Introduction

Studies in recent years have brought more and more evidence, such as that damage to cells by reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the most important factors leading to aging and degenerative diseases, such as cancer, cardiovascular and liver diseases, cataracts, chronic inflammatory processes, renal insufficiency and so on [1]. 

Identification, study and testing of new remedies for treatment approaches of diseases, resulting from imbalance between oxidants and antioxidants in favor of oxidants, with potentially destructive potential and pathogenesis in liver disorders is of particular interest due to the increase in incidence and severity of these pathologies.

Thus, it is certain the need to develop new compounds, which could serve as a basis for the development of drug preparations for the prevention and treatment of the above mentioned diseases, including liver diseases.

In this respect, derivatives of thiosemicarbazides are of particular interest, which could have a significant influence on metabolic processes.

Research carried out over the last decade has shown their therapeutic efficiency and perspective of valorification as raw material for obtaining medicinal remedies [2-5].

At the same time, their biochemical action mechanisms, in particular, on the peroxidative processes in the liver tissue, are not known in detail.

The aim of this study is to research the influence of new local copper coordination compounds, derivatives of thiosemicarbazides on free radical oxidation processes and antioxidant system in liver tissue, estimation, and selection of the most informative biomarkers for assessing the level of oxidative stress and which can be used to determine the effectiveness of new local remedies.

Material and methods

Study design

The study is preclinical experimental. The research was approved by the Research Ethics Committee of the Nicolae Testemitanu State University of Medicine and Pharmacy (favorable minute no. 73 from 26.04.2017).

Animals included in the study

In this study have been used new local copper coordination compounds, derivatives of thiosemicarbazides (CC) – 4 – ethyl – 2 - [phenyl (pyridine – 2 - yl) methylidene] hydrazine – 1 - carbothioamide (CMD-4) , chloro - {4 - (3 - methoxyphenyl) – 2 - [1 - (pyridine – 2 - yl) ethylidene] hydrazine – 1 - carbothioamide} copper (CMJ-33) and nitrato - {N – phenyl – N' - (pyridine - 2 - ylmethylidene) carbamohydrazonothioato} copper (CMT-67) synthesized in the Laboratory of Advanced Materials in Biopharmaceuticals and Technics at Moldova State University [5]. The autochthonous CC action on the liver tissue has been evaluated in experiments on a sample of 46 Wistar line male rats with 180 – 250 g mass, divided into 6 groups of 7-8 animals in each.

The first group – control, was made up of 8 intact animals, maintained at a normal diet of vivarium and whom was administered subcutaneous three times per week for 30 days physiological solution. Animals on the experimental groups 2 - 6 were administered the subcutaneous CC 3 times a week for 30 days during 30 days in the following sequence: 2nd group - CMD-4 (0.1 µM/kg body weight), 3rd group - CMD-4 (1.0 µM/kg body weight), 4th group - CMJ-33 (0.1 µM/kg body weight), 5th group - CMJ-33 (1.0 µM/kg body weight) and 6th group CMT-67 (0.1 µM/kg body weight).

Method of processing liver tissue

24 hours after the last administration of local CC, the animals were sacrificed under mild narcosis with sulfuric ether and the liver was collected. All operations were performed in glacial environment. The preparation of the material for the determination of the biochemical indices has been carried out as follows. The phosphate buffer solution 0.1 M (pH 7.4) containing 1 mm EDTA has been used as the dispersion medium, so that the final dilution of the homogenate constitutes 1:10. For complete destruction of the cell membranes the homogenate was processed with triton X-100 in the final concentration 0.1%. Subsequently, the tissue homogenates were subjected to centrifugation for 15 minutes at 5000 rpm, and the supernatant was transferred to clean tubes and until examined kept in the freezer at minus 40 ° C. The entire process of preparing tissue homogenates is performed under regulatory conditions for the assessment of biochemical parameters.

Tests of oxidative stress and antioxidant system tested

The intensity of oxidative stress was evaluated by determining the following laboratory parameters: malonic dialdehyde (MDA), nitric oxide (NO) derivatives, S-nitrosothiols (RSNO), advanced glycation end products (AGEs), advanced oxidation protein products (AOPP) and ischemia-modified proteins (IMP). 

