 × QT mix (Qiagen; Clifton Hill, Victoria, Australia), 0.4 uM each of forward and reverse primers, and 0.2 uM of probe to give a final volume of 25 μL. The cycling conditions for amplification were 95°C for 15 minutes, followed by 40 cycles of 94°C for 30 seconds and 60°C for 60 seconds in an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Sydney, Australia). All patient samples were amplified in the same assay for each gene of interest, and each assay was performed in duplicate.
The relative concentrations of mRNAs present were determined by the relative standard curve method. A dilution series and no-template controls were amplified in parallel with unknowns in each assay, and the concentration in each unknown was assessed by comparison to the standard curve. For each gene, the average of the duplicate assays was obtained and normalized to the average amount of glyceraldehyde-3-phosphate dehydrogenase for each sample to determine relative changes in mRNA expression.
Continuous normally distributed variables were represented graphically as mean ± SEM. Grade of steatosis, stage of fibrosis, and alcohol consumption were summarized by the median. A chi-square goodness of fit test was used to determine whether there was a difference in distribution of steatosis among the viral genotypes. To compare the means between groups, ANOVA or Student's t test was performed. To determine differences between groups not normally distributed, medians were compared using the Mann-Whitney U test. Pearson's correlation coefficient was used to measure the degree of association between continuous, normally distributed variables. The degree of association between non–normally distributed or ordinal variables was assessed using Spearman's nonparametric correlation. An ANCOVA was performed to identify independent predictors of normally distributed variables, correcting for age at biopsy, gender, viral genotype, stage of fibrosis, BMI, alcohol consumption, presence or absence of hepatic iron, grade of inflammation, and grade of steatosis. A backward elimination approach was used to remove nonsignificant variables and to determine the most parsimonious model. Interactions between variables were tested by including a multiplicative term in the model. Ordinal linear regression was used to assess the independent influence of variables on ordered categorical data (such as fibrosis and steatosis). All analysis was carried out using SPSS software version 11.0 (SPSS Inc., Chicago, IL). Statistical significance was taken at the 95% CI.
Clinical, Histological, and Laboratory Data.
The demographic and clinical characteristics of 125 patients with chronic hepatitis C are summarized in Table 2. Among the 125 patients, 83 (66%) were male and all were HCV RNA positive. Steatosis grade was 0 in 69 patients (55.2%), grade 1 in 29 patients (23.2%), and grades 2 or 3 in 27 patients (21.6%). The stage of fibrosis (Scheuer score) was 0 in 17 patients (13.6%), stage 1 in 57 patients (45.6%), stage 2 in 23 patients (18.4%), and stage 3 or 4 in 28 patients (22.4%). The prevalence of steatosis was higher in patients infected with viral genotype 3 (P < .001). The mean BMI was significantly higher in patients with steatosis (P < .001), but there was no significant difference in current or past alcohol consumption between steatosis-positive and steatosis-negative groups (P = .3 and P = .5 respectively). Patients with steatosis had a higher necroinflammatory score (P < .001) and more severe fibrosis (P < .001) than those without steatosis. The mean length of the paraffin sections was 14.1 ± 0.65 mm. There was no association between length of biopsy and stage of fibrosis, grade of steatosis, or viral genotype (data not shown).
Table 2. Demographic and Clinical Features of Patients With Chronic HCV
Liver Cell Apoptosis Is Increased in the Presence of Steatosis.
Liver cell apoptosis was assessed by both the TUNEL assay and the detection of active caspase-3. TUNEL-positive cells were observed in the liver sections from all patients with chronic hepatitis C (Fig. 1A). TUNEL-positive cells were present in the lobules and within CD68-positive Kupffer cells in the hepatic sinusoids. The TUNEL-positive cells did not express the T-lymphocyte marker CD3. Quantitation of TUNEL-positive cells revealed a significant increase of apoptosis in liver sections with increasing grade of steatosis (r = 0.42, P < .0001; Table 3). The mean apoptotic index in liver biopsies with or without steatosis is shown in Fig. 3A. By univariate analysis, AI was associated with stage of fibrosis, but with no other demographic or histological feature. (Table 3).
