Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The following techniques include the previous sacrifice of the animal, which was carried out in accordance with international ethical standards^46,47,48, and the subsequent dissection and ablation of the organs. The animals were captured under the authorization of the Ministry of Environment, Agriculture and Production of San Luis Province (Resolution 49-PMA2019). The methods of sacrifice and euthanasia of the animals were duly approved by the protocols of the Institutional Animal Care and Use Committee (CICUA, protocol Q-322/19) from the National University of San Luis. The procedures with anuran organisms were carried out according to guidelines detailed in Garber et al.⁴⁶, CONICET⁴⁷, and INTA⁴⁸. In addition, all the protocols presented here are for neotropical anuran species in their larval and adult life stages; they have already been widely accepted by local researchers and are carried out under a strict protocol and with authorization from the "Comité Institucional de Cuidado y Uso de Animales (CICUA)" of each university involved. A list of the materials and solutions used is presented in the Table of Materials and Table 1.

1. Individual level: Body condition and hepatic and gonadal indexes

1. Body condition index: Scaled mass index
   NOTE: This index can be used both with adults and with juveniles and larvae. This methodology is based on Peig and Green²⁷, with minor modifications for anurans according to Brodeur et al.^28,30and MacCracken and Stebbings²⁹.
   1. Record the body mass of each specimen using a precision analytical scale.
   2. Place each specimen on a millimeter sheet, and take a photograph at a distance of ~15 cm.
      NOTE: It is important to always take photographs from the same distance.
   3. Photograph analysis:
      NOTE: The photographs can be analyzed with the ImageJ program, which is freely available online (https://imagej.nih.gov/ij/index.html), or with any other image analysis program that allows measurements to be made from a scale included in the photograph.
      1. Measure the snout-vent length (SVL) of each specimen using the measure tool from the ImageJ program, and first refer to a known measure in the millimeter sheet.
         NOTE: In tadpoles, the measurement of the body length must be made up to the cloacal tube, disregarding the caudal fin.
   4. Scaled mass index (S)
      1. With the data obtained from the body mass and SVL of each anuran, construct a non-linear power function regression line of body mass (y-axis) against SVL (x-axis).
      2. Calculate the mean length with the SVL data of all the individuals measured from the studied population.
      3. Finally, calculate the scaled mass index (S) according to Peig and Green²⁷ and Brodeur et al.²⁸ using equation (1):
         S = M_i(L₀/L_i)^b    (1)
         where M_i and L_i are body mass and SVL from the individual i, respectively; b is the scaling exponent estimated by a non-linear power function regression²⁸; L₀ is the mean length for the studied population; and S is the predicted body mass for the individual i when its SVL is standardized to the L₀ value.
         ​NOTE: It is important to note that b is a species-specific constant that could also be sex-specific in sexually dimorphic species.
2. Hepatosomatic index: Scaled liver index
   NOTE: As for the body condition index, this index can be used with individuals at all stages of development. The methodology is based on Brodeur et al.²⁸.
   1. Record the SVL of each specimen according to step 1.1.2 and step 1.1.3.
   2. Euthanize individuals according to ethical standards for anuran amphibians^46,47,48.
   3. Remove the entire liver, and record its mass on a precision analytical scale to the nearest milligram.
   4. With the data obtained from the liver mass and SVL of each anuran, construct a non-linear power function regression line of liver mass (y-axis) against SVL (x-axis).
   5. Calculate the mean length with the SVL data of all the individuals measured from the studied population.
   6. Calculate the scaled liver index (SLI) according to Brodeur et al.²⁸ using equation (2):
      SLI = Lm_i (L₀/L_i)^b    (2)
      where Lm_i and L_i are the liver mass and SVL from the individual i, respectively; b is the scaling exponent estimated by a non-linear power function regression²⁸; L₀ is the mean length for the studied population; and SLI is the predicted liver mass for the individual i when its SVL is standardized to the L₀.
3. Gonadal index: Scaled gonadal index
   NOTE: This methodology is based on Brodeur et al.³⁰ and is useful only with adult individuals. It is important to note that male and female indices must be analyzed separately as they vary on distinct scales.
   1. Record the SVL of each specimen according to step 1.1.2 and step 1.1.3.
   2. Euthanize individuals according to ethical standards for anuran amphibians^46,47,48.
   3. Remove the right and left gonads, and record the mass on a precision analytical scale to the nearest milligram.
   4. With the data obtained from the gonad mass and SVL of each anuran, construct a non-linear power function regression line of gonad mass (y-axis) against SVL (x-axis), similar to step 1.1.4.1 and step 1.2.3.
   5. Calculate the mean length with the SVL data of all the individuals measured from the studied population.
   6. Estimate the scaled gonadal index (SGI) according to Brodeur et al.³⁰ for each animal using equation (3):
      SGI = Gm_i(L₀/L_i)^b    (3)
      where Gm_i and L_i are the gonad mass and SVL from the individual i, respectively, b is the scaling exponent estimated by a non-linear power function regression by Brodeur et al.³⁰, L₀ is the mean length for the studied population, and SGI is the predicted gonad mass for an individual i when its SVL is standardized to the L₀.

