Article

Prevention of Liver Cancer in Qidong, China: Lessons from Aflatoxin Biomarker Studies

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Abstract

Primary liver cancer has been the leading cause of cancer death in Qidong, China, with about 800 deaths annually in this region of 1.1 million residents. Epidemiological studies have highlighted the importance of infection with hepatitis B virus (HBV) and dietary exposure to hepatocarcinogenic aflatoxins as key, interactive determinants of risk in Qidong and other endemic areas. Identification of these risk factors was enabled through the development of biomarkers allowing for measures of their prevalence in case-control and other study cohorts. Vaccination against HBV began in pilot studies in the 1980s in Qidong but did not become universal for newborns until earlier this decade. Despite minimal impact on cancer mortality to date, vaccination programs are poised to blunt the development of liver diseases including cancer in this region over the next generations. Strategies for reducing aflatoxin exposure are also required, especially for those already infected with HBV. We have conducted a series of proof-of-principle clinical trials in which drugs, dietary supplements and foods have been used to alter the metabolism and elimination of aflatoxins following unavoidable exposures. Using aflatoxin biomarkers as intermediate endpoints, the efficacy of these agents (oltipraz, chlorophyllin, broccoli sprout beverages) has been demonstrated, highlighting roles for frugal approaches to chemoprevention against environmental carcinogenesis. Remarkably, recent evidence garnered from retrospective analysis of archived serum samples from the past quarter century demonstrates a 40-fold drop in aflatoxin exposure in Qidong, likely driven by changes in the primary dietary staple from maize to rice. Thus, primary prevention, evoked by changing economic and agricultural policies in the 1980s, heralds the unanticipated promise of elimination of liver cancer from this endemic region in a fore-shortened timeframe.

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... 8 It is a carcinogen in fish 9 and rodents. 10−12 Dietary exposures to AFB 1 are high in areas of Asia 13,14 and sub-Saharan Africa. 15,16 Epidemiological studies suggest that chronic exposure to AFB 1 is a contributing factor in the etiology of hepatitis B virus (HBV) associated hepatocellular carcinomas (HCC). ...
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Hepatitis B virus (HBV) had been considered as the main causative factor of primary hepatocellular carcinoma and universal immunization of newborns was recommended as the major approach to control hepatitis and hepatoma in areas of prevalence. As the initial phase of the first vaccination program for such a purpose, a pilot study was done from September 1983 to May 1984 in a high incidence rural area of China. In an area of 214,343 inhabitants, 1,703 newborns (99% of all births) were vaccinated. Ninety-seven percent of all vaccinees were followed up at 1 year. The vaccine used was Hep-B Vax, given intramuscularly at 0, 1, and 6 months after birth. Four immunization regimes were used: 5-μg or 2,5-μg doses with or without hepatitis B immune globulin (HBIG) added in the case of carriers children. These groups were defined by drawing lots at community level. A matched control was selected on a voluntary basis. Each group consisted of 400 infants. Vaccination was proven to be very safe and well accepted by the public. The prevalence of HBV infection in the area was further demonstrated by the high HBsAg-positive rate measured: 14.2% of the 1,180 mothers (3.9% were also e-antigen positive), 7.6% and 10.1% of the unvaccinated children at 6.5 months and 1 year of age, respectively. It was shown that vaccination with a 5-μg or 2.5-μg dose significantly lowered the HBsAg positives to a level close to 1.5% versus 10% in the control group at 1 year. An 85% protection was thus achieved. A 5-μg dose plus HBIG did not show additional benefit. A 2.5-μg dose plus HBIG gave less protection, and anti-HB levels were also significantly lower than in other groups. Among the 12 failures found in the 5-μg and 2.5-μg groups, 11 were born to HBsAg-positive mothers, nine of whom also had e-antigen. Available data showed that 29% of children born by e-antigen-positive and 2.7% of children born by e-antigen-negative carriers had the risk of becoming carriers during the first year of life following vaccination. The present study demonstrated the feasibility and rationale of conducting universal immunization of newborns in endemic rural area for controlling hepatitis and hepatoma. The significance of the possible use of the vaccination at lower dose had also been stressed.
