The occurrence and detection of aflatoxin-macromolecular conjugates in humans.

Abstract

Aflatoxin Bi (AFBi), a highly toxic fungal metabolite (mycotoxin) of certain strains of Aspergillus, has long been known to be carcinogenic in animal species. Accumulation of epidemiological evidence led to its classification, in 1993, by the International Agency for Research on Cancer as a Group I human carcinogen. Aflatoxin Bi contaminates the food supply in most tropical and sub-tropical countries, where it is associated with increased incidence of hepatocellular carcinoma (HCC). In these countries, AFBi is also linked to kwashiorkor, jaundice, and Rey's syndrome. The biological action of AFBi is through its oxidation to AFBi-8,9 epoxide (AFBiO). This epoxide binds to macromolecules like DNA, RNA and proteins as well as amino acids to form AFBi-macromolecular adducts. Quantitation of these adducts is thought to be the most promising approach in the development of methods to measure levels of exposure to aflatoxins. Aflatoxin Bi was produced, isolated and purified using preparative thin layer chromatography (TLC). The toxin was oxidised to AFBiO using dimethyldioxirane and the UV spectra of both the AFBi and AFBiO were determined. Reaction of selected Na-acetyl amino acids (AA) with AFBiO was studied and UV spectrophotometry, TLC, high performance liquid chromatography (FfPLC) and high performance capillary electrophoresis (CE) were used to characterise the reaction products. The epoxide was also reacted with albumin and DNA. Aflatoxin Bi-albumin reaction mixture was hydrolysed and characterised by TLC. Spectrum measurement of the oxidative product of AFBi gave peaks at 266 and 367nm. Qualitative TLC and the epoxide spray reagents confirmed that epoxidation was successful. The in vivo reaction of selected Na-acetyl AA with the epoxide gave peaks between 300 and 400 nm. Naacetyl-arginine, Na-acetyl-lysine and Na-acetyl-histidine showed reaction with AFBiO with maximum wavelengths at 392, 397 and 391 nm respectively. These results strongly suggest that AFBiO is able to covalently bind to lysine, histidine and arginine in albumin. A total of twenty nine blood samples were analysed by HPLC for the presence of AFBilysyl adduct. Of the twenty nine samples, ten were from HCC patients, ten from control patients and nine from kwashiorkor patients. The results show that AFBi-lysine does occur in patients at King Edward VIII Hospital (KEH) and the highest level was detected in HCC patients followed by kwashiorkor patients.