Maksoud A. H. A, Mahfouz M. K, Omnia M. A, Abdullah M. H, Eltabey M. E, Elharrif M. G. Biochemical Changes Followed Experimental Respiratory Distress by Benzene Vapours. Biomed Pharmacol J 2019;12(1).
Manuscript received on :9-Oct-2018
Manuscript accepted on :24-Nov-2018
Published online on: 05-03-2019
Plagiarism Check: Yes
Reviewed by: Sadegh Rajabi
Second Review by: Shaimaa Mutlak
Final Approval by: Dr. Ian James Martin

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Biochemical Changes Followed Experimental Respiratory Distress by Benzene Vapours

Abdel Maksoud H. A1, Mahfouz M. K1, Omnia M. A1, Abdullah M. H2, Eltabey M. E1 and Mohamed G. Elharrif3  

1Department of Biochemisry, Benha University, Egypt.

2Department of Biochemisry, 6th October University, Egypt.

3Department of Basic Medical Sciences, Shaqra university, KSA, Kingdom of Saudi Arabia.

Corresponding Author E-mail: al_harrif@yahoo.com

Abstract:

Monitoring of exposure to chemical matters is seriously needed  for evaluating health hazards resulted from its inhalation. The present study was carried out to determine the biochemical, immunological and oxidative stress parameters as well as the possible histological effects of exposure to benzene vapours in male albino rats. Results indicated that; Benzene vapours exposure  induced significant increasing in Myeloperoxidase (MPO) enzyme 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 different biochemical parameters including; Superoxide Dismutase  (SOD), Catalase (CAT) levels and Glutathione (GSH) content. Lung sections taken showed; Thickening of alveolar septa with chronic inflammation and /or fibrosis, Congested vessels/thick walled vessels and Peri-bronchiolar fibrosis. Hence, the study concluded that; prolonged benzene (BNZ) vapours exposure lead to biochemical, immune disterbance and histopathological changes  probably through potentiating oxidative stress and inflammation pathways.

Keywords:

Benzene Exposure; Inflammatory Pathways; Oxidative Stress

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Maksoud A. H. A, Mahfouz M. K, Omnia M. A, Abdullah M. H, Eltabey M. E, Elharrif M. G. Biochemical Changes Followed Experimental Respiratory Distress by Benzene Vapours. Biomed Pharmacol J 2019;12(1).

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Maksoud A. H. A, Mahfouz M. K, Omnia M. A, Abdullah M. H, Eltabey M. E, Elharrif M. G. Biochemical Changes Followed Experimental Respiratory Distress by Benzene Vapours. Biomed Pharmacol J 2019;12(1). Available from: https://bit.ly/2ENidfM

Introduction

Benzene is an aromatic hydrocarbon; toxic to the blood forming organs. The severity of the damage depends on the level, timing and pattern of exposure. This volatile compound in urban air pollution induces DNA oxidation (Uzma et al., 2010). Chronic benzene vapours exposure leads to decrease in antioxidant enzymes activity and hematologic disorders. Oxidative stress produces reactive oxygen species (ROS) in excess of the available antioxidant buffering capacity (Adly., 2010). 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 (Ghio et al., 2000).

Metals and other inorganic elements play important roles in a wide variety of biological processes of living systems. Several essential transition elements, such as zinc, magnesium, iron, copper, cobalt and manganese participate in the control of various metabolic and signaling pathways. However, their rich coordination chemistry and redox properties are such that they are capable of escaping out of the control mechanisms, such as homeostasis, transport, compartmentalization, and binding to the designated tissue and cell constituents. Breakdown of these mechanisms, caused by stimuli such as benzene exposure has been involved in a large variety of diseases (Jomova and Valko, 2011). Disruption of metal homeostasis is known to modulate gene expression by interfering with signal transduction pathways. This action may lead to uncontrolled metal-mediated formation of free radicals participating in the modification of DNA bases, enhanced lipid peroxidation and altered sulfhydryl homeostasis (Valko et al., 2007).

Repeated exposure to kerosene and petrol fumes causes degenerative changes in the ultra structural integrity of the hepatic cells which may impair the normal liver functions  (Uboh et al., 2005).

