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
2.1. Animals Fertilised eggs of Arctic charr of the Lake Inari strain were hatched and cultivated in
Ž
X X
. freshwater at the Laukaa Aquaculture and Fisheries Research Station 62830 N, 25830 E ,
Finland. After reaching an initial weight of 40 g, the fish were transferred into two Ž
. Ž
. constant temperatures: 10.38C 95 CL, 0.38C and 14.18C 0.38C . Fish were
acclimated 2 months prior to the experiment in round fibreglass tanks. Water flow to the tanks was maintained at 2 to 7.7 lrmin and the oxygen level was kept over 6 mgrl,
measured in the tank outlets. The fish were fed on excess with commercial dry food Ž
. Ž
. Tess Nutra G 3.0 mm which was dispensed 8 h daily from 4 AM to 12 PM by
beltfeeders. Constant light rhythm 18L:6D was used during the experiment. On the day of exposure, the fish were transferred in water to the ozone exposure tank
using the same ambient temperature where they were acclimated. The Arctic charrs Ž
. averaged 67.7 g in weight S.E. 11.8 g during the exposure.
2.2. Exposure system Ozone dose was selected to be high enough to inactivate Aeromonas sp., which,
Ž .
according to Wedemeyer and Nelson 1977 , is 0.1 mg O rl min, and according to
3
Ž .
Liltved et al. 1995 is 0.2 mg O rl min, but not to cause lethal damage to Arctic charr
3
with 30-min exposure. The Arctic charr were exposed to ozone in a recirculation system. The fish were kept for 15 or 30 min in the 50-l exposure tank covered with floating
plastic. Excess water from the exposure tank was led to the collection tank and pumped Ž
. up to the counter current contact column 1800 mm high, 12 l , where ozone gas was
Ž .
Ž .
mixed with water Fig. 1 . Two ozone generators Red Sea Fish pHarming, Israel produced adequate residual ozone concentration in the system. The residual ozone
concentration was measured in the exposure tank three times per exposure by the Ž
. indigo-trisulphonate method International Ozone Association with a Hach DR 2000
Ž .
colorimeter Hach, Loveland, CO . Due to the instability of the ozone at low concentra- tions, the reactions were allowed to stabilise for 30 min prior to the exposures. When the
Ž .
equilibrium was achieved 22.8 mg O rl, SD 2.6 mg O rl at both temperatures, 9 to
3 3
Fig. 1. Schematic illustration of water flow in the ozone exposure system used in the present study. Qs8.40.2 lrmin.
12 randomly selected fish were transferred to the exposure tank at the same time. Three to four fish were sampled before the exposure and three to four fish were taken after 15-
and 30-min ozone exposure. The calculated ozone dose was 0.34 mgrl min after 15 min and 0.69 mgrl min after 30-min exposure. The procedure was repeated three times in
both temperatures.
2.3. Sampling and sample preparation Fish were stunned by a blow to the head and weighed. Blood samples were collected
with Na-heparinized syringes from the caudal vein. Blood was acidified with 5 5 Ž
. Ž
. Ž
. sulfosalicylic acid 1:2 for total glutathione tGSH and oxidised glutathione GSSG
analysis. For hepatic glutathione and biotransformation enzyme analysis, the liver was removed and weighed. All the samples were frozen immediately in liquid nitrogen until
analysis.
Ž Blood samples were thawed and centrifuged at 28C at 10,000 = g for 5 min Sorvall
. Instruments RC5C . GSSG and tGSH were analysed immediately from supernatants.
Ž .
Ž .
Total glutathione tGSH is expressed as a sum of oxidised GSSG and reduced Ž
. glutathione GSH in GSH equivalents. Oxidative stress index describes the ratio of
Ž .
GSSG to tGSH in Eq. 1 Boehme et al., 1992 . An increased value is considered as a sign of oxidative stress.
Oxidative stress index s 2 = GSSG rtGSH = 100
1
Ž .
Ž .
Ž .
where GSSG s oxidised glutathione and tGSH s total glutathione. The values of GSSG are multiplied by two since 2 nmol of GSH are necessary to form 1 nmol of GSSG.
For measurements of GSSG and tGSH, the liver samples were homogenized in Ž
. ice-cold 5 5 sulfosalicylic acid 1:4 and 1:5, respectively with a Potter-Elvehjem-type
Ž homogenizer and centrifuged at 10,000 = g for 5 min at 28C Sorvall Instruments
. RC5C . Supernatants were stored at y808C until analysed.
Frozen liver samples for microsomal and cytosolic tissue fractions were weighed and Ž
. homogenised in 0.25 M sucrose 1:4 with Potter-Elvehjem-type homogenizer. The
Ž .
homogenates were centrifuged Sorvall Instruments RC5C for 20 min at 10,000 = g. Ž
Supernatant fractions were collected and centrifuged for 60 min at 105,000 = g Kontron .
TGA-65 . Microsomes were resuspended in 0.25 M sucrose containing 60 mM Tris, 5 Ž
. mM EDTA and 20 glycerol. Microsomes for 7-ethoxyresorufin EROD activity
determination and supernatant fractions for GST, GPX and GR analysis were prepared at 48C and stored at 808C until analysis.
2.4. Biochemical analysis Ž
. Total glutathione concentration was measured according the method of Tietze 1969
Ž .
X
modified by Adams et al. 1983 , where the rate of reduction of 5,5 -dithiobis-2-nitro- Ž
. Ž
. Ž
. benzoic acid DTNB is correlated to the sum of reduced GSH and oxidised GSSG
glutathione. Chemicals used were 85 mM KH PO 4.25 mM Na EDTA pH 7.5, 0.9
2 4
2
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
