There are increasing concerns over the detection of acetaminophen (APAP) in surface and ground waters across the globe. Its impacts on nontarget aquatic organisms have often been under monitored due to cost implications, with specific biomarkers in the exposed organisms increasing the underestimation of the impacts of APAP, thus generating some questions on the credibility of the choice of a single monitored marker. This study evaluated the possible multi-toxic effects of environmentally relevant concentrations of APAP in female catfish (Clarias gariepinus) exposed for 28 days using multiple biomarkers. Our results show that acetaminophen triggered oxidative stress, with superoxide dismutase (SOD), glutathione peroxidase (GPx) and total antioxidant capacity (TAC) activities significantly differing in the acetaminophen-exposed female catfish from the control. Histopathological alterations in the liver of acetaminophen-exposed female fish showed apoptotic hyperplasia, sinusoidal congestion, and necrosis of the hepatocytes, which were concentration-dependent. APAP exposure enhanced the fusion and shortening of some filaments, hyperplasia of the epithelial gill cells, aneurism, congestion and epithelial rupture of the gills and was ovarian toxic. Multivariate results indicated that there was a distinct response from the acetaminophen-exposed female catfish, with over 95% of the biomarkers significantly contributing to the discrimination between the acetaminophen-exposed female catfish and the control groups. Based on the data presented in this study for female catfish exposed to acetaminophen, it can be concluded that monitoring multiple biomarkers provides a more comprehensive picture of the exposed fish's health than single markers. As a result, pharmaceuticals may cause several alterations or multiple effects in nontargeted exposed organisms in the aquatic environment, yielding a less realistic risk assessment result in monitoring programs using a single marker. Our research provides evidence supporting the use of a multiple-biomarker approach to evaluate the impacts of drugs on the health status of female C. gariepinus over single markers.
Research Article
Multiple biomarker responses in female Clarias gariepinus exposed to acetaminophen
https://doi.org/10.21203/rs.3.rs-1011808/v4
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Acetaminophen
Immunotoxicology
Multiple biomarkers
female catfish
Sex hormone
Oxidative stress
• High levels of IL-6 and CRP confirm APAP-induced liver damage in exposed female fish.
• The histopathological evaluation of exposed organs provides evidence of the toxicity of APAP.
• Multivariate analysis indicates that multiple biomarkers offer more effective responses than a single biomarker.
Acetaminophen (APAP) (paracetamol) is the most widely used analgesic and antipyretic medication in the world, both alone and in combination with other medications to treat colds and pain (Guiloski et al., 2017), and accounts for over 458 deaths in the United States of America due to acute liver failure (ALF) (Lee 2004, Brune et al., 2014). The precise mechanism of action responsible for the analgesic effect of acetaminophen is still unknown despite its long history of use (Jozwiak-Bebenista and Nowak, 2014; Guiloski et al., 2017). Acetaminophen and its metabolites are endlessly introduced into aquatic habitats, mainly from clinics and domestic wastewater, personal use and waste discharges from drug production sites (Ebele et al. 2017; Zur et al., 2018). APAP reaches the environment mainly in an unaltered or slightly metabolized form (Zur et al. 2018), and due to the elevated and effective release rate, APAP and its associated metabolites (APAP, acetaminophen; APAP gluc, acetaminophen glucuronide; APAP-Cys, acetaminophen cysteine; NAPQI, N-acetyl-benzoquinone imine) are readily detected in treated wastewater, groundwater and rivers at varying concentrations (Mazaleuskaya et al., 2015; Folarin et al., 2019; Ebele et al., 2020).
Over the years, the demand for the consumption of acetaminophen has increased (Bloughz and Wu 2011, Gulmez et al., 2013; Moore and Moore, 2016), consequently leading to its high concentration of 1020 ng g-1-155.6 µg/L in our environment (Al-Mashaqbeh et al., 2019). APAP can react with hypochlorite during the water disinfection phase, leading to the occurrence of two extremely toxic compounds, 1,4-benzoquinone and hepatotoxic N-acetyl-p-benzoquinone imine, which can impact aquatic organisms (Bedner and MacCrehan, 2006; David and Pancharatna, 2009; Sung et al., 2014; Guiloski et al., 2017).
Several reports have shown that acetaminophen can act as a potential endocrine disruptor, induce oxidative stress, be hepatotoxic and neurotoxic and disrupt the physiological process in the exposed aquatic organism, hindering the usual embryonic development, growth, behaviour and survival of Daino rerio and Clarias gariepinus larvae (David and Pancharatna 2009, Erhunmwunse et al., 2021). Sublethal concentrations altered swimming behaviour, pigmentation and energy balance in Phalloceros harpagos (Matus et al., 2018). The drug induced endocrine disruption and altered the liver structure in male fish Rhamdia quelen (Guiloski et al., 2017) and caused oxidative stress in Clarias gariepinus (Folarin et al., 2018; Yakubu et al., 2020).
The need to assess the multiple effects associated with acetaminophen in a single exposed species is important and requires a holistic use of multiple biomarkers comprising haematological, biochemical, and histopathological indices. The collaborative use of a series of corresponding biomarkers that can specify exposure and measure the effects of acetaminophen on the exposed fish enables a more extensive and combined evaluation of biochemical and cellular impacts caused by acetaminophen (Linde-Arias et al., 2008; Cazenave et al., 2009). Hence, multibiomarker techniques have attracted significant attention in ecology and toxicological research and have been used in both uncontrolled and controlled experimental studies (Bacchetta et al., 2014, Javed et al., 2017). This study was therefore aimed at assessing multi-biomarker responses in Clarias gariepinus exposed to environmental concentrations of acetaminophen.
