Impact of HIF1-α/TGF-β/Smad-2/Bax/Bcl2 pathways on cobalt chloride-induced cardiac and hepatorenal dysfunction

Background: Cobalt chloride (CoCl2) is a ferromagnetic ubiquitous trace element extensively dispersed in the environment. Nevertheless, it may merit human hazard. Aim: Excess cobalt can harm vital organs this paves the way to elucidate the toxic impact of CoCl2 on the liver, kidney and heart. Method: CoCl2 was injected in a dose of (60 mg/kg, S.C.) proceeded via Carnosine (200 mg/kg) and/or Arginine (200 mg/kg) treatment 1 month, 24 and 1 h, prior to CoCl2-intoxication. Results: CoCl2 significantly alleviated hemoglobin concentration and BCl2; meanwhile, protein expression of transforming growth factor (TGF-β), hypoxia-inducible factor (HIF-1α), Mothers against decapentaplegic (Smad-2), AKT protein expression and Bax/Bcl2 ratio was noticeably elevated. Conclusion: The combination of the aforementioned antioxidants exerted a synergistic anti-apoptotic impact in all target tissues.

AKT, Smad-2, TGF-β, Bax/Bcl2 ratio, HIF-α were increased. The aforementioned antioxidants elucidated a synergistic anti-apoptotic and anti-mutagenic impact on all target organs which may be mediated via various growth factors signaling pathways.

Chemicals
All chemicals used were of high analytical grade, the product of Sigma and Merck companies. Carn, Argi and CoCl 2 were obtained from Sigma Chemical Co. (Sigma, MO, USA).

Animals
Adult male albino Wister rats (200-220 g; 6-8 weeks old) were obtained from the Animal House at the National Research Centre. Animals were kept in special cages and maintained on a constant 12 h light/dark cycle with a controlled temperature of 20-22 • C and humidity of 60%. Rats were fed a standard rat pellet chow with free access to tap water ad libitum for 1 week before the experiment for acclimatization. After one week of acclimation, the rats were kept fasting overnight before treatment and randomly divided into six groups, each of ten rats.

Experimental design
Fifty rats were randomly divided into five equal groups ten rats each.

Samples collection & preparation
After 6 h from CoCl 2 -intoxication, the experiment animals were fasted overnight and then sacrificed and blood samples were collected for Hb determination and serum analysis, liver, heart, and kidney tissues were collected thoroughly in ice-cold saline and were rapidly frozen in liquid nitrogen and stored at -80 • C for Western blotting analysis.

Determination of hemoglobin (Hb)
Hb was determined colorimetrically in blood using Drabkin's reagent according to the method of Kjeldsberg [35].

Assay of liver function
Serum transaminases (alanine transaminase; ALT and aspartate aminotransferase; AST) were assessed by Randox (Crumlin, UK) kits following the provided directions.

Assay of kidney function
Urea, and creatinine in serum were estimated, according to the instructions of the manufacturer. Diagnostic kits were bought from Randox Company Chemical CO.
Assay of heart function Troponin T (Trop. T) concentration was evaluated using a Siemens Dimension Xpand Plus instrument (IL, USA). Creatine kinase-MB (CK-MB) was assessed spectrophotometrically by a standard enzyme kit (Cat. No.1001055) bought from Spinreact, S.A.-Spain.
Western blot analysis for HIF1-α, AKT, Smad-2, TGF-β & Bax & Bcl2 Liver, kidney and heart tissues were homogenized in buffer according to radio-immunoprecipitation assay (RIPA), which contains phosphatase and proteinase inhibitors. Bradford reagent was used to determine protein concentration [36]. 40 g proteins were transferred to 10% SDS/PAGE. Transferring of the separated proteins to nitrocellulose  membranes occurred, and skimmed milk (5%) was used in tris-buffered saline/Tween-20 (TBST) for blocking. Then prodding the membranes with antibodies against HIF1-α, AKT, Smad-2, TGF-β and Bax and Bcl2 (Novus Biologicals, CO, USA) overnight at 4 • C. Washing the blots with TBST and incubating them with the 2ry antibodies (1 h/room temperature). Protein bands were visualized using the ECL-Plus detection system (Amersham Life Sciences, Little Chalfont, Buckinghamshire, UK).

