Research ArticleResearch Article

Synergistic Action of Clonorchiasis, HBV Infection and Alcohol Consumption on Primary Hepatocellular Carcinoma

Shengkui Tan, Xiaoqiang Qiu, Hongping Yu, Xiaoyun Zeng, Zengming Xiao, Lequn Li and Qiuan Zhong
Clinical Oncology and Cancer Research April 2009, 6 (2) 104-112; DOI: https://doi.org/10.1007/s11805-009-0104-0

Abstract

OBJECTIVE It has been recognized that HBV infection and alcohol consumption are two important risk factors for primary hepatocellular carcinoma (HCC). Recently, the role of clonorchiasis as a risk factor for HCC is controversial. We aimed to investigate whether these factors increase the risk of HCC in Guangxi, China.

METHODS A hospital-based, case–control study of HCC was conducted from July 2005 to July 2007. We enrolled 500 consecutive patients with HCC as an experimental group and 500 patients without tumor in liver as a control group. The risk factors that the patients were exposed to were assessed.

RESULTS Comparing the risks of developing the HCC, we found out the following results. The risk of developing HCC for the patients with clonorchiasis was 5 folds of that for the patients without clonorchiasis (OR = 5.0; 95% CI: 3.1-8.1), and the risk for the patients with alcohol consumption was 3 folds of that for the patients without drinking alcohol (OR = 3.4; 95% CI: 2.3-4.9), and similarly, the risk for the patients with HBV infection was 21 times of that for the patients without HBV infection (OR = 20.6; 95% CI: 14.3-29.7). According to crossover analysis, there was significant interaction among clonorchiasis, HBV infection and alcohol consumption, with synergistic indices greater than 1. The etiologic fractions attributed to these interactions [EF (A × B)] are 0.7465, 0.5789 and 0.5506, respectively.

CONCLUSION Clonorchiasis, HBV infection and heavy alcohol consumption are independent risk factors for developing HCC in our population in Guangxi, and as they can interact synergistically, the risk of developing HCC is increased. Data from this study may indicate new prevention strategies of developing HCC in high-risk individuals.

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Introduction

Primary hepatocellular carcinoma (HCC) is one of the most common cancers all over the world and is rarely detected at early stages. Patients usually survive for a few months after diagnosis of HCC. It has been recognized that there is a high incidence of HCC in sub-Saharan Africa, China, and the Far East, but a low incidence of HCC in the United States and Europe[1,2]. Guangxi is one of the areas in China, where there is a high incidence of HCC. As the effective prevention and treatment have been applied, the high incidence of HCC in some of these areas has been reduced. However, the total incidence in Guangxi remains increased considerably. In the past 20 years, incidence of HCC has been increased by 48.15% in Guangxi and the mortality rate has been the second highest in the country. HCC accounts for 40.04% of all the malignant tumors in Guangxi, and its mortality rate occupies the top among those caused by other tumors in the area[3]. HCC has been one of the endemic and common diseases in Guangxi.

Recent researches indicate that HCC can result from the synergistic action of multiple risk factors[4-7]. Hepatitis B virus (HBV) which causes chronic infection in liver is one of the established etiologic agents of HCC[6,8,9]. There is a remarkable geographic correlation between the incidence of HCC and the prevalence of HBV infection, which frequently occurs in China and the southeastern Asia. However, previous researches showed that the major risk factors of HCC in Guangxi include HBV infection, alcohol consumption, aflatoxin, and so on[10,11]. Among the potential risk factors, clonorchiasis as a common disease in Guangxi draws more and more attention from medical researchers. There is a controversy on whether clonorchiasis can induce hepatic cirrhosis, liver cancer and other liver diseases, but there is no report on whether clonorchiasis and other risk factors exert some synergistic action on the development of HCC.

In view of the increasing HCC incidence in Guangxi, we conducted this case-control study in order to precisely assess whether clonorchiasis, alcohol consumption and HBV infection are the potential risks for developing HCC, and to evaluate possible synergisms among the risk factors examined.

