ecotoxicological-methodologies-to-evaluate-biomarkers-at-different

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Instituto de Biociências - INBIO Universidade Federal de Mato Grosso do Sul

Amphibians have an extracutaneous pigmentary system composed of melanin-containing cells in various tissues and organs. The functional role of these pigment cells in visceral organs has not yet been determined, although several hypotheses have been proposed. Our aim was to describe the visceral pigmentation in the frog Eupemphix nattereri under conditions of endotoxemia induced experimentally with lipopolysaccharides (LPS) from Escherichia coli and to analyze the pigmentation on the organs' surface. We used 60 adult males of E. nattereri and analyzed the visceral pigmentation 2 (LPS 2h), 6 (LPS 6h), 12 (LPS 12h), 24 (LPS 24h) or 48 h (LPS 48 h) after the LPS inoculation. We observed pigmentation on the surface of several abdominal organs. The highest degree of pigmentation was found only on the testes of the animals in the LPS 2h, LPS 6h and LPS 12h groups. The pigmentation decreased in the animals of the LPS 24h and LPS 48 h groups. The LPS administration produced no changes in the pigmentation of the cardio-respiratory and digestive systems. Thus, the cells appear to have responded to LPS intoxication, producing a rapid increase of pigmentation on the surface of the testes and a subsequent decrease in the pigmentation. These changes are most likely related to the bactericidal role of melanin, which neutralizes the effects of LPS.

Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae).

Amphibians share with other ectothermic vertebrates an extracutaneous pigmentary system consisting of melanin-containing cells in various organs and tissues. We describe the interspecific variation in the visceral pigmentation in three anuran species (Scinax similis, S. fuscovarius, and S. fuscomarginatus). We analyzed the visceral pigmentation of 15 adult males from each species during the reproductive period. The individuals were weighed and measured, and the pigmented visceral cells were classified and documented in stereomicroscope. The shape and amount of pigment cells differed among organs and also among species. Significant differences were detected in the cardiorespiratory system, digestory system, urogenital system, and lumbosacral peritoneum. In the urogenital system, the main difference was observed in the testes, in which only S. fuscomarginatus had varying degrees of pigmentation.

Visceral pigmentation in three species of the genus Scinax (Anura: Hylidae): distinct morphological pattern.

The arrangement of the hepatic tissue is directly related to physiological characteristics of animals, such as ectothermy, diet, and reproductive status. Here, we describe the anatomy, histology, and ultrastructure of the liver in adult males of Eupemphix nattereri during the breeding season. The liver is an organ with an irregular shape, red in color, and occupies a large portion of the body cavity. Anatomically, it is divided into three lobes: right, mid, and left. Further subdivision into lobules is not observed. A thin capsule of connective tissue covers externally the organ, which is responsible for its support and protection. The hepatic parenchyma is formed by two layers of polyhedralshaped hepatocytes arranged in a double cordon. These cordons are filled with hepatic sinusoids that greatly vary in size and are closely associated with hepatocytes. There are also bile canaliculi and immune cells between hepatic cordons-the melanomacrophages. These canaliculi are covered with short microvilli, which protrude into the lumen. Melanomacrophages have several cytoplasmic substances, such as melanin, lipofuscin, and hemosiderin, which form due to liver metabolism and contribute to the typical coloration of the organ. A large amount of mitochondria and other organelles, such as a well-developed Golgi apparatus, smooth endoplasmic reticulum, and abundant glycogen are found in the cytoplasm of hepatocytes.

Liver anatomy, histochemistry, and ultrastructure of Eupemphix nattereri (Anura: Leiuperidae) during the breeding season.

