Evaluation of some biological parameters of gasoline station attendants in Damascus, Syria.

INTRODUCTION: Gasoline is a blend of organic compounds used in internal combustion engines. gasoline station attendants (GSA) are exposed to gasoline vapours, which promotes oxidative stress through the production of ROS, which can damage biological structures with the formation of new metabolites which can be used as markers of oxidant/antioxidant imbalance. This is a comparative cross-sectional study. This study aims to evaluate some biological parameters as indicators of toxicity as a result of exposure to gasoline in workers at gas stations in Damascus.
METHODS: Blood samples were collected from GSA (n = 30) and non-exposed (NE) (n = 30) with no history of occupational exposure, and the following markers of oxidative stress were analysed: malondialdehyde (MDA), advanced oxidation protein products (AOPP), catalase activity (CAT), CBC, ALT and AST.
RESULTS: We have found that the levels of MDA, AOPP, CAT, RBC and Hgb in GSA were significantly higher than NE (p = 0.000, p = 0.02, p = 0.002, p = 0.018 and p = 0.015 respectively). On the other hand, there were no statistically significant (p > 0.05) in HCT, MCV, WBC, PLT, ALT and AST between the two groups. In the GSA group, there was no effect of the smoking habit and the number of years of work on biological parameters, but alcohol consumption habit had a clear effect on increasing both levels of MDA and CAT (P = 0.021 and P = 0.036 respectively), in comparison to the non-consumers of the alcohol group. The results from our study showed that chronic gasoline exposure may result in long-lasting oxidative stress, as demonstrated by the presence of statistically significant correlations between gasoline exposure and levels of biomarkers (MDA, AOPPs, Catalase activity).
CONCLUSIONS: the early identification of these biomarkers can be very useful to promote programs on health protection and prevention for those populations more susceptible to the adverse effects of gasoline exposure.

Introduction

Gasoline is distilled from crude petroleum. The volatile nature of gasoline makes it readily available in the atmosphere any time it is dispensed, especially at gasoline stations and depots. Gasoline contains a mixture of volatile hydrocarbons and so inhalation is the most common form of exposure. Gasoline vapour can reach supra-lethal concentrations in confined or poorly ventilated areas, although such exposures are rare [1]. Fossil fuels used in motor vehicles consist of more than 500 saturated and unsaturated hydrocarbons. Hydrocarbons, primarily volatile organic compounds (VOCs), include alkanes, alkenes, and aromatic compounds, depending upon the production process, environmental regulations, manufacturing country, and base petroleum [2]. Exposure to automotive gasoline most likely occurs from breathing its vapour at a service station while filling a car's fuel tank [3]. This study is considered one of the first studies in Syria to study the effect of workers exposure to gasoline in gasoline stations. The number of gasoline stations in Damascus approximately 20 stations and the number of workers in each station depends on the size of the station, which ranges between 15-30 workers. In Syria, vehicle fueling is done by workers employed by gas stations exclusively to fill car tanks. This type of job typically requires an 8-hour shift where the worker is chronically exposed to solvents and other toxic substances found in gasoline. Most of them do not use protective devices. Despite the high number of employees at gas stations, there are no specific occupational exposure limits for gas station workers in Syria. Thus, this group of workers is likely to have grave ill effects on their health. Gasoline station attendants are constantly exposed to the polluted air and vapours of gasoline. Petroleum products and their exhaust cause significant health problem symptoms. Petrol pump workers are also likely to get exposed to such burnt petroleum products from vehicular exhaust [4]. Toxicological studies indicate that the light-chain volatile compounds (benzene, toluene, ethylbenzene, and xylene (BTEX)) are the components most toxic to humans [5]. Accumulated evidence indicates that acute and long-term exposure to gasoline compounds may be associated with several systemic health effects, including haematological, respiratory, reproductive, immunological, dermatological, renal, and central nervous system pathologies in humans. Other effects include hepatotoxicity, genotoxicity and carcinogenic potential. Cases of death following the inhalation of high concentrations of gasoline vapour have also been reported [5]. Oxidative stress is defined as an impaired balance between free radical production and antioxidant capacity resulting in excess oxidative products [6]. Oxidant compounds are extremely reactive species capable of independent existence that contains one or more unpaired electrons, named free radicals (FRs). FRs have a very short half-life (of the order of few seconds), and their measurement in vivo is faced with many challenges. However, oxy-radical derivatives (e.g., hydrogen peroxide or lipid hydroperoxides) are stable and have a long half-life (hours to weeks) and thus may be measured and monitored repeatedly [7]. In cell membranes, lipid peroxidation begins when electrons from lipids are kidnapped by unstable free radicals promoting a chain reaction with successive oxidations that results in lipid instability and formation of by-products such as malondialdehyde (MDA) [8]. MDA is a biomarker to prove that lipid peroxidation has occurred [9]. In 1996, a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPPs), were detected in the plasma of chronic uremic patients [10]. AOPP is a well-known biomarker of the oxidative modification of proteins. AOPP are formed in the reaction of chlorinated oxidants, such as chloramines and hypochlorous acid (HOCl), with plasma proteins, and act as the most commonly measured marker of oxidative stress in many diseases [11]. Catalase is one of the crucial antioxidant enzymes that mitigates oxidative stress to a considerable extent by destroying cellular hydrogen peroxide to produce water and oxygen [12]. Catalase is a common antioxidant enzyme found in nearly all living organisms that are exposed to oxygen. Catalase can also remove organic hydroperoxides [13].

