An integrative assessment to determine the genotoxic hazard of estuarine sediments: combining cell and whole-organism responses

Abstract

The application of the Comet assay in environmental monitoring remains challenging in face of the complexity of environmental stressors, e.g., when dealing with estuarine sediments, that hampers the drawing of cause-effect relationships. Although the in vitro Comet assay may circumvent confounding factors, its application in environmental risk assessment (ERA) still needs validation. As such, the present work aims at integrating genotoxicity and oxidative DNA damage induced by sediment-bound toxicants in HepG2 cells with oxidative stress-related effects observed in three species collected from an impacted estuary. Distinct patterns were observed in cells exposed to crude mixtures of sediment contaminants from the urban/industrial area comparatively to the ones from the rural/riverine area of the estuary, with respect to oxidative DNA damage and oxidative DNA damage. The extracts obtained with the most polar solvent and the crude extracts caused the most significant oxidative DNA damage in HepG2 cells, as measured by the formamidopyrimidine-DNA glycosylase (FPG)-modified Comet assay. This observation suggests that metals and unknown toxicants more hydrophilic than polycyclic aromatic hydrocarbons may be important causative agents, especially in samples from the rural part of the estuary, where oxidative DNA damage was the most significant. Clams, sole, and cuttlefish responded differentially to environmental agents triggering oxidative stress, albeit yielding results accordant with the oxidative DNA damage observed in HepG2 cells. Overall, the integration of in vivo biomarker responses and Comet assay data in HepG2 cells yielded a comparable pattern, indicating that the in vitro FPG-modified Comet assay may be an effective and complementary line-of-evidence in ERA even in particularly challenging, natural, scenarios such as estuarine environments.

The comet assay: past, present, and future

“…The use of plants as well as a wide range of terrestrial and aquatic species in the comet assay has dramatically increased in the last decade (Costa et al, 2014 ; de Lapuente et al, 2015 ; Santos et al, 2015 ), particularly in environmental risk assessment (ERA). A recent validation study has indicated that the in vitro comet assay combined with FPG may be an effective complementary line-of-evidence in ERA even in particularly challenging natural scenarios such as estuarine environments (Costa et al, 2014 ).…”

“…As with all biological assays, data integration is crucial to interpret the comet assay results within the bigger picture. Integration of information provided by the comet assay with other DNA-damage indicators and cellular responses (e.g., oxidative stress, cell division, or cell death) has been applied both in ERA (Costa et al, 2014 ; Santos et al, 2015 ) as well as human (biomonitoring) studies (e.g., Langie et al, 2010 ; Slyskova et al, 2012 ). Also including “omics” data will aid in unraveling the mode of action of genotoxic compounds (Slyskova et al, 2012 , 2014 ; Santos et al, 2015 )—though it is worth pointing out that several studies have shown that phenotypic measures of DNA repair do not necessarily correlate with genomic or transcriptomic data (Collins et al, 2012 ; Slyskova et al, 2012 , 2014 ); the different approaches should be regarded as complementary.…”

DNA damage in different Eisenia andrei coelomocytes sub-populations after in vitro exposure to hydrogen peroxide

“…Comet assay is a simple and reliable technique to assess DNA damage on the basis of altered electrophoretic mobility of DNA, at single cell level, that has received important attention over the last decade and has been shown to be a useful tool also in environmental risk assessment (Langie et al 2015 ; Costa et al 2014 ). Earthworms represent an interesting animal model and a useful candidate organism in soil ecotoxicology studies due to its direct exposure to soil contaminants.…”

Environmental risk assessment in a contaminated estuary: An integrated weight of evidence approach as a decision support tool

“…1). The presence of these potential pollution sources gave rise to a point and diffusively contaminated estuary, particularly in areas near industrial areas and the lower estuary where levels of concern for many contaminants, both organic and inorganic, with adverse toxicological consequences to biota, have been found in recent studies (see Caeiro et al, 2009;Costa et al, 2012Costa et al, , 2014Carreira et al, 2013;Costa et al, 2014).…”