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Liver cancer causes upwards of 1 million cancer deaths annually and is projected to rise by at least 55% over the next 15 years. Two of the major risk factors contributing to liver cancer have been well documented by multiple epidemiologic studies and the hepatitis B virus (HBV) and aflatoxin show a synergy that increases by more than 8-fold the risk of liver cancer relative to HBV alone. Using the population-based cancer registry established by the Qidong Liver Cancer Institute in 1972 and aflatoxin-specific biomarkers, we document that reduction of aflatoxin exposure has likely contributed to a nearly 70% decline in age-standardized liver cancer incidence over the past 30 years despite an unchanging prevalence of HBV infection in cases. A natural experiment of economic reform in the 1980s drove a rapid switch from consumption of heavily contaminated corn to minimally, if any, contaminated rice and subsequent dietary diversity. Aflatoxin consumption appears to accelerate the time to liver cancer diagnosis; lowering exposure to this carcinogen adds years of life before a cancer diagnosis. Thus, in 1990 the median age of diagnosis was 48 years, while increasing to 67 years by 2021. These findings have important translational public health implications since up to 5 billion people worldwide might be routinely exposed to dietary aflatoxin, especially in societies using corn as the staple food. Interventions against aflatoxin are an achievable outcome leading to a reduction in liver cancer incidence and years of delay of its nearly always fatal diagnosis., (© 2024 UICC.)

We evaluated whether aflatoxin B 1 (AFB 1 ) exposure was associated with later risk of developing gallbladder cancer (GBC). We measured AFB 1 -lysine albumin adducts in baseline samples from the Shanghai Cohort Study of 18 244 men aged 45 to 64 years (recruited 1986-1989). We included 84 GBC cases with sufficient serum and 168 controls matched on age at sample collection, date of blood draw and residence. We calculated adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for detectable vs non-detectable AFB 1 -lysine albumin adducts and gallbladder cancer. AFB 1 -lysine albumin adducts were detected in 50.0% of GBC cases, and risk of GBC was twice as high in those with detectable vs undetectable levels (OR = 2.0, 95% CI = 1.0-3.9). ORs ranged from 1.8 (95% CI = 0.75-4.3) for 0.5 to <1.75 pg/mg vs undetectable adduct levels to 2.2 (95% CI = 0.91-5.6) for >3.36 pg/mg vs undetectable, suggesting a dose-response (P trend  = .05). When restricted to cases diagnosed before the median time to diagnosis after blood draw (18.4 years), results were similar (OR = 2.2, 95% CI = 0.80-5.8) to those for the entire follow-up duration. The OR was 9.4 (95% CI = 1.7-51.1) for individuals with detectable AFB 1 -lysine albumin adducts and self-reported gallstones compared to individuals with neither. Participants with detectable AFB 1 -lysine albumin adducts at baseline had increased risk of developing GBC, replicating the previously observed association between AFB 1 exposure and providing the first evidence of temporality., (© 2023 UICC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can generate the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases (GST) and other cytoprotective enzymes. A broccoli sprout-derived beverage providing daily doses of 600 μmol glucoraphanin and 40 μmol sulforaphane was evaluated for magnitude and duration of pharmacodynamic action in a 12-week randomized clinical trial. Two hundred and ninety-one study participants were recruited from the rural He-He Township, Qidong, in the Yangtze River delta region of China, an area characterized by exposures to substantial levels of airborne pollutants. Exposure to air pollution has been associated with lung cancer and cardiopulmonary diseases. Urinary excretion of the mercapturic acids of the pollutants, benzene, acrolein, and crotonaldehyde, were measured before and during the intervention using liquid chromatography tandem mass spectrometry. Rapid and sustained, statistically significant (P ≤ 0.01) increases in the levels of excretion of the glutathione-derived conjugates of benzene (61%), acrolein (23%), but not crotonaldehyde, were found in those receiving broccoli sprout beverage compared with placebo. Excretion of the benzene-derived mercapturic acid was higher in participants who were GSTT1-positive than in the null genotype, irrespective of study arm assignment. Measures of sulforaphane metabolites in urine indicated that bioavailability did not decline over the 12-week daily dosing period. Thus, intervention with broccoli sprouts enhances the detoxication of some airborne pollutants and may provide a frugal means to attenuate their associated long-term health risks., (©2014 American Association for Cancer Research.)