2. Morphological-histological level

NOTE: For this analysis, it is necessary to use histological sections. The first step is to collect the tissue.

1. Fixing and dehydration
   1. Use a fixative solution to preserve the structures. For anuran tissues, preferably use methacarn or Bouin solution, recommended over the rest of the fixing solutions (Table 1).
      NOTE: All substances must be mixed at the time of use.
   2. Place tadpoles or a tissue fragment of 50-100 mg in a conical tube with 1-2 mL of fixative solution at 4 °C for 3 h. Wash the tissue in the same volume of 70% ethyl alcohol for 30 min.
   3. Place the tissue in a fresh 70% ethyl alcohol solution.
      NOTE: It is possible to stop the procedure at this point for some days before continuing with the dehydration.
   4. Dehydrate the tissue in a series of alcohol solutions: ethyl alcohol 80% (10 min), alcohol 90% (10 min), ethyl alcohol 100% (10 min), and ethyl alcohol 100% (10 min).
   5. Perform diaphanization (clearing) 2 x 10 min in xylene.
   6. Melt the paraffin in a beaker. Soak the tissue for 10 min. Remove the tissue, and place in a histological mold with melted paraffin. Wait for it to solidify at room temperature.
   7. Place 3 µm sections made on a rotating microtome on a glass slide.
2. Histometric analysis
   1. Stain the tissue sections in hematoxylin-eosin³¹.
   2. Use 10-20 photomicrographs for measurement with a 100x objective in a light microscope. Analyze 5-10 hepatic cells in each photomicrograph.
      NOTE: Here, liver was used as a model to describe the technique. It is possible to use other tissues; ensure that the objective of the light microscope to be used is suitable for the type of tissue. It is necessary to be able to see and identify the cells to be measured.
3. Histopathology
   1. After staining the tissue sections in hematoxylin-eosin³², examine 5-10 histological sections under 10x, 20x, 40x, and 100x objectives.
   2. Look for tissue changes and estimate the degree of tissue changes (DTC) index as described by Bernet et al.³² using equation (4):
      DTC =Σalt.(a × w)    (4)
      where Σalt is the sum of alteration; a represents the damage stage (0, absent; 1, low; 2, moderate; 3, high); and w represents the degree of damage reversibility (1, reversible; 2, partially reversible; 3, irreversible).
      ​NOTE: Here, liver was used as a model to describe the technique. It is possible to use other tissues; ensure that the objective of the light microscope to be used is suitable for the type of tissue.
4. Pigmentary system
   1. After staining the slides in hematoxylin-eosin, examine 10-20 photomicrographs with a 20x objective.
   2. Quantify the area occupied by melanin by measuring differences in the staining intensity using Image Pro-Plus software according to Santos et al.³³.
   3. Open the software Image Pro Plus 6.0®.
   4. Select Menu and Toolbar: Biological.
   5. Select the Spatial calibration of magnification used to take the photomicrographs.
   6. Open a histological image.
   7. Select Tool count | Measure objects.
   8. Click on the Select colors button.
   9. Select the dropper tool, and mark the coloring to be measured in the image.
   10. Close the window, and click on Count.
   11. Select View | Statistics to see the results.

