Gordon E. Neal

Mutations associated with in vivo aflatoxin B1-induced carcinogenesis need not be present in the in vitro transformations by this toxin

Ingestion of aflatoxin B1 is implicated in the high incidence of human liver cancers in several developing countries. An association has been detected between human exposure to aflatoxins, and mutations in the third base of codon 249 of the p53 gene in hepatomas. In vitro experiments using human cell line cells and aflatoxin B1 have demonstrated the induction of p53 mutations in codon 249 and adjacent codons. It was therefore of interest to see if this correlation between the in vivo and in vitro situations held for other species. The present study examined a rat liver-derived cell line, transformed in vitro with aflatoxin B1, for the presence of mutations associated with in vivo aflatoxin-induced hepatocarcinogenesis. In an in vivo rodent model systems using the aflatoxin B1-sensitive male F344 rat, previous studies have shown that hepatocarcinogenesis is accompanied by significant incidences of codon 12 mutations in K-ras and codon 13 mutations in N-ras genes, but in contrast to the human, apparently not by mutations in codon 243 of the p53 gene (which corresponds to codon 249 in the human gene). In contrast to the situation in humans, mutation in the third base of codon 243 in the rat would not result in any changes in amino acid sequence, but mutations in codon 250, as seen in in vitro human systems, would be expressed in the rat p53 protein. In the present study, an immortalised, non-transformed liver epithelial cell line derived from a male F344 rat was transformed in vitro by aflatoxin B1 as demonstrated by tumour formation in nude mice. The transformation was dependent on metabolic activation of the aflatoxin B1. Transfection of DNA, extracted from these tumours, into NIH 3T3 fibroblasts conferred a stable, malignant transforming capacity. However, no mutations in codon 12 of the K-ras or codon 13 of the N-ras genes were detected in any of these tumours. These results indicate that in vitro transformation does not necessarily involve the same mutations, as those observed in vivo. Also, no mutations in codon 243 or adjacent codons of the p53 gene, paralleling those observed in the human cell line treated with aflatoxin B1, were detected. The results serve to emphasise the in vivo and in vitro variation in the oncogene activation in the same target organ or cell lines derived from that organ, even when using a single carcinogen activated by a known metabolic pathway.

Further circumstantial evidence for the involvement of aflatoxin in primary liver cancer

Fifty-eight percent of hepatocellular carcinomas (HCCs) from Qidong, China, contain an AGG to AGT mutation at codon 249 of the p53 tumour suppressor gene, a mutation that is rarely seen in HCCs from Western countries. The population of Qidong is exposed to high levels of aflatoxin B1(AFB1), a fungal toxin that has been shown to induce the same mutation in cultured human HCC cells. To investigate the role of AFB1 and of these p53 mutations in hepatocarcinogenesis, normal liver samples from the United States, Thailand and Qidong (where AFB1 exposures are negligible, low and high, respectively) were examined for p53 mutations. The frequency of the AGG to AGT mutation at codon 249 paralleled the level of AFB 1 exposure, which supports the hypothesis that this toxin has a causative-and probably early-role in hepatocarcinogenesis.

The influence of partial hepatectomy on the biphasic response of gamma glutamyl transpeptidase to aflatoxin B1

We previously observed a biphasic response in rat hepatic gamma glutamyl transpeptidase (GGT) activity to aflatoxin B1 (AFB1) feeding [9]. We have extended this observation to examine the effect of partial hepatectomy (PH) on the activity and distribution of GGT at different stages of the feeding regime. In control-fed animals GGT levels were elevated 3-7 days after PH with increased activity in periportal hepatocytes. In animals fed a sub-carcinogenic dose of AFB1 (up to 4 weeks) the effect of PH on GGT activity was similar to that in control animals, but increased activity was mainly due to biliary hyperplasia. There was no obvious difference between animals returned to control diet after PH and those returned to toxic diet. In animals fed 4-15 weeks the percentage increase in GGT activity 1 week after PH correlated with length of time on AFB1 diet before operation, with an increase in number and size of altered foci. These results further support the idea that there is a preliminary toxic response in GGT activity followed by a secondary response more closely related to the carcinogenic process.

Biochemical and molecular aspects of mammalian susceptibility to aflatoxin B1 carcinogenicity.

Aflatoxin B, (AFB1) is a fungal toxin that has been implicated as a causative agent in human hepatic and extrahepatic carcinogenesis. In this review, the mechanisms involved in AFBI toxicity are delineated, in order to describe the features that make a specific cell, tissue or species susceptible to the mycotoxin. Important considerations include:(1) Different mechanisms for bioactivation of AFBI to its ultimate carcinogenic epoxide metabolite. (2) The balance between bioactivation to and detoxification of the epoxide. (3) The interaction of AFBI epoxide with DNA and the mutational events leading to neoplastic transformation. (4) The role of cytotoxicity in AFBI carcinogenesis. (5) The significance of non-epoxide metabolites in toxicity, and (6) The contribution of mycotoxin unrelated disease processes. Although considerable controversy remains about the importance of specific events, a great deal has been learned about biochemical and molecular actions of AFB1.