Interrelationship of apoptosis, mutation, and cell proliferation in N-methyl-N '-nitro-N-nitrosoguanidine (MNNG)-induced medaka carcinogenesis model

Abstract

The present study examined the interrelationship of GSH depletion, apoptosis, mutation. and cell proliferation following carcinogen exposure. Medaka (Oryzias latipes) were investigated following a 28 day, three times/week pulse exposure to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG). Fish (5 weeks old) were exposed to MNNG at concentrations of 0, 0.5, and 1 mg l(-1) and reared for 3, 5 and 7 more months after the last day of exposure. GSH levels were decreased in the higher concentration groups and longer-reared groups. Flow cytometric analysis revealed that fish from the groups reared 3 and 5 months showed active apoptotic changes in the dose- and time-dependent manner, but the group reared 7 months had fewer apoptotic, rather showed more necrotic and carcinogenic alterations. Mutational responses were detected by an arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting method using whole body DNA samples as templates and pBR primer. A mutational change was expressed by a loss or gain of a band. There was a time-dependent mutational change. but no distinctive concentration-dependent one. A band from normal fish sample that disappeared after treatment of MNNG was excised and sequenced. The band had an 869 base pair-long sequence, however, there was no putative protein-coding region based on an analysis by DNAsis. Spindle cell sarcomas invading muscle were detected on the whole body sections from three of ten fish examined, and immunohistochemical analysis with PCNA showed that tumor cells were actively proliferating. However, terminal deoxynucleotidyl transferase (TdT) assay showed that tumored fish still had active apoptotic cell changes in the tissues without tumor. This study shows not only the interrelationship of GSH depletion, apoptosis, mutation and cell proliferation, but also indicates that medaka is appropriate as a fish model for research on the passage of carcinogenesis, (C) 2000 Elsevier Science B.V. All rights reserved.