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The effect of dietary aflatoxin on wild turkey poults.

Aflatoxins, toxic metabolites of Aspergillus flavus or Aspergillus parasiticus, cause poor feed utilization, decreased weight gains, depressed immune function, liver dysfunction, coagulation abnormalities, and death in a wide variety of species including humans. Conservationists have become concerned that increasingly popular wildlife feeding or baiting practices could expose wildlife to toxic amounts of aflatoxin-contaminated grains. In particular, the effects of aflatoxins on the wild turkey (Meleagris gallopova silvestris) are of concern because the conspecific domestic turkey is highly susceptible to aflatoxins. To evaluate the effect of dietary aflatoxin on wild turkeys, four groups of 4-mo-old wild turkeys were fed diets containing either 0, 100, 200, or 400 micrograms aflatoxin/kg feed for 2 wk in September and October 1996. Aflatoxin-fed poults had decreased feed consumption and weight gains as compared with control poults. Decreased liver-to-body weight ratios, liver enzyme alterations, slightly altered blood coagulation patterns, and mild histologic changes indicated low-level liver damage. Compromise of cell-mediated immunity was indicated by decreased lymphoblast transformation. The effects were apparent in all treatment groups to variable levels, but significant differences most often were found at 400 micrograms aflatoxin/kg feed. This study shows that short-term aflatoxin ingestion by wild turkeys can induce undesirable physiologic changes; therefore, exposure of wild turkeys to feeds containing aflatoxin levels of 100 micrograms aflatoxin/kg feed or more should be avoided.

Aflatoxins↗

Immune response to aflatoxin B1-histone H1 complex.

Antibodies against aflatoxin B1 have been raised in rabbits using aflatoxin B1-histone H1 conjugates as immunogen. Aflatoxin B1 was coupled to histone H1 via the ultimate carcinogen aflatoxin B1-2, 3-epoxide. The antibodies are specific for aflatoxin B1. The average number of binding sites on the antibody molecules for aflatoxin B1 as obtained from Scatchard plot analysis of the binding data is 1.94 with < F degree = -6.19 Kcal/mol, while the average association constant for the binding is 34.5 x 10(3) M-1. Male wistar rats after immunization with aflatoxin B1-histone H1 showed lower mortality and reduction of acute toxic effects in the liver when challenged with a single dose of aflatoxin B1. The antibodies may be useful in immunoprophylaxis against aflatoxicosis.

Aflatoxin B1↗

Aflatoxin B1 DNA adducts in smeared tumor tissue from patients with hepatocellular carcinoma.

Aflatoxins are well-known animal hepatocarcinogens, but the association between aflatoxins and human hepatocellular carcinoma remains to be elucidated. A study method consisting of indirect immunofluorescence assay combined with densitometry was developed to quantitate aflatoxin B1 DNA adducts in smeared liver tissue obtained at the time of biopsy for diagnosis in 50 hepatocellular carcinoma patients in Taiwan. Monoclonal antibody 6A10, generated against the persistent form of the major N7 guanine adduct of aflatoxin B1, was used for detection of adduct. Thirty-five (70%) of the hepatocellular carcinoma samples had detectable levels of aflatoxin B1 DNA adducts (> or = 1/10(6) nucleotides). The detection rate was slightly lower in men (69%) than in women (75%), and younger patients had a significantly higher rate of adducts (83%) than did older ones (58%). Carriers of both HBsAg and HBeAg, carriers of HBsAg only and noncarriers had different rates of detection: 29%, 74% and 82%, respectively. Patients with family histories of hepatocellular carcinoma had a higher detection rate (100%) than did those patients without such histories (67%). No association was found between aflatoxin B1 DNA adducts in liver tissue and Child's score for severity of liver disease. The results suggest that aflatoxin B1 may be involved in the pathogenesis of hepatocellular carcinoma in Taiwan. Our immunohistochemical method for analysis of adducts in small numbers of cells from the target organ should improve results of monitoring for the biologically effective dose of aflatoxin.

Adult↗

Outbreak of aflatoxin poisoning--eastern and central provinces, Kenya, January-July 2004.

