Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading ...to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development.
Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters.
Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.
Glutathione
S
-transferase (GST) participates in the biotransformation of many xenobiotics including biocides. Its activity in plants is generally associated with their phytoremediation capabilities. ...Biocides have been used in agriculture and antifouling paints and they represent risks for the aquatic environment. The present study aimed to: (1) evaluate the basal GST activity in roots, stems, and leaves from thirteen plants (eleven aquatic macrophytes and two halophytes) collected at South Brazil wetlands; (2) estimate the biotransformation potential of
Nothoscordum gracile
for five biocides using competitive kinetic assays with 1-chloro-2,4-dinitrobenzene (CDNB), a typical GST substrate. The
N. gracile
,
Spartina alterniflora
and
Cakile maritima
presented the highest GST activities among the tested plants. The Lineweaver–Burk plot obtained from the GST competitive kinetic assays confirmed that the biocides chlorothalonil, 4,5-dichloro-
N
-octyl-3(2H)-isothiazolone (DCOIT), dichlofluanid, and diuron, but not irgarol, compete with the substrate CDNB for GST. Chlorothalonil and DCOIT showed the lowest IC
20
values (11.1 and 10.6 μM, respectively), followed by dichlofluanid (38.6 μM) and diuron (353.1 μM). The inhibition of GST-CDNB activity by 100 nM biocide was higher for chlorothalonil, DCOIT, and dichlofluanid (46.5, 49.0, and 45.1%, respectively) than for diuron (6.5%) and irgarol (2.2%). The present study indicates plant species that have significant GST activity and could be potentially used for phytoremediation. The competitive kinetic tests suggest that among the five biocides that were tested, chlorothalonil, DCOIT, and dichlofluanid are probably preferred for biotransformation via GST in plant.
Heavy metals pose a significant threat to animals in aquatic environments due to the adverse effects they exert. Species of the genus Artemia have been described as heavy metal tolerant, but the ...sensitivity/tolerance range for these species has not been established. In the present study, the toxicity of Cd, Cu, Zn and Ni as reported in the ECOTOX and Web of Science databases was examined for Artemia franciscana and compared with other species and taxonomic groups using an integrative ecotoxicity evaluation. The hazard concentration for 5% of the species (HC5) of acute toxicity tests (24–96 h), obtained through a species sensitivity distribution (SSD) indicated that Cu (0.02 mg/L) and Cd (0.03 mg/L) were the metals with the highest toxicity to aquatic animals followed by Zn (0.15 mg/L) and Ni (0.23 mg/L). In addition to the higher hazard of Cu and Cd to aquatic animals, the comparison of acute LC50 values for A. franciscana indicates lower toxicity of Cd followed by Cu, Zn, and Ni (200.0, 14.5, 9.5, and 0.6 mg/L, respectively). Using the SSD and physiological sensitivity (S) approaches, it was demonstrated that A. franciscana is relatively tolerant to Cd (SSD= HC99; S = 2.21), Cu (SSD= HC97; S = 2.00), Zn (SSD= HC90; S = 1.29) and Ni (SSD= HC83; S = 0.96) compared with other species and taxonomic groups. It appears that tolerance to the metals Cd, Cu, Zn and Ni is unique to the family Artemiidae within the order Anostraca, as the families Streptocephalidae and Thamnocephalidae are not tolerant (have negative S values). Our study confirmed that as expected, A. franciscana presents higher tolerance to Cd, Cu, Zn, and Ni than other aquatic animals. Our findings confirm that A. franciscana can be used as a model organism to understand mechanisms involved in tolerance to heavy metals, mainly Cd and Cu, which are considered highly toxic to other animals.
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•An integrative ecotoxicity evaluation was conducted on metals in aquatic species.•The metals Cu and Cd are more hazardous for aquatic animals than Zn and Ni.•Artemia franciscana has remarkable tolerance to Cd, Cu, Zn and Ni.•Metal tolerance is unique to the family Artemiidae within the order Anostraca.•A. franciscana is suggested as a model organism to study metal tolerance.
Atrazine (ATZ) is one of the most widely used herbicides in the world. A scientometric study was conducted to analyze the evolution of research on ATZ. The study also looked at the use of microalgae ...and cyanobacteria as biological models for toxicity tests during the period from 1959 to 2019, in the category of toxicology of Web of Science. The results show an increase in the number of scientific publications mainly in the USA, Canada, and China. The majority of papers was published in journals with high impact factors, demonstrating the relevance of such studies. About 83% of the studies aimed to evaluate the effect of ATZ on non-target organisms. From those, 7.5% included microalgae and cyanobacteria. The majority of them worked with chlorophyceae to perform toxicity bioassays of ATZ and analyze its sublethal effects. The gaps identified by this analysis included a small number of collaborations between research groups from different countries; the number of studies with terrestrial organisms, which are larger in comparison to aquatic organisms; and the fact that none of the studies with ATZ and microalgae was performed in the field. These findings can point out to researchers and funding agencies the gaps in knowledge on the toxic effects of ATZ and guide the development of new research projects as well as environmental policies.