Table 3. Association Between AI and Demographic and Histological Variables
Adjusted for age at biopsy, gender, viral genotype, BMI, alcohol consumption, stage of fibrosis, presence or absence of hepatic iron, grade of inflammation, and grade of steatosis.
Expressed as mean ± SEM.
0.50 ± 0.06
0.58 ± 0.06
0.55 ± 0.05
0.47 ± 0.08
Independent factors associated with the AI were assessed by multivariate analysis (Table 3). Variables independently associated with apoptotic index were steatosis (P < .0001) and previous ethanol consumption (P = .032). Apoptosis was independently associated with steatosis when past alcohol consumption was either <50 g/day (r = 0.42, P = .004) or >50 g/day (r = 0.49, P = .002). The AI was independently associated with steatosis in patients with either viral genotype 1 or 3 (P < .001 and P < .001 respectively).
To confirm the presence of increased apoptosis in patients with steatosis, IH for active caspase-3 was performed. Immunoreactive product was seen in liver tissue from all patients with chronic hepatitis C (Fig. 2A). Staining was present in both periportal and lobular areas, was seen predominantly in hepatocytes, and was localized to the cytoplasm. Immunoreactivity also was detected in cells lining the sinusoids. Most caspase-positive cells did not demonstrate obvious apoptotic nuclear morphological features, supporting the suggestion that IH detection of caspase-3 activation marks an early event in the apoptotic process that does not invariably progress to cellular apoptosis.
There was a significant correlation between the percentage area of active caspase-3 staining and the grade of steatosis (r = 0.42, P < .0001; Fig. 3B). This relationship between caspase expression and steatosis was seen in patients with either viral genotype 1 or 3 (r = 0.44, P = .002; r = 0.56, P < .001, respectively). After multivariate logistic regression, this relationship between steatosis and percentage area positive for active caspase-3 remained significant (P = .001).
Steatosis Is Associated With Decreased Bcl-2 mRNA Levels and Increased Bax/Bcl-2 Ratio.
The relationship between steatosis and the mRNA levels of the antiapoptotic factors Bcl-2 and Bcl-xL and the proapoptotic factor Bax was determined by real-time PCR. Hepatic Bcl-2 mRNA levels decreased significantly in patients with increasing grade of steatosis (r = −0.32, P = .007; Fig. 4A). This was not accompanied by a change in Bax mRNA levels (P = .5; Fig. 4B). To assess the direction of the Bcl-2 family-mediated apoptotic drive, the ratio Bax/Bcl-2 was calculated. This proapoptotic ratio increased significantly with increasing grade of steatosis (r = 0.27, P = .021; Fig. 4C).
There was no difference in Bcl-2 mRNA levels or Bax/Bcl-2 ratio between patients with viral genotype 1 or 3 (P = .79). After multivariate analysis, the relationship between steatosis and Bcl-2 mRNA levels and the Bax/Bcl-2 ratio remained significant (P < .001 and P = .028, respectively).
Bcl-xL mRNA levels were not associated with the grade of steatosis (data not shown).
Immunohistochemistry was performed to localize the expression of Bcl-2 and Bax. Immunoreactive Bcl-2 was not detected in hepatocytes or Kupffer cells in any of the liver sections. Bcl-2 staining was seen predominantly in bile ducts and inflammatory cells in portal areas and within lobules (Fig. 2B). In contrast to Bcl-2, Bax expression was detected readily in hepatocytes, Kupffer cells, inflammatory cells, and bile ducts (Fig. 2C).
In the Presence of Steatosis, Liver Cell Apoptosis Is Associated With Stellate Cell Activation and Increased Fibrosis.
