In Vivo and in Vitro Regulatory Effect of Silibinin on Some Metabolic Enzyme Activities against Cobalt Induced Toxicity in Rats: A Biochemical Approach

The study aimed to examine the in vivo inhibition effect of cobalt ion and silibinin on metabolic enzymes such as glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), and glutathione S-transferase (GST) and their in vitro inhibition effect on 6PGD. Twenty-four Wistar Albino rats weighing approximately 250–300 g were used in the study. The rats were divided into 4 groups as group 1 (control): isotonic serum (0.5 mL i.p), group 2 (cobalt): (150 mg kg/day cobalt), group 3 (silibinin): (100 mg/kg/day silibinin), group 4 (cobalt + silibinin). As a result of the in vivo applications, a statistically significant decrease was observed in the activities of G6PD (p < 0.05), 6PGD (p < 0.05), GR (p < 0.05), and GST (p < 0.05) enzymes in the groups that were administered cobalt compared to the control group. It was also found that the activities of G6PD (p < 0.05), 6PGD (p > 0.05), GR (p > 0.05), and GST (p > 0.05) enzymes increased in groups that were administered cobalt + silibinin compared to the group that was administered cobalt. As for in vitro applications, it was found that different Co2+ ions inhibited 6PGD enzyme which was obtained as a result of purification with IC50 = 346.6 μM value, while silibinin increased 6PGD enzyme activity within the concentration range of 100–750 μM by 40%. As a result, it was found that cobalt ions had an inhibition effect on G6PD, GR, and GST enzymes, which are vitally important for living metabolism, in vitro and in vivo and inhibited 6PGD enzyme activity in vitro, and silibinin increased these enzyme activities in vivo and 6PGD enzyme activity both in vivo and in vitro and decreased the inhibition effect.


INTRODUCTION
It has been stated that trace elements play a significant role in the metabolic process and functioning of many enzymatic systems by providing biochemical modulation in many living things, including the human body. 1−3 It has been reported that deficiency in physiological levels of trace elements leads to a decrease in vulnerability against the unstable antioxidant defense and oxidative stress, 4 and that overconcentration of certain trace elements could be among the reasons for elevated free radical development and lipid peroxidation. 5Cobalt (Co) is a precious metal that is utilized in producing metal alloys, batteries, and pigments. 6Co is essential for human health, as it is a component of the B12 vitamin complex that plays a role in the production of red blood cells and regulation of DNA synthesis, fatty acids, and amino acid metabolisms. 7However, high concentration of cobalt is toxic for humans, land and sea animals, and plants. 8Many researchers have reported that acute and chronic cobalt toxicity harms various organs. 9−12 Silibinin, which is the main active component of the silymarin complex, is usually obtained from the milk thistle.Silibinin, which has a wide area of use in order to protect the liver, kidneys, and heart tissue, has an antioxidant property. 13,14Recent studies have been oriented toward the anticancer effect of silibinin, and it has been shown that it could be used as a protective and therapeutic agent in cancer treatment. 15G6PD is the primary and speed-limiting enzyme of the pentose phosphate pathway that yields the production of ribose-5-phosphate and NADPH (β-nicotinamide adenine dinucleotide 2′-phosphate reduced). 16NADPH coenzyme is included in the synthesis of certain amino acids, protects the cells from oxidants, and plays a role in the detoxification of xenobiotics by way of the glutathione reductase-peroxidase system. 1 7 6PGD (E.C.1.1.1.44)is the third enzyme of the pentose phosphate metabolic pathway, and in the presence of NADP + , it behaves as a catalyzer in the conversion of 6-PGA (6-phosphogluconate) into D-ribulose-5-phosphate. 18,19NADPH protects the cell against oxidant agents by producing reduced glutathione (GSH). 20One of the important cellular antioxidant enzymes is GR, and it catalyzes the conversion of glutathione from its oxidized form into its reduced form. 21,22GSTs are multifunctional enzymes found everywhere and represent 10% of cytosolic proteins.They generate the conjugation of toxic xenobiotics and compounds that are produced oxidatively.Thus, they enable the elimination of metabolisms and provide protection from oxidants. 23In the study, it was aimed to examine the inhibition effects of the cobalt ion on G6PD, 6PGD, GR and GST enzymes and to determine whether silibinin prevents the side effects of the cobalt ion in vivo and in vitro.
2.2.In Vivo Effect of Silibinin and Cobalt.Ethical approval for the study was obtained from the Bingol University Animal Experiments Ethics Board (BUHADEK:18.05.2021-2021/02).All experiments were conducted in line with the ethical rules included in the Laboratory Animals Care and Use Guidelines.Twenty-four Wistar Albino rats that were 12 weeks old and weighed approximately 250−300 g were used in the study.The rats were divided into four groups.They were fed with standard feed (commercial food pellets; crude protein 24%, phosphorus 0.52%, crude fat 4%, sodium 0.17%, crude cellulose 6.28%, crude ash 7.06%, calcium 1.00%), and they were allowed to drink water.The environmental temperature was kept constant at 20−25 °C for the rats.Normal lighting and darkness (12L:12D) were arranged as a constant photoperiod.Group 1 (control) was administered isotonic serum (0.5 mL, i.p), group 2 (cobalt) (150 mg/kg/day/cobalt) (oral), 24 group 3 (silibinin): 100 mg/kg/day silibinin (oral), 25 and group 4 (cobalt + silibinin): (150 mg/kg/day-cobalt) + 100 mg/kg/day-silibinin (oral gavage).The animals fasted overnight (for about 12) on the last day of the experimental model before the induction of anesthesia or the collection of blood samples.At the end of day 7, the rats were anesthetized with (60 mg/kg ip) ketamine hydrochloride and 10 mg/kg Xylazine i.p.Kidney tissue was extracted following median laparotomy and was washed in phosphate-buffered saline and kept in deep freeze (−80 °C) until analysis time.

