Ž .
mM DTNB, 0.530 U GR and 0.18 mM NADPH. The method of Griffith’s 1980 was applied in determination of GSSG concentration. Final chemical concentrations were 70
mM KH PO 3.5 mM Na EDTA pH 7.5, 0.25 mM DTNB, 0.5 U GR and 0.22 mM
2 4
2
NADPH. Both glutathione analyses were measured at 208C with double-beam spectro- Ž
. photometer Perkin Elmer Lambda 2 UV , and the results were calculated using standard
curves. The catalytic properties of cytochrome P-4501A-dependent monooxygenases were
detected as ethoxyresorufin-O-dependent monooxygenases were detected as EROD Ž
Ž .
activities Burke and Mayer 1974 with final concentration of 1 mM ethoxyresorufin. Ž
. GST was measured by the method of Habig et al. 1974 , where 1 mM 1 chloro
Ž .
dinitrobenzene CDNB and 1 mM GSH were used at pH 6.5. GPX was assayed using cumene hydroperoxide as a substrate with cuvette concentrations of 0.08 mgrml
Ž .
cumene, 0.25 mM GSH, pH 7.6 Floche and Gunzler, 1984 . GR was measured
Ž .
spectrophotometrically according to Carlberg and Mannervik 1985 with final concen- trations of 0.12 mM NADPH, 0.4 mM GSSG, pH 7.6. In all of the above analyses,
protein contents were chosen to give linear changes in enzyme activities. The protein Ž
. contents of the samples were determined by the method of Bradford 1976 using bovine
Ž .
serum albumin Sigma as the standard. All analyses were performed at 188C and the activities expressed as units per milligram protein. All chemicals were purchased from
Sigma except 5-sulfosalicylic acid, which was obtained from Aldrich Chemicals.
2.5. Statistics Ž
Data were analysed using the SPSS for Win 7.5 computer program SPSS, Chicago, .
USA . Homogeneity of variances in variables was tested with Levene test and normality of variables with Shapiro–Wilk test. When homogeneity and normality of variances
prevailed, a parametric one-way analysis of variance test with a priori contrasts was used to test differences between groups. Non-parametric Kruskall–Wallis and Mann–Whit-
ney U-tests with Bonferroni’s correction were used in other cases. The differences were regarded as statistically significant when p - 0.05.
3. Results
As seen in Fig. 2, the whole blood GSSG concentrations increased significantly with ozone dose at both temperatures. After 15 min of ozone exposure, the GSSG concentra-
tions were 1.9 and 2.3 times elevated in the 108C and 148C groups compared to the Ž
values prior to the exposure P - 0.001 and 0.038, respectively, Mann–Whitney
. U-test . After 30-min exposure, GSSG concentrations were 2.4 times higher at both
Ž .
temperature groups P - 0.001, for both groups, Mann–Whitney U-test in comparison to respective control groups. There were no differences between temperature groups
during the exposure. The oxidative stress index in blood increased significantly during ozone exposure at
Ž .
both temperatures Fig. 3 . In the 108C group, the oxidative stress index after 15-min
Ž .
Ž .
Fig. 2. Oxidised glutathione GSSG concentrations mmolrl in whole blood of 1-year-old Arctic charr at 10.38C and 14.18C during ozone exposure. Ozone doses are 0.34 and 0.69 mgrl min after 15 and 30 min,
Ž .
respectively. Data are presented as meansSE ns9–12 ; a, aaa: significantly different compared to Ž
. control group
p- 0.05, p- 0.001, respectively by Mann–Whitney U-test with Bonferroni’s correction.
Ž .
exposure was 1.4 times higher P - 0.031; Contrast and after the 30-min exposure, it Ž
. was 1.7 times higher P - 0.0014; Contrast than the initial values. In the 148C group,
the oxidative stress index value after 30-min exposure was 2.5 times higher than the Ž
. control value P - 0.002; Contrast . The values of oxidative stress index in blood were
at the same level between temperature groups during the exposure. Ozone exposure significantly decreased GSH concentrations in blood after 15 min
Ž .
Ž .
P - 0.003; Contrast and 30 min of exposure P - 0.037; Contrast in the 148C group Ž
. Table 1 . However, no differences were observed in the 108C group caused by ozone
exposure. The whole blood tGSH concentrations tended to increase in the 108C group during ozone exposure but even after 30 min of exposure the difference was not
Ž .
significant P 0.072; Contrast when compared to control group. In the 148C group,
Fig. 3. Oxidative stress index in whole blood of 1-year-old Arctic charr at 10.38C and 14.18C during ozone exposure. Ozone doses are 0.34 and 0.69 mgrl min after 15 and 30 min, respectively. Data are presented as
Ž .
Ž meansSE
ns9–12 ; a, aa: significantly different compared to control group p- 0.05, p- 0.01,
. respectively by one-way analysis of variance; Contrast procedure.
Table 1 Ž
. Ž
. Effects of ozone exposure on whole blood reduced GSH and total tGSH glutathione concentrations in
Arctic charr at two temperatures Data represent mean activitySE; ns 7–12; Contrast procedure.