Statistical analysis
Data were expressed as means ± SEM for quantitative measures. Statistical comparison between different groups were performed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test using IBM SPSS Statistics program. Differences were considered significant at p < 0.05.

Argi & Carn attenuate CoCl 2 -altered Hb concentration
The present study revealed that Hb concentration was significantly decreased in the CoCl 2 -treated group compared with the normoxic group (p ≤ 0.001), while the administration of Carn and Argi either alone or in combination significantly increased Hb concentration with a mean value of 9.1, 8.5 and 9.07 respectively, compared with the CoCl 2 -treated group with the combination group revealing the most significant impact (Tables 1 & 2).

Argi & Carn attenuate CoCl 2 -induced liver injury
CoCl 2 -intoxicated rats demonstrated a significant elevation in serum ALT, and AST activities as illustrated in Table 3. Rats' pretreatment with Argi or/and Carn presented a significant improvement in the previously mentioned 10   TGF-E

Figure 2. (A-C) Representative immunoblots (Western blot analysis) of hepatic TGF-β, HIF1-α, and AKT in control and different treated groups.
expressions with a concomitant significant decrease in Bcl2 expression in hepatic tissue compared with the control value. Meanwhile, Carn and Argi administration alleviated the overexpression in HIF1-α, TGF-β, Smad-2, AKT and Bax, compared with the CoCl 2 -intoxicated group, while Bcl2 expression was significantly increased by the aforementioned treatments with the combination group revealing the most significant impact at P ≤ 0.05.

Argi or/& Carn attenuate CoCl 2 -induced renal injury
In the current study, Table 3 pointed that CoCl 2 induced an obvious increase in creatinine and urea levels indicating kidney dysfunction. However, antioxidants manipulation either solely or together depleted the aforementioned distorted biomarkers with a mean value of 1.23, 1.9 and 1.18 respectively for creatinine and 32.6, 32.05 and 29.7   respectively for urea compared with CoCl 2 intoxicated group with the combination regimen illustrating the most significant impact. The immunoblots and quantitative analysis of renal Bax, Bcl 2, Smad-2 TGF-β, HIF1-α, and AKT in control and different treated groups are presented in Figures 4-6.
CoCl 2-administration promoted a significant rise in protein expressions of Bax, HIF1-α, TGF-β, Smad-2 and AKT accompanied with a significant depletion in Bcl2 expression in renal tissue as compared with a non-treated group (p ≤ 0.001). While antioxidants treatment improved Bax, HIF1-α, TGF-β, Smad-2, and AKT overexpression as compared with the group intoxicated with CoCl 2 , while such treatment significantly increased Bcl2 expression with the combination group revealing the most significant impact.

Argi or/& Carn attenuate CoCl 2 -induced cardiac injury
Serum cardiac biomarkers including CK-MB and Trop-T in the control and other experimental groups intoxicated with CoCl 2 are revealed in   Figures 7-9 represent the immunoblots and quantitative analysis of cardiac Bax, Bcl 2, Smad-2 TGF-β, HIF1-α, and AKT, in control and different treated groups. Data of western blot analysis showed that CoCl 2-intoxication illustrated a significant raise in Bax, HIF1-α, TGF-β, Smad-2, and AKT protein expressions, while it significantly decreased Bcl2 expression compared with normal value (p ≤ 0.001). Whereas, Carn and Argi treatment attenuated Bax, HIF1-α, TGF-β, Smad-2 and AKT over expression in cardiac tissue. Even though Bcl2 expression was significantly improved via these treatments with the combination treatment showing the most significant impact.

