Metabolism of aflatoxin B1 in turkey liver microsomes: the relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B₁-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB₁ to the exo-AFBO and to detoxification products aflatoxin M₁ (AFM₁) and aflatoxin Q₁ (AFQ₁), respectively, complicates the kinetic analysis of AFB₁ in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB₁ in TLMs. In control TLMs, AFB₁ was converted to exo-AFBO in addition to AFM₁ and AFQ₁ confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (~ 0.1 μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 μM) AFB₁ concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM₁, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ₁ following the kinetic Hill equation. At 0.1 μM AFB₁, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM₁) compared to 3A37 (0.15: 1, exo-AFBO: AFQ₁), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB₁ bioactivation and metabolism at environmentally-relevant AFB₁ concentrations in turkey liver.     

Identification and reduction of urinary aflatoxin metabolites in dogs.

Hydrated sodium calcium aluminosilicate (HSCAS) is a phyllosilicate clay commonly used as an anticaking agent in animal feeds. HSCAS tightly and selectively adsorbs aflatoxin. In 1998, 55 dogs died in Texas after eating dog food containing aflatoxin (150-300 ppb). The corn in the diets was contaminated with aflatoxin. Six dogs were given a low-level, sub-clinical dose of aflatoxin B(1). On average, 71.5% of aflatoxin M(1) cleared within 6 h after dosing, increasing to 90.4% after 12 h. Aflatoxin M(1) was no longer detectable in urine after 48 h. Aflatoxin P(1) was not found in urine compared to large amounts of M(1) and trace amounts of Q(1). In a crossover study, six dogs randomly fed a commercial dog food (no-clay control) or coated with HSCAS (0.5% by weight) were subsequently administered a sub-clinical dose of aflatoxin B(1). Diets were switched and the process repeated. The HSCAS-coated diet significantly reduced urinary aflatoxin M(1) by 48.4%+/-16.6 SD versus the control diet. In conclusion, HSCAS protects dogs fed diets with even minimal aflatoxin contamination. Despite regular and careful ingredient screening for aflatoxin, low concentrations may reach the final product undetected. Therefore, HSCAS may provide the pet food industry further assurance of canine diet safety.     

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.     

Butylated hydroxytoluene chemoprevention of aflatoxicosis – Effects on aflatoxin B1 bioavailability, hepatic DNA adduct formation, and biliary excretion

The extreme sensitivity of turkeys to aflatoxin B₁ (AFB₁) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB₁ toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB₁ bioactivation. To test whether dietary BHT alters hepatic AFB₁–DNA adduct formation, excretion, and bioavailability of AFB₁ in vivo, turkeys were given diets with BHT (4000 ppm) for 10 days, given a single oral dose of [³H]-AFB₁ (0.05 μg/g; 0.02 μCi/g), then sampled at intervals up to 24 h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB₁–DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24 h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M₁ (AFM₁) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB₁, aflatoxin Q₁ or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB₁ bioavailability in addition to inhibition of bioactivation.     

The comparative metabolism and toxic potency of aflatoxin B1 and aflatoxin M1 in primary cultures of adult-rat hepatocytes