Some of the biomarkers used to determine hepatotoxicity levels include alanine and aspartate aminotransferase [14]. The haematotoxic effect of benzene has been reported to involve both bone marrow depression and leukaemogenesis that is caused by damage to multiple classes of haematopoietic cells with a variety of functions [3].

This work aims to study the effects of exposure to the gasoline on oxidative stress biomarkers (MDA, AOPP and CAT), hematologic parameters (RBC, Hgb, HCT, MCV, WBC and PLT) and biochemical parameters (ALT and AST) in gasoline station attendants. It also aims to study the correlation between smoking habit, alcohol consumption habit and number of years of work and (MDA, AOPP, CAT, RBC, Hgb, HCT, MCV, WBC, PLT, ALT and AST) in the group of gasoline station attendants.

Materials and methods

Study population

Characteristics of the study population, obtained through questionnaire interview, are provided in Table 1. 60 individuals were enrolled in this study. The exposed group consisted of thirty [30] gasoline station attendants (GSA) from Damascus, Syria. All subjects had been working in their current job position for at least 1 year. The non-exposed (NE) group who had no history of occupational exposure to gasoline consisted of thirty [30] subjects. Each participant was interviewed to obtain aspects of general health, lifestyle, smoking status, alcohol consumption and history of exposure. The average ages of the GSA and control groups were 39.53 ± 2.232 years and 41.43 ± 1.912 years, respectively. No significant difference between the GSA and control groups was found related to age. The mean exposure time in the GSA group was 10.50 ± 1.9864 years (range: 1–32 years). This study was approved by the research ethics committee of the University of Damascus and written informed consent was obtained from all participants before their enrollment in the study.

Table 1

Characteristics of the study population.

	GSA	NE
Age (years)	39.53 ± 2.232	41.43 ± 1.912
Duration of occupational exposure (years)	10.5 ± 1.9864	n.a.
Smokers [n (%)]	23 (76.7)	23 (76.7)
Alcohol - occasional drinkers [n (%)]	13 (43.3)	4 (13.3)

The values are expressed as mean ± SEM.

n.a.: not applicable.

[N (%)]: Total number found per group and, in parentheses, percentages.

Ethics approval and consent to participate

All procedures performed were following the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The local ethical review board approved this retrospective (No. 7 sessions 3 on September 5 ∖ 2016).

Sample collection

Venous blood samples were collected by venipuncture using vacuum tubes. EDTA-blood tubes were collected and divided into two parts, one to conduct a complete blood count CBC directly, and the other was added to BHT (butylated hydroxytoluene) and stored at - 80 °C until analysis of MDA content. vacuum blood tubes without anticoagulant were collected and divided into two parts, one to analyze ALT and AST directly, and the others were centrifuged at 2500 g for 20 min at room temperature and then stored at - 80 °C for the analysis of AOPP and Catalase activity.

