IARC MONOGRAPHS ON THE EVALUATION OF CARCINOGENIC ...

AFLATOXINS 239
coding for arginine. In four rhesus monkeys (Macaca mulatta) and four cynomolgus
monkeys (M. fascicularis), aflatoxin-induced HCC did not carry any mutation at codon
249, but only four HCC (two from one animal) were analysed (Fujimoto et al., 1992).
Preneoplastic lesions have been examined to define the time point in the natural
history of HCC when the TP53 mutation occurs. Hulla et al. (1993) examined six hyperplastic
nodules from rat liver following intraperitoneal treatment with 150 μg/kg aflatoxin
B 1 for 10 days (2 × 5 days with a two-day break in between, followed by partial
hepatectomy three weeks later and sacrifice after a further three weeks) and found no
mutations at the codon 249 equivalent.
Female AC3F 1 (A/J × C3H/He/J) mice [numbers unspecified], 5–7 weeks of age,
received intraperitoneal injections of aflatoxin B 1 three times per week for eight weeks
(total dose, 150 mg/kg bw). Mice were killed between 6 and 14 months later. Of the 71
lung tumours examined, 79% showed positive nuclear p53 staining. SSCP analysis of
microdissected tumour samples revealed mutations in different codons in exons 5, 6
and 7. Direct sequencing showed 26 mutations which included nine G:C to A:T transitions,
11 A:T to G:C transitions and five transversions (two G:C to T:A, two T:A to A:T
and one A:T to C:G). The high mutation frequency and heterogeneous staining pattern
suggested that TP53 mutations occur relatively late in aflatoxin B 1-induced mouse lung
tumorigenesis (Tam et al., 1999).
Lee et al. (1998) treated 10 male Sprague-Dawley rats with 37.5 μg aflatoxin B 1 five
times per week for eight weeks by gavage and a further 20 with the same treatment after
partial hepatectomy. Of the latter group, 13 of 17 rats that survived to 60 weeks after
treatment had either liver tumours (5), preneoplastic nodules (7) or both (1). Of the six
surviving rats from the group that received aflatoxin B 1 alone, two had liver tumours, one
had liver focal lesions and one had both. All rats were killed at 60 weeks and liver
tumours and preneoplastic lesions were excised. A total of 19 abnormal liver specimens
were obtained (10 liver focal lesions and nine liver tumours). The PCR SSCP method
was used to screen the TP53 gene; five rat livers (29%) exhibited abnormal conformational
polymorphisms, all five being from the group receiving aflatoxin B 1 after partial
hepatectomy. No TP53 alterations were detected in four samples from the group that
received aflatoxin B 1 alone. One liver tumour contained a silent mutation at codon 247
(CGG to CGT, Arg to Arg).
Aflatoxin B 1 activated with quail liver microsomes transformed BL9 rat epithelial
cells in vitro and these transformed cells induced liver tumours in nude mice. However,
the tumours did not contain mutations in codons 242–244 of the TP53 gene (spanning
the equivalent to codon 249 in human TP53) (Stanley et al., 1999).
Tree shrews (Tupaia belangeri chinensis), which can be infected with human HBV,
were used to study aflatoxin B 1–induced mutations in the presence or absence of viral
infection. Park et al. (2000) studied eight tree shrews, four infected with HBV at 12
weeks of age and four not infected. Two of the uninfected and all four infected animals
were treated with 400 μg/kg bw aflatoxin B 1 per day for six days. Five liver tumours
were detected upon sacrifice at age 2–3 years, four from HBV- and aflatoxin-treated