Secondary/microbial metabolite from Aspergillus flavus and A. parasiticus

Approximately half of hepatocellular carcinoma (HCC) from regions in the world with high contamination of food with the mycotoxin aflatoxin B1 (AFB1) contain a mutation in codon 249 of the p53 tumor suppressor gene. The mutation almost exclusively consists of a G-->T transversion in the third position of this codon, resulting in the insertion of serine at position 249 in the mutant protein. To gain insight into the mechanism of formation of this striking mutational hot spot in hepatocarcinogenesis, we studied the mutagenesis of codons 247-250 of p53 by rat liver microsome-activated AFB1 in human HCC cells HepG2 by restriction fragment length polymorphism/polymerase chain reaction genotypic analysis. AFB1 preferentially induced the transversion of G-->T in the third position of codon 249. However, AFB1 also induced G-->T and C-->A transversions into adjacent codons, albeit at lower frequencies. Since the latter mutations are not observed in HCC it follows that both mutability on the DNA level and altered function of the mutant serine 249 p53 protein are responsible for the observed mutational hot spot in p53 in HCC from AFB1-contaminated areas. Our results are in agreement with an etiological role of AFB1 in hepatocarcinogenesis in regions of the world with AFB1-contaminated food [2].

Tumor models can be created in animals by using aflatoxin B1.

Aflatoxin analysis by HPLC [2]
Detection and quantification of AFB1, AFB2, AFG1, and AFG2 levels in the samples was carried out by HPLC equipped with an autosampler using a fluorescence detector. The HPLC equipment was a Shimadzu system with Shimadzu LC-20AD pump, Shimadzu SIL-20 ADHT autosampler, CTO-20AC column oven, Shimadzu RF-10AXL fluorescence detector (FLD) set at 360-nm excitation and 460-nm emission. An ODS3 column (ODS3 250 mm × 5 μm × 4.6 mm) was used. The mobile phase was distilled water/acetonitrile (90:10), and the flow rate was 1 ml/min; injection volume was 100 μl (AOAC, 999.07).

Fungal cultures [1]
Two Aflatoxin B1 (AFB1) producing strains, Aspergillus flavus (NRRL 3357) and A. parasiticus (NRRL 465) were grown on Potato Dextrose Agar (PDA) individually at 27 °C for 5 days. The spore suspension of each strain was prepared in 5 ml aqueous solution of 0.05% Tween 80 and adjusted to 0.25 (OD540nm) which contained approximately 106–7 conidia/ml. Spore count in the inoculum was verified using an automated cell counter (TC 20, ....

Absorption, Distribution and Excretion
Four days after intraperitoneal injection in monkeys, 5.6% of the dose remained in the liver, primarily bound to liver proteins. In rhesus monkeys, approximately 20% of aflatoxin B1 was excreted as aflatoxin M1 on days 1–4; only a small portion was unmetabolized aflatoxin B1, while aflatoxin B1 β-glucuronide accounted for 5% (3.3% as glucuronide and 1.2% as sulfate conjugates). Another 5% of the dose was excreted in the feces as both aflatoxin B1 and aflatoxin M1. Aflatoxin alcohol, aflatoxin B1, and M1 were detected in the kidney, liver, and muscle tissues of finishing pigs fed with an estimated LD50 dose of aflatoxin B1 (0.1 mg/kg body weight, provided in the form of Aspergillus flavus rice culture) and market-weight pigs fed with a naturally contaminated diet containing 400 ng/g aflatoxin B1 (from corn) for 14 days. In the feeding trials, the levels of aflatoxin B1 and M1 were approximately the same in all tissues except the kidneys; in the kidneys, aflatoxin M1 was the most abundant aflatoxin. Aflatoxin is excreted in the milk of lactating animals in the form of its metabolite, aflatoxin M1. In cattle given a single oral dose of aflatoxin, 85% of the total toxin was detected in milk and urine within the first 48 hours after administration. Aflatoxin was not detected in milk after 4 days, and also not detected in urine and feces after 6 days. The total aflatoxin content in milk accounts for 0.39% of the ingested aflatoxin B1. ... Of the ingested aflatoxin B1, less than 0.6% is excreted through milk. The excretion of aflatoxin in milk is unrelated to milk production and is cleared from milk within three to four days after the feeding of toxic food is stopped.
Using ring-labeled or methoxy-labeled (14 carbon) aflatoxin B1, studies have shown that rats can excrete 70-80% of a single intraperitoneal injection dose within 24 hours.
For more complete data on the absorption, distribution, and excretion of aflatoxin B1 (18 types in total), please visit the HSDB record page.
Metabolism/Metabolites
Aflatoxins are expected to undergo biotransformation via the following four pathways: (i) hydroxylation of carbon atoms at the junction of two fused furan rings, a transformation of aflatoxin B1 to aflatoxin M1 that occurs to some extent in the mammalian liver; (ii) oxidative O-demethylation of a single aromatic ring. The methoxy substituent generates aflatoxin P1…(iii) hydration of the vinyl ether double bond generates a hemiacetal, aflatoxin B1… In the livers of guinea pigs, mice, and birds, it is converted to aflatoxin hemiacetal B2a, (iv) reduction of the cyclopentenone ring to dihydroaflatoxin alcohol, but this biotransformation appears to be limited to birds and may not be relevant to mammals. In rhesus monkeys, intraperitoneal injection… chloroform-soluble excretions in urine included aflatoxin M1 (2.3% of the dose) and at least three other unidentified compounds, as well as unmodified aflatoxin B1 (0.01–0.10%). Chloroform-insoluble metabolites in urine were separated by ion exchange; the major subcomponent was aflatoxin P1 β-glucuronide. Aflatoxin P1 β-glucuronide in urine accounted for approximately 20% of the administered dose; of which 17% was glucuronide, 3% was sulfate, and 1% was unconjugated phenol.
Studies on the in vitro metabolism of aflatoxin B1 in human liver homogenates showed the production of aflatoxin B1-2,3-epoxide…
The metabolism of aflatoxin B1 was detected in hepatocytes isolated from rainbow trout. The formation of intracellular DNA adducts was linearly correlated with the dose of aflatoxin B1 and was similar in nature to adducts formed in vivo. The metabolic rate of adduct accumulation remained constant in the first hour, then generally increased and gradually decreased. In the first hour after preparation, the relative production rates of the major free aflatoxin B1 metabolites (aflatoxin alcohol, aflatoxin M1, and their polar conjugates) remained constant, but subsequently changed in a manner consistent with changes in DNA binding.
For more complete data on the metabolism/metabolites of aflatoxin B1 (23 in total), please visit the HSDB record page.
Known metabolites of aflatoxin B1 include aflatoxin B1-exo-8,9-oxide, aflatoxin M1, and aflatoxin Q1.