Discussion
CoCl 2 is a ubiquitous trace element that is widely distributed in the environment and utilized in the production of alloys, catalysts and diamonds. Nonetheless, it is categorized as a human health hazard. Excess dietary cobalt can 10    impair various essential organs functions, which paves the way for understanding the toxicity of cobalt chloride in the liver, kidney and cardiac tissue. Previous evidence suggests that CoCl 2 -induced organ damage involves oxidative stress [37]. An imbalance between the antioxidant defense system and ROS levels brought on via excessive ROS production causes oxidative stress, inflammation, cell death, and DNA damage. Herein, the administration of CoCl 2 significantly increased the protein expressions of HIF-α. Meanwhile, a reduction in Hb concentration was significant. Nevertheless, Car and Arg significantly modulated these deviated parameters. According to some reports, CoCl 2 generates oxygen-derived free radicals, which worsen the effects of oxidative stress. Moreover, cobalt salts have been shown to increase the expression of many stress-responsive proteins, including heme oxygenase [10,13]. It is believed that increased heme oxygenase activity may have significant antioxidant importance by enhancing the body's ability to defend against oxidative stress [38,39].
CoCl 2 is a well-known chemical that mimics hypoxia; it causes the same effects in several aspects in various cultured cells, including loss of mitochondrial membrane potential, increase the release of ROS that causes cell death, and increased expression of HIF-1α and p53, which are implicated in the apoptotic pathway [    Erythropoietin transcription is activated by CoCl 2 therapy in normoxia, and it has been proposed that cobaltous ions replace ferrous ions under heme, changing the shape of a heme protein O 2 sensor [19]. HIF-1α is necessary for the induction of several genes during hypoxia, including erythropoietin and vascular endothelial growth factor. It causes Complex III's mitochondria to produce more ROS, which causes the HIF-1α protein to overexpress itself [48,49]. CoCl 2 induces apoptosis by triggering the caspase-3 and p38 mitogen-activated protein kinase (MAPK) pathways [50]. L-carnosine was observed to decrease HIF-1a protein expression influencing its stability and reduce the HIF-1 transcriptional impact. L-carnosine is involved in ubiquitin-proteasome system promoting HIF-1a degradation.
In the current study, hepatic injury was evaluated by the measuring hepatic biomarkers (AST and ALT). Administration of CoCl 2 induced a significant elevation in these enzymes' activity. These results coincide with other researches who revealed that CoCl 2 exposure induced hepatic necrosis accompanied with rise in the activities of hepatic enzymes [40,41]. Hepatic damage is usually marked by the consequence release of AST and ALT [42]. The present data revealed that CoCl 2 induced a noticeable elevation in creatinine and urea levels demonstrating renal dysfunction. CoCl 2 induced renotoxicity was corroborated by an increase in serum blood urea nitrogen and creatinine. Recent studies discovered a positive link between kidney injury and elevated blood urea nitrogen and creatinine levels [43].
Serum CK-MB and Troponin were significantly elevated in the CoCl 2 compared with normal group. Cobalt is present in the radical ions form in our body [44], it attacks cell membrane's unsaturated fatty acids and convert them into free radical (lipid and lipid peroxidation) [45]. Lipid peroxidation decreases fluidity of plasma membrane and elevates its fragility, with consequence impairing of membranous receptors and enzymes' function, affecting the membrane permeability, causing calcium ions' entry abnormalities, that causes destruction of the endoplasmic reticulum, and ends in cellular death [46]. Cellular death increase CK-MB, ALT and AST concentrations. It was reported that CoCl 2 significantly increased CK-MB and LDH levels [47].
In rat liver, CoCl 2 increases malondialdehyde, lowers glutathione (GSH), superoxide dismutase and catalase concentration. Alanine transaminase, aspartate aminotransferase, urea, creatinine and alkaline Phosphatase activity was elevated in CoCl 2 -induced hepatic and renal damage. Nonetheless, there was a decrease in lactate dehydrogenase activity [51]. The largest cobalt accumulation occurs in the liver. So, it is conceivable to hypothesize that cobalt could cause polyunsaturated fatty acid peroxidation, which would then cause phospholipid degradation and, ultimately, liver cell degeneration. When cobalt ions combine with ROS, they may produce cytotoxic hydroxyl radicals. The subsequent generation of free radicals by hydroxy radicals may lower cellular GSH levels, ascorbic acid concentrations and NADPH activity. Resultant ROS damages cellular proteins and DNA [52,53]. Novel experimental data suggest that the impact of hypoxia on renal cell survival, inflammatory cell recruitment and collagen gene expression may hasten the progression of chronic kidney disease.
The intoxication with CoCl 2 in the current study profoundly increased the protein expression of AKT, Smad-2 and TGF-α in the liver, kidney and heart tissue.
Phosphoinositide 3-kinase (PI3K) and receptor tyrosine kinases control the activity of the essential enzyme Akt, which is necessary for cell survival (RTKs). CoCl 2 enhances Akt phosphorylation in a manner that depends on both dose and time. The fact that Akt activation in response to CoCl 2 was reduced when the PI3K/Akt pathway was blocked suggests that PI3K is required for the phosphorylation of Akt by CoCl 2 [8,43,54].
Hepatic stellate cell (HSC) activation and fibrosis progression are two aspects of liver pathophysiology influenced by microRNA (miRNA)-mediated gene regulation. In the development of liver fibrosis, miR-942 expression was up regulated in activated HSCs and exhibited an inverse relationship with BAMBI expression. TGF-beta and lipopolysaccharide (LPS), two important factors in liver fibrosis and inflammation, cause HSCs to express miR-942 via Smad2/3 and NF-p50 binding to the miR-942 promoter, respectively. Mechanistically, the increased miR-942 degrades BAMBI mRNA in HSCs, making the cells more sensitive to fibrogenic TGF-signaling [55]. The relationship between active TGF and type II receptor (TGF-RII), which causes phosphorylated Smad2 and Smad3 to form a complex with Smad4 and migrate into the nucleus to regulate the expression of downstream genes, implicated in EMT [56].
Herein, the injection of CoCl 2 dramatically raised Bax protein expressions. Bcl2 was dramatically reduced in the meantime. These aberrant apoptotic biomarkers were dramatically regulated by Car and Arg. CoCl 2 increased oxidative stress, inflammation, and apoptosis. It has been documented that oxidative stress, inflammation, and apoptosis are connected to genotoxicity [57,58]. Yet, chronic inflammation brought on by persistent oxidative stress may be related to some chronic diseases, including cancer, diabetes, and cardiovascular conditions. The underlying etiology implicates the activation of various transcription factors such as NF-κB, p53, and NrF2 by oxidative stress ultimately leading to expressions of various genes including inflammatory cytokines, and chemokines [59,60].
Argi is an amino acid that plays an important role in immune function, wound healing, cell division, removing ammonia from the body, and hormone release. It is a precursor for nitric oxide (NO) synthesis, making it important in the regulation of blood pressure. Arginine is necessary for T-Cells to function in the body. It is known for its powerful antioxidant and anti-inflammatory power [21][22][23]. NO in its physiological values possess anti-apoptotic actions [24] and may promote healing during inflammation [61]. The small intestine and kidneys are the primary locations for arginine production.
Carn has important free radical scavenging ability, reduce lipid peroxidation, encourage the development of antioxidant enzymes and limit the production of pro-inflammatory cytokines [29,30]. It can protect cardiomyoblasts against HIF1-α induced hypoxia [31]. It can protect against ischemia/ reperfusion-induced acute renal failure in rats and lower rat mortality after cerebral ischemia [62].

