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 /IP/ injection into monkeys, 5.6% of the dose was still retained by the liver, principally bound to liver proteins. After oral dose of aflatoxin B1, rhesus monkeys excreted about 20% as aflatoxin M1 during days 1-4; unchanged aflatoxin B1 accounted only for a small proportion & aflatoxin B1 beta-glucuronide accounted for 5% (3.3% as glucuronide & 1.2% as sulfate conjugate). Another 5% of the dose was excreted as aflatoxin B1 & aflatoxin M1 in the feces.
Aflatoxicol & aflatoxin B1 & M1 were found in tissues of kidney, liver, & muscle of feeder pigs fed estimated LD50 dose of B1 (0.1 mg/kg body wt) provided as rice culture of aspergillus flavus & of market wt pigs, fed naturally contaminated feed containing aflatoxin B1 at level of 400 ng/g from corn for 14 days. B1 & M1, when found in the feeding experiment, were at about the same levels in all tissues except the kidney, in which M1 was the most dominant aflatoxin.
Aflatoxin is excreted in the form of its metabolite aflatoxin M1 in the milk of lactating animals. In cattle given a single oral dose of aflatoxin, 85% of the total amount found in the milk and urine were detected in the first 48 hours after treatment. There was none in the milk after four days, nor in the urine or feces after six days. The total aflatoxin found in the milk was 0.39% of that ingested. ... Less than 0.6% of administered aflatoxin B1 was excreted in the milk. The amount of aflatoxin excreted in milk is unrelated to milk yield, and it disappears from the milk three to four days after the feeding of toxic meal is discontinued.
Using aflatoxin B1, ring-labelled or methoxy-labelled with (14)carbon have shown that rats excrete 70-80% of a single ip dose within 24 hours.
For more Absorption, Distribution and Excretion (Complete) data for AFLATOXIN B1 (18 total), please visit the HSDB record page.

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Metabolism / Metabolites
Aflatoxins would be expected to undergo biotransformations by 4 routes: (i) by hydroxylation of carbon atom at junction of the two fused furan rings, aflatoxin B1 is converted into aflatoxin M1, & this occurs to some extent in mammalian liver, (ii) oxidative o-demethylation of single aromatic methoxy-substituent gives aflatoxin P1 ... (iii) hydration of vinyl ether double bond would afford hemiacetals, & aflatoxin B1 is ... converted into aflatoxin hemiacetal B2a in guinea pig, mouse, & avian livers, (iv) & by reduction of cyclopentenone ring, dihydroaflatoxicol, but this biotransformation seems to be confined to avian species, & may be irrelevant to mammals.
In rhesus monkeys, injected ip ... the chloroform soluble urinary excretory products included aflatoxin M1 (2.3% of dose) & at least 3 other, unidentified ... compounds, as well as unchanged aflatoxin B1 (0.01-0.10%). Chloroform insoluble metabolites in urine were separated by ion exchange methods; major sub fraction consisted of aflatoxin P1 beta-glucuronide. Urinary aflatoxin P1 beta glucuronide represented about 20% of the dose; 17% as glucuronide, 3% as sulfate ester, & 1% as unconjugated phenol.
Investigations of the in vitro metabolism of aflatoxin B1 by liver homogenates from humans ... indicate that aflatoxin B1-2,3 epoxide is produced ... .
Metabolism of Aflatoxin B1 was examined in isolated hepatocytes from rainbow trout. Intracellular DNA adduct formation was linearly related to aflatoxin B1 dose, & qualitatively similar to adducts formed in vivo. The rate of metabolism of adduct accumulation was constant during the first hr, after which an increased & gradual decrease in rate routinely occurred. Relative rates of production of the major unbound aflatoxin B1 metabolites aflatoxicol, aflatoxin M1 & polar conjugates, also remained constant over the 1st hr of preparation age, but subsequently changed in manner consistent with the changes in DNA binding.
For more Metabolism/Metabolites (Complete) data for AFLATOXIN B1 (23 total), please visit the HSDB record page.
Aflatoxin b1 has known human metabolites that include Aflatoxin B1- exo-8,9-oxide, Aflatoxin M1, and Aflatoxin Q1.

Interactions
Piperine is known to modify the biotransformation of drugs. The effect of piperine on the metabolic activation and distribution of (3)H-aflatoxin B1 in rats has been described. Piperine markedly inhibited liver microsome catalysed (3)H AFB1 binding to calf thymus DNA in vitro, in a dose dependent manner. Rats pretreated with piperine accumulated considerable (3)H AFB1 radioactivity in plasma and in the tissues examined as compared to the controls. However, piperine had no influence on hepatic (3)H AFB1-DNA binding in vivo, which could possibly be due to the null effect of piperine on liver cytosolic glutathione 5-transferase activity. Piperine treated rat liver microsomes demonstrated a tendency to enhance (3)H AFB1 binding to calf thymus DNA in vivo. The effect of piperine on AFB1 metabolism thus closely resembles the mode of action of SKF 525-A on biotransformation of foreign compounds.
