The use of the micronucleus test and comet assay in wild rodents: a historical review and future perspectives.

Rodents are considered good models for investigating genotoxic damage and mutagenic alterations caused by xenobiotic agents, due to their occupation of a wide variety of habitats. However, relatively few in situ studies have focused on DNA damage in wild rodents associated with environmental exposure. In this review, we investigate trends in the application of the micronucleus test and comet assay in in situ studies of wild rodents. A total of 33 papers were identified, distributed across 14 different countries. Brazil and Spain had the most published studies (six each), followed by Bulgaria (n = 5), Mexico (n = 4) and Italy (n = 3). Only 24 of the 2,652 recognized rodent species have been the subject of in situ studies, which have most frequently focus on species of the genus Mus. The protocols used for the micronucleus test and comet assay varied widely, although blood and bone marrow were the primary types of tissue used. Given the paucity of studies on wild rodents, we recommend further research, particularly focusing on the use of this group as bioindicators of environmental quality and the standardization of protocols.

Ecotoxicological consequences of urbanization: A multi-biomarker approach to assessing sewage treatment plant effects on free-living birds.

Birds are good bioindicators of disturbance in the environment. They are present in different habitats and trophic levels. In addition, rapid urbanization has led birds to use cities as shelter and for seeking food resources. Sewage treatment plants (STPs) are suitable locations for free-living birds within cities. However, few studies address the impacts of emerging pollutants from sewage treatment plants on wild birds. In this sense, the aim of this study was to analyze the genotoxic, mutagenic, and immunological impacts from metal and pollutant exposure on free-living birds collected at a STP. For comparison, birds were collected in a preserved environment, the Silvania National Forest (FLONA). To achieve this, we used non-destructive biomarkers sensitive to environmental changes. Birds were collected in both environments using mist nets. After collection, birds were weighed, measured, species-identified, and released. Blood was collected for comet assay, micronucleus test, and leukocyte profile, while feathers were collected for metal concentration analysis. Water physicochemical parameters were measured at both sites, and water samples were collected for metal analysis. Our results demonstrated that birds collected at the STP exhibit a higher frequency of genotoxic damage and erythrocyte abnormalities, and increased immune response compared to FLONA birds. Traces of potentially toxic metals, such as Hg and As, were found in the birds feathers from both environments, raising concerns about metal contamination in both environments. Trophic guilds appear to respond similarly to exposure. The parameters and metals found in the water reflect environmental characteristics and may be influencing pollutant availability. Finally, despite the advancement of our findings, studies linking these damages to detrimental effects on behavior and reproduction are encouraged.

Multi-taxon trends and the application of genotoxic biomarkers in the analysis of effluents from sewage treatment plant.

Sewage is a significant source of many contaminants, and the effectiveness of sewage treatment plants (STPs) is fundamental to ensure that the effluents produced by these plants have a minimal impact on aquatic environments and guarantee their long-term sustainability. The present study is based on a global scientometric survey of the published research on the application of genotoxicity biomarkers for the analysis of the effects of the contaminants found in the effluents and residues produced by STPs. The literature search focused on the Web of Science and Scopus databases. Research trends were investigated based on the year of publication of each study, the country in which it was developed, the type(s) of genotoxic assay applied, the model organism(s), the type of study (experimental or field study), the physicochemical parameters analyzed, and the principal findings of the genotoxic assays. A total of 134 papers, published between 1988 and April 2023, were selected for analysis. The studies were conducted in a total of 33 different countries, but primarily in Brazil, China, Germany. These studies employed 16 biomarkers to assess genotoxicity, of which, the micronucleus test was the most used. The studies reported on a number of genotoxic substances, such as pollutants, including pesticides, microplastics, metals, and drugs. The data produced by these studies provide important insights into the genotoxic effect of the xenobiotic agents found in STP effluents, which are capable of damaging the DNA of a range of different organisms.

