Occupational and Non-Occupational Exposure to Chromium Induces Oxidative Stress and DNA Damage in the Population Near Kanpur Tanneries

Abstract

Background:

The chrome tanning method is widely used in the Indian tannery industry. Workers from these tanneries are directly exposed to Cr compounds due to inadequate safety measures and knowledge.

Objective:

This study aimed to examine oxidative stress and DNA damage in tannery workers in Kanpur exposed to Cr both in the workplace and outside of it in terms of health.

Methods:

Blood samples were collected from the people of Jajmau, Kanpur, India. The study population was divided into three distinct categories. Group I comprised 100 individuals occupationally exposed to chromium (Cr) in tannery industries. Group II included 100 individuals who resided near tannery industries but were not occupationally exposed to Cr. Finally, Group III contained 100 healthy individuals who had never been exposed to Cr. Inductively coupled plasma‒mass spectrometry (ICP-MS) was used to measure Cr in all blood samples. Malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) concentrations were used to quantify oxidative stress. A comet assay was used to determine the extent of DNA damage, and the lengths of comet tails in both the exposed and control groups were measured.

Results:

Cr, MDA, SOD, and DNA damage levels were significantly greater in the exposed groups than in the control groups (P < 0.001), whereas GSH levels were significantly lower (P < 0.001).

Conclusion:

This study’s findings indicate that timely identification of the detrimental impacts of Cr on individuals employed in tanneries is highly important for mitigating health risks and minimizing exposure.

INTRODUCTION

Cr (III) and Cr (VI) are utilized in drum and finishing operations of the leather industry, respectively. The effluent drain from tanneries contains a large portion of Cr, which causes significant pollution of different components of the environment.[1] Therefore, the population living near tanneries is also indirectly exposed to Cr and its substituents. The primary ways in which workers in the tannery industry are exposed to Cr are through inhalation, oral intake of food and water contaminated with Cr while at work, or cutaneous contact during the tanning process.[2] Inhalation of Cr increases the chances of multiple nasal problems in chromium electroplating workers compared to zinc electroplating workers.[3] Metal levels in urine and blood were found to be high in occupationally exposed workers, necessitating the need for evaluation to reduce exposure and prevent harmful effects and the development of cancer.[4] Cellular membranes are resistant to Cr(III); however, Cr(VI) can pass through them. Cr(VI) is converted to Cr(III).[5] This form can interact with DNA and cause significant alterations in the polymerization activity and fidelity of DNA-duplicating enzymes, which can ultimately halt DNA replication and cause mutations.[6] Moreover, it results in breaks in both single-and double-stranded DNA,[7] which can cause chromosomal abrasion and mutation.[8] Cr treatment causes the formation of Cr(III) and Cr(II). Cr(II) reacts with H₂O₂ and releases free radicals that cause lipid peroxidation.[9] Lipid peroxidation triggers the antioxidant defense system, which induces the generation of antioxidant enzymes such as GSH and SOD to counteract the consequences of lipid peroxidation.[10] Nevertheless, it has been noted that Cr(III) compounds have hazardous activity comparable to that of Cr(VI) compounds at 1000-fold concentrations.[11]

Cr exposure at work affects millions of individuals globally, causing significant problems such as carcinoma of the lungs, pancreas, and nasal region, which are among the tumors associated with prolonged exposure to Cr.[12] According to Rastogi et al.,[13] it contributes to dermatitis, ulcerations, and nasal septal perforations. Further research demonstrating the impact of long-term exposure to Cr on tannery workers’ health is needed. Therefore, this study aimed to evaluate oxidative stress and DNA damage induced by prolonged Cr exposure in occupationally and non-occupationally exposed human populations.