...A Pekin duck model /was used/ to examine the effect of congenital duck hepatitis B virus infection and aflatoxin B1 (AFB1) exposure in the induction and development of liver cancer. AFB1 was administered to duck hepatitis B virus infected or noninfected ducks at two doses (0.08 and 0.02 mg/kg) by ip injection once a wk from the third month posthatch until they were sacrificed (2.3 yr later). Two control groups of ducks not treated with AFB1 (one of which was infected with duck hepatitis B virus) were observed for the same period. Each experimental group included 13-16 ducks. Higher mortality was observed in ducks infected with duck hepatitis B virus and treated with AFB1 compared to noninfected ducks treated with AFB1 and other control ducks. In the groups of noninfected ducks treated with high and low doses of AFB1, liver tumors developed in 3 of 10 and 2 of 10 ducks; in infected ducks treated with the high dose 3 of 6 liver tumors were observed and none in the low dose of AFB1. No liver tumors were observed in the two control groups. Ducks infected with duck hepatitis B virus and treated with AFB1 showed more pronounced periportal inflammatory changes, fibrosis, and focal necrosis compared to other groups. All duck hepatitis B virus carrier ducks showed persistent viremia throughout the observation period. An increase of viral DNA titers in livers and sera of AFB1 treated animals compared to infected controls was frequently observed. No duck hepatitis B virus DNA integration into the host genome was observed, although in one hepatocellular carcinoma from an AFB1 treated duck, an accumulation of viral multimer DNA forms was detected.
Indole-3-carbinol ((I3)C), a secondary metabolite from cruciferous vegetables, inhibits aflatoxin B1 (AFB1) hepatocarcinogenesis in trout and rats when given prior to and with carcinogen but promotes carcinogenesis in both species when given continuously following AFB1 initiation. Since human (I3)C intake may not be continuous, and the promotional stimulation may be reversible, we have assessed (I3)C promotion using delayed and discontinuous exposure protocols. Following initiation with AFB1, (I3)C was fed to trout for varying periods of time, with varying lengths of delay after initiation and continuous or intermittent patterns of (I3)C treatment. Promotional enhancement of tumor incidence by (I3)C was found to be significant when (I3)C treatment was delayed for several wk or months after the initial AFB1 challenge. Promotion also was found to increase with length of exposure to (I3)C treatment and to be decreased but still evident when (I3)C was given in alternating months or wk, or twice per wk only. These results do not support the idea that promotional stimulation in hepatocarcinogenesis is a reversible phenomenon. To quantify (I3)C promotional potency in terms of its dietary concn, a series of AFB1 tumor dose-response curves was established, each with a different level of (I3)C fed continuously following AFB1 initiation. The resultant tumor dose-response curves, plotted as logit percentage of incidence versus log AFB1 dose, were displaced parallel toward lower AFB1 50% tumor take (TD50) values with increasing (I3)C concn. The level of (I3)C that halves the AFB1 dose for 50% tumor incidence was calculated to be approximately 1000 ppm (I3)C, fed continuously, with no substantial threshold for promotion. By comparison, I3, when fed before and with AFB1, shows a 50% inhibitory value ((I3)C concn that doubles the dose of AFB1 for 50% tumor incidence) in trout of 1400 ppm (I3)C. Thus the potential for (I3)C as a dietary additive to promote prior hepatic initiation events when fed continuously is approximately as great as its potential to inhibit concurrent AFB1 initiation.
...This investigation examined the association between the development of gamma-glutamyl transpeptidase positive foci and alterations in indices of thermogenesis induced by feeding varying levels of dietary protein. Five days following the completion of AFB1 dosing, animals were assigned to groups fed 4%, 8%, 12%, 16%, or 22% dietary protein for 6 wk. Foci development (% liver volume occupied) was markedly reduced in animals fed the low-protein diet (4%, 8%), yet calorie consumption/100 g bw was greater. A modest negative linear trend was observed in oxygen consumption with increasing levels of dietary protein intake. Urinary norepinephrine levels were elevated in the groups fed 4% and 8% protein; urinary dopamine and norepinephrine turnover rates in brown adipose tissue were highest in animals fed 4% protein. These results suggest that gamma-glutamyl transpeptidase positive foci development occurs when a "critical level" (approx 12%) of dietary protein intake is reached. Inhibition of foci development at lower levels of protein intake is associated with several indicators of increased thermogenesis.
