Utilization of the zebrafish model to unravel the harmful effects of biomass burning during Amazonian wildfires

In vivo study: embryotoxicity test

Zebrafish embryos exposed to tested compounds developed lethal and sub-lethal alterations including different abnormalities and unhatching events. LC₅₀ (for mortality rate) and EC₅₀ (for abnormality and unhatching rate) values were extrapolated from concentration–response curves shown in Fig. 1. The rate of dead, abnormal, and/or unhatched specimens was concentration-dependent for all tested compounds (Fig. 1a–c). The lethality of the negative control group was less than 5%. Compounds 4NC and CAT showed the highest toxicity with LC₅₀ values of 8.16 and 10.95 mg/L, respectively, followed by 4,6DNG > 5NG > GUA. Experimental LC₅₀/EC₅₀ values and the predicted ones obtained by ECOlogical Structure Activity Relationship (ECOSAR) v2.0 software (https://www.epa.gov/tsca-screening-tools/ecological-structure-activity-relationships-ecosar-predictive-model) based on Quantitative Structure Activity Relationships (QSAR) models showed 4NC and CAT as the most toxic chemicals (Table 2). However, it is important to notice that experimental values for both compounds were approximately two times lower than the predicted ones. This led to the classification of 4NC into the group of molecules toxic to fish (1 < LC₅₀ < 10 mg/L), and not only harmful as predicted by ECOSAR (Fig. 2). Furthermore, our study showed that GUA, although considered as harmful to fish, classifies as not harmful to zebrafish. As a result, estimated ECOSAR values could not be used for quantitative environmental risk assessment nor for prediction on compound’s toxicity that could be applied for all fish species. In this study, ECOSAR-predicted values were provided for comparison to the obtained experimental values only. Differences between predicted and experimental values underline the increasing importance of accompanying in silico methods with in vivo toxicological tests, which is the only way to determine the compound’s realistic toxic potential and evaluate its impact on the aquatic ecosystem. These issues are presented schematically in Fig. 2.

Differences in toxicity between CAT, GUA, and nitrated intermediates were already observed elsewhere²⁴ and are highly dependent on the position of nitro groups on the benzene ring³³. In their study, Pflieger and Kroflič²⁴ observed an inhibitory effect on V. fischeri luminescence, which was 6-folds higher for bacteria exposed to 4,6DNG (EC₅₀ = 16.7 mg/L) than the ones exposed to GUA (100.6 mg/L). A similar, although more pronounced toxicity trend was observed within this work, showing an eightfold higher toxicity of 4,6DNG compared to its non-nitrated form (Fig. 1a; Table 2). Such findings confirm the high sensitivity of zebrafish embryos to nitrated MPs and their toxicity potential due to their specific highly-reactive functional groups and chemical properties. Furthermore, as ECOSAR uses only the partitioning coefficient (log P) to predict fish toxicity it could underestimate MPs and nitro-MPs real toxic potential for chemicals acting through specific interactions and non-covalent binding to enzymes. This potential mode of action of MPs is also addressed in “In silico study: molecular modeling”.

The most dominant abnormalities observed during the exposure to tested compounds were pericardial edema (Fig. 3e,h,j–l,n,o,s), blood accumulation in the yolk sac (Fig. 3n,p,s), and at later developmental stages (72, 96 hpf) skeletal deformities (Fig. 3f,h,i,r,s), undeveloped tail and necrosis of its apical part (Fig. 3g,h) and yolk sac edema (Fig. 3d,j,s). During the exposure to nitrated intermediates, blood accumulation in the brain region (Table S1, Fig. 3i) was also observed. Besides developmental abnormalities, the two most commonly observed endpoints were pigment formation failure (Fig. 3g,m) and non-hatching of larvae (Fig. 3e,h,n,o). Control group on artificial water developed normally (Fig. 3a–c). Based on Fig. 3 and Table S1 it can be concluded that the type of abnormality was not compound-related, but the sum of all obtained developmental abnormality types recorded on tested compounds (4NC > CAT = 4,6DNG = 5NG > GUA) mostly follows their toxic potential trend (4NC > CAT > 4,6DNG > 5NG > GUA).