Interactions
Piperine is known to alter drug biotransformation. Previous studies have described the effects of piperine on the metabolic activation and distribution of (3)H-aflatoxin B1 in rats. In vitro experiments showed that piperine significantly inhibited the binding of (3)H AFB1 to calf thymus DNA catalyzed by hepatic microsomes in a dose-dependent manner. Compared with the control group, the radioactivity of (3)H AFB1 in the plasma and tissues of rats pretreated with piperine was significantly increased. However, piperine had no effect on the binding of (3)H AFB1-DNA to the liver in vivo, possibly because it had no effect on the activity of glutathione 5-transferase in the hepatic cytoplasm. In vivo experiments showed that piperine-treated rat liver microsomes tended to enhance the binding of (3)H AFB1 to calf thymus DNA. The effect of piperine on AFB1 metabolism is very similar to the mechanism of action of SKF 525-A on the biotransformation of exogenous compounds. This study used a Beijing duck model to investigate the effects of congenital duck hepatitis B virus infection and aflatoxin B1 (AFB1) exposure on the development and progression of liver cancer. Starting from the third month after hatching, AFB1 was administered weekly via intraperitoneal injection to ducks infected or uninfected with duck hepatitis B virus, and to uninfected ducks until sacrifice (2.3 years later). Two control groups (one of which was infected with duck hepatitis B virus) were also observed for the same observation period. Each experimental group contained 13-16 ducks. Results showed that the mortality rate of ducks infected with duck hepatitis B virus and treated with AFB1 was higher than that of uninfected ducks treated with AFB1 and other control groups. In the uninfected duck group, after treatment with high and low doses of aflatoxin B1 (AFB1), 3 out of 10 ducks developed liver tumors; in the infected duck group, 3 out of 6 ducks in the high-dose AFB1 treatment group developed liver tumors, while no liver tumors were observed in the low-dose AFB1 treatment group. No liver tumors were observed in either control group. Compared with other groups, ducks infected with duck hepatitis B virus and treated with AFB1 showed more pronounced periportal inflammatory changes, fibrosis, and focal necrosis. All ducks carrying duck hepatitis B virus exhibited persistent viremia throughout the observation period. Compared with the infected control group, viral DNA titers in the liver and serum of the AFB1-treated group were frequently elevated. Although accumulation of viral multimeric DNA was detected in hepatocellular carcinoma of a duck treated with aflatoxin B1 (AFB1), no integration of duck hepatitis B virus DNA into the host genome was observed. Indole-3-carbinol ((I3)C), a secondary metabolite of cruciferous vegetables, inhibited the development of AFB1-induced liver cancer in trout and rats before or during treatment with the carcinogen AFB1; however, continued administration of (I3)C after AFB1 treatment promoted liver cancer development in both animals. Since human (I3)C intake may not be continuous and its promoting effect may be reversible, we assessed the promoting effect of (I3)C using delayed and intermittent exposure protocols. Trout were fed (I3)C for varying durations after AFB1 treatment, with different lengths of delay and continuous or intermittent (I3)C treatment patterns. The study found that iodine-3-cyclohexyl (I3C) treatment several weeks or months after initial aflatoxin B1 (AFB1) infection significantly promoted tumorigenesis. Furthermore, the longer the I3C treatment duration, the stronger the promoting effect; while administration of I3C every month or week, or only twice a week, resulted in a reduced but still present promoting effect. These results do not support the view that the promoting effect of I3C in hepatocellular carcinoma development is reversible. To quantify the tumor-promoting efficacy of I3C and its relationship with dietary concentration, researchers established a series of AFB1 tumor dose-response curves, each based on continuous administration of different concentrations of I3C after AFB1 infection. The results showed that with increasing I3C concentration, the tumor dose-response curve (plotted as the percentage of incidence against the log value of AFB1 dose) shifted parallel towards lower AFB1 half-cancer concentrations (TD50). Calculations showed that the I3C concentration required to achieve a 50% tumor incidence rate by halving the aflatoxin B1 (AFB1) dose was approximately 1000 ppm, and continuous feeding did not show a significant promotion threshold. In contrast, the 50% inhibition value (the I3C concentration required to achieve a 50% tumor incidence rate by doubling the AFB1 dose) in rainbow trout was 1400 ppm when fed I3 before and concurrently with AFB1 feeding. Therefore, the potential of I3C as a dietary supplement to promote prior liver tumor initiation events during continuous feeding is roughly equivalent to its potential to inhibit concurrent AFB1 tumor initiation events. This study investigated the association between changes in thermogenesis parameters induced by different protein levels of feeding and the development of γ-glutamyl transferase-positive lesions. Five days after AFB1 administration, animals were randomly assigned to four groups, fed 4%, 8%, 12%, 16%, or 22% dietary protein, respectively, for six weeks. Animals in the low-protein groups (4% and 8%) showed significantly reduced hepatic lesion formation (percentage of liver volume), but higher caloric expenditure per 100 grams of body weight. Oxygen consumption showed a slight negative linear trend with increasing dietary protein intake. Animals fed 4% and 8% protein had elevated urinary norepinephrine levels; the turnover rates of urinary dopamine and norepinephrine in brown adipose tissue were highest in animals fed 4% protein. These results indicate that gamma-glutamyl transferase-positive lesions appear when dietary protein intake reaches a “critical level” (approximately 12%). Lower protein intake inhibits lesion formation and is associated with several indicators of increased thermogenesis.
For more complete data on interactions with aflatoxin B1 (40 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats: 4800 μg/kg
Intraperitoneal LD50 in rats: 6 mg/kg
Oral LD50 in cats: 550 μg/kg
Oral LD50 in mice: 9 mg/kg
For more complete data on non-human toxicity values of aflatoxin B1 (out of 14), please visit the HSDB record page.