The changes in the antioxidant protection indices were evaluated by determination of the activity of superoxidismutase (SOD), catalase (CAT), histidine (His), as well as the level of total antioxidant activity that is based on the antioxidants inhibition of the absorption of the 2.2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS).

The assessment of oxidative stress and antioxidant system was performed according to the procedures described previously [6].

Statistical processing method

The statistical evaluation of the obtained data performed with use of the computer program StatsDirect. The arithmetic mean ± error of the mean (M ± m) was calculated. The nonparametric statistical test „U Mann-Whitney” and the significance threshold „p” (p < 0.05) were used to test the significant difference between the studied indices of the compared groups.

Results

The evaluation results of lipid peroxide indices: MDA, NO, AGEs, IMP, AOPP, and RSNO  in liver tissue when administering local CC are reflected in the statistical data of Table 1.

The study shows that MDA decreases statistical suggestive in the liver tissue by 14% - 40% under the influence of most of the studied preparations, except only the CMJ-33 compound (0.1 µM/kg), which shows a discrete tendency to decrease. Changes in the level of NO derivatives in liver tissue under the action of tested CC proved to be inconclusive. Administration of the tested CC results in a discrete reduction in the level of S-nitrosothiols in the liver, with the exception of the compounds CMD-4 and CMJ-33, which at maximum doses (1.0 µM / kg) have the property to veraciously decrease the level of S- nitrosothiols by 16% and 20% (p < 0.05) respectively compared to the control group. There was a discrete tendency to decrease the intensity of the AGEs product formation processes in the liver of white rats after the administration of the tested bioactive compounds. At the same time, compounds CMD-4 and CMJ-33 at the maximum dose of 1.0 µM/kg statistically significantly reduce the level of AGEs by 20% and 26% compared to the values attested in the control group.

Changes in the values of advanced oxidation protein products — AOPP in the liver were found to be without statistical relevance, these values remaining at the level of the reference parameters.

The results of the study show that the tested CC did not induce statistical relevance changes in the IMP content in the liver, except for the compound CMD-4 (0.1 µM / kg), which reduces this index by 28% compared to the control values.

The evaluation results of the antioxidant protection indexes: SOD, CAT, His and TAA in hepatic tissue on the action of local CC are reflected in the statistical data of Table 2.

The study shows that SOD decreases statistically conclusively by 24% (p <0.05) when administering CMD-4 (1.0 µM / kg), and the catalase function in this case is veracious decreasing by 23%. The changes in the content of His and TAA proved to be inconclusive, keeping them within the limits of the values recorded in the control.

Discussion 

In this study, the level of oxidative stress in the liver tissue of the laboratory animals subjected to the action of local CC, derivatives of thiosemicarbazide, was analyzed. It is evident the selective action of the studied compounds on the indices of oxidative stress and antioxidant system, which depends on the degree of their employment at different stages of the metabolic processes that occur in the liver tissue.

As free radicals have very short half-life periods, the in vivo assessment of oxidative stress is based on the measurement of different stable oxidized products of biomolecules in the cells and tissues of living organisms. Oxidative stress assessment methods are often referred to as fingerprinting methods, by which end products resulting from the interaction of ROS and RNS with various biomolecules such as membrane lipids, proteins and amino acids, carbohydrates, nitrogenous bases,  etc., are measured [7].

Primary products of the peroxidation of polyunsaturated fatty acids result in the formation of peroxyl and alkoxyl radicals, which are very reactive and have a short life, being further subjected to other reactions with the formation of different aldehydes, such as MDA, 4-hydroxyalkenals and acrolein. These aldehydes, because of their electrophilic nature, have a very high damage potential. The most abundant aldehyde resulting from the lipids peroxidation is the MDA and its accumulation may cause alterations in proteins, nucleic acids and many other biomolecules.