Preparation of Supernatant from Tissue Samples of Kidney.
Tris-HCl tampon of 20 mM at pH 7.4 was added to 200 mg of kidney tissue, and the mixture was homogenized by using a homogenizer device (Ultra Turrax-T25).Then, it was centrifuged at 15,000 rpm at 4 °C for 30 min.The supernatant on the surface was moved to a new tube. 26.4.Purification of 6PGD Enzyme.In line with the method determined by Temel et al. ( 2017), 2′,5′-ADP-Sepharose 4B column was prepared. 27,28The prepared supernatant was applied to the colon, along with 10 mL of the colon material.After that, the colon was washed by using 50 mM phosphate tampon with pH = 7.35.6PGD was washed by using 80 mM phosphate + 0.5 mM 1 mMEDTA + NAPD tampon with pH = 7.35.All applications were performed at 4 °C.

Measurement of Activity of Some Metabolic Enzymes.
In vivo activities of G6PD and 6PGD enzymes and in vitro activity of the 6PGD enzyme were measured according to the Beutler method. 29Activity of the GR enzyme was measured by using the Carlberg and Mannervik method. 30abig method was employed in measuring the activity of GST enzyme.Enzyme activities were expressed as U/mgprot. 312.6.Determination of Protein Quantity.Kidney tissue protein levels were made spectrophotometrically at 595 nm according to the Bradford method and it was formed by using standard graphic bovine serum albumin. 322.7.In Vitro Effect of Silibinin and Cobalt.In order to evaluate the effect of silibinin and cobalt on the activity of 6PGD enzyme, five different concentrations of silibinin (100, 200, 400, 500, and 750 μM) and five different concentrations of cobalt (38.5, 77, 192.5, 385, and 770 μM) were separately placed in tubes that contained purified enzyme.IC 50 values (inhibitor concentration that decreases the total enzyme activity by 50%) were determined over % activity − [I] graphs. 33.8.Analysis of Kinetic Data.The study data obtained were presented as mean ± standard deviation.The differences were defined as significant at p < 0.05.Shapiro−Wilk was done to evaluate the normality of the data, and Levene's tests were performed to evaluate the homogeneity of the data.In vivo effects of cobalt and silibinin on rat kidney G6PD, 6PGD, GR, and GST enzyme activity of groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.For statistical analyses, IBM SPSS Statistics for Windows, version 22.0 (IBM Corp., N.Y., USA) and GraphPad Prism for Windows ver.5.0 program (San Diego, CA, USA) were employed.