Primary liver cancer (PLC) is the third leading cause of cancer mortality globally. In endemic areas of sub-Saharan Africa and Asia, PLC largely arises from chronic infection with hepatitis B virus (HBV) and ingestion of aflatoxins. Although synergistic interactions between these two risk factors have been observed in cohort studies in China, here we determined the impact of agricultural reforms in the 1980s leading to diminished maize consumption and implementation of subsidized universal vaccination against HBV in the 2000s on PLC primary prevention. A population-based cancer registry was used to track PLC mortality in Qidong, China and was compared with the timeline of HBV immunization. Randomly selected serum samples from archived cohort collections from the 1980s to present were analyzed for aflatoxin biomarkers. More than 50% reductions in PLC mortality rates occurred across birth cohorts from the 1960s to the 1980s for Qidongese less than 35 years of age although all were born before universal vaccination of newborns. Median levels of the aflatoxin biomarker decreased from 19.3 pg/mg albumin in 1989 to undetectable (<0.5 pg/mg) by 2009. A population attributable benefit of 65% for reduced PLC mortality was estimated from a government-facilitated switch of dietary staple from maize to rice; 83% of this benefit was in those infected with HBV. Food policy reforms in China resulted in a dramatic decrease in aflatoxin exposure, which, independent of HBV vaccination, reduced liver cancer risk. The extensive HBV vaccine coverage now in place augurs even greater risk reductions in the future.

Epidemiological evidence has suggested that consumption of a diet rich in cruciferous vegetables reduces the risk of several types of cancers and chronic degenerative diseases. In particular, broccoli sprouts are a convenient and rich source of the glucosinolate, glucoraphanin, which can release the chemopreventive agent, sulforaphane, an inducer of glutathione S-transferases. Two broccoli sprout-derived beverages, one sulforaphane-rich (SFR) and the other glucoraphanin-rich (GRR), were evaluated for pharmacodynamic action in a crossover clinical trial design. Study participants were recruited from the farming community of He Zuo Township, Qidong, China, previously documented to have a high incidence of hepatocellular carcinoma with concomitant exposures to aflatoxin and more recently characterized with exposures to substantive levels of airborne pollutants. Fifty healthy participants were randomized into two treatment arms. The study protocol was as follows: a 5 days run-in period, a 7 days administration of beverage, a 5 days washout period and a 7 days administration of the opposite beverage. Urinary excretion of the mercapturic acids of acrolein, crotonaldehyde, ethylene oxide and benzene were measured both pre- and postinterventions using liquid chromatography tandem mass spectrometry. Statistically significant increases of 20-50% in the levels of excretion of glutathione-derived conjugates of acrolein, crotonaldehyde and benzene were seen in individuals receiving SFR, GRR or both compared with their preintervention baseline values. No significant differences were seen between the effects of SFR versus GRR. Intervention with broccoli sprouts may enhance detoxication of airborne pollutants and attenuate their associated health risks.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality with nearly 700,000 deaths occurring annually. Hepatitis B virus (HBV) is a major contributor to HCC and acquired mutations in the HBV genome may accelerate its pathogenesis. In this study, a matched case-control investigation of 345 men who died of HCC and 625 controls were nested within a cohort of male hepatitis B surface antigen (HBsAg) carriers from Qidong, China. Matched preserving odds ratios (ORs) were used as a measure of association and 95% confidence intervals (CIs) as a measure of precision. Real-time polymerase chain reaction allowed for a quantitative comparison of the levels of the HBV 1762(T)/1764(A) mutation in cases and controls. A total of 278 (81%) of the cases were positive for the HBV 1762(T)/1764(A) mutation compared with 250 (40%) of the controls. The matched preserving OR of 6.72 (95% CI: 4.66 to 9.68) strongly indicated that cases were significantly more probably than controls to have the mutation. Plasma levels of DNA harboring the HBV mutation were on average 15-fold higher in cases compared with controls (P < 0.001). Most strikingly, the level of the mutation in the 20 controls who later developed and died of HCC were on average 274-fold higher than controls who did not develop HCC. Thus, within this cohort of HBsAg carriers at high risk of developing HCC, individuals positive for the HBV 1762(T)/1764(A) mutation at enrollment were substantially more probably to subsequently develop HCC, with a higher concentration of the mutation in plasma enhancing predisposition for cancer development.

