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Abstract

Aim:

The aim of this work was to investigate the relationships between aluminium levels, oxidative status and DNA damage in workers occupationally exposed to aluminium.

Subjects and methods:

This study was conducted in a secondary aluminium smelter. It included 96 male workers occupationally exposed to aluminium fume and dust compared to 96 male nonexposed individuals. Full history and clinical examination were done for all participants. Laboratory investigations in the form of serum aluminium, total antioxidant capacity (TAC), urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) and comet assay test were performed.

Results:

Serum aluminium level ranged from 4 to 30 µg/L of median: 10 µg/L; urinary 8-OHdG ranged from 2.7 to 17.2 ng/mg creatinine of median: 7.6 ng/mg creatinine; comet tail length (CTL) ranged from 19.7 to 50.5 µm of median: 45 µm, were statistically significantly increased in the exposed group compared to nonexposed group. In exposed workers, a statistically significant positive correlations were found between serum aluminium level and urinary 8-OHdG (r = 0.75, p < 0.001); aluminium level and CTL (r = 0.71, p < 0.001); and urinary 8-OHdG and CTL (r = 0.71, p < 0.001). There was a statistically significant negative correlation between serum aluminium and TAC (r = −0.76, p < 0.001).

Conclusion:

Occupational exposure to aluminium in secondary aluminium smelters was related to the induction of oxidative stress and DNA damage. This may promote the development of adverse health hazards in the exposed workers

Keywords

Aluminium 8-OHdG total antioxidant capacity comet assay

Introduction

Aluminium (Al) is one of the most abundant elements in the environment. It causes adverse effects on various organs including brain, bones, kidneys and blood.^1

–4 It is implicated in causing multisystem toxicity, as it has no evidence of physiological role in the human body.⁵ At the workplace, workers are exposed to high aluminium levels through inhalation of dusts and fumes in contaminated air.⁶

Aluminium is trivalent cation that does not undergo redox changes.⁷ It is related to a variety of disorders that are associated with an increase in the formation of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and the superoxide radical (O₂ ⁻). These species are generated by electron leakage from enzymes involved in the mitochondrial electron transport chain, which contain transition metal ions at their active sites.⁷ Although the exact mechanism of aluminium toxicity is not known, however, different studies^8

–11 suggested that aluminium could potentiate oxidative and inflammatory events leading to tissue damage. Oxidative stress in turn may induce DNA damage in the cells. Aluminium toxicity decreases the repair capacity of cells, inhibiting DNA repair and increased DNA fragmentation.⁸

DNA damage can be considered as a biomarker of exposure to genotoxic agents, and it is also commonly known as a marker of cancer risk.¹²

In risk assessment, the comet assay can help in hazard identification. Comet assay seems to be a very rapid and sensitive method for detecting DNA damage in individual cells. It has become a reliable tool for the assessment of precancerous risk assessments.¹³ Cancer hazards associated with exposures in aluminium production industry were evaluated by the International Agency for Research on Cancer (IARC), 2010.¹⁴

Biological monitoring is known to be a useful tool to detect the effects of exposures on organs before distinct organ damage occurs. There are few reports about the associations between biological monitoring of aluminium concentrations in blood and clinical findings, except for associations with neurological findings.^15,16

This work was done to study some oxidative stress and DNA damage parameters in subjects occupationally exposed to aluminium, moreover to assess the possible relations between aluminium level, oxidative stress and DNA damage in exposed workers.

Subjects and methods

Study population

This study was carried out at one of the major aluminium factories in Helwan area, Southern Cairo, Egypt. The factory is manufacturing aluminium frames, long bars and evaporator panels for refrigerators, electrostatic powder-coated aluminium profiles for tubes, transportation systems including busses and trucks and many other aluminium applications. The factory contains many sectors; one of these sectors is secondary aluminium smelter. During the period from April 2016 to May 2016, a cross-sectional study was conducted on two groups: an exposed group and a control group. The first group included 96 male workers in the production line in secondary aluminium smelter. One hundred and twenty workers divided on three shifts were engaged in the production line in the aluminium smelter and accepted to participate in this study. The workers worked 8 h/day for 5 days, 40 h per week. Twenty-four workers were excluded from the study according to exclusion criteria. Participants included in the study had to be working in aluminium smelter for at least the preceding 2 years. Exclusion criteria for both exposed and control groups included following: alcohol intake and taking supplemental vitamins and aluminium containing antacids medications. Those having any chronic illness as cardiovascular, liver diseases or receiving antioxidant drugs during the last 6 months were excluded. The control group included 96 male workers in administering departments of the same company, who have never been occupationally exposed to aluminium. Both groups were matched for age, socio-economic status and smoking habit.