3. Biochemical level: Reactive oxygen species (ROS) and cholinergic enzymes

1. Sample homogenization
   NOTE: Once the experiment is over, preserve the samples (tissues or tadpoles) in phosphate-buffered saline (PBS) if they are not going to be processed at that time by freezing at −20 °C (for 2 months) or −80 °C (for 6 months). Alternatively, they can be homogenized at the time according to the recommendations proposed below.
   1. Tadpoles
      1. Weigh 1 g of a tadpole or pool of tadpoles on a precision analytical balance.
      2. Place the 1 g of tadpoles in a 15 mL conical tube containing 1 mL of PBS, and immerse it in an ice bath (4 °C).
      3. Homogenize with a teflon-tipped homogenizer adapted to a conical tube, and work in cold conditions (4 °C).
         CAUTION: Avoid using a high number of revolutions, as this may destroy proteins and raise the temperature.
      4. Transfer the same volume of the liquid homogenate of each sample into two labeled 2 mL microcentrifuge tubes, and work in cold conditions (4 °C).
      5. Centrifuge the samples with the liquid homogenate for 10 min at 4 °C and 9,520 × g.
      6. Finally, extract the supernatant, place it in microcentrifuge tubes, label it in aliquots of 0.5 mL to 1 mL, and preserve in a freezer at −80 °C (for 6 months) or −20 °C (for 2 months) until the enzyme assay.
   2. Adults
      1. Remove the tissue of interest (e.g., liver, muscle, kidney), and weigh 1 g on a precision analytical balance.
      2. Place the tissue in a 50 mL conical tube containing 1 mL of PBS, and immerse it in an ice bath (4 °C).
      3. Homogenize with a teflon-tipped homogenizer adapted to a conical tube, and work cold (4 °C).
         CAUTION: Avoid using a high rpm, as this may destroy proteins and raise the temperature.
      4. Transfer the same volume of the liquid homogenate of each sample into two labeled 2 mL microcentrifuge tubes, and work cold (4 °C).
      5. Centrifuge the tubes with the liquid homogenate for 10 min at 4 °C and 9,520 × g.
      6. Finally, extract the supernatant, place it in microcentrifuge tubes, label in aliquots of 0.5 mL to 1 mL, and preserve in a freezer at −80 °C (for 6 months) or −20 °C (for 2 months) until the enzyme assay.
2. Protein determination
   NOTE: The protein value is obtained to estimate the enzymatic activity in relation to the total amount of protein while avoiding the underestimation or overestimation of the enzymatic activity values. This methodology is based on Bradford³⁴ with minor modifications for anurans:
   1. Prepare Bradford reagent (see Table 1).
   2. Add 100 mL of H₃PO₄ at 85% (p/v) to Coomassie G-250 + ethanol, and bring to a volume of 1 L with distilled water.
   3. Prepare the calibration curve with bovine serum albumin (BSA) as a known protein standard (Table 1).
   4. Prepare albumin standards in increasing concentrations for a 3 mL spectrophotometer cuvette. For an example of the standard curve, see Table 2.
   5. Measure the absorbance using a spectrophotometer at 590 nm (this is the wavelength at which the complex of the reagent and the protein is formed).
      NOTE: Performing the Bradford reaction with the sample for reading in the spectrophotometer using a glass cuvette with a maximum volume of 3 mL and an optical path of 1 cm helps the protein determination.
   6. Place the following reactants in a cuvette in this order: 2,000 µL of the Bradford reagent + X µL of the sample (X = 20 µL of the homogenate sample of the organ of interest from an adult anuran, or 40 µL of the homogenate sample from a tadpole).
   7. Finally, measure the absorbance in the spectrophotometer at 590 nm.
      NOTE: It is not necessary to work at 4 °C.
3. Catalase