METHODS

Study design and study population

This was a comparative cross-sectional study. This study was conducted at Chaudhary Ehsan Kareem Hospital, located near the tannery industry in the Jajmau area of Kanpur, Uttar Pradesh, India, from August 2014 to May 2020. Of these 2200 tanneries in India, 402 are operated in Kanpur district, Uttar Pradesh.[14] Tannery waste is usually discharged from common effluent treatment plants (CETPs). However, most tannery industries do not have proper treatment plants; therefore, high concentrations of Cr discharged from these industries contaminate the nearest environment’s water, soil, and air. There were 300 human participants, split up into three groups for this study. Group I included 100 occupationally exposed workers employed in tanneries (mean age: 36.54 ± 13.4 years), Group II included 100 non-occupationally exposed individuals living near tanneries (mean age: 37.09 ± 12.02 years), and Group III included 100 control individuals without Cr exposure (mean age: 35.94 ± 9.92 years). The exposed workers belong to different tanneries in the Jajmau area of Kanpur. The base sample size calculation with a confidence level of 95%, margin of error of 5%, and estimated prevalence of health risk with special reference to oxidative stress and genotoxicity in Cr-exposed and unexposed subjects. The study’s inclusion criterion was that the workers had not less than 10 years of experience (exposure period) in the tannery industry.

Questionnaire

The questionnaire collected personal data, occupational details, and medical history from the individuals involved in this study. Factors such as smoking and drinking habits also induce oxidative stress and DNA damage and[15] therefore were included in this study apart from other variables such as age, sex, marital status, chronic illness, and location and duration of employment.

Laboratory investigations

Sample collection and ethical considerations

Each subject’s blood was collected in heparinized tubes, and a signed consent form was then sent to the University of Lucknow’s Institutional Human Ethics Committee (LU/IEC/ZOOL/2020/11/04).

Blood chromium analysis

Blood Cr levels were determined by inductively coupled plasma mass spectrometry (ICP-MS -7900 Agilent Technologies) at IIT, Delhi and results are expressed as μg/L.

Preparation of erythrocyte lysates

A total of 5 mL of blood was collected, and 0.5 mL was utilized for the Cr concentration analysis. The remaining blood was centrifuged for 10 min at 1000 rpm to separate the plasma and RBCs. Plasma was stored at 80°C until the MDA analysis. RBCs were centrifuged four times at 12,000 rpm for 15 min, and the supernatant was collected and subjected to various antioxidant assays. The Bradford method was used to estimate the protein content in blood.[16]

Malondialdehyde (MDA) concentration

Blood plasma MDA concentrations were measured spectrophotometrically using the method of Ohkawa et al.[17] and the values are presented in nmol/mL.

Reduced glutathione (GSH) concentration

The levels of GSH were measured spectrophotometrically following the method of Beutler et al.[18] and the values are presented as μg/mL.

Superoxide dismutase (SOD) activity

SOD levels were measured spectrophotometrically as previously described methods by McCord and Fridovich[19] and values are expressed as U/g Hb.

Comet assay

The level of DNA damage detected using the comet assay according to the method developed by Singh et al.[20] The damage was determined by visual scoring of cells (Leica Optiphase microscope) with software (Biovis). We quantified the extent of damage using the length of DNA migration (tail length) expressed in μm.

Statistical analysis

SPSS-16.0 was used for the analysis of the collected data. The means of various groups were compared using a one-way analysis of variance (ANOVA). Bivariate data were analyzed using the Chi-square (χ²) test. We used univariate and multivariate linear regressions to assess the effect of alcohol and smoking on oxidative stress and DNA damage. We considered P < 0.05 as a statistical significance value.

RESULTS

Demographic information of all groups is presented in Table 1. The marital status and year of exposure show significant differences. However, no differences were found in age, sex, smoking, and drinking habits in all groups.

Table 1.

Personal characteristics of the studied groups (occupationally exposed, nonoccupationally exposed, and control groups)

Variables	Controls (n=100)	Nonoccupationally exposed (n=100)	Occupationally exposed (n=100)	P
Age (yrs.)	35.94±9.92	37.09±12.02	36.54±13.4	0.791
Male (%)	72 (72.0)	58 (58.0)	66 (66.0)	0.112
Female (%)	28 (28.0)	42 (42.0)	34 (34.0)
Marital status
Married (%)	62 (62.0)	79 (79.0)	78 (78.0)	0.006*2
Single (%)	38 (38.0)	21 (21.0)	22 (22.0)
Alcohol consumption
Yes (%)	12 (12.0)	17 (17.0)	25 (25.0)	0.0542
No (%)	88 (88.0)	83 (83.0)	75 (75.0)
Smoking
Yes (%)	15 (15.0)	14 (14.0)	17 (17.0)	0.832
No (%)	85 (85.0)	86 (86.0)	83 (83.0)
Years of exposure	-	24.52 11.35	21.88±6.28	0.0013*