In May 2004, CDC Kenya, trainees of the CDC-supported Field Epidemiology and Laboratory Training Program (FELTP) in Kenya, the World Health Organization, and CDC were invited by the Kenya Ministry of Health (KMOH) to participate in the investigation of an outbreak of jaundice with a high case-fatality rate (CFR) in the districts of Makueni and Kitui, Eastern Province. Preliminary laboratory testing of food collected from the affected area revealed high levels of aflatoxin, suggesting that the outbreak was caused by aflatoxin poisoning, as was a previous outbreak in the same area in 1981. In the United States, aflatoxin concentrations are limited to 20 parts per billion (ppb), a level also adopted by Kenyan authorities. The 2004 outbreak resulted from widespread aflatoxin contamination of locally grown maize, which occurred during storage of the maize under damp conditions. Urgent replacement of the aflatoxin-contaminated maize with noncontaminated maize proved to be a critical intervention; however, as of July 21, a limited number of new cases continued to be detected. This report summarizes the preliminary results of the outbreak investigation. Aflatoxin poisoning likely will continue to be a public health problem until culturally appropriate storage methods for dry maize are implemented by the local population. In addition, enhanced surveillance for human aflatoxin poisoning and testing of commercially sold maize for aflatoxin levels will lead to long-term improvements in public health.

Adolescent↗

[Aflatoxin detection in urine in liver diseases in childhood].

During the last decade, a number of epidemiological and laboratory investigations have shown a close association among hepatitis B, dietary exposure to aflatoxin, and an increased incidence of hepatocellular carcinoma. An immunoenzymatic method for aflatoxin detection in urine was developed and assessed at the National Institute of Oncology. Fifty four children (age range 4-16 years) from the pediatric service at the National Institute of Gastroenterology were studied. Thirty patients had a diagnosis of chronic active hepatitis (CHA 17 of which 56%) were found aflatoxin- positive, 20 where HbsAg-positive carriers of which 7 of 35% were positive to aflatoxin, and 4 patients suffered from metabolic diseases from which 1 (25%) was found aflatoxin- positive. Controls and patients were matched by age, with 7.5% of aflatoxin-positive patients. Our results confirm the link between hepatitis B and aflatoxin in this type of patients compared to controls. The immunoenzymatic system proved to be specific for aflatoxin detection with a sensitivity of 100 picograms/milliliter.

Adolescent↗

Clinical implications of food contaminated by aflatoxins.

Aflatoxins have been incriminated, mainly on circumstantial evidence, in hepatocellular carcinoma, acute hepatic failure and Reye's syndrome, but other possible effects of continuous or intermittent dietary exposure to aflatoxins, which occurs widely in the tropics, have received little study. Over the past 10 years evidence has steadily accumulated that incriminates aflatoxins in the aetiology of kwashiorkor, a widespread and serious disorder of children in the tropics, previously believed to be caused by protein deficiency. Investigation of human breast milk, undertaken initially to elucidate the pathogenesis of kwashiorkor in breastfed infants, has revealed widespread and serious exposure to aflatoxins from this source. Extension of these studies to pregnant women, in turn, revealed widespread and serious prenatal aflatoxin exposure. In laboratory and farm animals, such exposure has serious implications for immune and hepatic functions, and is detrimental to growth and development. Recent analysis of heroin samples show that heroin addicts may also be exposed to these toxins. These findings show that human exposure to aflatoxins may begin prenatally, persist during breastfeeding, and continue into adult life. It is postulated that aflatoxins (i) play a role in the aetiology of kwashiorkor, (ii) increase neonatal susceptibility to infection and jaundice, (iii) increase childhood susceptibility to infections and malignant disease, (iv) compromise immune responses to prophylactic immunisations and (v) may play a role in the pathogenesis of diseases in heroin addicts. There are indications also that acute, fatal aflatoxin poisoning which masquerades as 'hepatitis' may occur more frequently than is currently appreciated.

Acute Disease↗

Survey of some cereal grains and legume seeds for aflatoxin contamination in the Sudan.