Patients and Methods

Subjects

We conducted a hospital-based, case-control study in which a total of 1,000 subjects (500 histologically confirmed HCC cases and 500 controls without tumor diseases) were enrolled at the First Affiliated Hospital and Tumor Hospital of Guangxi Medical University.

The 500 patients with their diagnosis of HCC underwent further investigations at the Department of Hepatobiliary Surgery in the First Affiliated Hospital and Tumor Hospital from July 2005 to July 2007. We routinely obtained lists of new HCC cases diagnosed by the Pathology Department and permission from the attending physician to contact each of the patients. The controls, matched with each case according to sex, age (± 3 years) and time of diagnosis, were recruited within 10 days of admission. All of the controls from Department of Spine Surgery and Traumatic Surgery at the First Affiliated Hospital had been confirmed as non-tumor patients. Written informed consent to participate in the study was obtained from all of the subjects, and the study was approved by Guangxi Medical University, the Health Department of Guangxi, and the National Science Foundation of China.

All of the subjects were interviewed using a questionnaire structured to yield basic demographic data and information about their history of liver disease, family history of liver cancer, history of smoking and drinking, and the history of clonorchiasis. Five milliliters of blood was taken from a peripheral vein in each participant and then separated. The blood samples were also tested for the presence of hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B core antigen (anti-HBc) using the ELISA. In addition, the excrement samples were tested for the egg of distoma hepaticum by method of direct smear. Some subjects had ultrasonic inspection or operation and were proved the existence of live polypide or the eggs of polypide flowed out from biliary drainage.

Measurement of exposure to alcohol consumption, HBV infection and clonorchiasis

The subjects were asked about the history of consumption of beer, wine, and hard liquor in their lifetime. Alcohol drinkers were de fined as subjects who consumed at least 100 servings of any of these alcoholic beverages during their lifetime. The alcohol drinkers were asked to estimate their prior frequency of alcohol consumption and were placed into 1 of 4 categories: (1) none or less than 100 servings ever, (2) infrequent use, (3) occasional use (at least one serving per week), and (4) regular use (at least one serving per day). The consumed-serving unit was defined as 12.0 oz for beer, 4.0 oz for wine, and 1.5 oz for hard liquor, which were all considered to be approximately equivalent to 12.0 ml of ethanol[12]. Moreover, the subjects were classified according to the volume of total ethanol consumed. This volume was obtained by multiplying the number of standardized servings by the number of milliliters of ethanol per drink and then adding the results for beer, wine, and hard liquor intake. In addition, all patients’ medical records were reviewed to confirm alcohol consumption and to estimate the consumed amount. Heavy alcohol consumption was defined as consumption of ≥ 80 ml of ethanol per day[4].

The blood samples were tested for the presence of hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B core antigen (anti-HBc) using the ELISA. The positive result of HBsAg was defined as HBV infection.

The diagnostic criteria of clonorchiasis include: having the habit of eating raw food; positive result of the eggs of polypide in the excremen; the symptoms of the patients having gone after treatment with praziquantel; and some subjects examed by ultrasonic inspection or operation having been proved the existence of live polypide or the eggs of polypide flowing out form biliary drainage. The positive result of the egg of distoma hepaticum was defined to clonorchiasis.

In addition, smokers were defined as subjects who had smoked at least 100 cigarettes during their lifetime and subclassified into current smokers and ex-smokers.

Statistical analysis

The SPSS10.0 system for windows Statistical Software was used for data procesing and statistical analysis. For univariate analysis, conditional logistic regression was used with maximum likelihood estimate of parameter values for assessing the risk of HCC, while for multivariate analysis, the significant variables (P < 0.05) were modeled using conditional logistic regression. The odds ratio (OR) and 95% of confidence interval (CI) for each variable were estimated using the logistic regression coefficient.