Article
Hepatocellular carcinoma (HCC) is one of the major cancers in China. Accordingly, the mortality rates in 1990 (per 100 000) were 20.10 in certain cities and 24.32 in certain counties. More than 90% of HCC cases and 70% of controls were infected with the hepatitis B virus (HBV) (Odds Ratio (OR) = 10–50). In the same group of patients, 8–27% of those with HCC and 0–11% of the healthy controls were also infected with hepatitis C (HCV) (OR = 2.11–17.29). There appears to be some correlation between HBV markers and the OR. The government requires that 85% of infants be immunized with HBV vaccine. In 1992, there were 3 million infants inoculated with HB vaccines. Aflatoxins have been found as contaminants in food, particularly in corn, peanut oil, soya sauce and fermented soya beans. The intake of aflatoxin B1, (AFB1) by people of ten different villages correlated with HCC mortality rates (r =0.55; P < 0.05). The concentration of AFB1-albumin adducts is an indicator of individual exposure to aflatoxins. These adducts are higher in hyperendemic HCC areas and cases. Most people have now changed their staple food and eat rice instead of corn. Six large epidemiological studies have confirmed that people who drink pond-ditch water experience higher HCC mortality rates than people who drink deep-well water. Recent research has found that the blue-green algal toxin microcystin (MCYST) was a contaminant of pond-ditch water. MCYST is a strong promoter of HCC and will induce severe intrahepatic haemorrhages and liver necrosis. More than 30% of people in Qidong County have already changed their sources of water from pond-ditches to deep wells. Therefore, a combined strategy of the prevention of hepatitis, control of crops and control of drinking water is advocated for the primary prevention of HCC in China.
Article
Prevention trials of whole foods or simple extracts offer prospects for reducing an expanding global burden of cancer effectively, and in contrast to promising isolated phytochemicals or pharmaceuticals, frugally. We use the term "green" chemoprevention to differentiate a food-centered approach that is sustainable in underserved populations. It can be applied to personalized medicine just as well as a pharmaceutical approach, but only green chemoprevention can be applied in both rich and poor settings. This MiniReview discusses some of the challenges of conducting food-based trials in developing countries, with particular emphasis on moving the limited number of promising phase II trials forward as placebo-controlled randomized trials, the gold standard for prevention studies. How does one define a placebo for a food? What is the regulatory context of such a food-based product? How can such products be produced and standardized to the benefit of a larger, individual trial, and importantly, the research community at large? What are the challenges and opportunities of conducting such trials in the international setting? Finally, how does one make the science practical?
Article
Chlorophylls, chlorins, and other porphyrins have been used clinically for many years, including photodynamic therapy of tumors. More recently, the cancer chemopreventive properties of chlorophylls have come to be recognized. Chlorophylls exhibit anti-mutagenic activity in short-term genotoxicity assays, and protect against various intermediate biomarkers of cancer in vivo. The anticarcinogenic activity of sodium-copper chlorophyllin (CHL), a clinically-used water soluble salt of chlorophyll, has been studied in several species. Collectively, the results from these studies support a chemopreventive role for CHL against aromatic carcinogens (aflatoxins, polycyclic aromatic hydrocarbons, heterocyclic amines) in various target organs of rats, mice, and rainbow trout. In vivo mechanism studies indicate that inhibition is most effective when CHL is administered simultaneously with the carcinogen, thereby allowing direct interaction (molecular complex formation) between CHL and the carcinogen. Studies of post-initiation treatment with CHL have provided conflicting results, with evidence for inhibition or promotion of carcinogenesis. These findings are discussed in terms of the inhibitory and promotional mechanisms of CHL, the relevance of such mechanisms to natural chlorophylls present in the diet, and the current use of CHL as a health supplement.