RESULTS
With respect to the control group, a statistically significant decrease in G6PD enzyme activity was observed in the cobalt group (p < 0.05).While a higher G6PD enzyme activity was found in the silibinin group compared to the cobalt group, G6PD enzyme activity in the silibinin group was determined to be statistically significant lower in comparison to the control group (p < 0.05).In the cobalt + silibinin group, G6PD enzyme activity displayed a statistically significant increase with respect to the cobalt group (p < 0.05), and G6PD enzyme activity in this group approximated the control group (p > 0.05) (Figure 1).
When compared with the control group, a statistically significant decrease in 6PGD enzyme activity was found in the cobalt group (p < 0.05).6PGD enzyme activity was higher in the silibinin group in to the cobalt group (p < 0.05), but the activity of this enzyme in the silibinin group was statistically significantly lower compared to the control group (p < 0.05).As for the cobalt + silibinin group, 6PGD enzyme activity was statistically insignificantly higher in comparison to the control group (p > 0.05), while a statistically significantly decrease was seen in 6PGD enzyme activity in this group with respect to the control group (p < 0.05) (Figure 2).
In comparison to the control group, a statistically significant decrease in GR enzyme activity was found in the cobalt group (p < 0.05).The comparison of the silibinin group with the cobalt group showed a higher GR enzyme activity in the silibinin group (p > 0.05), but GR enzyme activity in the silibinin group was statistically insignificantly lower when compared with the control group (p < 0.05).Regarding the cobalt + silibinin group, GR enzyme activity in this group was found to be statistically insignificantly higher compared to the cobalt group (p < 0.05).GR enzyme activity in the cobalt + silibinin group increased in comparison to the cobalt group and approximated the control group (p > 0.05) (Figure 3).
A statistically significant decrease in GST enzyme activity was observed in the cobalt group in comparison to the control group (p < 0.05).When the silibinin group was compared against the cobalt group, GST enzyme activity was found to be higher in the silibinin group (p > 0.05), but when compared with the control group, GST enzyme activity in the silibinin group was determined to be statistically significantly lower (p < 0.05).GST enzyme activity in the cobalt-silibinin group was determined to be statistically significantly lower in comparison to the control group (p < 0.05), while it increased with respect to the cobalt group (p > 0.05) (Figure 4).
In in vitro applications, 6PGD enzyme was obtained from rat kidney tissues by using 2′,5′-ADP-sefaroz-4B affinity chromatography.The effect of Co 2+ in different concentrations on 6PGD enzyme activity, which was obtained in pure form, was examined through the spectrophotometric method.When the data obtained were analyzed, it was found that Co 2+ ions inhibited the enzyme at the value of IC 50 = 346.6 μM.It was also determined that silibinin increased 6PGD enzyme activity by 40% within the concentration range of 100−750 μM (Figures 5 and 6).

DISCUSSION
Human beings are exposed to complicated toxic compound mixtures in their homes and professional environments.These chemical compounds create negative effects on aquatic biota, animals, and humans through water reservoirs, water courses, and rivers. 34As cobalt is utilized as a pigment in glass, ceramics, and paints and cobalt alloys are intensely employed in aircraft engines, magnets, and artificial connections, environmental exposure can occur at high levels, particularly in industrial environments.Workers employed in metal mining, melting, and refining can get exposed to higher amounts of cobalt. 35,36Being exposed to high levels of soluble cobalt salts are toxic, and the median varying between 150 and 500 mg kg −1 has been reported to be lethal dose in mammals. 37Cobalt in high doses, which is a cofactor for the activation of various enzymes and formation of B12 vitamin    and other creates various effects on the heart, thyroids, liver, and kidneys. 34Cobalt, which is among the important heavy metals that contribute to the production of free oxygen radicals, is known to be a highly toxic metal.Moreover, it has been reported in some papers that it is an important free radical H 2 O 2 generator. 38The cellular condition in which the generation of reactive oxygen species exceeds antioxidant capacity is defined as oxidative stress.It has been reported to significantly contribute to the pathogenesis of many human and animal diseases, including cardiovascular and renal disorders. 39The role played by oxidative stress in cobaltinduced organ pathologies have frequently been expressed in studies conducted. 8n the present study, the purpose was to investigate in vivo inhibition effects of cobalt ion and silibinin on metabolic enzymes such as G6PD, 6PGD, GR, and GST and their in vitro inhibition effects on 6PGD enzyme.When the study results were analyzed, it was seen that G6PD enzyme activity statistically significantly decreased in the cobalt group in comparison to the control group (p < 0.05).It was determined that the activity of the enzyme statistically significantly rose in the cobalt + silibinin group when compared to the cobalt group (p < 0.05), Regarding 6PGD enzyme activity, while a statistically significant drop in 6PGD enzyme activity was observed in the cobalt group with respect to the control group (p < 0.05), the enzyme activity in the silibinin group was found to be higher in the silibinin group compared to the cobalt group, but the increase was not statistically significant.6PGD enzyme activity in the cobalt + silibinin group statistically insignificantly increased in comparison to the cobalt group (p > 0.05).As regards GR and GST enzyme activities, a statistically significant decrease was observed in the enzyme activity in the cobalt group compared to the control group (p < 0.05), while an increase in the enzyme activities was found in the cobalt + silibinin group with respect to the cobalt group.GST enzyme activity in the cobalt + silibinin group was statistically significantly lower compared to the control group (p < 0.05).When the effects of cobalt ion on 6PGD enzyme, which was obtained from the rat kidney tissue through purification by using 2′,5′ ADP-sepharose-4B affinity chromatography, were evaluated, it was determined that Co 2+ ions restricted the enzyme at the value of IC 50 = 346.6 μM, and that silibinin increased 6PGD enzyme activity by 40% at the concentration range of 100−750 μM.
In different studies conducted, it was reported that exposure to CoCl 2 in Wistar rats led to significant increases in oxidative stress parameters (hydrogen peroxide, H 2 O 2 , and malondialdehyde, MDA), as well as causing a decrease in superoxide dismutase (SOD) activity in reduced glutathione (GSH) in kidneys and adaptive increases in GST and catalase (CAT). 40t was determined that high-dose cobalt application in rats considerably increased the concentrations of AST, ALT, and CK enzymes, and that no significant effect on the kidneys was observed in the pathological evaluation. 41In another study conducted, it was reported that cobalt amassed the most in the kidney, followed by the liver, blood, and lungs, in descending  order and depending on dose.Assessment of the liver and kidney function tests showed a sharp increase in serum urea and creatinine levels depending on the dose, and it was reported that the compound led to a higher degree of toxicity in the kidney compared to the liver. 42It was determined in a study that goldfish (Carassius auratus) that was exposed to Co 2+ at 50, 100, or 150 mg L −1 concentrations for 96 h, protein carbonyl content in the kidney increased, that catalase activity did not change, that superoxide dismutase enzyme activity increased, that glutathione peroxidase and glutathione S-transferase activities did not change, but that glutathione reductase activity increased by 70%. 8Silymarin and its main component, silibinin, are extracted from the healing plant Silybum marianum (milk thistle), and they have conventionally been used in treating diseases.In recent times, it has been shown that active flavonoid agents displayed considerable antineoplastic effects in various in vitro and in vivo cancer models, such as colon, breast, skin, bladder, prostate, and kidney carcinomas. 43In various studies conducted, it was reported that silibinin was effective in treating arsenic-induced nephrotoxicity by eliminating oxidative stress, inflammation, and apoptosis in rats, and that silibinin also activated GST enzyme activity. 44In the study conducted by Yassin et al.  (2021), in which they examined the effectiveness of silymarin and silibinin against experimentally induced renal carcinogenesis in male Wistar rats, it was reported that silymarin and silibinin application weakened toxicity indicators in serum, decreased lipid peroxidation, considerably strengthened renal antioxidant arsenal, as well as increased GST enzyme activity and decreased GR enzyme activity. 45In vitro experiments showed that silibinin nanoparticles protect liver cells and reduce cellular damage.This was shown by decreased serum ALP, ALT, and AST levels. 46In a different study, it was reported that doxorubicin treatment caused damage to the heart, but the coadministration of silymarin/silibinin protected the heart from this damage. 47In another study, it was reported that silibinin could have significant positive effects against nephrotoxicity. 48It was also reported that silibinin considerably reduced morphological changes observed in the S3 segment of the proximal tubule in the kidneys. 49It was determined that the results of the studies mentioned above were consistent with the results of the study and supported it.