The synergisms among risk factors were evaluated by considering the additive model in crossover analysis, since it is more appropriate to address biological interactions and public health concerns. Multiple logistic regression models were used to evaluate the departure from addictively. We matched the risk factors of HCC as the following: HBV infection and clonorchiasis, HBV infection and heavy alcohol consumption, heavy alcohol consumption and clonorchiasis and analyzed by crossover analysis. The additive model proposed by Rothman[13-15] was used to calculate the interactions. There are 3 calculating methods in the model:

  • (1) Relative excess risk of interaction, RERI: RERI= A−(B+C−1);

  • (2) Attributable proportions of interaction, API: API = [A−(B+C−1)]/A;

  • (3) The synergy index, S: S = (A−1)/[(B−1)+(C−1)].

A indicates the value of OR with the presence of 2 risk factors. B or C indicates the value of OR with the presence of each risk factor in the absence of other. The additive model proposed by Wang et al.[16] was used to calculate the χ2 value to evaluate whether the additive model has statistical significance.

Univariate analysis EF with the calculating formula:

EF = P (OR−1)/P (OR−1)+1; P stands for exposure rate of control group.

EF of interaction calculated by Walker’s method[17] fall into 3 categories:

  • (1) EF of 2 risk factors’ synergistic action: Embedded Image

  • (2) EF of 2 risk factors’ presence: Embedded Image

    R stands for relative risk; A or B indicates the effect of A or B; Ā or Embedded Image indicates the absence of the effect of A or B;

  • (3) The synergy index: I = EF (A×B)/EF (AB).

Results

Patient Characteristics

Table 1 shows the distribution of the patients with HCC and controls according to sex, age, nationality, residence, and level of education. Most of the subjects were Chuang people (HCC patients, 40.8%; controls, 36.4%); the rest were Han people (55.8%; 61.2%) and others (3.4%; 2.4%). Also, education levels of the subjects were: university or college or higher (HCC patients, 21.8%; controls, 18.8%); middle school (60.6%; 67.4%); primary school or no education received (17.6%; 13.8%). Additionally, Most of the subjects were residents of Nanning (HCC patients, 61.2%; controls, 66.3%). The city was the area of residence for 63.3% of the subjects, the rural area for 36.7%. HCC patients and the controls had a similar pattern of residence in Guangxi. In the aspects of nationality, level of education, and the distributions in the city and the countryside, the difference between the cases and the controls had no statistic significance (P > 0.05).

View this table:
Table 1.

Characteristics of subjects

Risk factors for developing HCC

Univariate analysis

An obviously significant difference of statistic in the prevalence of HBV infection between HCC patients and controls was observed (75.6% and 11.2%, respectively) (Table 2). The univariate OR for the association of HBV infection with HCC was 24.6 (95% CI, 17.4-34.7). In the study, because the rate of infection of HBV between HCC patients and controls was very low (patients 1.11% and controls 0.44%), the statistical analysis was unnecessary.

View this table:
Table 2.

Risk factors for HCC: univariate (crude) and multivariate (adjusted) OR and 95% CI

A history of consumption of more than 100 servings of alcohol in the lifetime of the subject was statistically significant (HCC patients, 55.6%; controls, 24.6%), resulting in an estimated univariate OR of 3.8 (95% CI, 2.9-5.0). HCC risk gradient (OR) was increased as the proportion of alcohol consumption was increased. A total of 240 HCC patients and 77 controls were classified as daily drinkers who consumed more than 80 ml of ethanol per day and got 5.3 (95% CI, 3.9-7.2) of the univariate OR (Table 2), whereas, those who consumed less than 80 ml of ethanol per day, the univariate OR was 1.4(95% CI, 0.9-2.2). The OR for daily alcohol consumption less than 80 ml was not statistically significant. Moreover, the risk of HCC in relation to preference for different types of alcoholic beverages and to the total amount of alcohol consumed was also assessed. A significant risk associated with HCC was consuming more than 80 ml of ethanol per day and having a preference for hard liquor (HCC patients, 28.9%; controls, 6.2% [OR, 7.3; 95% CI, 5.5-13.6]) or beer (HCC patients, 16.9%; controls 5.6% [OR, 4.2; 95% CI, 3.1-9.2]), compared with those in nondrinkers.

