Aflatoxin Carcinogenesis Research Articles

Study of aflatoxicosis reduction: effect of Alchornea cordifolia on biomarkers in an aflatoxin B1 exposed rats

The toxicity of aflatoxins results in cancer and liver disease. Several natural substances such as plants exhibited their ability to inhibit the initiation of aflatoxin carcinogenesis. The aim of this study was to evaluate the effect of Alchornea cordifolia on biomarkers in an aflatoxin B1 (AFB1) exposed rats. The contents of polyphenols, flavonoids and the antioxidant activity of A. cordifolia ethanolic leaf extract (EELac) were assessed. Groups of rats were treated orally with a daily dose of a mixture of AFB1 at a dose of 150 μg/kg body weight and EELac (50, 100 and 300 mg/kg body weight) for 21 days. Biomarkers of AFB1, such as the AFB1-lysine adduct and aflatoxin M1 were assayed in blood and urine, respectively, using an HPLC system with a fluorescence detector. The contents of polyphenols and flavonoids were 6783.23 ± 272.76 μg EAG/g and 10.54 ± 3.15% of dry matter, respectively. EELac showed a good antioxidant activity (IC50 = 12.65 ± 0.13 μg/mL). The administration of the mixture (AFB1 + EELac) at different doses significantly reduced the level of AFB1-lysine adduct from 14.04 ± 2.1 to 4.13 ± 0.9 ng/mg albumin and that of Aflatoxin M1 (AFM1) from 456 ± 16 to 220 ± 24 ng/mL (p <0.05). The rate of reduction was 70.58% for AFB1-lysine adduct and 51.75% for AFM1. A. cordifolia could be used in the prevention of toxicity induced by AFB1 on account of its high content in phenolic compounds.

Saudi Guidelines for the Diagnosis and Management of Hepatocellular Carcinoma: Technical Review and Practice Guidelines

Recognizing the significant prevalence of hepatocellular carcinoma (HCC) in Saudi Arabia, and the difficulties often faced in early and accurate diagnoses, evidence-based management, and the need for appropriate referral of HCC patients, the Saudi Association for the Study of Liver diseases and Transplantation (SASLT) formed a multi-disciplinary task force to evaluate and update the previously published guidelines by the Saudi Gastroenterology Association. These guidelines were later reviewed, adopted and endorsed by the Saudi Oncology Society (SOS) as its official HCC guidelines as well. The committee assigned to revise the Saudi HCC guidelines was composed of hepatologists, oncologists, liver surgeons, transplant surgeons, and interventional radiologists. Two members of the task force served as guidelines editors. A wide based search on all published reports on all aspects of the epidemiology, natural history, risk factors, diagnosis, and management of HCC was performed. All available literature was critically examined and available evidence was then classified according to its strength. The whole document and the recommendations were then discussed in details by members and consensus was obtained. All recommendations in these guidelines were based on the best available evidence, but were tailored to the patients treated in Saudi Arabia. We hope that these guidelines will improve HCC patient care and enhance the multidisciplinary care needed for these patients.

Protective Interventions to Prevent Aflatoxin-Induced Carcinogenesis in Developing Countries

The public health impact of aflatoxin exposure is pervasive in economically developing countries; consequently, we need to design intervention strategies for prevention that are practicable for these high-risk populations. The adverse health consequences of aflatoxins in populations are quite varied, eliciting acute effects, such as rapid death, and chronic outcomes, such as hepatocellular carcinoma. Furthermore, a number of epidemiological studies describe a variety of general adverse health effects associated with aflatoxin, such as impaired growth in children. Thus, the magnitude of the problem is disseminated across the entire spectrum of age, gender, and health status in the population. The aflatoxins multiplicatively increase the risk of liver cancer in people chronically infected with hepatitis B virus (HBV), which illustrates the deleterious impact that even low toxin levels in the diet can pose for human health. Thus other aflatoxin interactions, which likely contribute to the disease burden, still remain to be identified. Therefore, many diverse and appropriate strategies for disease prevention are needed to decrease the incidence of aflatoxin carcinogenesis in developing countries.

Mechanisms of aflatoxin carcinogenesis.