2.1. Fish
150 Female post-Juvenile C. gariepinus (20.25±1.76 cm (length) and 91.34±2.95 g (weight)) were selected from a pool of males and females obtained from the Faculty of Agriculture Farm Project and transferred to the aquatic trial section of the Ecotoxicology Laboratory, University of Benin, and conformed to laboratory conditions for 14 days in 250-L capacity aquaria (26 ± 1 °C). Female fish were used because endocrine disruptors have the potential to disrupt sensitive hormone pathways that control reproductive functions, which may result in decreased female fertility and egg production (Arcand-Hoy and Benson 1998) because estrogens are essential for normal ovarian development (Harding et al., 2013).
The fish were kept under a natural light/dark cycle and fed daily with Durante fish feed. The use of fish for this study was authorized by the Animal Experimentation Ethics Committee of the University of Benin, Faculty of Life Sciences, and all protocols followed the International Guidelines for Animal Use.
2.2 Chemicals purchase
Acetaminophen (≥ 98% purity, CAS Number 103-90-2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fish Vitellogenin ELISA (Cat. No. E0020Fi) was obtained from Biotechnology Laboratory (China). Interleukin 6 from CUSABIO Tech LLC (Catalogue No: CSB-E13258Fh TX, USA), SOD (CSB-E15929Fh), glutathione peroxidase (CSB-E15930Fh), total antioxidant capacity, high sensitivity C-reactive protein (CRP) ELISA, acetylcholinesterase, and catalase assay kits were obtained from Calbiotech Inc. (CA, USA).
2.3. Experimental Design
Experimental tests were carried out in 60-L glass aquaria in a static-renewal state under OECD guidelines No. 203; 229 and 230 (OECD, 1992, 2009, 2012) and APHA (1998). The experiments were carried out in 1:1 light-dark cycles over a 24-hr timeline. The water quality parameters of the experimental water were analysed by the methods of APHA (1998). The temperature had average values of 26.10±0.340°C, 6.50±1.01 (pH), 15.40±0.10 µs/cm (electrical conductivity), 6.20±0.34 mg/L (dissolved oxygen), 10.20±0.72 mg/L (total hardness), 0.03±1.50 mg/L (salinity), and 9.40±2.40 mg/L (total suspended solid). Fish (n = 7 per treatment) were exposed to five nominal concentrations of acetaminophen (15.5, 25.5, 35.5, and 45.5 µg/L µg/L) with two replicates each. A range-finding test was carried out by the procedure employed by Omitoyin et al. (1995) and Rahman et al. (2002) and was used to establish the concentration of acetaminophen for the definite test. A logarithmic dilution series was used for the range-finding assay. For the definitive test, stock solutions of the required concentrations were prepared. Acetaminophen powder (35.5 g) was dissolved in 1 L of distilled water. The stock was then serially diluted, and the required concentrations were used for the definitive test and prepared every 48 hrs.
2.4. Measurement of Biomarkers
Endpoints used to assess the sublethal toxicity of acetaminophen include alterations in physiological condition indices (condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI)), haematological alterations (full blood count), reproductive alterations (testosterone, 17β-oestradiol and plasma vitellogenin (VTG)), histopathological alterations (gills, liver and gonad (for post-juveniles) and alterations in oxidative stress and antioxidant defence markers (SOD/catalase, GST, glutathione peroxidase (GPx), acetylcholinesterase, total antioxidant capacity (TAC)) and immunological (IL-6, CRP). After 28 days of exposure, the fish were anaesthetized with 1% benzocaine. Blood was taken by perforating the genital opening with a heparin-coated 21 gauge ×0.5 inch needle attached to a 2 ml syringe of steroid hormones. The standard recovery assays were in the acceptable ranges as outlined by each test kit. The fish were put down by medullar section. The liver, gills and gonads were used for histopathological analysis
2.4.1 Condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI)
Calculation of gonadosomatic index (GSI): The GSI of the exposed female fish was calculated by the following formula.
2.4.2 Biomarkers of oxidative stress
2.4.2.1 Superoxide dismutase activity (SOD)
Superoxide dismutase levels in serum were measured using diagnostic reagent kits following the manufacturer's instructions (Cusabio Biotech Co., Ltd, China) instructions (Dawood, et al., 2021).
2.4.2.2 Catalase activity
Catalase was determined by measuring the H2O2 substrate remaining after the decomposition of hydrogen peroxide to H2O and O following the method of Ding et al., 2000. The assay reaction was performed at room temperature (25°C). Twenty-five microliters of blood agar was introduced into a test tube, followed by 75 µl of assay buffer. Another 25 µl of the substrate was added, mixed and incubated for 15 minutes, and then 825 µl of the stop solution was introduced into the mixture and dispersed. A 10 µl aliquot of the mixture was removed and introduced into another test tube. One milliliter of the chromogen reagent was added, mixed thoroughly and allowed to cool for 15 minutes for colour development. The resultant colour was read at 520 nm.
2.4.2.3. Glutathione peroxidase activity (GPx)
Glutathione peroxidase activity in plasma was determined by the oxidation of glutathione (GSH) by cumene hydroperoxides. The oxidized glutathione was reduced in the presence of glutathione reductase (GR) and NADPH. In this reaction, NADPH is oxidized to NADP+, and the absorbance was measured at 340 nm. The enzyme activity was expressed in μmol H2O2 min-1 mg of protein-1.
