Aflatoxin B1

Did You Know?

• Aflatoxin B1 can permeate through the skin, and dermal exposure under certain environmental conditions poses health risks.
• Aspergillus flavus prefers hot and dry conditions, with optimal growth at 37°C, which contributes to its pathogenicity in humans.
• Occupational exposure to aflatoxin B1 has been reported in swine and poultry production.

Biosynthetic pathway

Aflatoxin B1 is synthesized through a pathway that begins with a dedicated fatty acid synthase (FAS) and a polyketide synthase (PKS), together termed norsolorinic acid synthase. The FAS produces hexanoate, which serves as the starter unit for the iterative type I PKS. The PKS adds seven malonyl-CoA extenders to hexanoate, forming a C20 polyketide that cyclizes into the anthraquinone norsolorinic acid. A reductase then reduces the ketone on the norsolorinic acid side-chain to yield averantin. Averantin is converted to averufin via a hydroxylase and an alcohol dehydrogenase, which oxygenate and cyclize the side-chain to form a ketal. From averufin, the pathway becomes more complex and many enzymes remain uncharacterized. A P450-oxidase, AvfA, performs a Baeyer-Villiger oxidation on averufin, opening the ether rings and forming versiconal acetate after rearrangement. An esterase, EstA, hydrolyzes the acetyl group to produce versiconal. VERB synthase catalyzes cyclization of the versiconal side-chain, and VerB, a desaturase, reduces versicolorin B to form the dihydrobisfuran. Two enzymes, AflN (an oxidase) and AflM (a reductase), convert versicolorin A to demethylsterigmatocystin using molecular oxygen and two NADPH molecules. This step involves dehydration, ring opening, aldehyde formation, oxidation to a carboxylic acid, and decarboxylation to close a six-member ether ring. Two methyltransferases, OmtB and OmtA, then methylate the hydroxyl groups on the xanthone part using S-adenosyl methionine, yielding O-methylsterigmatocystin. Finally, an oxidoreductase, OrdA, catalyzes oxidative cleavage of the aromatic ring with loss of one carbon, followed by recyclization to form aflatoxin B1.

Mechanism of carcinogenicity

Aflatoxin B1 is a genotoxic hepatocarcinogen whose exposure is strongly associated with hepatocellular carcinoma, particularly in individuals co-infected with hepatitis B virus. The compound must be metabolized by cytochrome P450 into its reactive form, aflatoxin B1-8,9-exo-epoxide. This electrophilic species intercalates between DNA bases and forms adducts with guanine residues, most commonly aflatoxin B1-N7-Gua. These adducts can rearrange or be removed, creating apurinic sites that during DNA replication lead to mis-matched base insertion. Up to 44% of hepatocellular carcinomas in high-exposure regions show a GC→TA transversion at codon 249 of the p53 tumor suppressor gene, a characteristic mutation. Co-infection with hepatitis B virus greatly amplifies risk: individuals with urinary aflatoxin biomarkers face a threefold risk, those with hepatitis B alone a fourfold risk, and those with both a 60-fold risk compared to the normal population.

Toxicity

Acute toxicity studies across species report an oral LD50 range of 0.3–17.9 mg/kg body weight. In female rats the oral LD50 is 17.9 mg/kg, in male rats 7.2 mg/kg, and the intraperitoneal LD50 in male rats is 6.0 mg/kg. Symptoms include anorexia, malaise, and low-grade fever. Subacute toxicity in monkeys shows portal inflammation and fatty change. Chronic toxicity in chickens results in decreased hepatic microsomal cytochrome P-450 concentration, reduced feed consumption, and decreased weight gain. Subchronic toxicity in fish produces preneoplastic lesions along with changes in gill, pancreas, intestine, and spleen. Genotoxicity in human liver cells at 3–5 μmol/L leads to aflatoxin B1-DNA adducts, 8-hydroxyguanine lesions, and DNA damage. Carcinogenicity studies in rats report development of liver cell carcinoma. Embryotoxicity in chickens includes embryonic death and impaired development of the bursa of Fabricius. Teratogenic effects in rabbits include reduced fetal weights, wrist drop, enlarged eye socket, agenesis of caudal vertebrae, microphthalmia, cardiac defects, and lenticular degeneration. Immunotoxicity in fish shows reduced serum total globulin and reduced bactericidal activities.

Risk management and regulations

Exposure to aflatoxin B1 is managed through prevention of crop contamination in the field, post-harvest handling, and storage, as well as detection and decontamination of contaminated commodities or animal feed. Biological decontamination using Flavobacterium aurantiacum has been employed to remove aflatoxin B1 from peanuts and corn. Regulations vary by jurisdiction. In the United States, the maximum permitted level for aflatoxin B1 combined with other aflatoxins (B2, G1, G2) is 20 μg/kg in all foods except milk, which has a limit of 0.5 μg/kg; animal feeds may tolerate 100–300 μg/kg.