One of several challenges in design of clinical chemoprevention trials is the selection of the dose, formulation, and dose schedule of the intervention agent. Therefore, a cross-over clinical trial was undertaken to compare the bioavailability and tolerability of sulforaphane from two of broccoli sprout-derived beverages: one glucoraphanin-rich (GRR) and the other sulforaphane-rich (SFR). Sulforaphane was generated from glucoraphanin contained in GRR by gut microflora or formed by treatment of GRR with myrosinase from daikon (Raphanus sativus) sprouts to provide SFR. Fifty healthy, eligible participants were requested to refrain from crucifer consumption and randomized into two treatment arms. The study design was as follows: 5-day run-in period, 7-day administration of beverages, 5-day washout period, and 7-day administration of the opposite intervention. Isotope dilution mass spectrometry was used to measure levels of glucoraphanin, sulforaphane, and sulforaphane thiol conjugates in urine samples collected daily throughout the study. Bioavailability, as measured by urinary excretion of sulforaphane and its metabolites (in approximately 12-hour collections after dosing), was substantially greater with the SFR (mean = 70%) than with GRR (mean = 5%) beverages. Interindividual variability in excretion was considerably lower with SFR than with GRR beverage. Elimination rates were considerably slower with GRR, allowing for achievement of steady-state dosing as opposed to bolus dosing with SFR. Optimal dosing formulations in future studies should consider blends of sulforaphane and glucoraphanin as SFR and GRR mixtures to achieve peak concentrations for activation of some targets and prolonged inhibition of others implicated in the protective actions of sulforaphane. Cancer Prev Res; 4(3); 384-95. ©2011 AACR.

Chronic infection with the hepatitis B virus (HBV) is the most important risk factor for hepatocellular carcinoma (HCC). However, determinants of HCC risk in infected individuals are not well understood. We prospectively evaluated the association between acquired HBV 1762(T)/1764(A) double mutations and HCC risk among 49 incident HCC cases and 97 controls with seropositive hepatitis B surface antigen at baseline from a cohort of 18,244 men in Shanghai, China, enrolled during 1986 to 1989. Compared with HBV carriers without the mutations, chronic HBV carriers with the HBV 1762(T)/1764(A) double mutations experienced an elevated risk of HCC (odds ratio, 2.47; 95% confidence interval, 1.04-5.85; P = 0.04). Risk increased with increasing copies of the double mutations; men with > or =500 copies/microL serum had an odds ratio of 14.57 (95% confidence interval, 2.41-87.98) relative to those without the double mutations (P(trend) = 0.004). Thus, the HBV 1762(T)/1764(A) double mutation is a codeterminant of HCC risk for people chronically infected with HBV.

