Interactions of aflatoxin B1 and blood components of various species in vitro: Interconversion of aflatoxin B1 and aflatoxicol in the blood

The fate of aflatoxin B₁ (AFB₁) in the blood of various species of animals was studied in vitro. Examination of the distribution of radioactivity in blood incubated with [¹⁴C]AFB₁ at 37°C showed that high levels of radioactivity were associated with blood cells. The radioactivity was readily removed from the blood cells by washing with fresh plasma, indicating loose binding of AFB₁ to blood cells. Most of the radioactivity in plasma was bound to protein. These results suggest that a large part of the AFB₁ in blood in vivo may be carried not only by the plasma proteins but also by the blood cells. When chloroform extracts of plasma of [¹⁴C]AFB₁-treated mouse, rat, duckling, and hamster blood were developed by thin-layer chromatography, high levels of radioactivity were found in both the AFB₁ region and the aflatoxicol (AFL) region. Incubation of blood with nonradioactive AFB₁ and AFL showed marked interconversion of AFB₁ and AFL in the blood of rats, hamsters, mice, and Mongolian gerbils, but not in the blood of guinea pigs, rhesus monkeys, squirrel monkeys, or humans. Interconversion occurred in red blood cell suspensions but not in plasma, indicating that the red blood cells are responsible for AFB₁-AFL interconversion in the blood.     

Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheral blood mononuclear cells

Mycotoxins are secondary metabolites having a high cytotoxic potential. They are produced by molds and released in food and feed. To date, the mechanisms underlying the mycotoxin-induced cytotoxicity have not been fully clarified. The induction of oxidative stress, as a possible mechanism, has been postulated. This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB₁ (0, 5 and 20 μg/ml) and FB₁ (0, 35 and 70 μg/ml). Reactive oxygen metabolites (ROM), intracellular thiols (SH), malondialdehyde (MDA) and gene expression of cytoplasmic superoxide dismutase (SOD) and glutathione peroxidase (GSHPX-1) were measured on PBMC after incubation. The highest concentration of AFB₁ and all concentrations of FB₁ caused an increase (p < 0.05) of intracellular ROM without any time dependent effect. Intracellular SH decreased with 20 μgAFB₁/ml (p < 0.05) and the effect was particularly marked after 7 days of exposure. Intracellular SH were not affected by FB₁ even though a lower (p < 0.05) SH level after 2 days exposure than after 7 days was observed. MDA increased (p < 0.05) in AFB₁ or FB₁ treated PBMC. The exposure to FB₁ for 7 days increased MDA (p < 0.05) only in cells treated with 70 μg/ml. Exposure of PBMC to AFB₁ reduced SOD mRNA while FB₁ decreased both SOD and GSHPX-1 mRNA abundance. These results demonstrate that, even though by different mechanisms, AFB₁ and FB₁ may induce cytotoxicity through an impairment of the oxidative status of PBMC.     

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     

Butylate hydroxytoluene pretreatment protects against cytotoxicity and reduces covalent binding of aflatoxin B1 in primary hepatocyte cultures

Primary cultures of adult rat hepatocytes were used to characterize the effect of butylated hydroxytoluene (BHT) pretreatment on the metabolic disposition and cytotoxicity of a single dose of aflatoxin B₁ (AFB₁). Four male Sprague-Dawley rats were fed a control diet, and five were fed a diet containing 0.5% BHT. After 10 days, hepatocytes were prepared and cultured in chemically defined, hormone-supplemented medium. After 20–22 hr in culture, 120–150 ng of [¹⁴C]aflatoxin B₁ was added to dishes containing 2.5 × 10⁶ cells. By 10 hr, control cells had converted 59% of the AFB₁ to aqueous metabolites, while 15.5% was bound covalently. During the same interval, cells from BHT-fed rats produced 69% aqueous metabolites, and only 6.6% was bound covalently. The rate of AFB₁ disappearance in the two groups was not statistically different. AFB₁ produced marked cytotoxicity in hepatocyte cultures from control rats but had no apparent toxic effect on hepatocyte cultures from BHT-pretreated rats, as indicated by light microscopic examination and release of lactate dehydrogenase into the medium. These results suggest that reduction of cytotoxicity by BHT was associated with increased output of nontoxic, water-soluble metabolites and decreased binding of metabolites to macromolecules. These results also indicate that BHT may protect against the acute toxicity and carcinogenicity of aflatoxin B₁in vivo.     

