Urinary 15-F 2t -isoprostane, aﬂatoxin B 1 exposure and hepatitis B virus infection and hepatocellular carcinoma in Taiwan

To evaluate the role of oxidative stress and aﬂatoxin exposure on risk of hepatocellular carcinoma (HCC), a case–control study nested within a large community-based cohort was conducted in Taiwan. Baseline urine samples, collected from a total of 74 in-cident HCC cases and 290 matched controls, were used to deter- mine by enzyme-linked immunosorbent assays the level of urinary 15-F 2t -isoprostane (15-F 2t -IsoP), a biomarker of lipid peroxida- tion. These samples had been previously analyzed for urinary aﬂatoxin B 1 (AFB 1 ) metabolites and 8-oxo-7,8-dihydro-2 # -deoxy- guanosine (8-oxodG). Pearson partial correlation coefﬁcient analysis showed that urinary AFB 1 metabolites and 8-oxodG were signiﬁcantly associated with the level of urinary 15-F 2t -IsoP. After adjustment for potential confounding factors in a conditional logistic regression model, urinary 15-F 2t -IsoP was signiﬁcantly associated with risk of HCC [above versus below the mean odds ratio (OR) 5 2.53, 95% conﬁdence interval (CI) 5 1.30–4.93]. Moreover, when compared with subjects in the lowest tertile of 15-F 2t -IsoP, there was a trend of increasing risk of HCC ( P trend 5 0.0008), with adjusted ORs (95% CIs) of 3.87 (1.32– 11.38) and 6.27 (2.17–18.13) for the second and third tertile, respectively. In addition, the combination of urinary 15-F 2t -IsoP above the mean and chronic hepatitis B virus (HBV) infection resulted in an OR of 19.01 (95% CI 5 6.67–54.17) compared with those with low urinary 15-F 2t -IsoP and without HBV infection. These results suggest that elevated levels of urinary 15-F 2t -IsoP may be related to increasing level of aﬂatoxin exposure and are associated with an increased risk of HCC.


Introduction
In Taiwan, primary hepatocellular carcinoma (HCC) is the leading cause of cancer death for males and the second for females. Epidemiological evidence suggests that dietary exposure to aflatoxin B 1 (AFB 1 ) and chronic infection with hepatitis B virus (HBV) are major risk factors for HCC (1). In a previous prospective study in Taiwan, we demonstrated that the presence of AFB 1 -albumin adducts as well as urinary AFB 1 metabolites were associated with increased risk of HCC (2). A viral-chemical interaction was also observed (2). Similar results were observed in a study carried out in China (3).
Although a number of studies have demonstrated that increasing AFB 1 exposure results in increasing HCC risk, the underlying mechanisms leading to development of HCC are not fully understood. One possible mechanism of AFB 1 -related hepatocarcinogenesis is the induction of oxidative DNA damage in liver tissue (4,5). Reactive oxygen species (ROS) have also been suggested to be involved in the progression of chronic liver disease and the occurrence of HCC (6). In our recent study, we found that the level of urinary 8-oxo-7,8-dihydro-2#-deoxyguanosine (8-oxodG) was associated with AFB 1 exposure in a dose-dependent manner suggesting that AFB 1 -induced oxidative DNA damage may constitute an important pathway in AFB 1 hepatocarcinogenesis (7). Oxidative stress, mediated by ROS, may result in direct DNA damage as well as in lipid peroxidation (8). The measurement of F2-isoprostanes (IsoPs), produced during peroxidation of membrane lipids by free radicals and ROS, is a specific and sensitive marker of lipid peroxidation (9). IsoPs are compounds derived from arachidonic acid via a free radical-catalyzed mechanism. IsoPs are cleaved from the sites of their origin and then circulate in plasma and are excreted in urine (10). 15-F 2t -isoprostane (15-F 2t -IsoP), one class of isoprostanes, has been recognized as a specific, chemically stable, quantitative marker of oxidative stress and can be detected in peripheral blood and in urine (9,10).