Aflatoxins were detected in some cereals and leguminous seeds collected from 2 cities in the Sudan (Wad Medani and Khartoum). The levels of the aflatoxins in these seeds were below the reported hazard threshold. Sound intact seeds contained low or no aflatoxins compared to significantly higher levels detectable in damaged and mouldy seeds of the tested crop plants. The optimum temperature for aflatoxin production was variable for different isolates of A. flavus. The optimum is 30 degrees C for the maize isolate and only 20 degrees C for the haricot bean isolate. Large amounts of aflatoxins B1, B2 and G1 were produced by the 2 isolates while aflatoxin G2 was only produced by the haricot bean isolate at optimum temperature. Aflatoxin B1 had significantly affected seed germination and plant development in both maize and haricot bean plants grown from seeds soaked in aflatoxin solutions. The influence was increasing with increasing concentration of the toxin.

Aflatoxins↗

Aflatoxins in liver biopsies from Kenya.

The results of aflatoxin analysis of 15 needle liver biopsies from a rural hospital in Kenya are reported. Nine of the biopsies were from living subjects and six were post mortem. Blood and urine collected the same day was also analysed for aflatoxin content. Aflatoxins in the liver specimen from living subjects were only found in 3 out of 5 cases of hepatocellular carcinoma, but not in their blood. Aflatoxins were detected in 4 out of 6 post mortem liver specimens. In all three cases of cirrhosis aflatoxins were detected in blood and urine, but only one liver specimen showed aflatoxins. Our findings support the existing theory about the incrimination of aflatoxins in the aetiology of hepatocellular carcinoma, and the possible role aflatoxins may have in the pathogenesis of some forms of liver cirrhosis.

Adolescent↗

Aflatoxins in domestic and imported foods and feeds.

Aflatoxins, metabolic products of the molds Aspergillus flavus and A. parasiticus, may occur in foods and feeds. These toxins cannot be entirely avoided or eliminated from foods or feeds by current agronomic and manufacturing processes and are considered unavoidable contaminants. To limit aflatoxin exposure, the U.S. Food and Drug Administration (FDA) has set action levels for these toxins in foods and feeds involved in interstate commerce. FDA continually monitors food and feed industries through compliance programs. This report summarizes data generated from compliance programs on aflatoxins for the fiscal year 1986. Commodities sampled included peanuts and peanut products, corn and corn products, tree nuts, cottonseed, milk, spices, manufactured products, and miscellaneous foods and feeds. Correlations were highest between aflatoxin contamination and geographical areas for corn/corn products and cottonseed/cottonseed meal. Higher incidences of aflatoxin contamination in corn and corn products designated for human consumption were observed in samples collected in the southeastern states (32 and 28%, respectively). A higher incidence of contamination was observed in corn designated for animal feed from Arkansas-Texas (74%) than from the southeastern states (47%). Only 3% of feed corn from corn belt states contained detectable aflatoxins. All aflatoxin-contaminated cottonseed was collected in the Arizona-California area; 80% of cottonseed meal analyzed from this area also contained detectable levels of aflatoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxins↗

Aflatoxin B1 metabolism to aflatoxicol and derivatives lethal to Bacillus subtilis GSY 1057 by rainbow trout (Salmo gairdneri) liver.

Aflatoxicol, R0, was isolated from Mt. Shasta strain rainbow trout (Salmo gairdneri), and liver homogenates were incubated with aflatoxin B1. Its identity was confirmed by mass, infrared, and ultraviolet spectrometry. The structure was identical to one of the diastereomers prepared by chemical reduction of aflatoxin B1. Aflatoxicol was apparently formed by a reduced nicotinamide adenine dinucleotide phosphate-dependent soluble enzyme of the 105,000 x g supernatant from rainbow trout. Aflatoxicol was not lethal in phosphate buffer to Bacillus subtilis GSY 1057 (metB4, hisA1, uvr-1) nor were aflatoxins B1, Q1, and B2. In the presence of reduced nicotinamide adenine dinucleotide phosphate and trout liver microsomes, aflatoxicol reduced the viability of B. subtilis. Aflatoxin B2, which lacks the vinyl ether present in the other compounds, could not be activated. The product of aflatoxin B1 activation by trout liver microsomes was sought after incubation of 14C-labeled aflatoxin B1. The radioactivity was found in unaltered aflatoxin B1 and in three extremely polar metabolites. The quantity of the new metabolites and the level of microbial lethality was reduced by addition of cytosine and cysteine to the incubation medium. The vinyl ether configuration was a structural requirement for activation, and this finding and the nature of the enzymatic reaction were consistent with the hypothesis that the compounds were metabolized to highly reactive and unstable electrophilic products which bound to nucleophiles such as cytosine and were lethal to B. subtilis. The formation of aflatoxicol as the major product of trout liver metabolism is of great significance considering that it could be activated to a lethal compound and that rainbow trout are one of the most sensitive species to aflatoxin B1-induced carcinoma.