Melanocytes are found in various organs of ectothermic animals, playing a protective role against bacteria and free radicals. It is known that pigment cells from hematopoietic organs have immune functions. However, the role of visceral melanocytes is not well understood. Cutaneous melanocytes are responsive to α-melanocyte stimulating hormone (α-MSH), which is associated with the dispersion of melanin granules within melanocytes. α-MSH has also been reported to inhibit most forms of inflammatory responses by decreasing the pro-inflammatory cytokines and neutrophil migration. The present study evaluated the influence of an α-MSH analog (Nle(4), D-Phe(7)-α-MSH) and lipopolysaccharides (LPS) from Escherichia coli on the liver and testicular tissues of the anuran Eupemphix nattereri. The tested hypotheses were: (i) the pigmented area will increase following hormone and LPS administration, (ii) pre-treatment with α-MSH will decrease the number of mast cells, and (iii) the hormone will have protective effects against LPS-induced responses. We found that hormone administration did not change hepatic pigmentation, but increased testicular pigmentation. Testicular pigmentation quickly increased after LPS administration, whereas there was a late response in the liver. The response of enhanced pigmentation was delayed and the number of mast cells decreased in animals previously treated with the α-MSH analog when compared to the LPS group. Hemosiderin and lipofuscin were found in melanomacrophages, but not in testicular melanocytes. Although both the liver and the testes of E. nattereri have pigmented cells, these are distinct in morphology, embryonic origin, and pigmentary substances. These differences may be responsible for the different responses of these cells to the α-MSH analog and LPS administration.

Responses of melanocytes and melanomacrophages of Eupemphix nattereri (Anura: Leiuperidae) to Nle⁴, D-Phe⁷-α-melanocyte stimulating hormone and lipopolysaccharides.

Fish and amphibians have pigment cells that generate colorful skins important for signaling, camouflage, thermoregulation and protection against ultraviolet radiation (UVR). However, many animals also have pigment cells inside their bodies, on their internal organs and membranes. In contrast to external pigmentation, internal pigmentation is remarkably little studied and its function is not well known. Here, we tested genotoxic effects of UVR and its effects on internal pigmentation in a neotropical frog, Physalaemus nattereri We found increases in body darkness and internal melanin pigmentation in testes and heart surfaces and in the mesenterium and lumbar region after just a few hours of UVR exposure. The melanin dispersion in melanomacrophages in the liver and melanocytes in testes increased after UV exposure. In addition, the amount of melanin inside melanomacrophages cells also increased. Although mast cells were quickly activated by UVR, only longer UVR exposure resulted in genotoxic effects inside frogs, by increasing the frequency of micronuclei in red blood cells. This is the first study to describe systemic responses of external UVR on internal melanin pigmentation, melanomacrophages and melanocytes in frogs and thus provides a functional explanation to the presence of internal pigmentation.

Internal pigment cells respond to external UV radiation in frogs.

Amphibian populations have been experiencing a drastic decline worldwide. Aquatic contaminants are among the main factors responsible for this decline, especially in the aquatic environment. The linear alkylbenzene sulfonate (LAS) is of particular concern, since it represents 84% of the anionic surfactants' trade. In Brazil, the maximal LAS concentration allowed in fresh waters is 0.5mgL, but its potential harmful effects in amphibians remain unknown. Therefore, this study aimed to analyze the effects of a sublethal concentration of LAS (0.5mgL) for 96h on sensitive cardiac biomarkers of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802). For this, we measured the activity level (AL - % of animals), in situ heart rate (f - bpm), relative ventricular mass (RVM - % of body mass), in vitro myocardial contractility and cardiac histology of the ventricles. Tadpoles' AL and f decreased in LAS group. In contrast, the RVM increased, as a result of a hypertrophy of the myocardium, which was corroborated by the enlargement of the nuclear measures and the increase of myocytes' diameters. These cellular effects resulted in an elevation of the in vitro contractile force of ventricle strips. Acceleration in the contraction (TPT - ms) also occurred, although no alterations in the time to relaxation (THR -ms) were observed. Therefore, it can be concluded that even when exposed to an environmentally safe concentration, this surfactant promotes several alterations in the cardiac function of bullfrog tadpoles that can impair their development, making them more susceptible to predators and less competitive in terms of reproduction success. Thus, LAS concentrations that are considered safe by Brazilian by regulatory agencies must be revised in order to minimize a drastic impact over amphibian populations. This study demonstrates the relevance of employing cardiac biomarkers at different levels (e.g., morphological, physiological and cellular) to evaluate effects of xenobiotics in tadpoles.