Much progress has been made in elucidating the biochemical and molecular mechanisms that underlie aflatoxin carcinogenesis. In humans, biotransformation of AFB1 to the putative carcinogenic intermediate. AFB-8,9-exo-epoxide, occurs predominantly by cytochromes P450 1A2 and 3A4, with the relative importance of each dependent upon the relative magnitude of expression of the respective enzymes in liver. Genetic variability in the expression of these and other cytochromes P450 may result in substantial interindividual differences in susceptibility to the carcinogenic effects of aflatoxins. Detoxification of AFB-8,9-epoxide by a specific alpha class glutathione S-transferase is an important protective mechanism in mice, and it accounts for the resistance of this species to the carcinogenic effects of AFB. This particular form of GST is expressed constitutively only at low levels in rats, but it is inducible by antioxidants such as ethoxyquin, and it accounts for much of the chemoprotective effects of a variety of substances, including natural dietary components that putatively act via an "antioxidant response element" (ARE). In humans, the constitutively expressed GSTs have very little activity toward AFB1-8,9-exo-epoxide, suggesting that--on a biochemical basis--humans should be quite sensitive to the genotoxic effects of aflatoxins. If a gene encoding a high aflatoxin-active form of GST is present in the human genome, but is not constitutively expressed, and is inducible by dietary antioxidants (as occurs in rats), then chemo- and/or dietary intervention measures aimed at inducing this enzyme could be highly effective. However, as it is possible that human CYP 1A2 may also be inducible by these same chemicals (because of the possible presence of an ARE in this gene), the ultimate consequence of dietary treatment with chemicals that induce biotransformation enzymes via an ARE is uncertain. The balance of the rate of activation (exo-epoxide production) to inactivation (GST conjugation plus other P450-mediated non-epoxide oxidations) may be a strong indicator of individual and species susceptibility to aflatoxin carcinogenesis, if the experimental conditions are reflective of true dietary exposures. There is strong evidence that AFB-8,9-exo-epoxide binds to G:C rich regions of DNA, forming an adduct at the N7-position of guanine. Substantial evidence demonstrates that AFB1-8,9-epoxide can induce activating mutations in the ras oncogene in experimental animals, primarily at codon 12.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of Aflatoxin Carcinogenesis

Mechanisms of Aflatoxin Carcinogenesis

Aflatoxin is not a probable human carcinogen: The published evidence is sufficient

Since the early 1960s, when aflatoxin, the mold-produced contaminant of a number of important food commodities, was found to be a potent hepatocarcinogen for laboratory rats, there has been a sustained search for evidence to support the regulatory presumption that aflatoxin is a probable human carcinogen. The developing laboratory evidence of differences between species in metabolism of aflatoxin and susceptibility to its oncogenic effects indicated that humans were probably refractory to aflatoxin carcinogenesis, but the early epidemiological evidence indicated otherwise. That epidemiological evidence, however, contained flaws so that Working Groups of the International Agency for Research on Cancer (IARC) meeting in 1970, 1976, and 1982, although ignoring the biochemical evidence, did consider the available epidemiological evidence insufficient for a conclusion of human carcinogenicity. During the 1970s and 1980s, studies on the connection between chronic infection with hepatitis B virus (HBV) and primary liver cell cancer (PLC), the expected lesion from aflatoxin exposure, had established a very strong etiological relationship between HBV and PLC. Since all the epidemiological studies of aflatoxin and PLC conducted prior to 1982 had been of populations with endemic HBV infection, and, in addition to other flaws, had not been controlled for this confounding factor, there was a solid basis for their rejection. Most epidemiological studies in the 1980s of aflatoxin and PLC were either in the United States, where HBV-infected groups could be excluded from the study, or, when in areas of chronic HBV infection, attempts were made to include that factor. The study of U.S. populations showed no difference in mortality rates from PLC that could be attributed to aflatoxin exposure. The studies of populations with endemic HBV infection produced no convincing evidence to support a primary role for aflatoxin in the induction of human PLC, although an accessory role to HBV infection for aflatoxin could not be ruled out. However, the epidemiological studies of the HBV/PLC relation indicate that an accessory factor is not an essential condition, a conclusion supported by animal models and a laboratory study that specifically found no interaction between aflatoxin and a hepatitis virus in the duck, a species in which liver cancer can be induced by either agent. It was surprising that an IARC Working Group meeting in 1987 concluded, on the basis of much of this evidence that was available at that time, and citing other studies that appear to be irrelevant to the issue, that there was sufficient evidence to consider aflatoxin a probable human carcinogen.

Quantitation of carcinogen bound protein adducts by fluorescence measurements

A highly significant correlation of aflatoxin B 1 serum albumin adduct level with daily aflatoxin B 1 intake was observed in a molecular epidemiological study of aflatoxin carcinogenesis which used conventional fluorescence spectroscopy methods for adduct quantitation. Synchronous fluorescence spectroscopy and laser induced fluorescence techniques have been employed to quantitate antibenzo[ a]pyrene diol epoxide derived globin peptide adducts. Fast and efficient methods to isolate the peptide adducts as well as eliminate protein fluorescence background are described. A detection limit of several femtomoles has been achieved. Experimental and technical considerations of low temperature synchronous fluorescence spectroscopy and fluorescence line narrowing to improve the detection sensitivities are also presented.