Both aflatoxin B₁ (AFB₁) and a hydroxylated metabolite, aflatoxin M₁ (AFM₁), were potent cytotoxins and genotoxins to primary cultures of rat hepatocytes. However, AFB₁ stimulated the release of lactate dehydrogenase into the culture medium and the loss of viable cells from the monolayer at lower doses than did AFM₁. The lowest toxic doses of AFB₁ and AFM₁ were 0·05–0·1 and 0·6 μg/ culture, respectively. Genotoxicity, determined by an assay for stimulation of DNA repair, was apparent at lower doses than was cytotoxicity. AFB₁ was again more potent than AFM₁, stimulating DNA repair at 0·025 μg/culture. compared to the lowest genotoxic dose of AFM₁ of 0·05 μg/culture. At higher doses (1·2–2·4 μg/culture) the responses due to both aflatoxins in the cytotoxicity and DNA-repair assays were approximately equal. The metabolism of a low dose (c. 0·17 μg/culture) of [¹⁴C]AFB₁ and [³H]AFM₁ by cultured hepatocytes differed significantly. After 1 hr, 50% of the [¹⁴C]AFB₁ remained unchanged in the culture medium, whereas about 18 hr were required for the same amount of [³H]AFM₁ metabolism to occur. [¹⁴C]AFB₁ was metabolized to AFM₁, to polar metabolites recovered in the aqueous phase after chloroform extraction, and to metabolites covalently bound to hepatocyte macromolecules. [³H]AFM₁ was also metabolized to polar metabolites and to forms bound to macromolecules. The degree of covalent binding of the aflatoxins correlated with their cytotoxicity and genotoxicity at lower doses. After a 24-hr incubation, 12·5% of the dose of [¹⁴C]AFB₁ was covalently bound to macromolecules compared to 1·5% of [³H]AFM₁. Although AFM₁ was less potent than AFB₁ in cytotoxicity, DNA-repair and covalent-binding assays using primary cultures of hepatocytes, AFM₁ was still active at relatively low doses and therefore is probably a potent hepatotoxin in vivo.     

In vitro studies on the metabolism of aflatoxin B1 and aldrin

As Drosophila melanogaster occupies an important position within the test battery for mutagens and carcinogens, it is of interest to study the xenobiotics metabolism of this insect. Likewise, the genetic control of these important enzyme systems falls within this interest.Our attempt was to get new strains, which show changes in their xenobiotics metabolism. This was done by a mutagenization and selection procedure for the second chromosome. The 44 fertile homozygous inbred strains produced by this selection were first tested for DDT resistance. Some of them showed LT50 values which were remarkably higher than that of the original strain Berlin K.Aflatoxin B₁ metabolism in two of the new strains (H349 and H362), Berlin K, and Hikone-R was compared, whilst aldrin epoxidase activity was compared in strains H349, H362, Berlin K, vestigial, and Karsnäs-R. The metabolism studies were carried out in vitro with testes tissue of the different strains. The metabolism in testes is of specific interest because this tissue is most often used in mutagenicity testing.In the AFB₁ assays of the up to 12 observed metabolites three could be identified as AFB_2a, AFM₁, and AFR₀. Hikone-R produced mostly AFR₀ (3.43% of the initial AFB₁ concentration) and small amounts of AFM₁ (0.59% AF) and AFB_2a (0.36% AF). The strain Berlin K showed only a low production of AFB_2a (0.48% AF), while the strain H349 formed AFR₀ (6.02% AF) and AFM₁ (0.75% AF). The AFM₁ appeared in even higher amounts than with Hikone-R. On the other hand, H362 showed the lowest activity in AFB₁ metabolism. With this strain none of the determined metabolites could be detected in levels significantly higher than the control. The difference between H349 and the original strain Berlin K was highly significant. The production of AFR₀ and the binding of aflatoxin to macromolecules show a linear correlation. In both parameters measured, the strain H349 yielded the highest results. The determination of aldrin epoxidase activity gave the following results (in pmol dieldrin · mg^–1 protein · min^–1): H349: 0.74; Karsnäs-R: 0.57; vestigial: 0.57; Berlin K: 0.32; H362: 0.27. Again the difference between H349 and Berlin K was statistically significant. The measured activities match values obtained with extrahepatic tissue of mammals.It is concluded that the line H349 is a mutant in the xenobiotic metabolism. For the strains Hikone-R, Karsnäs-R, and H349 AFR₀ could be confirmed to be the main metabolite of AFB₁. The metabolism pattern was shown to differ strongly from strain to strain.Abbreviations AFB₁, AFB2_2a and AFM₁ aflatoxins b₁, B_2a, and m₁ - AFR₀ aflatoxicol - DDT 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane - EMS ethyl methanesulfonate     

Distribution and induction of aflatoxin B1-9a-hydroxylase activity in rat liver parenchymal and non-parenchymal cells