Article
Estimates of the worldwide incidence and mortality from 27 cancers in 2008 have been prepared for 182 countries as part of the GLOBOCAN series published by the International Agency for Research on Cancer. In this article, we present the results for 20 world regions, summarizing the global patterns for the eight most common cancers. Overall, an estimated 12.7 million new cancer cases and 7.6 million cancer deaths occur in 2008, with 56% of new cancer cases and 63% of the cancer deaths occurring in the less developed regions of the world. The most commonly diagnosed cancers worldwide are lung (1.61 million, 12.7% of the total), breast (1.38 million, 10.9%) and colorectal cancers (1.23 million, 9.7%). The most common causes of cancer death are lung cancer (1.38 million, 18.2% of the total), stomach cancer (738,000 deaths, 9.7%) and liver cancer (696,000 deaths, 9.2%). Cancer is neither rare anywhere in the world, nor mainly confined to high-resource countries. Striking differences in the patterns of cancer from region to region are observed.
Article
Previous studies have demonstrated that dietary administration of the schistosomicidal drug 5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione (oltipraz) ameliorates the hepatotoxicity of aflatoxin B1 (AFB1). Notably, mortality, altered hepatic function, hepatic AFB1-DNA adduct levels, and expression of hepatic enzyme-altered foci were markedly reduced in the rat by concurrent feeding of oltipraz during exposures to AFB1. Collectively, these studies prompted us to evaluate the chemoprotective properties of oltipraz against AFB1-induced liver cancer. In addition, preliminary molecular dosimetry studies were undertaken to determine the utility of measurements of urinary aflatoxin-N7-guanine excretion as a marker of relative risk for hepatocarcinogenesis in AFB1-exposed rats. For the carcinogenesis studies, 5-wk-old male F344 rats were randomly divided into two groups. One group (55 rats) received the AIN-76A diet, and the other group (56 rats) received the AIN-76A diet supplemented with 0.075% oltipraz. The oltipraz-supplemented diet was fed for 4 wk. Beginning 1 wk after starting the experimental diets, all rats in both groups received 25 micrograms of AFB1/rat/day by gavage for 5 days per wk over the next 2 wk. One wk following cessation of dosing with AFB1, oltipraz was removed from the diet, and all rats were fed the AIN-76A diet for the remainder of the experiment. At 3 mo after dosing, livers of ten sentinel rats from each group were analyzed for the burden of gamma-glutamyltranspeptidase-positive foci. In accord with previous findings, rats fed the oltipraz-supplemented diet exhibited substantial reductions in the focal burden (97% reduction; P less than 0.05) of these AFB1-induced lesions. The remaining rats were maintained for the cancer study until they became moribund or the termination of the experiment at 23 mo. Gross liver lesions were identified at autopsy and confirmed by microscopic evaluation. An 11% incidence of hepatocellular carcinoma was observed in the AFB1-treated, control diet-fed rats. An additional 9% of this group had hepatocellular adenomas. Oltipraz afforded complete protection against both AFB1-induced hepatocellular neoplasms. Using Kaplan-Meier survival analyses, rats in the oltipraz group had a significantly (P less than 0.02) longer life span and an increased survival free of liver tumors (P less than 0.0002). Molecular dosimetry studies used rats fed either the oltipraz-supplemented or control diet for 1 wk and then challenged with a single dose of AFB1 to examine the initial rates of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 excreted in the urine.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
Hepatocellular carcinoma is one of the five leading human cancers causing at least 250,000 deaths each year. One of the major risk factors for this disease is exposure to dietary aflatoxins, and the development of appropriate molecular dosimetry biomarkers would facilitate the identification of individuals at risk. This study was undertaken to explore the relationship between dietary intake of aflatoxins and the excretion of the major aflatoxin-DNA adduct and other metabolites into the urine of chronically exposed people. The following protocol was developed for this investigation in Guangxi Autonomous Region, People's Republic of China, where the diets of 30 males and 12 females (ages, 25-64 years) were monitored for 1 week and aflatoxin intake levels determined each day. Starting on the fourth day, total urine volumes were obtained in consecutive 12-h fractions for 3 or 4 days. High performance liquid