In this study we obtained significantly lower levels of the end product of lipid peroxidation - MDA in liver tissue under the influence of most of the preparations studied, except only CMJ-33 (0,1 µM / kg), which shows a discreet tendency to decrease. Reducing the level of MDA under the influence of most of the tested CC in liver tissue, possibly, is an expression of several factors: the intensity of the formation of the primary products of lipid peroxidation, the biosynthesis of MDA, the speed of the metabolic processes in the tissue and the ability of the body to eliminate this final product, as well as the level of substances with antioxidant role. We can admit that the tested compounds, due to their property of lowering the level of MDA by various mechanisms, can increase the efficiency of cellular protection against various peroxidants and cytotoxic agents.

Therefore, the hepatic level of MDA could be used as a test to assess the efficacy of tissue protection against lipid peroxidation by free radicals when testing different CC.

NO is a free radical that plays an important role in various physiological processes, and RSNO represent the natural reservoir (deposit) and form of transport of nitric oxide. Nitric oxide due to its high reactivity can cause S-nitrosylation of cysteine residues from proteins or peptides, which is an important redox signaling mechanism that regulates a wide range of biological, physiological and cellular functions in various tissues, including liver [8].

The tendency to decrease NO concentration, recorded in our research, even if it did not reach the statistical significance limit, is probably due to the inhibition by tested compounds of nitric oxide synthase (enzyme that increases the level of NO in tissues) and could be explained by the antitumor activity of these compounds [4-5]. These findings are consistent with the data of some authors, who found that nitric oxide synthase inhibitors that reduce NO production might have a therapeutic role in certain cancers due to their property of reducing angiogenesis, proliferation, and causes suppression of tumor invasion [9].

The decrease in the level of RSNO in the liver when administering the maximum doses (1.0 µM / kg) of the compounds CMD-4 and CMJ-33, recorded in our experiences is probably due to their intense decomposition by the transition metal ions, such as copper (II) ions, which are part of the tested compounds, and  by various redox active species, including reactive oxygen species [10], or by catalytic denitrosylation by specific enzymes, such as thioredoxin (Trx) and S-nitrozoglutation reductase (GSNOR) - enzymes that eliminate NO from nitrosylated proteins / peptides [11].

This could create a deficiency of NO signaling, which would decrease the proliferation intensity of the liver progenitor cells and cause the reduction of the processes of restoration in the hepatic lesions and, it is not excluded, could cause manifestations of liver toxicity. Indeed, according to some researchers, the increased NO and RSNO levels, in contrast to the low levels, induce the proliferation of liver progenitor cells and improve the liver restoration from partial hepatectomy [12].

In view of these aspects, it is important for the research of new bioactive compounds to evaluate the NO/RSNO system in order to identify potential changes of this system and to provide a better understanding of the mechanisms of action of these compounds, which will facilitate not only the discovery of new targets for the action of bioactive compounds, but also the development of new therapeutic agents.

Studies carried out over the last decades have brought more and more evidence that during the glycation process, namely the interaction of carbohydrates with the free amine groups of proteins, the early glycation products are formed first, and they are subsequently rearranged into final structures, called advanced glycation end products - AGEs through a series of complex chemical reactions. AGEs are involved in the pathogenesis of atherosclerosis, chronic inflammatory processes, aging, cancer, neurodegenerative diseases, such as Alzheimer's disease, etc. [13].

The obtained results indicated the discrete tendency to decrease the intensity of the AGEs product formation processes, and the compounds CMD-4 and CMJ-33 at the maximum dose of 1 µM / kg significantly decrease the level of AGEs in the liver tissue, compared to the values attested in the control group. This fact indicates the anti-AGEs activity, and capacity to prevent the formation of AGEs, exercised by studied CC, and can be qualified as a positive moment, because, AGEs can interact with the specific receptors of the cell surface - RAGE (receptor for advanced glycation end products), which in the liver are expressed in hepatic stellate cells and myofibroblasts - cells involved in the fibrogenesis of liver disease, and therefore, they may alter intracellular cell signaling, gene expression, enhance ROS production and activation of several inflammatory pathways, including the release of pro-inflammatory cytokines, growth factors and adhesion molecules by activating the NF-κB (nuclear factor κappa-light-chain-enhancer of activated B cells) pathway [14].