Study limitations
The number of animals and measured parameters caused some limitations to the experiment. Cobalt handling may cause asthma attacks with shortness of breath, coughing, wheezing, and chest tightness. It may affect the thyroid, heart, kidneys and liver. Repeated exposure can cause lungs fibrosis even if no signs are observed. Experiments on animals do not exactly mimic the way that the human body and diseases may respond to treatments, chemicals or drugs. Some compounds are highly toxic so small amounts may cause death. Future studies should investigate the effect of cobalt on different organs as the brain, testis and lung.

Conclusion
Intriguingly, the combined treatment with both Carn and Argi therapy was regarded as a potential candidate for halting the course of multi-organ dysfunction brought on by CoCl 2 via reversing the altered protein expression of growth factors and apoptotic biomarkers including HIF1-α, AKT, Smad-2, TGF-, Bax, and Bcl2 produced by CoCl 2 in the liver, kidney, and heart.

Summary points
• Induction of cardiac, hepatic and renal dysfunction via CoCl 2 in rats.
• Pretreatment with Arginine, Carnosine and their combination for one month.
• Monitoring the protective impact of Arginine and Carnosine.

Author contributions
All the authors are responsible for writing the contents in this article and jointly contributed to the study conception and design, writing of introduction, methodology, discussion, conclusion, recommendations, future perspective, executive summary, appropriate referencing and protocols, the literature review search, research, data collection, data analysis, data interpretation, result presentation and approval of the final version of this article.

Acknowledgments
Authors are grateful for the National Research center for all the facilities and tools provided.

Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research
Adult male albino Wister rats (200-220 g) were obtained from the Animal House at the National Research Centre The Animal