A major risk factor for hepatocellular carcinoma (HCC) is hepatitis B virus (HBV), whose pathogenesis is exacerbated by the acquisition of mutations that accelerate carcinogenesis. We examined, with mass spectrometry, the temporality of an HBV 1762(T)/1764(A) double mutation in plasma and tumors. Initial studies found that 52 of 70 (74.3%) tumors from patients residing in Qidong, People's Republic of China, contained this HBV mutation. Paired plasma samples were available for six of the tumor specimens; four tumors had the HBV 1762(T)/1764(A) mutation, whereas three of the paired plasma samples were also positive. The potential predictive value of this biomarker was explored by using stored plasma samples from a study of 120 residents of Qidong who had been monitored for aflatoxin exposure and HBV infection. After 10 years of passive follow-up, there were six cases of major liver disease including HCC (four cases), hepatitis (one case), and cirrhosis (one case). All six cases had detectable levels of the HBV 1762(T)/1764(A) mutation up to 8 years before diagnosis. Finally, 15 liver cancers were selected from a prospective cohort of 1,638 high-risk individuals in Qidong on the basis of available plasma samples spanning the years before and after diagnosis. The HBV 1762(T)/1764(A) mutation was detected in 8 of the 15 cases (53.3%) before cancer. The persistence of detection of this mutation was statistically significant (P = 0.022, two-tailed). We therefore found that a prediagnosis biomarker of specific HBV mutations can be measured in plasma and suggest this marker for use as an intermediate endpoint in prevention and intervention trials.

This chapter reviews the data that have been accumulated implicating aflatoxin ingestion as an important risk factor in the aetiology of hepatocellular carcinoma (HCC). Numerous epidemiological studies have observed a correlation between areas of high aflatoxin exposure and a high incidence of HCC. The use of experimental models and specific biomarkers for aflatoxin exposure, such as urinary metabolites or aflatoxin adducts, have validated these findings. Ongoing clinical trials in Qidong, China, have indicated that oltipraz, a chemopreventive agent, can lower the biologically effective dose of aflatoxins by decreasing the metabolism of aflatoxin to its carcinogenic form and increasing the detoxification pathways of these metabolites. Intervention with chemicals such as these, alongside hepatitis B virus immunization programmes and improved storage conditions of staple foods, are prevention measures that can be undertaken to reduce the incidence of HCC in high-risk regions.

Molecular biomarkers are becoming increasingly important tools to identify people who are at highest risk of developing cancer. For many years we have been studying residents of Qidong County, People's Republic of China, to examine the combined impact of aflatoxin exposure with other risk factors as contributors to the high liver cancer incidence rates in this region. This study was conducted to determine the effects of aflatoxin exposure, as measured by serum aflatoxin-albumin adduct levels, on somatic mutation frequency in the human hypoxanthine guanine phosphoribosyl transferase gene (HPRT). Subjects were assigned as low or high according to a dichotomization around the population mean of aflatoxin-albumin adducts. HPRT mutant frequency was determined in individuals by a T cell clonal assay and the samples were categorized as low or high according to mean values. Separate analyses were also conducted for the small set of hepatitis B virus surface antigen (HBsAg)-positive and the larger set of HBsAg-negative individuals, known risk factors for liver cancer. An odds ratio of 19.3 (95% confidence interval 2.0, 183) was demonstrated for a high HPRT mutation frequency in individuals with high aflatoxin exposure compared with those with low aflatoxin exposure. This association indicates that aflatoxin-induced DNA damage in T lymphocytes, assessed using the validated surrogate albumin adduct markers, leads to increased mutations reflected as elevated HPRT gene mutations. This cross-sectional study suggests the potential use of mutation frequency of the HPRT gene as a long-term biomarker of aflatoxin exposure in high risk populations.

The major risk factors for hepatocellular carcinomas (HCC) in high incidence areas include infection with hepatitis B and C viruses (HBV, HCV) and exposure to aflatoxin. Genetic alterations in 24 liver resection specimens from Shanghai and Qidong were studied. Hepatitis B virus was integrated in all patient samples, and a null phenotype for the GSTM1 enzyme was present in 63% of patients. Alteration of p53 was present in 95% (23/24) of cases: mutations of the p53 gene in 12 HCC, p53 overexpression in 13 and loss of heterozygosity (LOH) of chromosome 17p in 17. All seven HCCs with a p53 mutation from Qidong and three of five from Shanghai had the aflatoxin-associated point mutation with a G to T transversion at codon 249, position 3. No HCC had microsatellite instability. LOH of chromosome 4q, 1p, 16q and 13q was present in 50%, 46%, 42% and 38%, respectively, and 4q was preferentially lost in HCCs containing a p53 mutation: LOH of 4q was present in 75% (9/12) of HCC with, but only 25% (3/12) of HCC without, a p53 gene mutation (P = 0.01). These data indicate a possible interaction between p53 gene mutation and 4q loss in the pathogenesis of HCC.