Intestinal microbiota perform important functions for the health of fishes. Knowing the microbial composition and evaluating the possible effects caused by anthropogenic pollution in the intestinal ...microbiota of fish populations might represent an important step in defining microbial biomarkers for water pollution. This study evaluated the impact of environmental contamination on the gut microbiota of the livebearer killifish Phalloceros caudimaculatus. The 16S survey using the V4 region of the 16S rRNA gene was used to characterize and compare the microbiota of two P. caudimaculatus populations from streams with different levels of environmental contamination in Rio Grande, RS, Brazil. Twelve bacterial operational taxonomic units (OTUs) (around one-third of the total) were shared between both fish populations. They represent the core microbiota of the gut in this species. The dominant phyla were Protebacteria and Firmicutes, with more than 80% of relative abundance. The dominant genus was Burkholderia with more than 35% of the relative abundance irrespective of the environmental condition. We detected a lower microbial diversity (Shannon index and observed OTUs) in fish from the polluted stream compared to the reference stream. The PERMANOVA analysis showed that the intestinal microbial communities from fish living in the polluted stream were distinct from those found in the reference stream (p < 0.05). Finally, we identified Luteolibacter, Methylocaldum and Rhodobacter genera, which correlated strongly with the polluted stream. These taxa might represent potential microbial biomarkers of exposure to environmental contaminants in the guts of fish. Confirmation of these findings in other polluted environments might allow the development of a microbiota-based screening approach for environmental evaluation in ecotoxicological studies in aquatic ecosystems.
Gut microbiota, metabarcoding, next generation sequencing, guppy, pollution, 16S rRNA; Environmental Pollution; Water Pollution; Environmental Toxicology; Aquatic Biology; Microorganism; DNA Barcoding
•Cytochrome P450 1A mRNA was cloned from guppy P. caudimaculatus.•Waterborne exposure to BNF for 1-h induced CYP1A in several organs.•CYP1A induction also occurred in anal, caudal and dorsal ...fins.•Elutriate from PAH contaminated sites caused CYP1A induction in P. caudimaculatus.•P. caudimaculatus is a suitable model for environmental toxicology.
The cytochrome P450 1A (CYP1A) mRNA is induced by environmental contaminants such as PAHs, PCBs and dioxins. The present study cloned the CYP1A transcript from the guppy Phalloceros caudimaculatus, which represents a potential fish for toxicological studies in South America. The newly identified CYP1A encodes a protein with 521 amino acids that shared 96–70% identity with other fishes. The characterization of organ- and time-dependent induction of CYP1A using RT-qPCR was evaluated after waterborne exposure to beta-naphthoflavone (BNF; 1μM). The minimum exposure time that elicited significant CYP1A induction was 1h for liver, gill, gut, brain, anal fin and fingerlings; 2h for dorsal fin; and 4h for kidney and tail fin. CYP1A tended to reach peak induction in the first few hours (4h–8h) of experiment in most organs, although levels remained induced until the end of the experiment (96h). Validation of CYP1A use in environmental sample was performed by exposing P. caudimaculatus to elutriate made from sediment of three streams located in adjacent areas of the Patos Lagoon Estuary (RS, Brazil). CYP1A in liver, gills and anal fin was induced by elutriate made from urban (S1) and industrial (S2) sites; and not induced by a reference site located 22 Km from potential contaminant sources, suggesting that environmental contamination plays a role in this induction. The results suggest that fins could be used for CYP1A biomarker analysis and employed in non-lethal biopsy methods for environmental monitoring. The responsiveness of the newly identified CYP1A to BNF and elutriate indicates that the guppy P. caudimaculatus could be used for environmental toxicology investigations in South American environments.
Knowledge of the complement of cytochrome P450 (CYP) genes is essential to understanding detoxification and bioactivation mechanisms for organic contaminants. We cloned three new
CYP1 genes,
CYP1B1,
...CYP1C2 and
CYP1D1, from the killifish
Fundulus heteroclitus, an important model in environmental toxicology. Expression of the new
CYP1s along with previously known
CYP1A and
CYP1C1 was measured by qPCR in eight different organs. Organ distribution was similar for the two
CYP1Cs, but otherwise patterns and extent of expression differed among the genes. The AHR agonist 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) (31
pmol/g fish) induced expression of
CYP1A and
CYP1B1 in all organs examined, while
CYP1C1 was induced in all organs except testis. The largest changes in response to PCB126 were induction of
CYP1A in testis (∼700-fold) and induction of
CYP1C1 in liver (∼500-fold).
