Paracetamol has already been described as one of the most potent endocrine disruptors. Thus, it cannot only affect hormone levels, as commonly seen, but also promote adverse effects on various tissues and cellular systems of the endocrine system in fish (Kar et al. 2021).
The gonadosomatic (GSI) and hepatosomatic (HSI) indexes did not present significant alterations compared to control (Figure 1). However, fish exposed to 25 µg.L-1 of PCM have presented higher HSI than those exposed to 0.25 µg.L-1 (Figure 1B). Plasma 17β-estradiol and 11 – keto testosterone did not present significant alterations in fish exposed to PCM, but fish exposed to 2.5 and 25 µg.L-1 of PCM showed a reduction of approximately 66% in 17β-estradiol concentrations comparing to fish from control group (Table 1).
The hypothalamic cyp19a1b and the hepatic vtg expression were not significantly affected by PCM exposure after exposure for 14 days (Figure 2). However, it has been reported that exposure to PCM for 90 days resulted in concentration-dependent increase of the the vitellogenin protein concentration in liver of male medaka fish (Oryzias latipes), from 0.095 to 950 µg.L-1 (Kim et al. 2019). Male fish only produce large amounts of vitellogenin when they are exposed to substances that stimulate or mimic estradiol and thus the production of this protein, like some endocrine disruptors (Kleinkauf et al. 2004), as PCM. In Rhamdia quelen males, exposed to 0.25 µg.L-1 and 2.5 µg.L-1 of PCM for 21 days, a reduction in 11 – keto testosterone plasma levels and an increase in estradiol levels was observed (Guiloski et al. 2017). Altogether, these results demonstrate that exposure time can be related to the PCM endocrine disrupting capacity, at least in male fish, and it seems that it takes more than 14 days of exposure for these effects to be observed.
TABLE 1: Plasmatic 17β estradiol and 11 – keto testosterone of Rhamdia quelen males exposed to paracetamol for 14 days.
Hormone
|
Paracetamol concentration
|
Control
|
0.25 µg.L-1
|
2.5 µg.L-1
|
25 µg.L-1
|
17β estradiol
|
208.96±67.68a
|
115.75±51.76a
|
69.41±21.41a
|
70.8±6.02a
|
11 – keto testosterone
|
1754.74±145.44a
|
1615.36±261.56a
|
1615.74±344.57a
|
1953.01±97.57a
|
* Results are expressed as mean ± standard error. Different letters indicate significant differences (p <0.05). 17β estradiol and 11 – keto testosterone = pg.mL-1
However, some biochemical effects were observed in the R. quelen liver and gonads exposed to PCM for 14 days. In the liver, EROD and GST, enzymes related to the biotransformation process, were not significantly altered in the PCM presence. Although, GSH presented a significant increase in fish from 2.5 µg.L-1 group compared to the fish from solvent control and 0.25 µg.L-1 groups. Regarding the antioxidant system, CAT and GPx enzymes were not significantly affected in fish PCM exposed groups. However, SOD activity was reduced in fish from 0.25 µg.L-1 group compared to those from solvent control and 25 µg.L-1 groups. The fish from 0.25 µg.L-1 group presented lower LPO levels than those observed in the fish from other groups, including solvent control (Table 2).
In this study, 14 days were not enough to alter metabolism enzymes, unlike R. quelen males exposed to paracetamol for 21 days, that biotransformation enzyme activity was reduced and SOD was increased (Guiloski et al. 2017). During the PCM metabolism, the cell produces N-acetyl-p-benzoquinoneamine (NAPQI), a toxic bioactive metabolite that therefore must be excreted so that it does not cause oxidative stress (Santos et al. 2020). One way this neutralization occurs is through conjugation with glutathione, the GSH, which occurred in the liver of animals exposed to 2.5 µg.L-1 paracetamol for 14 days.
In gonads, GST activity decreased in fish from 25 µg.L-1 group compared to solvent control. SOD had its activity increased by 75% in fish from 0.25 µg.L-1 group compared to those from 25 µg.L-1. Fish from groups exposed to PCM showed a reduction in GPx activity when compared to the solvent control. LPO levels were not significantly altered by PCM exposure (Table 3). In this way, changes in the antioxidant system of the gonads were observed. Cytochrome P450 enzymes (CYP450) are important to pharmaceuticals detoxification, while glutathione protects the body against chemically reactive compounds (Burkina et al. 2015). Thus, paracetamol proved capable of altering the gonads biotransformation and antioxidant system after 14 days of exposure; however, these alterations were sufficient to prevent damage to macromolecules such as lipids.
Many factors can affect an endocrine disruptor effect. Some modulating factors must be taken into account, such as temperature, photoperiod, water quality, feeding rate, feed composition, population density, population sex ratio and individual genetics (Wang 2018). For example, differences of 48 h EC50 results from paracetamol to Daphnia magna have already been described. One of the possible justification for such results is the nutrition. This can affect glutathione metabolism, which is a metabolic pathway used for PCM metabolism and excretion, and feed composition is something that may vary between studies (Nunes et al. 2014).
