Biochemical Changes Followed Experimental Respiratory Distress by Benzene Vapours.
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 con thus 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 byatomic absorption spectra-photometry (PyeUnicum), 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 salineas 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), (G[P.sub.X])(Paglia and Valentine, 1967) and (MDA)(Satoh, 1978).
Statistical analysis:Data analysis was expressed as mean [+ or -] 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 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.
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 redoxactive 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 (Wintergerstet 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 (Jomovaand 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).
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.
(Received: 09 October 2018; accepted: 24 November 2018)
(1.) Adly, A. A. Oxidative stress and disease: An updated review. Res J Immunol. 23: 129-145 (2010).
(2.) Aebi, H., Catalase in vitro. Methods Enzymol., 105: 121-126 (1984).
(3.) Bauer, J.D. Clinical laboratory methods 9th Ed, the C.V. Mosby company, 11830, waistline industrial Missouri: 63146 U.S.A p: 112 (1982).
(4.) Beutler, E., O. Duron and B.M. Kelly, Improved method for the determination of blood glutathione. J. Lab. Clin. Med, 61: 882-888 (1963).
(5.) Eybl V, Kotyzova D, Leseticky L, Bludovska M, Koutensky J. The influence of curcumin and manganese complex of curcumin on cadmium-induced oxidative damage and trace elements status in tissues of mice. J Appl Toxicol; 26(3): 207-212 (2006).
(6.) Fossati P, Prencipe L, Berti G. (1980).Use of 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. Clin Chem.; 26(2): 227-31.
(7.) Ghio. A. J., Richards. J. H., Carter J. D., and Madden M. C. "Accumulation of iron in the rat lung after tracheal instillation of diesel particles," Toxicologic Pathology, 28(4): pp. 619-627 (2000).
(8.) Gurer H and Ercal N, Can antioxidants be beneficial in the treatment of lead poisoning. Free RadicBiol Med, 29: 927-945 (2000).
(9.) Ibitoroko GM, Adegoke O, Wachukwu KC. Effect of Vitamins C and E on Hematological Parameters in Albino Rats Treated with Gasoline. Global Veterinaria. 7(4): 347-352 (2011).
(10.) Jomova K, Valko M. Advances in metal-induced oxidative stress and human disease. Toxicology; 283: 65-87 (2011).
(11.) Korunburg, A. and Korecker, D. In "Methods in Enzymology". Acad. Press, New York, pp.323 (1955).
(12.) Marques R.E., Marques P.E., Guabiraba R. and Teixeira M.M. Exploring the Homeostatic and sensory roles of the immune system. Frontiers in Immunology, Mar.7(125):1-7 (2016).
(13.) Meuwese M.C., Stroes E.S., Hazen S.L., van Miert J.N., Kuivenhoven J.A., Schaub R.G., Wareham N.J., Luben R., Kastelein J.J., Khaw K.T., Boekholdt S.M. Serum myeloperoxidase levels are associated with the future risk of coronary artery disease in apparently healthy individuals. Journal of the American College of Cardiology, 50: 159-165 (2007).
(14.) Montgomery H. A. C and Dymock J. F. The determination of nitrate in water. Analyst; 86: 414-416 (1961).
(15.) Nishikimi, M., N.A. Rao and K. Yagi, The occurrence of superoxide anion in the reaction of reduced phenazinemethosulfate and molecular oxygen. Biochem. Biophys. Res. Commun., 46: 849-854 (1972).
(16.) Paglia, D.E. and W.N. Valentine, Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med., 70: 158-169 (1967).
(17.) Pan, Y. and Loo, G. Effect of copper deficiency on oxidative DNA damage in Jurkat Tlymphocytes. Free RadicBiol Med; 28: 824-830 (2000).
(18.) Plebani, M.; Bernardi, D.; Basso, D.; Borghesan, F. and Faggian, D. Measurement of specific immunoglobulin E: intermethod comparison and standardization. Clin. Chem. J. 44: 1974-1979 (1998).
(19.) Satoh, K., Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin. Chim. Acta, 90: 37-43 (1978).
(20.) Uboh FE, Ebong PE, Eka OU, Eyong EU, Akpanabiatu MI. Effect of Inhalation exposure to Kerosene and Petrol Fumes on some anaemia-diagnostic indices in rats. Global J. Environ. Sc.; 3(1): 59-63 (2005).
(21.) Uzma. N., Kumar. S. S., and Hazari. M. A. H., Exposure to benzene induces oxidative stress, alters the immune response and expression of p53 in gasoline filling workers, American Journal of Industrial Medicine, 53(12): pp. 1264-1270 (2010).
(22.) Valko M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol; 39: 44-84 (2007).
(23.) Valco M, Morris H, Cronin MTD. Metals, toxicity and oxidative stress. Current Med Chem; 12: 1161-1208 (2005).
(24.) Whicher, J.T.; warren, C. and Chambers, R.E. Immunochemical assays for immunoglobulines. Ann. Clin. Biochem. J. 21: 78-91 (1984).
(25.) Wiles, J.B. The determination of metals in blood serum by atomic spectrophotometery. Spectro. Chem. Acta 16, 259-272 (1960).
(26.) Wintergerst E.S, Maggini S, HornigD.H. Contribution of selected vitamins and trace elements to immune function. Ann Nutr. Metab, 51: 301-323 (2007).
H.A. Abdel Maksoud , M.K. Mahfouz , M.A. Omnia , M.H. Abdullah , M.E. Eltabey  and Mohamed G. Elharrif 
 Biochemisry Department, Benha University, Egypt.
