Mixed Aflatoxins Research Articles

A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products.

Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL-1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL-1 and 18 pg mL-1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.

Aflatoxin B -induced alterations in uptake and distribution of 65Zn-ZnCl2 by four Zea mays cultivars and toxin effects on root and stem elongations

Aflatoxin Bl has been recovered within seemingly healthy, intact seeds, which suggests that its transport from contaminated soil to the fruit can occur. Previously, we examined the effects of 19.7 µg•<sup>-3</sup> mixed aflatoxins (AFTs) on the abilities of three <em>Zea mays</em> cultivars to remove Zn<sup>++</sup> from Perlite and the influence of Zn<sup>++</sup> on the cultivars' capabilities to both take-up and distribute AFTs. Here, we report both 2.5 and 5.0 µg aflatoxin B<sub>1</sub> (AFB<sub>1</sub>)cm<sup>-3</sup> influences on time-dependent, uptake and organ distribution of <sup>65</sup>Zn-ZnCl<sub>2</sub> from liquid culture by <em>Zea mays</em>, cvs. 'Early Yellow', 'Silver Queen', 'Early White' and `Golden Queen'. In addition, time-dependent seed germination as well as root and stem elongation responses to AFB1 are described. Neither 2.5 nor 5.0 µg AFB<sub>1</sub>cm<sup>-3</sup> affected the cultivars' abilities to germinate. Whereas an analysis of variance revealed significant differences for the combination cultivars, AFB<sub>1</sub> concentration and time regarding root elongation, such analysis for stem elongation yielded only a significant interaction between AFB<sub>1</sub> concentration and one cultivar at a time course's completion. As for <sup>65</sup>Zn-ZnCl<sub>2</sub> uptake, an ANOVA indicated that there were significant differences for the combination organs, cultivars and AFB<sub>1</sub> concentration. Certain results suggest <em>Zea mays</em> cultivar variability in susceptibility to exogenous AFB<sub>1</sub>. The significance of these findings is discussed.

Naturally occurring level of mixed aflatoxins B and G stimulate toll-like receptor-4 in bovine mononuclear cells

Background: As common contaminants of agricultural commodities, aflatoxins (AFs) are highly hazardous carcinogenic mycotoxins originating from very common fungi present in the environment. Their effect on bovine key immune-surveillance molecules is unclear.Objective: To determine the in vitro effects of naturally occurring level of AFs on toll-like receptor-4 (TLR4) and viability in bovine peripheral blood mononuclear cells (PBMCs) as a model system for immunotoxicity.Methods: Ten healthy dairy cows were selected as a source of PBMCs. The mRNA expression of TLR4 was quantitatively evaluated in PBMCs treated with a mix of 1.0, 0.5, 0.25 and 0.25 ng/ml of AFB1, AFB2, AFG1 and AFG2, respectively, and 10 ng/ml of the well-known TLR4 agonist lipopolysaccharide (LPS) for 0.5, 2, 5 and 18 hours. We also analyzed the viability of PBMCs exposed to AFs by means of the trypan blue exclusion method and the tetrazolium bromide assay.Results: Compared with control PBMCs, transcription of TLR4 in AFs-treated PBMCs was significantly up-regulated at post-exposure hours (PEH) 2 and 5, but was unchanged at PEH 0.5 and 18. Unsurprisingly, much higher up-regulation of TLR4 transcript was observed in LPS-treated PBMCs at PEH 0.5, 2, 5 and 18. Conversely, the viability of post-AFs and/or LPS-exposed PBMCs revealed no significant changes.Conclusion: Naturally occurring levels of AFs result in increased expression of TLR4 mRNA in bovine PBMCs.Clinical importance: The increased expression of TLR4 mRNA in bovine PBMCs by AFs also emphasizes the need to reduce admissible levels of AFs in feed for preventive measures.