Erythrocyte Recovery in Oreochromis niloticus Fish Exposed to Urban Effluents.

Urban activities pollute aquatic ecosystems, and the integrity of organisms such as fish. The use of cytological techniques, such as the analysis of blood cellular integrity using the Micronucleus test, can help detect mutagenic damage as a result to urban effluents exposure. In this context, this study aimed to evaluate the frequency of micronucleus and other nuclear abnormalities in Oreochromis niloticus fish environmentally exposed to urban effluents in relation to their erythrocyte recovery capacity when exposed to clean water (30 and 45 days). The results indicated high copper, dissolved iron, nickel, and thermotolerant coliform levels in the urban stream. There was no difference in the frequency of micronuclei. In contrast, cells with nuclear nuclei, binucleates, kidney-shaped nuclei, notched nuclei, lobed nuclei, and segmented nuclei decreased according to the time the fish were exposed to clean water. When exposed to clean water, we conclude that urban fish recover from genotoxic and cytotoxic damage.

Genotoxic and mutagenic evaluation in Eisenia foetida annelids exposed to iron ore tailings from the region of Brumadinho, MG, Brazil.

Soils that have a disproportion of metallic elements due to anthropic activities endanger the terrestrial fauna. This study evaluated whether earthworms (Eisenia foetida) exposed to ore tailings from Brumadinho region presented a higher frequency of genotoxic and mutagenic damages than annelids from a reference area (control). The animals were exposed to substrates containing 0%, 25%, 50%, 75%, and 100% iron mining waste. The results indicated increased DNA damage (p < 0.05), detected by the comet assay at 25% and 50%. There was a three-fold increase in micronuclei in animals on the substrates with the highest concentrations (75% and 100%) [F = 3.095; p = 0.02]. The earthworms lost weight as the percentage of mining waste increased. We concluded that E. foetida presented DNA damage in the contaminated soils of Brumadinho. However, more research is fundamental, once the environmental disaster in Brumadinho was one of the biggest mining catastrophe in Brazil.

Damage on DNA and hematological parameters of two bat species due to heavy metal exposure in a nickel-mining area in central Brazil.

Exposure to heavy metals in mining zones is a significant threat, which can affect ecosystem services and contribute to the decline of wild bat populations. The present study investigated the impacts caused by mining on two bat species in central Brazil, the nectarivorous Glossophaga soricina and the frugivorous Carollia perspicillata. The bats were collected from a nickel-mining zone (treatment) and a protected area (control). The leukocyte profile of each species was compiled and genotoxicity (comet assay) and mutagenicity (micronucleus test) were determined using the appropriate procedures. Glossophaga soricina presented significantly higher frequencies of eosinophils and monocytes in the mining zone in comparison with the protected area, whereas C. perspicillata presented higher frequencies of lymphocytes in the mining zone, but significantly lower frequencies of monocytes. Concomitantly, G. soricina also presented a higher frequency of DNA damage, although no variation was found in this parameter in C. perspicillata when comparing environments. We also found no significant differences between populations in terms of the frequency of micronuclei and other nuclear abnormalities. Overall, the results of the study indicate that bats are susceptible to immunological disorders and DNA damage in mining zones, with the nectarivorous G. soricina appearing to be relatively more susceptible and thus a potentially effective bioindicator of the impact of contamination in these environments.

Genotoxic, mutagenic, and cytotoxic analysis in bats in mining area.

Pollution generated by the mining industry can cause harm to wildlife. This study aimed to evaluate the cytotoxicity, genotoxicity and mutagenicity in bats environmentally exposed to open pit mining. Thus, 62 bats of the following species, Carollia perspicillata, Glossophaga soricina, Phyllostomus hastatus, and Desmodus rotundus exposed to mining activities (ferronickel) were used in the analysis. The animals were obtained in samplings in July and November of 2021, totaling 8 days of sampling in the field. The results indicated that species differ in the frequency of genotoxic damage between sampling points within the mining landscape. Cytotoxicity was observed by scoring of karyorrhexis, pyknosis and karyolysis. The most captured species, C. perspicillata, showed differences in DNA damage between exposed and unexposed populations, but no differences were observed between males (n = 14) and females (n = 20). G. soricina was also a sensitive species for indicating a high frequency of DNA damages compared to the omnivore P. hastatus. Elements such as Mn, Cr, Pb, and Zn observed in water samples were at high levels in the mining area. We conclude that bats in mining areas are susceptible to increased DNA damage as already identified for other species.