¹ANOVA, ²Chi-square test, ³unpaired t-test, *P<0.05. Occupationally exposed group—tanners—nonoccupationally exposed group—the population living near the tanning industry

Cr levels from occupationally, non-occupationally exposed, and the control group with standard deviation (SD) are presented in Figure 1 and the means, standard deviations, and P values of Cr are summarized in Table 2. We found significantly greater Cr concentration suggested by NIOSH (0.5 mg/m³ on an 8-h workday) in occupationally exposed (69.37 ± 15.43 μg/L) and non-occupationally exposed subjects (51.32 ± 4.97 μg/L) than in controls (15.49 ± 5.31 μg/L).

Figure 1.

Mean concentration of chromium in blood samples from occupationally, non-occupationally exposed, and control subjects. The values are mean ± SD of data from 100 subjects of each group. Statistics: Tukey’s multiple comparison test, ***P <0.001.

Table 2.

Chromium concentrations in the studied subjects

Parameters	Control (mean±SD)	Non-Occupationally exposed (mean±SD)	Occupationally exposed (mean±SD)	P
Cr Concentration
Smoker	14.86±6.42	54.74±4.74	61.30±10.00	<0.001*
Non-smoker	15.60±5.13	50.77±4.81	70.81±15.81
Alcoholic	15.70±5.94	53.59±4.21	69.05±16.40	<0.001*
Non-alcoholic	15.47±5.26	50.86±5.01	69.49±15.21

ANOVA-analysis of variance, *Significant

In our study, the MDA concentration in the occupationally exposed group (9.72 ± 0.98) and non-occupationally exposed group (8.49 ± 0.93) was significantly greater [Figure 2] than that in the control group (6.39 ± 0.68).

Figure 2.

Malondialdehyde (MDA) concentration in occupationally, non-occupationally exposed, and control subjects. Each experiment was conducted in triplicate. Data are presented as the mean ± SD from 100 subjects of each group. Statistics: Tukey’s multiple comparison test, ***P <0.001

The GSH levels were quantified in all groups [Figure 3]. There was a significant (P < 0.001) decrease in the activity of GSH in the occupationally exposed group (50.69 ± 3.54) and the non-occupationally exposed group (62.73 ± 6.63) compared with the control group (73.72 ± 9.09).

Figure 3.

Reduced glutathione (GSH) concentration in exposed, moderate, and control subjects. Each experiment was conducted in triplicate. Data are presented as the mean ± SD from 100 subjects of each group. Statistics: Tukey’s multiple comparison test, ***P <0.001

The SOD activity in occupationally exposed subjects (102.4 ± 8.34) and non-occupationally exposed subjects (89.95 ± 4.41) was significantly greater [Figure 4] than that in control subjects (70.35 ± 8.34).

Figure 4.

Superoxide dismutase (SOD) activity in occupationally, non-occupationally exposed, and control subjects. Each experiment was conducted in triplicate. Data are presented as the mean ± SD from 100 subjects of each group. Statistics: Tukey’s multiple comparison test, ***P <0.001

DNA damage in exposed groups showed significantly (P < 0.001) higher comet tail length in occupationally exposed (24.81 ± 3.52 μm) and non-occupationally exposed subjects (14.45 ± 2.91 μm) when compared with controls (5.28 ± 2.05 μm) [Figure 5a].

Figure 5.

Comet assay detecting DNA damage in occupationally, non-occupationally exposed, and control subjects. (a) Comet tail length and (b) alkaline comet assay. Data are presented as the mean ± SD from 100 subjects of each group. Statistics: Tukey’s multiple comparison test, ***P <0.001

The univariate analysis model [Table 3] showed no significant relationship between MDA, GSH, SOD concentrations, and DNA damage with drinking or smoking behavior. Similarly, linear regression analysis of smoking and alcohol consumption on the MDA, GSH, SOD concentrations, and DNA damage were observed with no significant (P > 0.05) effect [Table 4].

Table 3.