Biologically relevant doses of mixed aflatoxins B and G up-regulate MyD88, TLR2, TLR4 and CD14 transcripts in human PBMCs

Context: Aflatoxins (AFs) are highly hazardous carcinogenic mycotoxins originated from very common fungi present in the environment. Their effect on key immune-surveillance molecules is unclear.Objective: We aimed to examine the effect of mixed AFs on immunologically relevant molecules and on viability in human peripheral blood mononuclear cells (PBMCs), in conditions similar to those occurring naturally, i.e. using a mixture of environmentally relevant levels of AFB1, AFB2, AFG1 and AFG2.Materials and methods: We evaluated the mRNA expression of MyD88, toll-like receptor (TLR)-2, TLR4 and CD14, in human PBMCs treated with a mixture of AFB1, AFB2, AFG1 and AFG2 at different doses for 2, 12 and 24 h. We used qRT–PCR to assess changes in transcripts of MyD88, TLR2, TLR4 and CD14 in PBMCs. We also evaluated the viability of PBMCs exposed to AFs.Results: Biologically relevant levels of mixed AFs elicited early immune modulation in human PBMCs. qRT–PCR results showed several folds increase of MyD88, TLR2, TLR4 and CD14 transcripts in PBMCs as early as 2 h post-exposure to mixed AFs. Kinetics and dose–response of the up-regulation differed for mentioned gene transcripts. Further, prolonged exposure to mixed AFs decreased PBMCs viability.Conclusion: Immunotoxicity of AFs on PBMCs may be mediated by up-regulation of key immune-surveillance molecule transcripts. The description of these effects induced by AFs on PBMCs are novel and should be taken into account when considering AF-related infectious and noninfectious diseases in areas highly exposed to AFs.

Embryotoxicity assay of aflatoxin produced by Aspergillus parasiticus NRRL 2999

1. The embryotoxicity of mixed aflatoxins (AF) and aflatoxin B1 (AFB1) were evaluated by a modified chick embryotoxicity screening test (CHEST). Adverse effects on the early embryonic development of thymus and bursa of Fabricius were also investigated by light microscopy. AF consisted of 83·06% AFB1, 12·98% AFB2, 2·84% AFG1 and 1·12% AFG2. 2. A total of 448 fertilised laying hens' eggs were used. AF and AFB1 were injected into the eggs at doses of 10, 100 and 1000 ng/egg. Embryonic developmental stages were evaluated according to the Hamburger-Hamilton scale (HH-scale). 3. The results showed that AFB1 given at 10 ng/egg had a significantly (P < 0·05) greater embryotoxic effect than AF given at a similar dose. The higher doses of both AF and AFB1 caused higher embryonic mortality and also an increase in early deaths. 4. In the groups receiving 100 ng/egg AF and AFB1 an abnormal development was seen, with a protruded central region, corresponding to the area pellucida of the blastoderm. No other developmental abnormality attributable to AF or AFB1 was found.

Short-Term Exposure to Subacute Doses of Aflatoxin-Induced Depressed Mitogen Responses in Young Mallard Ducks

Mallard ducklings were fed diets containing corn naturally contaminated with mixed aflatoxins, purified T-2 toxin, or no detectable mycotoxin in two trials. The aflatoxin level used was 12 ppb in the first trial and 33 ppb in the second. T-2 was added at 2 ppm in both trials. No pathology was associated with the aflatoxin used in this study, and T-2--induced lesions were described in a previous publication. The weights of primary (thymus and bursa of Fabricius) and secondary (spleen) lymphoid organs were significantly reduced in the T-2--treated birds. The total number of viable cells recovered from the thymus was significantly reduced in aflatoxin-treated birds. The numbers of viable cells recovered from thymus, bursa of Fabricius, and spleen were all significantly reduced after treatment with T-2. In each trial, significantly lower mitogenic responses were seen to pokeweed mitogen and concanavalin A in birds fed aflatoxin or T-2, representing reduction in both B-cell and T-cell mitogenesis. Birds fed aflatoxin also had significantly reduced Escherichia coli O55 lipopolysaccharide-induced mitogenic responses. These studies indicate that subacute oral exposure to aflatoxin caused a loss of normal lymphocyte reactivity in mallard ducklings. This finding supports the hypothesis that waterfowl that ingest even small quantities of mycotoxin-contaminated waste grain are likely to be more susceptible to bacterial or viral infections.

Disturbances in Tryptophan Metabolism in Rats Following Chronic Dietary Aflatoxin Treatment

Male Holtzman rats were fed 7.8 ppm mixed aflatoxins (AFT) for a 21-week period. At sacrifice there was extensive bile ductule cell proliferation and numerous precancerous changes evident in AFT-treated animals. The in vitro activity of hepatic holo-enzyme tryptophan 2, 3-dioxygenase was reduced in AFT-treated animals which correlated with a reduced urinary excretion of both kynurenine and kynurenic acid suggesting that this reduced tryptophan 2, 3-dioxygenase observed in vitro may be relevant to the in vivo response. The levels of serotonin and 5-hydroxyindole-3-acetic acid were found to be reduced in the pineal, pituitary, various brain regions and in duodenum of AFT-treated rats. It is proposed that the decreased levels of serotonin and 5-hydroxyindole-3-acetic acid in all tissues may be due to either a decreased free tryptophan pool or alterations in 5-hydroxyindole biosynthesis.

