Clinical research
Prevalence of cigarette smoking and smoking-related disease correlates in Iranian asymptomatic HBV carriers

Introduction
Hepatitis B virus (HBV) is a serious public health problem worldwide and is considered a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Approximately two billion people have serological evidence of past or present HBV infection, and more than 350 million are asymptomatic chronic carriers of HBV [1]. Around 75% of these chronic carriers live in Asia and the Western Pacific [2]. The prevalence of hepatitis B in the general population of Iran has been reported to be between 1 (Shiraz) to 9% (Toyserkan) [3], with a total of two million asymptomatic HBV carriers living in the country [4].
With regard to chronic hepatitis C, several studies have reported the negative impact of smoking on the aggravation of liver cirrhosis and response to treatment, health-related quality of life, and also mortality [5-12]. These studies have documented significant associations between smoking and elevated alanine transaminase (ALT) level [5], increased fibrosis, activity scores and severity of hepatic lesions [10], the risk for developing liver fibrosis [8], aggravation of cirrhosis in hospitalized patients [9], low response rates to interferon-alpha [6], and the risk of developing HCC [11, 12].
Within studies regarding smoking and HBV infection [10, 13-16], most have focused on possible effects of smoking on chronic active hepatitis [10, 14, 15]. A possible hepatotoxicity of cigarette smoking in HBV patients has been proposed by epidemiological and experimental studies [10]. In China, the high mortality rate of smokers through liver cancer has been attributed to the high prevalence of chronic HBV infection [15]. Furthermore, cigarette smoking is related to an increased expression of neuoncogene [13] and has adverse effects on liver cirrhosis development [16].
The aim of this study was to determine the prevalence of self report smoking in asymptomatic HBV carriers and evaluate some of its demographic, clinical and laboratory correlates.

Material and methods
In a cross-sectional study, we evaluated 1116 asymptomatic HBV carriers from patients of the hepatitis clinic of Tehran Blood Transfusion Organization between 2002 and 2006.
The state of asymptomatic HBV carrier was defined as persistent HBV infection (steady positivity for HBsAg in the serum) for more than six months, and without any significant, ongoing necroinflammatory liver disease [17]. The diagnosis of asymptomatic HBV carrier state was established by a hepatologist (SMA) or two infectious disease specialists (MK, BB). Patients with any other chronic liver diseases, a symptomatic infection, or a co-infection with hepatitis C (HCV), hepatitis D, or human immunodeficiency virus (HIV) were excluded.
Participants completed a structured questionnaire, which was then checked by an interviewer. The questionnaire collected extensive data on demographic data (sex, age, marital status and level of education), and also medical history (cigarette smoking, alcohol consumption, and a history of liver disease in self or family members).
The self report cigarette smoking was assessed according to two questions in the questionnaire: 1) ”Have you smoked at least 100 cigarettes in your entire life?” and 2) ”Do you now smoke cigarettes every day?”. Based on the first question, we divided our subjects into two groups: ever smokers (smoked at least 100 cigarettes), and never smokers (smoked less than 100 cigarettes per lifetime) [18]. The smoking index (cigarette pack-year), defined as the number of years of smoking twenty cigarettes a day.
The drinking pattern was assessed using the graduated frequency (GF) method: how often during the past 12 months did the subjects drink more than X amount of alcohol [19]. In the version of the GF used in this study, there were nine mutually exclusive categories of frequency, ranging from ”never’’ to ”every day’’, and seven mutually exclusive categories of alcohol dose (expressed in local units of beer, wine, and spirits), ranging from 10 γ to >160 of pure alcohol (details available from authors). Alcohol intake measures were recalculated into grams of ethanol. We used several definitions of binge drinking: >120 γ of ethanol at least once a month [20].
Paraclinical information, including complete blood count, liver function tests, ESR, PT, albumin, FBS and lipid profile (total cholesterol and triglyceride), were also collected. The presence of fatty liver, assessed by ultrasound imaging according to published literature, was also registered [21].
Data analysis was carried out using the statistical software of SPSS-13 for Windows. To study the correlates of ever cigarette smoking, we used chi-squared and to evaluate the correlates of smoking index, we used independent sample t-test. P<0.05 was considered significant.

Results
Patient population
The mean age of patients was 37.1±11.6 years. 857 (76.8%) subjects were male and 259 (23.2%) female. 895 (80.2%) subjects were married and 221 (19.8%) single. 331 (29.7%) patients had an educational level of below diploma and 785 (70.3%) had diploma or an academic degree.

