Environmental Risk Assessment of Dexamethasone Sodium Phosphate and Tocilizumab Mixture in Zebrafish Early Life Stage (Danio rerio)

Abstract

Pharmaceuticals are widely regarded as a menace to the aquatic environment. The constant consumption of biologically active chemicals for human health has been matched by an increase in the leaking of these compounds in natural habitats over the last two decades. This study was aimed to evaluate the molecular pathway underling the developmental toxicity of exposure in the ecological environment. Zebrafish embryos were exposed at doses of dexamethasone sodium phosphate (DEX) 1 μmol/L, tocilizumab 442.1 μmol/L and dexamethasone + tocilizumab (1 μmol/L and 442.1 μmol/L, respectively) from 24 h post-fertilization (hpf) to 96 hpf. This study confirmed that DEX exposure in association with tocilizumab 442.1 μmol/L at 1 μmol/L (non-toxic concentration) affected the survival and hatching rate, morphology score, and body length. Additionally, it significantly disturbed the antioxidant defense system in zebrafish larvae. Furthermore, a DEX 1 μmol/L and tocilizumab 442.1 μmol/L association also increased the production of apoptosis-related proteins (caspase-3, bax, and bcl-2).

1. Introduction

In the twenty-first century, coronaviruses have already caused three major epidemics: SARS-CoV (WHO, 2004), MERS-CoV (WHO, 2016), and, in the worst pandemic in over a century, Coronavirus disease 2019 (COVID-19) [1], caused by SARS-CoV-2, has symptoms, including pneumonia, fever, persistent cough, lung inflammation, and often diarrhea, acute kidney injury, and death [1]. COVID-19 comorbidities, vasoconstriction, salt and water retention, inflammation, and the generation of reactive oxygen species (ROS) are all promoted by the Angiotensin II (Ang II) 8-amino-acid peptide [2]. However, because the pathophysiology of COVID-19 is complex, treatment should involve the suppression of proinflammatory cytokine overproduction in addition to antiviral action and coagulopathy treatment. This so-called cytokine storm is thought to be the major cause of COVID-19-related mortality, causing multiorgan damage [3]. Dexamethasone sodium phosphate (DEX), which has some impact in advanced disease in patients requiring mechanical ventilation, was the first suggested approach for cytokine storm control. However, it is required to reduce significantly elevated levels of proinflammatory cytokines, such as interleukin (IL)-6, to prevent the development of this stage of COVID-19, which is marked by high mortality. It has been proven that greater levels of IL-6 are linked to a higher risk of death in patients. As a result, tocilizumab (TCZ), an IL-6 receptor inhibitor, was evaluated as a prospective treatment option. In 2021, Salama and colleagues found a survival benefit in individuals treated with TCZ in the most current and largest study; the rationale for using TCZ in patients with SARS-CoV-2 infection is based on its ability to block the IL-6 receptor (IL-6R), avoiding the cascade of proinflammatory effects [4]. The association of TCZ with DEX is a very recent therapy, nevertheless, it has already given very encouraging results in the treatment of COVID-19 patients suffering from an acute inflammatory phase [5,6]. In the aquatic environment, some of these medicines can be detected at ng/L⁻¹ concentrations and have the potential to be quite persistent. Only a few pharmaceuticals already had an environmental risk assessment, which is based primarily on projected data and excludes information on their metabolites and transformation products [7,8]. DEX is the most widely used glucocorticoid derivative of cortisone, and high levels have also been detected in sewage effluent [9]. Previous study detected a high concentration of DEX (10 μg/L) in river water collected downstream from a French pharmaceutical factory [10]. However, the concentration of DEX usually found in sewage effluent is about 0.3 μg/L [11]. The enormous variety of therapies involving DEX treatment make this glucocorticoid a persistent presence in the aquatic environment, especially due to hospitals wastewater [12]. Although to date, however, there are no studies of detected levels of TCZ in the environment, and its massive use in hospitals could pose a potential risk of environmental contamination. The zebrafish larvae are uniquely suited to identifying novel protective compounds through drug screening, combining the advantages of cell lines (the extremely small size is suitable for multi-well plates, ease of chemical exposure) and vertebrate models (functional organ systems, genetic conservation) [13,14]. Because surface water contains low quantities of environmental pollutants, this is a promising technique that suggests pharmaceuticals (and other compounds) and their toxicity processes could be monitored using fish lines at lower concentrations. Furthermore, zebrafish can quickly uncover drugs with clear developmental toxicity or absorption difficulties as a whole-animal drug screening platform [14,15]. Previous studies already highlighted the potential toxic effect of DEX on several aquatic organisms [16,17,18]. Moreover, DEX is able to induce hepatomegaly, steatosis, and osteoporosis in larval zebrafish [19,20]. Unfortunately, there is no comprehensive information on the toxicity of TCZ to fish and other aquatic organisms in the literature. In this study, we evaluated the potential toxicity of DEX alone and in combination with TCZ, looking at mortality and hatchability as well as the molecular involvement of the oxidative stress markers in larvae exposed to known concentrations of these substances.

