IARC MONOGRAPHS ON THE EVALUATION OF CARCINOGENIC ...

SOME TRADITIONAL HERBAL MEDICINES 105
tasis in the pancreas, an activated c-N-ras proto-oncogene was detected. All mutations
were A → T transversions at either the second or the third base of codon 61.
In mice, a mixture of aristolochic acids induced squamous-cell carcinoma in the
forestomach and adenocarcinoma in the lung. In the tumours analysed, one forestomach
squamous-cell carcinoma and 1/3 lung adenocarcinomas contained activated c-Ha-ras
proto-oncogenes both mutated by A → T transversions at the second base of codon 61.
(c) In-vitro studies (see Tables 6–8 for details of studies and references)
After metabolic activation, aristolochic acid I and aristolochic acid II form adducts
in vitro with calf thymus DNA, MCF-7 DNA, plasmids, polydeoxyribonucleotides,
oligodeoxyribonucleotides, deoxyribonucleotide-3′-monophosphates (purines), deoxyadenosine
and deoxyguanosine. In-vitro systems capable of activating aristolochic
acids I and II to reactive species that may form adducts are S9 mix from Aroclor 1254or
β-naphthoflavone-pretreated rats, xanthine oxidase, peroxidases (horseradish peroxidase,
lactoperoxidase, prostaglandin H synthase), zinc at pH 5.8 and microsomal preparations
from various species other than the rat. Aristolochic acid-specific adducts were
formed in calf thymus DNA after activation of aristolochic acids I and II with hepatic
microsomes from humans, mini-pigs and rats, as well as with microsomes containing
recombinant human CYP1A1 and CYP1A2. From studies with specific inducers and
selective inhibitors, it can be concluded that most of the microsomal activation of aristolochic
acids is due to CYP1A1 and CYP1A2.
Activated aristolochic acids I and II react with DNA to form three and two major
adducts, respectively. These major adducts co-chromatograph with 7-(deoxyadenosin-
N 6 -yl)aristolactam I (dA-AAI), 7-(deoxyguanosin-N 2 -yl)aristolactam I (dG-AAI), 7-
(deoxyadenosin-N 6 -yl)aristolactam II (dA-AAII) and 7-(deoxyguanosin-N 2 -yl)aristolactam
II (dG-AAII) (see Figure 6), indicating that aristolochic acid reacts preferentially
with the exocyclic amino group of purine bases. On the basis of the adduct structures, it
can be concluded that reduction of the nitro group is the main metabolic pathway for the
activation of aristolochic acid.
The major metabolites, the aristolactams, form DNA adducts in vitro after activation
by hepatic microsomes or horseradish peroxidase. Adducts with calf thymus DNA are
also formed by aristolochic acids I and II in vitro in the presence of rat faecal bacteria.
In explants of rat stomach tissue, both acids formed adducts in the DNA of the epithelial
layer. DNA adducts have been detected in MCF-7 cells after exposure to aristolochic
acid I and in opossum kidney cells after exposure to an aristolochic acid mixture.
After reaction of aristolochic acids with DNA, DNA synthesis by T7 DNA polymerase
and human DNA polymerase α is mainly blocked at the nucleotide 3′ to the
aristolochic acid-induced DNA adducts. This property has allowed the use of polymerase
arrest assays that revealed binding of aristolochic acids I and II in vitro to the c-Ha-ras
gene and the TP53 gene.
Aristolochic acids I and II and the aristolochic acid mixture induced SOS repair and
mutations in bacteria. In nitroreductase-deficient strains of Salmonella typhimurium,