On the occurrence of aflatoxin M1 in milk and dairy products

Aflatoxins are toxic fungal metabolites found in foods and feeds. When ruminants eat AFB₁-feedstuffs, they metabolise the toxin and excrete AFM₁ in milk. To control AFM₁ in foods it is necessary to reduce AFB₁ contamination of feeds for dairy cattle by preventing fungal growth and AFB₁ formation in agricultural commodities intended for animal use. Corn and corn-based products are one of the most contaminated feedstuffs; therefore risk factor analysis of AFB₁ contamination in corn is necessary to evaluate risk of AFM₁ contamination in milk and milk products. During the corn silage production, the aflatoxins production is mostly influenced by: harvest time; fertilization; irrigation; pest control; silage moisture; and storage practices. Due to the lower moisture at harvest and to the conservation methods, the corn grain is mostly exposed to the contamination by Aspergillus species. Therefore, it is necessary to reduce the probability of this contaminant through choice of: hybrids; seeding time and density; suitable ploughing and fertirrigation; and chemical or biological control. Grains harvested with the lowest possible moisture and conservation moisture close to or less than 14% are necessary to reduce contamination risks, as is maintaining mass to homogeneous moisture. Kernel mechanical damage, grain cleaning practices and conservation temperature are also factors which need to be carefully controlled.     

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.     

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.     

Occurrence of aflatoxin M1 in some samples of UHT,raw & pasteurized milk from Indian states of Karnataka and Tamilnadu

Aflatoxin M₁ (AFM₁) is a toxic metabolite found in the milk of lactating animals which have consumed feedstuffs contaminated with aflatoxin B₁. Ultra high temperature treated (UHT) milk is a product which is becoming popular in developing countries like India as there is a lack of proper cold storage or refrigeration facilities. In this study, 45 samples of UHT milk of popular brands prevalent in the market were analyzed for the presence of AFM₁ by reversed phase HPLC using fluorescent detector after cleanup of sample with immunoaffinity columns. All samples of plain UHT milk were positive for AFM₁ and 38% of these contained levels more than 0.5 μg/kg, the maximum permitted limit prescribed by the Codex Alimentarius Commission and by the mandatory regulations of the country, the FSSAI Regulations, 2011. In 62.5% of flavored UHT milk, AFM₁ was below detectable levels (0.02 μg L⁻¹). However, 12.5% of these samples also contained levels exceeding the maximum permitted limits. AFM₁ was present in 61.6% of the 52 raw milk samples analyzed from the two states of Karnataka and Tamilnadu with a range of 0.1–3.8 μg L⁻¹. 17.3% of these samples also exceeded the regulatory limits of the country.     

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.     

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     

Modification by beet and cabbage diets of aflatoxin B1-induced rat plasma α-foetoprotein elevation,hepatic tumorigenesis,and mutagenicity of urine

Weanling male Fischer rats were fed a purified diet or diets containing 25% (w/w) freeze-dried ground beets or cabbage with or without 1 ppm aflatoxin B₁ (AFB₁) for 26 wk. In 3–7 wk the cabbage diet diminished, while the beet diet enhanced AFB₁-induced plasma α-foetoprotein (AFP) elevation. When the experiment was extended to 42 wk by maintaining the animals on the purified (basal) diet for a further 16 wk the rats that had consumed AFB₁ in the beet diet had 72 ± 14 tumours/liver (mean surface diameter of tumours, 6·13 ± 4·69 mm); animals that had been given AFB₁ in the control diet had 30 ± 16 tumours/liver (mean surface diameter, 4·36 ± 3·16 mm); rats that had been given AFB₁ in the cabbage diet had 13 ± 5 tumours/liver (mean surface diameter, 4·28 ± 2·89 mm). In the Salmonella/ mammalian microsomal test, urine from rats fed AFB₁ with beets caused significantly (P < 0·05) more revertants in Salmonella typhimurium strain TA98 than did urine from rats fed AFB₁ with purified or cabbage diets. The beet- and cabbage-containing diets had no effect on the plasma AFP concentration, hepatic tumorigenesis, or the mutagenicity of urine in rats receiving no AFB₁. The evidence suggests that non-nutrient components of common vegetables may influence the response to chemical carcinogens, and that AFP determinations are useful in the rapid identification of dietary factors that modify carcinogenesis.     

