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Open Access | Published: 2023 - Issue 2

ANTI-OXIDATIVE BURST, CYTOTOXICITY, AND ADME STUDIES OF THIOUREA COMPOUNDS

Samreen Soomro1*, Sibghatullah Sangi2, Naira Nayeem3

 

  1. Department of Basic Health Sciences, Faculty of Pharmacy, Northern Border University, Rafha, KSA.
  2. Department of Clinical Pharmacy, Faculty of Pharmacy Northern Border University, Rafha, KSA.
  3. Department of Chemistry, Faculty of Pharmacy Northern Border University, KSA.

 


ABSTRACT

This oxidative stress is linked to number of immune related disorders like hypersensitivity, autoimmune disease and inflammation. In this context, many new compounds are being synthesized and screened for the purpose of immunomodulation. In current study, we are aiming to discover the potential of thiourea derivatives as potential immunomodulators via suppressing oxidative burst mechanism in macrophages and neutrophils. The immunomodulation studies were conducted using cell based spectrophotometric techniques. Fluorescent microscopy and Predictions using web-based tools. Results suggested that among three selected thiourea derivatives comp 3 significantly suppressed the superoxide anion with 86.94 ±1.2 percent inhibition. Additionally, comp 2 and 3 were significantly involved in inhibiting the myeloperoxidase dependent pathway that produce hypochlorite anion with IC50 value of 45.3±0.4 and 10.4±0.2 µg/mL, respectively. Similarly, intracellular oxidative stress was suppressed by comp 2 and 3 detected by fluoresce microscopy. In addition, the comp 1 and 3 showed moderate inhibitory activity with 36.9 and 33.8% respectively. No any compound has shown cellular toxicity at the 25 µg/mL tested fibroblast cell line. Physicochemical and Pharmacokinetic Predictions of comp 2 and 3 showed high blood brain barrier (BBB) permeability along with Gastrointestinal (GI) absorption. Prediction studies revealed the lethal dose (LD50) of 1700 mg/kg and 1400 mg/kg respectively. Additionally comp 3 showed activity against pancreatic carcinoma MIA PaCa 2, Colon adeno carcinoma SW620 and Non-small cell lung carcinoma NIH838. In conclusion, new compounds have potential of being immunomodulatory agent particularly comp 3 demonstrated intriguing biological potential that could be explored further for its mechanistic studies.

Keywords: Respiratory burst, Inflammation, Cytotoxic, ADMET


Introduction

Phagocytic NADPH oxidase, enzyme located on the membrane of immune cells like macrophages or neutrophils and become activated during the process of inflammation. Activation of this enzyme yields in the release of reactive oxygen free radicals (ROS) required to kill the invading pathogen. In addition to this another Enzyme inducible nitric oxide synthase (iNOS) is expressed in activated phagocytic cells such as macrophages and neutrophils, and it catalyzes the production of nitric oxide (NO), which is an important mediator of the immune response against pathogens. However, the excessive production of NO and other reactive oxygen species (ROS) and reactive nitrogen species (RNS) during phagocytosis can lead to destruction of macromolecules like lipids, DNA, and proteins. This is regarded as oxidative stress, and it can results to cellular dysfunction and tissue damage. ROS and RNS also behave as intracellular signaling molecules in biological processes, like cell development, differentiation, and apoptosis. However, when their levels are increased, they can initiate various pathological conditions, including inflammation, cancer, and neurodegenerative diseases [1-5].