Effects of smoking and alcohol consumption on genotoxic and oxidative stress parameters in controls and in nonoccupationally and occupationally exposed subjects

Parameters	Control (mean±SD)	Nonoccupationally exposed (mean±SD)	Occupationally exposed (mean±SD)	P
DNA damage
Smoker	5.34±1.98	15.29±2.81	24.07±2.22	0.19
Non-smoker	5.27±2.07	14.32±2.92	24.94±3.70
Alcoholic	4.31±1.71	16.09±2.52	25.25±3.15	0.52
Non-alcoholic	5.41±2.06	14.12±2.89	24.67±3.65
MDA
Smoker	6.03±0.60	8.38±0.82	10.15±1.04	0.06
Non-smoker	6.44±0.69	8.51±0.95	9.65±0.96
Alcoholic	6.30±0.82	8.31±0.79	9.87±0.83	0.18
Non-alcoholic	6.40±0.68	8.50±0.93	9.77±0.88
GSH
Smoker	72.54±12.16	60.43±5.86	51.02±2.71	0.35
Non-smoker	73.92±8.51	63.11±6.71	50.63±3.68
Alcoholic	71.53±10.89	63.01±5.87	48.90±3.02	0.62
Non-alcoholic	74.01±8.85	62.68±6.81	51.29±3.52
SOD
Smoker	67.68±4.91	88.67±3.45	102.0±6.95	0.23
Non-smoker	70.82±8.74	90.15±4.53	102.5±8.60
Alcoholic	68.87±5.58	89.18±4.42	102.2±9.10	0.78
Non-alcoholic	70.55±8.65	90.10±4.42	102.5±8.14

Univariate analysis of variance

Table 4.

Joint effect of smoking and alcohol consumption on DNA damage and oxidative stress parameters according to multivariate linear regression analysis

Parameters	Occupationally exposed Beta coefficient, P		Nonoccupationally exposed Beta coefficient, P
	Smoker	Alcoholic	Smoker	Alcoholic
DNA damage	1.56, 0.18	0.33, 0.80	3.94, 0.05	-7.11, 0.09
GSH	-0.31, 0.93	2.49, 0.57	-4.55, 0.08	2.64, 0.09
MDA	0.52, 0.21	-0.50, 0.31	0.84, 0.06	-1.20, 005
SOD	4.94, 0.06	-4.49, 0.14	7.12, 0.06	-1.51, 0.73

DISCUSSION

The Cr produced by the leather industry is in the form of Cr(III) because chromium sulfate is used as a tanning agent.[21] Earlier studies by Katiyar et al.,[22] Katiyar et al.,[23] Khan et al.,[24] and Ambreen et al.[25] reported that blood Cr concentrations (96.60 ± 113.95 mg/dL, 104.65 ± 77.21 μg/dL, and 157.59 ± 29.20 μg/L, respectively) in tannery workers were much greater than those in our study in Jajmau, Kanpur District, North India. Our findings were concordant with those of Qureshi et al.[26] and Zhang et al.,[27] who reported higher Cr concentrations (42.35 and 25.40 μg/L, respectively) in tannery workers in Pakistan and China. MDA is produced naturally by lipid peroxidation and the biosynthesis of prostaglandins. A high MDA concentration suggests a high oxidative stress rate in exposed populations. Our findings are in line with recent reports by Xu et al.[28] and Mozafari et al.[29] who reported higher MDA levels in the occupationally exposed group (3.62 ± 0.06 nmol mL^-1 and median: 3.54 μmol/L) in North China and Iran, respectively. Similarly, Khan et al.[24] and Ambreen et al.[25] observed increased MDA concentrations in occupationally exposed tannery workers at the same study site. The research by Khan et al.[24] and Ambreen et al.[25] showed a favorable association between MDA and Cr content following changes in drinking and smoking patterns. However, Xu et al.[28] reported no effect of drinking or smoking on the MDA concentration, which is consistent with our findings. The main reason for this lack of association is the considerable impact of Cr, which accounts for the effects of alcohol consumption and smoking on MDA concentration. Previous research has revealed several variations in MDA concentrations between men and women. In Cr(III)-exposed populations, women have a higher MDA concentration than men. In normal populations, however, women have lower MDA concentrations than men,[30] which is consistent with our study.