2. Materials and Methods

2.1. Selection of Preliminary Concentrations

In order to identify the suitable concentrations and time points for the following experiments, DEX concentrations of 0.1, 1 and 5 μmol/L were added into embryo water and were applied to observe the larvae morphology until 96 hpf. Next, we analyzed different concentrations of TCZ for lethal end points during embryonic development. TCZ doses of 221.05, 442.1 and 884.2 μmol/L were added to embryo water and used to observe the larvae morphology until 96 hpf in order to determine the best concentrations and time points for the next experiments.

2.2. Solutions Preparation for ZFET Assay

Soldesam was used to test the effects of dexamethasone sodium phosphate; excipients in the injectable formulation include phenol, sodium citrate dihydrate, and citric acid anhydrous. These substances have not been reported to have toxicity on aquatic organisms except at very high concentrations [21], such as for phenol 230 mg/kg [22], which are not those present in the drug formulation. Soldesam of 8 mg/2 mL was purchased from (Laboratorio Farmacologico Milanese SRL, Caronno Pertusella, 21042 VA, Italy), Tocilizumab (TCZ) Actemra/RoActemra of 20 mg/mL was purchased from (F. Hoffmann-La Roche Ltd., Via G.B. Stucchi 11020900—Monza, Italy). The solutions were diluted in embryo medium (NaCl (29.4 g/100 mL), KCl (1.27 g/100 mL), CaCl₂•2H₂O (4.85 g/100 mL), and MgSO₄•7H₂O (8.13 g/100 mL)) obtaining three concentrations: DEX 1 μmol/L, TCZ 442.1 μmol/L and one with the association of DEX 1 μmol/L with TCZ 442.1 μmol/L. These three solutions were dispensed in 24-well plates (2 mL each) (Labsolute, Th. Geyer GmbH & Co. KG, Dornierstr. 4–6 D-71272 Renningen, Germany), one for each concentration and one plate for the negative control (untreated).

2.3. Zebrafish Maintenance and Breeding

Wild type (WT) mature zebrafish with an age of 6 months were used for embryo production. Zebrafish were raised in the Department of Veterinary Sciences, University of Messina, Italy, in the Centre for Experimental Fish Pathology (Centro di Ittiopatologia Sperimentale della Sicilia—CISS). The fish were fed both dry and live food twice a day at 3% of body weight (BW). For a successful reproduction, mature females and males were mated at a 2:1 ratio. The day after, the eggs were collected, bleached and, afterwards, non-fertilized eggs were discarded. Only embryos which reached the blastula stage were used for the experiments.

