Aflatoxins can be produced by three species of Aspergillus—A. flavus, A. parasiticus, and the rare A. nomius—which contaminate plants and plant products. A. flavus produces only B aflatoxins, while the other two species produce both B and G aflatoxins. Aflatoxins M₁ and M₂ are the hydroxylated metabolites of aflatoxins B₁ and B₂ and can be found in milk or milk products obtained from livestock that have ingested contaminated feed. The main sources of aflatoxins in feeds are peanut meal, maize and cottonseed meal.

Aflatoxins were evaluated by the Committee at its thirty-first, forty-sixth, and forty-ninth meetings (Annex 1, references 77, 122, and 131). At its forty-ninth meeting, the Committee considered estimates of the carcinogenic potency of aflatoxins and the potential risks associated with their intake. At that meeting, the Committee reviewed a wide range of studies conducted in animals and humans that provided qualitative and quantitative information on the hepatocarcinogenicity of aflatoxins. The Committee evaluated the potency of these contaminants, linked those potencies to estimates of intake, and discussed the potential impact of hypothetical standards on sample populations and their overall risk. In its evaluation, the Committee stated that the carcinogenic potency of aflatoxin M₁ in sensitive species is about one order of magnitude less than that of aflatoxin B₁. In particular, the Committee noted that the carcinogenic potency of aflatoxin B₁ is substantially higher in carriers of hepatitis B virus (about 0.3 cancers per year per 100 000 persons per ng/kg bw per day), as determined by the presence in serum of the hepatitis B virus surface antigen (HBsAg⁺ individuals), than in HBsAg^– individuals (about 0.01 cancers per year per 100 000 persons per ng/ kg bw per day). Populations with both a high prevalence of HBsAg⁺ and a high aflatoxin intake might benefit from reductions in aflatoxin intake. The Committee also noted that vaccination against hepatitis B virus would reduce the number of carriers of the virus, and thus reduce the potency of the aflatoxins in vaccinated populations, leading to a reduction in the risk for liver cancer.

The Committee at its forty-ninth meeting concluded that changing the hypothetical standard for aflatoxin B1 from 20 µg/kg to 10 µg/kg would not result in any observable difference in rates of liver cancer.

At its present meeting, the Committee reviewed studies published since its forty-ninth meeting, as well as other information, to elucidate further the carcinogenic potencies of aflatoxin M₁ and aflatoxin B₁ and the differences between animal species in their sensitivity to aflatoxins.

The chemical structure of aflatoxin M₁ is shown in Figure 1. Aflatoxin M₁ is the 4-hydroxy derivative of aflatoxin B₁ and is secreted in the milk of mammals that consume aflatoxin B₁. Aflatoxin M₁ (CAS No. 6795-23-9) has a relative molecular mass of 328 Da and has the molecular formula C₁₇H₁₂O₇.

2.2.1 Biotransformation

A complete review of studies of the metabolism of aflatoxins conducted up to 1997 can be found in the report of the monograph on aflatoxins published after the forty-ninth meeting of the Committee (Annex 1, reference 132) and in a book by Eaton and Groopman (1994). The papers highlighted in this section address studies of matabolic differences among species in their sensitivity to aflatoxins, comparisons of the toxicity of aflatoxin M₁ and aflatoxin B₁, studies of the differences in the carcinogenic potency of aflatoxin M₁ and aflatoxin B₁, and papers published since the last report of the Committee.

The metabolism of aflatoxin B₁ and the extent to which it binds to cell macro-molecules were compared in liver slices from humans and rats, as rats are more sensitive to the carcinogenicity of aflatoxin B₁. Liver slices were prepared from three human liver samples and incubated with [³H]aflatoxin B₁ at 0.5 µmol/L for 2 h. The rates of formation of oxidative metabolites and of specific binding to cell macromolecules showed significant interindividual variation. The rates of oxidative metabolism of aflatoxin B₁ to aflatoxin Q₁, aflatoxin P₁, and aflatoxin M₁ in the human liver samples were similar to those previously observed in rat liver slices. No aflatoxin B₁–glutathione conjugate formation was detected in the human liver samples, and there was much less specific binding of aflatoxin B₁ to cell macromolecules in the human than in the rat liver slices. For example, the level of binding between aflatoxin B₁ and DNA ranged from 3 to 26% of that in control rats. These results suggest that humans do not form as much aflatoxin B₁ 8,9-epoxide as rats, but they also suggest that humans do not have glutathione-S-transferase (GST) isozymes with high specific activity towards this epoxide. Significant individual differences in aflatoxin B₁ metabolism and binding suggest the presence of genetic and/or environmental factors that may result in large differences in susceptibility.