 Biochemisry Department, 6th October University, Egypt.
 Basic Medical Sciences Department. Shaqra university, KSA.
Caption: Normal alveolar spaces and thin alveolar septum (black arrows). Uniform bronchiolar wall is also seen (Red arrow) (H&E, 10x)
Caption: Normal alveolar spaces and thin alveolar septum (black arrows). Uniform bronchiolar wall is also seen (Red arrow) (H&E, 20x)
Caption: There is preserved lung architecture with moderate thickening of alveolar septa (black arrows). There is congested blood vessel (Red arrow) (H&E, 10x)
Caption: There is moderate thickening of alveolar septa with chronic inflammatory cells (black arrows). There is congested blood vessel (Red arrow) (H&E, 20x)
Caption: There is marked thickening of alveolar septa with chronic inflammation (black arrows) (H&E, 20x)
Caption: There is focal disturbed lung architecture with marked thickening of alveolar septa with chronic inflammation (Black arrows) (H&E, 10x)
Caption: There is marked peri-bronchiolar inflammation and fibrosis in wall (Red arrows) (H&E, 10x)
Caption: There are thick walled blood vessels (Black arrows). Thickening of alveolar septa is seen (Red arrows), as well as chronic inflammation in bronchiolar wall (Arrow head) (H&E, 10x)
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 Groups [micro]g/dl [micro]g/dl Control 58.82 [+ or -] 42.25 [+ or -] normal 1.77 (a) 1.19 (a) Exposed After 44.89 [+ or -] 31.73 [+ or -] 3 months 1.16 (b) 0.97 (b) group After 36.27 [+ or -] 24.88 [+ or -] 6 months 1.01 (c) 1.06 (c) Parameters Fe Pb Groups [micro]g/dl [micro]g/dl Control 92.34 [+ or -] 6.58 [+ or -] normal 1.79 (a) 0.38 (c) Exposed After 53.97 [+ or -] 8.97 [+ or -] 3 months 2.16 (c) 0.30 (b) group After 59.92 [+ or -] 10.61 [+ or -] 6 months 2.08 (b) 0.34 (a) Parameters Cd NO Groups [micro]g/dl [micro]g/dl Control 1.61 [+ or -] 4.43 [+ or -] normal 0.12 (c) 0.21 (a) Exposed After 1.90 [+ or -] 2.59 [+ or -] 3 months 0.10 (b) 0.16 (b) group After 2.57 [+ or -] 2.07 [+ or -] 6 months 0.15 (a) 0.12 (c) Parameters MPO IgM Groups ng/ml mg/dl Control 7.72 [+ or -] 54.62 [+ or -] normal 0.49 (c) 1.85 (c) Exposed After 10.44 [+ or -] 76.72 [+ or -] 3 months 0.46 (b) 2.69 (b) group After 15.62 [+ or -] 95.56 [+ or -] 6 months 0.38 (a) 2.83 (a) Parameters IgG IgA Groups mg/dl mg/dl Control 494.30 [+ or -] 27.07 [+ or -] normal 3.35 (a) 0.91 (a) Exposed After 425.20 [+ or -] 18.03 [+ or -] 3 months 3.52 (b) 0.94 (b) group After 398.40 [+ or -] 10.88 [+ or -] 6 months 3.61 (c) 0.77 (c) Parameters IgE Groups IU/ml Control 2.99 [+ or -] normal 0.07 (c) Exposed After 8.32 [+ or -] 3 months 0.35 (b) group After 14.43 [+ or -] 6 months 0.54 (a) Data shown are mean [+ or -] 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 MDA Groups nmol/g. Hb Control 8.22 [+ or -] 0.41 (c) normal Exposed After 3-months 14.40 [+ or -] 0.76 (b) groups After 6-months 20.95 [+ or -] 1.06 (a) Parameter GSH Groups mg/dl Control 29.61 [+ or -] 0.75 (a) normal Exposed After 3-months 16.71 [+ or -] 0.56 (b) groups After 6-months 11.19 [+ or -] 0.63 (c) Parameter SOD Groups U/g. Hb Control 16.36 [+ or -] 0.81 (a) normal Exposed After 3-months 11.14 [+ or -] 0.86 (b) groups After 6-months 6.98 [+ or -] 0.72 (c) Parameter CAT Groups U/L Control 494.40 [+ or -] 3.63 (a) normal Exposed After 3-months 446.90 [+ or -] 2.52 (b) groups After 6-months 407.60 [+ or -] 2.45 (c) Parameter GPxm Groups U/ml Control 40.55 [+ or -] 1.07 (a) normal Exposed After 3-months 28.92 [+ or -] 0.91 (b) groups After 6-months 20.53 [+ or -] 0.83 (c) Data shown are mean [+ or -] 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 Patterns of injury Score Lung architecture 0 (Preserved) 1 (Disturbed) Thickening of alveolar septa with chronic 0 (None) inflammation and /or fibrosis Congested/thickened wall vessels 1 (Mild) Peri-bronchiolar inflammation and fibrosis 2 (Moderate) 3 (Severe) Parameter Lung Thickening of alveolar Groups architecture septa with chronic inflammation and /or fibrosis Control 0 0 3 months exposure 0 2 6 months exposure 1 3 Parameter Congested Peri- Groups vessels/ bronchiolar Thick walled fibrosis vessels Control 0 0 3 months exposure 2 0 6 months exposure 2 3