INTRODUCTION:

Painters are exposed to an assortment of organic chemicals, including ketone, alcohols, esters, and aromatic hydrocarbons (mostly toluene), as well as metals like aluminium, titanium, cobalt, chromium, and lead during their duties¹. Thus, when a lot of chemicals are utilised at work, employees may be exposed to occupational toxicity². Chemical exposure at work usually happens by breathing, but in some environments, skin exposure is also significant, particularly in the paint industry³. The International Agency for Research on Cancer (IARC) had classified the paint workers occupational exposure as a Group 1 carcinogen in 1989, leading to the paint and coating industries being regulated heavily all over the world⁴. Numerous research has examined the potential link between occupational paint exposure and various unfavourable health outcomes, including neurological disorders, hearing impairment, renal illnesses, skin allergies, hepatic disorders, and respiratory disorders⁵. Occupational exposure to chemical solvents and certain metals may cause induced oxidative stress and DNA damage⁶.

Toluene, xylenes, ethylbenzene, and styrene have been linked to oxidative and genotoxic effects on paint workers and painters in various studies⁷. There are numerous cases of paint exposure leading DNA damage^8,9. Currently, a variety of techniques are being employed in occupational contexts to identify the early biological impacts of chemicals that damage DNA^10,11. Single-cell gel electrophoresis, also referred to as the comet assay, has proven to be an incredibly sensitive method for assessing DNA damage in human biomonitoring and has shown to be an extremely sensitive technique for examining DNA damage include DNA crosslinking, excision repair, strand breaks, and alkali-labile sites¹². The Comet assay is utilised in ecotoxicological research in a range of sentinel organisms as well as in vitro and in vivo environmental genotoxins such as environmental pollution, pharmacological treatments, and occupational exposure¹³. Its use in the controlled laboratory setting of hazard characterisation advances knowledge of chemical mechanisms of action and enhances risk assessment¹⁴. The micronucleus (MN) test is another reliable cytogenetic method for determining DNA damage from occupational exposure¹⁵. This test has been used for exfoliated cells and enables the detection of both aneugenic and clastogenic substances^16,17. Since the MN test is thought to be a valuable indicator of genotoxic consequences in populations that have been exposed to genotoxicants, its application in exfoliated cells has significantly expanded^18,19. Peripheral lymphocytes are readily available and easy to isolate; they were chosen as sample cells. When assessing genotoxicity, lymphocytes are a great surrogate cell model to use. Consequently, as lymphocytes circulate throughout the body, they are exposed to both endogenous and exogenous DNA damage through a range of physio-chemical genotoxic lesions.^20,21.

Furthermore,Comet assay research mostly predicts human cancer caused by various substances using isolated lymphocytes as surrogate cells^22,23^,24. However, little research has been done on paint exposure at work. A review of the literature indicates that no studies using the Comet and MN assays have been conducted on Jordanian painters. The purpose of this study was to use the Comet assay and MN test, to analyse DNA damage in blood lymphocyte of painters in Jordan in order to explore the genotoxic effects related with workers’ exposure to paint chemicals.

MATERIALS AND METHODS:

Allchemicals used in this research were purchased from Sigma Aldrich. (Merck KGaA) including fetal bovine serum (FBS; cat. no. F7524), penicillin-streptomycin (cat.no.P4333), RPMI 1640 medium (RPMI‑1640; cat. no. R8758),Cell Counting Kit - 8(CCK-8; ca.no. NC1951040) and phytohaemagglutinin (PHA;ca.no. L1668).

Ethical approval:

The assays for Comet and micronucleus were performed with ethical consent in order to identify DNA damage in Jordanian painters' lymphocyte cells.It was given by the Jordanian Ministry of Health’s Scientific Research Ethics Committee (MBA/Ethics Committee/11470/ 16/9/2020).

Comet assay and sample preparation:

Following permission from 50 male painters and healthy volunteers in Jordan, whole blood samples were venipunctured using 9mL lithium heparin tubes that were clearly marked for identification. The samples were diluted 1:1 in RPMI-1640 Medium and then mixed with 10% dimethyl sulfoxide (DMSO). The diluted blood sampleswere placed into 1.5mL Eppendorf® tubes that were marked, tightly sealed, and stored at -80°C.