Aflatoxin M₁ is usually considered to be a detoxication by-product of aflatoxin B₁; it is also the metabolite present in the milk of nursing women who eat foods containing the toxin. Aflatoxin B₁ epoxide has been shown to exist as two stereoisomers—endo- and exo-epoxides—the latter being the DNA-reactive form, and a similar situation may apply to aflatoxin M₁ epoxide. In a study of the metabolism of aflatoxins M₁ and B₁ in vitro in human liver microsomes, they had a very limited capacity to catalyse epoxidation of aflatoxin M₁. The small amount of aflatoxin M₁ dihydrodiol formed from the epoxide also appeared to have a lower capacity to induce microsomal protein than did aflatoxin B₁ dihydrodiol. GST catalysed conjugation of the epoxides of both aflatoxins with glutathione; GST activity was present in mouse cytosol but not in the human liver fraction. The authors concluded that the difference between the genotoxic potency of the two toxins in vivo correlates with their mutagenicity in vitro, metabolic activation and DNA binding (Neal et al., 1998). In rats, however, activation of aflatoxin M₁ to the epoxide does not appear to be essential for its acute toxicity. Experiments in human cell lines indicated that cytochrome P450 (CYP) enzymes are involved in the cytotoxicity of aflatoxin B₁ but not of aflatoxin M₁. Studies of the toxicity of aflatoxin M₁ in human lymphoblastoid cell lines expressing or not expressing human CYP enzymes showed a direct effect in the absence of metabolic activation, in contrast to aflatoxin B₁. Aflatoxin M₁ is therefore not strictly a detoxication product of aflatoxin B₁ in biological responses in which cytotoxicity plays a significant role, such as immunotoxicity (Heinonen et al., 1996).

In studies of species sensitivity to the carcinogenicity of aflatoxin B₁, mice were resistant because they constitutively express an alpha-GST, which is strongly active against aflatoxin B₁ 8,9-epoxide, whereas rats, which do not express such a GST, were sensitive. Human hepatic alpha-class GSTs have little capacity to detoxify aflatoxin B₁ 8,9-epoxide. The nonhuman primate Macaca fascicularis showed significant constitutive hepatic GST activity towards aflatoxin B₁ 8,9-epoxide. GSTs were purified from liver tissue from this species and characterized, and GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (PCR). A protein, GSHA-GST, was purified by glutathione agarose affinity chromatography, which had stronger aflatoxin B₁ 8,9-epoxide-conjugating activity than other GST-containing peaks. The GSHA-GST was shown to belong to the µ class. The authors then showed that two distinct µ-class GST cDNAs have 97% and 98% homology with the human µ-class GSTs hGSTM4 and hGSTM2, respectively. µ-Class GSTs appear to be responsible for most of the conjugating activity of aflatoxin B₁ 8,9-epoxide in the liver of M. fascicularis. None of the known human µ-class GSTs acts preferentially on the ultimate genotoxic aflatoxin B₁ metabolite exo-aflatoxin B₁ 8,9-epoxide, but large interindividual differences in the expression of GST isoforms have been shown in various tissues, and few human livers have been evaluated. The authors concluded that identification of a potential human homologue of GSHA-GST would be relevant to the design of chemointervention strategies to reduce aflatoxin B₁-induced liver cancer in highly exposed populations. Nevertheless, induction of known human GSTs with little or no activity towards the epoxide of aflatoxin B₁ might be ineffective in reducing the genotoxicity of aflatoxin B₁ (Wang et al., 2000).

