Page 31 - Volume 4, Issue 2, July 2011

Emmanouil

et al.

60

digestive glands (< 0.2 g) were abstracted

from the animal and cut in small pieces with

surgical scissors. The pieces were gently dis-

aggregated in HEPES-NaOH buffered saline

(10 mL) and agitated on a rocking platform

(Innova2000, Brunswick Scientific) on ice for

2 h. Every 30 min aliquots of cell suspension

(2 mL) were taken out and replaced with

new HEPES-NaOH buffered saline. All the

aliquots from the same animal were final-

ly pooled (8 mL). After centrifugation a frac-

tion of the pellet was rediluted in saline (100

μL) and kept on ice until further processing.

The preparations provided whole and single

cells with negligible additional damage.

Single Cell Gel Electrophoresis on mus-

sel tissue (Comet assay)

A fraction of the cell suspension (15 μl)

was mixed with 150 μL low melting point

agarose. The mixture was spread on an aga-

rose-precoated slide and lowered in lysis

buffer (NaCl 2.5M, Na

2

EDTA 0.1M, Tris base 10

mM, Triton-X 1% v/v and DMSO 10% v/v, pH

10.0) for 1 h at 4º C in the dark. Two slides per

animal were prepared. The slides were then

rinsed in distilled water (1mL/slide) and left

in electrophoresis buffer (pH 13.0) in a hor-

izontal electrophoresis tank (Cleaver Scien-

tific, Ltd) for 30 min. The slides were subse-

quently subjected to electrophoresis at 25 V

for 20 min, neutralised with Tris buffer (Tris

0.4M, pH 7.5) and stained with propidium io-

dide (2.5 μg/mL) (Research Organics, Cleve-

land, USA). Each slide was analyzed using a

fluorescent microscope (Zeiss AxioCamMRC,

Carl Zeiss Inc., Germany) at 200 x magnifica-

tion, with an excitation filter of 515-560 nm

and a barrier filter of 590 nm and scored us-

ing an image analysis package (TriTekCom-

etScore

TM

). 40 randomly selected nucleoids

were analyzed per slide in two slides so that

a total of 80 cells (per animal) were scored.

Modified Single Cell Gel Electrophoresis

(Comet coupled with formamidopyrimi-

dine glycosylase)

The procedure was as described in 2.6

with the exception that, after lysis and be-

fore unwinding in high pH buffer, the slides

were rinsed 3 times in 1mL of Fpg buffer (KCl

0.1M, HEPES 40 mM, Na

2

EDTA 0.5mM, bovine

serum albumin 0.2 mg/mL, pH 8.0) each.

Four slides per animal were prepared. One

slide per pair was incubated with one unit of

Fpg enzyme (AMS Biotechnology, UK) in Fpg

buffer (50 μL) for 1 h as described by Collins

et al.

(14). The remaining slide of each pair

was incubated with 50 μL Fpg buffer only. 80

randomly selected nucleoids were analyzed

from the non-Fpg incubated slides and 80

randomly selected nucleoids were analyzed

from the Fpg incubated slides. A total of 160

cells were scored. The net difference (Fpg-in-

cubated minus non Fpg-incubated) is pro-

portional to oxidative DNA damage.

Modified Single Cell Gel Electrophoresis

(Halo assay)

The procedure was run as described in

2.6 with the complete omission of the elec-

trophoresis step. A positive apoptosis con-

trol was co-evaluated: staurosporine (Sig-

ma-Aldrich) was diluted in DMSO to a final

concentration of 1 μΜ (26) and was injected

(100 μL) in the adductor muscle directly be-

low the mantle of two individuals. The ani-

mals were returned to a plastic aquarium of

1 L and sacrificed 4 h post-injection. Select-

ed tissues were collected and processed to-

gether with the thiram samples. 50 random-

ly selected nucleoids were checked per slide

in two slides so that a total of 100 cells per

animal were scored. The percentage of char-

acteristic halo images of apoptotic cells (39)

was calculated.

Statistical Analysis

Differences between groups for SSB

were assessed using the parameter %DNA in

tail. Normality of data was tested by the Sha-

piro-Wilk

W

-test. Since data did not follow a

Gaussian distribution median values were

used for each set of cells (16). Data were an-

alyzed by 2-way ANOVA. Means were sep-

arated by Tukey’s HSD test (α=0.05). Differ-

ences between groups for % Fpg sensitive

sites and for % apoptotic cells were assessed

by Student’s

t

-test. Analyses were conduct-

ed using the statistical package JMP.