Smoking and its correlates
The prevalence of cigarette smoking in asymptomatic carriers of HBV was 13.5% (151 cases of 1116). Table I compares demographic, clinical and paraclinical characteristics of smoker and non-smoker asymptomatic HBV carriers. The relative risk for developing fatty liver in smokers versus non-smokers was 1.79 (95% CI: 1.16-2.75). Frequency of fatty liver was not significantly different between smokers with and without alcohol use (10.5 vs. 16.8%; p=0.44).
There was no association between the presence of fatty liver and age, marital status, level of education, and self-reported ethanol consumption, hypertension and diabetes mellitus. Also the comparison of complete blood count, liver function tests, ESR, PT, albumin, FBS and lipid profile (total cholesterol and triglyceride) between smokers and non-smokers were not significantly different.

Smoking index and its correlates
Mean smoking index in asymptomatic HBV carriers was 17.0±8.6 pack-years (for 151 smoker patients). Developing fatty liver was correlated with a higher smoking index (19.1±9.9 vs. 16.8±8.6 pack-years). Smoking index was correlated with age (p=0.001, r=0.302), and was not associated with marital status, level of education, self-reported ethanol consumption, complete blood count, liver function tests, ESR, PT, Albumin, FBS and lipid profile.

Discussion
In this study, we showed that the prevalence of cigarette smoking in Iranian asymptomatic HBV carriers was 13.5%. The prevalence of cigarette smoking in the general population is different between countries, and even different between provinces of a country. In Iran, for instance, this rate has been reported to be 16.8% in Shiraz [22] and 22.3% in Tehran [23].
Another important finding of this study was that men with chronic hepatitis had a higher rate of cigarette smoking compared with their female counterparts. Similar rates of smoking have been reported in the general population of Iran, with rates of 18.5-26% in men and 3.6-10.1% in women [22, 24, 25]. We also found that older patients tended to smoke more commonly compared with younger subjects, which is in line with a former report from our country [22]. With respect to the association between smoking and educational level, the results of this study contrast those of one report from Iran which has shown a significant link between these two items [26].
Our study showed an association between ethanol intake and cigarette smoking, in agreement with the results of Bierut et al. [27]. One study reported that up to 80% of persons who were alcohol dependent were also smokers [28]. This combination has been named as a deadly combination [29].
We could not draw any significant correlation between smoking and liver enzymes. In this respect, although smoking has been reported to be associated with elevated ALT levels among anti-HCV-seropositive individuals, such evidence is lacking in HBV infected persons [5] as well as in the general population [30].
From the numerous studies assessing the risk factors of fatty liver in the general population, some have neglected the possible effect of cigarette smoking [31, 32], some have shown smoking as an associated factor with metabolic disturbances [33] and risk factors of fatty liver [34], and some others have failed to demonstrate such links [35, 36]. Our results exhibited a link between fatty liver and cigarette smoking in asymptomatic HBV carriers. This may be considered a very important finding because fatty liver and asymptomatic HBV carrier state are shown to have a significant synergistic association with liver damage [37]. The findings that fatty liver sensitizes the host’s immune response to HBV infection have been reported by some studies [38, 39]; however, according to previous epidemiological studies, the coexistence of fatty liver and HBV carrier state is not uncommon in clinical practice [40]. Unfortunately, even the studies conducted in 2007 focusing on fatty liver in asymptomatic carriers of HBV have neglected the possible importance of smoking [37, 41].
Surprisingly, we could not find any association between fatty liver and alcohol consumption, which clearly contrasts literature. One possible explanations for this finding may be the relatively small sample size of this study. It is also important to note that the questionnaire used did not inquire about the amount of alcohol consumption. Previous reports suggest that low amounts of alcohol consumption does not increase risk of developing fatty liver [42].
Different mechanisms have been applied to explain the negative impacts of smoking in HBV carriers. N-acetyltransferase (NAT2) activity may be particularly critical in smoking related hepatocarcinogenesis among chronic HBV carriers [43]; N-acetyltransferase (NAT) is involved in the metabolic activation and detoxification of aromatic amines [43]. Nicotine, a major component of tobacco smoke, is rapidly absorbed through the lungs and released into the circulation, and is mainly metabolized by the liver. The effects of nicotine, both alone and combined with carbon tetrachloride (CCl4), have been studied on the liver of rats [44]. CCl4 induced liver lesions are also aggravated by tobacco nicotine, which has been ascribed to oxidative stress associated with lipid peroxidation both in vitro and in vivo [45-48]. Also, experimental studies on rats have shown that nicotine can cause deleterious effects to the liver, notably by aggravating the hepatotoxic effects of CCl4 [44].