 Chronic administration of aflatoxin B₁ (AFB₁) to rats gives rise to hepatocellular and cholangiocellular carcinomas without affecting Kupffer and endothelial cells. The enzymatic conversion of AFB₁ to AFB₁-8,9-epoxide is the critical step in the activation of the mycotoxin, while the conversion of AFB₁ to aflatoxin M₁ (AFM₁), catalyzed by the AFB₁-9a-hydroxylase, is considered to be a detoxication route for the toxin. In the present study the distribution and inducibility of AFB₁-9a-hydroxylase were analyzed in microsomes derived from freshly isolated liver parenchymal (PC) and nonparenchymal cells (i.e. Kupffer+endothelial cells, NPC). AFB₁-9a-hydroxylase activity was clearly measurable in NPC and similar to that of PC. In NPC the rate of formation of AFM₁ was higher (when incubating with 16 μM AFB₁) than or similar (with 128 μM AFB₁) to that of AFB₁-8,9-epoxide, while in PC it was significantly lower. Taken together, these results suggest that the AFB₁-9a-hydroxylase activity might be particularly important in NPC to protect these cells from AFB₁ by converting it to a significantly less mutagenic metabolite and by reducing the amount of AFB₁ available for epoxidation. Furthermore, it is shown that AFB₁-9a-hydroxylase activity is inducible by phenobarbital (only in PC), 3-methylcholanthrene, isosafrole and Aroclor 1254, thus indicating that in rat liver the conversion of AFB₁ to AFM₁ is catalyzed by members of the cytochrome 1A and 2B families. Received: 7 December 1995/Accepted: 13 February 1996     

Prevention of Maternal and Developmental Toxicity in Rats via Dietary Inclusion of Common Aflatoxin Sorbents: Potential for Hidden Risks

In earlier work, we have reported that a phyllosilicate clay(HSCAS or NovaSil) can tightly and selectively bind the aflatoxinsin vitro and in vivo. Since then, a variety of untested clayand zeolitic minerals have been added to poultry and livestockfeeds as potential "aflatoxin binders." However, the efficacyand safety of these products have not been determined. A commonzeolite that has been frequently added to animal feed is clinoptilolite.Our objectives in this study were twofold: (1) to utilize thepregnant rat as an in vivo model to compare the potential ofHSCAS and clinoptilolite to prevent the developmental toxicityof aflatoxin B₁ (AfB₁), and (2) to determine the effect of thesetwo sorbents on the metabolism and bioavailability of AfB₁.Clay and zeolitic minerals (HSCAS or clinoptilolite) were addedto the diet at a level of 0.5% (w/w) and fed to pregnant Sprague-Dawleyrats throughout pregnancy (i.e., day 0 to 20). Treatment groups(HSCAS or clinoptilolite) alone and in combination with AfB₁were exposed to sorbents in the feed as well as by gavage. Untreatedand AfB₁ control animals were fed the basal diet without addedsorbent. Between gestation days 6 and 13, animals maintainedon diets containing sorbent were gavaged with corn oil in combinationwith an amount of the respective sorbent equivalent to 0.5%of the estimated maximum daily intake of feed. Animals receivingAfB₁ were dosed orally (between days 6 and 13) with AfB₁ (2mg/kg body wt) either alone or concomitantly with a similarquantity of the respective sorbent Evaluations of toxicity wereperformed on day 20. These included: maternal (mortality, bodyweights, feed intake, and litter weights), developmental (embryonicresorptions and fetal body weights), and histologjcal (maternallivers and kidneys). Sorbents alone were not toxic; AfB₁ aloneand with clinoptilolite resulted in significant maternal anddevelopmental toxicity. Animals treated with HSCAS (plus AfB₁)were comparable to controls. Importantly, clinoptilolite (plusAfB₁) resulted in severe maternal liver lesions (more severethan AfB₁ alone), suggesting that this zeolite may interactwith dietary components that modulate aflatoxicosis. In metabolismstudies, adult male Sprague-Dawley rats, maintained on dietscontaining 0.5% (w/w) HSCAS or clinoptilolite, were dosed orallywith 2.0 mg AfB₁/kg body wt. The concentration of the majorurinary metabolite (AfM₁) was considerably decreased in thepresence of HSCAS. These results suggest that the mechanismof protection of AfB₁-induced maternal and developmental toxicitiesin the rat may involve adsorption and reduction of AfB₁ bioavailabilityin vivo. Importantly, this study demonstrates the potentialfor significant hidden risks associated with the inclusion ofnonselective aflatoxin binders in feeds. Aflatoxin sorbentsshould be rigorously tested individually and thoroughly characterizedin vivo, paying particular attention to their effectivenessand safety in sensitive animal models and their potential fordeleterious interactions.     

