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Article

Innovative Therapeutic Approach to Chemical Burns Produced by Vesicants; An Experimental Study

by
Cristina Anca Secară
1,
Otilia Liudmila Cinteză
2,
Diana Popescu
1,
Claudia Valentina Popa
1,2,
Camelia Andreea Hîrjeu
1,
Oana Cristina Voinea
1,3,
Luiza Georgia Șerbănescu
3,4,
Andreea Marilena Păuna
3 and
Mihail Silviu Tudosie
3,*
1
Cantacuzino National Medical-Military Research-Development Institute, 050096 Bucharest, Romania
2
Faculty of Chemistry, University of Bucharest, 030018 Bucharest, Romania
3
Carol Davila University of Medicine and Pharmacy, 020021 Bucharest, Romania
4
Prof. Dr. Al. Trestioreanu Oncological Institute, 022328 Bucharest, Romania
*
Author to whom correspondence should be addressed.
J. Mind Med. Sci. 2022, 9(2), 294-303; https://doi.org/10.22543/2392-7674.1355
Submission received: 23 June 2022 / Revised: 7 July 2022 / Accepted: 18 August 2022 / Published: 15 October 2022
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Versions Notes

Abstract

:
Vesicants are compounds that cause severe toxic effects on various tissues. Such chemical action causes tissue necrosis, with clinical expression of skin lesions with a burning character and characteristic blisters. Clinical toxic effects of cutaneous vesicles are correlated with the absorbed dose and exposure time. The goals of the study are to evaluate in vitro the skin toxicity produced by the vesicant chemical compound 2-chloroethyl-ethyl sulfide (CEES), to develop a complex antidote formula, and to optimize the therapeutic efficacy by inclusion in controlled release systems. The experimental protocol aims at the in vitro evaluation of the cytotoxicity of the vesicant compound CEES and of the optimized complex antidote, using the MTT cell viability test. Optimization of the complex antidote formula was achieved by developing and in vitro and in vivo testing of a fixed combination of active substances with anti-inflammatory and antioxidant effects, formulated as a solution with cutaneous administration. In vitro cytotoxicity tests on fibroblast cultures revealed the protective effect of the newly developed antidote solution, specifically a dose-related effect in the case of vesicant exposure.

Introduction

Vesicants are compounds that cause serious toxic effects to the eyes, lungs and skin, either by direct contact or through inhalation of vapors. This class of substances includes mustard gas, mustard nitrogen, halogenated oximes and arsenic agents [1,2]. Halogenated vesicants are a family of compounds based on sulfur, nitrogen and oxygen, with similar chemical and biological effects.
There are two types of mustard-based vesicants: nitrogen mustards, known for their use as chemotherapy drugs, and sulfur mustards, that are known only for their applications as warfare agents [3]. When dermally absorbed, molecules of sulfur-based vesicular compounds cyclize forming a highly reactive intermediate that binds to tissue proteins [4]. This chemical action causes tissue necrosis, clinically manifested as burn-like skin lesions and characteristic phlyctenules (blisters). Basal epidermal keratinocytes are the first target. After this, vesicant compounds cause cell death (apoptosis), followed by digestion of the anchoring filaments’ protease at the level of dermal-epidermal junctions, which leads to separation and the appearance of vesicles [5,6,7].
The present study performs an in vitro evaluation of skin toxicity produced by the vesicant chemical compound, 2-chloroethyl-ethyl sulfide (CEES). The objectives of this study are represented by the development of a complex antidote formulation, which pharmacodynamically should be able to antagonize the toxicity of vesicants. As a result of this action, a possible remission of severe lesions is expected, in order to delay the surgical procedure and thus to optimize the therapeutic effectiveness by including it in the systems with a possible controlled release (in the form of microemulsions).

