Aflatoxin B1-lysine adduct in dried blood spot samples of animals and humans

Dried blood spots (DBS) were proposed as potentially viable method for exposure assessment of environmental toxicants in infant and young children. For this study, we validated an experimental protocol to quantify AFB₁-lysine adduct in DBS samples of AFB₁-treated F344 rats, as well as samples from human field study. Significant dose-response relationships in AFB₁-lysine adduct formation were found in DBS samples of rats treated with single- and repeated-dose AFB₁. AFB₁-lysine levels in DBS samples were highly correlated with corresponding serum sample levels. The Person coefficients were 0.997 for the single-dose exposure, and 0.996 for the repeated-dose exposure. Levels of AFB₁-lysine adduct had also good agreement between DBS and serum samples as shown by Bland-Altman plot analysis. For human field study samples (n = 36), a Pearson correlation coefficient of 0.784 was found between AFB₁-lysine adduct levels of DBS and corresponding serum samples. Bland-Altman plots showed the distribution of the log differences between DBS and serum AFB₁-lysine levels are within 95% confidence intervals. These results showed AFB₁-lysine adduct levels in DBS cards and serum samples from animals and human samples are comparable, and the DBS technique and analytical protocol is a good means to assess AFB₁ exposure in infant and children populations.     

Synthesis of 5,5,6,6‐D4‐L‐lysine‐aflatoxin Bl for use as a mass spectrometric internal standard

Human exposure to the hepatocarcinogenic mycotoxin aflatoxin B_l results in modification of serum albumin lysine ε‐amino residues to form lysine‐aflatoxin adducts. A perdeuterated reference standard is now required to quantitatively measure this adduct in epidemiologic studies of liver cancer using isotopic dilution mass spectrometry. A convenient method for the preparation of D4‐L ‐lysine‐AFB_l using commercially available 5,5,6,6‐D4‐l ‐lysine is demonstrated for the first time. The application of two standard α‐amino protection methods is also reported that simplifies the production of natural isotopic abundance lysine‐AFB_l over the currently used method employing N_α‐acetyl‐l ‐lysine. t‐Boc‐N_α‐lysine was used to prepare lysine‐AFB_l; however, a preferred method for directing reaction of AFB_l‐dialdehyde to the ε‐amino group of 5,5,6,6‐D4‐l ‐lysine utilized cupric ions that were spontaneously removed during the reverse phase HPLC purification of D4‐lysine‐AFB_l using 1% HOAc. This strategy eliminates the need to otherwise synthesize and purify t‐Boc‐N_α‐ or N_α‐acetyl‐5,5,6,6‐D4‐lysine and then TFA or enzymatically deprotect overnight to obtain the target compound. Copyright © 2004 John Wiley & Sons, Ltd.     

Methods for chemical preparation of aflatoxin B1 adducts,AFB1-N7-guanine and AFB1-lysine

Abstract

Aflatoxin B₁ (AFB₁) is a hepatocarcinogen produced by fungi species of the genus Aspergillus. Dietary exposure to AFB₁ is associated with increasing incidence of hepatocellular carcinoma. The biotransformation of AFB₁ generates the adducts AFB₁-N⁷-guanine and AFB₁-lysine, which are used as biomarkers of human exposure to dietary AFB₁ in epidemiological studies. This article presents a review of main studies on the use of AFB₁ adducts as biomarkers of human exposure to AFB₁, and the current status of chemical procedures for the preparation of AFB₁-N⁷-guanine and AFB₁-lysine as analytical standards.     

Glycine N-methyltransferase affects the metabolism of aflatoxin B1 and blocks its carcinogenic effect

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B₁ (AFB₁) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB₁ treatment. Competitive enzyme immunoassays indicated that after AFB₁ treatment, the AFB₁-DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB₁-DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB₁ in the cultural supernatants of cells exposed to AFB₁ showed that the AFM₁ level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB₁. Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB₁. Automated docking experiments showed that AFB₁ binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT-AFB₁ interaction is 44.9 μM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB₁, resulting in reduced AFB₁-DNA adducts formation and cell death.     