chromatography and competitive radioimmunoassay analyses were done on each of the urine samples, and the relationships between excretion of total aflatoxin metabolites, aflatoxin-N7-guanine, aflatoxin M1, aflatoxin P1, and aflatoxin B1, and aflatoxin B1 intake values were determined. The average intake of aflatoxin B1 by men was 48.4 micrograms/day, giving a total mean exposure during the study period of 276.8 micrograms. The average daily intake by women was 77.4 micrograms/day, resulting in a total average exposure during the 7-day period of 542.6 micrograms aflatoxin B1. Initial efforts to characterize aflatoxin metabolites in urine samples were with an analysis by competitive radioimmunoassay. The analysis by linear regression of the association between aflatoxin B1 intake/day and total aflatoxin metabolite excretion/day showed a correlation coefficient of only 0.26. These findings stimulated the immunoaffinity/analytical high performance liquid chromatography analysis for individual metabolites. When the data were analyzed by linear regression analysis, the aflatoxin N7-guanine excretion and aflatoxin B1 intake from the previous day showed a correlation coefficient of 0.65 and P less than 0.000001. Similar analysis for aflatoxin M1 resulted in a correlation coefficient of 0.55 and P less than 0.00001, whereas there was no positive statistical association between exposure in the diet and aflatoxin P1 excretion, despite aflatoxin P1 being quantitatively a major metabolite. Analysis of the total aflatoxin-N7-guanine excretion in the urine during the complete collection period plotted against the total aflatoxin B1 exposure in the diet for each of the individuals, smoothing the day to day variations, revealed a correlation coefficient of 0.80 and P less than 0.0000001.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
Chlorophyllin (CHL), a food-grade derivative of the green plant pigment chlorophyll, has recently been shown in this laboratory to be a potent inhibitor in vivo of hepatic aflatoxin B1 (AFB1)-DNA adduction and hepatocarcinogenesis (Breinholt et al. (1995) Cancer Res. 55, 57-62). We report here that CHL forms a strong noncovalent complex with AFB1 in vitro (dissociation constant (Kd) by Scatchard analysis = 1.4 (+/- 0.4) microM based on copper chlorin content), which may contribute to its anticarcinogenic activity. Kd values for the related porphyrins chlorine e6, protoporphyrin IX, and zinc protoporphyrin IX were also of the same order of magnitude as that of the commercial CHL. Mole ratio analysis provided evidence that all porphyrins examined associate with AFB1 at an approximate one to one stoichiometric ratio. Energy minimization computer modeling of the complex indicates a favorable association energy of -20 kcal/mol, independent of oxidation state of the 8,9-double bond of AFB1. AFB1 incubated in vitro with liver microsomes in the presence of added CHL showed comparable levels of inhibition in the production of several phase 1 metabolites, including the postulated procarcinogenic metabolite AFB1 8,9-epoxide. Kinetic analysis of microsome-catalyzed AFB1-DNA adduction suggested a CHL inhibition constant near 10 microM chlorin. In vivo, addition of CHL to concentrated AFB1 solutions followed by gavage administration resulted in dose-dependent inhibition of hepatic AFB1-DNA adduction, whereas the same dosages of AFB1 and CHL incorporated into a single bolus of trout diet for gavage provided less protection at all CHL doses.(ABSTRACT TRUNCATED AT 250 WORDS)
To investigate the role of hepatitis B Virus (HBV) infection and aflatoxin (AF) exposure in the development of primary liver cancer (PLC). A 10-year prospective case-control study was carried out in 737 HBsAg carriers and 699 HBsAg negative cases, and the aflatoxin B1 albumin adducts (AFB1-Alb) were detected in the serum of the cohort including 30 HBsAg postive cases and 150 control individuals according to the case-control study model (ratio 1:5) at random in the high prevalance area of PLC. The average year-incidence rate was significantly higher in the HBsAg postive group (824.13/100,000) than in the control group (70.97/100,000, RR = 11.61). There was no significant difference in the incidence rate of other tumors between the two groups (P > 0.05). The serum positive rate of AFB1-Alb was significantly higher in the PLC group (76.67%) than in the control group (48.67%, OR = 3.47), and the serum concentration of AFB1-Alb was also significantly higher in the PLC group than in the control group (P < 0.01). HBV infection and AF exposure are important etiological factors in the development of PLC and both result in carcinogenic synergy.