The presence of the history of clonorchiasis was also statistically associated with the development of HCC (HCC patients, 32.2%; controls, 7.2%). Table 2 shows that patients with the history of clonorchiasis had a risk of HCC 6 times greater than that of patients never clonorchiasis. It indicated that clonorchiasis was the risk factor on the development of HCC. The further research showed that OR value of the patients with the history of clonorchiasis less than ten years was 5.096 and the OR value of the patients with the history of clonorchiasis more than ten years was 18.031. The results showed that as the history of clonorchiasis was extended, the risk for developing HCC would increase and it would be greater for the patients with the history of clonorchiasis more than ten years.

A history of having smoked once was reported by 61.8% of HCC patients and 44.0% of controls, causing a crude OR of 2.1 (95% CI, 1.6-2.7). Current smokers were subdivided according to the number of packs smoked (> 1 or < 1 pack/d) and the duration of their habit (> 10 or < 10 year smoking). No consistent dose dependent responses were found in current smokers who smoked more than one pack per day (OR, 1.5; 95% CI, 1.1-3.3) or smoked for more than 10 years (OR, 2.7; 95%CI, 1.5-4.6) compared with those in nonsmokers. In addition, the presence of income less than 500 RMB per month was also statistically associated with the development of HCC (HCC patients, 63.8%; controls, 26.6%). Table 2 shows that patients with income less than 500 RMB per month had a risk of HCC 2 times greater than that of patients with income of 500 RMB or more per month.

Multivariate analysis

The results from multivariate analysis did not meaningfully differ from those observed in univariate analysis, as the OR and 95% of CI were 20.6 (14.3-29.7), 2.2 (1.3-3.8), and 5.0 (3.1-8.1) for HBV infection, alcohol consumption and clonorchiasis, respectively. Daily alcohol consumption more than 80 ml also resulted in a greater risk of HCC as OR showed (3.4; 95% CI, 2.3-5.0). However, the adjusted OR for smoking and income status dropped to a nonsignificant (P > 0.05) (Table 2).

Analyzed by non-conditional logistic regression, EFs of 3 risk factors were positive. EFs of HBV infection, heavy alcohol consumption, and clonorchiasis were 0.6881, 0.2699, 0.2236, respectively.

Interaction among HCC risk factors

Table 3 shows the independent and joint effects of heavy alcohol consumption (≥ 80 ml of ethanol per day), HBV infection, and the history of clonorchiasis by crossover analysis. In particular, there was a synergism among HBV infection, heavy alcohol consumption and clonorchiasis. These interactions fited the additive model. The results of analyzing the relative risk factors for developing HCC using the OR showed that the risk for developing HCC in the patients who had two risk factors existed such as heavy alcohol consumption combined with HBV infection, or HBV infection combined with clonorchiasis, or heavy alcohol consumption combined with clonorchiasis was increased times than that of the patients who had only one risk factor exsited: 63.2−1> 25.0 + 4.4−2; 121.9−1 > 25.4 + 6.5−2 and 22.8−1 > 5.8 + 4.8−2, respectively. The estimated synergism index (S) was significantly greater than 1. The synergism index of heavy alcohol consumption plus HBV infection, and the history of clonorchiasis plus HBV infection, and the history of clonorchiasis plus heavy alcohol consumption was 2.27, 4.04 and 2.53 respectively, with relative excess risk of interaction (RERI) of 34.8, 91.0 and 13.2; attributable proportion of interaction (API) of 0.5506, 0.7465and 0.5789 respectively (Table 3). Their additive model had statistical significance by statistics analyses (P < 0.05). This indicated that heavy alcohol consumption, HBV infection and clonorchiasis, in addition to HBV infection’s own direct effects, may exacerbate the synergistic action on HCC.

