Cytogenotoxic potential and toxicity in adult Danio rerio (zebrafish) exposed to chloramine T

Experimental design

The zebrafish (wildtype AB lineage) used in this study were obtained from a quality-certified aquarium (Ogawa e Sato LTDA) acquired at 3 months of age, with a length of 2–3 cm and acclimated at the Aquaculture Station of the Federal University of Mato Grosso do Sul (CEUA establishment license no. 3.128/2021). They were housed in 10 L aquariums for the acclimatization period. In the test, a total of 40 specimens were used, males and females, 10 specimens were used per treatment (control, 70, 140 and 200 mg/L) and divided into three replicates. All samples were obtained from treated and control specimens of both sexes, with no recording of the sexual ratio. Only fish that survived until the end of the 96-hour exposure period were utilized. Two fish died when exposed to 140 mg/L and 200 mg/L concentrations, respectively. The water conditions were maintained with a waterfall filter, temperature of 28 °C, pH of 7 ± 0.5, conductivity of 55 ± 50 µS/cm, and dissolved oxygen levels equal to or above 95% saturation. A 12:12 h photoperiod cycle was followed. The adult zebrafish were fed twice daily with a commercial artificial diet (TetraMin flakes fish food, Germany). Sublethal concentrations of CL-T (C₇H₇CINNaO₂S. 3H₂O) (Halamid^®) for adult zebrafish were determined based on the LC₅₀ values obtained from previous evaluations in zebrafish embryos (143.05 ± 3.11 mg/L at 24 h and 130.97 ± 7.40 mg/L at 96 h) (Rivero-Wendt et al., 2023). CL-T (Halamid^®) with 98% purity was obtained from Western Chemical Inc. Exposure to CL-T was static for 96 h. The animals were euthanized following Concea (2018). All experimental procedures were conducted following the guidelines and regulations approved by the Ethics Committee for the Experimental Use of Animals (CEUA; approval no. 3.128/2021).

Nuclear cytotoxicity/genotoxicity and integrated optical density

The methodology employed for the test followed the procedure outlined by Hooftman & de Raat (1982). After euthanasia, peripheral blood samples were collected from the caudal vein of the zebrafish using a 10 µL micropipette containing 3% EDTA. Immediately after collection, slide smears were prepared from the blood samples. The slides were fixed in methanol for 5 min, air-dried, and then stained with May-Grünwald-Giemsa-Wright (MGGW) solution using the modified Rosenfeld method to enhance the visualization of erythrocyte nuclei for morphological analysis (Ranzani-Paiva et al., 2013).

Nuclear abnormalities (NA) were defined according to Carrasco, Tilbury & Myers (1990). Erythrocytes with two nuclei were considered binuclear; blebbed nuclei, when they exhibited a relatively small evagination of the nuclear membrane, containing euchromatin; lobed nuclei, when evaginations larger than the blebbed nuclei, which can have several lobes; and vacuolated nuclei, when an appreciable depth chromatin failure without nuclear material was observed. In this work, one thousand erythrocytes cells with complete cytoplasm were scored per fish and specific NA types were not discriminated (Carrasco, Tilbury & Myers, 1990).

For the analysis of micronucleus (MN), three thousand erythrocyte cells with complete cytoplasm were scored per fish. MN were identified when they were smaller than one-third of the main nuclei (a), did not touch the main nuclei (b), and exhibited a non-refractive circular or ovoid chromatin body with a similar staining pattern as the main nucleus (c) (Rivero-Wendt et al., 2020).

To calculate the IOD of the erythrocyte nuclei, ten images (RGB 4,096 × 3,286 pixels) were randomly captured from each blood smear slide at a magnification of 1,000× using bright-field microscopy (Opticam 500R^®) equipped with a LOPT1001^® camera. The images were transformed into 8-bit type and the threshold tool in ImageJ software version 1.53a (Ferreira & Rasband, 2012) was used for image analysis. In the “Analyze/Set Measurements” menu, the area (µm²), circumference (µm), roundness (0–1), and Ferret’s diameter (µm) of 15 randomly selected nuclei per image were measured. The calculation of IOD followed the method described by Hardie, Gregory & Hebert (2002), as follows: ${\displaystyle \sum _{i=0}^n-log10\left(\frac{IFi}{IBi}\right)}$

in which n = total number of pixels in the nucleus; IFi = intensity of the nuclear pixels; and IBi = intensity of background image pixel (Hardie, Gregory & Hebert, 2002).

Acetylcholinesterase (AChE) activity inhibitory assay

The inhibition of AChE was assessed following the Ellman’s assay protocol (Ellman et al., 1961). The primary modifications involved the use of a phosphate/HEPES buffer (50/5 mM, pH 7) to enhance the stability of 5,5-dithiol-bis-(2-nitrobenzoic acid) (DTNB), which was added after catalysis to minimize potential interference with AChE activity. Tetraisopropyl pyrophosphoramide (iso-OMPA) was employed as a selective inhibitor of butyrylcholinesterase (BuChE). After conducting the fish toxicity test, the brains of adult fish (n = 38) were crushed in 10 mM Tris buffer (pH 7.2) using a mortar and pestle to create homogenates, while keeping them on ice. The enzymatic activity analysis was performed in 96-well plates using the following experimental procedure, with a final volume of 300 µL. Briefly, 150 µL of phosphate/HEPES buffer (100/10 mM, pH 7), 80 µL of distilled water, 20 µL of homogenates, and 20 µL of 292 µM iso-OMPA were mixed. The plate was preincubated at 37 °C for 30 min. To initiate the reaction, 30 µL of 10 mM acetylthiocholine (ACSCh) was added and incubated at 37 °C for 30 min. Subsequently, 20 µL of 51 mM neostigmine bromide prepared in phosphate/HEPES buffer (100/10 mM, pH 7) was added to complete the reaction. Finally, 20 µL of 8.5 mM DTNB prepared in phosphate/HEPES buffer (100/10 mM, pH 7) was promptly added to induce the production of thiocholine. The hydrolysis of acetylthiocholine (ACSCh) by AChE leads to the formation of 5-thio-2-nitrobenzoate anion (TNB) through the reaction with DTNB. The concentration of TNB was determined at λmax = 412 nm using a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices LLC, USA) at room temperature. The activity of AChE was expressed as µmol hydrolyzed ACSCh/hr/mg protein. The protein content of the samples was determined using the method described by Bradford (1976) with bovine serum albumin (BSA) as the standard (Bradford, 1976).