The Use of Multiple Biomarkers to Assess the Health of Anuran Amphibians in the Brazilian Cerrado Savanna: An Ecotoxicological Approach.

Changes in the natural landscape and the indiscriminate use of pesticides can have a major impact on aquatic environments and have contributed to the worldwide decline of amphibian populations. In the present study, we sampled tadpoles of three anuran amphibians (Boana albopunctata, Physalaemus cuvieri, and Dendropsophus minutus) from ponds in six different agricultural landscapes of the Brazilian Cerrado savanna and evaluated whether and to what extent genotoxic and mutagenic damage was related to land use (the amount of forest and agricultural remnants, and related physicochemical factors) and the presence of pesticides in the water of the study ponds. We also evaluated the hepatotoxicity in P. cuvieri, which was the most abundant species at five of the six sampling points. Clomazone and atrazine were the most common pesticides found in the ponds. The B. albopunctata and P. cuvieri tadpoles presented similar patterns of DNA damage among the sampling points. The least DNA damage was found in the D. minutus tadpoles, although this species was present in only one of the study ponds. More binucleated and anucleated cells were observed in B. albopunctata, but there was no significant variation among species in terms of the number of micronuclei or other erythrocytic nuclear abnormalities. Land use and physicochemical factors did not explain the variation in the DNA damage observed in the three anurans. The hepatotoxicity analyses of P. cuvieri revealed the presence of a series of alterations, including the enlargement of the sinusoids, vacuolization of the hepatocytes, the infiltration of inflammatory cells, hepatic steatosis, and dilation of the blood vessels. The interaction between physicochemical factors and the biomarkers analyzed in the present study is complex. In particular, it will be important to better elucidate which factors are contributing, either directly or indirectly, to the decline of anuran amphibian populations, especially in threatened biomes, such as the Brazilian Cerrado. In this case, we would encourage further in situ studies that assess the ecotoxicology of the landscape, together with the systematic monitoring of aquatic environments, to guarantee the long-term integrity of amphibian populations, and those of other organisms that play an essential functional role in the ecosystem. Environ Toxicol Chem 2023;42:2422-2439. © 2023 SETAC.

Mutagenic effect of a commercial fungicide on Rana catesbeiana and Leptodactylus latrans tadpoles.

We have examined the mutagenic effects of the fungicide Elatus® on tadpoles of Rana catesbeiana and Leptodactylus latrans. Sixty-four tadpoles of each species have been exposed to three concentrations of Elatus® (10, 20, and 50 µg/L-1) during 96 hours. We've carried out the micronucleus test (MN) and erythrocyte nuclear abnormalities (ENAs) in 32 tadpoles of each species, the others 32 tadpoles of each species remained in a solution free of Elatus® during 96 hours, in order to assess the ability to recover from the damage caused by the fungicide. There was significant difference in MNs frequency between the treatment exposed to 50µg/L-1 and the control groups for R. catesbeiana, while for L. latrans, we've found difference between the treatment of 20 µg/L-1, followed by a period without exposure to the contaminant and the control group when all ENAs were analyzed. When we compared the two species, R. catesbeiana presented a higher frequency of MNs than L. latrans in the treatment exposed to 50 µg/L-1of the fungicide. Our findings highlight the need to monitor amphibians in places where this product is widely used.

Evaluation of genotoxicity in bat species found on agricultural landscapes of the Cerrado savanna, central Brazil.