View this table:
Table 3.

Interaction of heavy alcohol consumption, chronic HBV infection and Hepatic distomiasis: logistic regression analysis with OR

Etiologic fraction (EF) among risk factors of HCC

Among the risk factors of heavy alcohol consumption, HBV infection and clonorchiasis, the synergism between HBV infection and clonorchiasis was most significant, with a synergy index of 0.7527. The synergy index of heavy alcohol consumption and HBV infection was 0.5594 and that of clonorchiasis and heavy alcohol consumption was 0.6055. This indicated that 55%-75% of HCC patients resulted from the synergism of any two of risk factors above. The EFs of interaction for heavy alcohol consumption, HBV infection and clonorchiasis showed significance on the development of HCC. Table 4 shows that, among 190 cases related to both alcohol consumption and HBV infection, 187 (190 × 0.9842) cases resulted from the coexistence of these two factors, and 105 (190 × 0.5506) cases resulted from the synergism of them. Among 121 cases related to both HBV infection and clonorchiasis, 120 (121 × 0.9918) resulted from the coexistence of HBV infection and clonorchiasis, and 90 (121 × 0.7465) resulted from the synergism of these two factors. Among 85 cases related to both heavy alcohol consumption and clonorchiasis, 81 (85 × 0.9561) cases resulted from the coexistence of these two factors, and 49 (85 × 0.5789) cases resulted from the synergism of them.

View this table:
Table 4.

Etiologic fraction of interaction of HBV infection and heavy alcohol consumption, HBV infection and clonorchiasis, clonorchiasis and alcohol consumption

Discussion

This is one of the largest case-control studies in Guangxi, China, that has been conducted to investigate the potential risk factors of HCC and interaction among these factors. It has shown that chronic HBV infection, alcohol consumption, and clonorchiasis are significant risk factors for developing HCC. Moreover, significant synergy among alcohol consumption, HBV infection, and clonorchiasis was found in the population of this study. Compared to the controls without tumors, alcohol consumption, HBV infection and clonorchiasis could make the risk of HCC increased as 3-, 21-, and 5-fold, respectively. Particularly, the risk of chronic HBV infection on developing HCC was significantly higher than that of other risk factors. There is a high incidence of viral hepatitis B in China. Indeed, among 350 million carriers of viral hepatitis B have been found all over the world and 120 million of them inhabit in China[18]. In our study, 75.6% of HCC patients with HBV infection and 11.2% of the controls with HBV infection were observed, and the positive rate of HBsAg in HCC cases was significantly higher than that of the controls. This implies that HBV infection is an important independent risk factor for developing HCC in Guangxi. This result is consistent with that of the research reported in the literature[6-8]. The underlying mechanism for HBV-induced HCC remains unclear. Traditionally, the mechanism for carcinogenesis is believed that chronic HBV infection induces cirrhosis and HCC. A direct carcinogenic role of HBV infection has been identified. Integration of the HBV genome into cellular DNA has been found in the majority of HBV-induced HCC patients. It has strong cancerogenesis, resulting in 200-fold increase in the risk of HCC[19,20]. Integration of the HBV DNA into hepatic cell induces gene mutation of hepatic cell. The results from the recent research show that hepatitis B virus X protein (HBx) can induce genic transcription expression of matrix metalloproteinase-9 (MMP-9), which is important to the development of HCC[21].