[1]. Aflatoxin B1 (AFB1) production by Aspergillus flavus and Aspergillus parasiticus on ground Nyjer seeds: The effect of water activity and temperature. Int J Food Microbiol. 2019 May 2;296:8-13.

[2]. Aflatoxin B1 induces the transversion of G-->T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8586-90.

Aflatoxin B1 is a colorless to pale yellow crystal or white powder with blue fluorescence. (NTP, 1992)
Aflatoxin B1 is an aflatoxin with a skeleton of tetrahydrocyclopentano[c]furano[3',2':4,5]furano[2,3-h]chromene, containing oxygen functional groups at positions 1, 4, and 11. It is both a human metabolite and a carcinogen. It is an aflatoxin, aromatic ether, and aromatic ketone.
Aflatoxin B1 has been reported in licorice, Aspergillus flavus, and other organisms with relevant data.
Aflatoxin B1 is a member of a class of mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 is the most hepatotoxic and hepatocarcinogenic of aflatoxins and is commonly found in various foods.
It is a potent hepatotoxic and hepatocarcinogenic mycotoxin produced by Aspergillus fungi. It is also mutagenic, teratogenic, and can cause immunosuppression in animals. Aflatoxin B1 is found in peanuts, cottonseed meal, corn, and other grains. This fungal toxin requires epoxidation to form aflatoxin B1 2,3-oxide for activation. Microsomal monooxygenases biotransform the toxin into the less toxic metabolites aflatoxin M1 and Q1.