Aflatoxins↗

In vitro metabolism of aflatoxin B2 by animal and human liver.

The metabolism of aflatoxin B2 by postmitochondrial supernatant fractions of duck, rat, mouse, and human livers was studied in an in vitro system. Duck liver had a much higher level of activity than had tissues from other species. Postmitochondrial supernatant equivalent to 0.2 g whole liver metabolized 40 to 80% of the initial substrate in 30 min, compared to less than 6% for the other species. Among several metabolites formed by duck liver, aflatoxin B1 was produced in amounts equivalent to 2 to 8% of the initial substrate, and metabolites having chromatographic properties postualted for aflatoxicols 1 and 2 and aflatoxins M1 and M2 were also formed in small amounts. In contrast, rat, mouse, and human liver preparations produced no detectable aflatoxin B1 and only small amounts of compounds thought to be aflatoxins Q2 and P2. The greater susceptibility of duck liver to the toxicity of aflatoxin B2 may be attributable to its ability to form aflatoxin B2 may be attributable to its ability to form aflatoxin B1, which could then be activated through further metabolism.

Aflatoxins↗

Toxic effects of aflatoxin B1 on Bacillus brevis.

At a concentration of 50 microgram/ml and below, aflatoxin B1 inhibited Bacillus brevis (2611) bacteristatically, but at a concentration of 100 microgram/ml the inhibition was bactericidal. Bactericidal inhibition was further studied by determining the mean single survivor times of the bacterium in 100 microgram/ml aqueous solutions of aflatoxin B1 (18.2 mins.) and phenol (16.6 mins.). Further study on the bacteriostatic inhibition within the bacteriostatic concentration limit of 50 microgram/ml shows that the doubling time of B. brevis exposed to 5 microgram/ml aflatoxin B1 is approximately half that of the bacterium exposed to 10 microgram/ml. At the molecular level, 50 microgram/ml aflatoxin B1 inhibited the synthesis of DNA (78%), RNA (15%) and Protein (18%) in B. brevis. The strength of the affinity of subcellular fractions of B. brevis for aflatoxin B1 during inhibition was studied by assessing the resistance of aflatoxin B1 bound to cell fractions to removal by water. 66% (w/w) of the aflatoxin B1 bound to B. brevis after six successive washes in 50 ml distilled water, remained bound to the cell membrane fraction. The implication of this preferential strong binding to cell membrane was studied by investigating its effect on oxygen uptake. Results of inhibition of oxygen uptake is compatible with aflatoxin B1 binding to cell membrane. Vitamin k, however, reversed this inhibition.

Aflatoxins↗

Effects of aflatoxin B1 on chicken chondrocytes in culture.

