Chitosan-Glutathione Nanoparticles Modify Hepatic Cellular Response on Bovine Precision-cut Liver Slices Treated With Zilpaterol and Clenbuterol.

Zilpaterol and clenbuterol are two β-adrenergic agonist drugs used on animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries; clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used at unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proven yet of zilpaterol to produce reactive oxygen (ROS) and nitrogen species. Regarding glutathione which is the main intracellular antioxidant and plays detoxication functions on liver metabolism; case study is a primary interest to know the capacity of chitosan-glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures exposed to clenbuterol and zilpaterol. A single drug assay was performed in the rst instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both agonist β-adrenergic modify in a dose-dependent manner oxide-reduction response through ROS generation, glutathione peroxidase activity, and intracellular GSH content; and de release of liver enzymes associated with hepatocellular damage like gamma glutamyl-transpeptidase, aspartate aminotransferase, alanine aminotransferase. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.


Introduction
Zilpaterol and clenbuterol are two β-adrenergic agonists used to improve growth performance, feed e ciency, and carcass quality in cattle (Centner et  Previous reports demonstrate that antioxidants such as ascorbic acid and reduced glutathione (GSH) prevent reactive metabolites formation during clenbuterol metabolism (Brambilla et al. 2007).
GSH is a low-molecular-weight, non-protein thiol made up of glycine, cysteine, and glutamic acid synthesized and stored in the liver. As well as the main antioxidant of cellular origin that only can be synthesized in the cellular cytoplasm (Lu 2009), to exercise functions like reactive species scavenger, regulating the oxide reduction balance (Patlevič et al. 2016), participate in the xenobiotic detoxi cation processes by the conjugation with xenobiotic and their metabolites, cell signalling and others biochemical reactions; situations that limit the cellular GSH quantity many times; thus, the cell needs de novo synthesis and trigger the start-up of other enzymatic and non-enzymatic mechanisms to maintain cellular homeostasis (He et al. 2017).
Several research techniques to evaluate drug safety and toxicity effects; in vitro techniques are widely used to assess the effects of xenobiotic substances. Unlike in vivo studies with humans or animals, invitro techniques are versatile tools adaptable to ethical standards, with a short evaluation time, low cost, and high e cacy of the data generated (Ruoß et al. 2020). Particularly, precision-cut liver slice cultures (PCLS) have the advantage of reducing the number of experimental animals (Soldatow et al. 2013;Granitzny et al. 2017); better control in environmental conditions, a reduction in genetic heterogeneity, and due to the limited amount of the drug evaluated, there is less contamination by organic waste (Ruoß et al. 2020); additionally, the three-dimensional structure in the liver tissue is preserved, with good correlation on in vivo studies.
As mentioned above, GSH prevents the formation of reactive metabolites during clenbuterol metabolism; however, there is no information related to zilpaterol metabolism (Lu 2013; Lu 2020), so the use of chitosan nanoparticles offer an alternative to deliver GSH into the cell and counteract the adverse effects caused by GSH depletion; because chitosan is a biocompatible, biodegradable, and low toxicity polymer In this study, was utilized bovine PCLS and applied as a tool to evaluate clenbuterol and zilpaterol on hepatic cells through the release of hepatic enzymes and oxide-reduction markers (Masubuchi et al. 2016). Since clenbuterol and zilpaterol are metabolized in the liver, the bovine PCLS are an adequate strategy to explore the biological and toxicological effects of these β-adrenergic drugs, considering the combination of each one with chitosan-glutathione nanoparticles (CS/GSH-NP). GSH plays an essential role in hepatic metabolism and may reduce the hepatocyte damage caused by clenbuterol and zilpaterol treatment by considering the limited cytoplasmatic synthesis of GSH.

Nanoparticle synthesis and physicochemical characterization
Chitosan nanoparticles (CS-NP) and glutathione-chitosan nanoparticles (CS/GSH-NP) were prepared by the ionic gelation method (Koo et  and left under culture conditions for 6 hours ( Fig. 1), For good measure, cultures were harvested and stored at -80°C. An unthreaded group culture was considered as the negative control.
Combined drug-nanoparticles exposure PCLS cultures were exposed to a factorial arrangement 2x2. First, the cultures were exposed to 10 and 25 ng/mL of zilpaterol and clenbuterol for 3 hours, then each zilpaterol were added two amounts of the nanoparticles (zilpaterol level x CS-NP level; zilpaterol level x CS/GSH-NP level), and clenbuterol (clenbuterol level x CS-NP level; clenbuterol level x CS/GSH-NP level) after while were left under incubation another 3 h (Fig. 1). Untreated cultures were considered as negative controls. Three independent experiments were carried out by duplicate on each treatment.

