Maintenance of bivalve hemocytes for the purpose of delayed DNA strand break assessment using the comet assay

Abstract

The lack of appropriate methods for storing intact and viable cells for the purpose of delayed DNA strand break analysis has hitherto limited the application of the Comet assay to in vitro or in vivo laboratory studies and restricted ecologically more relevant field-collected samples to sites in proximity to suitable laboratory facilities. In the present article, osmotically corrected cell culture media Hanks Balanced Salt Solution (HBSS) and Leibovitz Media (L-15) were assessed for their suitability as temporary storage media of blue mussel (Mytilus edulis) hemocytes. It was found that hemocytes maintained in either HBSS or L-15 could be stored for at least 7 days at 4 degrees C without any significant deterioration in cell viability (Trypan blue) or increase in DNA strand breaks, expressed as % tail DNA. This approach allows the acquisition and examination of samples from organisms exposed in situ at previously unsuitable remote sites, thereby greatly increasing the potential ecological relevance of Comet assay-derived genotoxicity data.

Fluctuating estuarine conditions are not confounding factors for the Comet assay assessment of DNA damage in the mussel Mytilus edulis

“…2 a DNA damage, expressed as mean % tail DNA ± SD determined in the haemocytes and gill cells extracted from mussels held for 72 h in various fixed salinities; n = 5, b 72 hour positive control using haemocytes extracted from mussels exposed in vivo to 40 lMol CdCl 2 , mean % tail DNA ± SD; n = 5, asterisk indicates significant difference from control (P \ 0.05) Fig. 3 DNA damage, expressed as mean % tail DNA ± SD determined in the haemocytes and gill cells extracted from mussels held for 72 h in either fixed 100 % (32 %) salinity, or subjected to repeated partial tidal cycles, or emergence and exposure to air; n = 5 well-characterized site in the Firth of Forth (Hartl et al 2010), thus minimizing the inter-individual variability, and assuring the necessary resolution to identify subtle differences between treatment groups. This is particularly important because, with the exception of the positive control (Fig.…”

“…Specimens of the blue mussel Mytilus edulis L. of similar length (5 cm ± 0.5) were collected from the walkway to Cramond Island at the mouth of the River Almond, in the north-west outskirts of Edinburgh, an area showing good water quality after recovering from historical discharges, transported straight to the laboratory and left to acclimatize as previously described by Hartl et al 2010.…”

“…Haemocytes and gill cell suspensions were prepared according to the procedure described by Coughlan et al (2002) for the clam, Tapes semidecussatus, and adapted for mussels by Hartl et al (2010). Cell viability was assessed using the Trypan-Blue Exclusion method of Absolom (1986) and remained above 85 % in gills and 95 % in haemocytes.…”

“…The Comet assay procedure in this study was adapted from Woods et al (1999) with modifications as described for mussels by Hartl et al (2010). Fifty randomly chosen nucleoids per slide were examined using a Zeiss Axiophot epifluorescence microscope equipped with a monochrome CCD IEEE1394 FireWire video Camera (400 9 magnification).…”

“…The Comet assay is finding increasing application as a tool for assessing DNA damage as a biomarker for the genotoxic potential of contaminants, both in the laboratory and the field, and accordingly has been adapted for a variety of fish (Kilemade et al 2004;Hartl et al 2007) and invertebrate species, including bivalves (Coughlan et al 2002;Rank and Jensen 2003;Hartl et al 2004Hartl et al , 2010Richardson et al 2008;Pisanelli et al 2009). An emerging concern is the significance of natural environmental variables that may be interfering with results obtained with the Comet assay (Jha 2008;Baussant et al 2009;Frenzilli et al 2009).…”