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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 2  |  Issue : 2  |  Page : 129-135

Toxicological evaluation of the effect of phenol-contaminated water on the liver of albino rats


1 Department of Biochemistry, University of Ibadan, Ibadan, Oyo State; Department of Biochemistry, Salem University, Kogi State, Nigeria
2 Department of Chemistry, University of Ibadan, Ibadan, Oyo State, Nigeria
3 Department of Biochemistry, University of Ibadan, Ibadan, Oyo State, Nigeria

Date of Web Publication16-Mar-2015

Correspondence Address:
Aanuoluwa James Salemcity
Department of Biochemistry, Salem University, Kogi State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2348-3334.153257

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  Abstract 

Phenol is a constituent of coal tar and is formed during the natural decomposition of organic materials. The effect of phenol-contaminated water on the liver of rats was investigated. Activities of some liver enzymes, alkaline phosphatase, acid phosphatase, alanine transaminase, aspartate transaminase, and gamma-glutamyl transpeptidase were determined alongside some serum indices of liver function such as serum bilirubin, albumin, globulin and serum enzymes. The total bilirubin of rats treated with phenol-contaminated water was observed to be 8.4 ± 0.8 g/l while that of control rats was 5.6 ± 0.5 g/l. Serum albumin of test rats was found to be 15 ± 2 g/l while that of control rats was 7 ± 3 g/l. Activity of all the enzymes studied reduced significantly in the liver of test rats compared with the control (P < 0.05). However, serum enzymes activity, with the exception of serum aspartate transaminase, of test rats increased significantly (P < 0.05) relative to that of test rats. It is viewed that phenol-contaminated water is hazardous to health as it may be responsible for the leakage of enzymes into the serum and may impair liver function as portrayed by reduced serum globulin and albumin.

Keywords: Enzyme activity, phenol, toxicological evaluation, water


How to cite this article:
Salemcity AJ, Iyanda TA, Oladimeji O, Olajuyin AM. Toxicological evaluation of the effect of phenol-contaminated water on the liver of albino rats. CHRISMED J Health Res 2015;2:129-35

How to cite this URL:
Salemcity AJ, Iyanda TA, Oladimeji O, Olajuyin AM. Toxicological evaluation of the effect of phenol-contaminated water on the liver of albino rats. CHRISMED J Health Res [serial online] 2015 [cited 2020 Jul 10];2:129-35. Available from: http://www.cjhr.org/text.asp?2015/2/2/129/153257


  Introduction Top


Human life, as with all animal and plant life on the planet, is dependent upon water. Often, water that is of a sufficiently high quality at the point of collection is contaminated before it is used because it has to be carried and stored before use or because of unhygienic practices. Contaminants carried in water are dependent on solid waste composition and on the simultaneously occurring physical, chemical and biological activities within landfill. [1]

Phenol is identified as a contaminant that is used in the manufacture of phenolic resins, production of germicidal paints, pharmaceutical products, dyes, and disinfectant. Potential sources of phenol exposure include: The residential wood burning, cigarette smoke and presence of phenol in liquid manure. Increased phenol has been reported in sediments and ground water associated with industrial pollution and this is due to an increase in the use of synthetic chemicals, e.g. insecticides and herbicides, which can easily run off from agricultural land and industrial discharge into surface waters. [2]

Previous research reported that the phenol has serious health effect. For instance, phenol solutions are corrosive to the skin and eyes, while phenol vapor can irritate the respiratory tract. Also, drinking water with extremely high concentrations of phenol has caused muscle tremors, difficulty walking, and death in animals. [3]

The paucity of information on the effects of phenol-contaminated water (0.06 mg/l) on animal health [4] has necessitated the present study. The present study investigated the hepatotoxic effect of phenol-contaminated water on albino rats.


  Materials and methods Top


The experimental water for the study was collected from the water supply system of Adekunle Ajasin University, Akungba-Akoko, Ondo State, Nigeria and the study was conducted in the Department of Biochemistry of the same university after prior permission from the institutional animal ethics committee. Physiochemical properties of all water sample were determined in accordance with standard methods [5] and Atomic Absorption Spectrophotometer (Buck 210VGP) was used for the determination of heavy metals.