                    

Phagocytic NADPH oxidase is activated during phagocytosis, and catalyze the production of superoxide (•O-2). Another enzyme superoxide dismutase (SOD), converts the superoxide into hydrogen peroxide H2O2 [6, 7]. In the presence of myeloperoxidase further chloride reacts with H2O2 more making more toxic by product Hypochlorous acid, HOCl. Therefore, in abnormal condition, phagocytes can become over activated and stimulate the phagocytic NADPH oxidase and inducible nitric oxide synthase iNOs, leading to excessive production of ROS and reactive nitrogen species (RNS) respectively, contribute to inflammation and carcinogenesis. Furthermore, peroxynitrite (ONOO−), which is formed by the reaction between NO and superoxide (O2•−), is a highly toxic radical that can cause mutations by inducing breaks in double-stranded DNA or modifying DNA bases. Together, the excessive production of ROS and RNS during inflammation and infection have role in the growth of various pathological conditions, involving cancer, neurodegenerative diseases, and cardiovascular disorders. Therefore, it remains imperative to regulate the production as well as elimination of ROS and RNS to maintain cellular and tissue homeostasis and prevent oxidative stress [8-10].

Oxidative stress is one of leading cause of inflammation and considered as potent inflammatory mediator. Antioxidants are molecules that can neutralize and scavenge free radicals, protecting cells and tissues from their damaging effects. However, when free radical generation surpasses the body's antioxidant capacity, oxidative stress can occur [11]. Inflammation is a broad category involving a lot of immune cells, enzymes and chemical mediators resulting in the beginning of a mentioned disease. In the mentioned several mediators, the free radicals can also affect the tumor microenvironment and promote tumor growth, invasion, and metastasis by modulating the immune response, angiogenesis, and extracellular matrix remodeling. Therefore, having in-depth comprehension of the role free radicals in chronic inflammation and cancer is crucial for the development of effective strategies for precaution and management of of these diseases. To identify anti-oxidative stress activity of synthetic compounds we have selected thiourea derivatives, previously reported for their wide range of biological activity. It is reported that these derivatives possess Phytotoxic, Cytotoxic, Acetylcholinesterase and Butryl cholinesterase, glucuronidase inhibitory, α-chymotrypsin inhibitory, urease inhibitory activity and anti-microbial activity [12-18]. We have investigated these compounds for its effect on intracellular and extracellular oxidative stress in activate mouse macrophages and whole blood phagocytes and identified it’s in vitro cellular toxicity. Furthermore, in silico physicochemical and ADMET studies have done to predict the biological potential of the compounds.  

Materials and Methods

Collection of Compounds and Samples Preparation (1mg/mL Stock Solution)

Thiourea compounds were selected based on available literature for their potential biological activity and purchased from Bench chem and Sigma Aldrich. (Table 1) we used 1 mg/mL of stock solution by the addition of one milligram of compound in 50 µl DMSO, solubilize, if needed sonication is done till the compound solubilized completely. 950 µl phosphate buffer saline was added to get the final conc. of 1mg/mL, and stored in freezer 4°C until use.

Table 1. Selected thiourea compounds

S.no

Structure

IUPAC

Names

Compound code

purchased

1

N-(3-chlorophenyl)-N'-(3,4-dimethylphenyl)thiourea

Comp-1

Benchchem

C18H21NOS

M.W 299.4g/mol.

95%Pure

2

ChemSpider 2D Image | 1-Phenyl-3-(3-pyridinylmethyl)thiourea | C13H13N3S

1-Phenyl-3-(3-pyridinylmethyl) thiourea

Comp-2

Sigma Aldrich

C13H13N3S

M.W 243.3 g/mol.

95% pure

3

N-(3-Methyl-2-pyridinyl)-N'-Phenylthiourea

Comp-3

Benchem

C13H13N3S

M.W 243.33 g/mol.

95% pure

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Determination for Oxidative Burst by Chemilluminescence Assay

Chemilluminescence assay was performed as published before [19]. Different concentration of test compounds 1 µg/mL, 10 µg/mL and 100 µg/mL were set in 96 well white flat bottom plate, incubated at 37 ºC for fifteen mins in the thermostat chamber of luminometer with 1:20 dilution of whole blood in HBSS ++. Control wells established HBSS ++ and cells without the test compounds. Following incubation luminol (7 × 10 -5 M), and serum opsonized zymosan (SOZ) were added into each well except blank wells that contain HBSS ++ only to detect free radicals. Intracellular reactive oxygen detecting probe. Luminometer was used to detect oxidative burst ROS production. The level of the ROS was measured as relatively light units for fifty min using repeat scan mode.