Habitat loss and fragmentation together represent the most significant threat to the world's biodiversity. In order to guarantee the survival of this diversity, the monitoring of bioindicators can provide important insights into the health of a natural environment. In this context, we used the comet assay and micronucleus test to evaluate the genotoxic susceptibility of 126 bats of eight species captured in soybean and sugarcane plantation areas, together with a control area (conservation unit) in the Cerrado savanna of central Brazil. No significant differences were found between the specimens captured in the sugarcane and control areas in the frequency of micronuclei and DNA damage (comet assay). However, the omnivore Phyllostomus hastatus had a higher frequency of nuclear abnormalities than the frugivore Carollia perspicillata in the sugarcane area. Insectivorous and frugivorous bats presented a higher frequency of genotoxic damage than the nectarivores in the soybean area. In general, DNA damage and micronuclei were significantly more frequent in agricultural environments than in the control area. While agricultural development is an economic necessity in developing countries, the impacts on the natural landscape may result in genotoxic damage to the local fauna, such as bats. Over the medium to long term, then DNA damage may have an increasingly negative impact on the wellbeing of the local species.

Micronucleus and different nuclear abnormalities in wild birds in the Cerrado, Brazil.

Free-living birds play an important role as bioindicators in natural environments. In this study, we used the micronucleus test and nuclear erythrocyte abnormalities to investigate the difference in the genotoxic damage frequency between animals in agricultural areas (soybean planting) in relation to the conserved area. It was also discussed the bird's eating habits, which are important for ecotoxicological analysis. The results point to a difference between the insectivorous, granivorous, and omnivorous guilds. The omnivore Gnorimopsar chopi was the species that most exhibited micronucleus frequency in the protected area. In the agricultural area, the animals did not differ in the genotoxic damage frequency. In the comparison between common species in both environments, G. chopi from the agricultural area showed a micronuclei frequency almost three times higher in relation to specimens collected in the conserved area. Based on these results, this study adds to the efforts of using the micronucleus test as a simple and accessible tool for biomonitoring the wild fauna. It can be concluded that the passerine, G. chopi, due to its higher genotoxic damage frequency may be a strong candidate to indicate environmental health.

Cancer and occupational exposure to pesticides: a bibliometric study of the past 10 years.

Occupational exposure to pesticides has been identified as a major trigger of the development of cancer. Pesticides can cause intoxication in the individuals who manipulate them through either inhalation, ingestion, or dermal contact. Given this, we investigated the association between the incidence of cancer and occupational exposure to pesticides through a bibliometric analysis of the studies published between 2011 and 2020, based on 62 papers selected from the Scopus database. The results indicated an exponential increase in the number of studies published over the past decade, with most of the research being conducted in the USA, France, India, and Brazil, although a further 17 nations were also involved in the research on the association between cancer and pesticides. The principal classes of pesticides investigated in relation to their role in intoxication and cancer were insecticides, herbicides, and fungicides. The types of cancer reported most frequently were multiple myeloma, bladder cancer, non-Hodgkin's lymphoma, prostate cancer, leukemia, and breast cancer. Despite the known association between pesticides and cancer, studies are still relatively scarce in comparison with the global scale of the use of these xenobiotic substances, which is related to the increasing demand for agricultural products throughout the world.

Habitat differences affect the nuclear morphology of the erythrocytes and the hepatic melanin in Leptodactylus fuscus (Anura) in the Brazilian Cerrado savanna.