Serum albumin adducts in the molecular epidemiology of aflatoxin carcinogenesis: correlation with aflatoxin B1 intake and urinary excretion of aflatoxin M1.

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.

The use of monoclonal antibody affinity columns for assessing DNA damage and repair following exposure to aflatoxin B 1

Primary hepatocellular carcinoma is one of the most lethal and common cancers in the world. It is particularly prevalent on the continents of Africa and Asia. A number of epidemiological studies have associated the exposure status of people to aflatoxin B 1 as being important in the etiology of liver cancer. However, to date these studies have relied upon the criteria of presumptive intake data, rather than relying upon quantitative analyses of aflatoxin—DNA adduct and metabolite content obtained by monitoring biological fluids from exposed people. Information obtained by monitoring exposed individuals for specific DNA adducts and metabolites will define the pharmacokinetics of aflatoxin B 1 in people, thereby facilitating risk assessments. We have developed monoclonal antibodies specific for aflatoxin metabolites, especially the DNA adducts. These monoclonal antibodies are used in solid phase immunoassays for the preparative purification of these aflatoxin derivatives from biological fluids. These methods in conjunction with other analytical procedures have resulted in the development of rapid protocols used to quantitatively measure aflatoxins in urine obtained from people dietarily exposed to this carcinogen. The people examined live in the Gambia and high liver cancer regions in the People's Republic of China. We have recently completed a pilot study in China where we have now begun to identify the pharmacokinetic parameters associated with chronic exposure of aflatoxin B 1 in the diet. Despite the development of these new technologies, we must continue to define animal model systems which can be used to interpret the human data. Our work using animal models based on the differential effects of ethoxyquin on the kinetics of aflatoxin—DNA adducts and gamma glutamyl transpeptidase-positive foci formation indicate that the direct linear extrapolation of total adduct content in target tissues to dose may be inappropriate to assign risk to people (or rats). However, our findings do support the concept that measurement of the major, rapidly excised AFB 1- N 7-Gua adduct in tissues and fluids is an appropriate dosimeter for estimating exposure status and risk in individuals consuming this mycotoxin. Further studies employing different classes of modifiers of aflatoxin carcinogenesis in rodent models should better define the relationships between hepatic and urinary levels of AFB 1- N 7-Gua and susceptibility to neoplasia.

Defenses against aflatoxin carcinogenesis in humans.

Aspects of work on aflatoxin mainly from the author's studies are discussed, emphasizing procedures, strategies and insights related to protection against aflatoxin carcinogenesis in man due to transmission in the food chain. Five lines of defense are discussed. First, is the interfacing of the farm animal as a biologic filter between contaminated crop (feed) and human food derived from animal products. Second, is chemical detoxification of meals with ammonia and of contaminated milk by ultraviolet light. Third is prevention of mold growth and aflatoxin elaboration with ammonium carbonate for potential application during storage and drying of agricultural products. Fourth, is the development of ultrasensitive analytical methods for aflatoxins with structural proof of identity. And, fifth are natural defenses related to the role of hydroxylation of mammalian metabolism in detoxification, especially the pathway to aflatoxin Q1, a major detoxifying mechanism in primates, and the epoxidation pathway and its relation to prevention of aflatoxin residue transmission into edible tissues.

Development of resistance to cytotoxicity during aflatoxin carcinogenesis.

IT is now some time since Haddow first suggested that an early stage in chemical carcinogenesis might involve the induction of cells resistant to the toxic action of the carcinogen1. Although it seems that many carcinogens also have acutely toxic properties, the relationship between these activities is largely unknown. We have investigated this relationship by using the mycotoxin aflatoxin B1, and shown that resistance is linked with proliferation of specific hepatocytes.

Mode of action and human health aspects of aflatoxin carcinogenesis.

The toxicity and carcinogenicity of the aflatoxins have been studied extensively in the rat. In this species these compounds are recognized as some of the most active hepatocarcinogens. The early lesions induced by the aflatoxins have been studied in an attempt to recognize the developing malignant neoplasm prior to the presence of metastasis. The relationship of focal hyperplasia to neoplasia will be discussed in comparison with other recognized carcinogens. The pattern of human hepatic neoplasia will be compared with that of neoplasia induced by the aflatoxins in order to add weight to the "mycotoxin hypothesis" of the aetiology of hepatic neoplasia. The role of dietary modification will also be discussed.

Translational step inhibited in vivo by aflatoxin B1 in rat-liver polysomes.

