Ž .
were 1.4-fold higher P - 0.008; Contrast compared to the 108C group. GR revealed slightly higher activities in liver at 108C than 148C during the ozone exposure but the
Ž .
difference was significant only after 30-min exposure P - 0.002; Contrast . At the
Ž same time GR concentrations in the 148C group were significantly lower P - 0.015;
. Contrast in comparison to the 15-min exposure group at the same temperature.
The hepatic GST concentration in the 148C group was significantly lower after Ž
. 15-min ozone exposure in comparison to the control group P - 0.008; Contrast or the
Ž .
30-min exposure group P - 0.034; Contrast . However, the GST concentrations re-
Ž .
mained unchanged at 108C during the exposure Table 3 . Ozone exposure did not evoke any changes in hepatic EROD activities. After 30-min exposure, the EROD activity in
the 108C group was lower in comparison to the 148C group and the 15-min exposure group, but not in the control group.
4. Discussion
Ž .
In this study, as little as 15-min ozone exposure O dose: 0.34 mgrl min caused
3
symptoms of oxidative stress in Arctic charr. Oxidative stress was detected in whole blood samples as increased oxidised glutathione concentrations as well as in elevated
levels of oxidative stress index. Moreover, the basal levels of GSH did vary between liver and whole blood at different temperatures. This finding suggests the importance of
temperature in GSH biosynthesis and emphasises the differences between blood and liver tissues in the glutathione-dependent defence system.
Blood and liver were chosen to represent target organs since they play a significant role in glutathione homeostasis. Liver is known to serve as a storage site for GSH in fish
Ž .
Yokoyama and Nakazoe, 1991 , and in mammals, it can protect tissues from damage by Ž
. ROS by secreting GSH into the bloodstream Sen et al., 1992 . Furthermore, fish
erythrocytes are capable of producing GSH-dependent antioxidant enzymes and thus can Ž
take part in the glutathione-dependent antioxidant defence system Fukunaga et al., .
1992b . In this study, ozone exposure caused oxidative stress to Arctic charr after 15-min
exposure at both temperatures. This was detected by markedly elevated whole blood GSSG concentration and the oxidative stress index. The high GSSG levels probably
arose from the oxidation of blood GSH into GSSG, which can be considered as a sign of oxidative stress evoked by ROS. At the same time, oxidative stress index was elevated
presumably because the rate of GSSG formation exceeded the capacity of the cell to regenerate GSH from GSSG. The signs of oxidative stress in blood might indicate that
ozone exposure was so extensive that the first line of defence, the skin and gill epithelium cells, failed to provide complete protection to Arctic charr against ROS.
Elevated levels of GSSG and the oxidative stress index in whole blood, also after 30 min, were indicative of continuous presence of ROS in circulation during ozone
exposure. Further, GSSG in blood showed a partial ability to compensate for tempera- ture before the exposure as there were only slight differences in the concentrations.
Results of the current study verifies that Arctic charr were able to protect their tissues against ozone toxicity and ozone reaction by-products via activation of glutathione-de-
Ž pendent defence system. Since detrimental damage e.g., DNA-fragmentation, lipid
. peroxidation, protein oxidation caused by ozone exposure was not analysed in the
present study, it was not possible to assess the harmful dose of ozone, i.e., the point at which oxyradical generation poses a serious threat to the health of Arctic charr.
However, in oxidative stress, the struggle at the cellular level against pro-oxidant forces has already started and the maintenance of the defence system and the repair of possible
Ž .
damage requires energy Bell and Cowey, 1990 , which must be withdrawn from the growth budget.
The literature reveals that ozone is highly toxic to teleost fish though there is Ž
considerable variation between different species and life stages Wedemeyer et al., 1979; .
Asbury and Coler, 1980; Paller and Heidinger, 1980; Fukunaga et al, 1992a . Nothing is Ž
. known about ozone toxicity in Arctic charr. According to Wedemeyer et al. 1979 ,
Ž .
rainbow trout Oncorhynchus mykiss are vulnerable to ozone toxicity, since the LC50
96
value for juvenile rainbow trout was 9.3 mg O rl, whereas the lethal threshold level was
3
Ž .
slightly lower 8 mg O rl; calculated O dose: 46 mgrl min . On the other hand,
3 3
Ž .
Wedemeyer et al. 1979 have verified lethal histopathological changes in gill tissue Ž
. after ozone exposure of 29 mg O rl for 24 h calculated O dose: 41.8 mgrl min .