Distoma hepaticum is also named as clonorchiasis sinensis. It is a common parasitic disease resulting from the parasitism of clonorchiasis sinensis in the liver. Distoma hepaticum is common in the world, especially in middle of South America, Europe and Africa, etc. Presently, parasitic disease is an important disease which still harms the health of people in China. Guangxi province of China is one of the regions with the serious prevalence of parasitic disease. Clonorchiasis has been found in 60 counties and cities, especially in southern Guangxi. It has been estimated that the number of the patients infected by Distoma hepaticum in China has exceeded 12,000,000, among whom about 4,000,000 patients inhabit in Guangxi with an average infection rate of 9.76%. Department of Health had conducted a survey on the parasite affected in human body and the result showed that infection rate of clonorchiasis has been increased by 75% compared with the result of the investigation done 15 years ago, with the 164% increase in Guangxi[22]. Epidemiologic survey shows that the local people have the habits of eating raw fish or the porridge with raw piece of fish, which may lead to the infection of clonorchiasis[23]. Most people infected with distoma hepaticum do not show apparent symptoms. With the increasing of distoma hepaticum, secretion substance and their metabolic product in bile vessels oppress the wall of the bile vessels and obstruct the diminutive bile vessels, resulting in complications and even cholangiocarcinoma, hepatic cirrhosis and HCC[24-27]. Our study showed that 161 out of 500 HCC patients were accompanied with clonorchiasis and the rate reached up to 32.2%. Thirty six out of 500 control patients without tumor were accompanied with clonorchiasis and the rate was 7.2%. The prevalence of clonorchiasis in the control group was consistent with that of general population in China reported in the literature. OR value of HCC with the clonorchiasis was 6.1. It indicated that the rate of HCC patients with clonorchiasis was higher than that of the patients without tumors, and that the is one of the important risk factors for developing HCC. The study also showed that the OR value of HCC patients with the clonorchiasis less than ten years was 5.096, while that of HCC patients with the clonorchiasis more than ten years was 18.031. This indicated that with the extension of the history of clonorchiasis, the risk for developing HCC will increase and if the the history of clonorchiasis was over 10 years, the risk for developing HCC will be much increased.

Clonorchiasis is one of the important risk factors for developing HCC. Three factors contributed to this. First, the level of medical treatment and hygiene is relatively low. Second, the people who have been infected clonorchiasis are usually not aware, or ignore it even after they know that, or can’t afford to be treated, so, clonorchiasis in these patients repeatedly occurred. But infection rate of clonorchiasis is low in the developed countries such as Japan. There are some reasons for it. First, the level of medical treatment and hygiene in these counties are high. Second, people in these countries have realized the severity of this disease. Third, people infected by this disease are willing to be treated. In addition, the effectiveness of prevention and management is guaranteed. And the low rate of infection is also because of no repeated cases occurred after treatment.

The possible mechanisms include the followings. i) Mechanical injury. Internal migration of distoma hepaticum from intestine and liver can cause mechanical damage. Chronic excitation and inflammation in the bile duct caused by distoma hepaticum can result in epithelial proliferation of bile duct and transformation of adenoid tumor. The proliferative cells are damaged by carcinogen as a result, the DNA of cell multiplication are damaged, therefore, the malignant changes of epithelialis cells in bile duct, even hepatic cirrhosis and HCC may occur[24-27]. In addition, secretion substance and metabolic product of distoma hepaticum exert the toxic effect on the body of the patient and damage the liver[25]. ii) The increase of carcinogen of endogenous. In the bile duct with chronic inflammation, nitroso compound can be produced. Chronic cell nitrogen oxides and cellular epithelialis of bile duct is exposed to nitroso compound of high-concentration and precipitate the transformation of tumor[27]. iii) Activating the biocatalyst of drug. CYP-A6 (isoenzyme of CYTP450) metabolism can be found in hepatic carcinoma of the patients with distoma hepaticum. In tela the obvious metabolism of NDMA by CYP-E1 and CYP-A6 produces praeparatum of methylation damaging DNA[28]. iv) The increase of nitrogen monoxidum. In the section of chronic inflammation caused by distomatosis hepatic in the bile duct, macrophage is activated by specificity T-cell and cell factor of parasites and synthesize nitrogen monoxidum (L-arginine) which exerts toxicant to both cell and gene. The proliferating epithelium tissue in the bile duct is exposed to the product of genotoxicity inflammation, which creates the circumstance for the tissue developing malignant.

