Neurological risk assessment of co-exposure to heavy metals (chromium and nickel) in chromium-electroplating workers

Abstract

BACKGROUND:

Metal co-exposure of human subjects is an important matter of public health concern. It has been shown that Acetylcholinesterase activity is a suitable biomarker for the neurological risk assessment of some metals. A few studies have reported neurotoxicity risk among humans in co-exposure to chromium and nickel.

OBJECTIVE:

In this study, AChE activity was assessed in occupational exposure to chromium (VI) and co-exposure to nickel (II) and chromium (VI).

METHODS:

Air sampling was done in chromium electroplating workshops with the NIOSH 7600 and OSHA ID-121 methods for chromium and nickel assessment. Thirty-two workers from hard chromium plating and 30 from decorative chromium plating were evaluated, while AChE activity was measured by the Elman method.

RESULTS:

Personal exposure to chromium in 20% of the studied people exceeded the TWA set by ACGIH. Occupational exposure to nickel in 47% of the DCP subjects was found to be higher than TWA. Cholinergic inhibition in plating workers was marked by a decrease in AChE compared to controls. Subjects with chromium (VI) exposure contained significantly higher inhibition of AChE activity (p < 0.001) than workers with co-exposure to nickel (II) and chromium (VI).

CONCLUSIONS:

The chromium-matched electroplaters have no significant difference in AChE activity. It can be concluded that cholinergic inhibition with chromium (VI) is higher than nickel (II) exposure.

Keywords

Co-exposure hexavalent chromium (VI)nickel (II)acetylcholinesterase (AChE)chromium-electroplater

1 Introduction

In the present industrialized world, especially in the occupational setting, exposure to the mixture of pollutants is unavoidable [1]. In recent years, most of the risk assessment have focused on a chemical-by-chemical exposure evaluation, while co-exposure to other chemicals has not been considered [2, 3]. Co-exposure to metals is a serious hurdle in health concept. Exposure to a mixture of metals occurs in environmental contamination [4 –6] and occupational exposure such as welding, smelting and the plating processes [7 –9]. Chromium-plating is the application of a metallic coating to the material surface using inorganic salts of metals such as chromium(VI) and nickel (II). HCP and DCP are the most usable types of chromium-plating in which chromium (VI) is found in the HCP process, and nickel (II) and chromium (VI) are used in the DCP industry [10].

The International Agency for Research on Cancer (IARC) has classified hexavalent chromium as a human carcinogen (Group 1) [2]. There is evidence of the toxic effects of chromium on the nervous system [3]. Dizziness, headache, and weakness were reported due to exposure to hexavalent chromium [4]. Dashti demonstrated the toxicity of chromium VI on neural cell lines [7]. Moreover, some studies emphasize hexavalent chromium neurotoxicity and brain cancer [10, 11]. According to recent studies, there is a relationship between nickel exposure and neurological toxicity [12]. The developmental neurotoxicity of nickel is almost completely unexplored [13]. Although, the neurotoxicity of chromium and nickel has been documented, the risk assessment of the biochemical end point is less studied.

The cholinergic neurons play an important role in many functions of the central nervous system. Numerous studies have shown that some metals such as Pb²⁺, Hg²⁺, As³⁺, Cu²⁺, Zn²⁺ disrupt AChE activity in humans [14, 15]. It was presented that Pb²⁺, Hg²⁺, and Cd²⁺ activated presynaptic signals for neurotransmitter release [16]. Moreover, some based in vivo evidence, reported hexavalent chromium impairment of AchE [17]. Recently, a contradictory report has been published on the effect of nickel on AChE. Some in vitro studies have confirmed the effect of nickel on AChE [18] but other studies deny such effects [19].

In this study, we assessed the neurological risk by the evaluating of AChE activity in occupational exposure to chromium (VI) in HCP volunteers and then compared it with the co-exposure to nickel (II) and chromium (VI) in DCP workers.

