Sample preparation and antioxidant analyses

6.2.3.1 Antioxidant enzymes analyses

For the analysis of the antioxidant enzymes, each subsample pool were homogenized (1:10 w:v) in 100 mM of K-phosphate buffer (pH 7.5) containing NaCl (1.5%), 0.1 mg mL-1 phenylmethylsulphonyl fluoride (PMSF), 0.1 mg mL-1 bacitracin and 0.008 TIU mL-1 aprotinin as protease inhibitors (Aprotinine and Bacitracine). After centrifuging at 100,000 x g for 70 min at 4 °C, supernatants were collected and kept at -80 °C until required. All enzymatic measurements were carried out using a Varian (Model Cary 3) spectrophotometer at the constant temperature of 18° C.

Catalase (CAT) is a heme-containing protein that has a detoxifying effect towards the hydrogen peroxide (H2O2), catalysing the reaction which leads to the formation of water and oxygen:

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CAT was measured by the decrease in absorbance at 240 nm (ε = 0.04 mM−1 _cm−1_{) due} to H2O2 consumption (12 mM H2O2) in 100 mM K-phosphate buffer (pH 7.0).

Glutathione S-transferases (GST) are metabolic isoenzymes involved in detoxification of xenobiotic and endobiotic compounds. This antioxidant enzyme system detoxifies chemical compounds containing an electrophilic group, using the reduced glutathione (GSH) as cofactor:

GSH + CDNB → GS-DNBconjugate + HCl

GST were determined at 340 nm using 1-chloro-2,4 dinitrobenzene (CDNB) as substrate (ε = 9.6 mM−1 _cm−1_{). The assay was carried out in 100 mM potassium} phosphate buffer (pH 6.5), 1.5 mM CDNB, and 1.5 mM GSH.

Glutathione reductase (GR), also known as glutathione-disulphide reductase (GSR), catalyses the reduction of glutathione disulphide (GSSG) to the sulfhydryl form glutathione (GSH) which is a critical molecule in resisting oxidative stress and maintaining the reducing environment of the cell. GR use the NADPH as cofactor to make the reaction occurs:

GSSG + NADPH → 2GSH + NADP+

GR activity was measured by following the oxidation of NADPH at 340 nm during the reduction of GSSG (extinction coefficient, ε = 6.22 mM−1

cm−1). The assay conditions were 100 mM potassium phosphate buffer (pH 7.0), 1 mM GSSG and 60 μM NADPH.

Glutathione peroxidase (GPx) is an antioxidant system that is composed of glutathione dependent enzymes. GPx catalyse the reduction of organic and inorganic peroxides to the corresponding alcohol, using GSH as cofactor. Peroxides reduction takes place via glutathione transformation from its reduced form to the oxidized one:

2GSH + ROOH → GSSG + ROH + H2O organic peroxides 2GSH + H2O2 → GSSG + 2H2O inorganic peroxides The formed GSSG is reconverted to GSH by GR with NADPH consumption:

GSSG + NADPH + H+ _{→ 2GSH + NADP}+ GR

The activity of both Se-dependent and Se-independent GPx forms were measured in a coupled enzymatic assay, where GSSG is converted to the reduced form GSH. The consumption of NADPH was measured as decrease of absorbance at λ = 340 nm (ε = 6.22 mM−1 cm−1) in 100 mM K-phosphate buffer pH 7.5, 1 mM EDTA, 1 mM

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dithiothreitol (DTT), 2 mM GSH, 1 unit glutathione reductase, 0.24 mM NADPH, and 0.8 mM cumene hydroperoxide as substrate.

In order to obtain the specific antioxidant activities, data were normalized with the relative protein concentration according to Lowry method (1951) with bovine serum albumin used as standard.

6.2.3.2 Total oxyradical scavenging capacity (TOSC) assay

Polychaetes were homogenized on ice (1:10 w:v) using a glass plotter in 100 mM K- phosphate buffer (pH 7.5) containing NaCl (1.5%), PMSF (0.1 mg mL-1) and protease inhibitors as aprotinin (0.008 TIU mL-1_{), leupeptin (1 µg mL}-1_{), pepstatin (0.5 µg mL}-1_). After centrifuging at 100,000 x g for 70 min at 4 °C, cytosolic fractions were collected and maintained at -80 °C until they were used for the analysis.