Application of effective smoking cessation programs has been strongly encouraged for all populations. Self-help strategies for smoking cessation, individual and combined pharmacotherapy along with counseling, can help smokers quit smoking [49]. Although specific strategies for smoking cessation have been previously developed for pregnant women [50], patients with coronary artery disease [51], and subjects with chronic obstructive pulmonary disease [52]. It has been shown in one study that smoking cessation will provide a healthier lifestyle for HBV carriers, however, that study had not provide a smoking cessation strategy for this population [53].
The main limiting point of the current study was the minimal data regarding some risk factors of fatty liver such as body mass index. Another limiting point of the current study was that smoking habits were assessed by a questionnaire not with laboratory data on plasma nicotine or urinary nicotine levels to establish the levels of smoking or exposure to smoking.

Conclusions
Because of the link between cigarette smoking and fatty liver in asymptomatic HBV carriers, it is recommended that smoker HBV carriers be screened more closely for possible evidence of fatty liver. Furthermore, strategies for smoking cessation should be specifically designed for carriers of HBV.

References
1. Hepatitis B. World Health Organization Fact Sheet 204. World Health Organization; [2000]. Available at: http://www.pubmedcentral.nih.gov/redirect3.cgi?&&reftype=extlink&artid=1142225&iid=119118&jid=257&FROM=Article|CitationRef&TO=External|Link|URI&article-id=1142225 &journal-id=257&renderingtype=normal&&www.who.int/ mediacentre/factsheets/fs204/en/.
2. Gust ID. Epidemiology of hepatitis B infection in the Western Pacific and South East Asia. Gut 1996; 38 (suppl 2): S18-S23.
3. Zali MR, Mohamed K, Farhadi A, Masjedi MR, Zargar A, Nowroozi A. Epidemiology of hepatitis B in Islamic Republic of Iran. East Mediterr Health J 1996; 2: 290-8.
4. Sharifi MR, Ghoraishian SM. Epidemiology of chronic vector for Hepatitis B surface antigen in Yazd. J Sh Sad Univ Med Sci Heal Ser 1378; 2: 9-15.
5. Wang CS, Wang ST, Chang TT, Yao WJ, Chou P. Smoking and alanine aminotransferase levels in hepatitis C virus infection: implications for prevention of hepatitis C virus progression. Arch Intern Med 2002; 162: 811-5.
6. El-Zayadi A, Selim O, Hamdy H, et al. Impact of cigarette smoking on response to interferon therapy in chronic hepatitis C Egyptian patients. World J Gastroenterol 2004; 10: 2963-6.
7. Balfour L, Cooper C, Kowal J, et al. Depression and cigarette smoking independently relate to reduced health-related quality of life among Canadians living with hepatitis C. Can J Gastroenterol 2006; 20: 81-6.
8. Dev A, Patel K, Conrad A, Blatt LM, McHutchison JG. Relationship of smoking and fibrosis in patients with chronic hepatitis C. Clin Gastroenterol Hepatol 2006; 4: 797-801.
9. Pessione F, Ramond MJ, Peters L, et al. Five-year survival predictive factors in patients with excessive alcohol intake and cirrhosis. Effect of alcoholic hepatitis, smoking and abstinence. Liver Int 2003; 23: 45-53.
10. Pessione F, Ramond MJ, Njapoum C, et al. Cigarette smoking and hepatic lesions in patients with chronic hepatitis C. Hepatology 2001; 34: 121-5.
11. Mori M, Hara M, Wada I, et al. Prospective study of hepatitis B and C viral infections, cigarette smoking, alcohol consumption, and other factors associated with hepatocellular carcinoma risk in Japan. Am J Epidemiol 2000; 151: 131-9.
12. Chiba T, Matsuzaki Y, Abei M, et al. The role of previous hepatitis B virus infection and heavy smoking in hepatitis C virus-related hepatocellular carcinoma. Am J Gastroenterol 1996; 91: 1195-203.
13. Yu MW, Chen CJ, Luo JC, Brandt-Rauf PW, Carney WP, Santella RM. Correlations of chronic hepatitis B virus infection and cigarette smoking with elevated expression of neu oncoprotein in the development of hepatocellular carcinoma. Cancer Res 1994; 54: 5106-10.