Materials and Methods

Forty male albino rats, 12 weeks old were taken, and divided into three main groups; the first kept as a negative control group (Un-exposed), the second consisted of 15 rats subjected to benzene  vapors for 8 hours daily for 3 months and the third contained also 15 rats exposed to benzene vapours for 8 hours daily for 6 months. All rats were sacrificed and their lungs were immediately collected for analysis and placed in formalin solution 10.0 % to reserve organ cells for histopathological examination. All animal experiments were approved by the egyptian research institute.

Data Collection and Estimated Parameters

After 90 days of exposure to benzene vapours, blood samples will be collected from rats of each group. The samples will be taken in the early morning after overnight fasting from the medial conthus of the eyes by using the heparinized microhematocrite tubes to heparin and plain centrifuge tubes, then animals sacrificed. Plain tubes then allowed to be coagulate at room temperature for 30 minutes. The serum will be separated in dry sterile tube then kept in deep freeze until using for subsequent biochemical assays.

The sera were subjected to estimation of copper, zinc, iron, manganese, and lead by atomic absorption spectra-photometry (Pye Unicum), according the methods, described by Wilse, (1960) and Bauer (1982) as well as total protein immunoglobulines (IgM, IgG, IgA) (Whicker et al., 1984), IgE (Plebani et al., 1998) and nitric oxide. (Montgomery and Dymock, 1961) as well as Myeloperoxidse Enzyme activity (ELISA kit -Kamiya Biomedical Co.).

The remaining amount of blood were taken into clean dry tube containing heparin 0.5% and used for preparation of hemolysate by Digitonine after washing erythrocytes by physiological saline as described by Korrnburg and Korecker (1955). This hemolysate was subjected for quantitative determination of erythrocyte (CAT) (Aebi, 1984) and (Fossati et al., 1980), (SOD) (Nishikimi et al., 1972),, (GSH) (Beutler et al., 1963),(GPX) (Paglia and Valentine, 1967)  and (MDA) (Satoh, 1978).

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 (P<0.05) in serum Copper, Zinc and iron levels in rats exposed to benzene vapours for three months and highly significant decrease (P<0.05) in Cu, Zn after six months of exposure when compared to control. Also, a highly significant decrease was noticed in serum immunoglobulines IgG and IgA levels at (P<0.05) in spite of increasing of serum lead and cadmium metals, IgM and IgE in rats exposed to benzene vapours for three months and highly significant increase after six months of exposure when compared to control. Moreover, serum Nitric Oxide showed a significant decrease at (P<0.05) in rats exposed to benzene vapours for three months and highly significant decrease after six months of exposure in comparison with control. Serum MPO levels showed a significant increasing in rats exposed to benzene vapours for three months and highly significant increasing (P<0.05) after six months of exposure when compared to control.

The data recorded in table (2) showed; a significant reductions in RBCs SOD, CAT, GPx and GSH levels (P<0.05) in rats exposed to benzene vapours for three months and highly significant reduction in RBCs SOD, CAT, GPx and GSH levels after six months of exposure when compared to control. in spite of increasing of Lipid peroxidation, MDA levels in rats exposed to benzene vapours for three months and highly significant increase after six months of exposure when compared to control.

Table 1: Biochemical effect of benzene vapor on some serum biochemical parameters in male rats after 3 and 6 months of exposure.

Parameters Cu Zn Fe Pb Cd NO MPO IgM IgG IgA IgE
Groups µg/dl µg/dl µg/dl µg/dl µg/dl µmol/l ng/ml mg/dl mg/dl mg/dl IU/ml
Control 58.82 ± 1.77a 42.25 ± 1.19a 92.34 ± 1.79a 6.58 ± 0.38c 1.61 ± 0.12c 4.43 ± 0.21a 7.72 ± 0.49c 54.62 ± 1.85c 494.30 ± 3.35a 27.07 ± 0.91a 2.99 ± 0.07c
normal
Exposed group After 44.89 ± 1.16b 31.73 ± 0.97b 53.97 ± 2.16c 8.97 ± 0.30b 1.90 ± 0.10b 2.59 ± 0.16b 10.44 ± 0.46b 76.72 ± 2.69b 425.20 ± 3.52b 18.03 ± 0.94b 8.32 ± 0.35b
 3 months
After 36.27 ± 1.01c 24.88 ± 1.06c 59.92 ± 2.08b 10.61 ± 0.34a 2.57 ± 0.15a 2.07 ± 0.12c 15.62 ± 0.38a 95.56 ± 2.83a 398.40 ± 3.61c 10.88 ± 0.77c 14.43 ± 0.54a
6 months

 

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.