This study also shows that alcohol consumption is an independent risk factor for HCC. Although the positive association between alcohol consumption and HCC was observed in the ever-drinking group in our study, we believe that the true risk of alcohol consumption is related to the heavy consumption which was defined as daily consumption over 80 ml of ethanol. The results in our study, which indicated that HCC development was correlated with the magnitude of alcohol consumed is consistent with data from previous case-control studies[29]. In addition, the study shows that the risk of HCC is increased 6 times if the daily alcohol consumption is increased around 80 to 160 ml per day and 19 times if daily alcohol consumption exceeds 160ml. The result is consistent with that reported by Morgan et al.[30] It is usually believed that the risk for developing HCC can be attributed to recall bias in the HCC patients. However, both HCC patients and the controls were interviewed by the same interviewer, and the information collected about alcohol consumption was confirmed using their medical records, so we observed no discrepancies between the two groups. Most importantly, the prevalence of heavy drinking in the control group was consistent with that of general population in China reported by Li et al.[31]

In terms of the underlying mechanisms for alcohol-induced HCC, previous studies have suggested that heavy alcohol consumption could induce direct hepatic cellular injury and toxicity leading to the development of liver fibrosis and cirrhosis[32,33]. Recent study shows that the liver of human beings can only complete the metabolism of 80 ml alcohol, and over 80 ml of alcohol intake per day increases the concentration and accumulation of acetaldehyde (metabolic product of alcohol), which induces carcinogenicity and genic mutation. Therefore, the alcohol intake over 80 ml per day plays an important role in the process of carcinogenicity[34]. Moreover, oxidation and metabolism of ethanol in the liver by microsomal enzymes may contribute to the increased conversion of procarcinogens to carcinogens[35]. Alcohol induces cytochromeP450-E1 and increases free radicle, which can worsen hepatic injury, hepatic fibrosis and hepatic cirrhosis of the patients with type B hepatitis. They can also demolish the immune system, which results in the affectability of HBV [32,35,36]. The recent research in Japan shows that the integrity and fraction of HBV-DNA exist in DNA of HCC cell of alcoholist with negative HBsAg. The unapparent HBV infection in some alcoholists plays a significant role in HCC development[37]. The possible mechanism includes the chromosome elimination, oxidative stress, decrease of retinoic acid in hepatic tissue, DNA methylation and the increase of genetic predisposition[7,30].

Most importantly, this study shows that there exists the synergy (excess over additivity) between heavy alcohol consumption and chronic HBV infection, heavy alcohol consumption and clonorchiasis, and chronic HBV infection and clonorchiasis. This finding has biologic implication and indicates the status of the public health in the etiology of HCC[13,38].

The study analyzes the synergism among HBV infection, heavy alcohol consumption and clonorchiasis, and indicates the positive additive interaction by crossover analysis. By the hypothesis test in statistics, the synergism is statistically significant (P < 0.05). Our findings are consistent with those of previous studies[4-7,32]. Without synergism, the OR values of HBV infection, heavy alcohol consumption and clonorchiasis are 20.6, 3.4, and 5.0, respectively. With synergism, however, the OR values of HBV infection and heavy alcohol consumption, HBV infection and clonorchiasis, heavy alcohol consumption and clonorchiasis are increased sharply to 63.2, 121.9, and 22.8, respectively. EF of interaction shows that the cases resulted from the synergism among HBV infection, heavy alcohol consumption and clonorchiasis account for 55%-75% of those resulted from co-existing factors. This indicates that 55%-75% HCC patients result from the synergism among HBV infection, heavy alcohol consumption and clonorchiasis, especially that of HBV infection and clonorchiasis. It has shown that the synergism among HBV infection, heavy alcohol consumption and clonorchiasis plays an important role in the development of HCC.