Cardiac biomarkers as sensitive tools to evaluate the impact of xenobiotics on amphibians: the effects of anionic surfactant linear alkylbenzene sulfonate (LAS).

Ultra-Violet (UV) radiation is a stressor of the immune system and causes DNA damage. Leukocytes can change in response to environmental changes in anurans, making them an important biomarker of stressful situations. The initial barrier against UV in ectothermic animals is melanin-containing cells in skin and in their internal organs. Here, we tested the effects of UV exposure on immune cells and DNA integrity in pigmented and non-pigmented tadpoles of Lithobates catesbeianus. We used an inflammation model with lipopolysaccharide (LPS) of Escherichia coli to test synergic effects of UV and LPS. We tested the following hypotheses: 1) DNA damage caused by UV will be more pronounced in non-pigmented than in pigmented animals; 2) LPS increases leukocytes in both pigmented and non-pigmented animals by systemic inflammation; 3) The combined LPS and UV exposure will decrease the number of leukocytes. We found that the frequency of immune cells differed between pigmented and non-pigmented tadpoles. UV exposure increased mast cells and DNA damage in erythrocytes in both pigmented and non-pigmented tadpoles, while leukocytes decreased after UV exposure. Non-pigmented tadpoles experienced DNA damage and a lower lymphocyte count earlier than pigmented tadpoles. UV altered immune cells likely as a consequence of local and systemic inflammation. These alterations were less severe in pigmented than in non-pigmented animals. UV and LPS increased internal melanin in pigmented tadpoles, which were correlated with DNA damage and leukocytes. Here, we described for the first time the effects of UV and LPS in immune cells of pigmented and non-pigmented tadpoles. In addition, we demonstrated that internal melanin in tadpoles help in these defenses, since leukocyte responses were faster in non-pigmented animals, supporting the hypothesis that melanin is involved in the initial innate immune response.

UV-B affects the immune system and promotes nuclear abnormalities in pigmented and non-pigmented bullfrog tadpoles.

Brazil is an important consumer of herbicides. In sugarcane cultivation-the country's most extensive agricultural crop-atrazine-based formulations are the principal form of weed control. Several studies have investigated adverse effects of atrazine or their formulations on anurans, but not specifically on Brazilian species. Our aim was therefore to investigate the lethal and sublethal effects of an atrazine-based herbicide in Rhinella schneideri tadpoles and, in particular, effects on the pigmentation system as a new endpoint in ecotoxicological studies. Rhinella schneideri tadpoles at the Gosner-30 stage were exposed to the atrazine-based herbicide formulation, SIPTRAN 500 SC, in acute bioassays at concentrations of 1.5-25 mg/L. The lethal and sublethal effects induced were analysed at different ecotoxicological levels: organismal level (alterations in behaviour, growth, development, and body mass; morphologic abnormalities), histological level (liver histopathology), the pigmentation system (melanomacrophages and dermal-melanophores), and cellular level (erythrocyte micronucleus formation and other nuclear-abnormalities). This herbicide induced sublethal effects at the organismal level with alterations in swimming and growth and morphologic abnormalities. These results demonstrated that, in anuran tadpoles, the atrazine-based agrochemical increased the frequency of micronucleus formation and other nuclear-abnormalities in erythrocytes and caused liver damage. In addition, we demonstrated for the first time effects of an atrazine-based formulation on the pigmentation system of anuran tadpoles, specifically an increase in the number of melanomacrophages and dermal melanophores. This study is the first to use several widely differing endpoints at different ecotoxicological levels in a comprehensive manner for assessment of the effects of environmental stressors in order to determine the health status of Neotropical anuran species. In doing so, this study establishes a foundation for future ecological assessments.

Biomarkers at different levels of organisation after atrazine formulation (SIPTRAN 500SC) exposure in Rhinella schineideri (Anura: Bufonidae) Neotropical tadpoles.