Activities of tryptophan-metabolizing enzymes in liver and brain of rats treated with aflatoxins

Male Sprague-Dawley rats were treated ip twice daily for 10 days with mixed aflatoxins (10 μg/kg body weight) and then the activities of liver tryptophan 2,3-dioxygenase and brainstem tryptophan 5-hydroxylase were determined in vitro. Total tryptophan 2,3-dioxygenase activity was reduced by aflatoxin treatment while holoenzyme activity was not. Induction of tryptophan 2,3-dioxygenase activity by l-tryptophan was not altered by the treatment though induction by hydrocortisone was blocked. It is suggested that aflatoxin may alter the tryptophan 2,3-dioxygenase-haem bond in vivo rather than affect enzyme or cofactor synthesis. Kinetics studies performed in vitro on brain tryptophan 5-hydroxylase showed that aflatoxin treatment in vivo increased the K m of the hydroxylase when l-trytophan and synthetic 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin were used as the substrate and cofactor, respectively. However, aflatoxin treatment did not alter the V max of tryptophan-5-hydroxylase.

Histochemical Analysis of Liver Cells from Short Term, Aflatoxin-Dosed and Nondosed Coturnix coturnix japonica: 1. Aflatoxin-Sensitive Quail

Although aflatoxicosis in Coturnix coturnix japonica has been described, the histochemical localization of liver chemicals and the occurrence of ingested aflatoxins within blood, feces, and liver have not been described. Six to 8-week-old quail, which were intubated with a carrier with or without .3 mg mixed aflatoxins (AFB1, AFB2, AFG1, AFG2)/kg body weight were sacrificed within .25 to 5 days of intubation. Deparaffinized sections of livers were stained for lipids, nucleic acids, polysaccharides, and proteins. Other livers and excrement were homogenized and filtered homogenates as well as blood partitioned against chloroform. The aqueous phase was treated with pepsin and then partitioned, but the organic phase was analyzed directly. Organic phases of .25 to 5 day blood, feces, and liver lacked aflatoxins. Pepsin digesta of blood from males and females dosed 6 hr appeared to contain aflatoxicol (disappeared by 24 hr) and an unknown fluorescent compound, respectively. Whereas an unidentified fluorescent compound was observed within excrement of males dosed 6 hr, female excrement contained a fluorescent compound with an AFB1 Rf (disappeared by 24 hr). Although the liver of males dosed 6 hr had three fluorescent compounds (Rfs for AFB1 and AFB2a), only one was seen within dosed females. Ultra violet absorption spectra of presumed AFB2a and aflatoxicol failed to yield their reported absorption maxima. Livers from dosed quail exhibited bile duct proliferation, cellular necrosis, vacuolization, congestion, fatty changes and mild hepatitis. Sinusoidal membranes were thickened and contained abundant periodic acid-Schiff's (PAS)-positive substances. Although livers of nondosed quail abounded with regularly shaped and uniformly distributed, Sudan IV-positive droplets, livers of dosed quail accommodated few, irregularly-shaped and positioned droplets. Hepatocyte nuclei and nucleoli of dosed quail displayed marked affinities for the Feulgen reagent and toluidine blue O, respectively. Lobules of dosed quail possessed concentrations of cells in which their entire cytoplasm was PAS positive. Treatment of sections with α-amylase reduced staining suggesting the presence of glycogen. Ninhydrin-positive substances were distributed throughout the liver in both DQ and non DQ with no apparent difference in intensities between the two livers. Generally the DQ showed mild hepatitis due to aflatoxicosis and the toxin altered liver histochemistry for the major classes of cellular chemicals.

Uptake of Zn++ and aflatoxin from perlite and liquid culture by Zea mays seedlings.