The risk of interaction between HBV infection and clonorchiasis on the development of HCC is 91 times of that of each individual risk factor. Seventy four point six percent of HCC patients result from the interaction of HBV infection and clonorchiasis. The risk of the interaction of HBV infection and clonorchiasis is 4 times of that of the sum of the two independent factors. The risk of interaction between heavy alcohol consumption and clonorchiasis is 13 times of those of other individual risk factors. Fifth seven point nine percent of HCC patients result from the interaction of heavy alcohol consumption and clonorchiasis. The risk of the interaction of heavy alcohol consumption and clonorchiasis is 2.5 times of that of the sum of the two independent factors. Hence, with the coexistence of HBV infection and alcohol consumption, clonorchiasis may accelerate the hepatic injury and induce cancerization. More researches should be conducted to explain the carcinogenesis mechanism. With respect to the interaction between heavy alcohol consumption and HBV infection, both risk factors are significantly associated with HCC. The risk of coexistence of HBV infection and heavy alcohol consumption in the development of HCC is about 35 times of those of other individual factors. Fifty five point one percent of HCC patients result from the risk of interaction of HBV infection and heavy alcohol consumption. The risk of the interaction between heavy alcohol consumption and HBV infection is 2 times of that of the sum of the two risk factors. One common mechanism derived from which both HBV infection and heavy alcohol consumption may contribute to cirrhosis as well as hepatocarcinogenesis is oxidative stress. It has suggested that the increased level of free radical induced by the long term consumption of alcohol may promote HBV gene expression and favor the establishment of a chronic HBV carrier state following acute infection or reactivation of virus replication in a previously established infection. An increasing HBV load in liver cells may further intensify liver disease chronicity induced by heavy alcohol consumption toward advanced cirrhosis and/or liver cancer[39,40].

Our study simultaneously evaluates, for the first time, the relationship between HCC and all risk factors of heavy alcohol consumption, HBV infection and clonorchiasis. Like many other case–control studies, it is a hospital-based and not a population-based study. There may be several limitations. Firstly, the recruitment rate of cases is low because of the rapidly fatal feature of HCC (the median time interval between diagnosis and death is approximately 3 months)[41]. Most of eligible patients with HCC died before we attempted to contact. However, there is no evidence of a differential in demographic profile between eligible patients excluded from the study and those recruited. Secondly, there may be some selection bias. To minimize the selection bias, we selected cases and controls from clinics in the same institution to decrease potential differences such as the differences with regard to access to care, referral patterns, and socioeconomic status. Finally, the assessment of the participants exposed to alcohol consumption and clonorchiasis during their life time may be inaccurate due to recall bias. We tried to address this concern by using validated questionnaires administered by a trained interviewer. The interviewer was not blinded to case-control status and we did not eliminate the possibility that HCC patients may have been probed more thoroughly than the controls, leading to recall bias. In addition, we chose to use hospital controls and not population-based controls whose ages and sex were matched to those of HCC patients[42]. Conditional logistic regression was performed to keep the matching during the statistical analysis. To the extent that the proportions of the significant risk factors reflect the same pattern of the general population of Guangxi, China, the present study gives an accurate estimate of the independent effect of HBV infection, heavy alcohol consumption, and a history of clonorchiasis on HCC development in Guangxi.

In conclusion, we have shown that alcohol consumption, HBV infection, and clonorchiasis are independent risk factors for developing HCC. Public health considerations should prompt the study of HCC prevention strategies among high-risk individuals. Moreover, the three risk factors appear to act synergistically in increasing the risk of HCC when compared with those of the controls. Finally, our data needs to be validated with prospective studies in large multi-center. If confirmed, our data will provide a basis for the first-level precautionary measure in Guangxi, China.

Footnotes

  • This work was supported by a grant from the National Natural Science Foundation of China (No. 30660162).

  • Received October 14, 2008.
  • Accepted March 30, 2009.

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