in liver were elevated at 148C indicating increased ability of liver tissue to defend against reactive Ž
. Ž
. oxygen species ROS . Glutathione S-transferase GST , GSSG and cytochrome P-450 catalytic
Ž .
activity ethoxyresorufin-O-deethylase, EROD in liver were not influenced by ozone exposure. Ž
. Initially, both hepatic glutathione reductase GR and GSH were elevated at 108C, possibly
indicating ability to compensate for temperature. This study demonstrates that even the lowest Ž
. dose 0.34 mgrl min of ozone was high enough to cause oxidative stress in the blood of Arctic
charr and the signs of oxidative stress occur throughout the exposure. Furthermore, a blood sample is a reliable source of information when assessing the status of the glutathione-dependent
antioxidant system after a short-term ozone exposure. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Ozone; Oxidative stress; Glutathione; EROD; Temperature; Arctic charr
1. Introduction
Ž .
Ž .
Ozone O has been used for decades to disinfect drinking water Rice, 1985 and to
3
Ž .
treat sewage effluents Brink et al., 1991 . The ozonation techniques shown effective in handling large volumes of water in municipal water works are now being applied also in
aquaculture. Ozonation is proven useful in promoting the removal of solid matter Ž
. Ž
Rueter and Johnson, 1995 , stabilisation of water quality in recirculating systems Reid .
and Arnold, 1994; Summerfelt et al., 1997 and is advantageous in disease control Ž
. Liltved et al., 1995 . This indicates that ozonation could find a place in the intensive
culture of Arctic charr, which is an endangered species, but also a potential candidate for Ž
. commercial aquaculture
Jobling et al., 1993 . However, in spite of the apparent advantages of ozone in aquaculture, its physiological effects on the fish are still largely
Ž unknown since unexpected deaths have occurred even at low O concentrations Bullock
3
. et al., 1997 .
Decomposition of ozone in water can take place directly and involves reactions of Ž
molecular ozone in water with unsaturated aromatic and aliphatic compounds Bablon et .
al., 1991 . These types of reactions are predictable and can be controlled. On the other hand, indirect decomposition of ozone is rapid, unpredictable and produces harmful
Ž .
intermediates, the so-called reactive oxygen species ROS such as the superoxide Ž
P y
. Ž
. Ž
P
. radical O
, hydrogen peroxide H O , hydroperoxyl radical HO and ozonide
2 2
2 2
Ž
P y
. radical O
. These intermediates might be detrimental to the fish, leading to a state
3
called oxidative stress. In oxidative stress, pro-oxidant forces overwhelm the antioxidant defence system of
Ž animals. This has been detected after certain xenobiotics or ozone exposure Mustafa,
. Ž
. 1990 . According to Mustafa
1990 , ozone-induced ROS are capable of causing oxidative stress in mammals, which is reflected in peroxidation of lipids, loss of
functional groups and enzyme activities and alteration in membrane permeability. Therefore, it is probable that similar processes could occur in fish as well. Aquatic
organisms are protected against ROS by antioxidant enzymes and low molecular weight
Ž .
scavengers Winston and Di Giulio, 1991; Peters and Livingstone, 1996 . The most important scavengers are glutathione, vitamins E and C and total carotenoids, which
Ž .
have been detected in at least 15 fish species Lemaire and Livingstone, 1993 .
Ž .
Cellular glutathione
L
-g-glutamyl-
L
-cysteinylglycine is a tripeptide that plays a crucial role in oxidative stress. It is produced intracellularly and is involved in free
radical scavenging, detoxification of electrophiles, maintenance of thiol-disulfide status Ž
and signal transduction Meister and Anderson, 1983; Deneke and Fanburg, 1989; Droge
¨
. et al., 1994 . If potentially toxic H O
is present in a tissue, GSH is oxidised by
2 2
Ž .
Ž .
glutathione peroxidase GPX, EC 1.11.1.9 to glutathione disulfide GSSG . GSSG is Ž
. reduced back to GSH by glutathione reductase GR, EC 1.6.4.2 . GSH is also able to
Ž .
protect cells directly by scavenging radicals Meister and Anderson, 1983 . If toxic xenobiotics were not conjugated to GSH, they could combine covalently, e.g., with
DNA, RNA or cell proteins and thus cause serious cell damage. Several studies have shown that glutathione homeostasis can be used as a biomarker in a variety of fish
Ž species being exposed to different xenobiotics Otto and Moon, 1996b; Hasspieler et al.,
. 1994 . However, little or nothing is known of the piscine glutathione defence system in
response to ozone exposure. Ž
. In xenobiotic exposure e.g., polychlorinated biphenyls, PCBs cellular GSH has a
Ž .
notable influence on metabolism by controlling the cytochrome P-450 CYP1A path- Ž
. way in liver Otto et al., 1996a . Cytochrome P-450-dependent metabolism is a two step
Ž .
Ž process where xenobiotics are oxidised phase I reactions and further conjugated phase
. II reactions in order to facilitate their excretion or neutralise their bioreactivity. The first
Ž .
phase reactions are catalysed by cytochrome P-450 CYP -dependent enzymes, the monooxygenases. The catalytic activity of one of the main isoenzymes, CYP1A1, can be
Ž .
measured enzymatically by the ethoxyresorufin-O-deethylase EROD reaction. This activity has been widely found in aquatic organisms and used as a biomarker in
Ž .
environmental biomonitoring studies Lindstrom-Seppa et al., 1992; Tuvikene, 1995 .
¨ ¨
Although, after the following second phase reactions xenobiotics are transformed to be Ž
. more excretable by the action of, e.g., glutathione S-transferase GST, EC 2.5.1.18 ,
activation of more reactive metabolic products can occur. Production of electrophilic metabolites of the xenobiotic, free radicals or ROS can result in cytotoxic damage
Ž .
Lemaire and Livingstone, 1993 . During ozone exposure, both the glutathione-depen- dent defence system and the P-450 pathway might be recruited to resist the challenge by
ROS. The current study was carried out to evaluate the effects of an ozone dose on the
glutathione-dependent defence system and catalytic activity of cytochrome P-4501A in Arctic charr. Ozone exposures were carried out at two temperatures in order to evaluate
if there was a thermal influence on glutathione metabolism. Samples were taken from the liver to determine its responses to ROS. Blood was also sampled to see if it could
serve as a potential tissue for analysing the state of oxidative stress without necessitating the sacrifice of the fish.
2. Materials and methods