NBT Assay for Superoxide Production

Macrophages J774 (1 × 107) cells/ml were suspended in medium, 100µl of macrophages were placed in 96-well plates in the RPMI media with FBS. Nitro blue tetrazolium assay was done as prescribed earlier [20]. Cells were and test compounds were incubated for 30 min at 37oC, after incubation 100ul of 1mg/ml NBT solution was added and 50ul of phorbol-12- myristate-13-acetate (PMA) Diphenyleniodonium was used as a negative control after addition at a concentration of 10 µg/ml to activate the cells and after an additional 90 min incubation. Blue formazan was dissolved in DMSO and the absorbance was measured at 570 nm. Controls were set as cells in medium rather than without test compound. Percent inhibition of NBT reduction was calculated.

Determination of Intracellular Oxidative Stress

Intracellular ROS level was determined using the fluorogenic probe 2′,7′-Dichlorofluorescein (DCFH) a dye that is cell-permeable and oxidized by free radicals to highly fluorescent DCF particularly by H2O2. Briefly mouse macrophages J774.2 were adjusted to 5 × 104 /mL in 24 well transparent bottom plate and were activated using PMA (200 ng/mL) with or without compounds at 25 µg/mL for 30 min. Treated cells were further incubated with 20 μM DCFH for 30 mins at 37 °C. A fluorescent microscope detected fluorescence and we used Adobe Photoshop CS2 to process the images [21].

Measurement of NO in Cell Culture Supernatant

J774 macrophage cells were collected and adjusted to a concentration of 10^6 cells/ml, and 200 µl of cells were added to a 96-well microtiter plate. Lipopolysaccharide from E. coli was added at a final concentration of 30 µg/ml to activate the cells, inducing NO production. Next, test compounds were added to the Cells and plates at a concentration of 25 μg/ml were incubated at 37° C., 5% CO 2 for 24 hours. After incubation, cell culture supernatants were collected and immediately processed for nitrite accumulation studies using the Griess method. The Griess method used 50μl of 1% sulfanilamide in 2.5% phosphoric acid added to 50 µl of the cell culture supernatant, followed by 50μl of 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid. Absorbance was read at 550 nm after incubation for 10 minutes at 23°C. The micromolar concentration of sodium nitrite was calculated from a standard curve generated using sodium nitrite as the reference compound. The findings was described as means ± standard deviation (SD) of triplicate readings. This method provides a simple and reliable way to evaluate the ability of test compounds to modulate NO production in activated J774 macrophage cells, which can be useful in screening for potential anti-inflammatory or immunomodulatory agents [22].

In Vitro Cytotoxicity Assay

The MTT assay is a commonly used method to measure cell viability and proliferation. In this assay, 3T3 fibroblast cells were plated in a 96-well plate and treated with different concentrations of test compounds for forty-eight hours. After that, the cells were washed and MTT was added to the wells. MTT is reduced by mitochondrial dehydrogenases in viable cells to form a colored formazan product. The dissolution of formazan was done in DMSO, then the absorbance was measured at 570 nm using a microplate reader. The IC50 values, which represent the concentration of the compounds required to hinder cell viability by fifty percent, were calculated. Cyclohexamide, a known inhibitor of protein synthesis, was used as a positive control to compare the effect of the test compounds on cell viability. [23] The percentage of cell viability was calculated using the formula:

 

%Cell Viability = [Total Viable cells (Unstained) / Total cells (Viable +Dead)] × 100.

(1)

The viable cell count was calculated as Average viable cell count per square × Dilution Factor × 10.