14. Winter AP, Follett EA, McIntyre J, Stewart J, Symington IS. Influence of smoking on immunological responses to hepatitis B vaccine. Vaccine 1994; 12: 771-2.
15. Chen ZM, Liu BQ, Boreham J, Wu YP, Chen JS, Peto R. Smoking and liver cancer in China: case-control comparison of 36,000 liver cancer deaths vs. 17,000 cirrhosis deaths. Int J Cancer 2003; 107: 106-12.
16. Yu MW, Hsu FC, Sheen IS, et al. Prospective study of hepatocellular carcinoma and liver cirrhosis in asymptomatic chronic hepatitis B virus carriers. Am J Epidemiol 1997; 145: 1039-47.
17. Yalcin K, Degertekin H, Alp MN, Tekes S, Satici O, Budak T. Determination of serum hepatitis B virus DNA in chronic HBsAg carriers: clinical significance and correlation with serological markers. Turk J Gastroenterol 2003; 14: 157-63.
18. State-Specific Prevalence of Cigarette Smoking and Quitting Among Adults – United States, 2004. CDC. MMWR. November 11, 2005/54; 1124-1127. Available at: http://www. cdc.gov/mmwr/preview/mmwrhtml/mm5444a3.htm.
19. Greenfield TK. Ways of measuring drinking patterns and the difference they make: experience with graduated frequencies. J Subst Abuse 2000; 12: 33-49.
20. Bobak M, Room R, Pikhart H, et al. Contribution of drinking patterns to differences in rates of alcohol related problems between three urban populations. J Epidemiol Community Health 2004; 58: 238-42.
21. Kawai N, Kawai T, Kawai K. Ultrasonic and laboratory studies on fatty liver in white-collar workers [Japanease]. Nippon Shokakibyo Gakkai Zasshi 1995; 92: 1058-65.
22. Ahmadi J, Khalili H, Jooybar R, Namazi N, Mohammadagaei P. Prevalence of cigarette smoking in Iran. Psychol Rep 2001; 89: 339-41.
23. Azizi F, Rahmani M, Emami H, et al. Cardiovascular risk factors in an Iranian urban population: Tehran lipid and glucose study (phase 1). Soz Praventivmed 2002; 47: 408-26.
24. Kelishadi R, Ardalan G, Gheiratmand R, et al. Smoking behavior and its influencing factors in a national-representative sample of Iranian adolescents: CASPIAN study. Prev Med 2006; 42: 423-6.
25. Sarraf-Zadegan N, Boshtam M, Shahrokhi S, et al. Tobacco use among Iranian men, women and adolescents. Eur J Public Health 2004; 14: 76-8.
26. Roohafza HR, Sadeghi M, Kelishadi R. Cardiovascular risk factors in Iranian adults according to educational levels: Isfahan healthy heart program. Asia Pac J Public Health 2005; 17: 9-14.
27. Bierut LJ, Schuckit MA, Hesselbrock V, Reich T. Co-occurring risk factors for alcohol dependence and habitual smoking. Alcohol Res Health 2000; 24: 233-41.
28. Romberger DJ, Grant K. Alcohol consumption and smoking status: the role of smoking cessation. Biomed Pharmacother 2004; 58: 75-83.
29. Mihaltan F, Dragomir A, Ulmeanu R, Ciontu M, Munteanu I. Smoking and alcohol: a deadly combination [Romanian]. Pneumologia 2006; 55: 80-4.
30. Whitehead TP, Robinson D, Allaway SL. The effects of cigarette smoking and alcohol consumption on serum liver enzyme activities: a dose-related study inmen. Ann Clin Biochem 1996; 33: 530-5.
31. Chen CH, Huang MH, Yang JC, Nien CK, Yang CC, Yeh YH, Yueh SK. Prevalence and risk factors of nonalcoholic fatty liver disease in an adult population of Taiwan: metabolic significance of nonalcoholic fatty liver disease in nonobese adults. J Clin Gastroenterol 2006; 40: 745-52.
32. Zelber-Sagi S, Nitzan-Kaluski D, Halpern Z, Oren R. Prevalence of primary non-alcoholic fatty liver disease in a population-based study and its association with biochemical and anthropometric measures. Liver Int 2006; 26: 856-63.
33. Foy CG, Bell RA, Farmer DF, Goff DC Jr, Wagenknecht LE. Smoking and incidence of diabetes among U.S. adults: findings from the Insulin Resistance Atherosclerosis Study. Diabetes Care 2005; 28: 2501-7.