Therefore, the level of AGEs can serve as a valuable indicator when evaluating the action of new CC on cells and tissues, including the liver.

As is known, proteins by their complex structure, present numerous points where they can suffer oxidative attacks, making them one of the first targets of free radicals. 

The oxidative changes of proteins, called advanced oxidation protein products - AOPP, are protein products that contain dityrosine cross-linking bonds which can lead to various important functional consequences such as inactivation of enzymes, increased susceptibility to aggregation and resistance upon proteolysis, increased formation of free radicals, activation of the nuclear factor NF-κB and release of proinflammatory cytokines, adhesion molecules and growth factors [15,16].

For that reason, it is considered that AOPP may be a more accurate biomarker of oxidative stress than lipid peroxidation products [16-18].

In this study the changes in the values of the advanced oxidation protein products - AOPP in the liver, proved to be statistically unsuggestive, they keeping within the limits of the reference parameters, so the studied compounds do not cause excessive synthesis of FR with prooxidative character at the local tissue level and, it is not excluded that they, on the contrary, annihilate the FR action due to their antioxidant properties. This fact is in agreement with the data of researchers who have detected antioxidant properties characteristic of thiosemicarbazonic compounds [19].

As is known in hypoxic and ischemic conditions in blood serum increases the concentration of ischemia modified albumin (IMA) increases, characterized by a significant change in the ability to bind the transition metals, especially cobalt [20]. The IMA level may increase in certain circumstances, such as myocardial ischemia, malignant neoformations, chronic diseases, and inflammatory processes [21, 22].

In view of this, we investigated the influence of CC on the content of IMP in liver tissue. The results of the study show that the tested CC did not induce statistically significant changes in the IMP content in the liver tissue, except for the CMD-4 (0.1 µM/kg) compound, which statistical truthful reduces this index to the control values, suggesting that this compound has the property of diminishing tissue hypoxia. 

Thus, the results obtained demonstrate that the tested CC does not cause oxidative damage to the proteins of the liver tissue, which is manifested by the discrete tendency to decrease the intensity of the AGEs product formation processes under the influence of the majority of the investigated CC and to maintain the PPOA values within the normal values.

The conclusive reduction of the content of AGEs and IMP by the compound CMD-4 and AGEs by the CMJ-33 in the maximum dose of 1 µM/kg denotes their antioxidant effect.

Under the action of local CC studied the indices of antioxidant protection: SOD, catalase, His and TAA in the liver tissue of laboratory animals undergo changes of different intensity. Decrease of the activity of antioxidant enzymatic links SOD - enzymes that destroy superoxide anion O2- with the formation of O2 and hydrogen peroxide), catalase (which inactivates H2O2) in the liver after administration of compounds CMD-4, CMJ-33 and CMT-67 can result with weakening of the  cells enzymatic protection against the oxidation reactions with free radicals under conditions of a pronounced activation of this process. Not coincidentally, these two enzymes have the highest reaction rates (about 2 x 109 M-1 s-1), which allows them not only maximum efficiency, but also the possibility to act independently by providing substrates or coenzymes [23]. 

On the other hand, changes in the content of His (which has relevant antioxidant effects [24-25] and TAA (which includes various antioxidants, both enzymatic and non-enzymatic) have been shown to be inconclusive, maintaining them within the limits of the control values. This demonstrates that the studied compounds possess the ability to effectively remove free radicals (FR) and maintain the redox balance at local liver tissue level.

The antioxidant system, whereby aggressive compounds formed by the action of FR are converted into non-active compounds, play a decisive role in cell defending against the harmful action of FR. This system comprises a large number of elements, being as diverse as free radicals, cells containing a variety of scavenging-capacity substances for multiple radical species precisely to ensure maximum protection [26]. The high efficiency of antioxidants is explained by their wide diversity, the location in different cell compartments, the synergistic nature of their action, the summation of their combined action, each acting according to different mechanisms and varying levels of the chain of FR evolution in the body. Under normal conditions, antioxidants eliminate pro-oxidants, but under oxidative conditions, pro-oxidants predominate over antioxidants, which can lead to many inflammatory diseases, including cancer [27-28].