Cell viability:

Cell viability was determined using the CCK-8 from Sigma-Aldrich based on our earlier investigation. It was kept at 37˚C for four hours. A cell of viability ⩾75% were used in all experiments.

Comet Assay:

The preparation of the Comet assay followed^25,26. In brief, 1000μL of RPMI-1640 Medium (a liquid, sterile-filtered medium that is perfect for cell growth and contains sodium bicarbonate but not L-glutamine) was mixed with 100μL of whole blood samples. After that, the samples were incubated for 30minutes at 37°C. Following the incubation period, the samples were centrifuged at 3000rpm for five minutes. Following that, the process was followed in accordance with²⁷. The slides were coded before scored by using 20× magnification fluorescence microscope to score 100 nuclei on each slide. This was connected to a CCD camera using Komet 6 software and Kinetic Imaging (Andor Technology Ltd, Belfast). Olive tail moment (OTM) and % tail DNA were used concurrently to lower result variability.

The MN assay:

A T25 cm² Corning culture flask was filled with 4.5mL of RPMI-1640 Medium supplemented with 1% penicillin–streptomycin, 15% FBS, and L-glutamine and 25mM HEPES at final concentrations of 1% and 15%, respectively, after 500μL of whole blood had been added. Then, 100μL of 2.5% phythaemagglutinin (PHA). After adding 50μL of cytochalasin-B (6 μg•mL−1, Sigma) to each flask, it was incubated for 44 hours at 37°C and 5% CO2. FENECH's instructions were followed when performing the MN procedures²⁸. To evaluate DNA damage, a wide range of cytological scoring measures were employed. Both mononucleated (MonoNCs) and binucleated (BiNCs) cells had micronuclei (MNi) that were scored. The nuclear buds (NBUDs) and nucleoplasmic bridges (NPBs) were scored for BiNC ^.The nuclear division index (NDI) was calculated using the formula NDI=M1+2(M2) +3(M3)/N, where M1 denotes mononucleated cells, M2 denotes binucleated cells, M3 denotes multinucleated cells, and N is the total number of viable cells scored²⁹.

Statistical analysis:

Dunnett's post hoc test was performed by using one-way ANOVA in GraphPad Prism 8 (Dotmatics) for statistical analysis. The mean and SEM are used to present the data. Significant p-values were defined as p<0.05, p<0.01, and p<0.001.

RESULTS:

Table 1 displays the demographic characteristics of the two groups under study (the controls and the paint workers). The male individuals are nearly all the same age (32.76±1.130, 35.64±1.047, respectively). The exposed paint workers' mean daily paint exposure time (hours) was 6.740±0.2016. The paint workers' smoking habits were found to be different from the controls' (26%), with 40% of them smoking and none of them using any kind of self-protective equipment. Their socioeconomic backgrounds and educational levels were also low.

The general examination of the paint workers group revealed a statistically significant increase in DNA damage by using OTM and %Tail DNA (p<0.001) compared to control group as presented in figure 1.

Table 1: Study groups' demographic features

Characteristic

Control group

N=50

Paint workers

N=50

Age in years Range

Mean ± S.E.M

20-45

32.76±1.130

20-48

35.64±1.047

Working hours Range

Mean ± S.E.M

4-9

6.740±0.2016

Average working Range

Mean ± S.E.M time (years)

4-20

11.92±0.5847

Nonsmoker/smokerRange

Mean ± S.E.M

47/13

37/20

Use protection equipment

Figure 1: DNA damage measured as mean OTM and %Tail DNA in human lymphocytes from control group and paint workers in the Comet assay. n=50 in each group. NS: nonsignificant. *: p<0.05; **: p<0.01; ***: p<0.001.

In comparison to healthy control cells, figure 2 demonstrated a substantial increase in MNi frequency in the paint workers group (***p<0.001). Among the numerous cytological scoring factors are biomarkers of cell mitotic division, such as mononucleated cells (MoNC), binucleated cells (BiNC), and multinucleated cells (MultiNC). These parameter values were used to calculate the NDI for both the paint worker group and the healthy individuals. Both groups' mean NDI values fell within the normal range. (Table 2).