Materials and Methods

Materials and reagents
The following materials and reagents were used to perform the experimental study:
–
MTT cell viability determination kit – SIGMA ALDRICH Germany product;
–
2-chloroethyl-ethyl sulfur (CEES) 97% purity – Sigma Aldrich Germany product;
–
dexamethasone - CAS number 50-02-2 99% purity, produced by Sigma-Aldrich Chemie GmbH, Germany;
–
acetylcysteine - CAS number 616-91-1 99% purity, product of MERCK, Germany;
–
nicotinamide mononucleotide CAS number 1094-61-7 95% purity produced by Sigma Aldrich, Germany;
–
hydrolyzed hyaluronic acid - CAS number 9004-61-9. Purity ≥98%, produced by PARCHEM, USA;
–
recombinant human epidermal growth factor, expressed in E. coli, ≥98% packed as 0,1 mg, produced by Alomone Laboratories, Israel;
–
ketoprofen CAS number 22071-15-4 98% purity produced by Sigma-Aldrich Chemie GmbH, Germany.
Laboratory equipment
EnSight™ Multimode Microplate Reader multimodal reader, PERKIN ELMER, used for optical density reading in the MTT method for determining cell viability;
–
NANO ZETASIZER MALVERN PANALITICAL standard cell analysis system, used for dynamic light diffusion measurements in the study of microemulsions;
–
HPLC LC 4500 JASCO INC system, used to determine the analytical method for dosing antidote components in solutions and microemulsions;
–
ChromNAV 2.0 HPLC software.
The experimental design went through the following stages:
Pharmaceutical formulation of the complex antidote
A complex formulation was prepared in the form of a percutaneous solution whose active substances are capable to pharmacodynamically antagonizing the toxicity of the studied vesicant compound. The following composition is studied: 10% acetylcysteine (antioxidant); 5% ketoprofen (non-steroidal antiinflammatory compound); 4% dexamethasone (steroidal antiinflammatory compound); 10% nicotinamide nucleotide (PARP inhibitor); epidermal growth factor (EGF) 0,1 µg/mL (the stock solution dosage form, 10 µg/ml in concentration, was prepared in 0.9% w/v NaCl); PBS excipients ad 100 ml buffer containing disodium phosphate, sodium chloride, potassium phosphate and potassium chloride, achieving a pH of 7.8, necessary for faster transdermal absorption.
Experimental protocol
In vitro evaluation of 2-chlorodiethyl-ethyl sulfide (CEES) vesicant’s cytotoxicity and of the optimized complex antidote, using the MTT cell viability test.
An experimental model for in vitro toxicity assessment was performed by incubating a culture of fibroblasts (106 cells/ml) with doses of CEES 1: 7.78 × 10-2 M and CEES 2: 7.78 × 10-3M. The cell viability testing was performed using the MTT kit and 3T3 fibroblast cell line.
The 3T3 fibroblast cell line was cultured in 25 cm2 cell culture dishes using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 50 μg/ml gentamicin.
Cells in a 25 cm2 dish were trypsinized with 0,025% trypsin-EDTA and then centrifuged, while the pellet was resuspended in 5 ml of complete DMEM medium. Initial cell count was performed by Tripan Blue 1:1 staining (50 μl cell suspension + 50 μl Tripan Blue). The cell suspension has a concentration of 1.07 x 106 cells/ml.
To perform the cytotoxicity test, the cell suspension is cultured in a 96-well flat-bottomed microplate of 200 μl/well (2.14 x 105 cells/well) and incubated in a CO2 incubator (5%) at 370 C, for 24 hours.
The next day, the medium is changed as follows:
–
remove the medium from the control cell wells and add 200 μl of completely fresh DMEM medium;
–
in the wells with CEES toxic control, remove the medium and add 100 μl of fresh DMEM medium + 100 μl of CEES;
–
the medium is removed from the wells containing solutions to be tested and 100 μl of CEES + 100 μl of test treatment solution in different concentrations (dilutions) are added;
–
only 200 μl of complete DMEM medium is added to the blank wells; incubate the microplate in the CO2 incubator (5%) at 370 C, for 24 hours.
After 24 hours, the 3-(4,5-)dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT) test is performed to measure the conversion of MTT to a stained product in living cells. For this test, we used the MTT-based cell growth assay kit (Sigma) containing the MTT solution (5 mg/ml MTT in RPMI-1640 without phenol red) and the MTT solvent (0.1 N HCl in anhydrous isopropanol).