Estimation of lipid peroxidation as malondialdehyde (MDA)

Malondialdehyde levels were analyzed using Colorimetric Detection according to the manufacturer's instructions (OxiSelect™ TBARS Assay Kit, MDA Quantitation, STA-330, Cell Biolabs, Inc). The concentration of thiobarbituric acid reactive substances (TBARS) in plasma is an index of lipid peroxidation and oxidative stress. The unknown MDA containing samples or MDA standards were first reacted with TBA at 95 °C. After a brief incubation, the samples and standards were read spectrophotometrically at 532 nm. The MDA content in unknown samples was determined by comparison with the predetermined MDA standard curve.

Estimation of advanced oxidation protein products (AOPP)

AOPP levels were analyzed using Colorimetric Detection according to the manufacturer's instructions (OxiSelect™ AOPP Assay Kit, STA-318, Cell Biolabs, Inc). The unknown AOPP-containing samples or Chloramine standards were first mixed with an assay reaction initiator that began a colour development process. After a brief incubation, a stop solution was added and the samples and standards were measured with a standard colourimetric plate reader at 340 nm using quartz cuvettes. The AOPP content in unknown samples was determined by comparison with the predetermined Chloramine standard curve.

Estimation of catalase activity assay (CAT)

Catalase activity was analyzed using Colorimetric Detection according to the manufacturer's instructions (OxiSelect™ Catalase Activity Assay Kit, Colorimetric, STA-341, Cell Biolabs, Inc). The assay involved two reactions. The first reaction was the catalase induced decomposition of hydrogen peroxide H2O2 into water and oxygen. The rate of disintegration of hydrogen peroxide into water and oxygen is proportional to the concentration of catalase (See Reaction 1 in Figure). A catalase-containing sample was incubated in a known amount of hydrogen peroxide. The reaction proceeds for exactly one minute, at which time the catalase was quenched with sodium azide. The remaining hydrogen peroxide in the reaction mixture facilitates the coupling reaction of 3,5-dichloro-2-hydroxy-benzenesulfonic acid (DHBS) and 4-aminophenazone (4-aminoantipyrene) (AAP) in conjunction with a Horseradish Peroxidase (HRP) catalyst. The quinone imine dye coupling product was measured at 520nm, which correlated to the amount of hydrogen peroxide remaining in the reaction mixture.

Hematologic and biochemical analysis

Venous blood samples were obtained for hematologic and biochemical analysis. The hematologic parameters measured included RBC (red blood cell), Hgb (haemoglobin), HCT (hematocrit), MCV (mean corpuscular volume), WBC (white blood cell) and PLT (platelet). The measurements were performed within 2 h of sample collection using the Sysmex KX-21N Automated Hematology Analyzer (Japan). Biochemical parameters measured were liver enzymes (AST (aspartate aminotransaminase), ALT (alanine transaminase)). The measurements were performed within 2 h of sample collection according to the manufacturer's instructions using commercial kits ALAT (GPT) FS (IFCC mod.) at 340 nm and ASAT (GOT) FS (IFCC mod.) at 340 nm using OLYMPUS AU 400 Analyzer (Olympus Optical Co., Ltd. Japan).

Statistical analysis

The data analysis was performed using the IBM SPSS Statistics software (version 24). All study variables were tested for normality using the Kolmogorov – Smirnov test. Comparisons between groups were obtained using the Mann-Whitney U-test. The results were expressed as mean ± standard error of the mean (SEM) according to variable distribution. The significance level for all tests was p < 0.05.

Results

Oxidative stress biomarkers

In relation to oxidative stress biomarkers, the GSA group showed an increase in MDA, AOPP and CAT concentrations compared to the non-exposed group (p = 0.0001, p = 0.020, p = 0.002, respectively) (Figure 1 A-B-C respectively).

Figure 1

Biomarkers of oxidative stress in gasoline station attendants (GSA) (n = 30) and non-exposed (NE) group (n = 30). Data are expressed as mean. (A) Mean MDA levels; (B) Mean AOPP levels; (C) Mean CAT levels. ∗ indicates the statistical difference from the control group using the Mann-Whitney U-test p = 0.0001, ∗∗ indicates the statistical difference from the control group using the Mann-Whitney U-test p = 0.02, ∗∗∗ indicates the statistical difference from control group using the Mann-Whitney U-test p = 0.002.