Methods

The ethical committee of the Department of Occupational and Environmental Medicine, Cairo University, Egypt, approved the study. Prior to this study, a written consent to participate in the study and an approval to give blood samples from each individual were obtained after explaining to them the aim and the importance of the study. During the study, the ethical guidelines of good clinical practices have been followed. Strict confidentiality was observed throughout sample collection, coding, testing and recording of the results. The studied groups were subjected to a specially designed detailed questionnaire including sociodemographic data: age, residence, marital status, smoking habits (current cigarettes smoking), present, past and family history. Occupational history included current job and its nature, previous jobs, duration of employment in years, using protective equipment or not. Health complaints included onset, duration and relation to work. The study was conducted using face-to-face interviews in the local language of the country.

Sample preparation

Following an overnight 8 h, fasting peripheral blood samples (a total of 5 mL) were collected from an antecubital vein. Samples were divided into three parts: 1 mL of blood was collected into plain tube for serum aluminium assay. Two millilitres of blood was collected in heparinized vacuum tube to isolate mononuclear leucocyte to estimate DNA comet assay. Samples were handled under dimmed light to prevent DNA damage from ultraviolet light. The remaining 2 mL of blood was collected into EDTA tube and then centrifuged at 1500×g for 10 min in order to obtain the plasma. Plasma was then stored at −80°C until an analysis of total antioxidant capacity (TAC) levels could be performed.

Individuals were instructed to obtain midstream urine samples by voiding into polypropylene containers. Urine samples were centrifuged to remove particulate matter. Urine aliquots were transferred to polypropylene tubes and kept frozen at –80°C until analysis.

Determination of serum aluminium

It was performed using atomic absorption spectrometry with Zeeman background correction (Thermo elemental M6, Cambridge, England). The samples for aluminium were prepared by dilution of 0.5 mL of blood with 2 mL deionized water and then centrifuged. External calibrators were prepared by serial dilution of parent stock, using the diluents (deionized water). For the reading metals concentration of both samples and standard (Merck, Germany), it was important to choose proper wavelength, lamp current bandpass optimization for the aluminium. By plotting standard curve, the reading of the absorbance of the sample and calibrator on semi-log curve was done. The concentration of aluminium in samples was interpreted from the standard curve. Aluminium was measured as described by Valkonen and Aitio.¹⁷ The results were expressed in μg/L.

Measurement of the plasma TAC

The TAC was measured using an automated analyser (Abbott, Aeroset, Illinois, USA) with a TAC measurement kit developed by Erel.¹⁸ In this assay, a standardized solution of Fe²⁺−o⁻dianisidine complex reacts with a standardized solution of H₂O₂ via a Fenton-type reaction, producing hydroxyl radicals. At a low pH, these potent ROS oxidize reduced colourless-o-dianisidine molecules to yellow–brown coloured dianisidyl radicals. Oxidation reactions progress among the dianisidyl radicals and further oxidation reactions occur. Colour formations are increased with additional oxidation reactions. Antioxidants in the sample suppress oxidation reactions and colour formation. In the past, the assay has obtained a precision of less than 3% CV. The results are expressed as mmol/L.

Comet assay

Slides were prepared according to Singh et al.¹⁹ The cell suspension was centrifuged, and the pellet obtained was mixed with 0.7% low-melting agarose (LMA) and placed on fully frosted roughened slides previously coated with 1% normal-melting point agarose. To the solidified agarose, a third layer of 0.1% LMA was applied and was immersed in freshly prepared ice-cold lysis solution for 1 h. The slides were electrophoresed, neutralized, dried and stained with ethidium bromide. The electrophoresis was performed for 20 min by applying an electric field of 25 V (1 V/cm) and adjusting the current to 300 mA. After electrophoresis, the slides were first washed gently with 0.4 M Tris-HCl buffer (pH 7.5) to neutralize the alkali, and the DNA was then stained by adding 100 μL of ethidium bromide (2 μg/mL).

