Biochemical study on occupational inhalation of benzene vapours in petrol station.

inhalation of benzene vapours promote various and dangerous health problems. Fuel station workers are most susceptible to benzene inhalation toxicity. Samples were collected twice, at beginning of the study and 6 months later from 40 fuel station workers from different egyptian governorates and 10 control healthy volunteers. Fuel station workers were sub divided into four groups according to years working in the station. All of them are exposed to benzene vapours and exhausts during their duties, their work shifts were 8 hrs./day. Results indicated that; benzene vapours exposure induced significant increasing in serum Lead and Cadmium and Myeloperoxidase (MPO) enzyme activity levels. This goes with marked immunologic changes presented by decreases in immunoglobulins; IgA and IgG, along with increases in levels of IgM and IgE. Also, Malondialdehyde (MDA) levels were significantly increased. Meanwhile, reduction in some other biochemical parameters including; Copper, Zinc and Iron micronutrients, as well as; Superoxide Dismutase (SOD), Catalase (CAT) enzyme levels and Glutathione (GSH) content. Hence, the study inferred that prolonged benzene inhalation can lead to biochemical and immune disorders, probably through potentiating oxidative stress and inflammation pathways.

Introduction

Air pollution was increased with urbanization and rapid increasing number of automobiles in most of the towns and cities, Numerous epidemiological studies have documented decrements in pulmonary function and various other health problems associated with long term air pollution exposure [1].

Benzene long term exposures can give rise to effects that include haematotoxicity, genotoxicity, immunological and reproductive effects as well as various cancers [2].

Benzene is a volatile compound in urban air pollution induce DNA oxidation [3]. Moreover, iron and other transition metals leaching from particles or by their presence on particle surfaces play a role in the generation of ROS in biological systems [4]. when oxidative stress is relatively low, various transcription factors, such as the nuclear factor erythroid-2 (Nrf 2), induce a series of antioxidant and detoxification enzymes (e.g., CAT, SOD, GST that counteract ROS formation protecting from adverse biological outcomes [5].

The volatile nature of petrol makes it readily available in the atmosphere any time it is dispensed, especially at petrol filling stations and depots. People are exposed to gasoline fumes during fuelling and refueling at gas stations, but the gas station attendants are more at risk by virtue of their occupational exposure [6]. So, spotting on fuel station workers who are chronically exposed to gasoline seemed necessary, Workers were mostly suffering neurological symptoms including; headache, tiredness, irritability and disturbed sleeping, as recorded in the utilized questionnaires and remarked by Ref. [7].

Materials and methods

The study was carried out on 40 fuel station workers from different egyptian governorates and 10 control healthy volunteers after taking consent of them. Fuel station workers are exposed to benzene vapours and exhausts during their duties, their work shifts were 8 hrs./day and sub divided into four groups according to years working in the station. subgroup 1: Consists of 10 workers working in petrol station for 2 years. subgroup 2: Consists of 10 workers working in petrol station for 4 years. subgroup 3: Contains 10 workers working in petrol station for 6 years. subgroup 4: Compromise 10 workers working in petrol station more than 6 years. All attendants were subjected to medical and occupational history including age, marital status, respiratory and chronic diseases, history of operation, drug intake, duration of employment, location and use of protective equipment. Any individual with history of diabetes, renal or hepatic disease and atrophy or allergy was excluded.

Blood samples were collected twice, at beginning of study and 6 months later by withdrawing of ten cubic centimeter of venous blood through a vein puncture using sterile vacutainer tubes. Half of the blood samples taken in heparin tubes (green caped tubes). The other taken to plain tubes (red caped tubes) which allow to coagulate then centrifuge for 10 minutes at 4000 r.p.m for getting clear and non hemolyzed sera aspirated carefully by Pasteur automatic pipette and transferred into dry and sterile labeled vials.

The sera were subjected to estimation of the following parameters; Copper, Zinc, Iron, Cadmium, and Lead by using 5 pg atomic absorption spectra-photometry (Pye Unicum), according the methods, described by Wilse, [8] and Bauer [9]. as well as Myeloperoxidse enzyme activity (Myeloperoxidase ELISA kit, Kamiya Biomedical Company) and total protein immunoglobulines; IgM, IgG, IgA [10], IgE [11] and nitric oxide [12].

Heparin 0.5% tubes used for preparation of hemolysate by Digitonine after washing erythrocytes by physiological saline as described by Korrnburg and Korecker [13] This hemolysate was subjected for quantitative determination of erythrocyte catalase [14,15], total super oxide dismutase [16], reduced glutathione [17], glutathione peroxidase [18] and malondialdhyde [19].