Sample preparation
At the end of treatments, liver slices were washed with ice-cold PBS and transferred to polystyrene tubes containing 500 μL of PBS with cocktail protease inhibitors (Roche, IN., USA). After the homogenized samples were centrifuged at 13,000 g, 10 min, 4°C, the supernatant was divided into aliquots and stored at -80 °C for analysis. Determination of enzymes associated with liver damage Alanine transaminase; L-alanine:2-oxoglutarate aminotransferase (ALT), aspartate transaminase; Laspartate:2-oxoglutarate aminotransferase (AST), and γ-glutamyl transferase; (5-L-glutamyl)-peptide: amino-acid 5-glutamyltransferase (GGT) bode on the culture medium by using spectrophotometric kits (Spin React, Girona, Spain). The reagents from the diagnostic kits were mixed with the medium culture according to the manufacturer's instructions. The enzymatic kinetics were recorded, considering temperature, wavelength, and time kinetics for each enzyme. L-lactate dehydrogenase; (S)-lactate: NAD + oxidoreductase (LDH), release was determined by using the cytotoxic kit from Sigma-Aldrich, (St. Louis, MO., USA) the culture media was transferred to 96-well polystyrene plates, and the reagents of the kit were added according to the manufacturer protocol. Then, enzymatic kinetics were measured in a microplate reader.

Zilpaterol and clenbuterol quanti cation
Zilpaterol and clenbuterol content in cultures exposed to these beta-agonists were determinate by UPLC-MS/MS methodology. Aliquoted samples were defrosted, 100 µL and placed into 2 ml polystyrene tubes with 100 µL of NaOH 5M and mixed vigorously for 1 minute. Then, 800 μL of ZnOH 40% were added and mixed for one minute. After that, the samples were sonicated for 15 min at 40 °C, allowed to cool, pH  . 2a). The results showed an intracellular amount of zilpaterol and clenbuterol only in the cultures exposed to both β-adrenergic, and the amount increased according to the concentration used for each one (Fig. 2b). Subsequently, ROS production was determined to know if the β-agonist drugs and the nanoparticles understudy could modify the amount of these reactive species. The results for individual treatments show ROS generation in a dose-dependent manner on the groups treated with clenbuterol and zilpaterol (Fig. 3a); meanwhile, ROS generation on the groups treated with both types of nanoparticles understudy was lower or similar to the negative control.
ROS and GSH results are consistent with GPx activity; (Fig. 4a) an enzyme whose activity is promoted when there is an increase in peroxide radicals (Ghosh et al. 2019); therefore, an increase in GPx activity is expected when ROS production is elevated on the groups treated with zilpaterol and clenbuterol. In GGT activity, the levels also increased with clenbuterol and zilpaterol (Fig. 4b), which may be related to the need to synthesize GSH to counteract the effects of ROS generated by both drugs in the concentrations used. Similarly, as GPx and GGT, the results of the individual treatments in the determination of AST and ALT ( Fig. 5a-b) showed an increase in the high concentrations of clenbuterol and zilpaterol studied.
Regarding the combined effects, treatments in which both drugs were combined with CS-NP showed signi cant changes in ROS production; meanwhile, ROS levels increasing in the case of clenbuterol ( Fig. 6a), the combination of CS-NP with zilpaterol demonstrate an opposite effect (Fig. 6b). When CS/GSH-NPs were used, the induction of reactive species was not signi cantly modi ed. Therefore, these results suggest that exogen GSH delivered by nanoparticles in the cell could have been used to modulate oxidation-reduction stress or some events associated with liver biotransformation when the tissue was exposed to both drugs.
On the other hand, regardless of the type of nanoparticles used, promoted the response of GPx activity estimated in the combined groups with clenbuterol regardless of the type of nanoparticles used ( Fig. 7ab). Zilpaterol group combined with CS-NP also increased the response of GPx (Fig. 7c). However, the combined treatment of zilpaterol with CS/GSH-NP, was observed to reduce in GPx activity (Fig. 7d); that could be related to an increase in the amount of GSH (Fig. 8b). These ndings suggest that GSH can be incorporated into cells through nanoparticles, and the response of exposed PCLS to both agonists βadrenergic and exogenous GSH is different for each one. Also, CS/GSH-NP signi cantly reduces the GGT response in the PCLS treated with clenbuterol (Fig. 9b), while this effect in the combined treatments with zilpaterol was not signi cantly modi ed.
A signi cant response reduced ALT release in the groups exposed to clenbuterol combined with CS-NP (Fig. 10a) and CS/GSH-NP (Fig. 10b). This reduction also occurred in PCLS exposed to zilpaterol and CS-NP (Fig. 10c), surprisingly occurs the opposite in cultures exposed to zilpaterol with CS/GSH-NP (Fig. 10d). These results suggest a differential sensitivity of bovine liver tissue when is exposed to both drugs and the nanoparticles under study. In AST, the response also reduced on both agonists β-adrenergic combined with CS-NP ( Fig. 11a-b); whereas CS/GSH-NP does not modify the AST response during the combined treatment with zilpaterol or clenbuterol.

