19 Accumulation of Okadaic Acid and Detoxifying Enzymes in the Digestive. . .
219
Enzymatic Analysis
Following sex determination the digestive glands were pooled for each sampling
date and by sex and then homogenized in 20 mM Tris-HCl buffer pH7.6 (1:4,
w:v) containing 0.5 M sucrose, 0.15 M potassium chloride (KCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ˆ
a-mercaptoethanol and 0.1 mM of the
protease inhibitor phenylmethylsulfonyl fluoride (PMSF). The homogenate was
centrifuged at 4
ı C at 500 g for 30 min and the resulting supernatant further
centrifuged at 12,000 g for 45 min. This last supernatant was considered to be
the post-mitochondrial fraction, on which all enzymatic activities were assayed by
spectrophotometric methods at 25
ı C. Assays were performed in triplicate for each
sample.
Cytochrome P450 reductase (CPR) was determined as described by Livingstone
and Farrar (1984) using NADPH and NADH as electron donor and cytochrome c
as substrate. 7-ethoxi-resorufine-O-deetilase (EROD) was assayed by the method
of Burke and Mayer (1974). Xanthine oxidoreductase (XDH) was determinated
following the method described by Lallier and Walsh (1991). DT-diaphorase activity
(DTD) was determined as described by Ernster (1967) and Livingstone et al. (1992).
Glutathione-S-transferase (GST) was determined as the conjugation enzyme by
the method of Habig and Jakoby (1981) Among antioxidant enzymes, superoxide
dismutase (SOD), total and selenium-dependent glutathione peroxidase (GPx-tot;
GPX-Se); glyoxalases I and II (GLO I, GLO II) and Catalase (CAT) were assayed
respectively, by the methods described by Ewing and Janero (1995), Lawrence and
Burk (1976), Principato et al. (1983) and Aebi (1984). Glutathione reductase (GR)
was assayed by the Ramos-Mart´ ınez et al. method (1983), as an enzyme involved in
the redox cycle of glutathione, which yields an adequate concentration of reduced
glutathione for GPX and GLO I activities. Activities were expressed in UI or mUI
per gram of tissue.
Extraction and Analysis of Toxins
Toxin extraction for each sample date was carried out with 0.4 g of lyophilized
mussel (equivalent to 2 g of wet weight). Three extractions were carried out adding
80 % MeOH at a ratio of 1:4 (weight: volume) according to Quilliam 1995. Extracts
were clarified by centrifugation (10,000 g for 10 min at 20
ı C) and transferred into
a volumetric flask through syringe filters of 0.22 m. The extracts were evaporated
in a rotavapor, resuspended freshly in MeOH 80 %, and then filtered through
ultrafree centrifugal filters of 0.45 m.
Aliquots of each extract were hydrolysed at 75
ı C for 40 min. with 2.5 M NaOH,
and the reaction stopped by adding 2.5 M HCl.
The presence of okadaic acid in the samples was determined by HPLC–MS/MS
with a Surveyor MS HPLC system, coupled to a Deca XP plus ion trap mass
219
Enzymatic Analysis
Following sex determination the digestive glands were pooled for each sampling
date and by sex and then homogenized in 20 mM Tris-HCl buffer pH7.6 (1:4,
w:v) containing 0.5 M sucrose, 0.15 M potassium chloride (KCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ˆ
a-mercaptoethanol and 0.1 mM of the
protease inhibitor phenylmethylsulfonyl fluoride (PMSF). The homogenate was
centrifuged at 4
ı C at 500 g for 30 min and the resulting supernatant further
centrifuged at 12,000 g for 45 min. This last supernatant was considered to be
the post-mitochondrial fraction, on which all enzymatic activities were assayed by
spectrophotometric methods at 25
ı C. Assays were performed in triplicate for each
sample.
Cytochrome P450 reductase (CPR) was determined as described by Livingstone
and Farrar (1984) using NADPH and NADH as electron donor and cytochrome c
as substrate. 7-ethoxi-resorufine-O-deetilase (EROD) was assayed by the method
of Burke and Mayer (1974). Xanthine oxidoreductase (XDH) was determinated
following the method described by Lallier and Walsh (1991). DT-diaphorase activity
(DTD) was determined as described by Ernster (1967) and Livingstone et al. (1992).
Glutathione-S-transferase (GST) was determined as the conjugation enzyme by
the method of Habig and Jakoby (1981) Among antioxidant enzymes, superoxide
dismutase (SOD), total and selenium-dependent glutathione peroxidase (GPx-tot;
GPX-Se); glyoxalases I and II (GLO I, GLO II) and Catalase (CAT) were assayed
respectively, by the methods described by Ewing and Janero (1995), Lawrence and
Burk (1976), Principato et al. (1983) and Aebi (1984). Glutathione reductase (GR)
was assayed by the Ramos-Mart´ ınez et al. method (1983), as an enzyme involved in
the redox cycle of glutathione, which yields an adequate concentration of reduced
glutathione for GPX and GLO I activities. Activities were expressed in UI or mUI
per gram of tissue.
Extraction and Analysis of Toxins
Toxin extraction for each sample date was carried out with 0.4 g of lyophilized
mussel (equivalent to 2 g of wet weight). Three extractions were carried out adding
80 % MeOH at a ratio of 1:4 (weight: volume) according to Quilliam 1995. Extracts
were clarified by centrifugation (10,000 g for 10 min at 20
ı C) and transferred into
a volumetric flask through syringe filters of 0.22 m. The extracts were evaporated
in a rotavapor, resuspended freshly in MeOH 80 %, and then filtered through
ultrafree centrifugal filters of 0.45 m.
Aliquots of each extract were hydrolysed at 75
ı C for 40 min. with 2.5 M NaOH,
and the reaction stopped by adding 2.5 M HCl.
The presence of okadaic acid in the samples was determined by HPLC–MS/MS
with a Surveyor MS HPLC system, coupled to a Deca XP plus ion trap mass