Ochratoxin A induces DNA double-strand breaks and large deletion mutations in the carcinogenic target site of gpt delta rats.
Ochratoxin A (OTA) is a carcinogen targeting proximal tubules at the renal outer medulla (ROM) in rodents. We previously reported that OTA increased mutant frequencies of the red/gam gene (Spi(-)), primarily deletion mutations. In the present study, Spi(-) assays and mutation spectrum analyses in the Spi(-) mutants were performed using additional samples collected in our previous study. Spi(-) assay results were similar to those in our previous study, revealing large (>1kb) deletion mutations in the red/gam gene. To clarify the molecular progression from DNA damage to gene mutations, in vivo comet assays and analysis of DNA damage/repair-related mRNA and/or protein expression was performed using the ROM of gpt delta rats treated with OTA at 70, 210 or 630 µg/kg/day by gavage for 4 weeks. Western blotting and immunohistochemical staining demonstrated that OTA increased γ-H2AX expression specifically at the carcinogenic target site. In view of the results of comet assays, we suspected that OTA was capable of inducing double-strand breaks (DSBs) at the target sites. mRNA and/or protein expression levels of homologous recombination (HR) repair-related genes (Rad51, Rad18 and Brip1), but not nonhomologous end joining-related genes, were increased in response to OTA in a dose-dependent manner. Moreover, dramatic increases in the expression of genes involved in G2/M arrest (Chek1 and Wee1) and S/G2 phase (Ccna2 and Cdk1) were observed, suggesting that DSBs induced by OTA were repaired predominantly by HR repair, possibly due to OTA-specific cell cycle regulation, consequently producing large deletion mutations at the carcinogenic target site.
Division of Pathology.
Ken
K
Kuroda
Daisuke
D
Hibi
Yuji
Y
Ishii
Shinji
S
Takasu
Aki
A
Kijima
Kohei
K
Matsushita
Ken-Ichi
K
Masumura
Maiko
M
Watanabe
Yoshiko
Y
Sugita-Konishi
Hiroki
H
Sakai
Tokuma
T
Yanai
Takehiko
T
Nohmi
Kumiko
K
Ogawa
Takashi
T
Umemura
D000818:Animals; D001483:Base Sequence; D001835:Body Weight / Q000187:drug effects; D002273:Carcinogens / Q000008:administration & dosage / Q000633:toxicity*; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D029968:Escherichia coli Proteins / Q000235:genetics; D007668:Kidney / Q000187:drug effects / Q000473:pathology; D008297:Male; D009152:Mutagenicity Tests / Q000379:methods; D009793:Ochratoxins / Q000008:administration & dosage / Q000633:toxicity*; D009929:Organ Size; D010430:Pentosyltransferases / Q000235:genetics; D051381:Rats; D055647:Rats, Transgenic; D017384:Sequence Deletion / Q000187:drug effects*
D002273:Carcinogens; D029968:Escherichia coli Proteins; D009793:Ochratoxins; C025589:ochratoxin A; D010430:Pentosyltransferases; C071962:Gpt protein, E coli