2.4.2.4. Total antioxidant capacity (TAC)
The serum's total antioxidant capacity was determined using the ferric reducing antioxidant power (FRAP) method (Teimouri et al., 2019). In the presence of antioxidants, the reduction of ferric to ferrous ions results in the formation of the coloured complex ferrous–tripyridyl-S-triazine, which is the basis of the FRAP test, and a blue solution with a maximum absorbance at 593 nm. At 593 nm, the optical absorption of standards and specimens was measured. At 593 nm, a standard curve was created using absorbance values of 125, 250, 500, and 1000 µm of iron sulfate as standard solutions. Using the standard curve concentration, the absorbance values obtained from the specimens were converted to concentrations.
2.4.3 Immunotoxicity Biomarkers
2.4.3.1. Determination of C reactive protein (CRP)
C-reactive protein was measured using an ELISA kit from CalBiotech (USA) (Kindmark, 1972).
2.4.3.2 IL-6 activity
Plasma IL-6 intensity was calculated using ELISA with a reactivity of 12.5 pg/mL using the accessible IL-6 ELISA fish kit as instructed by the manufacturer.
2.4.4 Reproductive biomarkers/steroid hormones
2.4.4.1 Determination of Vitellogenin ELISA
Assessment of plasma VTG was determined using an ELISA kit (Bioassay Technology Laboratory, China). The plates were supplied pre-coated with VTG fish antibodies. Forty microliters of fish plasma (VTG) introduced into the plate binds to the antibodies coated with the well. Fifty microliters of biotinylated fish VTG was added and bound to the sample VTG, followed by the addition of 50 µl of streptavidin-HRP to the solution and incubation for 60 minutes at 37°C. Plates were rinsed 5 times with buffer followed by the addition of 50 µl of substrate A and B simultaneously and incubated for 10 minutes at 37°C. Fifty microliters of stop solution was added, and a colour change from blue to yellow was observed. The optical density was read at 450 nm.
2.4.4.2 17β-Estradiol and Testosterone
Plasma 17β-estradiol was tested using the Estradiol EIA Kit (Cayman Chemical, USA), and the color formation response was assessed using an enzyme-linked immunosorbent assay (ELISA) with an absorbance of 405 nm. Testosterone was measured using the enzyme immunoassay inhibition method (ELISA) defined by Larsen et al. (1992).
2.4.5 Neurotransmitter Biomarker
2.4.5.1 Determination of acetylcholinesterase
Blood samples were diluted 40-fold in assay buffer. Ten microliters of sample was introduced into a 96-well microplate. The blank well contained 200 µl of distilled water. Then, 160 µl assay buffer was introduced to the sample wells, followed by 20 µl ACHE chromogen. The absorbance was read after 2 minutes at 413 nm and recorded. Then, 10 µl of ACHE substrate was added, separated and allowed to stand for 10 min. The absorbance was read again at 412 nm, the initial absorbance was recorded, and the values of acetylcholine were calculated.
2.4.6. Haematological Biomarkers
Haematology indices were determined following the method of Banaee et al., 2008. Red cell indices: mean corpuscular volume (mcv:fl/cell), and mean corpuscular haemoglobin (mch: pg /cell) were calculated from RBC (106/mm3), Ht (%) and Hb (g/dl) according to Lee et al., (1998)
2.4.7. Histopathological Biomarkers
Histological slides of the studied organs were developed following the method of Erhunmwunse et al. (2014). Organs were harvested and rinsed in saltwater to remove the blood and fixed in Bouin’s fixative for 24 hours. The organs were dehydrated in a graded series of alcohol (ethanol) and fixed in paraffin wax (M.P. 58-680c). Alterations caused by acetaminophen exposure in the organs were analyzed and photographed under a photomicroscope along with the control group. Tissue samples were sent to the Faculty of Basic Medicine, Department of Anatomy, University of Benin, for final histological processing and preparation (HE staining) for light microscopy analysis”.
2.5. Statistical Analysis
Statistical calculations were carried out with GraphPad Prism 8 software. Data are presented as the mean ± standard error of the mean (SEM). The parameters were calculated using one-way ANOVA followed by Dunnett’s multicomparison test to denote the significant differences between acetaminophen concentrations and the control treatment. Differences were considered significant at p≤0.05. Due to a large number of biomarkers analysed, principal component analysis (PCA) was used to correlate acetaminophen-exposed fish with the control group with the responses of biomarkers.
3.1. Physiological Condition Indices
The fish morphometric indices (K and HIS) were not significantly affected (p>0.05) by exposure to acetaminophen (Fig. 1 a & b) but led to slight increases in all exposed groups, as the condition factors of the fish increased by 24.14, 6.89, 3.45 and 1.72% at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively, compared to the control. In HIS, increases of 33.83 and 30.61% were observed at 15.50 and 25.50 μg/L, respectively; however, decreases in HIS values were observed in fish exposed to 35.5 and 45.5 μg/L by 3.68 and 9.53%, respectively (Fig. 1). The gonadosomatic index of the fish was significantly reduced (p<0.05) by acetaminophen exposure compared to the control groups. The reductions were 51.90, 80.76, 73.99 and 78.09% at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively (Fig. 1).
3.2. Oxidative Stress Biomarkers
The SOD enzyme activities were significantly increased (p<0.05) in all acetaminophen-exposed groups compared to the control. Significant differential increases of 189.29, 89.29, 153.57 and 71.43% were observed at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively (Fig. 2A).
Acetaminophen exposure caused an increase in catalase activity (Fig. 2B). The catalase activity increased with an increasing concentration of acetaminophen, with the highest concentration of 45.5 µg/L having the highest catalase activity in all plasma samples studied. The differential increase in catalase activity was in the sequence of 45.5>15.50>25.50>35.50 µg/L. Increases of 23.25, 21.37, 14.90 and 25.49% at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively, were observed compared to the control.