Remove the microplate from the incubator, add 20 μl MTT/well (10% of the medium’s volume) and proceed with dark incubation for 4 hours, at 370C, in CO2. After 4 hours, remove the microplate from the incubator, remove the medium and add the MTT solvent, 200 μl/well. Read the optical density at a wavelength of 570 nm within one hour of adding the solvent using the PerkinElmer multimodal reader (EnSight™ Multimode Microplate Reader).
Obtaining and characterizing a delivery and controlled release system for the formulation of a chemical burn antidote
Obtaining microemulsions as a nanostructured delivery vector for antidote components. In order to control the release of the active substances that were proposed for the
formulation of the antidote, we suggested encapsulating them in the aqueous phase of microemulsions prepared from components with high biocompatibility. Skin absorption during transdermal administration of drugs is favored by the presence of surfactants that stabilize microemulsions.
Preliminary tests were performed for the preparation of uniphasic microemulsions with bicontinuous structure. Thus, we selected a composition which leads to obtaining bicontinuous structured Winsor IV microemulsion systems: sample 4 composition: 32% aqueous phase; 33% oil phase (IPM); 35% Surfactant-Cosurfactant (Tw80 + Sp80). The surfactant/cosurfactant molar ratio that gave the best results regarding the size of the microemulsion area on the phase diagram was 1:1, thus being selected for the final formula in the series. Dynamic light diffusion measurements were performed using the undiluted microemulsion, with a standard cell Nano Zetasizer Malvern instrument. The mean size of the liquid droplets was 19.54 ± 2.56 nm.
Establishment of the analytical dosing method for the antidote components in solutions and microemulsions
In order to study the release of active substances from the antidote formulations, we used the quantitative dosing of the antiinflammatory components of the proposed antidote preparation, namely dexamethasone and ketoprofen (inflammation mediation through oxidative stress being the main pathogenic mechanism involved in skin toxicity of vesicants). Tests were performed using the HPLC LC 4500 JASCO INC system. Chromatographic data were obtained using the ChromNAV 2.0 HPLC software.
Study of the release profile of antidote components
To evaluate the release profile of the antidote’s components during transdermal administration, a permeability study was performed for the aqueous solution and for the selected microemulsion, taking into consideration it had the composition with the highest biocompatibility, due to the characteristics of surfactants used for stabilization. The in vitro permeation study was performed using a Strat-M® membrane as a material that mimics the properties of the skin and the Franz cell technique. The in vitro skin permeability study using ketoprofen and dexamethasone was performed using a vertical Franz cell (PermeGear, Inc., Hellertown, PA, USA). Each experiment was performed three times.
The effective diffusion area of the membrane mounted on the Franz cell was 0.99 cm2. The 8 mL receptor compartment was filled with medium consisting of phosphate-buffered saline (PBS; pH 7.4). These experimental conditions are considered to be sink, thus assuming that the concentration of the drug in the receptor solution is negligible compared to that in the donor compartment. The diffusion cell was maintained at 37° C, and the receptor compartment was continuously homogenized using a magnetic stirrer. Then, 0.5 g of aqueous or microemulsion was added to the donor system compartment, which was sealed with Parafilm® to prevent evaporation of the sample. After certain time intervals (0.5, 1, 2, 4, 6, 10 and 24 h), 0.5 mL samples were extracted from the receptor medium and supplemented with a new receptor medium to maintain a constant volume. Samples of the analyte receptor medium extracted at said time intervals were analyzed using the methods described above, after appropriate prior dilution.