Experimental design

Twenty Albino rats (Rattus norvegicus) were obtained from the Animal Holding Unit of the Department of Biochemistry of University of Ilorin, Nigeria whereas the animals were transported to the Animal Holding Unit of the Department of Biochemistry, Adekunle Ajasin University, Akungba-Akoko, Nigeria where the study was conducted. The experimental sample size was determined using power analysis [6] and animals were assigned into two main groups of 10 animals each. Each group was further classified into two sub-groups detailed thus:

Group A: Rats placed on tap water (control)

Group B:

Rats placed on water contaminated with phenol (0.05 mg/l)

Group a: Treated as animals in Group A

Group b: Treated as animals in Group B

The phenol-contaminated water was prepared in the laboratory with concentration of 0.05 mg/l. The concentration of this contaminant is more than 10 times the permissible limits [7] and typifies the concentration found in the open runoffs. [8]

The feeding exercise lasted over a period of 65 days, a long-term standard for rats. [8] All animals were kept in a wooden cage and fed ad libitum after 10 days acclimatization.


  Blood sample collection and enzyme assays Top


The rats were fasted overnight, anesthetized in a jar containing chloroform and sacrificed by jugular puncture. The blood was collected in an anticoagulant free bottle and centrifuged at 3500 rpm for 15 min using refrigerated centrifuge RC650s, and the serum obtained was preserved at −8°C until required for use.

Liver samples were weighed and homogenized (1:10, w/v) with ice-cold 50 mM Tris-HCl, (pH 7.4) using Teflon and homogenizer (Janke and Kunkel, Germany). The homogenates were centrifuged at 3500 rpm for 15 min and the supernatant stored at −8°C until use. The protein concentration in the tissue of experimental rats was determined with Biuret assay using BSA as standard. [9]

The activity of ALP in serum and tissues of experimental rats was determined following the method described by Bessey et al.[10] as modified by Wright et al. [11] A volume of 0.1 M carbonate buffer (2.2 cm 3 ) was dispensed into test-tubes, 0.1 cm 3 MgSO 4 .7H 2 O was added and 0.2 cm 3 of either the homogenate or serum (sample) obtained from the experimental animals was added to the test tubes. Distilled water (0.2 cm 3 ) was added to the blank instead of the sample. The mixture was incubated at 37°C for 10 min after which 0.5 ml of 10 mM PNPP was added. The mixture was further incubated at 37°C for 10 min in water bath. The reaction was stopped by adding 2 ml of 1 N NaOH solution and the absorbance was read at 400 nm on a Spectrophotometer.

The activity of ACP in serum and tissues of experimental rats was determined following the method described by Bessey et al. as modified by Wright et al. A volume of 0.1 M acetate buffer (1.3 cm 3 ) was dispensed into two test-tubes labeled blank and sample. Sample (0.2 cm 3 ) obtained from experimental rats was added to the sample test-tube while the same volume of distilled water was added to the blank instead of the sample. The mixture was incubated at 37°C for 10 min after which 0.5 cm 3 of 10 mM PNPP was added. The mixture was further incubated at 37°C for 30 min in water bath, and the reaction was stopped by adding 2.0 ml of 1 N NaOH solution. The absorbance was thereafter read at 400 nm on a spectrophotometer.

The activity of aspartate aminotransferase (AST) in the serum and tissues of experimental animals was determined following the procedure reported by Reitman and Frankel [12] as modified by Schmidt and Schmidt. [13] A calibration of AST standard curve was first carried out by dispensing various volumes (0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 cm 3 ) of pyruvate standard into different test-tubes. The volumes were then made up to 1 cm 3 with AST buffered substrate. The mixture was shaken and incubated for 30 min at 37°C. 0.001 M 2,4-dinitrophenylydrazine (1 cm 3 ) was added to each of the test-tubes. The mixture was allowed to stand for 20 min at 25°C after which 5 cm 3 of 0.4 N NaOH was added. The absorbance of the mixture was read at 546 nm on a spectrophotometer and was plotted against the corresponding concentration of pyruvate.