CONCLUSIONS
It has been stated in the literature that cobalt and its alloys have a wide range of use in industrial fields, that higher levels of environmental exposure can occur, especially in industrialized countries, and that exposure to high levels of soluble cobalt salts creates a toxic effect in living things.In this study, we found that cobalt ions strongly inhibited G6PD, GR, and GST enzymes in vivo and inhibited 6PGD enzyme activity in vivo and in vitro.We also determined that silibinin, whose antioxidant properties have been demonstrated in previous studies, reduced the inhibitory effect of cobalt ions on these enzymes.These enzymes are important for antioxidant metabolism and are also associated with the formation of various types of cancer.For these reasons, we anticipate that the results of this study will shed light on the pathophysiology of diseases caused by cobalt toxicity and that silibinin may be a potential phytotherapy approach for the treatment of cobalt toxicity.

■ ASSOCIATED CONTENT Data Availability Statement
All data generated or analyzed during this study are included in this published article.

Figure 1 .
Figure 1.Effects of cobalt and silibinin on the activity of the G6PD enzyme in rat kidney in vivo.Different letters in (a−c) represent statistical differences between the groups (p < 0.05).

Figure 2 .
Figure 2. Effects of cobalt and silibinin on the activity of the 6PGD enzyme in rat kidney in vivo.Different letters in (a−c) represent statistical differences between the groups (p < 0.05).

Figure 3 .
Figure 3. Effects of cobalt and silibinin on the activity of the GR enzyme in rat kidney in vivo.Different letters in (a−c) represent statistical differences between the groups (p < 0.05).

Figure 4 .
Figure 4. Effects of cobalt and silibinin on the activity of the GR enzyme in rat kidney in vivo.Different letters in (a−c) represent statistical differences between the groups (p < 0.05).

Figure 5 .
Figure 5.In vitro effect of cobalt ions on the rat kidney 6PGD enzyme activity.

Figure 6 .
Figure 6.In vitro effect of silibinin on rat kidney 6PGD enzyme activity.