Distribution of aflatoxins in tissues of growing pigs fed an aflatoxin-contaminated diet amended with a high affinity aluminosilicate sorbent

The effect of hydrated sodium calcium aluminosilicate (HSCAS) added to the diet of swine fed an aflatoxin-contaminated diet on tissue aflatoxin levels was investigated. Pigs were fed control (less than 10 ng/g B1 + B2), contaminated (500-600 ng/g B1 + B2), and contaminated +0.5% HSCAS diets. Tissues analyzed for the presence of aflatoxin B1, B2, and M1 residues included liver, muscle, kidney, and adipose. Addition of HSCAS to the contaminated diet significantly reduced the amount of M1 in liver, kidney, and muscle tissue. Aflatoxin B1 was not reduced in liver or kidney, but was decreased in muscle.     

Modulation of aflatoxin B1 biotransformation by β-naphthoflavone in isolated rabbit lung cells

 The cytotoxic and carcinogenic mycotoxin aflatoxin (AF) B₁ (AFB₁) is biotransformed by the cytochrome P450 monooxygenases (CYP) to a number of relatively nontoxic metabolites, as well as to the ultimate toxic metabolite, AFB₁–8,9-epoxide. In a number of tissues and species, AFB₁ hydroxylation to the relatively nontoxic metabolite, AFM₁, is induced by β-naphthoflavone (BNF) treatment. Although the liver is the principal target organ for AFB₁ toxicity, the mycotoxin is also toxic and carcinogenic to respiratory tissues. To determine if BNF treatment alters the extent of pulmonary AFB₁ bioactivation by enhancing detoxification and thereby decreasing epoxidation, the effects of BNF on pulmonary AFB₁ metabolism were examined. Rabbit lung cells, isolated by protease digestion and centrifugal elutriation, were incubated with [³H]AFB₁. In nonciliated bronchiolar epithelial (Clara) cell-enriched (45–50%) fractions, [³H]AFM₁ production (pmol/mg DNA per 2 h) was increased by prior treatment of rabbits with BNF (80 mg/kg per day, 3 and 2 days before cell isolation) as follows: with 1.0 μM [³H]AFB₁; control, 10.6±2.3; BNF, 30.0±6.4; with 0.10 μM [³H]AFB₁; control, 9.4±4.7; BNF, 20.6±5.9. With 1.0 μM [³H]AFB₁, prior treatment of animals with BNF abolished formation of [³H]aflatoxicol (AFL) but not [³H]AFQ₁. The activation (epoxidation) of [³H]AFB₁ was measured indirectly as covalent binding to endogenous DNA. With 1.0 μM [³H]AFB₁, treatment of rabbits with BNF did not alter DNA binding (pmol/mg DNA per 2 h) in the Clara cell-enriched fraction: control, 103±41; BNF, 114±49. However, with 0.10 μM [³H]AFB₁, DNA binding in the same fraction was 47% lower in cells from BNF treated animals: control, 17.4±4.2; BNF, 9.3±3.9. Formation of 8,9-dihydro-8,9-dihydroxy- AFB₁, and the glutathione conjugate of the aflatoxin epoxide (AFB₁-GSH) were not detectable at the AFB₁ concentrations and time point studied, in cells from either BNF-treated or control rabbits. Incubation of isolated, unseparated lung cells from untreated rabbits with 5.0 to 50 μM BNF decreased [³H]AFB₁-DNA binding in the presence of 0.1 μM [³H]AFB₁ by 35 to 77%, while lower BNF concentrations did not alter DNA binding. In lung cells isolated from BNF treated rabbits, BNF was not detectable (i.e.<0.5 μM detection limit). Therefore, the amount of BNF present in isolated rabbit lung cells following in vivo treatment with BNF was below that required to directly inhibit AFB₁-DNA adduct formation. The decrease in AFB₁-DNA binding from rabbits treated with BNF is apparently due to the selective induction of CYP isozymes and related increases in AFM₁ formation, and not to direct inhibition of epoxidation or enhanced conjugation of AFB₁-8,9-epoxide with glutathione. Received: 5 March 1996/Accepted: 10 May 1996     

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.     