The morphometric measurements (Fig. 4) showed that all tested samples significantly affected sensorial (eye area), skeletal (head height), and physiological (yolk and pericardial sac area) parameters in zebrafish. Significant differences among all treatments with exact p values are presented in Table S2.

The highest impact was noted on the size of the yolk sac area (Fig. 4c). A statistically significant increase of yolk sac area was observed among all tested samples, thus indicating a potential decline in metabolic rate and consequent decline in yolk consumption. The highest values of the yolk sac area were noted in larvae exposed to CAT (169.1 × 10³ µm²) and GUA (170.3 × 10³ µm²) compared to the control group (85.9 × 10³ µm²). It is important to emphasize that yolk consumption delay was not caused by growth retardation. One of the most frequently recorded sublethal endpoints during CAT and GUA exposure was pericardial edema (Table S1) which resulted in pericardial sac enlargement (169.1 and 170.3 × 10³ µm², respectively, compared to the control value of 85.9 × 10³ µm²) (Fig. 4d). CAT and GUA significantly impacted both measured physiological parameters, while skeletal structural parameters (Fig. 4a, b) were severely affected by GUA and nitrated intermediates. Compared to the control of untreated specimens, GUA caused the highest decrease in the zebrafish eye area (13.1 × 10³ µm² compared to the control values of 29.9 × 10³ µm²) and head height (244.8 µm compared to the control values of 366.1 µm). The same decline in skeletal parameter values was noted during exposure to 4NC and 5NG, and 4,6DNG (10.1–11.3% decrease compared to the control values). This type of morphological measurement could serve as a valuable additional tool that has the potential to mitigate errors and limitations of qualitative analysis.

In silico study: molecular modeling

A recent study has shown that GUA and CAT effectively inhibit different human CA isoenzymes with K_i in mmol/L range³⁵. CA is a zinc-containing enzyme that catalyzes reversible interconversion between carbon dioxide and water into protons and bicarbonate ions. During zebrafish early development (24–48 hpf), embryos use CA for increased CO₂ excretion³⁶. The activity of zebrafish CA5 isozyme is shown to be essential for the regulation of acid–base balance during embryonic growth and its inhibition causes developmental abnormalities during embryonic development³⁷. It was already shown that phenol and 2-nitrophenol are inhibitors of a cytosolic, human CA isoform II (hCA II)^38,39. Namely, phenols bind to zinc-bound water through their OH moiety whereas the phenyl moiety is located in the hydrophobic part of the hCA II active site preventing binding of substrate CO₂, thereby acting as inhibitors⁴⁰.