Overall, there was no independent association between the extent of liver cell apoptosis and the stage of fibrosis (Table 3), suggesting that increasing apoptosis was not simply a consequence of more severe liver injury. However, in the presence of steatosis, there was a significant correlation between the AI and stage of fibrosis and percentage area positive for α-SMA (r = 0.33, P = .029; r = 0.349, P = .047, respectively; Fig. 5A, B). Similarly, in patients with steatosis, there was a significant correlation between active caspase-3 staining and the percentage area positive for α-SMA (r = 0.385, P = .025; Fig. 5C).
There was a trend toward a positive relationship between AI and increasing necroinflammatory activity; however, this did not reach statistical significance (r = 0.177, P = .097).
Association Between TNF-α mRNA Levels and Active Caspase-3 in Patients With Viral Genotype 3.
To investigate mechanisms of apoptosis in these liver sections, TNF-α mRNA levels were determined by real-time PCR. In patients with HCV genotype 3, there was a significant correlation between TNF-α mRNA levels and active caspase-3 (r = 0.539, P = .007; Fig. 6). This relationship was not seen in patients with viral genotype 1 (r = −0.075, P = .73). After multivariate analysis, the relationship between active caspase-3 and TNF-α remained significant (P = .009). The test for an interaction between viral genotype and TNF-α approached significance (P = .07).
In this study, we found that increasing steatosis in chronic hepatitis C was associated significantly with an increased AI and expression of active caspase-3 in hepatocytes. In addition, increasing steatosis was associated with decreased Bcl-2 mRNA levels and an increase in the proapoptotic Bax/Bcl-2 ratio. In the presence of steatosis, increased expression of active caspase-3 and the AI was associated with increased α-SMA staining and increased stage of fibrosis. These observations support an important role for steatosis in promoting liver cell apoptosis and fibrogenesis in chronic hepatitis C. It remains unclear whether these processes are occurring independently in the steatotic liver, or whether apoptosis is linked directly to fibrogenesis.
Although liver cell apoptosis has previously been demonstrated in chronic hepatitis C, the influence of steatosis on this process has not been determined. The number of TUNEL-positive cells and the extent of active caspase-3 staining in this study were similar to levels reported previously in liver biopsies with chronic hepatitis C.9, 10 Because there was no independent association between the extent of liver cell apoptosis and the stage of fibrosis, it is unlikely that the increased apoptosis seen in patients with steatosis was simply a consequence of more severe liver injury. In addition, factors contributing to steatosis such as increased BMI and viral genotype 3 were not independently associated with apoptosis. Instead, the significant independent association between steatosis and increased apoptosis suggests that hepatocyte lipid accumulation contributes to cell death.
Accumulation of lipid in nonadipose tissues has been reported to lead to cell dysfunction or cell death.28 Lipotoxicity in cardiac myocytes and pancreatic β-cells has been attributed to an excess of long-chain saturated fatty acids (FAs) that are surplus to the cell's oxidative requirements. Accumulation of FAs may result in the generation of specific proapoptotic lipid species or signaling molecules.28, 29 These signals include de novo ceramide synthesis, reactive oxygen species, nitric oxide generation, and a direct influence on mitochondrial structure or function. In addition, long-chain FAs have been shown to suppress antiapoptotic factors such as Bcl-2 in both rodent models and isolated human cells.30, 31 Prolonged exposure of isolated human islet cells to FAs was associated with a marked decrease in Bcl-2 mRNA expression and a significant increase in apoptosis. The proapoptotic effects were caspase-mediated and partially dependent on the ceramide pathway.31 Other human cells may have a similar response, as suggested by the recent finding that FA-induced apoptosis in human granulosa cells is accompanied by downregulation of Bcl-2.32 In our patients with chronic hepatitis C, the decrease in Bcl-2 mRNA levels with increasing steatosis suggests that the role of FAs and ceramide in liver cell death should be evaluated further. However, because Bcl-2 itself was not detected in hepatocytes by IH, it will be important to determine the role of other related antiapoptotic factors in hepatocytes. Conversely, relatively low expression of Bcl-2 in hepatocytes may be detected by real-time PCR but not by less sensitive methods such as IH.