Measurement of 15-F 2t -IsoP in urine or plasma has been shown to reflect the oxidative stress of the body in patients with a variety of disease conditions (11,12). It has also been suggested that the measurement of 15-F 2t -IsoP can be used as a biomarker of exposure to relevant carcinogens and may predict cancer risk (12)(13)(14). Increasing urinary 15-F 2t -IsoP has been demonstrated to be associated with breast cancer (14). There are no human data on the effect of AFB 1 exposure and the combined effect of chronic HBV infection and AFB 1 exposure on the level of urinary 15-F 2t -IsoP. The significance of AFB 1 -induced oxidative lipid damage in the carcinogenicity of AFB 1 has not been well investigated nor has any long-term follow-up study investigated the effect of oxidative lipid damage on the development of HCC. The specific aims of this study were to investigate, among subjects without HCC, whether oxidative lipid damage, as assessed by urinary excretion 15-F 2t -IsoP, is associated with AFB 1 exposure, as assessed by urinary AFB 1 metabolites, and to examine the role of urinary 15-F 2t -IsoP levels in HCC risk using data in an ongoing nested casecontrol study of susceptibility to AFB 1 -related HCC (2,7).

Study cohort
Subjects are from the community-based Cancer Screen Program cohort recruited in Taiwan. This study was approved by Columbia University's Institutional Review Board as well as the research ethics committee of the College of Public Health, National Taiwan University, Taipei, Taiwan. Written informed consent was obtained from all subjects, and strict quality controls and safeguards were used to protect confidentiality. The cohort characteristics and methods of screening and follow-up have been described in detail previously (2,7). Briefly, individuals who were between 30 and 64 years old and lived in seven townships in Taiwan, three located on Penghu Islets that has the highest HCC incidence rates in Taiwan, and the other four from Taiwan Island, were recruited between July 1990 and June 1992. A total of 12 020 males and 11 923 females were enrolled. Participants were personally interviewed based on a structured questionnaire regarding epidemiological information and donated a 20 ml fasting blood sample as well as a spot urine at recruitment. Biospecimen samples were transported on dry ice to a central laboratory at the National Taiwan University and stored at À70°C. Epidemiological information included sociodemographic characteristics, habits of alcohol intake and cigarette smoking, health history and family history of cancers. Habitual cigarette smoking was defined as having smoked .4 days a week for at least 6 months. Information about duration and intensity was also obtained. Habitual alcohol intake was defined as drinking alcohol-containing products .4 days a week for at least 6 months.
At enrollment, blood samples were tested in Taiwan for serological markers, including alanine transaminase, aspartate transaminase, a-fetoprotein (AFP), hepatitis B virus surface antigen (HBsAg) and antibody against hepatitis C virus (anti-HCV). HBsAg was tested by radioimmunoassay (Abbott Laboratories, North Chicago, IL). Anti-HCV and AFP were tested by enzyme immunoassay using commercial kits (Abbott Laboratories). Both alanine transaminase and aspartate transaminase levels were determined with a serum chemistry autoanalyzer (Hitachi Model 736; Hitachi Co., Tokyo, Japan) using commercial reagents (Biomerieux, Mercy I'Etoile, France). Anti-HCV and AFP were assayed in all males and females who resided in Hu-Hsi and Pai-Hsa on the Penghu Islets. The other assays were carried out on samples from all participants. Any participant who had an elevated level of alanine transaminase (!45 IU/ml), aspartate transaminase (!40 IU/ml) or AFP (!20 ng/ml) was positive for HBsAg or anti-HCV or had a family history of HCC or liver cirrhosis among first-degree relatives was referred for upper abdominal ultrasonography examination. Suspected HCC cases were referred to teaching medical centers for confirmatory diagnosis by computerized tomography, digital subtracted angiogram, aspiration cytology and pathological examination. The criteria for HCC diagnosis included the following: a histopathological examination, a positive lesion detected by at least two different imaging techniques (abdominal ultrasonography, angiogram or computed tomography) or by one imaging technique and a serum AFP level .400 ng/ml.