Gills and liver histopathology

Following euthanasia, the head of the fish was separated from the body for immediately immersed in Davidson’s solution for 24 h and subsequently subjected to decalcification for three days using a solution containing 0.7 g EDTA, 8 g sodium and potassium tartrate, 0.14 g sodium tartrate, 120 mL HCl, and distilled H₂O to a final volume of 900 mL. The fish specimens were then processed for histological analysis by embedding them in paraffin, cutting them into 3 µm-thick sections, and staining with hematoxylin and eosin for examination under bright-field microscopy (Suvarna, Layton & Bancroft, 2019).

Histopathological findings were classified based on the degree of tissue changes (DTC) as described by Bernet et al. (2001), which adopted the standard reaction (a) features and assigned importance scores (w) to these changes. Table 1 shows the histological alterations considered in this study and their corresponding importance degrees. The DTC was estimated using the following formula: (1)${\displaystyle DTC={\sum }_{\mathrm{alt}}\left(a\times w\right),}$ in which a represents the distribution of damage (0 = absent; 1 = minor; 2 = moderate; and 3 = marked occurrence) and w represents the reversibility degree of the damage (1 = easily reversible; 2 = moderate alterations with probable reversion after exposure; and 3 = irreversible alterations). The frequency (%) of each w value, adjusted according to the respective values, was calculated based on the overall sum (∑_alt) for each alteration using the following formula: F% = [(a × w)/∑_alt] ×100 (Silva et al., 2021).

Statistical analysis

The normal distribution of continuous variables was assessed using the Shapiro–Wilk test. Differences between the groups under analysis were evaluated using either ANOVA or the Kruskal–Walli’s test, depending on the data distribution. When ANOVA yielded significant results, the Bonferroni’s post-hoc test was used for pairwise comparisons between the treatments. For significant Kruskal-Wallis results, the Mann–Whitney U test was employed to examine differences between the treatments through pairwise comparisons.

Thus, the effect of CL-T concentrations on the erythrocyte MN test and histopathological findings DTC was compared using Kruskal–Wallis analysis of variance with the Mann–Whitney U test, as the data were not normally distributed. Total NA, erythrocyte nuclear morphometric data (area, circumference, roundness, and volume), and AChE activity inhibitory assay were compared using a one-way ANOVA (general linear model—GLM). The intensity over distance (IOD) curve fit was generated from the normalized values (y/mean). Box plots were used to represent the median, 25th and 75th quartiles, and the minimum and maximum interquartile intervals between concentrations. Radar graphs were employed to show the frequency (%) of the degree of reversibility between concentrations for gill and liver histopathology. The IBM SPSS Statistics version 22.0, GraphPad Prism 8.1 (San Diego, California, USA), and OriginPro version 2022 (OriginLab Corporation, Northampton, MA, USA) software packages were utilized for statistical analyses. A significance level of p < 0.05 was considered statistically significant.

Nuclear cytotoxicity/genotoxicity and IOD

The response pattern of CL-T was concentration dependence. After 96 h, a significant increase in total NA was observed at a concentration of 200 mg/L compared to the control group (Fig. 1) (p = 0.014).

The MN assay showed no genotoxic effects of CL-T. However, IOD decreased at concentrations of 70, 140, and 200 mg/L. At 200 mg/L, the curve fit displayed a different profile compared to 70 and 140 mg/L (Fig. 2). Notably, the increase in IOD was only detected in a specific pixel wave (3,5–5,8) at a concentration of 200 mg/L.

AChE inhibition

During the 96-h exposure to sublethal concentrations of CL-T, AChE activity was not significantly inhibited in the brain of adult zebrafish (Fig. 3).

Gill and liver histopathology

Figure 4 illustrates the DTC in the gills and liver, along with the frequency of respective importance scores (w1, w2, and w3). Figures 5 and 6 depict the control and exposed specimens, respectively. All fish exposed to CL-T exhibited higher DTC. In the gills, although the concentrations differed from each other, the occurrence of w2 changes was approximately 50% across all concentrations. W3 changes were observed in 25% of the samples exposed to a concentration of 200 mg/L. The most frequently observed alterations included dilatation of lamellar capillaries, edema in the secondary lamellae (sl) (Fig. 5B), and adhesion and complete fusion of the secondary lamellae (Figs. 5C and 5D).

In the liver, a high DTC was observed at 200 mg/L. Furthermore, a w2 degree of reversibility was found in all concentrations, with a frequency of approximately 50%. Liver structural changes ranged from vascular hyperemia to focal and diffuse necrosis, although these occurred less frequently. Hepatocellular changes were characterized by hypertrophy, vacuolar degeneration (hydropic and fatty), and the presence of typical degenerative nuclear figures (Fig. 6).