CYP1B1 in liver and gut,
CYP1A in brain and
CYP1C1 in gill also were induced strongly by PCB126 (>100-fold).
CYP1C1 expression levels were higher than
CYP1C2 in almost all tissues and
CYP1C2 was much less responsive to PCB126. In contrast to the other genes,
CYP1D1 was not induced by PCB126 in any of the organs. The organ-specific response of
CYP1s to PCB126 implies differential involvement in effects of halogenated aromatic hydrocarbons in different organs. The suite of inducible CYP1s could enhance the use of
F. heteroclitus in assessing aquatic contamination by AHR agonists. Determining basal and induced levels of protein and the substrate specificity for all five CYP1s will be necessary to better understand their roles in chemical effects and physiology.
Understanding the fate and effects of organic chemicals in animals requires knowledge of cytochrome P450 (CYP) genes, which thus far are poorly known in bivalve mollusks. We searched for
CYP ...sequences in EST databases for
Mytilus and
Crassostrea species, lophotrochozoan representatives of the protostomes. From ESTs averaging ca. 924
bp, we identified 58
CYP genes in
Mytilus californianus and 39
CYP genes in
Crassostrea gigas. The sequences fell in all known animal
CYP clans, and collectively they clustered in phylogenetic analysis with vertebrate
CYP families 1, 2, 3, 4, 17, 20, 26 and 27. As in deuterostomes, a majority of the sequences fell in Clan 2. The
CYP sequences found thus far in bivalves suggest a diversity consistent with that found in many other animal species. The present description of mollusk genes provides the overall framework for classification of any additional bivalve sequences. The sequences identified also will be useful in obtaining full-length sequences and in designing primers for analysis of expression of mussel and oyster
CYP genes, or for recombinant protein expression to identify potential substrates for the bivalve CYP proteins, and understand their roles in xenobiotic detoxification and physiology of bivalves.
Biological communities are currently facing multi-stressor scenarios whose ecological impacts are challenging to estimate. In that respect, considering the complex nature of ecosystems and types and ...interaction among stressors is mandatory. Microcosm approaches using free-living nematode assemblages can effectively be used to assess complexity since they preserve the interactions inherent to complex systems when testing for multiple stress effects. In this study, we investigated the interaction effects of three stress factors, namely i-metallic mixture of Cu, Pb, Zn, and Hg (control L0, low, L1 and high L2), ii- CO2-driven acidification (pH 7.6 and 8.0), and iii- temperature rise (26 and 28 °C), on estuarine free-living nematode assemblages. Metal contamination had the greatest influence on free-living nematode assemblages, irrespective of pH and temperature scenarios. Interestingly, whilst the most abundant free-living nematode genera showed significant decreases in their densities when exposed to contamination, other, less abundant, genera were apparently favored and showed significantly higher densities in contaminated treatments. The augmented densities of tolerant genera may be attributed to indirect effects resulting from the impacts of toxicity on other components of the system, indicating the potential for emergent effects in response to stress. Temperature and pH interacted significantly with contamination. Whilst temperature rise had potentialized contamination effects, acidification showed the opposite trend, acting as a buffer to the effects of contamination. Such results show that temperature rise and CO2-driven acidification interact with contamination on coastal waters, highlighting the importance of considering the intricate interplay of these co-occurring stressors when assessing the ecological impacts on coastal ecosystems.
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•Co-occurring stressors have significant interactive effects on nematode assemblages.•Metal contamination surpasses the effects of temperature rise and acidification.•Temperature rise intensified contamination effects on nematodes.•Acidification acted as a buffer to the contamination effects on nematodes.•Nematode genera showed variable responses to contamination.
Biochemical biomarkers were evaluated in the barnacle Balanus improvisus (Crustacea: Cirripedia) sampled from both polluted and reference sites in the Patos Lagoon Estuary, Southern Brazil. During ...winter, higher glutathione S-transferase (GST) activity was recorded in the barnacles from the polluted sites, indicating environmental exposure to contaminants. Relatively low lipid peroxide levels (LPO) were also observed in barnacles from polluted sites, indicating that oxidative stress by lipid peroxidation was not a major threat in barnacles from those sites. Seasonal differences in the GST and total oxyradical scavenging capacity (TOSC) could have contributed to the low LPO levels in the summer relative to the levels in the winter. Catalase activity and metallothionein levels were not affected by contamination or seasonality. The seasonal changes observed in biomarker responses were paralleled by the differences in temperature, which could have affected physiological responses, including the balance between pro-oxidants and antioxidants.
•Glutathione S-transferase activity was increased in barnacles from polluted sites.•GST, TOSC and LPO seasonal responses were associated to water temperature.•Lipid peroxide levels were decreased in barnacles from polluted sites.•Biochemical biomarkers were evaluated in the cosmopolitan barnacle Balanus improvisus
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