Endocrine disrupting chemicals are one of the most important factors contributing to worldwide amphibian decline. The 4-nonylphenol (NP) is a degradation product of several compounds, such as detergents and pesticides, affecting the aquatic environment. Here, we test whether treatment with NP has an effect on developing ovarian tissue, nuclear abnormalities in erythrocytes, and body darkness in pre-metamorphic tadpoles of the bullfrog Lithobates catesbeianus. Tadpoles were exposed for 14 days to three different concentrations of NP (1, 10, and 100 μg/L) besides the control group, which was maintained only with water. After determining body coloration, animals were euthanized and gonads and blood were collected and processed for histology and genotoxic analysis. Even though most animals were females, intersex tadpoles were observed in control and treated groups and there were no males in any group. The highest concentration of NP showed an increase in atretic oocytes, but the area corresponding to somatic compartment and early and late germ cells were not affected. Furthermore, all treated groups presented higher amount of nuclear abnormalities in erythrocytes and body darkening when compared with the control group. These results suggest that NP causes genetic damage and morphological alterations in L. catesbeianus tadpoles by disrupting oogenesis, inducing genotoxicity and increasing body coloration. Its effects on gonadal development could cause future impairments in reproduction, while its deleterious effects on genotoxicity and body pigmentation could be used as a biomarker of effect to this compound.

Gonadal, body color, and genotoxic alterations in Lithobates catesbeianus tadpoles exposed to nonylphenol.

Lambda-cyhalothrin is a synthetic pyrethroid that mimics the structure and insecticidal properties of pyrethrin, a natural insecticide derived from chrysanthemums. In fish, it disrupts the nervous system, causing motor paralysis and several other alterations associated with varying levels of mortality. This study aimed to evaluate osmoregulatory responses and histological changes in the gills of Oreochromis niloticus chronically exposed to a sublethal dosage (0.86 μg/L) of lambda-cyhalothrin. The mean serum values for Na, K, Cl, Ca, pH, lactate, H, HCO and glucose along to degree of tissue change (DTC) at 24, 96, 168, and 240 h post-exposure (hpe) were evaluated. Lambda-cyhalothrin affected the neuronal motor function at 24 hpe, followed by the increase of the K, Ca, H, and glucose levels in the exposed group, compared to the control group. Lactate and H levels in the exposed group were higher than those in the control group at 168 and 240 hpe respectively. HCO, and Cl- levels increased at 240 hpe, although there was no change in the pH values. DTC was higher in treated fish than in control fish, but there were no significant differences among time-exposure. The changes detected ranged from hyperemia of the branchial vasculature, eosinophilic granulocytic cell infiltration, mucous cell hyperplasia, and partial fusion of secondary lamellae at 24 hpe to vascular aneurysm formation, and necrosis of the lamellar epithelium at 240 hpe. Thus, a sublethal dosage of lambda-cyhalothrin in the long-term is toxic for Nile tilapia, characterized by hypokalemia, hypercalcemia, hyperglycemia, and respiratory alkalosis, followed by time-dependent histological changes.

Osmoregulatory profiles and gill histological changes in Nile tilapia (Oreochromis niloticus) exposed to lambda-cyhalothrin.

The liver of anurans play an important role in metabolism, including detoxification, the biotransformation of molecules, and the storage of metabolites. Surfactants are part of domestic and industrial effluents. The effects of linear alkylbenzene sulfonate (LAS) on anuran liver remain unknown, however, some studies have evaluated the effects of LAS on the skin, gills, heart, testes, and liver of fishes. Here, we tested the hypothesis that LAS is hepatotoxic, promoting morphometric alterations in hepatocytes along with inflammation in the tissue, altering hepatic catabolism. We evaluated the effects of a LAS concentration that is considered environmentally safe in Brazilian inland waters on the liver of Lithobates catesbeianus tadpoles, including studies on morphology, morphometry, immunology, and metabolism. LAS exposure promoted enlargement of liver sinusoids and vacuolization of hepatocytes. Exposure to LAS also increased the area of mast cells and melanomacrophages (MMs). Additionally, LAS exposure increased hemosiderin inside MMs, suggesting alterations in the catabolism and storage of iron. Hepatocyte size increased after exposure to LAS, suggesting cytotoxic effects. Integrative analyses (i.e., morphometric, metabolic, and immunological) demonstrated hepatotoxic effects of LAS. These types of studies are key to understanding the negative effects of these substances on tadpole health, as these liver alterations impair anuran homeostasis.

