In vitro and in vivo evaluation of toxic effect of benzene on lymphocytes and hepatocytes.
N Uzma, B Kumar, K Salar, A Madhuri, V Reddy
absolute lymphocyte count, benzene, liver function test, lymphocytopenia
N Uzma, B Kumar, K Salar, A Madhuri, V Reddy. In vitro and in vivo evaluation of toxic effect of benzene on lymphocytes and hepatocytes.. The Internet Journal of Toxicology. 2008 Volume 6 Number 2.
Benzene is a ubiquitous industrial solvent and widely distributed environmental contaminant that has been linked to adverse health effect in humans and animals [1-3]. Benzene ranks as the 17th chemical in terms of total annual production in 1994  and therefore represents a significant occupational hazard. Hematotoxicity is the most noted and characteristic systemic effect resulting from intermediate and chronic benzene exposure leading to aplastic anemia, leucopenia and thrombocytopenia [5,6]. Other health effects of benzene include immunological changes which appear largely related to decrease in circulating leukocytes and the ability of lymphoid tissue to produce mature lymphocytes necessary to form antibodies [7,8]. Chronic exposure results in consistent structural and numerical chromosomal aberrations in lymphocytes and bone marrow cells. Benzene metabolites covalently bind to cellular macromolecules (including DNA, RNA and proteins) thereby leading to dysfunction in the bone marrow and other tissues. Along with benzene, one or more reactive benzene metabolites are involved in the health effects. Majority of benzene metabolism occurs in liver by cytochrome P450 and a small amount is metabolized in the bone marrow [9-11], the site of characteristic benzene toxicity (Figure 1).
Benzene and its metabolites induce cell transformation, gene mutation and apoptosis in mammalian cells in culture . Liver function as the key organ of metabolism and excretion of benzene thus, constantly endowed with the task of detoxification, inducing various disorders to the organ. Cultured mammalian hepatocytes retaining differentiated hepatic functions would be great useful in toxicology [13,14]. Since identification of morphological and structural changes induced by benzene and its metabolites to the hepatocytes in living animals is a great challenge. Changes in the hepatic function induced by these metabolites in humans can be correlated with the changes in liver function test (LFT) [15,16]. Therefore, we conducted this study to evaluate the toxic effect of benzene by
Materials and Methods
Acetonitrile, Dimethyl Sulphoxide, Hepatocyte Incubation media and benzene were from Sigma, St Louis, MO, USA. Fetal calf serum purchased from Gemini Bio-Products, Inc., Calabasas, CA, USA. All other reagents used were analytical grade.
analysis of Benzene toxicity
Viability of Lymphocyte and Benzene exposure
Spleen cells were isolated from C57BL/6 mice as described earlier , FACS caliber analysis has done to conform the purity of the spleen cells and found 100% pure (Data not shown). Eight hours after incubation (5% of CO2 at 37oC) with different concentration of benzene (1ppm, 2ppm, 3ppm, 4ppm, 5ppm, 7ppm and 10ppm), viability of splenic lymphocytes (5 x 106 cells/ ml) was estimated by the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] dye conversion assay. Results were expressed as mean percentage viability ± SD for four replicates.
Viability of Hepatocytes and Benzene exposure
Parenchymal component of liver cells was isolated one day prior to the study and seeded in 24 well plates in Dulbecco’s modified Eagle’s medium supplemented with 10% heat inactivated fetal calf serum (FCS) and Pencillin as described elsewhere . After 2 hours the medium was replaced to remove floating cells. Eight hours after incubation cell viability was estimated with different concentrations of benzene (1ppm, 2ppm, 3ppm, 5ppm, 7ppm, and 10ppm) by MTT assay.
analysis of Benzene toxicity
The study was conducted on 154 male benzene exposed petrol filling workers (group I: <10 years of exposure, group II: >10 years) and 33 healthy control subjects were matched for demographic properties with the study group (Table1). Smokers and subjects suffering from any chronic illness were excluded from the study. Written informed consent was obtained from each subject prior to the study. Venous blood sample (3mL) was obtained in heparinised tubes from workers and controls. The study was approved by the Institutional Ethics Committee of Deccan College of Medical Sciences, Hyderabad.
Blood analysis for total Lymphocyte and Liver Function
Blood samples were analyzed by a Coulter TS40 blood counter which was calibrated daily. Approximately 104 white blood cells were counted to provide a total WBC count and a lymphocyte percentage, which are multiplied together to generate the absolute lymphocyte count. Differential leukocyte count was done according the method given elsewhere . All abnormal counts were repeated manually. Serum concentrations of total protein, albumin, total bilirubin, ALT, AST and ALP were determined using Beckman’s autoanalyser.