Figure 2. Comparison between control group individuals and paint workers group BiMNi

(n=10). *p<0.05; **p<0.01 ***p< 0.001 compared to control group, errors bars represent (SEM).

DISCUSSION:

Paint is a mixture of finely divided pigment molecules suspended in a liquid that is made up of water, a volatile solvent (such as resin), aliphatic hydrocarbons and aromatic hydrocarbons can make DNA damage to a number of reasons³⁰. Based on earlier research, painters were exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs), which have been connected to a higher risk of lung cancer death³¹. Paint containing Lead pigments have been increased risk of bladder, kidney, and urothelial malignancies in addition to multiple myeloma³². It is well established that PAH causes cancer and mutagenesis³³. It is common knowledge in the field of occupation that smoking, sex, and age all have an impact on DNA damage. These variables were not included in this study, though, as there were no significant variations in smoking habit between the paint workers' group and the healthy control group, and both groups' means were similar. Additionally, all of the individuals were male.

Workers in the Brazilian paint sector were shown to have DNA damage on their lymphocytes and buccal cells which measured by Comet assay^34,35. The formamidopyrimidine-glycosylase (Fpg) comet assay was used to measure the increase in oxidative DNA in the lymphocytes of automobile painters³⁶.

Our research suggesting that work painters have higher levels of genotoxicity by using OTM and % Tail DNA compared to healthy control which is consistent with previous studies that showed increased levels of micronuclei (MN) in lymphocytes and oral mucosa cells, sister chromatid exchange (SCE), chromosomal aberrations (CAs), and DNA damage found by the Comet assay in leukocytes of workers exposed to car coatings and painters^37,38. The micronucleus assay has been a vital assay in determining the genotoxicity of various substances by the measurement of MNi and other chromosomal abnormalities such NPBs, a dicentric chromosome biomarker, and NBUDs, a gene amplification biomarker³⁹. Because MNi are markers of chromosomal breakage, loss, rearrangement, necrosis, and apoptosis^40,41, special attention was given to their occurrence in the paint worker groups among the many cell types counted in this study. The MNi frequency in BiNC paint worker group was significantly high compared to healthy control group. Furthermore, after scoring 1000 cells, the percentage of BiNC in each person's lymphocyte cultures fell within the usual range. However, the low percentage of MultiNC in cell culture demonstrated that cytochalasin B prevented cell division following a single cell cycle. In the meantime, all studies' NDI levels were normal (Table 2). Few studies revealed there were no statistically significant variations in the frequency of MNi induction, despite some international studies demonstrating an increase in the frequency of MNi in paint workers' buccal cells which is compatible with this study⁴².

CONCLUSION:

The results of the Comet and micronucleus assays in this investigation suggested that occupational exposure to chemicals in paint workers may cause oxidative stress-induced DNA damage in workers because of increased lymphocyte DNA damage as detected by the Comet assay and elevated MNi in compared to a healthy control. It is difficult to pinpoint a single chemical or component as the source of oxidative and genotoxic injury in paint workers since they are frequently exposed to complex mixtures of heavy metals and organic solvents included in paints. Abrasive blasting and spray painting are two high-risk activities that should always be done with high-quality personal protective equipment (PPE) appropriate for the type of job in addition to higher safety level. The paint workers in this study did not use enough PPE.

ACKNOWLEDGEMENT:

Iwould like to thank the E. Positive Medical Lab Teams, Dr. Ahmad Z. Alsarayreh, Dr. Yaseen T. Al Qaisi for their help in this work.

CONFLICT OF INTEREST:

None declared.

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Received on 23.02.2024 Modified on 04.05.2024

Research J. Pharm. and Tech 2024; 17(9):4417-4421.

DOI: 10.52711/0974-360X.2024.00682

				Mean Per 1000 BiNC cells			Mean of % MNi Monc
	Mean of NDI	Mean of % BiNC	Mean of % MultiNC	BiMNi	BiNPB	BiBuds
Control group	1.90±0.04	61±1.42	17±1.13	6.5±0.17	0.2±0.06	0±0.00	3.5±0.04
Paint workers	2.01±0.15	60±0.27	20.3±0.82	17±1.06	0.6 ±0.02	0±0.00	6.3±0.46