The analysis of the total oxyradical scavenging capacity (TOSC) is a reliable tool for quantitatively assessing the biological resistance to toxicity of different forms of ROS, including peroxyl radicals, hydroxyl radicals and peroxynitrite decomposition products (Winston et al. 1998, Regoli and Winston 1999). The assay is based on the capability of cellular antioxidants to reduce the oxidation of α-keto-γ-methiolbutyric acid (KMBA) in presence of artificially generated oxyradicals. KMBA is oxidized to ethylene gas, and the time-course of its formation is monitored during the whole assay duration by gas-chromatographic analyses (measured at 10-12 min time intervals). The antioxidant efficiency of a sample is quantified by its ability to scavenge the produced oxyradicals thus inhibiting their reaction with KMBA and ethylene formation (Winston et al. 1998) (Fig. 6.1). TOSC assay was performed resorting to different forms of artificially oxidants generated at constant rate: peroxyl radicals (ROO•), hydroxyl radicals (HO•) and peroxynitrite (HOONO) (Fig. 6.1). Peroxyl radicals were obtained by the thermal decomposition of 2,2’-azo-bis-(2-methylpropionamide)-dihydrochloride (ABAP) according to Winston and Cederbaum (1983); hydroxyl radicals were generated thanks to the iron-ascorbate Fenton reaction; peroxynitrite were generated from 3-morpholinosydnonimine (SIN-1) following the procedure described by Lomonosova et al. (1998). Final assay conditions were: (i) 0.2 mM KMBA, 20 mM ABAP in 100 mM potassium phosphate buffer (pH 7.4) for peroxyl radicals; (ii) 1.8

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mM Fe3+, 3.6 mM EDTA, 0.2 mM KMBA, 180 mM ascorbic acid in 100 mM potassium phosphate buffer (pH 7.4) for hydroxyl radicals; and (iii) 0.2 mM KMBA and 80 mM SIN-1 in 100 mM potassium phosphate buffer (pH 7.4) with 0.1 mM diethylenetriaminepentaacetic acid (DTPA) for peroxynitrite (Regoli 2000). Reactions were conducted at 35 °C in 10 ml vials sealed with gas-tight Mininert valves (Supelco, Bellefonte, PA) in a final volume of 1 ml (Fig. 6.1). Aliquots of 200 ml of ethylene gas formed were taken from the head space of reaction vessels with a gas-tight syringe at 10–12 min intervals during the time course of the reaction. Then, they were injected into the gas chromatography instrument equipped with a Supelco SPB-1 capillary column (30 m × 0.32 mm × 0.25 µm) and a flame ionization detector (FID) (Regoli 2000). The oven, injection and FID temperatures were maintained constant at respectively, + 35, + 160 and + 220 °C; hydrogen flux of 30 mL min-1 _{and Helium flux} of 3 mL min-1. Each sample, according to this procedure, was analysed every 15 min, for 105 min.

Figure 6.1 Ethylene formation inside the vials of control reaction and sample reaction (Gorbi and Regoli 2012).

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The TOSC value was calculated using the formula:

TOSC = 100 – (∫SA / ∫CA x 100)

Where ∫SA and ∫CA are the integrated areas calculated under the kinetic curve produced during the reaction course for sample (SA) and control (CA) reactions, respectively (Winston et al. 1998). The experimental TOSC value falls between 0 and 100. In case of the absence of oxyradical scavenging capacity, ethylene formation is not reduced as compared to control (∫SA / ∫CA = 1) resulting in a TOSC value equal to zero. Alternatively, the total inhibition of ethylene formation throughout the assay (SA = 0) would corresponds to a theoretical maximum TOSC value of 100. In order to obtain the specific TOSC value, data were normalized with the relative protein concentration determined according to Lowry et al. (1951) method using bovine serum albumin as a standard.