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Mechanism of Action
The potent hepatotoxic fungal component aflatoxin B1 requires metabolic activation to exert its biological effects and covalently binds to liver macromolecules in rats.
Among the four major aflatoxins tested, the order of inhibition against RNA polymerase II was: B1 > G1 > B2 > G2.
Aflatoxin B1 (AFB1), a suspected human hepatotoxic carcinogen, is a known potent inducer of liver tumors in rainbow trout (Oncorhynchus mykiss). AFB1 can induce hepatocellular carcinoma and mixed hepatocellular/cholangiocarcinoma in rainbow trout, with the mixed type being more common. Two c-ras genes had previously been isolated from trout liver cDNA. This study analyzed DNA from 14 aflatoxin B1 (AFB1)-induced trout liver tumors, detecting point mutations in exon 1 of these two genes. Using polymerase chain reaction (PCR) and oligonucleotide hybridization, activation point mutations in the trout c-Ki-ras gene were found in a high proportion (10/14) of the AFB1-induced tumor DNA. Of these 10 mutant ras genes, 7 were GGA-GTA transversions at codon 12, 2 were GGT-GTT transversions at codon 13, and 1 was a GGA-AGA transversion at codon 12. Nucleotide sequence analysis of the clonal PCR products from these four tumor DNAs provided conclusive evidence of two GGA-GTA mutations at codon 12, one GGA-AGA mutation at codon 12, and one GGT-GTT mutation at codon 13, which was completely consistent with the oligonucleotide hybridization results. No mutations were detected in exon 1 of the second trout ras gene expressed in the liver, nor in the DNA of the control liver. This is the first report of experimentally induced point mutations in the ras gene in a lower vertebrate fish model. The results indicate that the c-Ki-ras gene mutation induced by the hepatotoxic carcinogen AFB1 in trout is similar to the mutation in rat liver tumors. Aflatoxin B1 is considered a pathogenic factor causing the G-to-T mutation at codon 249 of the p53 gene in human hepatocellular carcinoma in South Africa and Qidong, China. To verify this hypothesis, we analyzed the p53 gene mutations in nine non-human primate tumors induced by aflatoxin B1. These tumors included four hepatocellular carcinomas, two cholangiocarcinomas, one cholangiocarcinoma spindle cell carcinoma, one hepatic angioendothelial sarcoma, and one tibial osteosarcoma. Cleavage analysis at the HaeIII restriction site at codon 249 revealed no mutations at the third site of codon 249 in any of the tumors. Sequencing analysis of the four conserved domains (II to V) of the p53 gene revealed a point mutation (G-to-T transversion) at codon 2 of 175 in one hepatocellular carcinoma. These data suggest that aflatoxin B1-induced hepatocellular carcinoma in non-human primates does not necessarily require p53 gene mutations. The mutation at codon 249 of the p53 gene in specific human hepatocellular carcinoma samples may indicate the involvement of other environmental carcinogens besides aflatoxin B1, or that hepatitis B virus-associated hepatitis is a prerequisite for aflatoxin B1-induced G-to-T transversion at codon 249. For more complete data on the mechanisms of action of aflatoxin B1 (11 in total), please visit the HSDB record page.

C17H12O6

312.28

312.063

C, 65.39; H, 3.87; O, 30.74

1162-65-8

Aflatoxin B1-13C17;1217449-45-0

186907

White to yellow solid powder

1.6±0.1 g/cm3

528.2±50.0 °C at 760 mmHg

268-269 °C

237.7±30.2 °C

0.0±1.4 mmHg at 25°C

1.687

0.45

0

6

1

23

650

2

COC1=C2C3=C(C(=O)CC3)C(=O)OC2=C4[C@@H]5C=CO[C@@H]5OC4=C1

OQIQSTLJSLGHID-WNWIJWBNSA-N

InChI=1S/C17H12O6/c1-20-10-6-11-14(8-4-5-21-17(8)22-11)15-13(10)7-2-3-9(18)12(7)16(19)23-15/h4-6,8,17H,2-3H2,1H3/t8-,17+/m0/s1

(3S,7R)-11-methoxy-6,8,19-trioxapentacyclo[10.7.0.02,9.03,7.013,17]nonadeca-1,4,9,11,13(17)-pentaene-16,18-dione

NSC-529592; AFLATOXIN B1; 1162-65-8; AFB1; AFBI; (-)-Aflatoxin B1; NSC 529592; CCRIS 12; EINECS 214-603-3; NSC 529592; Aflatoxin B1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMF : 33.33 mg/mL (~106.73 mM)
DMSO : ~30 mg/mL (~96.07 mM)

Solubility in Formulation 1: 3 mg/mL (9.61 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)