Discussion
Previous works demonstrate that clenbuterol metabolism promotes the formation of reactive oxygen and nitrogen metabolites or species that react with biomolecules to form covalent adducts (Brambilla et al. 2007; The results obtained in this work suggest that the hepatic biochemical mechanisms on bovine PCLS dependent on GPx, GGT, ALT, and AST can be modi ed by delivering antioxidant molecules such as glutathione into the cells by using nanoparticles. Therefore, the impact of those compounds on redox markers and transaminases release could be associated with the formation of reactive species; from here, the importance of extending the cellular and molecular evaluation of drugs like clenbuterol and zilpaterol, which are employed in animal production to contribute to the knowledge of the toxicological features at the cellular level of both agonists β-adrenergic. Figure 1 Single drug and nanoparticles exposure Single treatments were carried out on PCLS treated with two concentrations of zilpaterol (10 and 25 ng/mL), clenbuterol (10 and 25 ng/mL), CS-NP (10 and 25 µg/mL), and CS/GSH-NP (10 and 25µM GSH) and left under culture conditions for 6 hours ( Fig. 1), For good measure, cultures were harvested and stored at -80°C. An unthreaded group culture was considered as the negative control.  2a). The results showed an intracellular amount of zilpaterol and clenbuterol only in the cultures exposed to both β-adrenergic, and the amount increased according to the concentration used for each one (Fig.   2b).

Figure 3
Subsequently, ROS production was determined to know if the β-agonist drugs and the nanoparticles understudy could modify the amount of these reactive species. The results for individual treatments show ROS generation in a dose-dependent manner on the groups treated with clenbuterol and zilpaterol (Fig. 3a); meanwhile, ROS generation on the groups treated with both types of nanoparticles understudy was lower or similar to the negative control. ROS and GSH results are consistent with GPx activity; (Fig. 4a) an enzyme whose activity is promoted when there is an increase in peroxide radicals (Ghosh et al. 2019); therefore, an increase in GPx activity is expected when ROS production is elevated on the groups treated with zilpaterol and clenbuterol. In GGT activity, the levels also increased with clenbuterol and zilpaterol (Fig. 4b), Figure 5 synthesize GSH to counteract the effects of ROS generated by both drugs in the concentrations used.
Similarly, as GPx and GGT, the results of the individual treatments in the determination of AST and ALT (Fig. 5 a-b) showed an increase in the high concentrations of clenbuterol and zilpaterol studied. Regarding the combined effects, treatments in which both drugs were combined with CS-NP showed signi cant changes in ROS production; meanwhile, ROS levels increasing in the case of clenbuterol (Fig. 6 a), the combination of CS-NP with zilpaterol demonstrate an opposite effect (Fig. 6 b). When CS/GSH-NPs were used, the induction of reactive species was not signi cantly modi ed. Therefore, these results suggest that exogen GSH delivered by nanoparticles in the cell could have been used to modulate oxidation-reduction stress or some events associated with liver biotransformation when the tissue was exposed to both drugs. On the other hand, regardless of the type of nanoparticles used, promoted the response of GPx activity estimated in the combined groups with clenbuterol regardless of the type of nanoparticles used (Fig. 7 ab). Zilpaterol group combined with CS-NP also increased the response of GPx (Fig. 7c). However, the combined treatment of zilpaterol with CS/GSH-NP, was observed to reduce in GPx activity (Fig. 7d); that could be related to an increase in the amount of GSH (Fig. 8b).

Figure 8
However, the combined treatment of zilpaterol with CS/GSH-NP, was observed to reduce in GPx activity (Fig. 7d); that could be related to an increase in the amount of GSH (Fig. 8b).

Figure 9
These ndings suggest that GSH can be incorporated into cells through nanoparticles, and the response of exposed PCLS to both agonists β-adrenergic and exogenous GSH is different for each one. Also, CS/GSH-NP signi cantly reduces the GGT response in the PCLS treated with clenbuterol (Fig. 9b), while this effect in the combined treatments with zilpaterol was not signi cantly modi ed.

Figure 10
A signi cant response reduced ALT release in the groups exposed to clenbuterol combined with CS-NP ( Fig. 10 a) and CS/GSH-NP (Fig. 10 b). This reduction also occurred in PCLS exposed to zilpaterol and CS-NP (Fig. 10 c), surprisingly occurs the opposite in cultures exposed to zilpaterol with CS/GSH-NP (Fig.  10 d).

Figure 11
These results suggest a differential sensitivity of bovine liver tissue when is exposed to both drugs and the nanoparticles under study. In AST, the response also reduced on both agonists β-adrenergic combined with CS-NP (Fig. 11 a-b); whereas CS/GSH-NP does not modify the AST response during the combined treatment with zilpaterol or clenbuterol.