Haematological profile

The mean level of red blood cell (RBC) of the GSA group (5.3940 ± 0.08205) was significantly higher (P = 0.018) in comparison to the NE group (5.0780 ± 0.06873) (Figure 2A). Also, the statistical analysis showed significant increases (P = 0.015) in hemoglobin (Hgb) content of the GSA's blood (15.0867 ± 0.18262) in comparison to NE group (14.3433 ± 0.22918) (Figure 2B). Oppositely, no significant differences were found between the hematocrit value (HCT) (P = 0.072) (Figure 2C), mean corpuscular volume (MCV) (P = 0.900) (Figure 2D), blood platelet count (PLT) (P = 0.228) (Figure 2E), and total and differential white blood cells (WBC) (P = 0.297) (Figure 2F). The data are presented in Table 2.

Figure 2

Hematological profile in gasoline station attendants (GSA) (n = 30) and non-exposed (NE) group (n = 30). Data are expressed as mean. (A) Mean RBC levels; (B) Mean HGB levels; (C) Mean HCT levels; (D) Mean MCV levels; (E) Mean WBC levels; (F) Mean PLT levels. ∗indicates the statistical difference from the control group using the Mann-Whitney U-test p = 0.018, ∗∗ indicates the statistical difference from the control group using the Mann-Whitney U-test p = 0.015.

Table 2

Mean values of oxidative stress biomarkers, hematological and biochemical parameters of both studied groups.

	GSA	NE	Mann–Whitney U test: P = values
MDA)nmol/ml (	13.5627 ± 1.13973	6.7738 ± .64227	.000
AOPP (μMol/L)	41.8935 ± 4.39199	28.7157 ± 4.7152	.020
CAT (kU/L)	22.5545 ± .64501	17.4106 ± 1.1891	.002
RBC (106 mm-3)	5.3940 ± .08205	5.0780 ± .06873	.018	4.3–5.7
HGB (g dL−1)	15.0867 ± .18262	14.3433 ± .22918	.015	13.2–17.3
HCT (%)	45.2567 ± .58212	43.4767 ± .65425	.072	39–49
MCV fL	84.8633 ± 1.17281	85.4483 ± .51592	.900	80–90
WBC (103 mm−3)	8196.67 ± 372.117	7652.00 ± 366.60	.297	4000–11000
PLT (103 mm−3)	252.73 ± 8.790	275.17 ± 11.476	.228	150–450
ALT U/L	27.93 ± 1.524	28.27 ± 1.684	.882	5.0–45
AST U/L	19.4 ± 0.994	18.17 ± 0.825	.320	5.0–45

The values are expressed as mean ± SEM.

p < 0.05.

Bold is used for statistically significant values only.

Biochemical parameters (Liver function tests)

AST and ALT levels were measured in the serum of all participants. The activities of these liver enzymes in GSA and NE groups are shown in Table 2. There was no statistical difference in ALT levels between the GSA group (27.93 ± 1.524 U/L and NE group (28.27 ± 1.684 U/L) (Figure 3A). Also, mean levels of AST of the GSA group (19.4 ± 0.994), were not statistically different (P = 0. 320), in comparison to the NE group (18.17 ± 0.825) (Figure 3B).

Figure 3

Biochemical parameters in gasoline station attendants (GSA) (n = 30) and non-exposed (NE) group (n = 30). Data are expressed as mean. (A) Mean ALT levels; (B) Mean AST levels.

Comparisons in the group of GSA

, the group of GSA was divided according to the smoking habit into two groups: The first group of smokers (N = 23). The second is a non-smoking group (N = 7). There were no statistically significant differences between the two groups (Table 3).

Table 3

Comparisons of hematological and biochemical parameters of both studied groups in the group of GSA.

	SMOKE	ALCOHOL	YEARS OF WORK
N	YES: 23	YES: 13	15≥: 21
	NO: 7	NO: 17	>15 : 9
Mann–Whitney U test
	Asymp. Sig. (2-tailed)	Asymp. Sig. (2-tailed)	Asymp. Sig. (2-tailed)
MDA	.177	.021	.197
AOPP	.349	.946	.256
CAT	.864	.036	.856
RBC	.607	.167	.964
HGB	.883	.785	.441
HCT	.806	.706	.803
MCV	.659	.250	.651
WBC	.589	.325	.230
PLT	.364	.258	.342
ALT	.169	.121	.196
AST	.376	.785	.650

P-value is significant at the level of <0.05.