The measured GST activities of fish are shown in Fig. 2C. Acetaminophen exposures did not cause any noticeable change (p>0.05) in GST activities and were found to fluctuate as GST activities were reduced in 15.50 and 35.50 µg/L by 21.00 and 14.28% and increased in 25.50 and 45.50 µg/L by 28.57 and 50.0% increase, respectively (Fig. 2C).
Fish exposed to acetaminophen revealed a remarkable reduction in GPx activity compared to the control (Fig. 2D). The differential decrease in the GPx did not show a dose-response pattern, with 74.00, 36.42, 70.71 and 68.57% increases in 15.50, 25.50, 35.50 and 45.50 µg/L, respectively, compared to the control.
Acetaminophen caused an increase in TAC activities in all acetaminophen-exposed female catfish compared to the control. Significant increases were observed at 25.5 and 45.5 µg/L (Fig. 2E). Increases of 122.72, 404.54, 309.09 and 400% were recorded at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively.
3.3. Neurotransmitter
In terms of AChE induction, there were no significant increases (p>0.05) in the acetaminophen-treated groups compared to the control. Extraneous increases in AChE activities at 15.50, 35.50 and 45.50 µg/L were 08.09, 11.51 and 7.80%, respectively, and a nonsignificant reduction of 06.38% was observed at 25.50 µg/L (Fig. 3A).
3.4. Immunological Markers
Immunological analysis of IL-6 demonstrated that exposure to acetaminophen caused nonsignificant increases of 21.02, 6.25, 34.09 and 21.02% at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively (Fig. 3B). C-reactive protein (CRP) activities were significantly increased (p<0.0001) in all acetaminophen-exposed groups compared to the control (Fig. 3D). The increases were 136.59, 131.71, 136.59 and 136.59% at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively.
3.5. Haematological parameters
There were increases in white blood cells (WBCs) of acetaminophen-exposed fish relative to the control. Increases of 101.28, 113.86, 112.57 and 61.00% were reported in WBCs at different concentrations of 15.50, 25.50, 35.50 and 45.50 μg/L (Table 1). Red blood cells (RBC), haemoglobin (HGB) and haematocrit (HCT) fluctuated in the acetaminophen-exposed groups as opposed to the control. The HGB responses were found to fluctuate by 118.41, 11.64, 71.98 and 27.93% at concentrations of 15.50, 25.50, 53.50 and 45.50 μg/L, respectively. An increase of 60 and 26.65% at 15.50 and 35.50 μg/L and a decrease of 35.09 and 5.81% at 25.50 and 45.50 μg/L were documented in HCT. Acetaminophen caused increases in mean cell volume (MCV) and mean cell haemoglobin (MCH) in all exposed groups, while the MCHC count was not affected. Platelet count (PLT) increased in all exposed fish and is predicted to decrease at 15.50 μg/L. Lymphocytes increased by 81.49, 90.34 and 48.03% in fish exposed to 15.50, 35.50 and 43.50 μg/L, respectively, while decreases in the community exposed to 25.50 μg/L were observed.
Table 1: Haematological indices responses in female catfish exposed to acetaminophen
|
Acetaminophen (µg/L) |
|||||
|
Parameters |
0.00 |
15.50 |
25.50 |
35.50 |
45.50 |
|
WBC |
35.00±1.00 |
70.45±16.50 |
74.85±20.15 |
74.40±19.40 |
56.35±2.95 |
|
RBC |
1.07±0.01 |
1.66±0.45 |
0.67±0.39 |
1.33±1.13 |
0.98±0.07 |
|
Lym (%) |
36.75±1.05 |
66.70±14.40 |
30.90±21.90 |
69.95±15.95 |
54.40±2.60 |
|
HGB |
4.65±1.58 |
10.15±2.69 |
4.10±2.40 |
7.99±6.84 |
5.95±0.68 |
|
HCT |
18.95±0.25 |
30.45±8.05 |
12.30±7.20 |
24.00±20.50 |
17.85±2.10 |
|
MCV |
173.20±3.19 |
183.8±1.32 |
186.4±2.42 |
178.30±3.32 |
181.5±7.90 |
|
MCHC |
57.91±1.06 |
61.23±0.43 |
62.14±0.82 |
59.27±1.27 |
60.47±2.67 |
|
PLT |
29.00±14.00 |
23.50±9.50 |
50.00±34.00 |
39.50±9.50 |
45.00±17.00 |
3.6. Reproductive Biomarkers
Acetaminophen caused a significant increase (p<0.05) in the VTG of exposed fish. Increases of 98.21, 91.34, 14.03 and 970.75% at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively, were recorded in the acetaminophen-exposed fish. The differential increases in VTG induction were in the sequence of 45.5>15.50>25.50>35.50 µg/L. The levels of testosterone were significantly reduced in all acetaminophen-exposed fish by 19.71, 13.84 and 37.12% at 25.50, 35.50 and 45.50 µg/L, respectively (Fig.4). Acetaminophen exposure caused 17β-estradiol increases in all exposed groups compared to the control. Fish from the 35.5 µg/L acetaminophen-exposed group had a significantly lower level of 17β-estradiol (p < 0.05) than the controls. The levels of 17β-estradiol in exposed fish increased by 41.58, 33.22, 30.82 and 31.74% at 15.50, 25.50, 35.50 and 45.50 µg/L, respectively, compared to the control.
