Page 37 - Volume 4, Issue 2, July 2011

Emmanouil

et al.

66

the low dose group.

Thiram, in the present experiments, has

caused a significant increase in oxidative

DNA damage as measured through Fpg in-

cubation, which quantifies mainly 8-oxo-dG

but also other damaged purines according

to Collins

et al.

(14). This increase was most

prominent in gill cells rather than in haemo-

cytes and in digestive gland cells. The meta-

bolic system of bivalves, even though mark-

edly different from the mammalian ones is

capable of producing oxidizing intermedi-

ates (28) which in their turn affect the cell’s

metabolism and components (46). For ex-

ample, in a similar way to the present re-

sults, the common aquatic pollutant BaP

which necessitates metabolic activation

to redox quinones to exert its toxic action,

caused formation of 8-oxo-dG in

Mytilus gal-

loprovincialis

digestive gland (2, 29).

An oxidative mode of action is not un-

usual for thiram as it has been shown in oth-

er animal models. Any imbalance between

prooxidant substances and antioxidant de-

fenses in favor of the prooxidants causes ox-

idative stress associated with damage to cel-

lular macromolecules (40). Thiram has acted

as a potent oxidative agent in the liver of

broilers fed with this fungicide. The activi-

ties of the antioxidant enzymes superoxide

dismutase (SOD) and glutathione peroxi-

dase (GSH-Px) were decreased and this low-

ering of antioxidant defenses has led to lip-

id peroxidation in the liver of broilers (27).

Thiram has also caused deregulation in ac-

tivities of key antioxidant enzymes (cata-

lase, GSH-Px, SOD, glutathione reductase)

in an

in vitro

model of V79 Chinese hamster

ovary cells (22). These results may be part-

ly explained by glutathione (GSH) depletion

caused by thiram (13, 23). GSH offers one of

the most efficient non-enzymatic protective

mechanisms by its conjugation with elec-

trophilic and/or oxidised components. Thi-

ram however possesses a reactive disulfide

bond which may react with thiols of critical

cellular proteins such as GSH forming mixed

disulfides and other products (23), thus ren-

dering them inactive. Furthermore, the GSH

redox cycle offers the reducing equivalents

for thiram reduction in the cell, minimiz-

ing in this way the regeneration of reduced

GSH. In more detail, it is postulated that di-

thiocarbamates undergo oxidation by Cu

2+

ions within the cell to their corresponding

thiuram disulfides. These intermediates are

then reduced by GSH, regenerating the par-

ent compound and oxidized glutathione

(11). GSH aberrant metabolism due to thi-

ram was further corroborated in studies in

V79 CHC which showed a decrease in total

GSH/oxidised GSH ratio (22). Therefore, the

pro-oxidant effects of thiram are considered

to be indirect and mainly due to the lower-

ing of antioxidant defenses.

Finally, a series of important experiments

in aquatic organisms showed that thiram ox-

idative effects are not peculiar to terrestrial

organisms. Namely, incubation of

Oncho-

rhynchus mykiss

liver with thiram led to loss

or decrease of activity of SOD and GSH-Px

respectively (6). Exposure of the mussel

Unio

tumidus

to thiram has also caused decrease

of activity of selenium-dependent glutathi-

one peroxidase and glutathione reductase

as well as decrease in reduced and oxidised

GSH in both gills and digestive gland (15).

Regarding testing for apoptosis, apop-

totic cells give a characteristic image of large

fan-like tail and small head (ghost cells) in

the conventional comet assay. However

due to their extensive fragmentation, they

may become lost during the electropho-

resis step (32). In contrast, omission of the

electrophoresis step but retention of alka-

line unwinding in the alkaline halo assay de-

picts successfully the unique morphology of

apoptotic cells which present a difuse, spot-

ted halo and a pin-like head clearly delin-

eated from the halo (39). The assay has here

revealed a significant increase in apoptotic

cells in gills and digestive glands but no in-

crease in haemolymph when mussels were

exposed to 1.00 mg/L thiram (approximate-

ly 4 μM) for 48 h. In related bibliography, tri-

closan (3 nM) caused more extensive dam-

age (approximately 16%) after 48 h exposure

to

Dreissena polymorpha

haemolymph (45).

This difference cannot be readily explained,

however it may be linked to the apoptotic