The Presence of Aflatoxin M1 in Milk and Milk Products in Bangladesh

As milk provides both micro- and macronutrients, it is an important component in the diet. However, the presence of aflatoxin B₁ (AFB₁) in the feed of dairy cattle results in contamination of milk and dairy products with aflatoxin M₁ (AFM₁), a toxic metabolite of the carcinogenic mycotoxin. With the aim to determine AFM₁ concentrations in milk and milk products consumed in Bangladesh, in total, 145 samples were collected in four divisional regions (Sylhet, Dhaka, Chittagong, and Rajshahi). The samples comprised these categories: raw milk (n = 105), pasteurized milk (n = 15), ultra-high temperature (UHT)-treated milk (n = 15), fermented milk products such as yogurt (n = 5), and milk powder (n = 5). AFM₁ levels in these samples were determined through competitive enzyme-linked immunosorbent assay (ELISA). Overall, AFM₁ was present in 78.6% of milk and milk products in the range of 5.0 to 198.7 ng/L. AFM₁ was detected in 71.4% of raw milk (mean 41.1, range 5.0–198.7 ng/L), and in all pasteurized milk (mean 106, range 17.2–187.7 ng/L) and UHT milk (mean 73, range 12.2–146.9 ng/L) samples. Lower AFM₁ levels were found in yogurt (mean 16.9, range 8.3–41.1 ng/L) and milk powder samples (mean 6.6, range 5.9–7.0 ng/L). About one-third of the raw, pasteurized, and UHT milk samples exceeded the EU regulatory limit (50 ng/L) for AFM₁ in milk, while AFM₁ levels in yogurt and milk powder samples were well below this limit. Regarding regions, lower AFM₁ contamination was observed in Chittagong (mean 6.6, max 10.6 ng/L), compared to Sylhet (mean 53.7, max 198.7 ng/L), Dhaka (mean 37.8, max 97.2 ng/L), and Rajshahi (mean 34.8, max 131.4 ng/L). Yet, no significant difference was observed in AFM₁ levels between summer and winter season. In conclusion, the observed frequency and levels of aflatoxin contamination raise concern and must encourage further monitoring of AFM₁ in milk and milk products in Bangladesh.     

The Efficacy of a Smectite-Based Mycotoxin Binder in Reducing Aflatoxin B1 Toxicity on Performance,Health and Histopathology of Broiler Chickens

The aim of the experiment was to investigate the efficacy of a smectite-based clay binder (Toxo-MX) in reducing the toxicological effects of aflatoxin B₁ (AFB₁) in commercial broiler chickens. A total of 450 one-day old male broiler chickens were randomly allocated into three treatment groups with ten replicates of 15 birds each in a 42-day feeding experiment. The dietary treatments included a negative control (NC, a basal diet with no AFB₁ and binder), a positive control (PC, a basal diet contaminated with 500 ppb of AFB₁) and a smectite-based mycotoxin binder(Toxo-MX, PC with smectite clay binder). AFB₁ challenge resulted in 14 to 24% depression in growth performance, elevated levels of aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT), organ enlargement and immuno-suppression.As compared to PC, feeding of Toxo-MX improved the final weight (15%; p < 0.0001), average daily gain (ADG) (15%; p < 0.001) and feed efficiency of broilers (13%; p < 0.0003) but did not have any effects on liver enzyme activities. Supplementation of smectite claysignificantly increased serum globulin levels and reduced the weight of the liver (p < 0.05) as compared to AFB₁-fed broiler chickens. The severity of lesions (inflammatory and degenerative changes) observed in the liver, kidney, heart, pancreas, and lymphoid organs in PC birds was reduced by feeding smectite clay. The immuno-suppression caused by AFB₁ was moderately ameliorated in Toxo-MX groupby stimulating the production of antibodies against IBD at day 42 (p < 0.05). In conclusion, dietary supplementation of a smectite-based mycotoxin binder to the diet containing AFB₁ improved growth performance, reduced toxicological effects in liver and improved humoral immune response in broilers, suggesting its protective effect against aflatoxicosis.     