Mycotoxins are toxic fungal metabolites which are structurally diverse, common contaminants of the ingredients of animal feed and human food. To date, mycotoxins with carcinogenic potency in experimental animal models include aflatoxins, sterigmatocystin, ochratoxin, fumonisins, zearalenone, and some Penicillium toxins. Most of these carcinogenic mycotoxins are genotoxic agents with the exception of fumonisins, which is currently believed to act by disrupting the signal transduction pathways of the target cells. Aflatoxin B1 (AFB1), a category I known human carcinogen and the most potent genotoxic agent, is mutagenic in many model systems and produces chromosomal aberrations, micronuclei, sister chromatid exchange, unscheduled DNA synthesis, and chromosomal strand breaks, as well as forms adducts in rodent and human cells. The predominant AFB1-DNA adduct was identified as 8, 9-dihydro-8-(N7-guanyl)-9-hydroxy-AFB1 (AFB1-N7-Gua), which derives from covalent bond formation between C8 of AFB1-8,9-epoxides and N7 of guanine bases in DNA. Initial AFB1-N7-guanine adduct can convert to a ring-opened formamidopyrimidine derivative, AFB1-FAPY. The formation of AFB1-N7-guanine adduct was linear over the low-dose range in all species examined, and liver, the primary target organ, had the highest level of the adduct. Formation of initial AFB1-N7-guanine adduct was correlated with the incidence of hepatic tumor in trout and rats. The AFB1-N7-guanine adduct was removed from DNA rapidly and was excreted exclusively in urine of exposed rats. Several human studies have validated the similar correlation between dietary exposure to AFB1 and excretion of AFB1-N7-guanine in urine. Replication of DNA containing AFB1-N7-guanine adduct-induced G-->T mutations in an experimental model. Activation of ras protooncogene has been found in AFB1-induced tumors in mouse, rat, and fish. More strikingly, the relationship between aflatoxin exposure and development of human hepatocellular carcinoma (HHC) was demonstrated by the studies on the p53 tumor suppressor gene. High frequency of p53 mutations (G-->T transversion at codon 249) was found to occur in HHC collected from populations exposed to high levels of dietary aflatoxin in China and Southern Africa. Furthermore, AFB1-induced DNA damage and hepatocarcinogenesis in experimental models can be modulated by a variety of factors including nutrients, chemopreventive agents, and other factors such as food restriction and viral infection, as well as genetic polymorphisms., (Copyright 1999 Elsevier Science B.V.)

Background: Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of foods contaminated with aflatoxins, which require metabolic activation to become carcinogenic. In a randomized, placebo-controlled, double-blind phase IIa chemoprevention trial, we tested oltipraz, an antischistosomal drug that has been shown to be a potent and effective inhibitor of aflatoxin-induced hepatocarcinogenesis in animal models., Methods: In 1995, 234 adults from Qidong were enrolled. Healthy eligible individuals were randomly assigned to receive by mouth 125 mg oltipraz daily, 500 mg oltipraz weekly, or a placebo. Sequential immunoaffinity chromatography and liquid chromatography coupled to mass spectrometry or to fluorescence detection were used to identify and quantify phase 1 and phase 2 metabolites of aflatoxin B1 in the urine of study participants. Reported P values are two-sided., Results: One month of weekly administration of 500 mg oltipraz led to a 51% decrease in median levels of the phase 1 metabolite aflatoxin M1 excreted in urine compared with administration of a placebo (P = .030), but it had no effect on levels of a phase 2 metabolite, aflatoxin-mercapturic acid (P = .871). By contrast, daily intervention with 125 mg oltipraz led to a 2.6-fold increase in median aflatoxin-mercapturic acid excretion (P = .017) but had no effect on excreted aflatoxin M1 levels (P = .682)., Conclusions: Intermittent, high-dose oltipraz inhibited phase 1 activation of aflatoxins, and sustained low-dose oltipraz increased phase 2 conjugation of aflatoxin, yielding higher levels of aflatoxin-mercapturic acid. While both mechanisms can contribute to protection, this study highlights the feasibility of inducing phase 2 enzymes as a chemopreventive strategy in humans.