The extreme sensitivity of turkeys to aflatoxin B₁ was studied by measuring microsomal activation of aflatoxin B₁ to the 8,9-epoxide, the putative toxic intermediate, cytosolic GST-mediated detoxication of aflatoxin B₁ 8,9-epoxide, and hepatic phase I and phase II enzyme activities in 3-week-old male Oorlop turkeys. Liver microsomes prepared from these turkeys activated aflatoxin B₁ in vitro with an apparent K_m of 110 µmol/L and a V_max of 1.25 nmol/mg per min. The involvement of CYP 1A2 and, to a lesser extent, 3A4 in the activation of aflatoxin B₁ was assessed with specific mammalian CYP inhibitors. The possible presence of avian orthologues of these CYPs was indicated by activity towards ethoxyresorufin and nifedipine, as well as by western immunoblotting with antibodies to human CYPs. GST-mediated conjugation of 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene was demonstrated in cytosol prepared from the turkey livers, but the rate was much lower than that observed in other species. The presence of alpha- and µ-class GSTs and another aflatoxin B₁ detoxifying enzyme, aflatoxin B₁ aldehyde reductase, was shown by western immunoblotting. Quinone reductase activity was also present in the cytosol. Furthermore, the cytosol showed no measurable GST-mediated detoxication of microsomally activated aflatoxin B₁. Thus, turkeys are deficient in the most crucial aflatoxin B₁ detoxication pathway. The authors concluded that the extreme sensitivity of this species to aflatoxin B₁ is due to a combination of efficient aflatoxin B₁ activation and deficient detoxication by phase II enzymes such as GSTs (Klein et al., 2000).

The carcinogenicity of aflatoxin B₁, aflatoxicol (aflatoxin L), aflatoxin M₁, and aflatoxicol M₁ (aflatoxin LM₁) was compared in terms of their binding to target organ DNA in rainbow trout. Tritiated compounds were synthesized, dose–response curves for DNA binding were established, and liver DNA binding indices were calculated for the four aflatoxins after a 2-week dietary intake by trout fry. The adduct levels increased linearly with dietary concentration, with relative DNA binding indices of 21, 20, 2.4, and 2.2 x 10³ (pmol/mg of DNA)/(pmol/g of diet) for aflatoxin M₁, aflatoxin L, aflatoxin M₁, and aflatoxin LM₁, respectively.

In a similar protocol, over 7200 trout fry with an average initial body weight of 1.2 g were used to establish full carcinogen dose–response curves for each aflatoxin and an estimate of the DNA binding index after a single dose. Since trout are very sensitive, < 180 µg of each aflatoxin were required. Data analysed on logit incidence versus L_n dose coordinates generated four curves, which were modelled as parallel in slope over most or all the doses studied. In this analysis, the relative tumorigenic potencies were 1.0 for aflatoxin B₁, 0.94 for aflatoxin L, 0.086 for aflatoxin M₁, and 0.041 for aflatoxin LM₁. When the data were plotted as logit incidence versus L_n adducts (effective dose received), dose–response relationships were found for all aflatoxin adducts, indicating that they are equally tumorigenic, except for aflatoxin LM₁, which was two to three times less potent. Differences in the tumorigenicity of the four aflatoxins are largely or entirely accounted for by differences in uptake and metabolism leading to DNA adduction, rather than to any inherent difference in tumour initiating potency per DNA adduct (Bailey et al., 1998).

Since most people are exposed to carcinogens in food intermittently, the effects of intermittent intake of aflatoxin B₁ on hepatic and testicular glutathione was studied in male Fischer 344 rats fed diets containing aflatoxin B₁ at a concentration of 0, 0.01, 0.04, 0.4, or 1.6 ppm at 4-week intervals up to 20 weeks. The control animals were fed an aflatoxin B₁-free NIH-31 diet. Rats eating diets containing 0.01 ppm aflatoxin B₁ did not show induction of hepatic or testicular GST activity, but intermittent intake of concentrations of 0.04–1.6 ppm significantly increased GST activity. The increase in enzyme activity was proportional to the dose and the length of intake of aflatoxin B₁ (Sahu et al., 2000).

MAXIMUM LEVEL FOR AFLATOXIN M1 IN MILK

(March, 2000)

The CCFAC is charged with establishing or endorsing permitted maximum or guideline levels for naturally occurring toxicants in foodstuffs and animal feeds.

Aflatoxin M₁ is a toxic metabolite of aflatoxin B₁. It is produced in the livers of animals and humans that have ingested aflatoxin contaminated commodities, primarily cereal grains. It is normally excreted in the urine and also the milk of dairy cattle and other lactating mammals. The occurrence of aflatoxin M₁ in milk is transitory in nature, usually reaching a peak within 2 days after the ingestion of the contaminated commodity and disappearing within 4-5 days after the withdrawal of the contaminated source.