Results

In vitro evaluation of the cytotoxicity of the vesicant compound 2-chlorodietylethyl sulfide (CEES) and of the complex antidote optimized solution (A1), using the MTT cell viability test.
The following formula is used to interpret the results:
% cell viability = (DO positive control – DO blank)/(DO negative control – DO blank) x 100
Where:
DO = optical density read at 570 nm
positive control = cells + CEES and treatment + CEES + MTT + MTT solvent
negative control = cells + MTT + MTT solvent
blank = medium (complete DMEM) + MTT + MTT solvent
Antidote treatment stock solution was formed by: Acetylcysteine: 10%; Dexamethasone: 4%; Ketoprofen: 5%; 1/5 and 1/10 dilutions were prepared from the stock antidote solution (Table 1).
The samples (positive control) were processed in 2 wells for each treatment concentration. At the end, the arithmetic mean of the optical densities readings read at a wavelength of 570 nm was calculated. The negative control and the blank were distributed in duplicate, and the arithmetic mean of the read optical densities was calculated afterwards (Table 2). The average values of the cell viability (calculated in percentages) and which are determined by the tested compounds are represented in Table 3, as well as in Figure 1 and Figure 2.
The CEES 2 formulation (concentration 7.78 * 10-3M) showed a lower toxicity compared to CEES1 (concentration 7.78 * 10-2M). The 1/1 dilution treatment reduced cyto-toxicity of the toxic compound for both CEES formulations. The reduction of the treatment effect with the increase of its dilution (1/5 and 1/10 respectively) can be observed.
The evaluation of the statistically significant difference between the averages of the optical density values correlated with cell viability for the groups intoxicated with 2-chloroethyl-ethyl sulfides and the control group, performed by applying the T Student test, highlighted the following aspects presented in Table 4 and Table 5.
Optical density correlated with cell viability differs in a statistically significant way in groups exposed to CEES concentrations of 7.78 * 10-2 M and 7.78 * 10-3 M and untreated groups compared to the control cell group. The evaluation of the statistically significant difference between the average density values for the groups that were intoxicated with the two concentrations of 2-chloroethyl-ethyl sulfides and those that received treatment was performed by applying the Student T test, these aspects being highlighted in Table 6 and Table 7.
Optical density correlated with cell viability differs in a statistically significant manner in the groups exposed to CEES1 7.78 * 10-2 M and CEES 2 7.78 * 10-3 M compared to the groups exposed to the same CEES concentrations and treated with the optimized antidote stock solution (in a dilution ratio of 1/1). The obtained data therefore demonstrate the effective therapeutic effect of the studied antidote in vitro.
Obtaining and characterizing a delivery and controlled release system for the formulation of an antidote for the treatment of chemical burns caused by vesicants
Ketoprofen was dosed using the Mediterranean Sea8 chromatographic column (Teknokroma), a column with a stationary phase of silica gel type modified with C8 groups. Chromatograms were recorded by measuring the absorbance at 233 nm (Figure 3 and Figure 4).
Dexamethasone Dosing
The dosing method for dexamethasone in the receptor liquid was spectrophotometric, implemented using a UV-VIS Jasco 650V spectrophotometer. Measurements were made in quartz cells, reading the maximum absorption at a wavelength of 244 nm. A calibration curve linear over the range 4–16 µg/ml was drawn, with these data of the fitting equation: y = 10.973x + 39.695, R2 = 0.9902 (Figure 5).
Study of the release profile of antidote components
Experimental data were used to determine the cumulative release of the active substances (KET and DEX), calculated using specialized literature equations. The total amount of release drug at harvest, expressed as a percentage of the total amount in the sample that was subjected to analysis (Figure 6 and Figure 7).