Induction of meiotic micronuclei in spermatocytes in vivo by aflatoxin B1: Light and transmission electron microscopic study in Swiss mouse

Dietary aflatoxins produce a disease state known as aflatoxicosis, and disruption of spermatogenesis is one of its serious consequences. Towards finding the cellular targets in spermatogenic compartment for aflatoxin toxicity, aflatoxin B₁ (AFB₁) was administered to 90-day-old Swiss mouse through i.p. route at a daily dose of 20 μg per kg body weight for 7, 15, 35 and 45 days. The testis and epididymis were subjected to light- as well as transmission electron microscopic analysis. One of the newer observations was occurrence of meiotic micronucleate giant spermatocytes in seminiferous epithelium and epididymal lumen. The origin of these cells could be traced to imminent disruption of spindle apparatus during meiotic division of spermatocytes, resulting in lagging of chromosome bivalents or replicated univalents. Such chromosomes appeared to undergo condensation and become micronuclei. Thus, this study reports that aflatoxin exposure would result in generation of meiotic micronucleate giant spermatocytes.     

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.     

Effect of aluminosilicates and bentonite on aflatoxin-induced developmental toxicity in rat.

Numerous studies have established that aflatoxin is a potent developmental toxin in animals. Previous research has demonstrated that a phyllosilicate clay, hydrated sodium calcium aluminosilicate (HSCAS or Novasil), tightly binds and immobilizes aflatoxins in the gastrointestinal tract of animals and markedly reduces the bioavailability and toxicity of aflatoxin. Our objective in this study was to utilize the pregnant rat as an in vivo model to compare the potential of HSCAS and bentonite to prevent the developmental toxicity of aflatoxin. Aluminosilicates (HSCAS) and bentonite were added to the diet at a level of 0.5% (w/w) and fed to the pregnant rat throughout pregnancy (i.e. days 0-20). Test animals were fed an aflatoxin-contaminated diet (2.5 mg kg(-1) diet) with or without sorbents during gestation days 6-15. Evaluations of toxicity were performed on day 20. These included maternal (mortality, body weights, feed intake and litter weights), developmental (embryonic resorptions and fetal body weights) and biochemical (ALT, AST and AP) evaluations. Sorbents alone were not toxic and aflatoxin alone resulted in significant maternal and developmental toxicity. Animals treated with phyllosilicate (plus aflatoxin) were comparable to controls following evaluations for resorptions, live fetuses and fetal body weights, as well as biochemical parameters. While bentonite plus aflatoxin resulted in significant reduction in fetal body weight, none of the fetuses from HSCAS or bentonite plus aflatoxin-treated groups had any gross, internal soft tissue or major skeletal malformations.     

The Effect of Aflatoxin-B1 on Red Drum (Sciaenops ocellatus) and Assessment of Dietary Supplementation of NovaSil for the Prevention of Aflatoxicosis

Aflatoxin B₁ (AFB₁) is a potent carcinogen that causes growth stunting, immunosuppression and liver cancer in multiple species. The recent trend of replacing fishmeal with plant-based proteins in fish feed has amplified the AFB₁ exposure risk in farm-raised fish. NovaSil (NS), a calcium montmorillonite clay, has previously been shown to reduce AFB₁ bioavailability safely and efficaciously in several mammalian species. This study was designed to: (1) evaluate AFB₁ impact on cultured red drum, Sciaenops ocellatus, over the course of seven weeks; and (2) assess NS supplementation as a strategy to prevent aflatoxicosis. Fish were fed diets containing 0, 0.1, 0.25, 0.5, 1, 2, 3, or 5 ppm AFB₁. Two additional treatment groups were fed either 5 ppm AFB₁ + 1% NS or 5 ppm AFB₁ + 2% NS. Aflatoxin B₁ negatively impacted red drum weight gain, survival, feed efficiency, serum lysozyme concentration, hepatosomatic index (HSI), whole-body lipid levels, liver histopathological scoring, as well as trypsin inhibition. NovaSil inclusion in AFB₁-contaminated diets improved weight gain, feed efficiency, serum lysozyme concentration, muscle somatic index, and intraperitoneal fat ratios compared to AFB₁-treated fish. Although not significant, NS reduced AFB₁-induced histopathological changes in the liver and decreased Proliferating Cell Nuclear Antigen (PCNA) staining. Importantly, NS supplementation improved overall health of AFB₁-exposed red drum.     