The activity of alanine aminotransferase (ALT) in the serum and tissues of experimental animals was determined following the procedure reported by Reitman and Frankel as modified by Schmidt and Schmidt.

The activity of gamma-glutamyl transpeptidase (GGT) was determined following the method described by Tietz. [14] Sample (0.2 ml) and distilled water (0.2 ml) was pipetted into different cuvettes (for sample and blank respectively) and 2 ml of reagent (tris buffer, glycylglycine, L-γ-glutamyl-p-nitroanilide and surfactants) was added to each. The reactants were mixed thoroughly, and the absorbance was read, against blank, at 400 nm at time 0, 1, 2 and 3 min.

The bilirubin in the serum was determined by method described by Tietz. [15] Sample/distilled water (1.5 ml) was dispensed into test-tubes, 0.4 ml diazoA and diazo B was added to the sample and blank test-tubes respectively. Absorbance was read after 5 min at 540 nm.


  Results Top


The effect of phenol-contaminated water on the liver of albino rats was studied in this research. Various indices of liver function (bilirubin, albumin and globulin) were determined in the serum of rats as shown in [Table 1].
Table 1: Effect of phenol-contaminated water on some indices of liver function in the serum of rats

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Generally, the indices of liver function namely bilirubin (direct and total), albumin and globulin in test group of rats were observed to be significantly higher (P < 0.05) than those of control group A.

[Figure 1] shows the growth pattern of rats placed on phenol-contaminated water over a period of 65 days. The weight of rats in group B were significantly lower (P < 0.05) than those of rats in group A. This reduction in weight of group B rats began to manifest from day 25.
Figure 1: Growth pattern of rats placed on phenol-contaminated water over a period of 65 days. Plotted results are means of fi ve determinations ± standard deviation (A and B are the group of rats)

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[Figure 2] shows the liver/body weight ratio of rats placed on phenol-contaminated water over a period of 65 days. The liver/body weight ratio of test group B rats was significantly lower (P < 0.05) than that of the control group A rats.
Figure 2: Liver/body weight ratio of rats placed on phenol-contaminated water over a period of 65 days. Plotted results are means of fi ve determinations ± standard deviation bars with same notations are not signifi cantly different (P > 0.05) (A and B are the group of rats while a and b are the bar notations)

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[Figure 3] shows the specific activity of ALP of liver and serum of rats placed on phenol-contaminated water over a period of 65 days. The specific activity of ALP of liver of rats in group B was found to be significantly lower (P < 0.05) than that of group A. Conversely, the specific activity of ALP of serum of rats is observed to be significantly higher (P < 0.05) in test group B relative to control group A.
Figure 3: Specific activity of ALP of liver of rats placed on phenol-contaminated water over a period of 65 days. Plotted results are means of fi ve determinations ± standard deviation bars with same notations are not signifi cantly different
(P > 0.05) (A and B are the group of rats while a and b are the bar notations)


Click here to view


[Figure 4] shows the specific activity of ACP of liver and serum of rats placed on phenol-contaminated water over a period of 65 days. The specific activity of ACP of liver of rats was found to be significantly lower (P < 0.05) in test group B relative to control group A whereas the specific activity of ACP of serum of rats is also significantly higher (P < 0.05) in test group B compared to control group.
Figure 4: Specific activity of ACP of liver of rats placed on phenol-contaminated water over a period of 65 days. Plotted results are means of fi ve determinations ± standard deviation bars with same notations are not significantly different
(P > 0.05) (A and B are the group of rats while a and b are the bar notations)


