Interrelationship of apoptosis, mutation, and cell proliferation in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced medaka carcinogenesis model
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1Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, 441-744, Suwon, Korea
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.
Insights
This study shows N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) exposure in medaka fish leads to glutathione (GSH) depletion, apoptosis, and mutations. Medaka fish are a suitable model for studying carcinogenesis progression.
Area of Science:
- Environmental toxicology
- Molecular biology
- Carcinogenesis research
Background:
- Carcinogen exposure can induce complex cellular responses including DNA damage, altered cell proliferation, and cell death.
- Understanding the sequential events in carcinogenesis is crucial for developing effective prevention and treatment strategies.
- Medaka fish (Oryzias latipes) are a valuable model organism for aquatic toxicology and cancer research due to their sensitivity and genetic tractability.
Purpose of the Study:
- To investigate the interrelationship between glutathione (GSH) depletion, apoptosis, mutation, and cell proliferation following exposure to N-methyl-N itro-N-nitrosoguanidine (MNNG) in medaka fish.
- To evaluate the medaka fish as a model organism for studying the progression of carcinogenesis.
Main Methods:
- Medaka fish were exposed to MNNG at varying concentrations (0, 0.5, 1 mg l(-1)) for 28 days.
- GSH levels were measured, and apoptotic/necrotic changes were assessed using flow cytometry at 3, 5, and 7 months post-exposure.
- Mutational analysis was performed using arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting.
- Tumor development was assessed through histological examination and immunohistochemistry for proliferating cell nuclear antigen (PCNA).
Main Results:
- MNNG exposure led to decreased GSH levels in a dose- and time-dependent manner.
- Apoptotic changes were observed in a dose- and time-dependent manner at 3 and 5 months, with increased necrosis and carcinogenic alterations at 7 months.
- Time-dependent mutations were detected, but no clear concentration-dependent mutational response was observed.
- Spindle cell sarcomas were identified in some fish, with actively proliferating tumor cells, alongside ongoing apoptosis in non-tumored tissues.
Conclusions:
- The study elucidates the interconnectedness of GSH depletion, apoptosis, mutation, and cell proliferation in the context of MNNG-induced carcinogenesis.
- Medaka fish demonstrate a robust response to MNNG, supporting their utility as a model for investigating the stages of carcinogenesis.
- The findings highlight the dynamic interplay of cellular events during cancer development, from initial damage to tumor formation and cell death.
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