3.7.0. Histopathology
3.7.1. Gills
Acetaminophen exposure caused histopathological alterations in the gills of exposed fish (Fig. 5). The common changes observed in the gills of acetaminophen-exposed fish were large elevation of the lamellar epithelium, edema of the lamellae, blood congestion, exfoliated epithelium of lamellae, lamellar disorganization, hypertrophy and hyperplasia of the epithelial cell, which resulted in the incomplete merging of the secondary lamellae and a reduced water space at 45.50, 35.50 and 25.50 µg/L. Hemorrhagic telangiectasia, fatty lobes, biliary atresia, congestion, Kupffer cells, apoptotic hepatocytes and inflammatory cell infiltration characterized the gills of fish exposed to 15.50 µg/L.
3.7.2. Liver
The alterations in the liver of acetaminophen-exposed C. gariepinus are shown in Fig 6. Fish in the control group showed normal hepatic structure and parenchyma. Histological evaluation of prepared slides revealed all exposed groups had various degree of alterations compared to the control which showed normal architectural structure of the liver with the central vein and hepatocytes intact. Acetaminophen-exposed fish showed histological alterations which include; IFP: Infiltration in liver parenchyma; SCN: Single cell necrosis; TTA: thickening of the tunica adventitia; VH: vacuolation of hepatocytes; FT: Fatty tissue; CMMC: Congestion of melanomacrophage centers; C: Congestion/Blood congestion; DCV: Dilation of central vein; ALPA: Aggregation of lymphocytes in portal area characterized the female fish liver exposed to 15.50-45.50 µg/L.
3.7.3. Gonads
Acetaminophen exposure caused histological changes in the gonads of the acetaminophen-exposed catfish (Fig. 7). Ovarian examination of catfish and the corresponding changes were observed at various concentrations of acetaminophen. There were no reported alterations in the gonads of fish in the control group (Figure 7a), as the photomicrograph showed oocytes in the perinucleolar phase (primary growth oocytes). Acetaminophen exposure caused several alterations in the exposed groups compared to controls and degenerative nuclei; degenerative follicles were noticed in the ovarian structure in the experimental groups. In the 15.50 µg/L group, histopathological alterations were observed to a minimum degree and included disintegrative follicles, follicle atresia (Figure 7b). An increase in the primary oocyte numbers and degenerating oocytes or atretic oocytes (AO) were observed at 25.50 µg/L (Figure 7c). The female gonad exposed to 35.50 µg/L showed alterations, including increased atretic follicles, the appearance of fat deposits (yellow patches), degenerative nuclei and ovarian hyperplasia (Figure 7d). Disintegration in ovarian structures was observed for the highest concentration. (Figure 7d). Others include the large occurrence of atretic follicles, yellow patches (fat)
3.7.4 Multivariate analysis
The results of the PCA of the fish exposed to various concentrations of acetaminophen plotted against the fish in the control are shown in Figure 8. There was a clear distinction between the fish in the control on the left side of the plot and the acetaminophen-exposed fish on the right side of the plot. Figure 8 also reveals that the pattern of most biomarkers in acetaminophen-exposed fish is strongly correlated with increasing concentration.
4.1 Effect of acetaminophen on physiological condition indices and oxidative stress biomarkers
Exposure to acetaminophen at environmentally relevant concentrations had distinct effects in female post-juvenile Clarias gariepinus. Morphometric analyses demonstrated that the HIS and K of the acetaminophen-exposed fish did show any significant change, which was similar to the report of Stancova et al. (2014), who observed no changes in the growth parameters of Tench (Tinca tinca) exposed to a combination of ibuprofen, diclofenac, and carbamazepine for 35 days. Acetaminophen significantly reduced the gonadosomatic index of exposed female C. gariepinus and agrees with the report of Mills et al. 2011, who showed a significant decrease in the gonadosomatic index of the marine fish Cunner (Tautogolabrus adspersus) exposed to an anticancer drug, anastrozole.
The biotransformation pathway of APAP has shown that the toxicity is a result of the formation of the very active metabolite N-acetyl-p-benzo-quinone Imine (NAPQ1) by cytochrome P450s (Macherey, 2015; Ramos-Tavar and Muriel, 2019) when consumed at nonpharmacologic doses. At high doses, the pathways for the transformation of APAA (sulfation and glucuronidation) become saturated, and a portion of APAP is excreted in the parent form, leading to excess NAPQ1, which further reacts with other protein groups to form protein adducts (McGill et al., 2013), leading to oxidative stress and mitochondrial dysfunction and resulting in the production of free radicals.
The increased SOD enzyme activities in the exposed female catfish must have been triggered by the presence of acetaminophen, which produced reactive oxygen species (ROS) (Halliwell and Gutteridge, 1999). As the levels of ROS increase, the biological system creates a first-line defence mechanism by altering the behaviours of SOD (Roberts and Oris, 2004; Bagnyukova et al., 2006). Increased SOD activity is an indication of increased antioxidant status that attempts to neutralize the ROS effect (Kurutas, 2016). The elevated CAT activity in our study might be a result of the functioning of the defense mechanism, which counteracts the oxidative stress induced by acetaminophen metabolism in exposed female catfish (Li et al., 2010; Shukla et al., 2017). Acetaminophens have been reported to disturb the redox status of the organism (Trachootham et al. 2008), and CAT levels are an excellent marker of protein oxidation and lipid peroxidation (Bohn, 2019).