A total of 100 randomly captured comets from each slide were examined at 400× magnification using an epifluorescence microscope (Zeiss, Oberkochen, Germany) connected through a black and white camera to an image analysis system (Comet Assay II; Perceptive Instruments Ltd, UK). Computerized image analysis system acquires images, computes the integrated intensity profiles for each cell, estimates the comet cell components and then evaluates the range of derived parameters. To quantify the DNA damage, tail length (TL) and tail moment (TM) were evaluated. The TL (length of DNA migration) is related directly to the DNA fragment size and is presented in micrometres. It was calculated from the centre of the cell. The TM was calculated as the product of the TL and the fraction of DNA in the comet tail. An undamaged cell resembles an intact nucleus without a tail, and a damaged cell has the appearance of a comet. The length of the DNA migrated in the comet tail, which is an estimate of DNA damage, was measured. Similarly, the images were analysed manually to evaluate the percentage of cells with DNA damage as damaged cell frequency (DCF).

Urine analysis for 8-hydroxy-2′-deoxyguanosine (8-OHdG)

It was determined using a competitive enzyme-linked immunosorbent assay kit (MyBioSource Inc, San Diego, California, USA), as it is a more sensitive method and generally provides higher 8-OHdG levels than the other methods as high-performance liquid chromatography with electrochemical detection. Urine samples were thawed and centrifuged at 2000 rotations per minute (rpm) for 10 min to remove the particulate matter. Fifty microliters (µL) of sample or standard and 50 µL of primary monoclonal antibodies were added to microtiter plates precoated with 8-OHdG. Then, the plates were incubated at 37°C for 1 h with continuous mixing at 100 rpm. The antibodies bound to the 8-OHdG in urine were then washed with 250 µL of phosphate-buffered saline three times. With 100 µL of horse: radish peroxidase-conjugated secondary antibody added to each well, samples were incubated at 37°C for 1 h with mixing. The urinary 8-OHdG concentration was adjusted to the urinary concentration of creatinine (nanograms 8-OHdG/milligram creatinine) to control for the variability in urine dilution.²⁰

Workplace monitoring

Air sample of total aluminium dust levels was assessed by the industrial hygienist of the factory. Measurements were taken during shift time from different places in production units. Samples of indoor air were collected in the production units by active sampling on 8 × 110 mm² adsorbent tubes containing activated charcoal at a flow rate of 200 mL/min, using an air sampling pump with electronic flow control. The flow of the pump was calibrated using a mini-BUCK Calibrator M-30 Electronic Primary Gas Flow Standard, Orlando, Florida, United States. After 4 h, the sampling was stopped by placing caps on both ends of the tubes. The tubes were covered with aluminium foil and stored at 4°C until analysis. Analysis was performed by the atomic absorption spectrophotometry, spectrophotometer: a nitrous oxide–acetylene burner head and aluminium hollow cathode lamp for the aluminium, according to National Institute for Occupational Safety and Health (NIOSH) method 7013.²¹

The geometric means of the measurement of aluminium dust 0.5 ± 0.02 mg/m³ ranged from 0.07 to 0.37 mg/m³. These values are within the occupational exposure limit of occupational safety and health administration (15 mg/m³) standards and procedures.²² These measurements also within the maximum allowable limits according to Egyptian Environmental Law²³ (3 mg/m³).

Statistical analysis

Data obtained from the study were coded for both groups and entered using the statistical package SPSS version 16. The mean values, standard deviations and ranges were estimated for quantitative variables; qualitative data were represented as frequencies and percentages. Comparisons between exposed and control groups were carried out using χ² and the independent sample t-tests for quantitative variables, which were normally distributed variables and nonparametric Mann–Whitney test for quantitative variables, which were not normally distributed. The correlations between individual variables were calculated using Spearman correlation coefficient; p values <0.05 and <0.001 were considered statistically significant, and p value >0.05 was not considered statistically significant.