Statistical analysis: Data analysis was expressed as mean ± S.E. and analyzed for statistical significance by one-way ANOVA followed by Tukey's post-hoc test for multiple comparisons, using SPSS program for Windows version 22.0 (SPSS Inc., Chicago, USA). Values were considered statistically significant at P < 0.05 correlations between the measured variables were assessed by linear regression analysis by the least squares method.

Results

Results obtained in Table 1 showed a significant decrease in serum Copper, Zinc and Iron levels (P < 0.05) in fuel station workers exposed to benzene vapours for 2 and 4 years and highly significant reductions in Cu, Zn after 6 and more than 6 years of exposure when compared to control. This significant decrease was still noticeable in the 2nd sample after 6 months of the first one. Also, a highly significant decrease was found in serum immunoglobulines IgG and IgA levels at (P < 0.05) in spite of the increase in of IgM and IgE and serum Lead and Cadmium levels in workers exposed to benzene vapours for 2 and 4 years and highly significant increase after 6 and more than 6 years of exposure when compared to control, these significant changes were persisted in the 2nd sample. Moreover, serum nitric oxide showed a significant decrease at (P < 0.05) in workers exposed to benzene vapours for 2 and 4 years and highly significant decrease after 6 and more than 6 years of exposure in comparison with control and also, NO levels were still decreased significantly in 2nd sample after 6 months. Serum MPO levels showed a significant increasing in workers exposed to benzene vapours for 2 and 4 years and highly significant increasing (P < 0.05) after 6 and more than 6 years of exposure when compared to control in the first and second samples.

Table 1

Biochemical effect of benzene vapour on some serum biochemical parameters in Fuel station workers after 2, 4, 6 and more than 6 years of exposure.

IgEIU/ml	IgA mg/dl	IgG mg/dl	IgM mg/dl	MPO ng/ml	NOμmol/l	Cdμg/dl	Pbμg/dl	Feμg/dl	Znμg/dl	Cuμg/dl	Parameters & Groups
28.28 ± 0.74e	95.66 ± 1.31a	763.70 ± 3.71a	95.00 ± 0.87e	20.32 ± 1.51e	58.68 ± 0.94a	26.19 ± 1.19e	27.62 ± 1.01e	110.22 ± 5.62a	81.29 ± 2.76a	78.60 ± 1.72a	1st sample	Control normal
31.08 ± 0.79e	92.46 ± 1.23a	752.80 ± 3.57a	96.21 ± 0.91e	22.13 ± 1.44e	56.20 ± 1.14a	27.34 ± 1.03e	28.99 ± 0.94e	106.72 ± 4.35a	87.47 ± 2.55a	86.79 ± 1.76a	2nd sample
46.94 ± 1.10d	83.03 ± 1.35b	653.80 ± 3.14b	118.34 ± 0.92d	26.89 ± 1.06d	53.06 ± 1.77b	28.91 ± 2.26d	33.43 ± 2.64d	68.73 ± 1.13c	69.34 ± 1.83b	70.82 ± 2.17b	1st sample	2 years working	Fuel station workers
52.23 ± 1.06d	77.42 ± 1.32b	620.30 ± 3.65b	124.90 ± 1.22d	33.61 ± 1.13d	45.76 ± 1.83b	31.12 ± 1.95d	35.93 ± 2.41d	62.83 ± 1.36d	65.44 ± 2.51b	68.81 ± 2.25b	2nd sample
66.57 ± 0.90c	74.67 ± 0.87c	600.80 ± 2.24c	139.50 ± 1.24c	39.55 ± 0.93c	40.63 ± 1.96c	36.62 ± 1.76c	47.23 ± 2.98c	56.07 ± 2.04e	61.48 ± 2.67c	63.77 ± 2.59c	1st sample	4years working
73.63 ± 1.00c	62.87 ± 1.08c	564.50 ± 2.56c	158.18 ± 1.65c	46.36 ± 1.15c	33.86 ± 2.12c	41.30 ± 2.65c	53.21 ± 1.25c	51.12 ± 2.76e	57.08 ± 2.58c	58.41 ± 1.96c	2nd sample
81.09 ± 1.04b	62.51 ± 1.14d	537.40 ± 2.11d	168.54 ± 1.82b	49.02 ± 1.49b	30.55 ± 2.00d	44.91 ± 2.17b	57.02 ± 1.37b	63.68 ± 1.51d	49.88 ± 2.86d	51.77 ± 1.94d	1st sample	6 years working
90.22 ± 1.04b	55.06 ± 1.07d	511.40 ± 2.31d	177.84 ± 1.55b	53.19 ± 0.88b	26.65 ± 2.56d	49.89 ± 2.25b	61.94 ± 1.67b	71.29 ± 2.02c	42.07 ± 2.02d	48.32 ± 1.43d	2nd sample
102.55 ± 1.93a	45.67 ± 1.35e	486.40 ± 3.04e	187.30 ± 1.20a	59.20 ± 1.03a	23.14 ± 1.96e	53.94 ± 3.63a	66.51 ± 1.22a	74.59 ± 1.96b	39.27 ± 2.96e	40.22 ± 1.09e	1st sample	More than 6 years working
123.67 ± 1.55a	36.38 ± 1.06e	463.90 ± 2.35e	195.94 ± 1.42a	65.45 ± 1.04a	19.80 ± 1.98e	59.12 ± 3.41a	72.42 ± 1.41a	78.98 ± 1.53b	35.47 ± 2.85e	37.07 ± 1.11e	2nd sample