Several authors have hitherto detailed the significance of GST, GPx, and GSH in averting cellular destruction (Livingstone, 2001; Guiloski et al., 2015). The depletions in GPx activities observed in acetaminophen-exposed female catfish may have been a sign of the induced distress in the fish and agrees with the findings of Guiloski et al. (2015), who reported a decline in GPx activities in gonads of H. malabaricus exposed to dexamethasone.
GST is a phase II detoxifying enzyme that catalyzes the conjugation of the reduced form of glutathione to xenobiotics for detoxification (Stancova et al., 2017). GST is known to be the first line of defence preventing oxidative stress damage, decomposition of superoxide radicals and hydrogen peroxide before interacting to form reactive hydroxyl, which has many unfavourable biological consequences when present in high concentrations (Kaur et al., 2017). The increase in GST enzyme activity in exposed female catfish may have represented the occurrence of a defensive mechanism to prevent the impact of acetaminophen, as indicated by Sayeed et al. (2003).
The increase in TAC activity in exposed female catfish could have been triggered by oxidative stress in the exposed fish, which may be due to an increase in antioxidant activity employing a cellular defensive mechanism to achieve homeostasis and, in some way, adaptation in the management of antioxidant stress (Kohen et al., 2000, Castillo et al., 2006). A similar report of an increase in TAC activity is comparable to the report of Mantle et al. (2000), who observed an increase in TAC activity in humans administered the perioperative anesthetics dopamine, propofol, dobutamine and noradrenaline.
4.2 Immunological Biomarker Alteration
C-reactive protein (CRP) is an acute inflammatory protein that can increase its activity at infection or inflammation sites (Sproston and Ashworth, 2018). While it is mainly generated by hepatocytes in the liver, it can also be produced by smooth muscle cells, macrophages, and endothelial cells. CRP is involved in inflammatory processes and host responses to infection, such as the complement pathway, apoptosis, phagocytosis, and the development of cytokines (IL), specifically interleukin-6 and tumour necrosis factor-α (Sproston and Ashworth, 2018; Pathak and Agrawal 2019).
It has been established in the previous paragraph that in the metabolism of APAP, abundant NAPQI use up the glutathione (GSH) of the cell and bond the proteins covalently, leading to mitochondrial dysfunction (Fig 6) and eventually the death of cells (Hinson et al., 2010; McGill et al., 2012). When parenchymal hepatocytes are impaired, innate immunity plays a role in the development and enhancement of liver injury triggered by APAP. The induction is marked by the release of proinflammatory cytokines and chemokines by activated Kupffer cells (resident hepatic macrophages), which leads to signal propagation marked by the facilitation of neutrophil adhesion and transmigration into the hepatic vasculature, which aggravates liver injury (Liu et al., 2004; Liao et al., 2016).
Increased C-reactive protein (CRP) in all acetaminophen-exposed female catfish may have resulted from the entrance of proinflammatory cytokines into the circulatory system of the exposed fish due to the presence of acetaminophen (Gani et al. 2009). Our study revealed that acetaminophen induces the expression of CRP in exposed fish, which may be an indication that the synthesis of CRP is part of the host defence reaction to the toxic effect of acetaminophen. In the recent past, the innate immune system of fish has drawn attention and is considered to be crucial in primary defence and adaptive immunity in fish (Whyte, 2007). Acetaminophen exposure caused elevated hepatocyte injuries, as seen in the histopathological analysis of the liver of the exposed fish, and the increased level of CRP shows a protective function (Sproston and Ashworth 2018). Similarly, Ghosh et al. (1993) and Kodama et al. (2004) documented a rise in CRP activity in fish exposed to carbaryl and formalin.
Interleukin-6 (IL-6) is one of the most pleiotropic cytokines due to its function in both innate and adaptive immune responses and other physiological processes (Varela et al. 2012). Our study showed increased IL-6 activity in acetaminophen-exposed female catfish. This increase may be an indication that IL-6 returned the exposed fish to a homeostatic condition, and it has been stated that IL-6 regulates the magnitude of tissue inflammatory responses (Gabay, 2006, Hong et al., 2013). Interleukin (IL)-6 is developed at the site of inflammation and plays a significant role in the acute phase response as defined by a variety of diagnostic and biological characteristics, such as the production of acute-phase proteins (Gabay, 2006). IL-6 is an inflammatory cytokine that is freed from monocytes and lymphocytes at sites of tissue injury and may have been freed from the severe liver and gill injuries observed in this study (Gani et al., 2009). Several authors (Kopf et al. 1994, Xing et al. 1994, Romani et al., 1996, Ruzek et al., 1997, Gabay, 2006) have noted that chronic disorders, usually evidenced by immune stressors such as chronic intracellular infection and tumours, IL-6 not only induce acute phase reactions but also perform an important role in producing cellular immune responses to affected cells and mucosal humoral responses to re-infection.
4.4 Neurotransmitter Biomarker
The inhibition of AChE in all acetaminophen-exposed female catfish suggests an adverse consequence in cholinergic neurotransmission and probably in nervous and neuromuscular function (Ribeiro et al., 2017). The normalization of the enzyme activity in the exposed fish compared to the control can be due to the compensatory response of the fish cell to the toxic effects induced by acetaminophen exposure. This study has shown the susceptibility of AChE activities in the plasma of acetaminophen-exposed female C. gariepinus (Mdegela et al., 2010). These results indicate that the female catfish (C. gariepinus) is a model organism for pollutant monitoring and evaluation (Mdegela et al., 2006) and that the activity of AChE in C. gariepinus is a prospective diagnostic tool for the environmental pollution assessment of anticholinesterases.