   NOTE: This methodology is based on Aebi³⁵ with minor modifications for anurans.
   1. Incubate 1,900 µL of PBS + 40 µL of hydrogen peroxide (H₂0₂) + 20 µL of the sample (pure in tadpoles and diluted 1/25 for adult tissue) in a 3 mL quartz cuvette with a 1 cm path length.
      CAUTION: Follow the order given above for the addition of the reagents and the sample during the incubation. The reactants can be incubated at room temperature (25 °C) since anurans are physiologically ectothermic.
   2. Read the catalase kinetics at 240 nm for 2 min in a UV spectrophotometer.
   3. Calculate the enzyme activity with the following equation (5)³⁵:
      k (mmol∙min^-1∙mg^-1 Prt) = (Δ absorbance/1,000)/protein concentration    (5)
      ​NOTE: In studies with purified enzyme preparations, the specific activity k'0 is obtained by dividing k by the molar extinction coefficient, (e = 43,6); k' = (k/e).
4. Glutathione S-transferase (GST)
   NOTE: This methodology is based on Habig et al.³⁶ with minor modifications for anurans.
   1. Incubate 300 µL of GST + 10 µL of 1-choro-2, 4-dinitrobenzeno (CDNB) (0.1 M) + 10 µL of the sample (pure in tadpoles and diluted 1/25 for adult tissue) in the cuvette.
      CAUTION: Respect the order of addition of the reagents and the sample given above during the incubation. The reagents can be incubated at room temperature (25 °C) since anurans are physiologically ectothermic.
   2. Prepare the GST (Table 1).
   3. Read the GST kinetics at 340 nm for 2 min in the spectrophotometer.
   4. Calculate the enzyme activity with the following equation (6):
      Activity rate of GST (mmol/per minute/mg protein) = ([Δ absorbance/1,000]/9.6 × 10⁴)/protein concentration    (6)
      ​Where 9.6 mM⁻¹ cm⁻¹ = molar extinction coefficient.
5. Lipid peroxidation (TBARS)
   NOTE: This methodology is based on Buege and Aust³⁷ with minor modifications for anurans.
   1. Construct a calibration curve with MDA as the standard and 0.7% TBA (Table 3).
   2. Prepare the standard MDA solution (10 mM) (Table 1). Carry out THP hydrolysis to MDA by placing the solution in a 50 °C water bath for 1 h.
   3. Prepare a 0.7% (w/v) solution of TBA (Table 1) using a magnetic hot plate stirrer.
   4. Then, centrifuge the sample homogenates for 30 min at 9,520 × g and 4 °C.
   5. Extract 500 µL of the supernatant, add 100 µL of 6 M NaOH, and place in a thermal bath at 60 °C for 30 min.
   6. After the thermal bath, add 250 µL of 35% HClO₄, and then place in an ice bath (4 °C) for 30 min.
   7. After that time, centrifuge at 9,520 × g for 12 min, and extract 300 µL of the supernatant.
   8. Add 300 µL of 0.7% TBA to the supernatant (sample), and place in a thermal bath (97.5 °C) for 30 min.
   9. Read the absorbance of the complex formed by the sample and TBA at 532 nm, and determine the MDA concentration in the samples by substituting the absorbance values in the equation of the curve (Figure 1).
6. Acetylcholinesterase (AChE)
   NOTE: This methodology is based on that proposed by Ellman et al.³⁸ with minor modifications for anurans.
   1. At the moment of measurement, set up the DTNB and acetylthiocholine iodide (ATCh) reagents.
   2. Prepare the DTNB reaction (Table 1).
   3. Prepare the AChE reaction (Table 1).
   4. In a glass cell with an optical path of 1 cm, prepare the AChE reaction by placing these reagents in the following order: 150 µL of PBS buffer (pH = 8) + 150 µL of DTNB + 50 µL of ATCh + 10 µL of the sample (pure in tadpoles and diluted 1/10 in adult anurans).
   5. Read the AChE kinetics at 412 nm for 2 min at room temperature.
   6. Obtain the value of AChE activity using the following equation (7):
      Activity rate of AChE (mmol∙min^-1∙mg^-1 protein) = ([Δ absorbance/1,000]/1.36)/protein concentration    (7)