Discussion

The main pathogenic mechanism of vesicant compounds is correlated with their alkylating property [8] and with their effect on glutathione (GSH), a nucleophilic antioxidant whose depletion leads to oxidative stress and macromolecular aggressions [9,10].
Another important pathogenic mechanism is the activation of some inflammation mediators: prostaglandins derived from cyclooxygenases [11]. Histopathologically, vesicant compounds mainly affect the epidermis. Cytotoxic effects have been observed mainly in basal keratinocytes (proliferative basal layer). Separation of the epidermis from the dermis and the appearance of edema was observed a few hours after exposure. The morphology of the basal layer was characterized by karyolysis and pyknosis. The dermis was less affected and showed only signs of discrete necrosis, along with a low number of fibroblasts and histiocytes. The erosive areas did not present an epidermis, but necrosis and massive cellular infiltration, with a lower degree of leukocyte infiltration [9,10]. Several studies found that serum levels of inflammatory mediators such as Il-1, Il6, IL-8, IL-10, IL-12, IL-13 mustard vesicants may be correlated with the severity of lesions induced by vesicants [12]. Interleukin-6 targets multiple cell types and induces a broad array of responses [13]. IL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein that indirectly initiates the cascade of events such as skin irritation and blister formation observed in the pathophysiology of HD injury [14]. Exposure to vesicants can be fatal, especially when pulmonary airways are affected, causing profound inflammation, hyper-coagulation, and oxidative stress. Inflammation can influence coagulation by increasing cytokine levels of IL-6, IL-1, and IL-12, diminishing activated protein C (APC), decreasing fibrinolysis, and increasing platelet activation [15,16,17]. With an interdependent relation between inflammation and coagulation, the increased cytokine production led to the expression of the tissue inhibitor of plasminogen-1, and a tissue factor, and subsequently triggering the coagulation system through binding to the clotting factor VIIa [18]. The fluido-coaglant balance is extremely important in managing inflammation and wound healing. The inadequate removal of fibrin can hinder the normal healing process of wounds and may lead to the formation of fibrous adhesions [19]. Modeling coagulation and fibrinolysis could be future direction of research in wound healing [20].
The cutaneous toxic clinical effects of vesicants are correlated with the absorbed dose and the time of exposure, appearing late (7-24 hours after exposure) and being represented by edema, hyperpigmentation and blisters evolving into phlyctenules [21,22].
The usual medical countermeasures in case of cutaneous exposure to vesicants mainly involve the removal of the toxic compound by applying general decontamination: removal of clothes, thorough rinsing of the contaminated region with 0.1% benzalkonium bromide solution, applying antiseptics (silver sulfadiazine) and local antibiotics, occlusive bandages, and surgical treatment in case of severe injuries [23,24,25,26]. Current studies indicate that the effect of monotherapy is clearly inferior to therapies targeted at the multiple pathogenic mechanisms of vesicants. Thus, it is considered that the antioxidant antiinflammatory medication, anticytokines, represents a beneficial alternative in the treatment of this type of lesions [5,27,28]. In addition, the use of acetylcysteine, sodium thiosulfate and vitamin E were used in experimental studies and proved potential benefits due to their antioxidant properties [29,30,31]. The treatment of scars is still a controversial issue, mainly due to unpredictable results. Therapeutic options include lasers and fractioned radio frequency [32].
To elucidate pathogenic mechanisms and identify effective therapeutic solutions, studies were performed using a chemical vesicant, namely 2-chloroethyl-ethyl sulfide (CEES) [1,33,34]. It has pathogenic mechanisms similar to those seen with acute exposure to vesicants (induction of oxidative stress and inflammatory response), but is less toxic, constituting a valid experimental alternative for scientific research studies in the laboratory [35,36,37].
By evaluating the experimental data obtained for the drug release through the skin-like model membrane, we found that for both tested substances, ketoprofen and dexamethasone, the transfer is facilitated by their incorporation in microemulsions. In the case of ketoprofen, the release occurs with the presence of the initial acceleration zone from both aqueous solution and microemulsion. As expected for the chemical structure of the ketoprofen molecule, very little is released from the aqueous vehicle, up to a maximum 32%, with the release occurring rapidly, with the plateau stabilizing after about 8-10 hours. From the microemulsion, it is released relatively quickly in the first 6 hours, up to approximately 60%, and within 24 hours it is released to up to 79% of the total amount. For dexamethasone, the release from the aqueous solution has no acceleration zone and has an almost linear appearance. A total amount of approximately 50% of what is present in the sample is released after the first 10 hours from the antidote solution. The amount released from the microemulsion is significantly higher, up to 75%, and the transfer through the skin is slightly prolonged, up to 24 hours.
The novelty element is represented by the therapeutic association of epidermal growth factors and niacinamide-type Poly (ADP-ribose) polymerase (PARP) inhibitors, as well as the inclusion of the complex antidote in microemulsions.
The epidermal growth factor (EGF) family is important in regulating growth, maturation, function, and maintenance in epithelial tissues. EGF stimulates the growth of various epidermal and epithelial tissues in vivo and in vitro1 and of some fibroblasts in cell culture [10,38] The EGF family is comprised of 13 members, which are all membrane-anchored proteins, in addition to their properties as ligands that activate the epidermal growth factor receptor (EGFR), bearing tyrosine kinase activity [39].
The PARP-1 monoclonal antibody recognizes native and/or cleaved polymerase. PARP cleavage is an early indicator of apoptosis (the cleaved form of caspase) as well as of DNA repair. The administration of optimized complex antidote treatment is correlated with inhibition of PARP expression, which can be explained by its effect on maintaining the integrity of the nuclear apparatus, which, under the protection of the pharmacological agents, is no longer stimulated to secrete PARP for its repair [40,41].
Microemulsions are the best novel drug delivery system due to their improved drug solubilization, and ease of preparation and administration [20,27,28,42]. They have emerged as novel vehicles for drug delivery, which allow controlled or sustained release for topical, transdermal administration of drugs [43,44,45]. Microemulsions are bicontinuous systems that are essentially composed of bulk phases of water and oil separated by a surfactant/ cosurfactant-rich interfacial region [46]. With the ability to carry both lipophilic and hydrophilic drugs, the dispersed phase, which is lipophilic or hydrophilic (O/W, or W/O microemulsions), can act as a potential reservoir for lipophilic or hydrophilic drugs, respectively. The use of microemulsions as delivery systems can improve the efficacy of a drug, allowing the total dose to be reduced, thus minimizing side effects [47].