Article
Oltipraz is currently undergoing clinical evaluation as a cancer chemopreventive agent, especially with respect to aflatoxin-associated hepatocarcinogenesis. The agent's ability to induce phase II xenobiotic enzymes that detoxify the ultimate carcinogen formed in vivo is thought to be an important mechanism by which disease risk may be attenuated. However, an additional mechanism could be a reduction in the activation of environmental procarcinogens by certain cytochrome P450 (CYP) isoforms. This hypothesis was tested with respect to CYP1A2, by using the clearance of caffeine by N-demethylation as a phenotypic trait measurement of the isoform's catalytic activity. Subjects received a single oral dose of caffeine (200 mg) on five separate occasions: on the day prior to oltipraz administration (day 0), 2 h after the first (day 1) of eight daily oral doses of oltipraz (125 mg) and 2 h after the last dose (day 8). In addition, CYP1A2 activity was also measured 2 and 14 days (days 10 and 22, respectively) after discontinuation of oltipraz administration. Plasma concentrations of caffeine and its N-demethylated metabolite, paraxanthine, over 24 h after drug administration, were determined by HPLC. A single 125-mg dose of oltipraz markedly reduced CYP1A2 activity by 75 +/- 13% in nine healthy subjects, resulting in a higher caffeine plasma level and prolongation of the in vivo probe's elimination half-life. Daily administration of 125 mg oltipraz for 8 days resulted in further inhibition so that only 19 +/- 13% of the original baseline level of activity was present. However, 2 days after discontinuation of oltipraz treatment, CYP1A2 activity had returned to 66 +/- 33% of its original level and complete recovery was achieved within 14 days of the chemopreventive agent being stopped. These results demonstrate that oltipraz is a potent, in vivo inhibitor of CYP1A2 in humans and, because this isoform is importantly involved in procarcinogen activation, they also indicate that such inhibition probably contributes to oltipraz's cancer-chemopreventive effect. In addition, the findings also suggest the likelihood of significant drug interactions between oltipraz and drugs whose metabolism is mediated by CYP1A2.
Article
We assessed the separate and combined effects of hepatitis B virus (HBV), hepatitis C virus (HCV), and aflatoxin in causing hepatocellular carcinoma (HCC) in Qidong, China. A consecutive series of 181 pathologic-diagnosed HCC cases were studied for hepatitis B surface antigen (HBsAg), anti-HBc, HBV X gene sequence, anti-HCV, the 249ser-p53 mutation, and chronic hepatitis pathology. Each of the 181 incident HCC cases had markers for HBV infection and hepatitis pathology; only 6 of 119 cases were coinfected with HCV. The 249ser-p53 mutation was found in 54% (97/181) of HCC cases and in all 7 cases with tissue for analysis from the hepatitis cohort but in none of 42 matched cases from Beijing. The estimated cumulative dose of aflatoxin B1 in these 7 cases ranged from 0.13 to 0.49 mg/kg. Follow-up data through 13.25 years on a cohort of 145 men with chronic HBV hepatitis showed that the relative risk from aflatoxin exposure was 3.5 (1.5-8.1). A similar relative risk was found using 249ser-p53 mutation as a marker for aflatoxin exposure. In conclusion, HBV hepatitis is ubiquitous in Qidong HCC cases, whereas HCV contributes little to its risk. The 249ser-p53 mutation appears to result from coexposure to aflatoxin and HBV infection. Even modest levels of aflatoxin exposure tripled the risk of HCC in HBV-infected men.
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