2 Methods

2.1 Chemicals

In this research, acetylthiocholine iodide (Fluka, Germany), ethylene diamine tetra acetic acid (EDTA), 5,5′ dithiobis-nitrobenzoic acid (DTNB), triton×-100, sodium chloride, quinidine sulfate salt dehydrate (Sigma, USA), potassium hydrogen phosphate, potassium dichromate, diphenylcarbazide solution and sulfuric acid, nickel (II) chloride, kalium dihydrogen phosphate, nitric acid, hydrochloric acid, perchloric acid, hydrogen peroxide (Merck, Germany) all of extra pure grade were used.

2.2 Subjects

A total of 62 workers from chrome plating workshops were selected, of whom which 32 were from HCP and 30 from DCP. Age and sex were matched in controls (n = 65) from food industry who were not occupationally exposed to chromium (VI) and nickel (II), or any other physical or chemical hazardous compounds. Control and platers were matched socioeconomically. Demographic specifications of the workers (HCP: hard chromium plating, DCP: decorative chromium plating) based on a questionnaire is presented in Table 1.

Table 1
Demographic characteristics of studied population

Variable HCP workers DCP workers

Age* Exposed Unexposed Exposed Unexposed

35.88±10.44 35.80±7.58 36.00±11.08 36.13±8.43

Years of work experience^* 9.69±10.10 11.00±9.1 6.20±5.39 8.53±8.42

Gender

Male 33 35 26 26

Female – – 4 4

Total number 33 35 30 30

Smoking habit

Smokers 10 10 7 7

Nonsmokers 23 25 23 23

Variable	HCP workers	DCP workers
	35.88±10.44	35.80±7.58	36.00±11.08	36.13±8.43
Years of work experience^*	9.69±10.10	11.00±9.1	6.20±5.39	8.53±8.42
Gender
Male	33	35	26	26
Female	–	–	4	4
Total number	33	35	30	30
Smoking habit
Smokers	10	10	7	7
Nonsmokers	23	25	23	23

^*Mean±SD

Shahid Beheshti University of Medical Sciences (SBMU) Institutional Review Board (IRB) approved all study procedures and ethical issues.

2.3 Assessment methods

2.3.1 Occupational exposure to metals

Personal monitoring for chromium was evaluated in HCP workshops, while exposure to chromium and nickel was assessed in DCP employees. Airborne hexavalent chromium was evaluated by the National Institute of Occupational Safety and Health (NIOSH) method 7600. Nickel exposure was assessed according to the Occupational Health and Safety Administration (OSHA) method ID-121. Air samples were collected regular working days using a PVC filter. The amount of chromium (VI) in the samples was determined using diphenylcarbazide after acidic extraction. Filters in DCP samples, because of nickel interference, was extracted after elution with NaOH/Na2CO3 solution. Finally, the reaction of chromium (VI) with diphenylcarbazide was determined by a visible spectrophotometer (CECIL 2021) at 540 nm. Nickel concentration was evaluated by flame atomic absorption spectrophotometry (ANA180) after acid digestion.

2.3.2 AChE activity

The Elman method was used for Erythrocyte AChE activity determination [20]. Two milliliter of heparinized blood sample was obtained from all subjects and was transferred to the laboratory on ice for analysis. AChE activity was evaluated by spectrophotometry using acetylthiocholine iodide as a substrate. For this assay, acetylthiocholine iodide (75 mmol/L) and 5, 5-dithiobis-2-nitrobenzoic acid (0.127 mg/mL DTNB) in a phosphate buffer were added to lysed blood samples. AChE activity was monitored by absorbance changes at 405 nm with a spectrophotometer at 30 s intervals for 5 min.

2.4 Statistical analysis

AChE activity in exposed and control groups was compared using two sample student t-test in normal distribution and Mann–Whitney U-test in abnormal distribution of samples. The P value of 0.05 was regarded as the level of statistical significance. All statistical analyses were carried out by SPSS software.

3 Results

Chrome-platers in HCP workshops had significantly higher levels of chromium (p < 0.0001) than DCP population (Fig. 1). The mean±SD values in HCP and DCP workers were 0.046±0.023 mg.m^–3 and 0.023±0.012 mg.m^–3, respectively.