4.5 Haematological Indices
There were increased responses in most haematological parameters of exposed female catfish except MCV and MCH after the 28-day exposure period. Acetaminophen-exposed female catfish showed an increase in WBC counts, which indicates an immune and protective response (Saravanan et al., 2011) to the presence of acetaminophen. The increased WBC counts show that C. gariepinus develops a protective mechanism to control the toxic stress caused by acetaminophen exposure (Mohammod Mostakim et al., 2015). Similarly, ibuprofen and clofibric acid increased WBCs in exposed C. carpio and Cirrhinus mrigala (Saravanan et al., 2011, 2012). Again, the observed increased WBC count may reveal physiological and immunological (inflammatory) challenges in response to the toxic impact of acetaminophen (Alimba et al., 2019). The observed reduction in RBC counts at 25.50 µg/L might be due to the inhibition of RBC production by acetaminophen. The reduction in RBC counts in fish may show the anaemic state of the fish under stress conditions (Sigbojorn and Tatjana, 2015). Acetaminophen exposure resulted in increased HGB, HCT, lymphocyte, and platelet counts, which may be ascribed to the stimulation of the hematopoietic system of the female catfish caused by the exposure.
4.6 Reproductive Markers
Vitellogenin (VTG) is a known biomarker for the confirmation of endocrine activity in fish, and it is used in numerous OECD test guidelines (TGs) to detect the activities of chemicals on hormonal pathways (Baumann et al. 2019). It is typically found only in the plasma of female fish, although males do have the VTG gene, which can be easily induced by exogenous estrogens (Hutchinson et al., 2006). There was an increased induction in the levels of VTG in all acetaminophen-exposed female catfish, which agrees with the acetaminophen concentration range previously reported by Ayabohan et al., 2020, where female zebrafish exposed to 79 µg/L acetaminophen for 21 days had increased VTG compared to the control. According to Tyler et al. (1990), plasma VTG concentrations can increase up to a millionfold, from nanograms per millilitre to milligrams per millilitre depending on the specificity and magnitude. After a 29-day exposure, significant increases in VTG (4 times the control level) levels were observed in juvenile female rainbow trout caged in the impact and postimpact areas in a pulp mill discharge area (Barra et al., 2021). Induction of VTG has been reported in fish following exposure to pharmaceuticals, heavy metals and pesticides (Versonnen et al. 2003, Hook et al. 2014). The increased VTG activities in all exposed catfish correspond to the fact that estradiol induces high levels of VTG (Versonnen et al. 2003). This study thus suggests that further studies are required to determine whether analgesic drugs (acetaminophen) are capable of endocrine disruption via estrogenic effects in female fish. As expected in female fish, there was a concentration-dependent increase in 17-estradiol with a corresponding decrease in T. However, these increases did not occur in the absence of the acetaminophen-exposed female fish when compared to the control.
4.7 Histopathology Biomarkers
Histological changes are associated with complex biochemical and physiological responses to any stressor (Lushchak et al. 2018). Tissue histology is seen as a sign of exposure to contaminants and is an essential apparatus for assessing pollution levels, especially for short-term exposure and prolonged effects (Mostakim et al. 2015). Changes in fish tissue histology have been commonly used, both in controlled experiments and field studies, to assess fish health (Mela et al. 2007, Erhunmwunse et al. 2014, Alimba et al. 2015, Alimba et al. 2019).
Injury to gill tissue can impede the gas exchange capacity of the gill and cause respiratory disturbances, ion modulation, osmoregulation dysfunction, and inefficiency in the removal of waste nitrogen metabolites in exposed fish (Nero et al. 2006; Cengiz and Unlu, 2006; Velmurugan et al. 2007; Banaee, 2013). The gill morphology showed disruptions in the lamellar epithelium, gill hyperplasia, cell proliferation, and lamellar fusion in the gills of acetaminophen-exposed fish. In addition, secondary lamellae often had vascular lesions, such as aneurysms, caused by cell disruption and hemorrhage. These findings indicate a reduced respiratory potential and the development of osmotic imbalances due to acetaminophen-induced stress. Lamellar fusion occurs as a result of the excessive proliferation of filament epithelial cells and is an innate defensive mechanism to shield the lamellar epithelium from direct interaction with acetaminophen (Flores-Lopes and Thomaz 2011; Hadi and Alwan 2012). The fusion and hyperplasia of gill lamellae can be caused by the effects of acetaminophen altering glycoprotein in the cell-covered mucus, influencing the negative charge of the epithelium and favoring adhesion to adjacent lamellae (Hadi and Alwan 2012; Malarvizhi et al., 2012). The presence of lamellar telangiectasia in the gills of exposed female catfish may have resulted from the break-up of elongated bodies and capillaries under the effect of acetaminophen, which leads to the accumulation of erythrocytes in the distal section of the secondary lamellae (Randi et al. 1996; Hadi and Alwan 2012). Changes such as epithelial lifting, hyperplasia and hypertrophy of the epithelial cells, in addition to partial fusion of some secondary lamellae, are examples of defence mechanisms, since in general, these result in an increase in the distance between the external milieu and the blood (Hadi and Alwan 2012). Our findings corroborate those of Kavitha et al. (2011), who found that acetaminophen causes gill proliferation, lamellae fusion, and vascular congestion in exposed Oreochromis mossambicus. In acetaminophen-exposed fish, Oncorhynchus mykiss Choi et al., 2018 reported filament end swelling, whole filament swelling, and lamellae swelling.
