Development and validation of the in vivo alkaline comet assay for detecting genomic damage in marine flatfish

Abstract

Biomonitoring is an important subject within environmental sciences. Biomonitoring tests are required to be quick, relatively inexpensive, accurate, and reproducible. No genetic test currently fulfils all of these requirements. The chromosome aberration and sister chromatid exchange tests are very time consuming, the DNA adduct technique is rather expensive, and the micronucleus test has not inconclusively proven its use as a reliable monitoring tool. This work is focused on the validation of the comet assay as a candidate for monitoring marine ecosystems. For the comet assay, this work deals with the effectiveness of tissue dissociation, storage of cells in lysing buffer and in liquid nitrogen, different electrophoretic conditions, neutralisation and fixation of slides, interindividual variation between samples, and responsiveness of four tissue types to ethyl methanesulphonate (EMS). The main conclusions are: (i) dissociation of solid tissues in a phosphate buffer supplemented with 200 mM N-t-butyl-alpha-phenylnitrone provides cells with an acceptable background DNA damage; (ii) freezing of cells or tissues in liquid nitrogen generally leads to an increase in DNA breakage, especially for liver, gill and kidney tissue; (iii) storage of slides in the lysing solution for up to one week gives minor changes in comet tails; (iv) differences in protocols for neutralisation and fixation may influence the results; (v) high intra- and interindividual variations in comets (length and DNA content) may obscure the interpretation of comet results; (vi) blood, gill, liver and kidney all showed a statistically significant increase of DNA damage after exposure to 50 mg EMS/l; (vii) electrophoresis at low voltage for longer periods is to be preferred to high voltage and short electrophoresis times. The simplicity and sensitivity of the comet assay make it an adequate test system for biomonitoring of chronic low level exposure. However, protocols and experimental conditions have to be chosen carefully.

Avaliação da mutagênese e genotoxicidade em Eigenmannia virescens (Teleostei: Gymnotiformes) expostos ao benzeno

“…A simplicidade e sensibilidade do Ensaio do Cometa fazem dele um sistema adequado de teste para biomonitoramento a níveis crônicos de exposição, podendo ser utilizado em inúmeras análises onde se podem avaliar células viáveis (Belpaeme et al, 1998). Além das vantagens citadas e do relativo baixo custo, o Ensaio do Cometa difere de outros ensaios que detectam danos no DNA por requerer células viáveis, mas não em divisão, permitindo, assim, sua aplicação a qualquer tipo de tecido dos quais células vivas possam ser obtidas (Collins et al, 1997;Ribeiro et al, 2003).…”

Zinc Phosphide Induced DNA Damage in the Blood Cells of <i>Gallus</i> sp. using Comet DNA Assay

“…Belpaeme et al (1998) stated that the comet assay can also be used to complement studies of chromosome aberrations, micronuclei and sister chromatid exchanges.…”

Morphological and molecular effects of cortisol and ACTH on zebrafish stage I and II follicles

“…Frosted microscope slides (VWR, Carnaxide, Portugal) were covered with 1% normal melting point agarose (NMA) in PBS and dried overnight at room temperature (Belpaeme et al 1998, Lacaze et al 2010. Follicles of stages I and II were collected and mixed in 1% low melting point agarose (LMA).…”