SYMPOSIA PAPER Published: 01 January 1996

Quantification of Dna Adducts in Small Fish Exposed to Alkylating Agents


It is widely believed that most chemical carcinogens act by binding to cellular genetic material and causing somatic mutations. Chemical modification of DNA (e.g., the formation of DNA adducts) is the first in a series of stages that lead to mutation, cell transformation, and tumor development.

Sensitive methods for detection of DNA adducts are essential for mechanistic studies of mutagenesis and carcinogenesis and for biomonitoring of populations at risk for environmentally caused cancer. DNA adducts may be detected with such methods as 32Ppostlabeling, immunoassays, HPLC with fluorescence detection, and gas chromatography/mass spectrometry, with varying degrees of sensitivity and structural specificity. Alkylation at the N-7 position of guanine is a preferential site of attack for most alkylating agents, but may be of little biological consequence, while adduct formation at the O6 position of guanine (less common) is most highly correlated with carcinogenesis.

We have examined the suitability of western mosquitofish (Gambusia affinis) as a native sentinel species for monitoring environmental exposures to mutagens and carcinogens. Mosquitofish are a native species to the U.S. and are also widely distributed in warm waters throughout the world. These fish are ideal for monitoring local exposures because they are non-migratory and are found in a wide variety of habitats. In the laboratory, they are easily cultured using techniques developed for other small fish species and appear resistant to infectious diseases. Thus, methods may be directly assessed under controlled conditions before being applied to field situations.

In these studies, mosquitofish were exposed to a model alkylating agent, methylazoxymethanol acetate, at 10 ppm in the ambient water for 2 h. Fish were then transferred to clean water and held for up to 72 h. Livers were excised, pooled for 5 fish, and DNA was extracted and prepared for analysis. Liver DNA extracts were assessed for levels of N-7 and O6-methylguanine by isotope dilution GC/mass spectrometry, using deuterated analogs of methylguanine adducts as standards. Detection limits for the assay were approximately 1.6 femtomoles of adduct per mg DNA (about 1 adduct per 2 million bases). Approximately 55–185 pg (330–1100 femtomoles) O6 -methylguanine per mg DNA were measured in exposed fish in the first 72 h after exposure. These lesions were correlated with a 33% liver tumor incidence after 25 weeks in parallel experiments.

Author Information

Law, JM
North Carolina State University, College of Veterinary Medicine, Raleigh, NC
McMillin, DJ
Louisiana State University, School of Veterinary Medicine, Baton Rouge, LA
Swenson, DH
Louisiana State University, School of Veterinary Medicine, Baton Rouge, LA
Means, JC
Louisiana State University, School of Veterinary Medicine, Baton Rouge, LA
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Developed by Committee: E47
Pages: 117–137
DOI: 10.1520/STP11703S
ISBN-EB: 978-0-8031-5345-5
ISBN-13: 978-0-8031-2031-0