Experiment with benzo[a]pyrene
Rainbow trout juveniles (n=24) with an average weight and length of 70 g and 190 mm, originating from Vänneåns Laxodling, Halmstad, Sweden, were used for the exposure experiments with BaP (Sigma Aldrich, St Louis). The experimental design consisted in two treatments, one received BaP and the other receiving only the vehicle (control), and two exposure times of 48 hours and 7 days. Therefore, the group 48h received a single intraperitoneal injection of BaP (2 mg/kg, dissolved in peanut oil), while the group denominated 7d received 3 injections with 2-days intervals. The control groups received the same injections, which contained only the peanut oil. The animals were kept in a water-recirculation system with constant aeration (12 ºC) under a natural night and daylight regime (12h/12h) in glass tanks without being fed. The chosen concentration of BaP was based on the study developed by Padrós et al. (2003), with adaptations.
At the end of the experiment, the animals were euthanized with a blow to the head and had their weight and length recorded. Blood was collected from the caudal vein and the liver excised. The liver was shock-frozen and stored on liquid nitrogen until preparation for analysis. All procedures described in this study were conducted according to Swedish ethics guidelines, in accordance with the Ethics permit 15986-2018.
EROD assay
Livers were homogenized in a cold homogenizing buffer solution (0.1 M Na/P - phosphate buffer containing 0.15 M KCl, pH 7.4). The homogenate was centrifuged at 10,000 g during 20 min at 4 °C. The supernatant was centrifuged again for 100,000 g for 60 min at 4 °C, in order to prepare the microsomal fraction. Subsequently, the supernatant (cytosol) was stored at -80 °C until use. The pellet (microsomes) was re-suspended in the homogenizing buffer solution containing 20% glycerol and stored at -80 °C.
The EROD activity was measured in the microsomal fraction of the liver, according to the method described by Förlin et al. (1986) using rhodamine as standard. The reaction mixture contained sodium phosphate buffer (0.1 M, pH 8.0), ethoxyresorufin (0.5 mM), and 25 to 50 ml of sample in a final volume of 2 ml. The reaction began with the addition of 10 ml of NADPH (10 mM). The increase in fluorescence was monitored at 530 nm (excitation) and 585 nm (emission). EROD activity was expressed as picomoles of resorufin formed per minute and per milligram of protein (pmol/min/mg protein). For this purpose, the protein content was quantified according to Lowry et al. (1951) using bovine serum albumin (BSA) as standard.
Quantification of melanin
In regard to the histological analysis, liver fragments were fixed in Metacarn (60% methanol, 30% chloroform, 10% acetic acid) for 3 hours, dehydrated in alcoholic series, embedded in paraffin and stained with Hematoxylin-Eosin. 25 random pictures were taken from each animal under a light microscope, with the aid of an image capture system. Then, the quantification of the pigmented area was carried out by the difference in color intensity observed in MMs as suggested by Santos et al. (2014), using the software Image Pro-Plus (version 6.0).
Cytoskeleton analysis
Liver samples were frozen at -20 °C prior to analysis, and after thawing, fragments of the organ were placed in 2 mL of an EDTA solution (2 mM) for up to 24-48 h at 4 °C, in order to dissociate liver cells and separate MMs. This step allows cells to be dissociated by mechanical action of continuous suctioning of its contents. Subsequently, the content was centrifuged at 1,500 g for 10 min at 4 °C for pellet formation. Pellets containing MMs were dissolved in KCl (0.56% w / v) for 20 min and mixed during 10s to suspend the pellet.
Regarding microtubules immunostaining process, the cells were fixed in ice methanol for 6 min and then washed three times during 5 min with PBS (phosphate buffered saline), before being incubated with the primary antibody Rabbit polyclonal to beta tubulin - Loading control (Abcam) for 60 min at room temperature. The cells were then washed three times during 5 min with PBS and incubated in the darkness with the secondary antibody Goat polyclonal to rabbit IgG - H&L (Alexa Fluor® 488) (Abcam) for 45 min. Finally, cells were again washed three times during 5 min with PBS and underwent a final rinse in miliQ water, in the attempt to avoid the formation of salt crystals before they were allowed to dry (Hedberg and Wallin 2010).
The analysis of actin filaments was performed by initially rinsing cells in PBS, followed by fixation in a 3.7% formaldehyde solution for 10 min, then washing it twice with PBS, and permeabilized with 0.1% Triton X-100 for 5 min. The slides were then washed again twice for 5 min in PBS, and blocked with 1% BSA for 20 min. The cells were then stained with Rhodamine-Phalloidin (Thermo-Fisher) for 20 min, washed three times for 5 min in PBS, rinsed in miliQ water and dried (Hedberg and Wallin 2010).
A total of 50 cells per animal were counted under a Nikon Eclipse E100 fluorescence microscope, using the ACT-1 (version 2.0) software. The quantification was made by the measurement of the fluorescent cell area, using the software Image Pro-Plus (version 6.0).
Nuclear abnormalities analysis
Upon euthanasia, blood was collected from the caudal vein with a heparinized syringe and needle, dripped onto a slide and blood smears were made. After drying, slides were fixed in methanol for 20 min and stained with Giemsa 7.5%, for 30 min. The following nuclear abnormalities in erythrocytes were analyzed: anucleate, binucleated, bud and MN. For each animal, 1000 erythrocytes were counted, as suggested by Pérez-Iglesias et al. (2014).
Statistical analysis
The experimental design consisted of two treatments (control and one BaP concentration) and two exposure times (48 hours and 7 days), which characterizes a 2 x 2 factorial design. Six animals were used in each treatment for the quantification of EROD activity, cytoskeleton, MMs and erythrocytes abnormalities.
A Kruskal-Wallis test was applied in the EROD assay, in order to determine whether significant differences among groups existed, seen that the data did not present a normal distribution.
A Linear Mixed-Effects Model (package lme4; Bates et al. 2015) (Zuur et al. 2009) with restricted maximum likelihood (REML; Bolker et al. 2009) considering treatment and time of exposure as fixed factors along with their interaction, was applied to model microtubules, actin filaments and MMs area, considered as continuous responses variables. Sampling units (cells for microtubules and actin filaments; pictures for MMs area), in which we estimated the response variables, were nested within each animal (true replicate). To control the dependency among 50 cells or 25 pictures from the same animal (Crawley 2012: 703), we included a random intercept for animal (categorical with 6 levels; Moen et al. 2016). Then, in order to test the model’s assumptions, we used diagnostic plots with the R package sjPlot (Lüdecke 2016) and to assess for differences between treatment and exposure time we used least-squares means with R package lsmeans (Lenth 2016). Microtubule analysis data were log-transformed, aiming to attend the assumptions of normality and homogeneity. Then, the statistical models were summarized and, as suggested by Kenward and Roger (1997), P values were estimated based on conditional F-tests, with the approximations of degrees of freedom being made with the aid of the sjPlot.
A Generalized Linear Model (GLM) was used to model erythrocytes abnormalities. A binomial distribution and log link function included treatment and exposure time, along with their interactions. To test model assumptions we used diagnostic plots from the R (Team Core 2016) sjPlot package (Lüdecke 2016). All analysis were performed using software R v. 3.3.2 (R Core Team 2016).