In this study, a relationship was demonstrated between previous ethanol consumption and the apoptotic index. Because in our patient cohort, current median alcohol consumption was only 3 g/day, it is possible that patients underreported their current alcohol intake, and this may have led to a misrepresentation of genuine consumption. The mechanism whereby prior alcohol consumption may induce apoptosis remains unclear.
Although our results suggest that steatosis contributes to both apoptosis and fibrosis, it remains to be determined whether apoptosis contributes directly to fibrogenesis. Interestingly, our results show that in the absence of steatosis, increased liver cell apoptosis is not associated with stellate cell activation or fibrosis (Fig. 5). In contrast, in the presence of steatosis, increasing apoptosis is associated with activation of stellate cells and increased stage of fibrosis, suggesting that steatosis both increases apoptosis and also decreases the threshold at which apoptosis becomes pathogenic. The association between steatosis, apoptosis, and fibrosis can be interpreted in several ways. Steatosis may promote cellular injury and death independently from its effect on fibrosis, or apoptosis itself, driven by steatosis, may enhance fibrogenesis. We favor the latter for several reasons. Engulfment of apoptotic bodies by hepatic stellate cells has been reported and was associated with activation of these cells, resulting in increased TGF-β and collagen production.33 Additionally, inhibition of hepatocyte apoptosis has been shown to reduce fibrogenesis in animal models of cholestasis.34
Hepatocyte apoptosis also may contribute to the production of cellular mediators causing inflammation and fibrosis. Although originally regarded as a noninflammatory event, ongoing apoptosis in the liver may evoke an inflammatory response, and this could cause stellate cell activation secondarily.35 A recent study has shown that Kupffer cell engulfment of apoptotic bodies results in the secretion of TNF-α along with increased expression of the death ligands TRAIL and Fas ligand.36 It has been suggested that steatosis is accompanied by Kupffer cell dysfunction or activation,37 and it is possible that the Kupffer cell response to apoptotic bodies may differ between patients with or without steatosis. In our liver sections, many apoptotic cells were identified within CD68-positive Kupffer cells in the hepatic sinusoids. In our patients with viral genotype 3, we identified a significant association between active caspase-3 staining and TNF-α mRNA levels. This proinflammatory cytokine may lead to further tissue damage, promoting inflammation and fibrogenesis.38
In addition to differences in the prevalence of steatosis and response to antiviral therapy, the mechanisms responsible for liver injury may differ between the viral genotypes. In a recent preliminary report, patients with HCV genotype 3 had higher hepatic c-myc and Fas mRNA levels than patients infected by other HCV genotypes.39 Similarly in our study, patients with genotype 3, but not genotype 1, demonstrated an increase in TNF-α mRNA levels with increasing active caspase-3 expression. It remains unclear whether these differences in virus behavior in the host are the result of diversity in amino acid sequence between the viral genotypes.40 Information about the properties of HCV proteins has been largely acquired from studies using viral genotype 1 strains. Further study of proteins encoded by genotypes other than type 1 will be required to elucidate differences in the behavior of HCV infection in the host.
In summary, liver cell apoptosis was readily detected in our patients with chronic hepatitis C. Hepatic steatosis and previous heavy alcohol consumption were the only two variables independently associated with the apoptotic index. In the presence of steatosis, liver cell apoptosis was associated with stellate cell activation and increased fibrosis. These observations suggest a mechanism whereby steatosis contributes to the progression of liver injury and fibrosis in chronic HCV. Further investigation will be required to determine the molecular pathways responsible for the proapoptotic effect of steatosis and whether this increase in apoptosis contributes directly to fibrogenesis.