Hepatotoxicity of the anionic surfactant linear alkylbenzene sulphonate (LAS) in bullfrog tadpoles.

The digestive tract of fish has many morphological adaptations related to habitat and nutrition. Intestinal biometry may reflect these adaptations. Here, we aimed to describe histometric patterns in farmed fish and their relationship with feeding by using a standardized protocol considering cell density by tissue area. Five juvenile specimens of each species (Pseudoplatystoma corruscans, Piaractus mesopotamicus, and Oreochromis niloticus) were used. O. niloticus possessed higher intestinal weight and length besides higher intestinal quotient and intestinal somatic index than the other species. The general histological composition was similar between species. However, P. corruscans showed differences in thickness between the anterior and posterior segments. O. niloticus had thinner serosa and muscularis layers than the other species. The cell density was distinct in both species and segments. Comparing the intestinal segments, O. niloticus displayed the lowest count of granulocytes. Goblet cell density was lower in P. mesopotamicus in all segments. However, the volume of these cells was higher in the anterior and middle anterior segments. Our data demonstrated that intestinal structural plasticity is associated with the difference in feeding habits. Here, we used quantitative standardized histometric criteria to understand the morphophysiological diversity of the fish digestive tract, and this technique can be applied in future studies to evaluate changes in the digestive tracts of vertebrates.

Article	Year
Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae). Zoology (Jena, Germany)\| PubMed ID: 21885266	2011
Visceral pigmentation in three species of the genus Scinax (Anura: Hylidae): distinct morphological pattern. Anatomical record (Hoboken, N.J. : 2007)\| PubMed ID: 22190535	2012
Liver anatomy, histochemistry, and ultrastructure of Eupemphix nattereri (Anura: Leiuperidae) during the breeding season. Zoological science\| PubMed ID: 23215976	2012
Responses of melanocytes and melanomacrophages of Eupemphix nattereri (Anura: Leiuperidae) to Nle⁴, D-Phe⁷-α-melanocyte stimulating hormone and lipopolysaccharides. Zoology (Jena, Germany)\| PubMed ID: 24035001	2013
Internal pigment cells respond to external UV radiation in frogs. The Journal of experimental biology\| PubMed ID: 26944494	2016
Cardiac biomarkers as sensitive tools to evaluate the impact of xenobiotics on amphibians: the effects of anionic surfactant linear alkylbenzene sulfonate (LAS). Ecotoxicology and environmental safety\| PubMed ID: 29351853	2018
UV-B affects the immune system and promotes nuclear abnormalities in pigmented and non-pigmented bullfrog tadpoles. Journal of photochemistry and photobiology. B, Biology\| PubMed ID: 29413693	2018
Biomarkers at different levels of organisation after atrazine formulation (SIPTRAN 500SC) exposure in Rhinella schineideri (Anura: Bufonidae) Neotropical tadpoles. Environmental pollution (Barking, Essex : 1987)\| PubMed ID: 30384079	2019
Gonadal, body color, and genotoxic alterations in Lithobates catesbeianus tadpoles exposed to nonylphenol. Environmental science and pollution research international\| PubMed ID: 31152429	2019
Osmoregulatory profiles and gill histological changes in Nile tilapia (Oreochromis niloticus) exposed to lambda-cyhalothrin. Aquatic toxicology (Amsterdam, Netherlands)\| PubMed ID: 32911328	2020
Hepatotoxicity of the anionic surfactant linear alkylbenzene sulphonate (LAS) in bullfrog tadpoles. Chemosphere\| PubMed ID: 33250231	2021
Intestinal morphometry and cell density features in tropical farmed fish: A methodological approach. Anatomia, histologia, embryologia\| PubMed ID: 36651672	2023

Lilian Franco-Belussi

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