Conclusions

Optimization of the complex antidote formula was achieved by developing and in vitro and in vivo testing of a fixed combination of active substances with anti-inflammatory and antioxidant effects, formulated as a solution with cutaneous administration Also, the complex antidote was formulated as a gel in 2020. In vitro cytotoxicity tests on fibroblast cultures revealed the protective effect of the newly developed antidote solution, a dose-related effect, in case of exposure to vesicants. Treatment applied as a 1/1 dilution reduced cytotoxicity for both CEES formulations.
The complex curative antidote that was developed and optimized in the study has superior therapeutic efficiency in case of exposure to sublethal doses of vesicants simulating mustard gas in experimental conditions. The novelty element is represented by the realization of a complex antidote formula, with anti-inflammatory, antioxidant, healing, hydrating and epithelizing effects following daily percutaneous administration for 7 days on the cutaneous chemical burn. Experimental studies have shown the optimization of transdermal transfer through encapsulation in microemulsion-type controlled-release vectors.

Highlights

✓
Chemical burns induced by vesicant agent are severe and the therapeutic management is challenging.
✓
Using a fixed combination of active substances with anti-inflammatory and antioxidant effects in a topically administrated solution revealed a protective effect on fibroblast cultures exposed to different concentrations of CEES solution.

Conflict of Interest disclosure

There are no known conflicts of interest in the publication of this article. The manuscript was read and approved by all authors.

Compliance with ethical standards

Any aspect of the work covered in this manuscript has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.