The present paper reports our attempts to determine whether the inclusion of 0.0014 mM Zn++ within a hydroponic culture medium affects the ability of 12-day-old Zea mays, cv. ‘SS-522’ to take-up [3H]-aflatoxin B1. Data from the corollary experiment, i.e., whether inclusion of aflatoxin affects the ability of Zea mays, cvs. ‘Truckers White’, ‘X-Sweet’ and ‘Merit’ to take-up 65ZnCl2 are presented also. This report is a preliminary to one regarding an in-progress analysis of whether pollutant levels of Zn++ affect aflatoxin uptake and distribution. In the absence of irrigating seedlings, which were grown in Perlite containing 65ZnCl2, with a solution containing mixed aflatoxins, the stem contained the greatest amount of label with root plus seed the next highest and the leaf the least for each of the cvs. In contrast, when the seedlings were irrigated with a solution containing mixed aflatoxins, the root plus seed contained either an amount nearly identical to (cv. ‘Truckers White’) or in excess of that within the stem (cvs. ‘X-Sweet’ and ‘Merit’). Calculation of the percentages of aflatoxin-induced diminutions in leaf, stem and root label suggested that the aflatoxins interfered with the translocation of 65ZnCl2 from the root to the stem and leaf, at least for cvs ‘X-Sweet’ and ‘Merit’. When 0.0014mM Zn++ as ZnSO4 was added to an incubation medium in which 12-day-old seedlings were suspended and plant growth assessed over 72 hours, a 15% increase (significant at 0.05 level) in seedling height over that of Zn++-deficient plants was observed. No differences in [3H]-aflatoxin B1 uptake were noted between those seedlings which were grown in either Zn++-containing or lacking media. Less than one % of the[3H]-aflatoxin B1 which was taken-up was recovered within chloroform extracts of the seedlings. The distributions of radioactivity from [3H]-aflatoxin B1 for leaf, stem, seed and root were 0, 57, 26 and 19% and 0, 26, 58 and 18% for Zn++-containing and -lacking media, ‘respectively’.

Aflatoxin‐induced alteration in soybean membrane protein

AbstractIsolates of aflatoxin‐producing strains ofAspergillus can grow on Glycine max beans. Furthermore, both mixed aflatoxins and aflatoxin B1 inhibit the elongation of both attached and excised G. max roots. In addition, the toxin inhibits the capacity of the excised roots to take up [14C]leucine. This suggests that the toxin may inhibit the activity of an uptake system responsible for uptake of low‐molecular‐weight (MW) compounds by possibly altering membrane structure and/or function. In this connection, incubation of excised roots in media containing either mixed aflatoxins or aflatoxin B1 results in a diminution of acid‐insoluble protein, but neither sterol nor lipid phosphorus levels, of an 80,000 X g pellet (purported “crude” plasmalemma fraction). To provide preliminary evidence that mixed aflatoxins can decrease the amount of a specific plasmalemma protein (which might regulate the uptake of low‐MW compounds), we incubated roots with and without mixed aflatoxins and then gel‐filtrated integral proteins which were released by detergents from 80,000 X g pellets that had been obtained by differential centrifugation of Miracloth filtrates. The released proteins were gelfiltrated on Sephadex G‐100 columns. Sodium dodecyl sulfate, Triton X‐100 and Tween 20 each solubilized greater than 85% of the pellet's protein. Gel filtration yielded 280 nm absorbing void volume and retarded peaks for substances which were either precipitated by trichloroacetic acid (total protein) or solubilized by detergents (integral protein) from pellets that were derived from aflatoxin‐treated and nontreated roots. The amounts of protein which were recovered within column void volumes following gel filtration of 80,000 X g, acid‐precipitable protein were not significantly different for treated and nontreated roots, suggesting that incubation of roots with aflatoxins does not reduce the pellet's content of an acid‐precipitable, high‐MW (100,000 or more) protein. However, this conclusion is highly tentative as less than 15% of the acid‐insoluble protein which was layered onto the column was recovered. The amplitude of the void volume peaks for detergent‐released proteins from treated roots was consistently less than that for nontreated roots, suggesting that treatment of roots with aflatoxins diminishes the “crude” plasmalemma fraction's content of high‐MW protein(s). This suggestion was supported through calculation of the amounts of protein which were recovered within both the void volume and retarded peaks and comparing these to the total protein levels that were recovered from the columns. The amount of protein which was found within the void volume peak following gel filtration of either Triton X‐100 or Tween 20‐released proteins from pellets that were derived from treated roots was less than that for nontreated roots.