Physicochemical and Pharmacokinetic Predictions of Thiourea Compounds

SwissADME (http://www.swissadme.ch/index.php) and PreADMET (https://preadmet.bmdrc.kr/) predicted the physicochemical and pharmacokinetic profiles of thiourea compound. The cell-line cytotoxicity was predicted by CLC-Pred (http://way2drug.com/Cell-line/) The toxicity of the compound was identified using webtool ProTox II (http://tox.charite.de/).

Statistics

To determine the statistical significance of the results, a paired t-test was used to compare the mean values of the experimental test conditions with their respective controls. A p-value of 0.05 was regarded in order to signify a considerable difference amongst the two groups, denoted as * p<0.05. A p-value of less than 0.005 was regarded to signify a highly considerable difference, denoted as ** p<0.005.

Results and Discussion

Effect of Compounds on Superoxide Production O2•−

Phagocytic NADPH Oxidase is activated by Phorbol Myristate Acetate PMA in mouse macrophages and initiate the protein kinase C (PKC) pathway. PKC pathway lead to the generation of superoxide anion [24]. PMA activated cells were treated exposed to 25µg/mL of compounds and effect on superoxide was detected by NBT reduction assay. Comp 3 was found to suppress the superoxide production significantly p<0.005 with percent inhibition of 86.94 ±1.2 %. In contrast, comp 1 and 2 moderately enhanced the superoxide generation (Table 2).

 

Effect of Compounds on HOCl Production

During myeloperoxidase-dependent intracellular killing process of phagocytosis HOCl, hypochlorous acid is produced that can be detected with Luminol enhanced chemilluminescence technique [25]. The study demonstrated that compounds 2 and 3 have significant inhibitory activity against ROS production in whole blood cells activated with zymosan. The IC50 value for comp 2 was found to be less than 5.3 ± 0.4 µg/mL, indicating a strong suppressive effect on ROS. Comp 3 also showed significant inhibitory activity with an IC50 value of 10.4 ± 0.2 µg/mL, which is lower than the IC50 value of ibuprofen (43.4 ± 0.9 µg/mL). The inhibitory effect of these compounds on ROS production suggests their potential as anti-inflammatory therapeutic agents (Table 2).  

 

Table 2. Effect of Compounds on ROS, NO, and O-2 Production in activated macrophages and neutrophils and cytotoxicity analysis

Compounds

ROS (HOCl)

IC50 ± SD

µg/mL

Superoxide O-2

%inhibition ±SD

Nitric oxide (NO)

%inhibition ±SD

Cytotoxicity

IC50 ±SD

µg/mL

Comp-1

>100

-29.2± 0.51

36.9±0.7

>25

Comp-2

5.3 ± 0.4**

-49.63± 0.90

-17.9±0.66

>25

Comp-3

10.4 ± 0.2 *

86.94 ±1.2**

33.8±0.80

>25

Standard

drugs

Ibuprofen

43.4±0.9ug/mL

Diphenyl iodonium

92±0.94%

NG monomethyl L arginine

64±0.05%

Cyclohexamide

0.13±0.02µg/mL

The findings are shown as the mean ± standard deviation (SD) of triplicate measurements. A paired t-test was employed in determining the statistical significance of the difference between the experimental and control groups A p-value of less than 0.005 was considered to indicate a highly significant difference, denoted as ** p<0.005, while a p-value of less than 0.05 was considered to indicate a significant difference, denoted as * p<0.05.

 

Effect of Compound on Intracellular Oxidative Stress (H2O2)

Intracellular oxidative stress was also investigated using fluorescence microscopy technique and effect of comp 2 and 3, was studied on intracellular ROS using DCFH dye. Reaction of this dye with ROS, primarily hydrogen peroxide (H2O2), results in the production of fluorescent molecule DCF, so that DCF fluorescence can be used as a measure for intracellular ROS levels [26]. Intensity of green fluorescence is directly proportional to the production of ROS.  We compare the results with positive control in which J774.2 Mouse macrophages cells activated with PMA and negative control in which Diphenyliodonium was used to suppress ROS. Compound 2 and 3 significantly decreased the intensity of fluorescence in the cells, at 25 µg/mL we can conclude that compound 2 and 3 selectively suppress the H2O2 therefore they also suppress the HOCl production detected by chemilluminescence technique (Figure 1).