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.     

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.     

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.     

Effects of phenobarbital pretreatment on the metabolism and toxicokinetics of aflatoxin B1 in the rhesus monkey

The in vitro and in vivo metabolism and toxicokinetics of [¹⁴C]aflatoxin B₁ (AFB₁) were determined in the rhesus monkey with and without phenobarbital pretreatment. Metabolic induction with phenobarbital increases an animal's resistance to the carcinogenic effects of AFB₁. Phenobarbital pretreatment reduced plasma levels of total aflatoxins, AFB₁, aflatoxin M₁, and water-soluble metabolites; enhanced the circulation and elimination of less toxic, less mutagenic, and more water-soluble urinary products; and increased the in vitro formation of aflatoxin Q₁ and aqueous metabolites. These changes are consistent with increased resistance to AFB₁, based on previously proposed correlations between metabolism and species susceptibility. Phenobarbital did not evoke significant toxicokinetic changes in rate constants for distribution and elimination, which have been associated with susceptibility to the acute effects of AFB₁. Phenobarbital protection may be attributable to the enhancement of detoxification pathways.     

Toxicity,biochemical effects and residue of aflatoxin B1 in marine water-reared sea bass (Dicentrarchus labrax L.)

Aflatoxicosis and resulting epizootic hepatoma have been reported among a wide range of fish where Aspergillus species-contaminated foodstuffs are incorporated into the diet. Aflatoxin B₁ (AFB₁) is among the most potent known hepatotoxins and carcinogens. Therefore, it is an important potential toxicant to the most of the popularly cultured fish species. The present study was undertaken to assess the susceptibility and toxicity of AFB₁ to sea bass (Dicentrarchus labrax L.), by behavioral and biochemical evaluations. The estimated oral acute median lethal concentration (96 h LC₅₀) of AFB₁ for sea bass was 0.18 mg/kg bwt. The abnormal behavioral responses and signs of toxicity were described. The prolonged oral administration of 0.018 mg/kg bwt AFB₁ to sea bass for 42 successive days induced a significant increase in serum transaminases and alkaline phosphatase activities, and significant decrease in plasma proteins. Residual AFB₁ was detected at high levels (≈5 ppb) in fish musculature at the end of the experimental period. We conclude that marine water sea bass is a species highly sensitive to AFB₁. In addition, consumption of sea bass reared on AFB₁-contaminated diet could have a negative health impact on human health.     

Metabolism of aflatoxin B1 (AFB1), aflatoxin G1 (AFG1) and vitamin C intake by guinea pig liver preparation in vitro

Male and female guinea pigs weighing 150–200 g were divided into three groups, with equal number of males and females in each group. They were fed an experimental diet which varied as follows: group I, 0 mg vitamin C/g of diet; group II, 1.08 mg/g and group III, 5.4 mg/g, for 28 days. Twenty-four hours after the last feeding, liver slices and 9000 g supernatant were prepared from each group, according to sex, and used for enzyme assays. For the demethylation assay, enzyme activity expressed as amount of formaldehyde produced from AFB₁, or AFG₁/hr/g fresh liver was seen to increase with the two levels of ascorbic acid intake in females. Males showed an enhancement of activity only in group II and remained with the same production of formaldehyde as above in group III. Although in each dietary group, the activity was higher in males than in females the variation in the amount of formaldehyde produced from one group to another was higher with females than with male guinea pigs. However with both sexes, the production of formaldehyde from AFG₁, was greater than from AFB₁. For the hydroxylation assay, enzyme activity was expressed as amount of metabolites (a) and (b) produced. Compared to group II, which offered a control level of ascorbic acid, group I fed without vitamin C showed a decreased production of metabolite (a) and (b) with males and females. Moreover, high intake of ascorbic acid in group III decreased the production of metabolite (a) and (b) in males, while in female guinea pigs the reduction was observed only with (b).     

Effects of inducers and inhibitors on the metabolism of aflatoxin B1 by rat and mouse.