The effect of dietary fibre sources on aflatoxicosis in the weanling male rat.

1. Two experiments with male weanling rats were conducted in which they received individually and restrictedly either a basal semi-purified diet containing starch as the principal carbohydrate or the same diet to which mixed aflatoxins were added in quantities providing from 0.13 to 0.4 mg aflatoxin B1/kg diet. Various natural ingredients, or semi-purified sources of dietary fibre were substituted for a portion of the starch in the basal diet containing aflatoxin. The diets were fed for 13-14 weeks after which the rats were given ad lib. a commercial rodent diet until they were killed at 109 weeks of age. 2. Two further experiments were conducted in which twenty-four rats in each experiment received the basal diet plus aflatoxin, or diets in which a portion of the starch was replaced by gum arabic or by wheat offal. After 13 or more weeks the absorption, retention and excretion of 14C-labelled aflatoxin B1 was measured in each rat. 3. The addition of gum arabic or wheat bran to the diet decreased the effects of the toxin in the first two experiments, but as measured by several characteristics, only wheat bran provided an effect which persisted during the period when neither it nor the toxin was given. The effects included an apparent reduction in tumour incidence. The change in the content of starch in the basal diet, occurring as a consequence of adding the test ingredients is also considered to be an associated cause of the effects observed. 4. In comparison to starch, wheat offal increased the total 14C in the faeces and the proportion of the total found during the first 48 h after dosing. Rats receiving starch excreted more 14C in their urine and retained more 14C in their livers. The differences between gum arabic and starch were not significant as measured by 14C excretion and retention. Liver size as a proportion of carcass weight was less in rats receiving wheat offal or gum arabic, and rats receiving wheat offal had a lower incidence of fat-loaded hepatocytes. 5. The interaction of dietary toxins, or drugs, with the ingredient composition of diet may affect animal response even when the diets are adequate and similar in nutrient composition. This may be of significance both in drug-safety studies and in animal production.

Effects of dietary copper on developing aflatoxicosis in Syrian hamsters.

The possible suppression of toxic and carcinogenic activities of aflatoxin by the cupric ion were investigated. Weanling, male Syrian hamsters received diets of either ground meal, ground meal plus 22.9 ppm mixed aflatoxins (9.2 ppm AFB1, 0.4 ppm AFB2, 12.8 ppm AFG1, 0.5 ppm AFG2), ground meal plus copper acetate (0.5% by weight) and 22.9 ppm mixed aflatoxins, or ground meal plus copper acetate (0.5%). Experimental parameters measured included: food and water consumption, body weight responses, mortality, gross pathology and histopathological changes, as well as protein synthesis in the liver tissue during incubation in Krebs' medium. The results indicate that copper may act as a protective agent against low levels of these mycotoxins. Significantly, more animals undergoing combined treatment (aflatoxin plus copper) survived and had higher body weights. Less severe liver pathology was seen in these animals as compared to those receiving aflatoxin without a copper supplement.

Toxic responses of germinating pollen and soybeans to aflatoxins

Aflatoxin producing strains of Aspergillus grow on soybeans thereby contaminating the latter through secretion of the toxin. Investigations dealing with either soybean seed germination or intact seedling growth responses to aflatoxin (B1) are lacking. Similarly, a possible interaction of aflatoxins with phosphate in the germination and elongation of both soybeans and pollen as well as roots of the former and tubes of the latter has not been examined. Imbibition of Glycine max, cv. ‘Essex’ seeds for 18 hours in solutions containing 0.38, 2.90, 5.80 or 11.60 μg/ml (AFB1) yielded% germination inhibitions of 5, 20, 40 and 80, respectively. By 36 hours these were 6, 4, 13 and 19 % for the same toxin concentration series. At 140 hours attached root elongation was inhibited 26, 35 and 50 % for 2.90, 5.80 and 11.60 μg/ml AFB1. No effect was noted at 0.38 μg/ml AFB1. Incubation of excised roots in medium containing 3.0 mM KH2PO4 stimulated their elongation 3.2 fold. Addition of 33.28 μg/ml mixed aflatoxins together with KH2PO4 resulted in only a 1.5 fold stimulation. When KH2PO4 was added to a culture medium lacking AFB1, Lilium longiflorum, cv. ‘Ace’ pollen germination was enhanced 50%. Withholding KH2PO2 but supplying AFB1 did not markedly affect germination. However, supplementing the medium with KH2PO4 while simultaneously adding AFB1 did not inhibit germination at 5 and 10 μg/ml but caused 27.3 and 45.1 % declines at 25 and 30 μg/ml. In the absence of KH2PO4 AFB1 stimulated pollen tube elongation 7.5, 14.3, 16.5 and 13.2 % at 5, 10, 15 and 20 μg/ml but 30 μg/ml inhibited it 11.1%. In contrast, tube elongation was suppressed at all AFB1 concentrations (maximum 36.1% at 30 μg/ml) tested upon KH2PO4 addition. Results derived from germinating pollen in medium supplemented with KH2PO4 or NaH2PO4 indicate that the phosphate anion does not preferentially promote aflatoxin-induced inhibition of tube elongation.