X-ray studies on crystalline complexes involving amino acids and peptides. X. Head-to-tail sequences in the crystal structure of l-lysine acetate

l -Lysine acetate crystallises in the monoclinic space group P2₁ with a = 5.411 (1), b = 7.562(1), c= l2.635(2) Å and β = 91.7(1). The crystal structure was solved by direct methods and refined to an R value of 0.049 using the full matrix least squares method. The conformation and the aggregation of lysine molecules in the structure are similar to those found in the crystal structure of l -lysine l -aspartate. A conspicuous similarity between the crystal structures of l -arginine acetate and l -lysine acetate is that in both cases the strongly basic side chain, although having the largest pK value, interacts with the weakly acidic acetate group leaving the α-amino and the α-carboxylate groups to take part in head-to-tail sequences. These structures thus indicate that electrostatic effects are strongly modulated by other factors so as to give rise to head-to-tail sequences which have earlier been shown to be an almost universal feature of amino acid aggregation in the solid state.     

Prevention of Aflatoxin B1-Induced DNA Breaks by β-D-Glucan

Aflatoxins are a group of naturally-occurring carcinogens that are known to contaminate different human and animal foodstuffs. Aflatoxin B₁ (AFB₁) is the most genotoxic hepatocarcinogenic compound of all of the aflatoxins. In this report, we explore the capacity of β-d-glucan (Glu) to reduce the DNA damage induced by AFB₁ in mouse hepatocytes. For this purpose, we applied the comet assay to groups of animals that were first administered Glu in three doses (100, 400 and 700 mg/kg bw, respectively) and, 20 min later, 1.0 mg/kg of AFB₁. Liver cells were obtained at 4, 10 and 16 h after the chemical administration and examined. The results showed no protection of the damage induced by AFB₁ with the low dose of the polysaccharide, but they did reveal antigenotoxic activity exerted by the two high doses. In addition, we induced a co-crystallization between both compounds, determined their fusion points and analyzed the molecules by UV spectroscopy. The data suggested the formation of a supramolecular complex between AFB₁ and β-d-glucan.     

X-ray studies on crystalline complexes involving amino acids and peptides. XXXII. Effect of chirality on ionisation state,stoichiometry and aggregation in the complexes of oxalic acid with dl- and l-lysine

Crystals of the oxalic acid complex of dl -lysine (triclinic Pl; a= 5.540(1), b= 10.764(2), c= 12.056(2) Å, α= 77.8(1), β= 80.6(1), γ= 75.6(1)°; R = 4.7% for 2023 observed reflections) contain lysine and semioxalate ions in the 1:1 ratio, whereas the ratio of lysine and semioxalate/oxalate ions is 2:3 in the crystals of the l -lysine complex (monoclinic P2₁; a= 4.906(1), b= 20.145(4), c= 12.455(1) Å, β= 92.5(1)°; R = 4.4% for 1494 observed reflections). The amino acid molecule in the l -lysine complex has an unusual ionisation state with positively charged α- and side-chain amino groups and a neutral carboxyl group. The unlike molecules aggregate into separate alternating layers in the dl -lysine complex in a manner similar to that observed in several of the amino acid complexes. The l -lysine complex exhibits a new aggregation pattern which cannot be easily explained in terms of planar features, thus emphasizing the fundamental dependence of aggregation on molecular characteristics. Despite the differences in stoichiometry, ionisation state and long-range aggregation patterns, the basic element of aggregation in the two complexes exhibits considerable similarity.     

Is the activation of aflatoxin B1 catalysed by the same form of cytochrome P-450 as that 4-hydroxylating debrisoquine in rat and/or man?

A possible association between the metabolic activation of aflatoxin B₁ (AFB₁) to a mutagen and the 4-hydroxylation of debrisoquine, which shows genetic variation both in man and in the rat, was investigated. Hepatic microsomal fractions from female DA and Fischer rats catalyse, at the same rate, the conversion of AFB₁ to a mutagenic and arylating metabolite, that bound covalently to microsomal proteins. Debrisoquine was without effect on either of these reactions. In contrast, metyrapone did inhibit the mutagenic activation of AFB₁. Microsomal fraction from human liver was also capable of activating AFB₁ to a mutagenic and arylating metabolite. Again, debrisoquine was without appreciable effect on these reactions. In contrast, cimetidine caused profound inhibition of the mutagenic activation of AFB₁. It is concluded that the activation of AFB₁ to a mutagenic, and presumably carcinogenic, metabolite is catalysed by a different form of cytochrome P-450 from that catalysing the 4-hydroxylation of debrisoquine, both in rat and in man.     

