2-3-dihydro-2-(n(7)-guanyl)-3-hydroxyaflatoxin-b1 and Liver-Neoplasms

Chemical-specific markers have been developed for a number of environmental carcinogens for use as molecular dosimeters of individual exposure. In addition to contributing substantially to the specificity and sensitivity of epidemiological studies aimed at determining the role of environmental agents in the etiology of human cancers, some of these biomarkers may prove to be useful endpoints for assessing the efficacy of preventive interventions including exposure avoidance or remediation and chemoprevention. Biomarkers of the biologically effective dose may be particularly useful in this context in that they provide a mechanistic linkage between exposure and disease outcome. The biologically effective dose reflects the amount of toxicant that has interacted with its critical molecular target and can be measured through a variety of analytical techniques as either carcinogen-DNA or -protein adducts. Approaches for the development and validation of aflatoxin adduct biomarkers are presented as a paradigm for the application of carcinogen-specific markers for cohort selection and as modifiable endpoints for assessing efficacy in chemoprevention trials.

Topics: Aflatoxin B1; Aflatoxins; Aged; Anticarcinogenic Agents; Biomarkers, Tumor; Carcinogens; Clinical Trials as Topic; Cohort Studies; DNA Adducts; Guanine; Hepatitis B; Humans; Liver Neoplasms; Lung Neoplasms; Male; Middle Aged; Proteins; Pyrazines; Risk Factors; Thiones; Thiophenes

In vitro and in vivo studies suggest that selected strains of probiotic bacteria can form tight complexes with aflatoxin B(1) and other carcinogens.. The aim of the present study was to determine whether administration of probiotic bacteria could block the intestinal absorption of aflatoxin B(1) and thereby lead to reduced urinary excretion of aflatoxin B(1)-N(7)-guanine (AFB-N(7)-guanine), a marker for a biologically effective dose of aflatoxin exposure. Elevated urinary excretion of this aflatoxin-DNA adduct is associated with an increased risk of liver cancer.. Ninety healthy young men from Guangzhou, China, were randomly assigned to 2 groups; one group received a mixture of Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii subsp. shermanii strains 2 times/d for 5 wk, and the other group received a placebo preparation. The subjects provided 4 urine samples: at baseline, at 3 and 5 wk after starting the supplementation, and at the end of the 5-wk postintervention period.. The percentage of samples with negative AFB-N(7)-guanine values tended to be higher in the probiotic group than in the placebo group during the 5-wk intervention period (odds ratio: 2.63, P = 0.052), and a statistically significant decrease in the concentration of urinary AFB-N(7)-guanine was observed in the probiotic group. The reduction was 36% at week 3 and 55% at week 5. The geometric means for the probiotic and placebo groups were 0.24 and 0.49 ng AFB-N(7)-guanine/mL, respectively, during the intervention period (P = 0.005).. A probiotic supplement reduces the biologically effective dose of aflatoxin exposure and may thereby offer an effective dietary approach to decrease the risk of liver cancer.

Topics: Aflatoxin B1; Biomarkers, Tumor; Carcinoma, Hepatocellular; China; DNA Adducts; Double-Blind Method; Guanine; Humans; Lacticaseibacillus rhamnosus; Liver Neoplasms; Placebos; Probiotics; Propionibacterium; Risk Factors

Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part from consumption of foods contaminated with aflatoxins. Chlorophyllin, a mixture of semisynthetic, water-soluble derivatives of chlorophyll that is used as a food colorant and over-the-counter medicine, has been shown to be an effective inhibitor of aflatoxin hepatocarcinogenesis in animal models by blocking carcinogen bioavailability. In a randomized, double-blind, placebo-controlled chemoprevention trial, we tested whether chlorophyllin could alter the disposition of aflatoxin. One hundred and eighty healthy adults from Qidong were randomly assigned to ingest 100 mg of chlorophyllin or a placebo three times a day for 4 months. The primary endpoint was modulation of levels of aflatoxin-N(7)-guanine adducts in urine samples collected 3 months into the intervention measured by using sequential immunoaffinity chromatography and liquid chromatography-electrospray mass spectrometry. This aflatoxin-DNA adduct excretion product serves as a biomarker of the biologically effective dose of aflatoxin, and elevated levels are associated with increased risk of liver cancer. Adherence to the study protocol was outstanding, and no adverse events were reported. Aflatoxin-N(7)-guanine could be detected in 105 of 169 available samples. Chlorophyllin consumption at each meal led to an overall 55% reduction (P = 0.036) in median urinary levels of this aflatoxin biomarker compared with those taking placebo. Thus, prophylactic interventions with chlorophyllin or supplementation of diets with foods rich in chlorophylls may represent practical means to prevent the development of hepatocellular carcinoma or other environmentally induced cancers.

Topics: Adult; Aflatoxin B1; Aflatoxins; Aged; Animals; Biomarkers; Carcinoma, Hepatocellular; China; Chlorophyllides; DNA Adducts; Female; Food Contamination; Guanine; Humans; Liver Neoplasms; Male; Middle Aged; Risk Factors

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B(1) (AFB(1))-induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB(1) to alter repair of AFB(1)-DNA damage was determined. Plasmid DNA containing AFB(1)-N(7)-guanine or AFB(1)-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and approximately 6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB(1)-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB(1) i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB(1)-N(7)-guanine damage was approximately 3.5-fold higher in liver of AFB(1)-treated mice relative to control. This is the first study to show that in vivo treatment with AFB(1) can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB(1), and selective induction of repair in liver contribute to the susceptibility of mice to AFB(1)-induced lung tumorigenesis relative to hepatocarcinogenesis.

Topics: Aflatoxin B1; Animals; Carcinogens; Cocarcinogenesis; DNA; DNA Repair; Female; Genetic Predisposition to Disease; Guanine; Liver; Liver Neoplasms; Lung; Lung Neoplasms; Male; Mice; Pyrimidines; Rats; Rats, Sprague-Dawley; Species Specificity

A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B(1) (AFB(1)). Hypotheses have been put forth that AFB(1), in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation. The primary DNA adduct of AFB(1) is 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxyaflatoxin B(1) (AFB(1)-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB(1)-formamidopyrimidine (AFB(1)-FAPY) adduct. AFB(1)-FAPY is detected at near maximal levels in rat DNA days to weeks after AFB(1) exposure, underscoring its high persistence in vivo. The present study reveals two striking properties of this DNA adduct: (i) AFB(1)-FAPY was found to cause a G to T mutation frequency in Escherichia coli approximately 6 times higher than that of AFB(1)-N7-Gua, and (ii) one proposed rotamer of AFB(1)-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer.

Topics: Aflatoxin B1; Bacteriophage M13; Carcinoma, Hepatocellular; DNA Adducts; DNA, Neoplasm; DNA, Viral; Guanine; Humans; Liver Neoplasms; Molecular Structure; Mutagenesis; Mutagens; Pyrimidines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Epidemiological evidence indicates that aflatoxin B1 (AFB1) intake is associated with an increased risk of hepatocellular carcinoma (HCC). The hepatocarcinogenesis is initiated by covalent binding of AFB1 to cellular DNA. To determine whether nutritional factors and hormonal status may influence the binding of AFB1 to hepatic DNA, a cross-sectional study was performed on a total of 42 male asymptomatic hepatitis B surface antigen (HBsAg) carriers and 43 male non-carriers in a cohort study on the multistage development of HCC in Taiwan. The major AFB1-DNA adduct in vivo, AFB1-N7-guanine, was measured by high-performance liquid chromatography in urine. Urinary AFB1-N7-guanine was detectable in 40% of the subjects. HBsAg carriers had a higher detection rate of urinary AFB1-DNA adducts than non-carriers and the difference was statistically significant after multivariate adjustment. After taking into account the total AFB1 urinary metabolite level, chronic HBsAg carrier status, and other potential confounders, plasma levels of cholesterol, alpha-tocopherol, and alpha- and beta-carotene were positively associated with the detection rate of the AFB1-DNA adducts in a dose-dependent manner, whereas plasma lycopene level was inversely related to the presence of the adducts in urine. The association of urinary AFB1-DNA adducts with the plasma levels of cholesterol, alpha-tocopherol, lycopene, and alpha- and beta-carotene was observed at both low and high exposure levels of AFB1. There was a synergistic interaction of plasma alpha-tocopherol with alpha- and beta-carotene on the adduct levels. No association with the adducts was found for plasma levels of retinol and testosterone. This study demonstrated different associations of antioxidant vitamins with AFB1-DNA adduct formation. The data consistent with our previous finding in cultured woodchuck hepatocytes that alpha-tocopherol and beta-carotene enhanced AFB1-DNA adduct formation suggest that prospective investigation of the relationship between plasma micronutrients and risk of AFB1-related HCC is warranted.