Intensive follow-up, including abdominal ultrasonographic screening and serum AFP level determination every 3 months, was carried out on those with ultrasonographic images indicative of liver cirrhosis, whereas others were examined annually. Any suspected HCC cases identified during follow-up were referred for confirmatory diagnosis as described above.

Study subjects
Between February 1991 and June 2004, a total of 241 cases were newly diagnosed with HCC. Cases were primarily identified through linkage to the National Cancer Registry and death certification systems. A total of 1246 controls were randomly selected from cohort subjects who were not affected with HCC through the follow-up period by matching to each case by age (±5 years), gender, residential township and date of recruitment (±3 months). The number of matched controls per case varied depending on the number of eligible controls with available specimens and ranged from 2 to 6. Baseline urine samples were shipped to Columbia University on dry ice from 56 cases diagnosed between 1991 and 1995 and from 220 controls and previously assayed for AFB 1 metabolites (2). More recently, 74 cases diagnosed between 1996 and 2004 and 290 controls were assayed for urinary 8-oxodG as well as AFB 1 metabolites (7). The urine samples from these 74 cases and 290 controls were available for the present study. The characteristics of subjects with and without baseline urine have been described in detail (7). Briefly, they were similar except that the frequency of positive anti-HCV was significantly higher in cases when compared with cases without baseline urine samples. Controls with baseline urine samples were significantly younger than controls without and also had a low frequency of HbsAg-positive status. Among those with baseline urine available, the distribution of either habitual smoking or alcohol consumption was similar for cases and controls.

8-OxodG and AFB 1 metabolites in urine
Before examination, urine samples were centrifuged at 2000g for 10 min to remove any suspended cell debris. The supernatants were used for the determination of 8-oxodG, 15-F 2t -IsoP and AFB 1 metabolite levels. Urinary AFB 1 metabolites were determined by competitive enzyme-linked immunosorbent assay using monoclonal antibody AF8E11. Briefly, 2.5 ml of urine was adjusted to pH 5.0 with 1 N HCl and then digested with 500 U b-glucuronidase (Sigma Chemical, St. Louis, MO). Urine was extracted by Sep-PAK C-18 cartridges (Waters, Milford, MA), previously washed with chloroform, methanol and water. After extraction, cartridges were rinsed with 10 ml water and 5 ml 5% methanol in water and eluted with 5 ml 80% methanol. Eluants were dried under vacuum and redissolved in 0.5 ml phosphate-buffered saline. Concentrations of urinary metabolites were determined using a standard curve of serially diluted AFB 1 .
Urinary 8-oxodG levels were determined by competitive enzyme-linked immunosorbent assay. Briefly, wells were coated with 5 ng of conjugated 8-oxoG. Serially diluted 8-oxodG was used for the standard curve (concentration range 5-80 ng/ml) and urine samples were diluted 1:1 with phosphatebuffered saline before incubation with primary antibody 1F7.

15-F 2t -IsoP in urine
Urinary 15-F 2t -IsoP levels were analyzed using competitive enzyme-linked immunosorbent assay kits form Oxford Biomedical Research (Oxford, MI) according to the manufacturer's directions. Samples were analyzed in duplicate blinded to case-control status. According to the manufacturer, the lower limit of reliable detection is 0.2 ng/ml. A quality control sample consisting of urine pooled from five controls was analyzed with each batch of test samples. The coefficient of variation was 19% (n 5 10). Urinary creatinine was measured with commercial kit from BioAssay System (Hayward, CA), as directed by the manufacturer.

Statistical methods
To characterize the levels of urinary excretion of 15-F 2t -IsoP and the potential factors modulating these levels in a Taiwanese population, multivariateadjusted linear regression models were used to compute regression coefficients among cases and control, separately. Levels of urinary 15-F 2t -IsoP, 8-oxodG, creatinine and urinary AFB 1 metabolites were natural log transformed (ln) to normalize the distribution. Pearson partial correlation coefficient was used to determine the correlation of urinary 15-F 2t -IsoP with either AFB 1 metabolites or 8-oxodG adjusted by age, gender and urinary creatinine. To evaluate the dose-response relationship between the levels of urinary 15-F 2t -IsoP and AFB 1 metabolites, subjects were divided into quartiles based on the distribution of urinary AFB 1 metabolites for all control subjects (,2.26, !2.26 to ,3.64, !3.64 to ,6.14 and !6.14 fmol/mmol creatinine). Then, a multivariate logistic regression model adjusted for potential confounding factors was constructed to determine whether there was a trend. Wald's test with consecutive score 1, 2, 3 and 4 assigned to the first, second, third and fourth quartiles of urinary AFB 1 metabolites was used to test for trend of adjusted odds ratios (ORs) across strata.