Fig.1

Occupational exposure to hexavalent chromium in electroplaters.

The mean±SD values of nickel concentration for DCP workers was 0.107±0.0.035 mg.m^–3 (Table 2). Occupational exposure to nickel in 47% of DCP subjects was evaluated to be higher than TWA suggested by ACGIH (0.1 mg.m^–3). Nickel exposure was randomly assessed in 20% (n = 7) of the HCP groups. The concentrations of nickel exposure in all studied people were lower than the limit of detection (LOD) where LOD was 0.015 ppm.

Table 2

Personal monitoring of chrome-platers (mg.m^–3)

Occupational exposure Mean±SD (mg.m^–3)	HCP workers	DCP workers
Chromium	0.046±0.023	0.023±0.012
Nickle	Not detectable	0.11±0.013

There were four occupational tasks in chromium plating factories, namely (1) polishing, (2) washing, (3) sorting and (4) supervising operations. As shown in Table 3, exposure to hexavalent chromium in all the HCP operators except the supervisor was significantly higher than in the DCP task groups.

Table 3

Occupational exposure to chromium VI in different tasks

Occupational tasks	Mean±SD HCP workers (mg.m–3)	Mean±SD DCP workers	P-value (mg.m–3)
Polishing	0.051±0.019	0.016±0.007	0.001
Washing	0.043±0.03	0.014±0.002	0.01
Sorting	0.034±0.002	0.016±0.005	0.001
Supervising	0.048±0.024	0.034±0.012	0.45

The AChE activity in chromium-platers is shown in Table 4. HCP and DCP workers contained significantly lower levels of AChE activity compared to the unexposed population.

Table 4

AChE activity in chromium electroplaters and unexposed subjects

Groups	Mean±SD HCP workers (IU/L)	Mean±SD DCP workers(IU/L)
Exposed	33.8±26.5	46.6±26.4
Unexposed	44.9±2.5	62.7±26.7
P-value	0.008	0.023

HCP subjects showed significantly higher inhibition of AChE activity (p < 0.001) compared to the HCP workers (Fig. 2). The ACGIH established a threshold of 30% reduction from the baseline as a Biological Exposure Index (BEI) for AChE inhibitors [18]. In this study, the mean value of the AChE activity in the control population was considered as the baseline value. It was found that 36% of the HCP groups and 21% of DCP workers had AChE activity lower than BEI of AChE inhibitors.

Fig.2

Inhibition AChE activity in chrome-platers.

Inhibition of cholinergic receptors in individual of HCP workers was more (OR-odds ratio) (95% CI [confidence interval]) = 3.375 (1.23–9.11) than DCP subjects (Table 5).

Table 5

OR for cholinergic inhibition in subjects

Groups	Odds ratio	B Regression	Standard error	Confidence interval 95%	P-Value
HCP	3.37	1.21	0.509	1.2–9.1	0.017
DCP	0.21	–1.58	0.56	0.07–0.63	0.005

In order to compare the neurotoxicity risk of chromium and nickel, we found the AChE activity difference in subjects with chromium exposure to be lower than TWA proposed by ACGIH (0.05 mg.m^–3). The results show that despite a variation in nickel exposure in HCP subjects and DCP population, there are any significant differences between chromium exposure (p = 0.04) like the AChE activity (p = 0.03) in Table 6.