Data shown are mean ± standard deviation of number of observations within each group. Mean values with different superscript letters in the same column are significantly different at (P < 0.05).Small letters are used for comparison between the means within the column.

The data recorded in Table 2 showed; a significant decrease in RBCs SOD, CAT, GPx and GSH levels (P < 0.05) in fuel station workers exposed to benzene vapours for 2 and 4 years and highly significant reduction in RBCs SOD, CAT, GPx and GSH levels after 6 and more than 6 years of exposure when compared to control. This significant decreasing still registered in the 2nd sample after 6 months from first sample. Despite of increasing of Lipid peroxidation, MDA levels in workers exposed to benzene vapours for 2 and 4 years and highly significant increase after 6 and more than 6 years of exposure when compared to control and also, MDA levels were still increased significantly in 2nd sample.

Table 2

Biochemical effect of benzene vapours on RBCs' lipid peroxidation and antioxidant parameters in Fuel station workers after 2, 4, 6 and more than 6 years of exposure.

GPx mU/mL	CATU/L	SODU/g. Hb	GSH mg/dl	MDA nmol/g. Hb	Parameters & Groups
121.92 ± 1.33a	135.25 ± 3.41a	96.55 ± 1.84a	72.90 ± 1.34a	46.42 ± 1.36e	1st sample	Control normal
118.11 ± 1.22a	130.26 ± 3.30a	92.41 ± 1.05a	69.23 ± 1.45a	43.52 ± 1.28e	2nd sample
105.93 ± 1.09b	122.85 ± 3.12b	80.09 ± 1.66b	49.94 ± 0.78b	53.32 ± 1.30d	1st sample	2 years working	Fuel station workers
100.04 ± 0.98b	115.92 ± 2.02b	71.12 ± 1.81b	41.81 ± 0.96b	58.12 ± 1.39d	2nd sample
94.28 ± 1.77c	109.11 ± 3.44c	76.58 ± 1.83c	42.65 ± 1.04c	64.84 ± 1.28c	1st sample	4years working
88.48 ± 0.97c	98.06 ± 1.95c	68.41 ± 1.64c	36.41 ± 0.92c	74.02 ± 1.24c	2nd sample
83.28 ± 1.09d	97.52 ± 1.96d	65.28 ± 1.90d	34.92 ± 1.05d	75.32 ± 1.15b	1st sample	6 years working
77.50 ± 1.10d	91.02 ± 1.84d	59.08 ± 1.92d	29.12 ± 0.83d	81.72 ± 1.39b	2nd sample
76.78 ± 1.05e	88.92 ± 1.08e	60.02 ± 1.76e	28.91 ± 1.26e	80.82 ± 1.34a	1st sample	More than 6 years working
70.48 ± 0.84e	83.02 ± 1.04e	53.15 ± 1.91e	24.52 ± 1.01e	88.30 ± 1.48a	2nd sample

Data shown are mean ± standard deviation of number of observations within each group. Mean values with different superscript letters in the same column are significantly different at (P < 0.05).Small letters are used for comparison between the means within the column.

Discussion

Risk of benzene exposure is mostly related to four main components; benzene, toluene, ethylene and xylene that known as BTEX [20]. However, benzene was found to be the most hazardous component due to mutagenic and carcinogenic effects of its metabolites [22]. Spotting on fuel station workers who are chronically exposed to benzene vapours seemed necessary, as properly achieved in the present study.