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Jmms 09 00033 g001
Figure 1. Cell viability after exposure to CEES-1.
Figure 1. Cell viability after exposure to CEES-1.
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Figure 2. Cell viability after exposure to CEES-2.
Figure 2. Cell viability after exposure to CEES-2.
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Figure 3. Chromatogram obtained for ketoprofen, the associated retention time being 4.902 minutes.
Figure 3. Chromatogram obtained for ketoprofen, the associated retention time being 4.902 minutes.
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Figure 4. Calibration line associated with ketoprofen.
Figure 4. Calibration line associated with ketoprofen.
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Figure 5. Calibration curve for dexamethasone.
Figure 5. Calibration curve for dexamethasone.
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Figure 6. Ketoprofen release profiles from aqueous antidote solution compared to P6 microemulsion.
Figure 6. Ketoprofen release profiles from aqueous antidote solution compared to P6 microemulsion.
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Figure 7. Dexamethasone release profiles from aqueous antidote solution compared to P6 microemulsion.
Figure 7. Dexamethasone release profiles from aqueous antidote solution compared to P6 microemulsion.
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Table 1. The distribution in wells on the cell culture plate.
Table 1. The distribution in wells on the cell culture plate.
1234567891011
BlankCells controlCells + Stock treatmentCells+ CEES1Cells+ CEES1Cells+ CEES1+ stock treatmentCells+ CEES1+ stock treatmentCells+ CEES1+ 1/5
treatment		Cells+ CEES1+ 1/5
treatment		Cells+ CEES1+ 1/10
treatment		Cells+ CEES1+ 1/10
treatment
BlankCells controlCells+ Stock treatmentCells+ CEES2Cells+ CEES2Cells+ CEES2+ stock treatmentCells+ CEES2+ stock treatmentCells+ CEES2+ 1/5
treatment		Cells+ CEES2+ 1/5
treatment		Cells+ CEES2+ 1/10
treatment		Cells+ CEES2+ 1/10
treatment
Table 2. The average values of optical density (DO) at 570 nm.
Table 2. The average values of optical density (DO) at 570 nm.
Toxic1234567891011
CEES 10,054
±0,0037		1,578±
0,067		1,389±
0,049		0,064 ±0,0020,823 ± 0,0040,587± 0,00250,291± 0,001
CEES 20,070 ±0,00150,944 ± 0,00350,700± 0,00250,343± 0,003
Footnote. CEES 1: mean optical density (DO) values for cell exposure to CEES 1, concentration 7.78 × 10-2M; CEES 2: mean optical density (DO) values for cell exposure to CEES 2, concentration 7.78 × 10-3M; Column 1 blank DO; Column 2 control untreated cells DO; Column 3 cells + stock treatment; Columns 4, 5 average DO values: CEES + cells; Columns 6, 7 average DO values: CEES + stock treatment + cells; Columns 8, 9 average DO values: CEES + 1/5 dilution stock treatment + cells; Columns 10, 11 average DO values: CEES + 1/10 dilution stock treatment + cells.
Table 3. Cell viability in different solutions.
Table 3. Cell viability in different solutions.
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CEES 1 10087,570±2,670,668 ±0,0550,432 ± 0,4934,989 ± 0,8915,20± 0,9
CEES 21,010±0,0750,387± 0,5542,384± 0,9818,980± 0,73
Footnote. CEES 1 mean cell viability values for cell exposure to CEES 1, concentration 7.78 × 10-2M CEES 2 is the mean cell viability values for cell exposure to CEES 2, concentration 7.78 × 10-3M; Column 2 was considered reference (100%) for the value of cell viability for control untreated cells; corresponding DO 1,578 ± 0,067; Column 3 cells + stock treatment; Columns 4, 5 average cell viability values: CEES + cells. Columns 6, 7 average cell viability values: CEES + stock treatment + cells. Columns 8, 9 average cell viability values: CEES + 1/5 dilution stock treatment + cells. Columns 10, 11 average cell viability values: CEES + 1/10 dilution stock treatment + cells.
Table 4. Highlighting the cytotoxicity of the CEES solution 1.
Table 4. Highlighting the cytotoxicity of the CEES solution 1.
No.ControlAverage optical density values (nm)CEES ConcentrationP Probability (T test)Observations
1Cells Control1,578±0,06700,002< 0,05
2CEES 1 Positive Control0,064 ±0,0027,78 *10 -2M
Table 5. Highlighting the cytotoxicity of the CEES solution 2.
Table 5. Highlighting the cytotoxicity of the CEES solution 2.
No.ControlAverage optical density values (nm)CEES ConcentrationP Probability (T test)Observations
1Cells Control1,578±0,06700,0035< 0,05
2CEES 2 Positive Control0,070 ±0,00157,78 *10 -3M
Table 6. Comparative cytotoxicity of CEES-1 solution in the absence and in the present of treatment stock solution.
Table 6. Comparative cytotoxicity of CEES-1 solution in the absence and in the present of treatment stock solution.
No.ControlAverage optical density values (nm)CEES
Concentration		Treatment stock solution (percentage concentration)P Probability (T test)Observations
1CEES1 Positive Control and treatment0,823 ± 0,0047,78 *10 -2MAcetylcysteine: 10%;
Dexamethasone: 4%;
Ketoprofen: 5%;		0,00039<0,05
2CEES 1 Positive Control0,064 ±0,0027,78 *10 -2M0
Table 7. Comparative cytotoxicity of CEES-2 solution in the absence and in the present of treatment stock solution.
Table 7. Comparative cytotoxicity of CEES-2 solution in the absence and in the present of treatment stock solution.
No.ControlAverage optical density values (nm)CEES
Concentration		Treatment stock solution (dilution)P Probability (T test)Observations
1CEES2 Positive Control and treatment0,944 ± 0,00357,78 *10 -2MAcetylcysteine: 10%;
Dexamethasone: 4%;
Ketoprofen: 5%;		0,00018<0,05
2CEES 2 Positive Control0,064 ±0,0027,78 *10 -2M0