The sensitivity of anuran to the effects of habitat destruction and contamination has led to a preoccupying global decline in their populations. Morphological biomarkers such as micronuclei and other erythrocyte nuclear abnormalities (ENAs), as well as the occurrence of hepatic melanin, can be used to evaluate the effects of habitat impacts. In the present study, these two parameters were combined for the in situ assessment of the effects of soybean cultivation on the grassfrog, Leptodactylus fuscus. Specimens were also collected from a protected area to provide a reference site (non-agricultural environment). The frequency of some of the nuclear abnormalities in the animals from the soybean plantation was much higher than that recorded at the reference site, in particular micronuclei, which were 3.6 times more frequent in the plantation, lobulated nuclei (3.4 times more frequent), and reniform nuclei, which were four times more common than at the reference site. The combined analysis of all the ENAs together also revealed a frequency approximately 1.4 times higher in the animals from the soybean plantation, in comparison with the protected area. Smaller areas of hepatic melanin were observed in the specimens from the soybean plantation. These results provide further evidence of the sensitivity of anurans to habitat impacts and indicate that animals found in soybean plantations are susceptible to systematic alterations of their cells.

Genotoxic effect of heavy metals on Astyanax lacustris in an urban stream.

Uncontrolled urbanization growth contributes to the pollution of aquatic environments. Heavy metals released by domestic and industrial effluents can negatively affect aquatic organisms. This study aimed to evaluate the effect of environmental pollutants, such as metals, on fish DNA damage, in stretches of an urban stream. Specimens of the Neotropical fish, Astyanax lacustris, were exposed in situ for 96 h along the Antas stream, a Brazilian aquatic system deteriorated by anthropogenic factors. Water and sediment samples were collected simultaneously for physicochemical and heavy metal analyses. The comet assay was performed as a biomarker of genotoxicity. Fish located downstream had a higher frequency of DNA damage than in the reference site. We found concentrations of Cr and Ni above acceptable levels in sediment samples. Generally, Ba, Mn, Mg, Zn, Cr, and Ni were the elements most associated with genotoxic damage. Water and sediment of the Antas stream showed genotoxic potential in A. lacustris according to the urbanization gradient, demonstrating the importance to prevent the release of environmental pollutants, especially heavy metals in urban areas.

Micronucleus test in tadpole erythrocytes: Trends in studies and new paths.

The micronucleus test has been applied for more than three decades in tadpoles, generating an early warning of environmental quality. In this study, we reviewed 48 articles on the micronucleus test in tadpoles, published between 1987 and 2018. The findings reveal that pesticides have been the main topic discussed in the induction of micronucleus and other nuclear abnormalities in anuran larvae to the detriment of the widespread use of compounds used in agriculture. In addition to pesticides, a number of other xenobiotic agents have been targeted for genotoxic damage, such as heavy metals, radiation and wastewater. An appeal is reported to environmental contaminants, which when released naturally into the environment or because of human activities may contaminate aquatic habitats, threatening populations of tadpoles that depend on these environments for their survival. Larvae can bioaccumulate these contaminants that cause progressive impacts, ranging from DNA damage to metamorphosis delays, as well as malformations. We found that Argentina is the main driving force for the application of this test in anuran larvae along with Brazil. Different erythrocyte malformations have been reported for the erythrocyte nuclear abnormalities test, binucleated cells, nuclear buds, notched, lobed, reniform, nuclear bebbled, anucleated, picnotic and apoptotic cells are the most cited. In summary, the presence of chemical or physical agents, along with other disturbances of the habitat, can have a significant impact on the life history of the species, contributing to the decline of anuran populations.

Genotoxic Evaluation in Tadpoles Associated with Agriculture in the Central Cerrado, Brazil.

Many agricultural practices cause environmental degradation that affects the cellular integrity of anurans. In the present study, we provided in situ data of Dendropsophus minutus, Physalaemus cuvieri, and Scinax fuscovarius collected in soybean/corn and conservation units in the Brazilian Cerrado. The in situ data showed no significant variation in the micronucleus frequency between the sites, only the reniform cells had a higher rate for the agricultural environment. A combined analysis of all nuclear erythrocyte abnormalities (ENAs = nuclear buds, reniform nuclei, apoptotic cell, binucleated, and anucleated cells) was recorded higher frequencies in farmland. Overall, Scinax fuscovarius was considered the best potential bioindicator for soybean/corn plantations. Finally, we recommend expanding the micronucleus test for in situ studies to expand our understanding of the sensitivity of native anuran species and provide a more systematic assessment of the adverse effects of environmental pollutants on wildlife.