Aflatoxin B1 strongly inhibits protein synthesis in rat liver cells. We previously demonstrated that this inhibition could be divided into two steps: up to 5 h aflatoxin blocks protein synthesis directly and specifically at the polysome level; beyond 7 h protein synthesis inhibition appears chiefly as a consequence of transcription impairment due to drug action. This paper confirms the foregoing results and represents an attempt to localize the translational step inhibited in vivo by aflatoxin B1. We used the simulation study developed by Li, Kisilevsky, Wasan and Hammond, 1972 (Biochim. Biophys. Acta, 272, 451-462) to determine precisely the site inhibited in vivo after drug intoxication. This analysis is based on two parameters: the kinetics of polysome labeling to follow the nascent peptide synthesis, and the kinetics of supernatant labeling to follow the completed protein synthesis. Up to 5 h after dosing, aflatoxin specifically inhibits the elongation and/or termination steps during protein synthesis; after longer periods of time inhibition occurs essentially at the initiation step. When the intracellular concentration of aflatoxin is too high, particularly 2 h after dosing, each step of protein synthesis is blocked. Polypeptide synthesis by the postmitochondrial supernatants isolated from aflatoxin-treated animals is impaired in the same proportion as protein synthesis in vivo. The damage caused by aflatoxin is mostly observed on microsomes. However, purified polysomes isolated from aflatoxin-treated rats synthesize proteins in vitro to the same extent as those from controls. These results suggest that aflatoxin metabolite(s) are bound to polysomes with noncovalent bonds. These active metabolites are probably lost during polysome isolation procedures. Finally, relationships between protein metabolism and aflatoxin carcinogenesis are discussed.

Carcinogenic effects of low dietary levels of aflatoxin B1 in rats

Aflatoxin B1 was added to a semi-synthetic diet at levels of 1, 5, 15, 50 and 100 μg/kg (ppb). These diets were fed to male Fischer rats, beginning when their body weights were 80 g and continuing for up to 105 wk. A control group received the same diet without aflatoxin. Hepatocellular carcinomas were induced in a high incidence by levels of 50 and 100 ppb, and in a lower incidence by the other dietary levels. Tumours were induced in two of 22 animals fed 1 ppb, and pathological lesions were present in the livers of seven of the remaining survivors. These results suggest that, under these experimental conditions, the sensitivity of the Fischer rat to aflatoxin carcinogenesis is approximately the same as that previously reported for the rainbow trout.

Nutrition and Aflatoxin Carcinogenesis

ALTHOUGH malnutrition and aflatoxins coexist in many areas of the world where there is a high incidence of liver disease and hepatocarcinoma, animal experiments so far have not precisely duplicated the disease syndrome and the morphological picture observed in man1–8. We have examined interactions between nutritionally induced liver disease and experimental aflatoxin carcinogenesis and found that diets marginal in lipotropes result in remarkable differences in the response of rats to aflatoxin. This work was suggested by observations that diets low in protein had variable effects7,9 on aflatoxin toxicity and carcinogenicity. Furthermore, dietary lipotropes, expecially methionine and vitamin B12, may be critically low in many areas where liver carcinoma and mycotoxins coexist, and so we considered marginal lipotropes to be of more practical significance than low protein per se in the overall evaluation of interactions between nutrition and aflatoxin and their relation to carcinogenesis.

Inhibition of aflatoxin carcinogenesis by diethylstilbestrol in male rats.

Charles River Cesarean-Derived male rats were fed diets contaminated with aflatoxin, a potent hepatotoxin and liver carcinogen recently found to be contaminating staple food products. Rats fed diethylstilbestrol, in addition to the aflatoxin-contaminated diet, had decreased body weight and decreased incidence of tumors. Paired-feeding and feeding-to-weight studies indicated that the decreased tumor incidence associated with diethylstilbestrol was not the result of decreased food intake. These studies illustrate the extreme potency of aflatoxin and indicate that it represents a potential environmental hazard to man and domestic animals in many areas of the world. The male is more sensitive than the female to the effects of aflatoxin.

Aflatoxin carcinogenesis: inhibition of liver cancer induction in hypophysectomized rats.

AbstractIntact and hypophysectomized male rats were fed aflatoxin at a dose (4 μg/g in diet) sufficient to produce liver cancers in all of the 14 intact rats surviving 49 weeks. Fourteen hypophysectomized rats also survived this latent period, but no liver tumors were induced although the animals received aflatoxin at a higher rate than the intact control animals. However, tumors of extrahepatic tissues—particularly of the retro‐orbital lacrimal glands—were induced in the hypophysectomized rats. The selective blockade of hepatocarcinogenesis by hypophysectomy previously known with aminoazo dyes and fluorenamines is therefore extended to a new class of chemicals, the aflatoxins. It is suggested that aflatoxins may require metabolic activation before becoming effective carcinogens for rat liver.