Results

As shown in Table 1, no statistically significant differences were found between the exposed and control groups as regards demographic characteristics. Serum aluminium, urinary 8-OHdG, DCF and comet tail length (CTL) were found to be significantly increased in the aluminium exposed group than nonexposed, (p < 0.001), while plasma TAC was significantly decreased (p < 0.001; Table 2). Different correlations between serum aluminium, DNA damage, comet assay and duration of employment were presented in Table 3. Multiple linear regressions were done to assess aluminium level and duration of exposure as predictors of the level of 8-OHdG. The model was significant, explaining 35.2% of the variability in 8-OHdG. Duration of exposure is the significant predictor in this model (β = 0.137, p < 0.05; Table 4). Another model was used to assess 8-OHdG, CTL and duration of exposure as predictors of aluminium level. The model was significant, explaining 47.4% of the variability in aluminium level. The CTL and duration of exposure were significant predictors (β = −0.641, p < 0.001), (β = −0.327, p < 0.001), respectively, but 8-OHdG was not significant predictor for aluminium level in this model (Table 4).

Table 1.

Demographic characteristics in exposed and nonexposed groups.^a

	Exposed (n = 96)	Control (n = 96)	p value
Age (years)	36.41 ± 11.50	34.80 ± 11.1	0.07
Duration of work (years)	14.6 ± 9.6	–	–
Smoking habits (%)
Yes	50	48	0.88
No	46	48	0.93
Smoking index (no. of cig./day × no. of years)	405.20 ± 130.85	403.25 ± 104.65

^aData represented by Mean ± SD. χ² and the independent sample t-tests were used. Duration of employment (3 min–34 max) in years.

Table 2.

Serum aluminium, plasma TAC, urinary 8-OHdG, DCF and CTL in exposed and nonexposed groups.^a

	Exposed (n = 96)	Control (n = 96)	p value
Serum Al (μg/L)	10 (4–30)	0.3 (0.01–2.1)	<0.001
8-OHdG (ng/mg creatinine)	7.6 (2.7–17.2)	3.5 (2–7)	<0.001
TAC (mmol/L)	0.9 (0.5–3.4)	4.7 (1.2–8.2)	<0.001
DCF (%)	100 (60–130)	30.9 (12.9–40.6)	<0.001
CTL (µm)	45 (19.7–50.5)	5.1 (3–8.8)	<0.001

TAC: total antioxidant capacity; 8-OHdG: 8-hydroxy-deoxyguanosine; CTL: comet tail length; DCF: damage cell frequency.

^aData represented by median and range. Mann–Whitney test was used.

Table 3.

Correlations between serum aluminium, DNA damage, comet assay and duration of employment.

	Serum Al (µg/dL)	DCF (%)	CTL (µm)	TAC (mmol/L)	8--OHdG (ng/mg creatinine)
Duration of employment(years)	0.681^b	0.834^b	0.833^b	−0.750^b	0.830^b
TAC (mmol/L)	0.762^b	−0.661^b	−0.671^b	–	−0.615^a
8-OHdG (ng/mg creatinine)	0.755^b	0.724^b	0.716^b	−0.711^b	–
CTL(µm)	0.711^b	0.621^b	–	−0.617^b	0.760^b

TAC: total antioxidant capacity; 8-OHdG: 8-hydroxy-deoxyguanosine; CTL: comet tail length; DCF: damage cell frequency.

^a p < 0.05.

^b p < 0.001.

Table 4.

Multiple regression analysis to test predictor for 8-OHdG and aluminium level in exposed workers.

Dependent variable	Independent variables	R ²	β coefficient	p value
8-OHdG	Al level	0.352	0.002	0.087
	Duration of exposure		0.137	<0.05
Al level	8-OHdG		0.005	0.868
	CTL	0.474	−0.641	<0.001
	Duration of exposure		−0.327	<0.001

8-OHdG: 8-hydroxy-deoxyguanosine; CTL: comet tail length.

Discussion

In this study, although environmental measurement of aluminium dust was within the acceptable levels, there were increased oxidative stress and DNA damage among exposed worker compared to their controls. The significantly increased levels of aluminium in exposed workers were in accordance with the results of other studies,^15,16,24,25 during their occupational exposure to aluminium dust. Aluminium levels represented a wide variation in healthy nonexposed subjects. The exposure to aluminium in humans could be from different sources, for example, drinking water, leached Al from utensils, eating processed cheese and cosmetics.²⁶ In the current study, the aluminium level in the control group ranged from 0.01 to 2.1 µg/L of median: 0.3 μg/L which is similar to the finding of Mahdi et al.^17,27 who concluded that the upper reference limit for aluminium in a healthy, nonexposed population was estimated to be 0.1 mmol/L in serum.