TABLE 2: Biochemical biomarkers in the liver of Rhamdia quelen males exposed to paracetamol for 14 days.
Biomarker
|
Paracetamol concentrations
|
Solvent control
|
0.25 µg.L-1
|
2.5 µg.L-1
|
25 µg.L-1
|
EROD
|
129.86±23.56a
|
98.56±14.69a
|
94.64±8.47a
|
83.49±9.90a
|
GST
|
120.15±6.06a
|
130.39±5.91a
|
113.86±4.47a
|
118.49±4.15a
|
GSH
|
16.06±3.21a
|
13.36±1.91a
|
33.69±4.73b
|
19.95±2.48ab
|
SOD
|
200.11±12.45a
|
159.81±7.42b
|
189.05±14.15ab
|
245.66±12.32a
|
CAT
|
36.01±3.32a
|
45.44±2.82a
|
36.28±4.15a
|
38.52±2.83a
|
GPx
|
167.90±17.34a
|
198.13±6.61a
|
175.16±22.71a
|
180.52±15.66a
|
LPO
|
15.66±2.73a
|
7.03±1.55b
|
25.89±4.44a
|
20.85±1.71a
|
* EROD activity = ƒmol of resorufin.min-1.mg protein-1; GST and GPx activity = nmol.min-1.mg protein-1; SOD activity = U.mg protein-1; CAT activity = µmol. min-1.mg protein-1; GSH concentration = µg.mg protein-1; LPO concentration = μmol of hydroperoxides.mg protein-1. The results are expressed as mean ± standard error. Different letters indicate significant differences (p <0.05).
TABLE 3: Biochemical biomarkers in the gonads of Rhamdia quelen males exposed to paracetamol.
Biomarker
|
Paracetamol concentrations
|
Solvent control
|
0.25 µg.L-1
|
2.5 µg.L-1
|
25 µg.L-1
|
GST
|
177.82±14.75a
|
152.35±15.87ab
|
118.70±19.33ab
|
111.75±15.79b
|
SOD
|
425.04±50.25ab
|
303.37±24.47a
|
283.42±44.78a
|
533.07±87.24b
|
GPx
|
63.98±5.81a
|
45.67±2.84b
|
38.05±3.38b
|
41.13±1.50b
|
LPO
|
129.98±17.01a
|
111.89±10.89a
|
77.29±10.33a
|
86.53±8.75a
|
* SOD activity = U.mg protein-1; GST and GPx activity = nmol.min-1.mg protein-1; LPO concentration = μmol of hydroperoxides.mg protein-1.he results are expressed as mean ± standard deviation. Different letters indicate significant differences (p < 0.05)
Most of the gonads were in development, with no sperm formation (Figure 3). Some animals, both in control and PCM treatments groups, were either able to sperm (seminiferous tubules full of sperm, Figure 4) or had already sperm (seminiferous tubules were not so full of sperm, especially in the center of the gonad). For Danio rerio, after 6 weeks to 0.5 and 10 μg.L−1 of PCM exposure, the males showed all spermatogenesis stages, including spermatozoa, with no observable effect on testis morphology (Galus et al. 2013). For the same species of this study, R. quelen, exposed for 21 days at 0.25 μg.L−1, males showed similar conditions with cells at different stages of development. However, at exposure to 2.5 μg.L−1, spermatogenesis inhibition was observed in some animals, showing once again that with increased exposure time, for R. quelen, PCM can become estrogenic (Guiloski et al. 2017).
The nMDS presented a stress of 0.2248. The paracetamol groups that were most spatially distant from the solvent control group, focusing on the right quadrants (Figure 5).
Vitellogenin and gonadal histology are the most sensitive endpoints for fathead minnow, medaka and zebrafish in 21-day assays for most endocrine disruptors (Dang et al. 2011). This can be different for different organisms. For crustaceans, for example, the young number per adult or per brood were used as disruption biomarker. For Moina macrocopa, after 7 days of exposure to PCM, adults had no change in the offspring number, but between concentrations of 950 and 8580 μg.L-1, the offspring number was lower (Kim et al. 2012). For fish, concentration and exposure time are also important for the biochemical responses of detoxification and the antioxidant system in the liver of Oncorhynchus mykiss, an important reproductive organ for producing vitellogenin (Ramos et al. 2014).
For R. quelen, the endpoints, vitellogenin and gonadal histology, were not sensitive for PCM exposure. However, at concentrations of 0.25 and 2.5 μg.L-1, after 14 days, biochemical changes in liver and gonads were observed. For Danio rerio, hepatotoxicity was not related to changes in endocrine disruption. Even after observing hepatotoxicity after exposure to 1000 and 10000 μg.L-1 for 21 days, with liver necrosis, no change in vtg1 and vtg3 genes expression were observed (Baumann et al. 2020). However, comparing our results to those obtained by Guiloski et al. (2017), fish size and exposure time are important factors for PCM toxicity. The difference of the studies were seven more days of exposure, 128 g and 12 cm more of the weight and length of the fish respectively. Rhamdia quelen males showed changes in plasma hormone levels, more hepatic biochemical changes and changes in gonadal stage.