The v 2 test was used to examine differences in the distributions of variables between cases and controls. To examine the independent and combined effects of the level of urinary 15-F 2t -IsoP on HCC risk, cases and controls were compared using conditional logistic regression models. Urinary 15-F 2t -IsoP was used to divide subjects into two groups: those with levels above the mean value (0.53 nmol/mmol creatinine) for all control samples versus those below the mean. To evaluate the dose-response relationship between urinary 15-F 2t -IsoP and HCC risk, subjects were divided into tertiles based on control values (,0.33, !0.33 to ,0.55 and !0.55 nmol/mmol creatinine). HBsAg, smoking, alcohol consumption, body mass index (BMI) and urinary 8-oxodG and AFB 1 metabolite-adjusted ORs and 95% confidence intervals (CIs) were derived from conditional logistic regression models stratified on the matching factors to estimate the association between levels of urinary 15-F 2t -IsoP and HCC risk. Wald's test with consecutive scores 1, 2 and 3 assigned to the first, second and third tertiles of urinary 15-F 2t -IsoP was used to test for trend of adjusted ORs across strata. To evaluate the combined effect of urinary biomarker levels and HCC risk, subjects were divided into different groups based on the levels of urinary biomarkers: those with urinary 8-oxodG levels above the mean value (24.70 nmol/mmol creatinine) for all control samples versus those below the mean and those with AFB 1 metabolite levels above the mean value (5.12 fmol/mmol creatinine) for all control samples versus those below the mean. The interaction terms for urinary 8-oxodG and 15-F 2t -IsoP, AFB 1 metabolites and 15-F 2t -IsoP were assessed in multivariate conditional logistic regression models. All analyses were performed with SAS software 9.0 (SAS Institute, Cary, NC). All statistical tests were based on two-tailed probability.

Results
Results of the linear regression analysis of multiple factors associated with levels of urinary 15-F 2t -IsoP indicated that, after multivariate adjustment, gender, age, BMI, HBsAg, smoking and alcohol consumption were not associated with urinary 15-F 2t -IsoP level among either cases or controls (data not shown). The levels of urinary AFB 1 metabolites as well as 8-oxodG were positively associated with urinary 15-F 2t -IsoP levels. Each 1 fmol/mmol creatinine of urinary AFB 1 metabolites was associated with a 1.37 nmol/mmol creatinine increase in urinary 15-F 2t -IsoP concentration, whereas each 1 nmol/ mmol creatinine of urinary 8-oxodG was associated with a 1.26 nmol/mmol creatinine increase in urinary 15-F 2t -IsoP concentration. The levels of ln urinary 15-F 2t -IsoP were correlated with ln urinary AFB 1 metabolites as well as 8-oxodG, with Pearson partial correlation coefficients of 0.24 (P , 0.0001, Figure 1) and 0.12 (P , 0.0001, Figure 2), respectively.
Subjects were divided into quartiles based on the distribution of urinary AFB 1 metabolites in controls to evaluate the dose-response relationship with urinary 15-F 2t -IsoP (Table I). When compared with control subjects in the lowest quartile of urinary AFB 1 metabolites, there was an increase in detection of high level of urinary 15-F 2t -IsoP, with adjusted ORs of 0.70 (95% CI 5 0.29-1.70), 3.69 (95% CI 5 1.68-8.10) and 7.50 (95% CI 5 3.14-16.46) for subjects in the second, third and fourth quartile, respectively, (P trend , 0.0001).