2.4. Zebrafish Embryo Toxicity (ZFET) Assay

The toxicity of all three solutions was established following the OECD guidelines (OECD, Test No. 236: fish embryo acute toxicity (FET) test) [23]. Two solutions, one of DEX and one of TCZ (1 μmol/L, 442.1 μmol/L, respectively) and one solution with the association of both drugs (DEX 1 μmol/L + TCZ 442.1 μmol/L), were prepared using embryo medium and placed into 24-well plates (1 embryo each well). Fertilized eggs (n = 24 in each plate, 20 exposed to the drug and 4 used as negative control) were transferred into 24-well plates with the test solutions and incubated at 26 °C at a 14:10 h day/night light regime as seen in a previously [24,25]. For the preliminary experiments aimed at finding non-toxic concentrations of DEX and TCZ, n = 20 larvae were used for each experimental group and the experiment was repeated 3 times. Whereas, for the study of toxic effects in combination of DEX and TCZ, n = 40 larvae were used for each experimental group and the experiment was repeated 4 times. The entire mortality and developmental abnormalities of embryos and larvae were monitored and recorded at 24, 48, 72 and 96 h post-fertilization (hpf) [26]. Coagulation, lack of somites, non-detachment of the tail and no heartbeat were considered as the lethal endpoint. Furthermore, malformations of the embryos during development were evaluated as a teratogenic endpoint. In addition, the percentage of hatchability and mortality were estimated. A stereo microscope was used to capture images and video (Leica M205 C). Every 24 h, four separate endpoints were checked to see any malformations:

(a)

Embryo coagulation—can also occur within a few hours of the start of exposure and indicates a generic acute toxic effect;

(b)

Lack of somite formation—somite should be visible 12 h after fertilization. If absent, the embryo will not develop further, thus causing its death;

(c)

Non-detachment of the tail—detachment of the tail from the yolk can be observed 24 h after fertilization, indicating a normal growth of the embryo;

(d)

Absence of heartbeat—the heartbeat is easily detectable 30 h after fertilization; its absence indicates the death of the embryo. Embryo coagulation and absence of heartbeat were considered as endpoints of mortality.

2.5. Western Blot

Western blot analysis was carried out according to a previously established protocol [27,28]. All treated zebrafish larvae (n = 20 for each experimental group), after being washed two times with PBS (pH 7.4), were homogenized in ice-cold RIPA buffer (Tris-HCl, pH 7.4, 50 mM; NaCl, 150 mM; Sodium Deoxycholate 1 percent; Triton-X1000 0.05 percent; EDTA 0.5 mM, Na₃VO₄ 1 mM; NaF 50 mM, 0.1% SDS) to extract proteins (WuhanBoster Biological Technology, Wuhan, China). For protein preparation, each plate of larvae (n = 20 per experimental group of each experiment) was pooled, so that n = 1 refers to protein from these 20 larvae. The homogenates were centrifuged for 10 min at 14,515 g and the supernatants were collected. The Bradford method [29,30] was used to determine protein concentrations. To reach denaturation, the samples were heated to 95 C for 5 min. SDS-PAGE (10% gels) was used to separate equal amounts of proteins (40 μg) from each sample for 2 h at 100 V under denaturing conditions. The proteins were transferred from gels onto a 0.42 mm polyvinylidene fluoride (PVDF) membrane after electrophoresis (GEAmersham, Casoria, NA, Italy). At 35 V for 10 min, the proteins were transferred onto PVDF membranes in a Tris-glycine transfer buffer. After blocking with 5 percent skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 2 h, the membrane was incubated with primary antibodies against caspase-3 (Abcam ab13847, 1:500), Bax (Abcam ab32503, 1:800), bcl-2 (Abcam ab18285, 1:800), NF- B (Antibodies A323357, 1:500), and IκBα (ANAWA pS32/S36, 1:500) and the expression of the proteins was compared to beta actin expression (Bioreagents F52293 NSJ, 1:1000). The membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit lgG or goat anti-mouse lgG (diluted at 1:5000) for 2 h at room temperature after being washed three times with TBST. Finally, the immunoreactive bands were detected using ECL techniques, and the protein bands were quantified using BIORAD ChemiDoc TM XRS + (Bio Rad, Hercules, CA, USA) software utilizing densitometry. The density ratio of target proteins to beta actin expression was used to determine protein expression.

2.6. MDA, SOD, and CAT Measurements

The larvae from each plate (n = 20 for each experimental group) were defrosted and homogenized on ice, with 180 μL of ice-cold physiological saline. The supernatant was obtained by centrifuging the homogenate at 4000 g for 15 min at 4 °C. As previously mentioned, the content of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in the supernatant was determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [31,32]. Lipid peroxidation products (measured as MDA) were quantified by the thiobarbituric acid (TBARS) method, and the MDA concentration was expressed as nanomoles per mg protein as seen in a previous study [33]. One unit of SOD activity was defined as the amount of enzyme required to inhibit the oxidation reaction by 50% and was expressed as U/mg protein. One unit of CAT activity was defined as the amount of enzyme required to consume 1 μmol H₂O₂ in 1 s and was expressed as U/mg protein. Protein concentration was assayed using the Bradford assay using bovine serum albumin as a standard.