3 3
Consequently, the toxicity of ozone in fish is a product of the concentration and Ž
. exposure time cumulative O
dose , which should be used in establishing dose–re-
3
sponse-type relationships. At least in rainbow trout, it seems that there is a conservative Ž
. margin between the lethal dose of ozone 41.8 mg O rl min compared to that needed
3
Ž .
to kill pathogens 0.2 mg O rl min and, therefore, O can be used in disease control. It
3 3
is apparent, however, that the practical therapeutic safety margin is narrower than in the Ž
. laboratory tests and should be elucidated on site Wedemeyer, 1996 . Hence, another
concern related to toxicity testing might be the wide variability among individuals. In the present study, inter-individual variation was extensive, which could not be explained
by prevailing conditions, culture history or measurement accuracy. One explanation could be genetic susceptibility to O , which has been observed in mammalian studies as
3
Ž .
reviewed by Kleeberger 1995 . Ozone exposure caused a rapid depletion of GSH in whole blood in the 148C group
but not in the 108C group. The fact that the GSH concentration was unaltered and GSSG was enhanced in the 108C group caused by ozone exposure suggest that the decrease of
GSH at 148C with a concomitant increase in GSSG concentration, was due to enhanced GSH utilisation rather than impaired GSH synthesis. The reason for the markedly higher
initial GSH concentrations in blood at 148C in comparison to 108C remain unresolved in this study but one explanation might be linked to higher metabolic activity, since the
Ž optimum growth temperature for Arctic charr is between 13.78C and 15.18C Lyytikainen
¨
. et al., 1997 . The finding that initial GSH concentrations in blood and in liver were not
equal in different temperature groups but tended to equalise during ozone exposure, raises the question of how temperature can influence the glutathione-dependent antioxi-
dant defence system in Arctic charr. The changes in hepatic GSH in the 148C group, low initial levels vs. elevated levels during ozone exposure, are most probably related to
temperature-dependent differences in basal concentrations of GSH and the role of liver as a backup GSH generator. In particular, high levels of GSH in tissues correspond to an
Ž .
increased ability of a tissue to defend itself against oxyradicals Hasspieler et al., 1994 .
Hepatic GSH and tGSH concentrations stayed at the same level in the 108C group during ozone exposure revealing no signs of oxidative stress in liver. However, the
initial GSH and tGSH concentrations in liver were markedly lower at 148C group but increased to the same level as seen in the 108C group after 15-min ozone exposure. At
that point, the increased GSH generation in the 148C group was verified by elevated total GPX activity in comparison to the 108C group. According to our results, it is
probable that even 15-min ozone exposure was sufficient to alert liver cells to the environmental changes provoked by ozone. Most likely the information was transmitted
Ž .
in the circulation by H O or organic hydroperoxides ROOH and detected in liver as
2 2
increased GPX activity. Total GPX is known to detoxify H O and ROOH produced in
2 2
Ž .
lipid peroxidation in fish Peters and Livingstone, 1996 . Since hepatic GSSG was not increased at 108C or 148C concomitantly with GPX activity in ozone exposure and the
level of GR was unaltered, it can be assumed that the capacity of glutathione synthesis was not overloaded.
The prevailing temperature might have an essential role on glutathione-dependent enzymes GPX and GR, since they performed different responses in the control groups. It
is worth noting that in the current study the highest GR enzyme activities in liver did not Ž
. Ž
occur at the optimum temperature for growth 13.7–15.18C of Arctic charr Lyytikainen
¨
. Ž
. and Jobling, 1999; Lyytikainen et al., 1997 . According to Snegaroff and Bach 1990 ,
¨
the highest EROD activities were observed in rainbow trout at 118C whereas the Ž
. optimum growth rate can be achieved at 178C Jobling, 1983 . Temperature compensa-
tion in Arctic charr should be assessed in future to better understand xenobiotic processes in cold water species.
The effect of ozonation on hepatic GST activity was not consistent. If GST reactions were activated in ozone exposure, elevated levels of GST should be indicative of the
activation of GST-dependent xenobiotic metabolism. The results of our study reveal that GST was not recruited in liver during 30-min ozone exposure. It is possible that the
exposure time used in the present study was not long enough to elevate hepatic GST. On the other hand, GSH-dependent GST might not be needed to any great extent in the
xenobiotic metabolism of ozone.
In the current study, the level of hepatic EROD activity was the same in both temperature groups and it did not show any elevation in response to ozonation.
Presumably, protein synthesis in liver will require longer than 30 min since no increase in EROD activity could be detected. However, the basal levels of hepatic EROD activity
found in the present study were consistent with the results obtained by Jørgensen et al. Ž
. 1999 . They showed that fasting does not have any effect on hepatic EROD activities in
Arctic charr, but on the other hand, xenobiotics are able to cause considerable induction in CYPA1 activity.
5. Conclusions