X-ray studies on crystalline complexes involving amino acids and peptides

l -Lysine d -glutamate crystallizes in the monoclinic space group P2₁ with a= 4.902, b= 30.719. c= 9.679 Å. β= 90° and Z = 4. The crystals of l -lysine d -aspartate monohydrate belong to the orthorhombic space group P2₁2₁2₁ with a= 5.458, b= 7.152, c= 36.022 Å and Z = 4. The structures were solved by the direct methods and refined to R values of 0.125 and 0.040 respectively for 1412 and 1503 observed reflections. The glutamate complex is highly pseudosymmetric. The lysine molecules in it assume a conformation with the side chain staggered between the α-amino and the α-carboxylate groups. The interactions of the side chain amino groups of lysine in the two complexes are such that they form infinite sequences containing alternating amino and carboxylate groups. The molecular aggregation in the glutamate complex is very similar to that observed in l -arginine d -aspartate and l -arginine d -glutamate trihydrate, with the formation of double layers consisting of both types of molecules. In contrast to the situation in the other three ld complexes, the unlike molecules in l -lysine d -aspartate monohydrate aggregate into alternating layers as in the case of most ll complexes. The arrangement of molecules in the lysine layer is nearly the same as in l -lysine l -aspartate, with head-to-tail sequences as the central feature. The arrangement of aspartate ions in the layers containing them is, however, somewhat unusual. Thus the comparison between the ll and the ld complexes analyzed so far indicates that the reversal of chirality of one of the components in a complex leads to profound changes in molecular aggregation, but these changes could be of more than one type.     

Removal of Aflatoxin B1 by Edible Mushroom-Forming Fungi and Its Mechanism

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B₁ (AFB₁), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB₁ removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB₁ removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB₁ analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB₁ removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24–48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB₁ removal activity by whole cells was mainly due to AFB₁ binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB₁ binding process onto cell wall components.     

Recombinant Expression of Trametes versicolor Aflatoxin B1-Degrading Enzyme (TV-AFB1D) in Engineering Pichia pastoris GS115 and Application in AFB1 Degradation in AFB1-Contaminated Peanuts

Aflatoxins seriously threaten the health of humans and animals due to their potential carcinogenic properties. Enzymatic degradation approach is an effective and environmentally friendly alternative that involves changing the structure of aflatoxins. In this study, Trametes versicolor aflatoxin B₁-degrading enzyme gene (TV-AFB₁D) was integrated into the genome of Pichia pastoris GS115 by homologous recombination approach. The recombinant TV-AFB₁D was expressed in engineering P. pastoris with a size of approximately 77 kDa under the induction of methanol. The maximum activity of TV-AFB₁D reached 17.5 U/mL after the induction of 0.8% ethanol (v/v) for 84 h at 28 °C. The AFB₁ proportion of 75.9% was degraded using AFB₁ standard sample after catalysis for 12 h. In addition, the AFB₁ proportion was 48.5% using AFB₁-contaminated peanuts after the catalysis for 18 h at 34 °C. The recombinant TV-AFB₁D would have good practical application value in AFB₁ degradation in food crops. This study provides an alternative degrading enzyme for the degradation of AFB₁ in aflatoxin-contaminated grain and feed via enzymatic degradation approach.     

Influences of Aflatoxin B1 on main intestinal bacteria communities and enzyme activities in mice