Click here to view


[Figure 5] shows the specific activity of ALT of liver and serum of rats placed on phenol-contaminated water over a period of 65 days. The specific activity of ALT of liver of rats was observed to be significantly lower (P < 0.05) in test group compared to control group A. Also, the specific activity of ALT of serum of rats is significantly higher (P < 0.05) in test group B relative to control group A.
Figure 5: Specific activity of alanine aminotransferase of liver of rats placed on phenol-contaminated water over a period of 65 days. Plotted results are means of fi ve determinations ± standard deviation bars with same notations are not
significantly different (P > 0.05) (A and B are the group of rats while a and b are the bar notations)


Click here to view


[Figure 6] shows the specific activity of AST of liver and serum of rats placed on phenol-contaminated water over a period of 65 days. The specific activity of AST of liver of rats is significantly lower (P < 0.05) in test group B compared to control group A. In contrast, there exists no significant difference (P < 0.05) between the specific activity of AST in the serum of group A and group B rats.
Figure 6: Specific activity of aspartate aminotransferase of liver of rats placed on phenol-contaminated water over a period of 65 days. Plotted results are means of five determinations ± standard deviation bars with same notations are
not significantly different (P > 0.05) (A and B are the group of rats while a and b are the bar notations)


Click here to view


[Figure 7] shows the specific activity of GGT of liver and serum of rats placed on phenol-contaminated water over a period of 65 days. The GGT specific activity of the liver is significantly lower (P < 0.05) in test group B compared to control group A. In contrast, the GGT specific activity of the serum was observed to be significantly higher (P<0.05) in test group B than that of control group A.
Figure 7: Specific activity of gamma-glutamyl transpeptidase of liver of rats placed on phenol-contaminated water over a period of 65 days. Plotted results are means of five determinations ± standard deviation bars with same notations
are not significantly different (P > 0.05) (A and B are the group of rats while a and b are the bar notations)


Click here to view



  Discussion Top


Some indices of liver function in the serum of rats namely bilirubin, albumin and globulin have responded to phenol-contaminated water [Table 1]. Bilirubin protects tissues against oxidative damage caused by free radical and other reactive oxygen species. Baranano et al. [16] reported that the statistical analysis of people with high normal bilirubin levels in the blood shows that they have a lower risk of developing cardiovascular diseases. Mild rises in bilirubin may be caused by an increased breakdown of red blood cells while a very high level is also caused by severe liver failure with cirrhosis.

Hemoglobin transports oxygen from the lungs or gills to the rest of the body such as muscle. Decrease of hemoglobin with the absolute decrease of red blood cells leads to symptoms of anemia. It was reported that the decrease in hemoglobin can cause renal failure. Human serum albumin maintains oncotic pressure, transport thyroid hormones and fatty acids to the liver. Low level of human serum albumin causes hypoalbuminemia. A report showed that glycosylation has the potential to alter the biological structure and function of the serum albumin protein. [17]

The observation on body weight [Figure 1] may be associated with the high concentrations of phenol in the phenol contaminated water which may make utilization of the nutrients inefficient or complexing with the nutrients that should have been available for use by the rats causing malabsorption of the nutrients in the gastrointestinal tract. Dolk et al. [18] reported that stunted growth is observed in children who consume contaminated water containing high levels of organics.

Liver-body weight ratio of experimental rats [Figure 2] revealed that, within the period of the experiment, the concentration of phenol in the contaminated water may have affected the liver- body weight ratios of the animals in those groups. However, this may be because the level of phenol in contaminated water is several times higher than the safe level for drinking water and have the potential to produce desirable effects on tissues, particularly liver.

Alkaline phosphatase is essential for the hydrolysis of nucleotides of ATP, ADP and AMP in human liver. It is a marker enzyme for the plasma membrane and endoplasmic reticulum. Reduction in the liver ALP specific activity and subsequent increase in the serum of rats with phenol-contaminated water [Figure 3] may seem to be due to malfunctioning of the liver membrane. The reduction could also be attributed to inhibition of the enzyme by the contaminated water as is the case with the consumption of metasulfate [19] or inactivation of the enzyme molecules in situ.[17] Furthermore, the membrane of the liver may have been damaged by the pollutants present in the phenol contaminated water thereby allowing the leakage of ALP into the extracellular fluid.