2.7. Data Analysis

All values in the figures and text are expressed as the mean ± standard error (SD) of N number of experiments. The results were analyzed by two-way for graphs where there were two variables, such as time and different exposures (e.g., Survival rate), or one way ANOVA followed by a Bonferroni pos hoc test for multiple comparisons. The data were tested for normal distribution with the Shapiro–Wilk test (p < 0.05) and they were represented as mean ± standard deviation (SD), (alpha value of 0.05). Statistical analysis was performed using Graphpad Prism 8.

3. Results

3.1. Viability and Morphology of Zebrafish Embryos after Preliminary DEX and TCZ Exposure

As presented in Figure 1, the DEX concentrations of 0.1 and 1 μmol/L did not alter the zebrafish morphology after 96 hpf compared to the control group (Figure 1). The DEX 5 μmol/L group induced a rise in mortality (about 55% of the entire group) in the zebrafish embryos during the 96h exposure. No hatched larvae were observed in the zebrafish exposed to DEX at concentrations as low as 5 μmol/L, and this condition was observable in 100% of larvae at the DEX concentration of 5 μmol/L. Time interaction for survival was [F (3, 304) = 1402], and for the hatching rate was [F (3, 64) = 120,895].

Figure 2 shows that TCZ doses of 221.05 and 442.1 μmol/L had no effect on zebrafish morphology after 96 h when compared to the control group (Figure 2). In zebrafish embryos, the TCZ 884.2 μmol/L group caused a 100% mortality and no hatched larvae at 96 hpf (Figure 2). Time interaction for survival was [F (3, 304) = 18,343], and for the hatching rate was [F (3, 64) = 94,565].

3.2. Mortality, Hatching Rate and Malformations after ZFET Assay

Figure 3A depicts the development of DEX 1 μmol/L -exposed, TCZ 442.1 μmol/L -exposed and DEX + TCZ-exposed zebrafish embryos from 24 to 96 hpf. At 96 hpf, the association group (DEX 1 μmol/L + TCZ 442.1 μmol/L) presented several morphogenetic defects, including spinal curvature, pericardial edema, and tail deformity. The 1 μmol/L DEX-treated group and TCZ 442.1 μmol/L group showed no statistically significant changes at 96 hpf. Figure 1B depicts the embryos’ cumulative survival after exposure to all three solutions; survival rate was documented at 24, 48, 72 and 96 hpf. Because hatching is a critical time in zebrafish embryogenesis, the hatching rate is one of the most important indices for determining developmental toxicity in zebrafish. According to the studies conducted, embryos started to hatch by 48 hpf and finished by 72 hpf. Our results showed that 80% of the TCZ 442.1 μmol/L group for all embryos had hatched by 72 hpf, while in groups with concentrations of DEX 1 μmol/L and both DEX 1 μmol/L and TCZ 442.1 μmol/L, the hatching rate was 16% and 0%, respectively. Therefore, as shown in Figure 1C, the embryo-hatching rate was reduced in the DEX-treated groups (DEX 1 μmol/L and DEX 1 μmol/L + TCZ 442.1 μmol/L), mostly in the association group. These data showed a slight decrease in the hatching rate in both DEX-treated groups compared with the control group (100% hatching at 72 hpf). Time interaction for survival was [F (3, 282) = 144.1], and for the hatching rate was [F (3, 64) = 25,232].