34. Mendez-Sanchez N, Chavez-Tapia NC, Uribe M. An update on non-alcoholic fatty liver disease [Spanish]. Rev Invest Clin 2004; 56: 72-82.
35. Chavez-Tapia NC, Lizardi-Cervera J, Perez-Bautista O, Ramos-Ostos MH, Uribe M. Smoking is not associated with nonalcoholic fatty liver disease. World J Gastroenterol 2006; 12: 5196-200.
36. Bedogni G, Miglioli L, Battistini N, Masutti F, Tiribelli C, Bellentani S. Body mass index is a good predictor of an elevated alanine transaminase level in the general population: hints from the Dionysos study. Dig Liver Dis 2003; 35: 648-52.
37. Lin YC, Hsiao ST, Chen JD. Sonographic fatty liver and hepatitis B virus carrier status: synergistic effect on liver damage in Taiwanese adults. World J Gastroenterol 2007; 13: 1805-10.
38. Shabot JM, Jaynes C, Little HM Jr, Alperin JB, Snyder N. Viral hepatitis in pregnancy with disseminated intravascular coagulation and hypoglycemia. South Med J 1978; 71: 479-81.
39. Canbay A, Bechmann LP, Best J, Jochum C, Treichel U, Gerken G. Crohn’s disease-induced non-alcoholic fatty liver disease (NAFLD) sensitizes for severe acute hepatitis B infection and liver failure. Z Gastroenterol 2006; 44: 245-8.
40. Gordon A, McLean CA, Pedersen JS, Bailey MJ, Roberts SK. Hepatic steatosis in chronic hepatitis B and C: predictors, distribution and effect on fibrosis. J Hepatol 2005; 43: 38-44.
41. Bondini S, Kallman J, Wheeler A, et al. Impact of non-alcoholic fatty liver disease on chronic hepatitis B. Liver Int 2007; 27: 607-11.
42. Thomopoulos KC, Arvaniti V, Tsamantas AC, et al. Prevalence of liver steatosis in patients with chronic hepatitis B: a study of associated factors and of relationship with fibrosis. Eur J Gastroenterol Hepatol 2006; 18: 233-7.
43. Yu MW, Pai CI, Yang SY, et al. Role of N-acetyltransferase polymorphisms in hepatitis B related hepatocellular carcinoma: impact of smoking on risk. Gut 2000; 47: 703-9.
44. Yuen ST, Gogo AR, Luk IS, et al. The effect of nicotine and its interaction with carbon tetrachloride in the rat liver. Pharmacol Toxicol 1995; 77: 225-30.
45. Gogo AR, Cho CH, Yuen ST, et al. The cytotoxic effect of nicotine on the liver and its modulation of hepatotoxicity induced by carbon tetrachloride in rats. Eur J Gastro Hepatol 1993, 5: 859-65.
46. Bagchi M, Bagchi D, Hassoun EA, Stohs SJ. Smokeless tobacco induced increases in hepatic lipid peroxidation, DNA damage and excretion of urinary lipid metabolites. Int J Exp Pathol 1994; 75: 197-202.
47. Panda K, Chattopadhyay R, Chattopadhyay DJ, Chatterjee IB. Vitamin C prevents cigarette smoke-induced oxidative damage in vivo. Free Radic Biol Med 2000; 29: 115-24.
48. Baskaran S, Lakshmi S, Prasad PR. Effect of cigarette smoke on lipid peroxidation and antioxidant enzymes in albino rat. Indian J Exp Biol 1999; 37: 1196-200.
49. Ranney L, Melvin C, Lux L, McClain E, Lohr KN. Systematic review: smoking cessation intervention strategies for adults and adults in special populations. Ann Intern Med 2006; 145: 845-56.
50. Wisborg K, Obel C, Henriksen TB, Hedegaard M, Secher NJ. Strategies for smoking cessation among pregnant women. Ugeskr Laeger 1994; 156: 6881-2.
51. Hobbs SD, Bradbury AW. Smoking cessation strategies in patients with peripheral arterial disease: an evidence-based approach. Eur J Vasc Endovasc Surg 2003; 26: 341-7.
52. Wagena EJ, van der Meer RM, Ostelo RJ, Jacobs JE, van Schayck CP. The efficacy of smoking cessation strategies in people with chronic obstructive pulmonary disease: results from a systematic review. Respir Med 2004; 98: 805-15.
53. Tan NC, Cheah SL, Teo EK. A qualitative study of health-seeking behavior of Hepatitis B carriers. Singapore Med J 2005; 46: 6-10.