The specific activity of acid phosphatase of liver of rats placed on phenol-contaminated water is shown in [Figure 4]. ACP is a lysosomal enzyme and very hydrolytic in nature. It occurs when there is a fall in cytosol pH due to apoptosis. ACP is also needed to trigger specific chemical reactions and hence, used to monitor carcinogenicity in the prostate. [20],[21] Reduction in ACP activity in the liver of the test rats may be as a result of the interactions between the constituents of the contaminated water and the components of the membrane causing the leakage of the enzyme into the serum of the extracellular fluid, hence, the high value observed in the serum. This leads to malfunctioning of the liver.

The specific activities of the transaminases (AST and ALT) of liver tissue and serum over phenol- contaminated water are presented in [Figure 5] and [Figure 6]. Transaminases are used to monitor the activity of the liver especially in the serum and also, it predicts liver diseases. The reduction in the specific activities of AST and ALT in the liver implies that constituents of phenol-contaminated water may be inhibiting the enzymes or causing their leakages into the serum. Previous study (Schimidt and Schmidt, 1963) reported that elevated serum level of ALT may be due to leakage of enzymes from the liver and may significantly impair liver function. Conversely affected by the treatment suggests the inhibition of the enzyme rather than leakage of enzymes from the cell into the extracellular space. Kumar et al. [22] reported that AST and ALT activities reduction in the liver is inversely proportional to the concentration of pollutants, e.g. ammonia in a polluted or contaminated water.

Gamma-glytamyl transpeptidase (γ-GT) is considered to be an index of hepatobiliary dysfunction and alcohol abuse. Recent epidemiological and pathological studies have suggested its independent role in the pathogenesis and clinical evolution of cardiovascular diseases brought on by atherosclerosis. [23] Lower γ-GT and higher serum level of γ-GT in [Figure 7] show that the role of γ-GT in replenishing intracellular glutathione, and possibly in controlling apoptosis and proliferation of atheromatous plaques may give it added significance. Also, elevated serum γ-GT has a prognostic impact on fatal events of chronic forms of coronary heart disease, congestive heart failure, and ischemia or hemorrhagic stroke. [24]


  Conclusion Top


The data generated from this study suggest that: Phenol-contaminated water may possibly result to loss of weight (as evident in the test rat) as well as reduction in the liver body weight ratio which may be an indication of possible damage to the organ.

Phenol-contaminated water may impair liver function as supported by the increased serum bilirubin levels and decreased albumin and globulin levels; and elevated activities of ALT, ACP, ALP and GGT in serum and decrease activities of the same enzymes in the liver as observed in the test rats.

However, agencies involved in setting standards for drinking water are advised not to rely only on routine chemical analysis of water alone but go further to examine the hematological and histopathological parameters before water is certified safe for drinking.

 
  References Top

1.
Raju MV. Contamination of ground water due to landfill leachate. Int J Eng Res 2012;1:48-53.  Back to cited text no. 1
    
2.
Kale SS, Kadam AK, Kumar S, Pawar NJ. Evaluating pollution potential of leachate from landfill site, from the Pune metropolitan city and its impact on shallow basaltic aquifers. Environ Monit Assess 2010;162:327-46.  Back to cited text no. 2
    
3.
Agency for Toxic Substances and Disease Registry (ATSDAR). Toxicological Profile for Phenol (Update). Atlanta, GA: US Public Health Service, US Department of Health and Human Services; 2009.  Back to cited text no. 3
    
4.
Warner MA, Harper JV. Cardiac dysrhythmias associated with chemical peeling with phenol. Anesthesiology 1985;62:366-7.  Back to cited text no. 4
    
5.
APHA. Standard Methods for Examinations of Water and Waste Water. 16 th ed. Washington, D. C: American Public Health Association; 1992. p. 268.  Back to cited text no. 5
    