Bold is used for statistically significant values only.

, the group of GSA was divided according to the alcohol consumption habit into two groups: The first group of alcohol consumers (AC) (N = 13). The second is a non-consumers of alcohol (NCA) group (N = 17). The mean level of MDA of the AC group (14.4024 ± 1.15255) was significantly higher (P = 0.021), in comparison to the NCA group (12.9205 ± 1.82578). Also, the statistical analysis showed significant increases (P = 0.036) in CAT of the AC group (24.0484 ± 0.61656) in comparison to the NCA group (21.4121 ± 0.96148). There were no statistically significant differences between the two groups for other values (Table 3).

, the group of GSA was divided according to the number of years of work in the gasoline stations into two groups: The first group: 15≥ (N = 21). The second group: 15< (N = 9). There were no statistically significant differences between the two groups (Table 3).

Discussion

In this study, we have observed the influence of exposure to gasoline compound on the oxidative stress biomarkers, haematological parameters, and biochemical parameters in gasoline attendants who spend 8 h a day on the job.

Gasoline contains aromatic hydrocarbons like benzene, toluene, ethylbenzene, and xylenes (BTEX), among which benzene has the greatest toxicological risk [15]. The enzymatic bio-activation of absorbed benzene leads to the formation of ROS, decreases antioxidant activity and hence increases oxidative stress [16], this leads to damage to the cell components like proteins, lipids and DNA [17]. MDA is one of the by-products of this process, a widely used index to estimate oxidative stress [8]. the quantification of primary lipid hydroperoxide products is difficult due to their instability and reactivity. For this reason, quantification of lipid peroxidation is usually estimated by measuring the concentration of secondary oxidation products derived from these initial hydroperoxides, which are mostly aldehydes, such as MDA [18]. This study has shown a high statistically significant difference among the GSA group compared to the NE group regarding the level (MDA) which was higher among exposed. This is in agreement with Hegazy RM et al. (2014) and Georgieva T et al. (2002) which illustrate that benzene exposure has been associated with increases in the overall formation of MDA [19, 20]. The presence of plasma AOPP indicates the effects of oxidative stress on plasma proteins. AOPP is a very important biomarker of oxidative stress because the proteins are the major targets of free radicals, being present and abundant in cells, plasma, and most tissues [7]. Our findings are in agreement with Costa C et al. (2016) [21]. The current study has shown a significant increase in the CAT in the GSA group compared to the NE group. Our findings are also in agreement with Rekhadevi PV et al. (2010) [22]. Superoxide anion is eliminated by superoxide dismutase and the accumulation of toxic hydrogen peroxide is prevented by catalases and peroxidases. Physiological adaptation to oxidative stress is typically complex and tightly regulated. This can be seen in the control of catalase synthesis which is an important component of the oxidative stress response. The increase in catalase synthesis in response to either hydrogen peroxide or stationary phase protects microorganisms against high concentrations of hydrogen peroxide which would be lethal were it not for a previous adaptive response [23].

The results of the study have shown that the haemoglobin levels and RBCs count in the GSA group were significantly higher than those of the NE group. Other haematological parameters were either higher (HCT, white blood cells count) or lower (platelets and MCV count) in the GSA group compared to the NE group, but was not statistically different and all values were in the normal reference range. There is a gradual increase in the RBC count and Hgb levels among the attendants exposed to gasoline as well as air pollutants like CO.

In a previous study in Syria conducted by Waed et al. (2011), the concentrations of BTX compounds in the air of gasoline stations were as follows (Benzene 0.02 PPM, toluene 1.29 PPM and xylene (o-xylene 0.32 PPM and m-, p-xylene 0.69 PPM) [24] while the permissible exposure limits according to OSHA are: OSHA PEL (permissible exposure limit) for benzene = 1 ppm (averaged over an 8-hour work shift) [25], OSHA PEL for toluene = 200 ppm (averaged over an 8-hour work shift) [26] and OSHA PEL for xylene = 100 ppm (averaged over an 8-hour work shift) [27]. Consequently, the concentrations to which workers are exposed are far below the permissible limits. Erslev et al. (1990) showed that exposure to CO is causing tissue hypoxia and stimulation of RBC formation. The CO emitted mainly by internal combustion engines of motor vehicles readily enters the blood through the respiratory system and binds over 200 times more firmly to Hgb than oxygen, forming carboxyhaemoglobin and seriously interfering with blood's oxygen transport capability, which ultimately leads to hypoxia. Tissue hypoxia is the most potent stimulus for erythropoiesis, so it leads to the stimulation of erythropoietin and subsequently to the production of more RBC cells, thus elevating Hgb levels in the circulating blood [28]. These results also agree with Ahmadi z et al. (2019) [2].