The Joint Expert Committee on Food Additives (JECFA) has established an acceptable level of risk of 0.5 mg/kg for aflatoxin M₁ in fluid milk. The International Grocery Manufacturers Associations (ICGMA) supports this assessment.

The 30^th CCFAC forwarded a draft ML of 0.05 mg/kg to the 23^rd CAC (1999) for adoption at step 8. However, the CAC could not reach a consensus and returned the Draft ML to the CCFAC for further consideration in regards to the public health and the potential economic implications of a higher or lower level than the proposed 0.05 mg/kg.

The 32^nd CCFAC (March 2000) requested JECFA to conduct a quantitative risk assessment at its 56^th Session to compare the levels of 0.05 mg/kg and 0.5 mg/kg using monitoring data from all regions of the world. The 33^rd CCFAC (2001) discussed the summary report of the 56^th JECFA (2001). The summary report showed that the additional risk for liver cancer predicted for an ML between 0.05 to 0.5 mg/kg was negligible. The JECFA representative commented that the data, as analyzed, assumed that all milk was contaminated; if a distribution analysis of the data had been used, the risk would be even smaller.

The 33^rd CCFAC forwarded an ML of 0.5 mg/kg M₁ in milk to the CAC, based on:

(a) Risk assessment by the JECFA;

(b) A level of 0.5 mg/kg was both adequate for the protection of consumer health and reasonably achievable for all countries,

(c) a reduction in the maximum level might entail a significant reduction in the availability of milk in developing countries and therefore would have negative implications from a nutritional point of view; and

(d) The level of 0.05 mg/kg seemed not to be achievable in some regions of the world.\

Source: http://www.icgma.com/AFLAmilk.HTM

ICGMA supports establishing an ML of 0.5 mg/kg for aflatoxin M₁in milk because:

· Risk assessments conducted by the US FDA support establishment of an ML of 0.5 mg/kg M₁ in milk. The most recent assessment revealed an insignificant upper bound lifetime risk even for 90^th percentile “eaters-only” group. This upper bound risk estimate indicates that the US system, which relies on a combination of good agricultural and good manufacturing, practices and FDA’s action level of 0.5 mg/kg for aflatoxin M₁in milk provides adequate public health protection.

· Aflatoxin risk assessment performed by the 49^th (1997) and 56^th JECFA (2001), support the level of 0.5 mg/kg as being adequate to protect public health. The JECFA noted that no significant public health gain would be achieved by setting a level lower than 0.5 mg/kg for aflatoxin M₁ in milk. JECFA compared the predicted risk reduction under worst case assumptions (life time exposure to all milk at the highest level) at MLs of 0.05 and 0.5 mg/kg and concluded that the additional risks were insignificant.

· The Codex’s health and safety decisions should be grounded on science based risk assessments. Therefore, setting an ML for M₁ in milk other than 0.5 mg/kg would be inconsistent with Codex’s stated principles of establishing standards based on risk assessments.

· In regions of the world where low levels of aflatoxins are virtually unavoidable in grains used for dairy feed, an ML of 0.5 mg/kg for aflatoxin M₁ in milk is practical and allows an adequate supply of rations for milk producing animals. Endorsement of an ML below 0.5 mg/kg is technically difficult for many regions of the world to achieve and could comprise the availability of healthy and productive milk producing animals and adequate supplies of milk for human nutrition.

· An ML below 0.5 mg/kg for aflatoxin M₁in milk could have a negative public health effect by reducing the availability of a safe, low cost food that is a good dietary source of high quality protein, calcium and other valuable nutrients. Reduction in availability of otherwise safe milk could also create an unnecessary health risk by depriving infants and young children of a dietary source of essential nutrients necessary for normal growth and development.

· The imposition of a maximum level less than 0.5 mg/kg would restrict international trade of feed ingredients because the level of aflatoxins in the feed would need to be controlled to lower levels than currently used in order to assure that the milk met the lower standard. This issue is further complicated by the fact that the animals for which the feed components will be used are not known at the time of international shipments. Therefore, the milk level may result indirectly in an unwritten lower limit for aflatoxins in feed ingredients for all animals, even though animal feed is a good use of the more aflatoxin contaminated ingredients for many animal species without significant risk to the animals or consumers from derived foods.

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AFLATOXIN M1

2.1 Chemical structure

2.2 Biochemical aspects

2.2.1 Biotransformation

MAXIMUM LEVEL FOR AFLATOXIN M1 IN MILK

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