All the data was expressed as Mean ± Standard deviation. Statistical comparison between different groups were performed using one-way analysis of variance (ANOVA) and tukey test for average comparison, significance was accepted at p < 0.05. Statistical analysis was performed using ORIGIN version 6 and SPSS version 10.0.
analysis of Benzene toxicity
To examine the lymphocyte and hepatocyte toxicity of benzene and its metabolites, we performed
analysis of Benzene toxicity
Figure 3 shows the benzene toxicity to Lymphocytes in petrol attendants. Percentage of lymphocyte count in control subjects was found to be 37.92±1.89 whereas a border line significant decrease (32.3±1.65,
Table 2 shows the mean ± SD levels of total protein, albumin, ALT, AST, ALP and total bilirubin in petrol attendants with corresponding value in controls. The mean value of ALP (135 ± 23) and ALT (41±11) in study group was significantly lowered (
The aromatic hydrocarbon benzene is used in many different industrial activities and is present in fuels like petrol at a concentration of 0.5- 1.5%. The physiochemical properties of benzene include low evaporation temperature and vapor pressure allows its incorporation into the environment directly from the petrol industry or from automobiles. The chronic toxicity of benzene is the result of a series of biotransformation events that initiate with the generation of reactive intermediates which form covalent adducts with diverse critical macromolecules such as proteins and nucleic acids in liver, kidney, spleen and blood. The liver has the highest concentration of enzymes to metabolize carcinogens and toxicants. The most of the enzymatic reactions that convert lipophilic substances to hydrophilic conjugates in liver.
Majority of benzene metabolism occurs in the liver by cytochrome p4502E1. The major hepatic benzene metabolites are phenol, cathecol, hydroquinone and
Lymphocytes are uniquely sensitive to the toxic effects of benzene and its metabolites and are possibly one of the more sensitive indices to assess benzene associated toxicity. Depending on the dose and duration of exposure or time of exposure it covers a variety of effect that in general may be terms as toxic. Previous study indicated that exposure to benzene acutely affect human hematopoiesis system which leads to increased risk of leukemia at levels below 10ppm . Kirkeleit et al (2008) also indicated that the benzene exposure to 10ppm in shoe manufacturing workers lead to deleterious effect on the lymphocytes . To investigate this possibility, we proformed
The liver as the key organ of metabolism and excretion, is constantly endowed with the task of detoxification. Hepatotoxicants including environmental pollutants and industrial solvents can induce various disorders of the organ. Metabolism in the liver protects tissues in higher organism from potentially harmful blood-borne environmental chemicals. Ironically, the metabolic products of detoxification reactions that protects other tissues from effects of the primary toxicant can be destructive to the liver when in excess or chronically present.
No studies have reported the effect of benzene on the human liver; however several animal studies were identified. The study conducted by Constan et al. (1996) evaluated apoptosis in the livers of Fischer-344 rats following exposure to drinking water containing seven contaminants, one of which was benzene . Repeated exposure to these contaminants lead to the apoptosis which was directly correlated with changes in cell proliferation. Pawar and Mungikar (1975) reported an increase in liver weight after administering benzene to rats . They also reported a decrease in protein in the post mitochondrial supernatant fractions, changes in hepatic drug metabolism and lipid peroxidation in the benzene exposed animals. Ugurnal et al. (1995) investigated the effect of acute and chronic benzene treatment on the lipid peroxidation and antioxidant system in mouse liver . Malondialdehyde and diene conjugate levels were found to be increased in liver homogenates and microsomes of chronically exposed mice and were unchanged in the acutely exposed; glutathione levels remained unchanged in liver homogenates of all treatment groups. Whereas negative results were reported by Shell (1992), no adverse hepatic effects were observed in B6C3F1 mice exposed benzene in drinking water for 30 days .
The regular panels of LFTs are estimation of total protein (TP), albumin (Alb), (ALT), (ALP), total bilirubin (Tbil), (AST), gamma glutamyl transpeptidase and coagulase tests. Hepatic damage induced by any toxins can be observed by evaluating serum TP, AST, ALP and ALT levels. As these enzymes are cytoplasmic in nature, upon liver injury, these enzymes enter into the circulatory system due to altered permeability of membrane . The present study reports that significant decrease in these two enzyme levels that may be due to inflammatory conditions in the benzene exposed groups. The liver produces most of the plasma proteins in the body and albumin is a protein made specifically by the liver. Albumin is decreased in chronic liver disease, nephritic syndrome, a state of poor nutrition and during protein catabolism. TP and Alb were slightly raised within the normal range in petrol attendants, thus revealing that they are not having any of the above diseases. ALP, an enzyme in the cells lining the biliary ducts of the liver, which will increases in the serum during obstructive liver disease. Significant low levels of ALP was observed in study group, which indicates absence of bile duct obstruction. During inflammatory conditions and acute liver damage, ALT rises dramatically. The statistically significant changes in the levels of ALT in study group than in controls indicate changes in the liver function which may be due to inflammation and it is supported by our
In summary, this is the first study to have found a consistent relation between exposure to benzene and reduced hepatocyte functioning across a number of petrol attendants who were exposed to benzene. The results presented here also suggest significant toxicity to lymphocyte by