The liver is primarily involved in the metabolism, detoxification, storage, and removal of xenobiotics and their metabolites (Mela et al. 2007, Alimba et al. 2019). The liver of the female C. gariepinus exposed to acetaminophen showed histopathological changes, according to our findings (Fig 6A-E). The current study's findings can be used as a baseline study, and the results may be used in future pharmaceutical monitoring studies in Nigerian waters by river basin authorities. The liver tissues of female C. gariepinus showed a wide range of histopathological changes (infiltration, hepatocyte vacuolization, increased in MMCs, congestion and fat deposits). The degree of change in the liver tissues of female catfish is not unrelated to acetaminophen exposure. In the liver of Danio rerio exposed to much lower concentrations of acetaminophen, similar changes (congestions, infiltrations, macro vascular fat degeneration) were observed (0.125, 0.250 and 0.500µg/L) (Elizalde-Velazquez ´ et al., 2022). In exposed fresh water fish (Oreochromis mossambicus) acetaminophen-treated liver cells show hepatocellular vacuolization and blood vessel congestion (Kavitha et al., 2011). Furthermore, hepatocellular vacuolization and degeneration characterized the liver tissues of freshwater fish Labeo rohita exposed to acetaminophen (Raja and Bharathi 2018). Iftikhar et al., 2022 reported similar histopathological alterations (vacuolization, melanomacrophages and blood congestion) in Cyprinus carpio exposed to sulfamethoxazole for 28days. Although in male fish, Guiloski et al., 2017 reported similar alterations (blood congestion and multifocal leukocyte infiltration) in the liver tissues of male Rhamdia quelen exposed to acetaminophen.
According to Elizalde-Velázquez et al., 2022 and Guiloski et al., 2017, the ability of acetaminophen to disrupt cell-to-cell integrity and cause liver injury in both humans and fish may have caused the observed changes in liver tissues of exposed female C. gariepinus. As shown in experiments with rainbow trout, Japanese medaka Oryzias latipes, and guppies Poecilia reticulata exposed to organotins and tri-n-butyltin chloride (TBTC), toxic exposure can result in fat accumulation (yellow patches) in the liver (Wolf and Wolfe, 2005). Fat/glycogen accumulation in our study could be due to decreased glycogen breakdown as a result of acetaminophen's hepatocellular toxicity.
Gonad histological analysis proved useful in understanding and evaluating the impacts of possible endocrine-disrupting compounds on aquatic organisms (Leino et al. 2005). Acetaminophen-exposed female catfish showed several altered ovarian structures with patches of degenerative follicles, follicular atresia and degenerative nuclei. Atresia follicles were lower in lower concentrations of acetaminophen exposure. Increased follicle atresia in the ovaries of estrogen-exposed fish has been reported by some authors (Länge et al., 2001; Metcalfe et al., 2001; Zillioux et al., 2001; Kidd et al., 2007; Kaptaner and Ünal, 2011). Acetaminophen caused degenerative nuclei and follicles, eccentric nuclei, ovarian cell hyperplasia and destructive ovarian walls. Similarly, some classes of phthalates have been shown to cause atresia of the follicle and degenerative follicles in both humans and fish (Hannon and Flaws 2015).
4.8 Multivariate statistical analysis
Cazenave et al., 2009 highlighted the significant use of arrays of biomarkers when assessing biological effects in polluted environments, since one marker may not show the true status of exposed organisms. Beliaeff and Burgeot (2002) have stressed the need for researchers to carefully select suitable fusion of markers that can yield data about universal detrimental environmental effects. The statistical analyses of our results distinctly confirm that the use of a single biomarker analysis was not enough to detect alterations induced by acetaminophen in the exposed fish. Nevertheless, the assessment of multiple biomarkers in physiological, biochemical, oxidative, reproductive, immunological and neurological activities in C. gariepinus validates the visible differentiation between the control groups and the acetaminophen-exposed groups. Multivariate statistical analysis (Figure 8) indicated that female catfish in the control groups exhibited a distinct response from the acetaminophen-exposed female catfish and that over 95% of the biomarkers significantly contributed to discriminating between the acetaminophen-exposed fish and the control group. Figure 9 denotes that acetaminophen-exposed female catfish may have undergone several stresses, possibly due to exposure to different acetaminophen concentrations.
The consideration of multiple biomarkers in female catfish for the evaluation of a single pharmaceutical will be a useful tool in assessing the effects of pharmaceuticals in exposed fish. The multiple biomarker approach in this study provides insight into the capacity of APAP to cause multiple changes from the cellular to organismal level and reduces the possibility of bias in single biomarker evaluation. The multiple biomarkers provided more information on the overall health status of the APAP-exposed fish, including the induction of oxidative stress, activation of the immune response system, alteration of the haematological profile and oxidative damage in many tissues. The studied organs (gills, liver and gonads) provided better information on the hepatotoxicity, neurotoxicity and immunomodulatory effect of APAP at varying concentrations. Our study has shown that multiple biomarker evaluation revealed consistent evidence on the health status of the female catfish and provides better vigorous information that is obvious in justifying the multiple effects of APAP previously misjudged and documented. The multivariate analysis further justifies the use of multiple biomarkers in the potential unknown impact of APAP in exposed fish and agrees with the summation of Georgieva et al., 2021 that in order to develop a protocol for the aquatic assessment of pollutants; a multi-biomarker approach must be used. The said protocol can be used in risk evaluation and water monitoring programs to provide a legal basis for the presence of organic pollutants in aquatic ecosystems and biological responses at concentrations that are equal to or lower than those allowed by country legislation. Therefore, we recommend the use of multiple biomarkers for the evaluation of fish fitness and toxicological impacts of PPCPs.
Acknowledgements
This study was supported in part by the TETFUND Internal Based Research (TBR) 2017/2018.
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