Alterations of brain and intestine serotonin levels in hamsters pretreated with dietary aflatoxin

Male Syrian hamsters were fed 30.3 ppm mixed aflatoxins (AFT) for a 45-day period followed by a 123-day recovery period. Tests made at the end of the recovery period indicated a reduction in whole brain and duodenal serotonin and a slight but not statistically significant elevation in brain 5-hydroxyindoleacetic acid. There was a high incidence of liver cell hyperplasia in the aflatoxin fed animals. Following toxin withdrawal, the experimental animals showed significant gains in body weight but they never attained a body weight equal to the controls. Aflatoxin apparently interferes with serotonin production and the return to normal body weight even after withdrawal of the animal from the contaminated diet.

Chemosterilant and insecticidal activity of mixed aflatoxins against Anthonomus grandis (Coleoptera)

A mixture of the four major aflatoxins at 0.06 ppm in food supplied to adult boll weevils, Anthonomus grandis, was an effective chemosterilant.

Excretion of aflatoxin in a lactating cow

After oral administration of a single dose (0·5 mg/kg) of mixed aflatoxins(B1, 44%; G1, 44%; B2, 2%) to a lactating cow, the excretion of fluorescent aflatoxin components was determined in the milk, urine and faeces over a period of 9 days. About 85% of the total aflatoxin detected in the milk and urine was excreted in the first 48 hr. None could be detected in the milk after 4 days and none in the urine and faeces after 6 days. Only 4·52% of the total dose of 300 mg was identified, the milk containing 0·18%, the urine 1·55% and the faeces 2·79%.Aflatoxin M1 was the only component found in the milk, the total being 0·35% of the amount of aflatoxin B1 ingested. M1 was also the major compound excreted in the urine, whereas aflatoxin B1 was the component present in greatest amount in the faeces. Aflatoxin G1 was excreted in both urine and faeces, the greater amount being found in the urine, but none was detected in the milk.A comparison of the pattern of excretion in the cow with that in the ewe showed that although the two were similar, there were some distinct differences, the chief one being a greater excretion of unchanged B1 in the cow than in the ewe.

Crystalline Aflatoxin M1 from Urine and Milk

Allcroft and Carnaghan1 and de Iongh et al.2 reported the appearance in milk of a compound related to aflatoxin B1, and Allcroft et al.3 showed by thin-layer chromatography and ultra-violet studies that a similar factor appeared in the urine of sheep given mixed aflatoxins. Holzapfel et al.4 isolated the urinary compound and established that it was aflatoxin M1 (Fig. 1).

Metabolism of aflatoxin in sheep: Excretion pattern in the lactating ewe

The excretion of aflatoxin components was determined in milk, urine and faeces of a lactating ewe after oral administration of a single dose (1 mg/kg) of mixed aflatoxins (B 1, 36%; G 1, 52%; B 2, 3%; G 2, 2%). About 90% of the total aflatoxin excretion in the milk and urine occurred in the first 48 hr. None could be detected in milk after 6 days and none in urine and faeces after 8 and 9 days respectively. Only 8.1% of the total dose was recovered in an identifiable form, the milk containing 0.1%, the urine 6.4% and the faeces 1.6%. Some unchanged aflatoxin B 1 was present in the faeces but only trace amounts were found in milk and urine. Aflatoxin M 1 was excreted in relatively large amounts by all three routes, but mainly in the urine, and was the major identifiable aflatoxin constituent in milk and faeces. Aflatoxin G 1 was excreted chiefly in the urine and faeces with only traces in the milk.