For these reasons, indices of antioxidant protection may be useful as diagnostic markers or therapeutic targets.

Because excessive FR formation can cause multiple cellular and tissue damage, their effects can be analyzed by local tissue markers of oxidative stress and antioxidant system. Further studies are needed to confirm the therapeutic utility of these bioactive tested compounds.

Conclusions 

The most informative biomarkers of functionality of the prooxidant and antioxidant system have been estimated and selected to assess the level of oxidative stress in liver tissue to experimental exposure through the administration of local CC to laboratory animals and which can be used to determine the efficiency of new local drug preparations.

The reduction of oxidative stress indices (MDA, AGEs, S-nitrosothiols, IMP) when using local CC denotes their antioxidant effect.

The elucidation of molecular mechanisms who stay on the basis of the CC action expand the theoretical knowledge of the biological properties of a range of chemical compounds and also offers new possibilities to explore prospective objects for the purpose of obtaining new efficient drug preparations.

Competing interests

None declared

Authors` contribution 

The authors have equally contributed to the manuscript drafting, design and paper editing. All authors approved the final version of manuscript.

 Authors’ ORCID IDs

Valeriana Pantea. https://orcid.org/0000-0002-8835-6612
Veaceslav Popa. https://orcid.org/0000-0003-3139-2652
Olga Tagadiuc. https://orcid.org/0000-0002-5503-8052
Lilia Andronache. https://orcid.org/0000-0002-8781-8037
Valentin Gudumac. https://orcid.org/0000-0001-9773-1878

References                                                                            

1. Liguori I., Russo G., Curcio F., Bulli G. et al. Oxidative stress, aging, and diseases. Clin Interv Aging. 2018; 13: 757–772.

2. Pavan F. R., da S Maia P. I., Leite S. R., Deflon V. M. et al. Thiosemicarbazones, semicarbazones, dithiocarbazates and hydrazide/hydrazones: anti-Mycobacterium tuberculosis activity and cytotoxicity. Eur J Med Chem. 2010 May; 45 (5): 1898-1905.

3. Stefani C., Jansson P. J., Gutierrez E. et al. Alkyl substituted 2'-benzoylpyridine thiosemicarbazone chelators with potent and selective anti-neoplastic activity: novel ligands that limit methemoglobin formation. J Med Chem. 2013 Jan 10; 56 (1): 357-370.

4. Ma J., Ni X., Gao Y., Huang K. et al. Semicarbazone Derivatives Bearing Phenyl Moiety: Synthesis, Anticancer Activity, Cell Cycle, Apoptosis-Inducing and Metabolic Stability Study. Chem Pharm Bull (Tokyo). 2019 Apr 1; 67 (4): 351-360.

5. Gulea A., Poirier D., Roy J., Stavila V., Bulimestru I., Ţapcov V., Bârcă M., Popovschi L. In vitro antileukemia,antibacterial and antifungal activities of some3d metal complexes: Chemical synthesis and structure activity relationships // Journal of Enzyme Inhibition and Medicinal Chemistry, 2008; V. 23. Nr.6, p. 806-818.

6. Gudumac V., Rîvneac V., Tagadiuc O., et al. Metode de cercetare a metabolismului hepatic. Elaborare metodică / Chișinău: S.n., 2012 (Tipogr. „Tehnica-Info”). – 162 p.

7. Persecă O., Orăsan R., Pârvu AE., Moldovan R. Studiul markerilor de stres oxidativ în expunerea experimentală la izocianați. Clujul Medical, 2012, vol.85 (3), p. 358-362.

8. Neelam Shahani, Akira Sawa. Protein S-nitrosylation: role for nitric oxide signaling in neuronal death. Biochim Biophys Acta. 2012 Jun; 1820 (6): 736–742.

9. Lala P. K., Chakraborty C. Role of nitric oxide in carcinogenesis and tumour progression. Lancet Oncol. 2001 Mar; 2 (3): 149-56.