3.3. Effect of DEX and TCZ on Lipid Peroxidation and Stress Oxidative Pathway

The results showed an increase in SOD and CAT expression levels related to oxidative damage after the co-exposure of DEX and TCZ (Figure 4). At the same time, the co-exposure of DEX and TCZ showed increased MDA levels in the larval zebrafish significantly at 96 hpf compared to the CTRL group (Figure 4C) Contrary, both the TCZ 442.1 μmol/L and DEX 1 μmol/L groups did not show effects on oxidative stress in larval compared to the CTRL group (Figure 2). (Figure 4A,B). Furthermore, the single exposure of DEX and TCZ did not show an increase in MDA levels at 96 hpf compared to CTRL (Figure 4)

3.4. Apoptotic Process

When compared to the control group, the protein levels of apoptosis-related genes (caspase-3, and bax) rose following DEX 1 μmol/L + TCZ 442.1 μmol/L treatment (Figure 5). On the contrary, bcl-2 expression was downregulated. The cell death was forcefully increased after DEX + TCZ exposure. Expression levels of caspase-3 and bax were reduced in the TCZ 442.1 μmol/L and DEX 1 μmol/L alone groups compared to the DEX + TCZ association group; on the other hand, gene expression of bcl-2 was upregulated compared to the DEX + TCZ association group. The results indicate that DEX + TCZ induced cell death in zebrafish embryos. Moreover, when compared to the control group, DEX 1 μmol/L + TCZ 442.1 μmol/L exposition dramatically increased caspase-3 and Bax protein expression (Figure 5) (p value < 0.0001). In the DEX 1 μmol/L + TCZ 442.1 μmol/L group, however, the anti-apoptotic protein bcl-2 expression was considerably reduced (p value < 0.0001). Furthermore, when compared to the CTRL group, DEX + TCZ increased the Bax/bcl-2 ratio in larvae (Figure 5E).