For more Interactions (Complete) data for AFLATOXIN B1 (40 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 4800 ug/kg
LD50 Rat ip 6 mg/kg
LD50 Cat oral 550 ug/kg
LD50 Mouse oral 9 mg/kg
For more Non-Human Toxicity Values (Complete) data for AFLATOXIN B1 (14 total), 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 b-1 appears as colorless to pale yellow crystals or white powder. Exhibits blue fluorescence. (NTP, 1992)
Aflatoxin B1 is an aflatoxin having a tetrahydrocyclopenta[c]furo[3',2':4,5]furo[2,3-h]chromene skeleton with oxygen functionality at positions 1, 4 and 11. It has a role as a human metabolite and a carcinogenic agent. It is an aflatoxin, an aromatic ether and an aromatic ketone.
Aflatoxin B1 has been reported in Glycyrrhiza uralensis, Aspergillus flavus, and other organisms with data available.
Aflatoxin B1 is a member of a group of mycotoxins produced by Aspergillus flavus and A. parasiticus. Alflatoxin B1 is the most hepatotoxic and hepatocarcinogenic of the aflatoxins and occurs as a contaminant in a variety of foods.
A potent hepatotoxic and hepatocarcinogenic mycotoxin produced by the Aspergillus flavus group of fungi. It is also mutagenic, teratogenic, and causes immunosuppression in animals. It is found as a contaminant in peanuts, cottonseed meal, corn, and other grains. The mycotoxin requires epoxidation to aflatoxin B1 2,3-oxide for activation. Microsomal monooxygenases biotransform the toxin to the less toxic metabolites aflatoxin M1 and Q1.

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Mechanism of Action
The potent hepatocarcinogenic fungal constituent aflatoxin B1, requires metabolic activation to yield its biological effects & is covalently bound to hepatic macromolecules in the rat.
With the 4 principal aflatoxins tested, the order of inhibitory effect on RNA polymerase II was: B1 greater than G1 greater than B2, G2.
The suspect human hepatocarcinogen aflatoxin B1 (AFB1) is a well-known potent initiator of hepatic tumors in rainbow trout (Oncorhynchus mykiss). Both hepatocellular carcinomas and mixed hepatocellular/cholangiocellular carcinomas are induced by AFB1 in trout, with the mixed form predominating. Previously two c-ras genes were isolated from trout liver cDNA, and in the present study DNA was analyzed from 14 AFB1-induced trout liver tumors for point mutations in exon 1 of both genes. Using the polymerase chain reaction and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1 initiated tumor DNAs showed evidence of activating point mutations in the trout c-Ki-ras gene. Of the 10 mutant ras genotypes, seven were codon 12 GGA - GTA transversions, two were codon 13 GGT - GTT transversions, and one was a codon 12 GGA - AGA transition. Nucleotide sequence analysis of cloned polymerase chain reation products from four of these tumor DNAS provided definitive evidence for two codon 12 GGA - GTA mutations, one codon 12 GGA - AGA mutation, and one codon 13 GGT - GTT mutation, in complete agreement with the oligonucleotide hybridization results. No mutations were detected in exon 1 of a second trout ras gene also expressed in liver, nor in DNA from control livers. This is the first report of experimentally induced ras gene point mutations in a lower vertebrates fish model. The results indicates that the hepatocarcinogen AFB1 induces c-Ki-ras gene mutations in trout similar to those in rat liver tumors.
Aflatoxin B1 has been suggested as a causative agent for a G to T mutation at codon 249 in the p53 gene in human hepatocellular carcinomas from southern Africa and Qidong in China. To test this hypothesis, nine tumors induced by aflatoxin B1 in nonhuman primates were analyzed for mutations in the p53 gene. These included four hepatocellular carcinomas, two cholangiocarcinomas, a spindle cell carcinoma of the bile duct, a hemangioendothelial sarcoma of the liver, and an osteogenic sarcoma of the tibia. None of the tumors showed changes at the third position of codon 249 by cleavage analysis of the HaeIII enzyme site at codon 249. A point mutation was identified in one hepatocellular carcinoma at the second position of codon 175 (G to T transversion) by sequencing analysis of the four conserved domains (II to V) in the p53 gene. These data suggest that mutations in the p53 gene are not necessary in aflatoxin B1 induced hepatocarcinogenesis in nonhuman primates. The occurrence of mutation in codon 249 of the p53 gene in selective samples of human hepatocellular cancers may indicate involvement of environmental carcinogens other than aflatoxin B1 or that hepatitis B virus-related hepatitis is a prerequisite for aflatoxin B1 induction of G to T transversion in codon 249.
For more Mechanism of Action (Complete) data for AFLATOXIN B1 (11 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.)