10. Lo Conte, Mauro & Carroll, Kate. (2013). The Chemistry of Thiol Oxidation and Detection. 10.1007/978-94-007-5787-5_1.

11. Min Sik Choi. Pathophysiological Role of S-Nitrosylation and Transnitrosylation Depending on S-Nitrosoglutathione Levels Regulated by S-Nitrosoglutathione Reductase. Biomolecules & Therapeutics 2018; 26 (6): 533-538.

12. Andrew G., Cox, Diane C. Saunders, Peter B. Kelsey, Jr., Allie A. Conway, et al. S-Nitrosothiol Signaling Regulates Liver Development and Improves Outcome following Toxic Liver Injury. Cell Reports January 16, 2014; 6: 56–69.

13. Ajith A., Vinodkumar P. Advanced Glycation End Products: Association with the Pathogenesis of Diseases and the Current Therapeutic Advances. Curr Clin Pharmacol. 2016; 11 (2): 118-27.

14. Hyogo H., Yamagishi S. Advanced glycation end products (AGEs) and their involvement in liver disease. Curr Pharm Des. 2008;14 (10): 969-72.

15. Zuwała-Jagiełło J., Pazgan-Simon M., Simon K., Warwas M. Elevated advanced oxidation protein products levels in patients with liver cirrhosis. Acta Biochimica Polonica, Vol. 56 No. 4/2009, 679–685.

16. Capeillère-Blandin C., Gausson V., Descamps-Latscha B. et al. Biochemical and spectrophotometric significance of advanced oxidized protein products. Biochim Biophys Acta 2004; 1689: 91-102.

17. Witko-Sarsat , Friedlander M., Capeillère-Blandin C., Nguyen-Khoa T. et al. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int. 1996 May;49 (5): 1304-13.

18. Dalle-Donne I., Rossi R., Giustarini D., Milzani A., Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta. 2003 Mar; 329 (1-2): 23-38.

19. Reddy DHK, Lee SM et al. Synthesis, characterization of thiosemicarbаzone metal complexes and their antioxidant activity in different in vitro model systems. J. Serb. Chem. Soc. 2012. 77 (2) 229–240.

20. Bar-Or D., Curtis G., Rao N., Bampos N., Lau E. Characterization of the Co(2+) and Ni(2+) binding amino-acid residues of the N-terminus of human albumin. An insight into the mechanism of a new assay for myocardial ischemia. Eur J Biochem. 2001, 268(1), p.42-47].

21. Mustafa Ilker Inan, Omer Deniz, Ergun Ucar.et al. The role of serum ischemia modified albumin level for the diagnosis of pulmonary thromboembolism. European Respiratory Journal 2013; 42: P4115; DOI:

22. Caner Karahan, Mehmet Sonmez, Fatma Saglam. et al . Can ischemia-modified albumin be a valuable indicator of tissue ischemia in polycythemia vera? Hematology 2010, 15 (3), p. 151-156

23. Olinescu R. Radicali liberi în fiziopatologia umană. Bucureşti, 1994. – 215p.

24. Lee JW., Miyawaki H., Bobst EV. et al. Improved functional recovery of ischemic rat hearts due to singlet oxygen scavengers histidine and carnosine. J Mol Cell Cardiol 1999; 31: 113–21.

25. Velez S,Nair NG,Reddy VP. Transition metal ion binding studies of carnosine and histidine: biologically relevant antioxidants. Colloids Surf Biointerfaces 2008; 66: 291–294

26. Srdic-Rajic T., Ristic KA. Antioxidants: Role on health and prevention. Encyclopedia of Food and Health, 2016, pp. 227-233.

27. Ferrari CKB. Oxidative stress pathophysiology: Searching for an effective antioxidant protection. International Medical Journal. 2001; 8: 175-184.

28. Kolanjiappan K. et al. Measurement of erythrocyte lipids, lipid peroxidation, antioxidants and osmotic fragility in cervical cancer patients. Clinica Chimica Acta, 2002, 326 (1-2): 143-149.