Intestine microbiota and enzyme activities have crucial effects on host's health and behavior. In order to provide evidences for the toxicology of Aflatoxin B₁ (AFB₁) on human’s health objectively, the influence of AFB₁ on intestine microbiota and enzyme activities was conducted. Thirty-two Kunming mice were randomly placed into control group, low-dosage group, middle-dosage group, and high-dosage group after being fed for 4?days adaptively. Then they were fed intragastrically with 0.4?mL of 0?mg/L, 2.5?mg/L, 4?mg/L, and 10?mg/L of AFB₁ solutions, twice a day for 62?days. Following this, the intestine contents were collected and the microorganisms and enzyme activities were analyzed. The results showed that the number of bacteria in the low-dosage (p?=?.024), the middle-dosage (p?=?.016) and the high-dosage (p?=?.000) groups and the number of Bifidobacterium spp. in the high-dosage group (p?=?.001) increased significantly in comparison with the control group. Compared to the control group, the activities of amylase in all AFB₁ groups increased significantly (p?=?.000), the activities of xylanase (p?=?.005) and cellulase (p?=?.002) increased also in the high-dosage group. These results indicated that AFB₁ destroyed the intestinal microecological balance and increased the activities of intestinal enzymes, but there were no significant effects on the weight of mice.     

Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B1 and its dependence on p53 genotype

Aflatoxin B₁ (AFB₁) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53^tm1BrdN5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB₁ for 26 weeks. NER activity was assessed with an in vitro assay, using AFB₁-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB₁–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB₁ respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB₁ (p < 0.05). In heterozygous p53 knockout mice, repair of AFB₁–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB₁ (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB₁ or in liver extracts from mice treated with either AFB₁ concentration. p53 genotype did not affect basal levels of repair. AFB₁ exposure did not alter repair of AFB₁-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB₁ increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage.     

Degradation of Aflatoxin B1 during the Fermentation of Alcoholic Beverages

Aflatoxin B₁ (AFB₁) is a contaminant of grain and fruit and has one of the highest levels of carcinogenicity of any natural toxin. AFB₁ and the fungi that produce it can also contaminate the raw materials used for beer and wine manufacture, such as corn and grapes. Therefore, brewers must ensure strict monitoring to reduce the risk of contamination. In this study, the fate of AFB₁ during the fermentation process was investigated using laboratory-scale bottom and top beer fermentation and wine fermentation. During fermentation, cool wort beer samples and wine must samples were artificially spiked with AFB₁ and the levels of AFB₁ remaining after fermentation were analyzed. AFB₁ levels were unchanged during both types of fermentation used for beer but were reduced to 30% of their initial concentration in wine. Differential analysis of the spiked and unspiked wine samples showed that the degradation compound was AFB_2a, a hydrated derivative of AFB₁. Thus, the results showed that the risk of AFB₁ carryover was still present for both types of beer fermentation but was reduced in the case of wine fermentation because of hydration.     

Trp266 determines the binding specificity of a porcine aflatoxin B1 aldehyde reductase for aflatoxin B1-dialdehyde

Aflatoxin B₁ (AFB₁) is a severe threat to human and animal health. The aflatoxin B₁ aldehyde reductase (AFAR) family specifically catalyzes AFB₁-dialdehyde, a toxic metabolic intermediate of AFB₁, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB₁-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB₁-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB₁-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB₁-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π–π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB₁-dialdehyde.     

NMR and Radical Scavenging Activities of Patuletin from Urtica urens. Against Aflatoxin B1

Abstract

In a previous study, we reported six flavonoids isolated from the methanol extract of Urtica urens L. Patuletin, the major component, was found to have powerful anti-inflammatory and antimicrobial activity. We have now investigated whether patuletin also has antioxidant activity and free radical scavenging effects in rats treated with aflatoxin B₁ (AFB₁). Male Sprague-Dawley rats (60) were assigned to 6 experimental groups including a control group and groups treated for 10 days with patuletin at low dose (7.5 mg/kg b.w) or high dose (10 mg/kg b.w) with or without AFB₁ (2 mg/kg b.w). Blood samples were collected at the end of the experimental period for biochemical analysis. The results showed significant changes typical to aflatoxicosis in rats treated only with AFB₁. Patuletin at the two tested doses caused an increase in glutathione peroxidase (GPx) and superoxide dismutase (SOD); high doses also caused an increase in liver and kidney enzymes, except total bilirubin (TB), which was in the normal value of the control animals. On the other hand, these enzymes were comparable to the controls in rats treated with low doses. Cotreatment of AFB₁ and patuletin resulted in a significant improvement in all parameters tested. We conclude that patuletin possesses an antioxidant activity and free radical scavenging effects in AFB₁-treated rats that can be observed at a dose as low as 7.5 mg/kg b.w.     

