Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

While the techniques for evaluating biochemical biomarkers are extensively utilized, this protocol present crucial details for standardizing and replicating the methodology in neotropical anurans. The main advantage of this technique is its low cost, replicability, and its application. This technique can be applied to obtain quantitative data to evaluate alterations in the internal melanin.

The comet assay is a sensitive, versatile, and simple technique that allows the determination of genotoxic effects induced by a specific compound. It can be applied to any type of eukaryotic cells to monitor DNA damage, DNA repair, and assess the antioxidant status of cells. To begin, open the software Image-Pro Plus 6.0 and select menu and toolbar as biological.

To quantify the area occupied by melanin, select measure, then select the spatial calibration of magnification used to take the photo micrographs. After opening the histological image, select tool count and then measure objects. Mark the coloring to be measured in the image using the select colors and dropper tool, and then close the window.

Visualize the results by clicking on count, view, and statistics. Incubate a mixture of PBS, hydrogen peroxide, and a sample in a three milliliter quartz cuvette with a one centimeter path length. Then remove the sample kept in a 500 microliter centrifuge tube and add it to the quartz cuvette.

Read the catalase kinetics at 240 nanometers for two minutes in a UV spectrophotometer and calculate the enzyme activity using the equation shown on the screen. Dilute the collected blood sample with one milliliter of PBS. Centrifuge it at 381 G for nine minutes, and resuspend the pellet with 50 milliliters of PBS.

Mix 30 microliters of the blood sample in 70 microliters of low melting point agarose or LMPA at a 0.5%concentration of agarose to form layer two. Place 50 microliters of layer two on a slide previously coated with layer one containing 100 microliters of 0.5%normal melting point agarose. Cover the slide with a coverslip and place it in the cold at four degrees Celsius for 10 minutes to solidify layer two.

After solidification, remove the coverslip. Form the third layer by laying down 100 microliters of 0.5%LMPA and cover the slide again. After the third layer solidifies, remove the coverslip and immerse each slide in a 100 milliliter coplin jar containing the lysis solution prepared on the same day.

Place the coplin jars in a cold bath at four degrees Celsius for one hour in the dark for lysis to occur. At the end of the lysis, remove the slides and immerse them in the electrophoresis buffer solution in the electrophoresis tank for 15 minutes at four degrees Celsius to allow the DNA to unwind. After nuclear DNA unwinding, perform electrophoresis on the same buffer at four degrees Celsius for 10 minutes at 25 volts and 250 milliamperes.

At the end of the electrophoresis, neutralize the samples in the slides by adding two milliliters of tris hydrochloric solution three times for five minutes. Place the samples in 100 milliliter coplin jars containing 95%alcohol at four degrees Celsius to dehydrate the samples. Stain the samples by adding 10 microliters of DAPI to the center of the slide.

Then spread the dye throughout the samples with a coverslip. Examine the slides under a fluorescence photomicroscope with an NU filter. 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. Then classify the DNA damage into four classes where zero to one is for no damage, two is for minimum damage, three is for medium damage, and four is for maximum damage. Express the data as the mean number of damaged cells and the mean comet score for each treatment group.

Finally, from these data, calculate the genetic damage index or GDI for each test organism using the equation shown on the screen. The scaled mass index of Boana pulchella and Leptodactylus luctator adults are shown in this figure. For the analysis of the scaled mass index, a non-linear regression analysis was performed that demonstrates the power function relationship between the SVL and body mass for the species to determine the scaling exponent.

The scaled mass index is useful for comparing groups of adults or juveniles from the same species. The histological sections of the liver of Boana raniceps are presented here. A greater amount of c and greater dimensions of the hepatocyte and nucleus of the hepatocytes can be observed from these sections.

Different and complementary responses of cytogenetic biomarkers in an herbicide scenario exposure in Boana pulchella are shown in this figure. Here, the black bars indicate 48 hours, and the gray bars indicate 96 hours. The time of exposure and the concentration should always be considered as a factor here since a particular time or concentration may not be enough to induce damage.

The representative image describes how the biomarkers are related to the biomarker response index. Here, the blue indicates the control group, while the orange, yellow, and green colors indicate the exposure scenarios to BDE 209. This data indicates that catalase is an effective biomarker for stress situations in which the stressor is present at the highest concentrations.

When calculating the body condition index, consistently take photographs from the same distance. In tadpoles, measure the SVL body length up to the cloacal tube, excluding the collar fin. This approach safely addresses amphibian physiology and the impact of the environmental changes.

While alternative potentially invasive biomarkers exist, these methods offer insights into oxidative stress, toxicodynamics, and toxicogenetics. Additional enzyme measurements related to oxidative stress can serve as alternative methodologies. The color difference analysis technique evaluates tissue responses in photomicrographs based on color disparities.

For the pigmentary system, ensure the program's calibration aligns with the photomicrograph. This advanced technique is instrumental in investigating whether xenobiotics directly affect DNA in diverse cell types, addressing queries regarding genetic material oxidative damage and repair processes that other cytogenetic techniques are unable to address.