In screening for possible effects of hepatotoxicants, it is important to select the liver enzymes tests with the best combination of specificity and sensitivity. The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are the most commonly measured enzymes that detect hepatocellular injury due to the toxicant's effect on all or part of the hepatocyte, including the cell membrane [19]. In this study, the median value of ALT was less among GSA than the NE group, while the median value of AST was higher among GSA than the NE group, but ALT and AST showed a non-significant difference. In agreement with the present results, Kari Kurppa et al. (1982) have shown that exposure to a mixture of organic solvents does not affect the levels of the liver enzymes, such as ALT, and AST [29]. Also, contradictory, several studies proved that liver enzymes were significantly elevated in attendants exposed to organic solvents compared to controls [19, 30]. According to the study of Al-Mahbashi et al. (2020) that conducted at the same time on gasoline station attendants, it indicated that the mean blood lead concentration of GSA was not significantly different from the non-exposed group in Damascus; Syria. Therefore, the normal serum concentration of AST and ALT of the exposed group in this investigation may be because blood lead level is still below the threshold that causes liver dysfunction [31].

Alcohol consumption was correlated with MDA and CAT levels. Although alcohol is primarily metabolized by alcohol dehydrogenase and acetaldehyde dehydrogenase, the alternative pathway, alcohol-inducible cytochrome P-450 2E1 (CYP2E1) plays an important role at high alcohol levels and in chronic alcoholics. The high rate of CYP2E1 oxidative activity enhances the formation of reactive oxygen species (ROS) and alcohol derived (hydroxyethyl) free radicals and subsequently initiates lipid peroxidation [32]. Ethanol ingestion increases the permeability of the intestine for Gram-negative bacterial endotoxin (lipopolysaccharide, LPS). In the blood, LPS binds to LPS-binding protein (LPS-BP), which can activate cells in an LPS-BP-dependent or -independent manner. Specifically, LPS is a potent activator of Kupffer cells in the liver, which release both ROS and pro-inflammatory cytokines, including tumour necrosis factor-α (TNFα). One of the major ROS produced is superoxide anion radical, which dismutases to hydrogen peroxide. The latter is rapidly inactivated by both catalase and GS-Px, which are both abundant in the liver [9].

Conclusion

From the results of this study, we conclude that occupational exposure to gasoline found to have hazardous effects on RBCs count and Hgb, CAT, AOPP, MDA concentrations. In the GSA group, there was no effect of the smoking habit and the number of years of work on biological parameters, but alcohol consumption habit had a clear effect on increasing both levels of MDA and CAT. So, the early identification of these biomarkers can be very useful to promote programs on health protection and prevention for those populations more susceptible to the adverse effects of gasoline exposure. Gasoline induces oxidative stress through excessive production of ROS. These observations support the need for preventive action that will improve conditions in the job environment and micronutrient status since several studies have indicated that an increase in health toxicity effects is associated with increased oxidative stress. Thus, the study provides further evidence to dysregulation of antioxidant/oxidant balance in gasoline attendants.

Limitation of the study

Since the study was conducted among small sample size, this might reduce the statistical power of the study. On the other hand, the cross-sectional nature of the study design makes it difficult to establish a cause-effect relationship. However, the overall results of the current study are generally consistent with many previous studies. It might be much more reliable if we evaded some limitations in the method we applied such as the sample size, cost and multi-source gasoline.

Declarations

Author contribution statement

Mohammad Alses: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Samar Alzeer: Conceived and designed the experiments; Wrote the paper.

Funding statement

This work was supported by Damascus University, Syria.

Data availability statement

Data will be made available on request.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.