The mean level of urinary 15-F 2t -IsoP was statistically significantly higher in HCC cases than in controls (0.67 ± 0.34 and 0.53 ± 0.59 nmol/mmol creatinine, respectively; P 5 0.04). The association of urinary 15-F 2t -IsoP with HCC risk is given in Table II. After adjustment for HBsAg status, smoking, alcohol drinking, BMI and urinary AFB 1 metabolites and 8-oxodG, the OR for those with urinary 15-F 2t -IsoP levels above the mean compared with those with levels below the mean was 2.53 (95% CI 5 1.30-4.93). When urinary 15-F 2t -IsoP levels were stratified into tertiles based on control values, HCC risk increased with adjusted ORs of 3.87 (95% CI 5 1.32-11.38) and 6.27 (95% CI 5 2.17-18.13; P trend 5 0.0008) for subjects with 15-F 2t -IsoP in the second and third tertile, respectively, compared with those in the lowest tertile.
The combined effect of urinary 15-F 2t -IsoP and HBsAg is given in Table III. HBsAg carriers with urinary 15-F 2t -IsoP above the mean had a significantly increased HCC risk (OR 5 19.01, 95% CI 5 6.67-54.17) compared with non-carriers with urinary 15-F 2t -IsoP below the mean (P for linear trend , 0.0001). The combined effect of urinary 15-F 2t -IsoP, 8-oxodG and AFB 1 is given in Table IV. In our previous study, we found that increasing level of urinary 8-oxodG was associated with a non-significant decrease in HCC risk. Thus, in the present analysis, subjects with levels of 15-F 2t -IsoP below the mean and 8-oxodG above the mean were considered as the low-risk group. Among subjects with urinary 15-F 2t -IsoP above the mean, the ORs (95% CIs) were 2.99 (1.00-8.98) and 2.69 (0.97-7.67; P trend 5 0.008) for those with urinary 8-oxodG above and below the mean, respectively. Among subjects with urinary 15-F 2t -IsoP above the mean, the OR was 1.58 (95% CI 5 0.58-4.31) for those with urinary AFB 1 above the mean compared with those with AFB 1 below the mean. Among subjects with urinary 15-F 2t -IsoP above the mean, the ORs (95% CIs) were 2.76 (1.24-6.13) and 2.86 (1.26-6.49; P trend 5 0.004) for those with urinary AFB 1 below and above the mean, respectively.

Discussion
We investigated the relative contributions of environmental determinants to urinary levels of 15-F 2t -IsoP in a well-characterized Chinese adult population living in an area with high AFB 1 exposure. We found that the major factors determining urinary excretion of 15-F 2t -IsoP in this population were levels of urinary AFB 1 metabolites and 8-oxodG. In addition, we observed a statistically significant increased trend in HCC risk with increasing tertiles of urinary 15-F 2t -IsoP levels (OR 5 3.87, 95% CI 5 1.32-11.38 and OR 5 6.27, 95% CI 5 2.17-18.13, for subjects in the second and third tertile, respectively, compared with those in the lowest tertile; P trend 5 0.0008). Our results provide   In the present study, we failed to find an effect of smoking or alcohol intake on the urinary excretion of 15-F 2t -IsoP. Alcohol intake may result in increased oxidative stress due to ROS generation through induction of CYP450 2E1 (8). But no effect of alcohol in the level of urinary 15-F 2t -IsoP has been observed in some previous studies (14,15). Substantially higher doses of alcohol consumption were required to produce a statistically significant elevation in urinary 15-F 2t -IsoP (16). While smoking has been associated with increased levels of 15-F 2t -IsoP in some prior studies (12,14), our negative results may partly be due to misclassification. Information on smoking and alcohol intake in our study was based on self-report. Data on the correlation of urinary 15-F 2t -IsoP with either age of subjects or BMI are conflicting. Whereas a positive association with age was observed in one small study with only 19 health controls (17), no effect of age was observed in our and other two studies with larger sample sizes (14,15). Our observation of the lack of an age effect is consistent with a hypothesis that unlike oxidative damage to DNA which is related to age, oxidative lipid damage is related to physiologic conditions concurrent with aging (15). Although obesity is associated with oxidative stress, urinary 15-F 2t -IsoP levels do not seem to be associated with BMI in our and other studies (14,15).