4. Discussion

In SARS-CoV-2 infection—the causative agent, COVID-19—one of the main pitfalls is the excessive inflammation that the immune system puts in place to defend itself against the infection. In severe cases, when the response becomes uncontrollable, the patient faces serious complications that can lead to death [2,3] Counteracting excessive inflammation, particularly that occurring in the lungs, is of crucial importance in reducing the symptoms of COVID-19. In this regard, the use of DEX and its association with TCZ were found to be a valuable tool to reduce the symptoms of COVID-19 [34]. Recent findings show that pharmaceuticals are evenly dispersed in aquatic ecosystems, necessitating more investigation into their potential effects on aquatic species [35,36]. Pharmaceuticals are different from other pollutants, as they are designed to react with specific pathways at low concentrations, which can cause considerable changes in aquatic species. The purpose of this research was to assess the environmental impact and the toxicity of DEX and its association with TCZ, as well as the possible processes involved in its toxicity pathway. The first step in this study was to evaluate, through an embryo toxicity assay, the mortality and hatchability of zebrafish larvae from 24 to 96 hpf. According to Roche’s (F. Hoffmann-La Roche Ltd.) environmental risk assessment performed on TCZ, the lethal concentration of 50 (LC50) on zebrafish larvae was estimated to be 100 mg/L (884.2 μmol/L). Therefore, we decided to use a concentration that was reduced by half to ensure that we did not have an acute toxic response on our larvae. Regarding DEX, we also decided to use (for a pilot study) a halved concentration compared to the one already used in previous papers (10 μM), which demonstrated its actual toxicity [37]. We have conducted some preliminary experiments using different concentrations of DEX (0.1 μmol/L, 1 μmol/L and 5 μmol/L) to find a minimal full non-toxic dose. From our results, we inferred that larvae exposed to DEX 5 μmol/L showed malformations and developmental toxicity, in contrast to DEX 1 μmol/L and 0.1 μmol/L concentrations, which showed no statistically significant signs of developmental toxicity. Following these findings, we decided to use the highest non-toxic concentration of DEX (1 μM) and observe its possible harmful effects in combination with a still non-toxic dose of TCZ (442.1 μmol/L). In accordance with the results obtained by the observation of exposed larvae to the three solutions (DEX 1 μmol/L, TCZ 442.1 μmol/L and DEX 1 μmol/L + TCZ 442.1 μmol/L), we found an increase in mortality in the DEX-TCZ association group compared to the other two exposure groups, which was even more marked compared to the control group. Specifically, it was observed that in the group exposed to DEX 1 μmol/L and TCZ 442.1 μmol/L together, mortality was approximately 30% at 96 hpf. On the other hand, the TCZ 442.1 μmol/L and the solo DEX 1 μmol/L groups showed no particular differences compared to the control group at 96 hpf. No statistically significant malformations were found in the treated groups; only the DEX 1 μmol/L + TCZ 442.1 μmol/L group showed abnormalities in larvae embryogenesis. Spinal cord teratogenesis was the most common type of malformation detected in this group. The larvae hatching rate also showed a clear collapse in the group exposed to the DEX-TCZ group, compared to all other experimental groups. In fact, the DEX-TCZ group showed no hatched larvae at 72 hpf and an almost 40% reduction in the hatching rate at 96 hpf. The group containing the solo DEX 1 μmol/L showed a 10% decrease in hatching compared to the control, whereas the hatching of the TCZ-only group showed no appreciable difference compared to the control, and all larvae hatched at 96 hpf. Because hatching is such a crucial step in zebrafish development, structural and functional abnormalities throughout the embryonic stage were ascribed for the reduced hatching rate [38,39]. The difficulties of emerging larvae breaking the eggshell, [40] as well as the inhibition of mitosis or embryogenesis [41] all contributed to the developmental delay. The groups that showed significant abnormalities were the DEX 1 μmol/L-only group and the association group with TCZ 442.1 μmol/L. As noted in several previous studies, DEX triggers an inflammatory response and increases oxidative stress [42,43]. Oxidative stress occurs when the steady-state link between the formation of reactive oxygen species (ROS) and the body’s antioxidant defense capacity is disrupted. ROS is continuously produced by active metabolic activities under normal physiological conditions. Tissues have developed a complex array of antioxidant enzymes and free radical scavengers as a defense strategy against ROS [44,45]. Increased lipid peroxidation and DNA breakage in germ cells may be caused by reactive oxygen species (ROS). Antioxidant enzymes are the first line of defense against the harmful effects of reactive oxygen species (ROS) [29]. SOD is an enzyme that catalyzes the conversion of naturally-occurring but harmful superoxide radicals into molecular oxygen and hydrogen peroxide. Whereas, CAT catalyzed the decomposition of hydrogen peroxide to water and oxygen [30,46]. One of the most common processes resulting from oxidative stress is lipid peroxidation [47,48]. In the current study, we showed that, despite individually at the same concentrations statistically relevant toxic effects are not caused, DEX-TCZ co-treated larvae had considerably higher SOD and CAT activity, which resulted in dramatically enhanced lipid peroxidation. This was consistent with the results of other studies in different animal models [49], suggesting an increase in the oxidative pathway after co-exposure. Furthermore, the increased activity of the SOD and CAT antioxidant enzymes observed in our results has already been highlighted in other research, which also demonstrated that DEX is directly involved in cell death mechanisms [50]. Several studies have largely highlighted the involvement of oxidative stress in the activation of apoptosis processes [51,52]. These correlations between oxidative damage and cell death have also been demonstrated in several studies conducted in both larvae and adult zebrafish [53,54]. We employed Western Blot analysis to assess the protein levels of larvae exposed to the three solutions (DEX, TCZ, and DEX + TCZ) at 96 hpf to determine the possible causes of the harmful effects generated by DEX and its association with TCZ. The expression levels of apoptosis-related proteins (caspase-3 and bax) rose in DEX + TCZ exposure dose, according to our data, while anti-apoptotic protein bcl-2 expression was significantly inhibited in DEX 1 μmol/L + TCZ 442.1 μmol/L group larvae (p value < 0.0001).

5. Conclusions

In conclusion, co-exposure of DEX and TCZ showed altered antioxidant defenses, as well as increased lipid peroxidation, while the individual exposures showed no significant effects compared to the control group. The toxicity caused by co-exposure of DEX and TCZ not only led to defects in embryonic development, accompanied by an imbalance in antioxidant defenses, but also to an increase in the apoptotic process, in contrast to the single concentrations that reported no toxic effects. The presence of both drugs in the environment could result in increased toxicity to species living there. It is not yet clear on a mechanistic level, however, how these two drugs might interact in co-toxicity. Therefore, further studies will also be needed to assess any long-term effects.