Topics: Adult; Aflatoxin B1; Aged; Antioxidants; beta Carotene; Carotenoids; Chronic Disease; DNA; DNA Adducts; Guanine; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Testosterone; Vitamin E; Vitamins

Earlier work in this laboratory and that carried out by others demonstrated that after a single dose of aflatoxin B1 (AFB) the resulting liver AFB-DNA adduct levels were directly proportional to dose. Earlier work also showed that after ten daily doses the AFB dose-response relationship with gamma-glutamyl transpeptidase (GGT) positive preneoplastic foci measured at 3 months was sublinear, with a threshold at a dose of about 150 micrograms/kg body weight/day. The objective of this study is to determine the factors influencing the shift in AFB dose-response between AFB-DNA adducts and GGT foci. Male Fisher 344 weanling rats were orally administered one or ten doses of AFB ranging from 50 to 350 micrograms/kg body weight/day. The animals were killed 2 or 24 h after the first AFB dose, or after the tenth AFB dose. The first and tenth doses were tritiated in these animals and 3H-AFB-guanine adducts isolated from liver DNA were measured by HPLC. Another group was killed 3 months after receiving ten doses in order to measure GGT foci development. AFB-guanine adduct levels were directly proportional to dose after the first dose, but after the tenth dose were much lower in the 200-350 micrograms/kg groups than after a single dose. The GGT foci response confirmed earlier work concerning a sublinear response. Among the individual animals in the 200-350 micrograms/kg groups there was a positive relationship, after controlling for dose, between GGT foci development and weight gained both during dosing (P = 0.018) and also to a lesser extent during the early promotional period (P = 0.066). Enzyme activity levels of GGT in liver homogenates were higher in the highest dose groups and reflected biliary proliferation rather than histological GGT stained foci. Urinary levels of AFB metabolites changed proportions in the high dosage multiply dosed animals reflecting alteration in AFB metabolism or excretion. The differences between the linear adduct and the sublinear foci dose-response curves may be the result of non-adduct effects of higher multiple AFB doses on foci formation including acute cytotoxicity, altered AFB metabolism and disposition, enhanced weight gains, or shortened foci latency but not through enhanced guanine adduct levels. Other studies that showed a linear relationship between AFB dose and liver tumor development used continuous feeding of maximal doses an order of magnitude less than the lowest dose in this study and thus avoided acutely toxic effects.

Topics: Administration, Oral; Aflatoxin B1; Animals; Body Weight; Carcinogens; Chromatography, High Pressure Liquid; DNA Adducts; Dose-Response Relationship, Drug; gamma-Glutamyltransferase; Guanine; Liver; Liver Neoplasms; Male; Precancerous Conditions; Rats; Rats, Inbred F344

The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin. The G --> T mutations of AFB1-N7-Gua, unlike those (if the AFB1-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern matching that in intact cells treated with the toxin. It is concluded that the AFB1-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells. While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine. In contrast, the apurinic site-containing genome gave rise only to targeted mutations. The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face of the guanine residue. These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1.