4. Genetic and cellular level: Micronucleus and comet assay

1. Micronucleus (MN) assay
   NOTE: The methodology is in agreement with that proposed by Fenech³⁹ with slight modifications for neotropical anurans.
   1. Anesthetize the specimens by immersion in ice water (4 °C), and obtain blood samples by sectioning behind the operculum in tadpoles or by cardiac puncture in adults.
   2. Smear two drops of peripheral blood from the specimens onto precleaned slides.
   3. Afterward, air-dry the slides, fix with 100% (v/v) cold methanol (4 °C) for 20 min, and then stain with 5% Giemsa solution for 12 min.
   4. Have one researcher code and blind-code the slides at 1,000x magnification.
   5. Determine the frequency of MNs by analyzing 1,000 mature erythrocytes from each specimen, and express as the total number of MNs per 1,000 cells.
   6. Use the following criteria for the correct identification of MNs:
      1. Look for MNs that have diameters smaller than 1/3 of that of the main nucleus.
      2. Ensure that the coloration of the MN is not refractable but has a staining intensity that is the same or lighter than that of the main nucleus.
      3. Look for an MN boundary that is distinguishable from the main nucleus boundary without any connection to the core or overlapping with the main nucleus.
      4. Ensure that the number of MNs in the cells does not exceed more than four MNs associated with the nucleus.
2. Comet assay or single-cell gel electrophoresis (SCGE)
   1. Anesthetize specimens by immersion in ice water, and obtain blood samples by sectioning behind the operculum in tadpoles or by cardiac puncture in adults.
   2. Dilute the blood with 1 mL of PBS, centrifuge (381 × g, 9 min), and resuspend in a final volume of 50 mL of PBS.
   3. Mix 30 µL of the blood sample in 70 µL of low-melting point agarose (LMPA) at a 0.5% concentration of agarose to form layer 2. Subsequently, place 50 µL of layer 2 on a slide previously coated with layer 1 containing 100 µL of 0.5% normal-melting point agarose.
   4. Cover the slide with a coverslip, and place in the cold (4 °C) for 10 min to solidify layer 2.
   5. After solidification, remove the coverslip, and form the third layer by laying down 100 µL of 0.5% LMPA.
   6. When the third layer solidifies, remove the coverslip, and immerse each slide in a 100 mL Coplin jar containing lysis solution (Table 1) that has been prepared on the same day and kept at 4 °C. Place the Coplin jars in a cold bath for 1 h in the dark (4 °C) for lysis to occur.
      NOTE: The experiment can be paused and restarted later. The lysis period has been reported to last from 1 h to 1 month.
   7. At the end of the lysis, remove the slides, and immerse in the electrophoresis buffer solution (Table 1) in the electrophoresis tank. Perform this step in the dark for 15 min at 4 °C to allow the DNA to unwind. After nuclear DNA unwinding, perform electrophoresis on the same buffer at a temperature of 4 °C for 10 min at 25 V and 250 mA.
   8. At the end of the electrophoresis, neutralize the samples contained in the slides 3 x 5 min by adding 2 mL of Tris-HCl solution (Table 1).
   9. In the last step of the comet assay technique, dehydrate the samples by placing the samples in 100 mL Coplin jars containing alcohol (95%) at 4 °C.
   10. Finally, stain the samples by placing 10 µL of 4′,6-diamino-2-phenylindole (DAPI) in the center of the slide, and place a coverslip that spreads the dye throughout the samples. Examine the slides under a fluorescence photomicroscope with a WB filter.
       CAUTION: The whole process must be carried out in the dark to avoid DNA photodegradation.
   11. Quantify the DNA damage by evaluating the length of DNA migration from the nucleoid. In this case, visually determine it on 100 randomly selected cells without overlap. Classify the DNA damage into four classes: 0-I (no damage), II (minimum damage), III (medium damage), and IV (maximum damage). Express the data as the mean number of damaged cells (sum of classes II-IV) and the mean comet score for each treatment group. From these data, calculate the genetic damage index (GDI) for each test organism using the following equation (8):
       GDI = (1[I] + 2[II] + 3[III] + 4[IV])/N[I-IV])     (8)
       where I-IV represent the type of nucleoid damage, and NI-NIV represent the total number of nucleoids scored according to Pitarque et al.⁴⁰.

5. Correlated biomarkers

NOTE: In recent times, biomarkers can be integrated at all levels by using the biomarker response (IBR) index proposed by Beliaeff and Burgeot⁴⁹ and adapted for neotropical anurans. The IBR provides a numeric value that integrates all biomarker responses; higher IBR values indicate higher stress levels⁴⁹. In terms of the IBR estimation for a given station or treatment of a given survey, the successive data-processing steps to determine the final score are as follows:

1. Compute the mean estimate (X) when individual results are available; otherwise, use the value from the pooled sample at each sampling station.
2. For each biomarker, compute the general mean (m) and standard deviation (s) of X for all the stations and/or surveys, depending on the comparisons to be made.
3. Standardize X to obtain Y. Calculate a value called Y: Y = (X– m)/s.
4. Compute the Z value as follows: Z = Y or Z = −Y, in the case of a biological effect corresponding, respectively, to inhibition or activation in response to a stressor.
5. Finally, compute the score (S), with S = Z + |Min|, where S ≥ 0 and |Min| is the minimum absolute value determined.
6. Plot a spider or star diagram after calculating the value of S for each biomarker (Si) and performing the summatory to obtained the IBR.
   NOTE: refer to the original paper⁴⁹ for any details of the calculations.