In vitro effects of aflatoxin B1 on DNA and proteoglycan synthesis were assessed by measuring tritiated thymidine and radiosulfate incorporation by chicken chondrocyte cultures. Chondrocytes were harvested from the growth-plate cartilage of a 35-day-old chicken, cultured in triplicate, and treated with 0, 1, 5, 10, or 25 micrograms aflatoxin B1/ml of culture medium. The cultures were then spiked separately with [3H]thymidine or [35S]sulfate. After the cells, matrix, and medium were harvested, radioactivity was measured by a scintillation counter. Aflatoxin B1 resulted in a dose-dependent inhibition of DNA synthesis, as measured by tritiated thymidine incorporation by chicken chondrocytes: 54.6%, 30.9%, 25.9%, and 5.9% of control in cultures treated with 1, 5, 10, and 25 micrograms aflatoxin B1/ml of culture medium, respectively (P < 0.05). Sulfated proteoglycan synthesis, as measured by radiosulfate incorporation in chondrocyte cultures, was decreased in the media and intercellular matrix by all aflatoxin B1 levels. Compared with controls, 21% less (P < 0.05) radiosulfate was incorporated into non-matrix macromolecules of the medium fraction from chondrocytes exposed to 1 microgram aflatoxin B1/ml. However, 5 micrograms aflatoxin B1/ml was necessary to produce a significant decrease in radiosulfate incorporation in proteoglycans of intercellular matrix (34% less than controls). The ratios of the means of radiosulfate incorporation into intercellular macromolecules to the means of tritiated thymidine incorporation indicated that aflatoxin B1 inhibited DNA synthesis more than proteoglycan synthesis.

Aflatoxin B1↗

Predicting the distribution of aflatoxin test results from farmers' stock peanuts.

Suitability of the negative binomial function for use in estimating the distribution of sample aflatoxin test results associated with testing farmers' stock peanuts for aflatoxin was studied. A 900 kg portion of peanut pods was removed from each of 40 contaminated farmers' stock lots. The lots averaged about 4100 kg. Each 900 kg portion was divided into fifty 2.26 kg samples, fifty 4.21 kg samples, and fifty 6.91 kg samples. The aflatoxin in each sample was quantified by liquid chromatography. An observed distribution of sample aflatoxin test results consisted of 50 aflatoxin test results for each lot and each sample size. The mean aflatoxin concentration, m; the variance, S2 mean among the 50 sample aflatoxin test results; and the shape parameter, k, for the negative binomial function were determined for each of the 120 observed distributions (40 lots times 3 sample sizes). Regression analysis indicated the functional relationship between k and m to be k = 0.000006425m0.8047. The 120 observed distributions of sample aflatoxin test results were compared to the negative binomial function by using the Kolmogorov-Smirnov (KS) test. The null hypothesis that the true unknown distribution function was negative binomial was not rejected at the 5% significance level for 114 of the 120 distributions. The negative binomial function failed the KS test at a sample concentration of 0 ng/g in all 6 of the distributions where the negative binomial function was rejected. The negative binomial function always predicted a smaller percentage of samples testing 0 ng/g than was actually observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxins↗

Predictive value of molecular dosimetry: individual versus group effects of oltipraz on aflatoxin-albumin adducts and risk of liver cancer.

Studies in animals and humans have established serum aflatoxin-albumin adducts as biomarkers of exposure to aflatoxin B1 (AFB1), a food-borne hepatocarcinogen. To assess the utility of measurements of aflatoxin-albumin adducts to predict risk of hepatocellular carcinoma (HCC), 123 male F344 rats were dosed with 20 microg of AFB1 daily for 5 weeks after randomization into three groups: no intervention; delayed-transient (500 ppm of oltipraz, weeks 2 and 3 relative to AFB1); or persistent (500 ppm oltipraz, weeks -1 to 5). Serial blood samples were collected from each animal at weekly intervals throughout aflatoxin B1 exposure and assayed for levels of aflatoxin-albumin by radioimmune assay. Area under the curve (AUC) values for aflatoxin-albumin adducts decreased 20 and 39% in the delayed-transient and persistent oltipraz intervention groups, respectively, as compared to no intervention. Similarly, the total incidence of HCC dropped from 83 to 60% (P = 0.03) and 48% (P < 0.01) in these groups. Tumor multiplicity was also reduced in the two oltipraz intervention groups, whereas time to HCC was increased. Mononuclear cell leukemia, a common neoplasm in F344 rats, was seen in 39% of the control animals, whereas the two oltipraz interventions reduced incidence to 18% (P = 0.05) and 13% (P = 0.01), respectively. Overall, a significant association was seen between biomarker AUC and risk of HCC (P = 0.01). However, when the predictive value of aflatoxin-albumin adducts was assessed within treatment groups, there was no association between AUC and risk of HCC (P = 0.56). Thus, aflatoxin-albumin adducts can be useful for monitoring population-based changes induced by interventions, such as in chemoprevention trials, but have limited utility in identifying individuals destined to develop HCC. As a consequence, the use of this biomarker in quantitative risk assessment should be pursued cautiously.