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Secară, C.A.; Cinteză, O.L.; Popescu, D.; Popa, C.V.; Hîrjeu, C.A.; Voinea, O.C.; Șerbănescu, L.G.; Păuna, A.M.; Tudosie, M.S. Innovative Therapeutic Approach to Chemical Burns Produced by Vesicants; An Experimental Study. J. Mind Med. Sci. 2022, 9, 294-303. https://doi.org/10.22543/2392-7674.1355

AMA Style

Secară CA, Cinteză OL, Popescu D, Popa CV, Hîrjeu CA, Voinea OC, Șerbănescu LG, Păuna AM, Tudosie MS. Innovative Therapeutic Approach to Chemical Burns Produced by Vesicants; An Experimental Study. Journal of Mind and Medical Sciences. 2022; 9(2):294-303. https://doi.org/10.22543/2392-7674.1355

Chicago/Turabian Style

Secară, Cristina Anca, Otilia Liudmila Cinteză, Diana Popescu, Claudia Valentina Popa, Camelia Andreea Hîrjeu, Oana Cristina Voinea, Luiza Georgia Șerbănescu, Andreea Marilena Păuna, and Mihail Silviu Tudosie. 2022. "Innovative Therapeutic Approach to Chemical Burns Produced by Vesicants; An Experimental Study" Journal of Mind and Medical Sciences 9, no. 2: 294-303. https://doi.org/10.22543/2392-7674.1355

APA Style

Secară, C. A., Cinteză, O. L., Popescu, D., Popa, C. V., Hîrjeu, C. A., Voinea, O. C., Șerbănescu, L. G., Păuna, A. M., & Tudosie, M. S. (2022). Innovative Therapeutic Approach to Chemical Burns Produced by Vesicants; An Experimental Study. Journal of Mind and Medical Sciences, 9(2), 294-303. https://doi.org/10.22543/2392-7674.1355

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