Topics: Aflatoxin B1; Bacteriophage M13; Base Sequence; Carcinoma, Hepatocellular; DNA Adducts; DNA, Viral; Escherichia coli; Guanine; Humans; Liver Neoplasms; Models, Molecular; Molecular Sequence Data; Mutagenesis; SOS Response, Genetics; Structure-Activity Relationship

Covalent binding of aflatoxin B1 (AFB1) with hepatic DNA may be a critical step in hepatocarcinogenesis. The extent of the AFB1 binding to DNA may depend on various endogenous factors and concurrent exposure to other environmental agents. This study was performed to determine whether any individual characteristics correlated with the formation of AFB1-DNA adducts. The major AFB1-DNA adduct, AFB1-N7-guanine, was measured using a high performance liquid chromatographic assay in urine samples from 43 asymptomatic hepatitis B virus surface antigen carriers and 43 noncarriers randomly selected from a cohort study in Taiwan. The total aflatoxin metabolite level was associated with the detection rate of urinary AFB1-N7-guanine adducts in a dose-dependent manner. The AFB1-DNA adduct excreted in the urine was detectable in 60% of individuals who smoked cigarettes but abstained from alcohol, 64% of individuals who had a habit of drinking alcohol but not smoking cigarettes, and only 29% of those who neither smoked nor drank alcohol. The association between urinary AFB1-DNA adduct level and habits of smoking cigarettes and drinking alcohol remained statistically significant when adjustment was made for potential confounders. There was a significant increase with age for the detection rate of urinary AFB1-N7-guanine adducts. Age and habits of cigarette smoking and alcohol drinking were also found to be associated with a higher percentage of AFB1-N7-guanine in total AFB1 metabolite excretion, indicating an increased activation of AFB1. No significant association with the AFB1-DNA adduct level was observed for hepatitis B virus surface antigen carrier status, educational level, and ethnicity. These data suggest a potential role of age, cigarette smoking, and alcohol drinking in AFB1-induced hepatocarcinogenesis.

Topics: Aflatoxin B1; Age Factors; Alcohol Drinking; Biomarkers; Carcinogens; Carcinoma, Hepatocellular; Carrier State; DNA Adducts; Guanine; Hepatitis B; Hepatitis B Surface Antigens; Humans; Liver Neoplasms; Logistic Models; Male; Multivariate Analysis; Prospective Studies; Risk Factors; Smoking; Taiwan

Two major etiological agents, hepatitis B virus and aflatoxin B1, are considered to be involved in the induction of liver cancer in Africa. In order to elucidate any synergistic effect of these two agents we conducted a study in various parts of Kenya with different liver cancer incidence in order to establish the rate of exposure to aflatoxin and the prevalence of hepatitis infections. Of all tested individuals 12.6% were positive for aflatoxin exposure as indicated by the urinary excretion of aflatoxin B1-guanine. Assuming no annual and seasonal variation, a regional variation in the exposure was observed. The highest rate of aflatoxin exposure was found in the Western Highlands and Central Province. The incidence of hepatitis infection nationwide as measured by the presence of the surface antigens was 10.6%, but a wide regional variation was observed. A multiplicative and additive regression analysis to investigate if hepatitis and aflatoxin exposure had a synergetic effect in the induction of liver cancer was negative. However, a moderate degree of correlation between the exposure to aflatoxin and liver cancer was observed when the study was limited to certain ethnic groups. The study gives additional support to the hypothesis that aflatoxin is a human liver carcinogen.

Topics: Aflatoxin B1; Aflatoxins; Carcinoma, Hepatocellular; DNA Damage; Female; Guanine; Hepatitis B; Hepatitis B Surface Antigens; Humans; Kenya; Liver Neoplasms; Male; Sex Factors