Mechanisms of butylated hydroxytoluene chemoprevention of aflatoxicosis--inhibition of aflatoxin B1 metabolism

Chemoprevention of toxicoses and/or cancer through the use of nutrients or pharmacologic compounds is the subject of intense study. Among the many compounds examined, food additives such as antioxidants are being considered due to their ability to reduce disease formation by either induction or inhibition of key enzyme systems. One such compound, butylated hydroxytoluene (BHT), has been found to protect against cancer formation caused by exposure to aflatoxin B₁ (AFB₁) in rodents. We have shown that dietary BHT protects against clinical signs of aflatoxicosis in turkeys, a species that is very susceptible to this mycotoxin. In this study, the effect of BHT on AFB₁ metabolism and other cytochrome P450 (CYP)-related enzyme activities in turkey liver microsomes was examined to discern possible mechanisms of BHT-mediated protection against aflatoxicosis. Ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), prototype activities for CYP1A1 and 1A2, respectively, were decreased in the BHT fed (4000 ppm) animals, while oxidation of nifedipine, a prototype activity for CYP3A4, was increased. However, BHT added to microsomal incubations inhibited these CYP activities in a concentration-related manner. Importantly, BHT inhibited conversion of AFB₁ to the reactive intermediate AFB₁-8,-9-epoxide (AFBO), exhibiting Michaelis-Menton competitive inhibition kinetics (Ki = 0.81 μM). Likewise, microsomes prepared from turkeys fed BHT were significantly less active in AFBO formation compared to those from control birds. When turkeys were fed BHT for up to 40 days, residual BHT was present in liver, breast meat, thigh meat and abdominal fat in concentrations substantially below U.S. FDA guidelines for this antioxidant, but in concentrations greater than the Ki, likely sufficient to inhibit bioactivation of AFB₁in vivo. BHT-induced hydropic degeneration in the livers of BHT fed animals was significantly greater in birds that remained on BHT treatment for up to 30 days, but this lesion diminished in animals fed for 40 days or when returned to a control diet. The data indicate that the observed chemopreventive properties of BHT in turkeys may be due, at least in part, to its ability to inhibit hepatic AFB₁ epoxidation and also that the BHT-induced hydropic degeneration is reversible and does not appear to cause long-term effects.     

Molecular Mechanisms of Lipoic Acid Protection against Aflatoxin B1-Induced Liver Oxidative Damage and Inflammatory Responses in Broilers

Alpha-lipoic acid (α-LA) was evaluated in this study for its molecular mechanisms against liver oxidative damage and inflammatory responses induced by aflatoxin B₁ (AFB₁). Birds were randomly allocated into four groups with different diets for three weeks: a basal diet, a 300 mg/kg α-LA supplementation in a basal diet, a diet containing 74 μg/kg AFB₁, and 300 mg/kg α-LA supplementation in a diet containing 74 μg/kg AFB₁. In the AFB₁ group, the expression of GSH-P_X mRNA was down-regulated (p < 0.05), and the levels of lipid peroxide and nitric oxide were increased (p < 0.05) in the chicken livers compared to those of the control group. Additionally, the mRNA level of the pro-inflammatory factor interleukin-6 was up-regulated significantly (p < 0.05), the protein expressions of both the nuclear factor kappa B (NF-κB) p65 and the inducible nitric oxide synthase were enhanced significantly (p < 0.05) in the AFB₁ group. All of these negative effects were inhibited by α-LA. These results indicate that α-LA may be effective in preventing hepatic oxidative stress, down-regulating the expression of hepatic pro-inflammatory cytokines, as well as inhibiting NF-κB expression.     

Adsorbents Reduce Aflatoxin M1 Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B1 in Diet and Its Exposure Risk for Humans

This study investigated the effect of moderate risk level (8 µg/kg) AFB₁ in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM₁ content of healthy lactating cows and the AFM₁ residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB₁ and adsorbents (CON), CON with 8 μg/kg AFB₁ (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB₁-challenge phase (day 1 to 14) and an AFB₁-withdraw phase (day 15 to 19). Results showed that both AFB₁ and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM₁ concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB₁ into milk AFM₁ (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2–4 years old had the highest exposure risk to AFM₁ in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB₁ in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM₁ residue in milk and its exposure risk for humans.