In 1995, 234 adults from Qidong, Jiangsu Province, People's Republic of China, where hepatocellular carcinoma is the leading cause of cancer deaths and exposure to dietary aflatoxins is widespread, were enrolled and followed in a Phase II chemoprevention trial. The goals of the study were to define a dose and schedule of oltipraz for reducing levels of validated aflatoxin biomarkers and to characterize dose-limiting toxicities. Healthy eligible individuals, including those infected with hepatitis B virus, were randomized to receive either 125 mg of oltipraz daily, 500 mg of oltipraz weekly, or placebo. Blood and urine specimens were collected to monitor toxicities and evaluate biomarkers over the 8-week intervention period and subsequent 8-week follow-up period. Unique trial aspects included a synchronous follow-up schedule, daily observed administration of all medications, timely international data transference, and use of biomarkers as outcomes. One hundred thirty-two participants took their medications without interruptions, approximately 77% contributed all nine urine samples, and 78% contributed all seven blood samples. Fifty-one participants (21.8%) reported clinical adverse events. An extremity syndrome, developing soon after the start of treatment, was the only event that occurred more frequently (P = 0.002) among the active groups (18.4 and 14.1% of the daily 125 and weekly 500 mg arms, respectively) compared with placebo (2.5%). The oltipraz arms did not differ in symptom type or severity, and there were no indications of exacerbated drug intolerance among the few participants infected with hepatitis B virus. The good compliance with an intense follow-up schedule shows that chemoprevention trials with biomarker end points may be conducted in such populations.

Oltipraz has been used clinically in many regions of the world as an antischistosomal agent and is an effective inhibitor of aflatoxin hepatocarcinogenesis in rats. This chemopreventive action of oltipraz results primarily from an altered balance in aflatoxin metabolic activation and detoxication. In 1995, a randomized, placebo-controlled, double-blind intervention was conducted in residents of Qidong, People's Republic of China, who are at high risk for exposure to aflatoxin and development of hepatocellular carcinoma. The major study objectives were to define a dose and schedule for oltipraz that would reduce levels of aflatoxin biomarkers in biofluids of the participants, and to further characterize dose-limiting side effects. Two hundred thirty-four healthy eligible individuals, including those infected with HBV, were randomized to receive either 125 mg oltipraz daily, 500 mg oltipraz weekly, or placebo. Blood and urine specimens were collected to monitor potential toxicities and evaluate biomarkers over the 8-week intervention and subsequent 8-week follow-up periods. Overall, compliance in the intervention was excellent; approximately 85% of the participants completed the study. Objective evaluation of adverse events was greatly facilitated by inclusion of a placebo arm in the study design. A syndrome involving numbness, tingling, and pain in the fingertips was the only event that occurred more frequently among the active groups (18 and 14% of the daily 125 mg and weekly 500 mg arms, respectively) compared to placebo (3%). These symptoms were reversible and could be relieved with non-steroidal antiinflammatory agents. A more complete understanding of the chemopreventive utility of oltipraz awaits completion of an assessment of the efficacy of oltipraz in modulating levels of aflatoxin biomarkers.