Moderate and long term exposures to benzene which carry the risk of numerous health problems including inorganic elements in such hazards. Our results was in agreement with [23] who found that, all of the accumulated metals are proposed to contribute to oxidative stress by different mechanisms. The underlying mechanisms of their toxicity involve formation of the superoxide radicals, hydroxyl radicals and other reactive oxygen species (ROS). Increased formation of ROS overwhelms body antioxidant protection [24], which may be the underlying cause of the decreased antioxidant activity observed in the present study, leading to possible induction of numerous conditions detrimental to health.

Results obtained in this study showed significant decrease in copper and zinc metal levels in comparison to control group. This came in agreement with Mona et al. [25], who reported that; both trace elements copper and zinc were significantly lower compared to peoples that are not exposed to fuels. These results are consistence with [21] who reported that; long to moderate benzene exposed individuals under oxidative stress due to decreased levels of antioxidants, including copper and zinc, in the plasma and red blood cells. In this study, copper and zinc were reduced by 51% and 56%, respectively, than lower limit of the accepted reference intervals and amounted 63% and 64% decrease to control individuals. Zinc and Copper are essential antioxidants in the human body [26]. These trace elements are essential component of many enzymes and proteins involved in protection against oxidative stress damage. Zinc deficiency associated with reduced immunity [27], DNA damage and increases other metals induced oxidative toxicity [28]. Copper is essential micronutrients for normal growth and protection of many organs and electron transport [29]. Copper deficiency increases risk of cellular oxidative damage due to decreased activity of superoxide dismutase (SOD) [30]). The balance between antioxidant, such as copper and zinc, and rate of free radicals levels are essential factor to prevent organs and tissue from oxidative damages [31]. The deficiency of copper and zinc antioxidants observed in petroleum fuel stations workers indicates the increasing of oxidative stress in such populations showed in previous results of our study.

The most risky toxic metal those lead which can cause abnormal alterations in the functioning of many vital organs [32] and associated with increased risk of hematological malignancies. Unfortunately, the majority of the workers are neglecting or lacking the protective safety measures such as; facemasks, protective cloths, this carelessness makes them more susceptible to those toxic fumes. Lead exposure may increase the susceptibility of membranes by altering their integrity via causing deterioration of their components [33]. Cadmium metal was found to be elevated in the blood benzene exposed individuals. Increase in lipid peroxidation represented by increased MDA level, has been observed in experimental animals treated with cadmium [34]. While cadmium itself is unable to generate free radicals directly, indirect generation of various radicals has been reported. Such mechanism involves displacement of other redox-active metals from their binding sites thus increasing their free form and enhancing their capability of producing free radicals [35]. found that; Fuel station workers who have exposed to benzene showed highly significant reductions in total antioxidant capacity (TAC) activities and high significant increased plasma MDA levels [36]. Malondialdehyde (MDA) is one of the end-products of the peroxidation of membrane lipids caused by ROS formation, especially by the superoxide ion. This is in agreement with other studies which illustrated that Benzene exposure has been associated with increases in the overall formation of MDA [37].

MPO is one of the most promising biomarkers of oxidative stress for clinical cardiologists [38]. Elevated circulating MPO levels have been found to be associated with the presence of CAD. In our present study, high MPO levels were able to predict increased risk of developing CAD in benzene vapours exposed workers, this was in agreement with Meuwese et al., [39] who stated that; high MPO levels were able to predict increased risk of developing coronary artery diseases (CAD) in healthy individuals. Most of attention was directed toward gasoline related immunotoxicity through decreasing number of immunoglobulins (IgA, IgG) which are often measured to give information about immune system homeostasis [40]. Current study recorded remarkable decreases in IgA and IgG levels with significantly elevated levels of serum IgM and IgE in fuel stations individuals compared to the unexposed ones. It was found that people with primary immunodeficiency have decreased levels of serum IgG and IgA and normal or elevated levels of IgM [41]. Decreased levels of immunoglobulins in gasoline exposed workers were explained to either suppression of immunoglobuline producing cells or decreased cell mediated immunity [3].

Conclusion

By the end of this study we concluded that, benzene vapours inhalation lead to potential biochemical and immunological disorders appeared as elevation of serum Lead, Cadmium, (MPO) and (MDA) levels. Also, markedly decreases in IgA and IgG, along with increases in levels of IgM and IgE. Meanwhile, reduction in some other biochemical parameters including; Copper, Zinc and Iron, (SOD), (CAT) enzyme levels and glutathione (GSH) content. This probably through potentiating oxidative stress and inflammatory pathways.