Control Ozone exposure
15 min 30 min
GSH m mol r l 10.38C
355.629.9 372.029.5
375.729.6
UU
aa a
14.18C 485.417.8
382.124.0 420.022.0
tGSH m mol r l 10.38C
398.028.3 456.429.5
477.926.3
UU
14.18C 526.017.6
475.721.4 518.618.3
U
Significantly different from 108C group, p- 0.05, by one-way analysis of variance.
UU
Significantly different from 108C group, p- 0.01, by one-way analysis of variance.
a
Significantly different from control group, p- 0.05, by one-way analysis of variance.
aa
Significantly different from control group, p- 0.01, by one-way analysis of variance.
blood tGSH concentrations did not change during ozone exposure. In control groups, both GSH and tGSH concentrations, measured from whole blood samples, were
significantly higher in 148C than in 108C control groups. GSH values in 148C group
Table 2 Ž
. Ž
. Ž
. Effects of ozone exposure on reduced GSH , oxidised GSSG and total tGSH glutathione concentrations
and oxidative stress index in Arctic charr liver at two temperatures Data represent mean concentrationsSE; ns 7–12; Contrast procedure for GSSG and by Mann–Whitney
U-test with Bonferroni’s correction for the other variables.
Control Ozone exposure
15 min 30 min
y 1
GSH m mol g wet tissue 10.38C
1.440.11 1.360.09
1.380.09
U
14.18C 1.080.05
1.510.16 1.570.25
y 1
GSSG nmol g wet tissue
a
10.38C 53.06.6
36.45.8 45.74.3
14.18C 52.510.2
50.111.4 36.36.8
y 1
tGSH m mol g wet tissue 10.38C
1.550.10 1.430.09
1.470.09
U
a
14.18C 1.190.04
1.610.15 1.640.24
OxidatiÕe stress index 10.38C
6.81.2 5.10.6
6.21.0 14.18C
8.81.9 6.22.1
4.41.2
U
Ž .
Significantly different from 108C group p- 0.05 by one-way analysis of variance.
a
Ž .
Significantly different from control group p- 0.05 by one-way analysis of variance.
Ž .
were 1.4 times elevated P - 0.004; Contrast and tGSH values were 1.3 times higher Ž
. P - 0.003; Contrast in comparison to 108C.
Initially, hepatic GSH and tGSH concentrations were notably higher in the 108C Ž
. groups than in the 148C groups
P - 0.026 for both; Mann–Whitney U-test . GSH tended to increase during the exposure in the 148C group, but the difference was not
Ž .
significant P 0.084; Mann–Whitney U-test due to the wide variability. At 108C,
GSH levels were uniform during the exposure. The tGSH concentration stayed at the same level in the 108C group during ozone exposures, but showed an increasing trend in
Ž .
148C groups Table 2 . However, only the difference after 15-min exposure was
Ž .
statistically significant P - 0.030; Mann–Whitney U-test when compared to control. As seen in Table 2, hepatic GSSG concentrations were initially at the same level. In
the 108C group, GSSG concentrations tended to decrease during the exposure. However, Ž
. only the decrease after 15-min exposure was significant
P - 0.046; Contrast in
comparison to the control group. No significant changes in hepatic GSSG concentrations at 148C were observed. Similarly, there were no observable changes in the hepatic
oxidative stress index in Arctic charr during ozone exposure. Hepatic GPX concentrations in the 108C groups tended to increase during the ozone
Ž .
Ž exposure Table 3 . After 30-min exposure, GPX concentrations were 1.3-fold
P - .
0.016; Contrast higher than in the control group. The only difference between tempera- ture groups was observed after 15-min exposure. GPX concentrations in the 148C group
Table 3 The activities of glutathione-dependent enzymes; GPX, GR, GST and EROD activity in liver
Data represent mean activitySE; ns 7–12; Contrast procedure. Control
Ozone exposure 15 min
30 min
y 1 y 1
GPX nmol min mg protein
a
10.38C 9.00.7
10.10.6 11.40.6
UU
14.18C 11.31.1
13.71.1 11.20.8
y 1 y 1
GR nmol min mg protein
10.38C 22.41.7
33.85.7 26.31.7
UU
a
14.18C 20.61.3
24.22.1 18.31.2
y 1 y 1
GST nmol min mg protein
10.38C 128.13.9
124.24.1 139.76.1
aa a
14.18C 133.15.8
112.64.4 121.03.9
y 1 y 1
EROD pmol min mg protein
a
10.38C 10.11.9
12.81.9 7.21.5
U
14.18C 10.82.1
7.51.6 11.91.0
a
Ž .
Significant difference between 15 min and 30 min groups p- 0.05 by one-way analysis of variance.
U
Significantly different from 108C group, p- 0.01, by one-way analysis of variance.
UU
Significantly different from 108C group, p- 0.05, by one-way analysis of variance.
a
Significantly different from control group, p- 0.05, by one-way analysis of variance.
aa
Significantly different from control group, p- 0.01, by one-way analysis of variance.
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