Several studies provided evidence that AFB 1 -related HCC is associated with lipid peroxidation. An in vivo study demonstrated increases in malondialdehyde in plasma and lipid peroxide levels in liver of AFB 1 -treated rats (18,19). In our previous study, we observed a positive correlation between AFB 1 exposure, based on urinary excretion of aflatoxin metabolites, and urinary 8-oxodG (7). In the present study, we observed a linear association of urinary 15-F 2t -IsoP with  8-oxodG as well as AFB 1 exposure. Moreover, the ORs for detection of urinary 15-F 2t -IsoP above the mean showed a dose-dependent increase with level of urinary AFB 1 (Table I). These results provide evidence that AFB 1 exposure increase oxidative stress in humans. ROS generated in inflamed tissues can cause injury to target cells and also damage DNA and may be involved in the progression of viral hepatitis as well as hepatocarcinogenesis (6). Chronic HBV infection is a major risk factor associated with the development of HCC. Although the underlying mechanisms that lead to malignant transformation of infected cells remain unclear, several direct and indirect mechanisms have been described for HBV-induced hepatocarcinogenesis [reviewed in ref. 20]. HBV has a direct oncogenic effect through integration of HBV DNA into the cellular DNA resulting in genetic alterations. In a transgenic mouse model, the accumulation of toxic levels of HBsAg is followed by liver injury, inflammation and HCC formation (21). The continuous generation of ROS due to chronic inflammation may be part of indirect mechanisms of HBV-induced HCC. We did not observe an association between chronic viral infection and urinary 15-F 2t -IsoP level; however, the combination of urinary 15-F 2t -IsoP above the mean and chronic HBV infection resulted in an OR of 19.01 (95% CI 5 6.67-54.17) compared with those with low urinary 15-F 2t -IsoP and without HBV infection, supporting the multiple mechanisms of HBV-related HCC.
It is well known that the formation of trans-8,9-dihydro-8-(N7guanyl)-9-hydroxy-AFB 1 adducts is a critical step in AFB 1 genotoxicity (22). Recently, we reanalyzed the effect of AFB 1 exposure on HCC risk with additional cases and controls and a longer period of follow-up compared with that in our previous study (2). The effect of AFB 1 on HCC risk remains statistically significant with an OR of 1.76 (95% CI 5 1.22-2.55) for those with urinary AFB 1 level above the mean compared those with ABF1 level below the mean (data not shown). Our demonstration that AFB 1 is associated with oxidative DNA damage (7) and lipid peroxidation (current study) suggests that increased oxidative damage may be partly responsible for AFB 1 -induced hepatocarcinogenesis. This is also supported by the analysis of the combined effects of urinary AFB 1 and 15-F 2t -IsoP (Table IV). We observed that the 15-F 2t -IsoP effect was more prominent among subjects with high 8-oxodG and low AFB 1 levels. These observations are consistent with the notion that the three factors, namely, AFB 1 , 8-oxodG and F2-IsoPs, are reflective of a common mechanistic pathway showing a threshold dose in relation to risk. However, our findings should be interpreted with caution since the small sample size in the present study might result in an unstable estimation of the combined effect.
Although 8-oxodG is a biomarker indicating oxidative DNA damage, a recent study (23) suggests that urinary 8-oxodG level is an index measuring both oxidative DNA damage and oxidative DNA repair capacity. In a previous study, we showed a slightly decreased risk of HCC in those with a higher level of urinary 8-oxodG (7). In the present study, we found ORs (95% CIs) of 2.99 (1.00-8.98) and 2.69 (0.97-7.67) for those with 8-oxodG above and below the mean among those with urinary 15-F 2t -IsoP above the mean. In order to fully understand the relationship between urinary 8-oxodG and 15-F 2t -IsoP on human diseases, DNA repair capacity should be taken into consideration.