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.     

Effect of Low Protein Diet on Low Dose Chronic Aflatoxin B1 Induced Hepatic Injury in Rhesus Monkeys

Abstract

The role of dietary protein in low dose chronic alfatoxin B₁ (AFB₁) liver injury in Rhesus monkeys has been studied using 0.16 and 0.5 ppm of AFB₁ in diet. The high and low protein diet contained 20% and 5% casein, respectively. Following 32 weeks of intoxication with 0.16 ppm of AFB₁, histological examination revealed mild alterations in low protein group only. However, distinct histochemical and ultra-structural alterations indicative of toxic liver injury are present in both the groups fed AFB₁, though more prominent in animals fed a low protein diet. Monkeys on low protein diet surviving for 90 weeks or more show foci of preneoplastic lesions, whereas those on high protein diet reveal no such alterations at the corresponding time interval. With 0.5 ppm of AFB₁ in diet, the pattern of toxic liver injury is similar to that of 0.16 ppm of AFB₁ in diet. However, the liver damage is more prominent with this dose. The hepatic injury again is more accentuated in the low protein group as compared with the high protein group. No preneoplastic lesions are observed, possibly due to a poor survival (less than 70 weeks) in the low protein animals with this dose. The animals in the high protein group surviving even beyond 90 weeks do not show any preneoplastic/neoplastic lesions. It appears that in the simian model used by us, the liver injury caused by AFB₁ is accentuated by simultaneous restriction of dietary protein and in animals on such combined regimen preneoplastic lesions appear around 90 weeks of experiment. These observations suggest a synergism between protein calorie malnutrition and aflatoxin induced hepatocarcinogenesis and may explain the higher incidence of hepatocellular carcinoma in certain areas of the world where contamination of foods with aflatoxin and malnutrition are prevalent.     

Aflatoxin B1 Degradation by a Pseudomonas Strain

Aflatoxin B₁ (AFB₁), one of the most potent naturally occurring mutagens and carcinogens, causes significant threats to the food industry and animal production. In this study, 25 bacteria isolates were collected from grain kernels and soils displaying AFB₁ reduction activity. Based on its degradation effectiveness, isolate N17-1 was selected for further characterization and identified as Pseudomonas aeruginosa. P. aeruginosa N17-1 could degrade AFB₁, AFB₂ and AFM₁ by 82.8%, 46.8% and 31.9% after incubation in Nutrient Broth (NB) medium at 37 °C for 72 h, respectively. The culture supernatant of isolate N17-1 degraded AFB₁ effectively, whereas the viable cells and intra cell extracts were far less effective. Factors influencing AFB₁ degradation by the culture supernatant were investigated. Maximum degradation was observed at 55 °C. Ions Mn²⁺ and Cu²⁺ were activators for AFB₁ degradation, however, ions Mg²⁺, Li⁺, Zn²⁺, Se²⁺, Fe³⁺ were strong inhibitors. Treatments with proteinase K and proteinase K plus SDS significantly reduced the degradation activity of the culture supernatant. No degradation products were observed based on preliminary LC-QTOF/MS analysis, indicating AFB₁ was metabolized to degradation products with chemical properties different from that of AFB₁. The results indicated that the degradation of AFB₁ by P. aeruginosa N17-1 was enzymatic and could have a great potential in industrial applications. This is the first report indicating that the isolate of P. aeruginosa possesses the ability to degrade aflatoxin.