Our results coincided with those of Qureshi et al.,[26] Elhosary et al.,[31] Ambreen et al.,[25] and Khan et al.,[24] who reported that occupationally exposed groups showed a significantly lower concentration of GSH in the control group. Thus, the decrease in GSH can result from Cr-induced toxicity. In this study, levels of SOD were significantly higher in the exposed groups. Our finding is in line with earlier studies by Khan et al.,[24] Ambreen et al.,[25] and Junaid et al.[32] Conversely, certain studies[28,33] have shown that occupationally exposed workers have lower SOD activity. Decreased SOD activity in the serum may alter antioxidant defense mechanisms.

Our study reported significantly higher tail length in exposed groups than in control. The amount of tail damage to the DNA was 6-fold greater than that in a previous report.[34] The study revealed that occupationally exposed workers had considerably greater tail length than workers in the non-occupationally exposed and control groups [Figure 5b]. According to Xu et al.,[28] Zhang et al.,[35] and Bagchi et al.,[36] exposed individuals exhibit significantly greater oxidative DNA damage levels in urine than unexposed individuals. This finding is consistent with earlier research on occupational exposure, which revealed that exposure to Cr(VI) promotes DNA damage. Zhang et al.[27] reported substantially more significant tail DNA damage in occupationally exposed workers than in control workers, indicating that Cr(III) causes high genetic damage in tannery workers. Prolonged occupational exposure to Cr(III), as reported by Medeiros et al.,[37] causes tannery workers to have significantly greater levels of DNA‒protein crosslinks and micronuclei than controls.

According to some studies, smoking is linked to a variety of cancers.[38] Setyaningsih et al.[39] reported that alcohol consumption and smoking increase oxidative DNA damage in electroplating workers exposed to Cr. Xu et al.[28] reported that smokers and drinkers in both the Cr-exposed and unexposed groups had higher levels of 8-OHdG than people who did not smoke or drink. We also studied the effect of smoking and drinking on genotoxic and oxidative stress parameters in the control and exposed groups. However, there was no clear link between smoking and drinking in terms of genotoxicity and oxidative stress.

CONCLUSION

The current investigation indicated that the concentration of Cr in both the Cr-exposed groups increased with exposure duration. We also found that the Cr-exposed groups had elevated levels of oxidative stress and DNA damage, suggesting a correlation with Cr concentrations. Occupationally exposed workers experience Cr exposure higher concentrations of Cr over longer periods. This research raises awareness of the dangers of Cr to tannery workers, who need to be provided with safe working conditions or encouraged to switch to a different tanning procedure. Primarily, the tannery owners are responsible for safeguarding the health of their employees as the workers are potentially at risk of not just chemical toxicants but also many psychosocial, biological, and physical hazards. Early intervention could be education regarding the harmful effects on the workers coming in contact with various chemicals and the use of protective measures. Authorities on a government level, NGO personnel, employers, and employees themselves should advocate for the proper implementation of “the Occupational Safety, Health, and Working Conditions Code, 2020” under the labor law of India[40] and primary healthcare facilities for them. Another measure could be the use of PPE while handling the chemicals. Time-to-time health monitoring and health education are also required for workers. Education regarding safe work practices and hygiene before eating should also be practiced.

This industry needs to be sustainable by following the government guidelines. Tanneries are stringently instructed to regulate and treat all kinds of waste and energy consumption to limit its harm to the environment. Also, the industry should prioritize the use of syntans (eco-friendly synthetic tanning salts) and environment-friendly raw materials for leather production, which is of utmost importance in mitigating health risks and minimizing exposure. The case of small and medium-scale tanneries in the area of Kanpur-Unnao, Uttar Pradesh are proving to be landmarks of sustainable and responsible practices such as physio-remediation of wastewater, recycling and recirculation of lime water produced while tanning, aiming to zero waste discharge using electro-oxidation, using environment-friendly salt-free tanning technology, and sulfide-enzyme based unhairing. Lastly, this industry needs a major modern transformation in terms of raw materials, methods of tanning, amount of water consumption, and other safety measures to reduce environmental pollution.

This industry is indeed very large and gives extensive employment and thus it becomes of utmost importance to train and educate both the employees and the employer to work safely and regarding the use of chemicals.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.