Table 6

AChE activity in subjects with chromium exposure lower than TWA

Mean ±SD	HCP workers (n = 22)	DCP workers (n = 23)	P-Value
Chromium exposure(mg.m^–3)	0.03±0.006	0.02±0.012	0.04
Nickle exposure(mg.m^–3)	Not detectable	0.11±0.03	–
AChE activitiy(IU/L)	42.6±22.2	30.4±20.3	0.03

4 Discussion

In the context of human health assessment, there is evidence of some metals having the potential to cause neurotoxicity [1]. There are several studies on nickel and chromium’s neurological effects [12 , 21–23]. Nickel (II) interferes with cellular iron metabolism and inhibits the activities of the respiratory chain enzyme complexes. It may cause ROS production and mitochondrial dysfunction, which could relate to the neurological effects of nickel. In this regard, the neurotoxicity of nickel highly depends on the dose and time of exposure [22]. However, the risk assessment of chromium and nickel by the biochemical endpoint of the neurological system is less studied. Furthermore limited report was evaluated the effect of binary exposure to these metals. Cholinergic inhibition has been proposed as a neurotoxicity mechanism of Pb, As, Mn [19]. The present survey was designed in order to find the effects of hexavalent chromium and co-exposure of chromium and nickel on the AChE activity as a neurological risk assessment.

According to the air sampling results, 31% of occupational groups in the HCP workshops and 8% of DCP subjects had a higher exposure to hexavalent chromium than TWA set by ACGIH (0.05 mg.m^–3). The mean value of hexavalent chromium in the HCP and DCP workshops were lower than the occupational exposure levels. However, the application of a thicker layer of chromium coating on the material surface in HCP workshops causes a significantly higher airborne exposure than the DCP workshops. Between the four occupational tasks in chromium-plating factories, polishing workers in the HCP population had a higher exposure to hexavalent chromium than TWA.

In recent years, some studies reported dysfunction of motor activities and brain impairment in-vivo with trace chromium oral exposure. In a survey by Singh and Chowdhuri, the Drosophila melanogaster was treated by hexavalent chromium, which led to the locomotor impairment and increased inhibition of AChE activity in adult flies [21]. Soudani et al. found an increasing inhabitation of the AChE in female rats after treatment by K₂Cr₂O₇ [24]. The results of the present work is in line with the previously reported cholinergic effect of chromium from in vitro and in vivo studies.

The AChE activity in chromium-plating workers was observed to decrease significantly compared to the control groups. The activity of the AChE in 65% of all workers was lower than BEI of cholinergic inhibitors. This finding revealed the occupational neurotoxicity of chromium (VI) in the HCP workers to be the same as the co-exposure to chromium (VI) and nickel (II) for DCP workers by interfering with the inhibition of AChE activity. The activity of AChE in the HCP workers was significantly inhibited compared to DCP workers. On the other hand,in the chromium-matched subjects of DCP and HCP workers (p < 0.099), there were no significant difference in AChE activity. According to the results, cholinergic inhibition in chromium (VI) exposure was higher than nickel (II).

5 Conclusion

Excessive and continuous exposure to several metals can lead to neurological dysfunction or neurological enzyme inhabitations. The present study shows that the occupational exposure to chromium (VI) and co-exposure to chromium (VI) and nickel (II) inhibits AChE activity. It can be concluded that there is not cholinergic risk in nickel (II) exposure in the concentration of TWA (0.1 mg.m^–3), while chromium (VI) exposure at lower levels than the suggested TWA (0.05 mg.m^–3) inhibits AChE activity.

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Shahid Beheshti University of Medical Sciences.

Footnotes

Acknowledgments

This research was financially supported by the School of Public Health, Shahid Beheshti University of Medical Sciences.

References

Andrade

, et al., Urinary delta-ALA: A potential biomarker of exposure and neurotoxic effect in rats co-treated with a mixture of lead, arsenic and manganese. Neurotoxicology. 2013;38:33–41.

Wise

, et al., Hexavalent chromium induces chromosome instability in human urothelial cells. Toxicology and Applied Pharmacology. 2016;296:54–60.

Ray

, Ray

MK.

Bioremediation of heavy metal toxicity-with special reference to chromium. Al Ameen J Med Sci. 2009;2(2):57–63.

Guertin

, Avakian

, Jacobs

JA.

Chromium (VI) handbook. (2016): CRC press.

Malakootian

, et al., ZnO nanoparticles immobilized on the surface of stones to study the removal efficiency of 4-nitroaniline by the hybrid advanced oxidation process (UV/ZnO/O3). Journal of Molecular Structure. 2019;1176:766–76.