Uniprot database possesses one manually annotated structure of zebrafish CA (CAH-Z), EC 4.2.1.1. (UniProt ID Q92051) that consists of 260 amino acids. Titration of this zebrafish CA isozyme with ethoxzolamide (EZA), a sulfonamide inhibitor, resulted in the subnanomolar K_i which was an indication that this zebrafish CA is a homolog to hCA II, which is also inhibited by sulfonamide inhibitors⁴¹. Alignment of zebrafish CA (CAH-Z) and hCA II, whose structure has been solved, resulted in 63% identities and 76% positives in primary sequences that account for highly homologous enzymes (Fig. S1). In addition to sulfonamide inhibitors, the previous study showed that catechols also inhibit hCA II isoform and because of high conservation in primary sequence between hCA II isoform and zebrafish CA isozyme we considered that catechol also inhibits zebrafish CA⁴². Therefore, we have modeled a 3D structure of zebrafish CA by using the I-TASSER server. With the obtained model we performed docking of the compounds tested in this study—CAT, GUA, 4NC, 5NG, and 4,6DNG. Docking proposed the same mode of binding as was previously explained for phenol, CAT, and GUA intermediates^35,40. The corresponding predicted binding free energies of the docked structures (CAT, GUA, 4NC, 5NG, and 4,6DNG) obtained by AutoDock Vina are − 4.4, − 4.5, − 5.3, − 5.5, − 5.9 kcal/mol and by SwissDock are − 6.2, − 6.1, − 6.3, − 6.2, − 6.6 kcal/mol, respectively. Lignin pyrolysis products and their nitrated intermediates bind within the D. rerio CA binding site in the same manner as was described for hCA II. Namely, in the case of GUA, interactions are made between GUA –OH moiety and water bound to the Zn cation. Methoxy moiety of GUA makes interactions with side-chain of Thr199, which is conserved amino acid among all CA, whereas methyl group from methoxy moiety and phenol part of the GUA make hydrophobic interactions with Leu197, Trp208, Val121, and Val142 (Fig. 5a). According to the docking results, all docked compounds have comparable binding free energies (within the standard error of the scoring function) to 4,6DNG showing slightly better affinities towards D. rerio CA. The obtained pose from 4,6DNG is virtually the same as described for GUA (Fig. 5b). It is known that nitro compounds (−NO₂) have a positive electrostatic potential named π-hole which can establish favorable interactions with lone-pair electrons⁴³. Nitro group at the 6-position is directed towards the lone pairs of Ser56 −OH of the side chain, and side chains of Asn62 and Gln67 making favorable interactions.

In addition to inhibition of CA, recent studies have shown that our tested compounds can inhibit other enzymes shown to be essential for zebrafish embryo development^44,45,46,47. Embryotoxicity results obtained within this study indicated that 4NC, followed by CAT, GUA, 4,6DNG, and 5NG notably reduce or even prevent the formation of melanin (Table S1). Such a reduction/absence of pigmentation is usually caused by the ability of a compound to inhibit copper-containing enzyme tyrosinase, thus preventing the conversion of tyrosine into melanin⁴⁸. CAT, GUA, and nitrated intermediates can serve as alternative substrates for tyrosinase, competing with tyrosine, which in turn results in reduced or even inhibited synthesis of melanin^44,45,46,47. For instance, it has been already observed that tyrosinase from S. glaucescens is strongly inhibited by 4NC⁴⁹. Therefore, the delay and absence of pigmentation observed within this study is most certainly the result of tyrosinase inhibition due to specific interactions with tested compounds (Table 2).

It has also been shown that 4NC strongly inhibits the iron-containing enzyme lipoxygenase that catalyzes the oxidation of unsaturated fatty acids to yield fatty acid hydroperoxides⁵⁰. When the gene for lipoxygenase is subjected to the targeted knock-down, the zebrafish embryo displays an abnormal phenotype characterized by malformation of the brain, the eyes, and the tail as well as pericardial and yolk sac edema⁵¹. Therefore, compounds that inhibit lipoxygenase lead to the above-mentioned severe phenotype, which was also observed within this study.

GUA, CAT, and their nitrated derivatives 4NC, 4,6DNG, and 5NG act jointly on different enzymes as targets (i.e. tyrosinase, lipoxygenase, and CA), leading to the abnormal phenotype that has been observed. Previous studies have shown that these enzymes are indeed important for the normal development of zebrafish specimens, whereas their inhibition or decreased expression leads to concurrent abnormalities that have been reported.

Our study has pinpointed 4NC and CAT as the most toxic compounds in the embryotoxicity test followed by 4,6DNG, 5NG, GUA, respectively. On the other hand, our docking study has revealed that all tested compounds in interaction with D. rerio CA II have binding free energies within the standard error of the scoring function making those compounds equally good inhibitors of this enzyme. Thus, tested compounds most likely act jointly on many different enzymes resulting in the abnormal phenotype that has been observed within our experiments.

Source: Ecology - nature.com