To the best of our knowledge, no published studies have measured urinary 15-F 2t -IsoP to investigate the effect of AFB 1 exposure on lipid peroxidation in humans. The use of urinary 15-F 2t -IsoP as a biomarker of oxidative damage resulting from AFB 1 exposure has several advantages: (i) urinary 15-F 2t -IsoP is a specific product of lipid peroxidation; (ii) the antibody-based method of quantitation does not require extraction or purification of the biological samples prior to analysis making it suitable for application in large clinical or experimental studies; (iii) there is no evidence of artifactual formation of isoprostanes during handling and storage of urine, unlike plasma (10); (iv) urinary 15-F 2t -IsoP excretion is not confounded by the lipid content of the diet (24) and (v) since 15-F 2t -IsoP is metabolized rapidly in the body and a large quantity of this compound is excreted from the circulation within a few minutes, it is adequate to measure the compound in urine (25).
Although the use of urinary 15-F 2t -IsoP as an index of lipid peroxidation remains attractive, our results must be interpreted with caution. First, the putative causal role of AFB 1 exposure in increasing urinary 15-F 2t -IsoP could not be verified. The association between urinary 15-F 2t -IsoP and AFB 1 metabolites was examined using a cross-sectional study design that only measured levels of both analytes at baseline, making temporal separation of cause and effect difficult. A longitudinal rather than a cross-sectional study should be conducted to ascertain the possible association between AFB 1 exposure and lipid peroxidation. Nevertheless, the strong association indicates the presence of oxidative lipid damage in persons with high AFB 1 metabolites. Further investigations, incorporating prospective and dietary intervention studies, are required to confirm AFB 1 -related HCC via oxidative lipid damage. Second, levels of urinary 15-F 2t -IsoP represent a steady-state concentration that is dependent on production (degree of lipid peroxidation) versus metabolism and excretion (26). Third, measuring the levels of urinary 15-F 2t -IsoP by single spot samples might not be representative of individual levels of oxidative lipid damage. There was, however, no diurnal variation in the urinary of 15-F 2t -IsoP (27), suggesting that the collection of a single urinary sample would be adequate. Currently, there is no information available pertaining to the stability of isoprostanes in urine and the urine samples examined in our study were collected nearly 15 years ago. Nevertheless, since we treated urine samples of cases and controls identically in terms of process, storage and assay, any effect on the biomarker levels might be disease blind. Fourth, there is concern that because of the small sample size in our case-control study, the statistically significant finding may be due to the chance. However, given the strong linear correlation with urinary AFB 1 metabolites, as well 8-oxodG, and statistically significant high mean level of 15-F 2t -IsoP among cases, the possibility of a false-positive finding may be small. Also, due to the small sample size, the results of the combined effect of urinary 15-F 2t -IsoP with either HBV infection (Table III) or other urinary biomarkers (Table IV) must be interpreted with additional caution. In addition, the controls with available urine may not be representative of the general reference population due to the higher frequencies of younger as well as HbsAg-negative subjects. However, the cases and controls were comparable with regard to sociodemographic characteristics such as age or gender (7) that may affect the HCC risk or levels of urinary 15-F 2t -IsoP. In this case, selection bias is unlikely to occur. Finally, levels of 15-F 2t -IsoP are not a quantitative marker of damage to lipid by all reactive species (13). Future epidemiological studies should measure multiple oxidative stress markers in order to understanding the role of oxidative stress in HCC.
In summary, we found, among controls, a statistically positive association of urinary 15-F 2t -IsoP, a biomarker of oxidative stress with urinary AFB 1 metabolites, a biomarker of AFB 1 exposure and with urinary 8-oxodG, a biomarker of oxidative DNA damage. These results strongly suggest that AFB 1 exposure may result in an increased risk of oxidative damage. In terms of HCC risk, a significant positive relationship between urinary 15-F 2t -IsoP was observed. Our results provide information on the application of biomarkers in human populations at high risk for cancer and that AFB 1 -induced lipid peroxidation may, in addition to the formation of AFB 1 -DNA adducts, have an important role in AFB 1 carcinogenicity.

Funding
National Institutes of Health (RO1ES05116 and P30ES09089).