Table 2: Biochemical effect of benzene vapor on erythrocyte MDA, GSH, SOD, CAT and GPx in male rats after 3 and 6 months of exposure.

Parameter Groups MDA nmol/g. Hb GSH mg/dl SOD U/g. Hb CAT U/L GPx mU/ml
Control normal 8.22 ± 0.41c 29.61 ± 0.75a 16.36 ± 0.81a 494.40 ± 3.63a 40.55 ± 1.07a
Exposed groups After  3 – months 14.40 ± 0.76b 16.71 ± 0.56b 11.14 ± 0.86b 446.90 ± 2.52b 28.92 ± 0.91b
After  6 – months 20.95 ± 1.06a 11.19 ± 0.63c 6.98 ± 0.72c 407.60 ± 2.45c 20.53 ± 0.83c

 

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.

Histopathological Results of Lung Section

Table 3

Patterns of injury Score
Lung architecture 0 (Preserved) 1(Disturbed)
Thickening of alveolar septa with chronic inflammation and /or fibrosis 0 (None)

1 (Mild)

2 (Moderate)

3 (Severe)

Congested/thickened wall vessels
Peri-bronchiolar inflammation and fibrosis

 

Table 4

Parameter Groups Lung architecture Thickening of alveolar septa with chronic inflammation and /or fibrosis Congested vessels/Thick walled vessels Peri-bronchiolar fibrosis
Control 0 0 0 0
3 months exposure 0 2 2 0
6 months exposure 1 3 2 3

 

Figure 1 Figure 1

 

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Figure 2 Figure 2

 

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Discussion

Monitoring of exposure to chemical matters is seriously needed for evaluating health hazards and providing suitable strategies for making safe work environment. Among the most toxic chemicals is gasoline and its most constituent, benzene that is strongly and causally related to wide spread of health problems (Ibitoroko et al., 2011).

The disturbance in many inorganic elements are essential for a multitude of biological processes and their homeostasis, which is maintained within strict limits, is critical for life (Valco, et al., 2005). Disruption of such homeostasis may lead to oxidative stress. The generation of free radicals in living systems is closely linked with the participation of redox active metals which undergo redox cycling reactions and possess the ability to produce reactive radicals in biological systems. Some redox-active metals including cadmium and lead may increase the susceptibility of membranes by altering their integrity via causing deterioration of their components (Gurer and Ercal, 2000).

Elevated cadmium level and increased lipid peroxidation represented by increased MDA level in benzene vapour exposed rats were found in  this study came in accordance with (Eybl et al., 2006) who reported that, cadmium itself is unable to generate free radicals directly. Copper deficiency alters the role of cellular constituents involved in antioxidant activities, such as iron, selenium and glutathione, so increased cellular susceptibility to oxidative damage and leads to decreased capability to produce (SOD), thus increasing their propensity to oxidative damage (Pan and Loo, 2000). Mild to moderate zinc deficiency can depress immune function through impairment of macrophage and neutrophil, natural killer cell and complement activity (Wintergerst et al., 2007). This redox-inert metal is an essential component of numerous proteins involved in defense against oxidative stress, as for example (SOD). Besides it possesses neuroprotective properties. Depletion of zinc may enhance DNA damage via impairment of DNA repair mechanisms (Jomova and Valko, 2011).

Our result was in agreement with Meuwese et al., (2007) 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 (Marques et al., 2016).

Conclusion

By the end of this study, we concluded that, the experimental exposure to benzene vapours in male albino rat followed by cascade of Biochemical, immunological and histopathological  alteration represented by significant changes in (MPO), immunoglobulins; IgA and IgG, IgM, IgE, (MDA), (SOD), (CAT) and  (GSH) content along with significant changes in lung histopathology. These changes might be  through initiation of ROS and promotion of oxidative stress and inflammatory pathways.

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