Khatami

, et al., Super-paramagnetic iron oxide nanoparticles (SPIONs): Greener synthesis using Stevia plant and evaluation of its antioxidant properties. Journal of Cleaner Production. 2019;208:1171–7.

Dashti

, Soodi

, Amani

Evaluation of Cr (VI) induced neurotoxicity and oxidative stress in PC12 cells. Pathobiology Research. 2015;18(1):55–65.

Khatami

, Alijani

, Sharifi

Biosynthesis of bimetallic and core–shell nanoparticles: Their biomedical applications–a review. IET Nanobiotechnology. 2018;12(7):879–87.

Khatami

, et al., Rectangular shaped zinc oxide nanoparticles: Green synthesis by Stevia and its biomedical efficiency. Ceramics International. 2018.

10.

Wesseling

, et al., Cancer of the brain and nervous system and occupational exposures in Finnish women. Journal of Occupational and Environmental Medicine. 2002;44(7):663–8.

11.

Verhage

, Cheong

, Jeejeebhoy

KN.

Neurologic symptoms due to possible chromium deficiency in long-term parenteral nutrition that closely mimic metronidazole-induced syndromes. Journal of Parenteral and Enteral Nutrition. 1996;20(2):123–7.

12.

, et al., Melatonin protects against Nickel-induced neurotoxicity in vitro by reducing oxidative stress and maintaining mitochondrial function. Journal of Pineal Research. 2010;49(1):86–94.

13.

Slotkin

, et al., Screening for developmental neurotoxicity using PC12 cells: Comparisons of organophosphates with a carbamate, an organochlorine, and divalent nickel. Environmental Health Perspectives. 2006;115(1):93–101.

14.

Patlolla

, Tchounwou

PB.

Serum acetyl cholinesterase as a biomarker of arsenic induced neurotoxicity in sprague-dawley rats. International Journal of Environmental Research and Public Health. 2005;2(1):80–3.

15.

Bolognesi

, et al., Multi-target-directed drug design strategy: From a dual binding site acetylcholinesterase inhibitor to a trifunctional compound against Alzheimer’s disease. Journal of Medicinal Chemistry. 2007;50(26):6446–9.

16.

Cooper

, Manalis

. Influence of heavy metals on synaptic transmission: A review. Neurotoxicology. 1983;4(4):69–83.

17.

Domingues

, et al., Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2010;152(3):338–45.

18.

Najimi

, et al., Use of acetylcholinesterase in Perna perna and Mytilus galloprovincialis as a biomarker of pollution in Agadir Marine Bay (South of Morocco). Bulletin of Environmental Contamination and Toxicology. 1997;58(6):901–8.

19.

Frasco

, et al., Do metals inhibit acetylcholinesterase (AChE)? Implementation of assay conditions for the use of AChE activity as a biomarker of metal toxicity. Biomarkers. 2005;10(5):360–75.

20.

Worek

, Eyer

, Thiermann

Determination of acetylcholinesterase activity by the Ellman assay: A versatile tool for in vitro research on medical countermeasures against organophosphate poisoning. Drug Testing and Analysis. 2012;4(3-4):282–91.

21.

Singh

, Chowdhuri

DK.

Environmental presence of hexavalent but not trivalent chromium causes neurotoxicity in exposed Drosophila melanogaster. Molecular Neurobiology. 2017;54(5):3368–87.

22.

M-D

, et al., L-carnitine protects against nickel-induced neurotoxicity by maintaining mitochondrial function in Neuro-2a cells. Toxicology and Applied Pharmacology. 2011;253(1):38–44.

23.

Halder

, et al., Quercetin modulates the effects of chromium exposure on learning, memory and antioxidant enzyme activity in F 1 generation mice. Biological Trace Element Research. 2016;171(2):391–8.

24.

Soudani

, et al., Ameliorating effect of selenium on chromium (VI)-induced oxidative damage in the brain of adult rats. Journal of Physiology and Biochemistry. 2012;68(3):397–409.