A cohort of 18,244 mostly middle-aged (45-64 years) men residing in four small geographically defined areas of Shanghai was accrued between January 1986 and September 1989. In addition to an in-person interview regarding dietary and other past exposures, each subject donated a single void urine sample at recruitment so that the presence of aflatoxins in urine could be assessed. In addition, a 1-year survey of market foods in Shanghai was conducted to quantitatively estimate the extent of aflatoxin exposure in the study population. After close to 70,000 person-years of follow-up, 55 incident cases of hepatocellular carcinoma (HCC) had been identified. Levels of urinary aflatoxin B1 and the oxidative metabolites, including the major aflatoxin nucleic acid adduct, aflatoxin-N7-guanine, were determined for 50 of the 55 identified cases of HCC. Two hundred sixty-seven controls were chosen randomly from the cohort; they were matched to the 50 cases by age (within 1 year), time of specimen collection (within 1 month), and residence. After integrating the high-pressure liquid chromatography chromatograms to measure aflatoxin-N7-guanine, aflatoxin M1, aflatoxin P1, and aflatoxin B1, 49, 67, 53, and 71 of the urine samples had detectable levels of these compounds, respectively. The aflatoxin metabolite detected at the highest concentration was aflatoxin P1; the range was 0.59-16.0 ng/ml. The range of aflatoxin M1 in the urine was 0.17-5.2 ng/ml. The aflatoxin-N7-guanine adduct range was 0.3-1.81 ng/ml in the 49 positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Human epidemiology and experimental animal data have provided the statistical association and biological information necessary to propose that aflatoxins are risk factors for human liver cancer. As liver cancer causes at least 200,000 deaths per year, prevention measures must be developed to ameliorate this nearly always fatal disease. Preventive strategies will be facilitated by the identification of individuals at high risk. It is the goal of the molecular dosimetry field to provide facile and accurate biomarkers to identify people at high risk for carcinogen exposure and consequent adverse health effects. We have developed methods to defect the major aflatoxin DNA adduct, aflatoxin N7-guanine (AFB-N7-guanine), in urine, examined the dose-response characteristics in people living in China and The Gambia, and have found an excellent association of this biomarker with exposure. In addition to exposure studies in people, our laboratories have monitored AFB-N7-guanine excretion in the urine of rats whose risk for developing cancer has been modulated with dietary chemoprotective agents such that independent groups of animals receiving the same dosage of aflatoxin B1 were at either high or low risk for tumorigenesis. The production of DNA damage by aflatoxins is not the exclusive mechanism for liver cancer. Many other factors, including hepatitis B virus, cell proliferation, and nutritional status, can exert strong modification effects in human disease. Thus, molecular epidemiological investigations that examine only one biomarker may greatly underestimate or overestimate the risk for an individual.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

We have used monoclonal antibody technology to produce antibodies that recognize aflatoxins in order to develop noninvasive methods in conjunction with other chemical analytical techniques to monitor human exposure to environmental carcinogens. These methods require the ability to quantitate aflatoxins and their metabolites, including DNA and protein adducts, in readily accessible compartments such as serum and urine. The techniques permit efficient analysis of many samples in a relatively short time. Also, these monoclonal antibody affinity columns have been extremely useful for rapid isolation of aflatoxins from food and grain samples, as well as aflatoxin M1 from milk. Monoclonal antibody affinity methods are nondestructive to the aflatoxin molecule, so the sample aliquot can be used for confirmation. The use of monoclonal antibody preparative affinity columns represents a major, substantive breakthrough for analytical chemists and will be a generally applicable technology for isolation of many different substances.

Aflatoxin-serum albumin adducts in the blood of 42 residents of Guangxi Province, People's Republic of China, were determined and compared with intake of aflatoxin B1 (AFB1) and excretion of aflatoxin M1 (AFM1) in urine. Blood specimens were obtained during the same period that urine was collected and that diet was sampled. Serum albumin was isolated from blood by affinity chromatography on Reactive Blue 2-Sepharose and subjected to enzymatic proteolysis using Pronase. Immunoreactive products were purified by immunoaffinity chromatography and quantified by competitive radioimmunoassay. A highly significant correlation (r = 0.60, P less than 0.00003) of adduct level with AFM1 excretion was observed. An equally highly significant correlation of adduct level with intake (r = 0.69, P less than 0.000001) was also observed. From the slope of the regression line for adduct level as a function of intake, it was calculated that 1.4-2.3% of ingested AFB1 becomes covalently bound to serum albumin, a value very similar to that observed when rats are administered AFB1.