Aflatoxin B1↗

Influence of dietary aflatoxin on the severity of coccidial infection in quails.

One hundred-sixty one day old Japanese quail-chicks were divided into four groups and kept for 6 weeks. The first group was kept as control. Group 2 received 1 ppm of dietary aflatoxin from day zero of chick life till the end of experiment. Group 3 was inoculated with 5 x 10(4) sporulated oocysts of Eimeria alone. Quail-chicks of group 4 were inoculated with coccidial oocysts orally and received aflatoxin in their rations. The combination of aflatoxin and coccidia produced higher mortality rate, higher faecal scores with increased oocysts output and reproductive potential than those quails received aflatoxin or coccidia alone. Body weight and efficiency of feed utilization decreased in all treated groups. The maximal losses of body gain and efficiency of feed utilization were recorded in quail-chicks infected with Eimeria and received aflatoxin in their ration. The lowest erythrocytic count, hemoglobin values, PCV and leukocytic count were recorded in quails that infected with coccidia and received aflatoxin. The levels of total serum proteins, gamma globulins, calcium and phosphorus were decreased in case of quails infected with coccidia and received dietary aflatoxin. Total bilirubin and GOT activity appeared to be higher in quails infected with Eimeria and received aflatoxin.

Aflatoxins↗

Rapid immunochemical screening method for aflatoxin B1 in human and animal urine.

A method has been developed to determine the presence of aflatoxin B1 in the urine of animals (including humans) by utilizing commercial immunochemical kits that can be used in the field. Urine is treated with diatomaceous earth and filtered to clarify the sample; 2-3 ppb aflatoxin B1, corresponding to about 300 ppb in the ingested feed/food, can be detected in the filtered urine without further purification. To improve sensitivity, the urine filtrate is passed through a C18 solid phase column to extract the aflatoxin. The column is washed with acetonitrile-water (15 + 85) and water, aflatoxin B1 is eluted with methanol-water (7 + 3), and water is added to the eluate, which is then tested for aflatoxin with the test kit. The limit of detection is 0.2 ppb, reflecting consumption of 40 ppb or more aflatoxin in the feed/food. When the initial sample volume is adequate, purification through the C18 column step is usually sufficient. For limited sample volumes, the eluate from the C18 column is mixed with water, added to an immunosorbent affinity column, and washed with water to remove excess sample matrix and impurities. Aflatoxin B1 is eluted with acetonitrile. The extract is evaporated under nitrogen and the residue is redissolved in methanol-water (25 + 75). At this purification stage, the limit of detection is reduced to 0.05 ppb.

Aflatoxin B1↗

Zinc, copper, manganese and iron in rat organs after the administration and withdrawal of aflatoxin B1.

The in vivo levels of zinc, copper, manganese and iron were determined in the testis, kidney and three brain regions (cerebellum, cerebrum and medulla oblongata) from rats intraperitoneally injected with 20 micrograms per kg body weight of aflatoxin B1 for 6 weeks. Similar investigations were made after a recovery period of 6 weeks. The results obtained were compared with those of pair-fed control rats. The aflatoxin B1-induced decrease in testicular zinc was not reversed after the recovery of the rats. On the contrary, the significantly increased zinc concentration in the other organs following aflatoxin B1 exposure, reverted to the control level, as judged by significant levels. Only in the kidney was there accumulation of copper, even after the cessation of aflatoxin B1 administration. The marked increase in the manganese concentration of all the organs following repeated toxin administration, persisted only in the brain regions after the recovery. Aflatoxin B1 treatment significantly decreased the iron concentration in the testis, kidney, cerebellum and cerebrum. Recovery did not alter this trend. Iron concentration in the medulla oblongata was, however, not significantly changed in the presence of aflatoxin B1. The present data suggest that the repeated administration of aflatoxin B1 to rats could result in either a permanent or temporary re-distribution of these elements in the organs examined.

Aflatoxin B1↗