The microsomal metabolism of aflatoxin B₁ (AFB₁) via various pathways and the induction and inhibition specificities of these pathways were examined in Sprague-Dawley rats, inbred strains of mice, and recombinant inbred lines derived from AKR/J and C57L/J cross. The data suggests that the metabolism of aflatoxin B₁ is catalyzed by at least three different enzymes of the microsomal mixed function oxygenase system: one that mediates conversion to aflatoxin M₁ (AFM₁-hydroxylase) is cytochrome P-448-linked and is associated with the Ah locus at the level of regulatory genetic factors: the other two enzymes which are cytochrome P-450-linked, mediate the metabolism of aflatoxin B₁ to aflatoxin Q₁ (AFQ₁-hydroxylase) and to AFB₁-2,3-oxide (metabolic activation). Although AFQ₁-hydroxylase and metabolic activation, measured in vitro as the formation of DNA-alkylating metabolite(s), are not clearly distinguishable on the basis of induction and inhibition responses, the two activities can be distinguished on the basis of their kinetic parameters and genetic regulation: (a) the apparent K_m of the metabolic activation pathway is almost three orders of magnitude (19.7 × 10^?4 M) higher than that of AFQ₁-hydroxylase (0.7 × 10^?4), [B. D. Roebuck and G. N. Wogan, Cancer Res.37, 1649 (1977)]; and (b) while both activities are induced by phenobarbital (PB) and depressed by 3-methylcholanthrene (3-MC), unlike the metabolic activation, differences in the 3-MC-induced depression of AFQ₁-hydroxylase activity were noted in Ah responsive and nonresponsive strains. Kinetic studies revealed that metabolic activation of AFB₁ is linear with the time of incubation up to 20 min, with up to 2 mg of microsomal protein in the incubation, and has a pH optimum of 7 to 7.4. While PB pretreatment, relative to control, enhanced the apparent V_max 3-fold, 3-MC depressed it about 60 per cent; however, the apparent K_m values of the three microsomal preparations were of the same order of magnitude (1.3 to 2 × 10^?3 M). Under the conditions of the incubation, the minimum number of binding sites in DNA was estimated to correspond to a resultant specific activity of 8–9 nmoles AFB₁-metabolite bound/μmole DNA-P.     

Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1

It has been proposed that the consumption of lactic acid bacteria capable of binding or degrading foodborne carcinogens would reduce human exposure to these deleterious compounds. In the present study, the ability of eight strains of Lactobacillus casei to bind aflatoxin B₁ in aqueous solution was investigated. Additionally, the effect of addition of bile salts to the growth medium on aflatoxin B₁ binding was assessed. The eight strains tested were obtained from different ecological niches (cheese, corn silage, human feces, fermented beverage). The strains exhibited different degrees of aflatoxin binding; the strain with the highest AFB₁ binding was L. casei L30, which bound 49.2% of the available aflatoxin (4.6 μg/mL). In general, the human isolates bound the most aflatoxin B₁ and the cheese isolates the least. Stability of the bacterial–aflatoxin complex was assessed by repeated washings. Binding was to a limited degree (0.6–9.2% release) reversible; the L. casei 7R1–aflatoxin B₁ complex exhibited the greatest stability. L. casei L30, a human isolate, was the strain least sensitive to the inhibitory effects of bile salts. Exposure of the bacterial cells to bile significant increased aflatoxin B₁ binding and the differences between the strains was reduced.     

Tissue disposition and excretion of C-labelled aflatoxin B1 after oral administration in channel catfish

The pharmacokinetics, tissue distribution and excretion of ¹⁴C-labelled aflatoxin B₁ (AFB₁) were examined after oral administration (250 μg/kg body weight) in channel catfish (Ictalurus punctatus). Plasma concentrations of parent AFB₁ were best described by a one-compartment pharmacokinetic model, in which peak plasma concentration (503 ppb) occurred at 4.1 hr after dosing. The absorption and elimination half-lives were 1.5 and 3.7 hr, respectively. AFB₁ was highly bound (95%) to plasma proteins. Concentrations of ¹⁴C (in AFB₁ equivalents) measured in the tissues were highest at 4 hr, ranging from 596 ppb in the plasma to 40 ppb in the muscle. AFB₁ residues were rapidly depleted; at 24 hr the concentrations in the plasma and muscle were 32 and <5 ppb, respectively. Concentrations in the bile exceeded 2000 ppb (at 24 hr), whereas the highest concentration in the urine was 51 ppb (4–6-hr collection interval). Renal and biliary excretion accounted for <5% of the administered dose, indicating incomplete absorption. Pharmacokinetic modelling and tissue data demonstrate a very low potential for the accumulation of AFB₁ and its metabolites in the edible flesh of channel catfish through the consumption of AFB₁-contaminated feed.