Monitoring the morphological integrity of neotropical anurans.

Amphibians are considered to be excellent bioindicators to their morphophysiological characteristics and life cycle. In this context, the present study investigated the morphological integrity of anuran larvae collected in preserved environments in the Emas National Park, in the municipality of Mineiros (Goiás state, Brazil), and in environments representative of the agricultural matrix of the Rio Verde region, also in Goiás, where there is a long history of the use of agricultural pesticides. Samples of water from temporary ponds, permanent dams, and veredas were analyzed for the presence of pesticides and, especially atrazine (5350 µg/L), found at significantly higher concentrations in the agricultural matrix. We observed a high percentage (approximately 10%) of morphological malformations including alterations of the fins in Boana albopunctatus and Scinax fuscovarius; alteration in oral structures in B. albopunctatus, Dematonotus muelleri, Physalaemus centralis, Physalaemus cuvieri, and Leptodactylus fuscus mainly in the tadpoles collected in the agricultural environment in comparison with those from the protected area (3.5%; P < 0.0001, χ2 31.75). However, changes in the eyes, mouth, intestines, and nostrils, as well as amelia were observed only in the agricultural environment. The vast majority of the observed malformations were associated with the tail and oral disc, which suggests that these anatomical parameters may be used as sensitive morphological biomarkers. Given these findings, we reinforce that areas of agricultural land may have a deleterious effect on the morphological integrity of the tadpoles and consequently, on their development, and that these features may be used as indicators of environmental quality and health.

Genotoxicological analyses of insectivorous bats (Mammalia: Chiroptera) in central Brazil: The oral epithelium as an indicator of environmental quality.

The micronucleus (MN) test of the human buccal mucosa was developed more than 30 years ago, although this technique has only recently been applied to wild mammals. This paper presents a pioneering study in the genotoxicological evaluation of the exfoliated cells of the buccal mucosa of bats. The assay was applied to two insectivorous bat species (Noctilio albiventris and Pteronotus parnellii) sampled in riparian corridors located in the city of Palmas (capital of the Brazilian state of Tocantins), with the results being compared with those obtained for a third insectivorous species (Nyctinomops laticaudatus), which has established a colony under a road bridge in the same region. This colony represents one of the largest molossidae populations ever recorded in Brazil. A significantly higher frequency of micronuclei was recorded in this colony, as well as a number of other nuclear abnormalities, including binucleated cells, cells with condensed chromatin and karyolysis, in comparison with the bats from the riparian corridors, indicating that the bats from the bridge colony are more susceptible to genotoxic damage. Thus, it is demonstrated the importance of the biomarker (MN) for use in wild animals and allows to conclude that colony bats are more susceptible to genotoxic damages.

The micronucleus test for the oral mucosa: global trends and new questions.

This study reviews global trends in the publication of papers on the micronucleus test of the exfoliated cells of the oral mucosa in mammals as an approach for environmental biomonitoring. This test has been widely used due to its precision for the detection of chromosome damage. Our temporal analysis showed a significant increase (p < 0.05) in the publication of papers on the oral mucosa over the past 33 years. Brazil was the country that published most papers (24% of the total), followed by India (16%), the USA (10%), Mexico (7%), and Turkey (6%). A further 30 countries contributed the other 37% of the papers. Overall, 99% of the micronucleus studies involved human subjects, and only 1% involved other mammals. As many wild mammals are subject to the same environmental pressures as humans, in particular chemical pollution, it seems likely that many species are equally susceptible to genotoxicogical damage. We emphasize the importance of applying this technique to the analysis of the oral mucosa of wild mammals, as well as the evaluation of its effectiveness, as observed in humans, and the expansion of the available approaches to the monitoring of environmental quality.