 

Figure 1. Effect of natural compound on intracellular oxidative stress measured with fluorescence microscopy as described in material and methods. Results are presented as images processed with adobe Photoshop.

 

Effect of Compounds on Nitric Oxide (NO)

The statement is accurate. Lipopolysaccharide (LPS) activate toll-like receptor 4 (TLR4) on macrophages, leading to the production of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO) [27]. In the study mentioned, the researchers investigated the effect of the test compounds on NO production in macrophages activated with LPS. The results showed that comp1 and 3 moderately suppressed NO production, with a percentage of inhibition of 36.9% and 33.8% at a concentration of 25 μg/mL, respectively. These findings suggest that the compounds may have a potential therapeutic effect on conditions that involve excessive NO production, such as cardiovascular diseases. However, further studies are needed to confirm their efficacy and safety in vivo. In contrary, comp 2 stimulated the production of nitric oxide with % stimulation of 17.9% (Table 2).

 

Effect of Compound on Cytotoxicity

Viability of 3T3 Fibroblast cells were studied in presence of compounds at several concentrations 50, 5 and 0.5 µg/mL. Results were compared with Cyclohexamide, which is used as standard cytotoxic drug [28]. The data is presented in Table 2.  All of the tested compound were seen to be nontoxic for fibroblast cells with IC50 value of >25µg/mL therefore are reliable to be further researched as compared to the Cyclohexamide with IC50 value of 0.13±0.07µg/mL.

 

Physicochemical and Pharmacokinetic Predictions of Thiourea Compounds

Compound 2 and 3 were studied for it in-silco ADMET analysis, physicochemical and toxicity effect using web based tools [29]. Prediction studies have shown that compound exhibit the significant potential of therapeutic agent with GI absorption and blood brain barrier permeability. Both compounds showed bioavailability of 55 % and does not prove to be inhibitor of major drug metabolizing enzymes except CYP2C19 and CYP3A4. Skin permeation was found to be in the range of −0.769 to −5.818 cm/s, which falls under the acceptable range. Predicted value of toxicity for both compounds showed that compounds are nontoxic to liver and possess high lethal dose (LD50) of 1700 mg/kg and 1400 mg/kg respectively, refer the Tables 3 and 4.

 

Table 3. Predicted ADMET properties of 1-Phenyl-3-(3-pyridinylmethyl) thiourea (comp2)

Physicochemical Properties

Formula C13H13N3S, Molecular weight 243.33 g/mol Num. heavy atoms17, Num. arom.12, heavy atoms Fraction Csp3 0.08, Num. rotatable bonds 5, Num. H-bond acceptors 1, Num. H-bond donors 3, Molar Refractivity73.43, TPSA 69.04 Ų. Water solubility Moderately soluble.

Pharmacokinetic Properties

GI absorption = high; BBB permeant = yes; P-gp substrate = no; CYP1A2 inhibitor = yes; CYP2C19 inhibitor = yes; CYP2C9 inhibitor = no; CYP2D6 inhibitor = no; CYP3A4 inhibitor = yes; Log Kp (skin permeation) = −6.4 cm/s

Drug Likeness

Lipinski = yes, 0 violation; Ghose = yes; Veber = yes; Egan = yes; Muegge = yes; bioavailability score = 0.55

Medicinal Chemistry

PAINS = 0 alert; Brenk =  1 alert: thiocarbonyl_group ; leadlikeness =  No; 1 violation: MW<250; synthetic accessibility = 2.07

Toxicity

AMES test = mutagenic; carcinogenicity in mouse= none; carcinogenicity in rats =yes; predicted LD50 = 1700 mg/kg; hepatotoxicity = (none)