6.
Dell RB, Holleran S, Ramakrishnan R. Sample size determination. ILAR J 2002;43:207-13.  Back to cited text no. 6
    
7.
Adeyemi O, Oloyede OB, Oladiji AT. Physicochemical and microbial characteristics of leachate-contaminated groundwater. Asian J Biochem 2007a; 2:343-8.  Back to cited text no. 7
    
8.
Adeyemi O, Oladiji AT. Physicochemical and microbial characteristics of leachate-contaminated groundwater. Asian J Biochem 2007;2:343-8.  Back to cited text no. 8
    
9.
Gornall AG, Bardawill CJ, David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem 1949;177:751-66.  Back to cited text no. 9
    
10.
Bessey OA, Lowry OH, Brock MJ. A method for the rapid determination of alkaline phosphates with five cubic millimeters of serum. J Biol Chem 1946;164:321-9.  Back to cited text no. 10
    
11.
Wright PJ, Leathwood PD, Plummer DT. Enzymes in rat urine: Alkaline phosphatase. Enzymologia 1972;42:317-27.  Back to cited text no. 11
    
12.
Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957;28:56-63.  Back to cited text no. 12
    
13.
Schmidt E, Schmidt FW. Determination of serum GOT and GPT. Enzyme Biol Clin1963;3:1.  Back to cited text no. 13
    
14.
Teitz NN. Assay for gamma glutamyl transpeptidase activity in serum. In: Fundamentals of Clinical Chemistry. 3 rd ed. Philadelphia, USA: W.B. Saunders Co.; 1987. p. 391.  Back to cited text no. 14
    
15.
Tietz NW. Serum triglyceride determination. In: Clinical Guide to Laboratory Tests. 2 nd ed. Philadelphia, USA: W.B. Saunders Co.; 1990. p. 554-6.  Back to cited text no. 15
    
16.
Baranano DE, Rao M, Ferris CD, Snyder SH. Biliverdin reductase: A major physiologic cytoprotectant. Proc Natl Acad Sci U S A 2002;99:16093-8.  Back to cited text no. 16
    
17.
Mendez DL, Jensen RA, McElroy LA, Pena JM, Esquerra RM. The effect of non-enzymatic glycation on the unfolding of human serum albumin. Arch Biochem Biophys 2005;444:92-9.  Back to cited text no. 17
    
18.
Dolk H, Vrijheid M, Armstrong B, Abramsky L, Bianchi F, Garne E, et al. Risk of congenital anomalies near hazardous-waste landfill sites in Europe: The EUROHAZCON study. Lancet 1998 8;352:423-7.  Back to cited text no. 18
    
19.
Akanji MA, Olagoke OA, Oloyede OB. Effect of chronic consumption of metabisulphite on the integrity of the rat kidney cellular system. Toxicology 1993;81:173-9.  Back to cited text no. 19
    
20.
Umezawa H, Hooper IR. Amonoglycosides Antibiotics, New York: Spanger Verky, Berlin, Heidelberg; 1982.  Back to cited text no. 20
    
21.
Moul JW, Connelly RR, Perahia B, McLeod DG. The contemporary value of pretreatment prostatic acid phosphatase to predict pathological stage and recurrence in radical prostatectomy cases. J Urol 1998;159:935-40.  Back to cited text no. 21
    
22.
Kumar T, Anitha K, Muralimohan E, Pillai KS, Murthy RP, Sekar BH. Toxicity of combination of a neen based pesticide and an organophosphorus pesticide to Wistar rat. J Exp India 1998;1:35-41.  Back to cited text no. 22
    
23.
Salemcity AJ, Adeyemi O, Iyanda TA, Oladimeji O. Toxicological assessment of consumption of Benzene-polluted water on the heart and blood of Abino rats. Int J Pharm Sci Res 2014;5:572-7.  Back to cited text no. 23
    
24.
Paolicchi A, Minotti G, Tonarelli P, Tongiani R, De Cesare D, Mezzetti A, et al. Gamma-glutamyl transpeptidase-dependent iron reduction and LDL oxidation - A potential mechanism in atherosclerosis. J Investig Med 1999;47:151-60.  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1]



 

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