In this study, there was decrease in the TAC in exposed workers, indicating oxidative stress. Cumulative exposures to aluminium are not easy to estimate, considering many cases exposure started long before the toxic kinetics of aluminium to be of any significance.²⁸ In this study, the levels of serum aluminium were statistically significantly increased in exposed workers than the control group. They were correlated with duration of employment (r = 0.68, p < 0.01). These findings agreed with the finding of Siha et al.,²⁹ who found positive correlation between serum aluminium level and duration of exposure among Egyptian foundry workers exposed to aluminium. Experimental studies^30,31 showed that aluminium exposure induced oxidative stress and DNA damage as indicated by fragmentation and increase in the appearance of comets; however, the human studies at this issue are limited.

The current study found increased risk of genotoxicity in exposed workers. These results are in accordance with Balasubramanyam et al.,³² who studied the genotoxic effects of aluminium oxide nanomaterials in rat peripheral blood cells using the micronucleus test and the comet assay. They reported increase in the frequency of micronuclei and the percentage of tail DNA (% tail DNA) migration. 8-OHdG is a biomarker of oxidative DNA damage. In occupational setting, urinary 8-OHdG has been widely studied as a biomarker of oxidative stress, due to easy collection of samples. Recently, Wang et al.,³³ suggested that there is a positive relation between the biomarkers of plasma and urinary 8-OHdG. In the current study, we found a statistically significant increase in urinary 8-OHdG in exposed workers than controls. On contrary to these results, Ogawa and Kayama³⁴ found no significant difference between aluminium handling workers and nonaluminium workers as regards urinary 8-OHdG a marker of oxidative DNA damage. In the current study, we found statistically significant positive correlation between duration of employment and serum aluminium, 8-OHdG and comet assay test. However, Ogawa and Kayama³⁴ found no significant relationships between aluminium concentration and work duration among aluminium handling workers. This can be explained by the longer duration of employment of the exposed workers in the current study. Chronic occupational exposure is associated with a decreased rate of clearance of aluminium from the body. This is thought to be a consequence of the systemic absorption of aluminium and its incorporation into compartments having more prolonged turnover rates. In this study, we found statistically significant negative correction between TAC and duration of employment, serum aluminium and the comet test results. The model that included 8-OHdG explained about 35.2% of variants; of these two variables, duration of exposure made a statistically significant contribution to predication of the model. A statistically significant model (β = 47.4%) allows us to predicate aluminium level using three variables; two of these variables, CTL and duration of exposure, had statistically significant contribution to this model, with the CTL had the largest contribution of the two but still both were statistically significant. In support to these findings, experimental studies by Achary et al.,^31,35 concluded that Al-induced oxidative burst through the generation of ROS leads to cell death and DNA damage in the root cells of Allium cepa. There are limited studies regarding occupational exposure to aluminium and oxidative stress. A study by Celik etal.,²⁶ found increased plasma aluminium concentrations among persons using aluminium containers and utensils daily. They found positive correlation between increased oxidative stress and increased DNA damage in aluminium-exposed humans.

Conclusion

The results of this study concluded that increased aluminium levels in exposed workers induce oxidative DNA damage. The disruption of homeostasis induced by oxidative stress manifested by decreased TAC. DNA damage indicated by increased 8-OHdG in urine and comet assay test results was correlated with the duration of employment. Longer duration of exposure of workers to aluminium manifested by increased serum aluminium might be the cause of possible health risk among exposed workers; however, further studies are needed.

The induced oxidative stress and DNA damage found in the current study may promote the development of health hazards with continued occupational exposure to aluminium dust at workplace.In the current study, although environmental measurements at workplace are within the maximum allowable limits according to Egyptian Environmental Law, there is a clear evidence of increased aluminium levels and oxidative DNA damage in exposed workers. Therefore, considering revising these limits at work place is very important.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflict of interest with respect to the research, authorship and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship and/or publication of this article.

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