ORIGINAL RESEARCH

published: 14 March 2017

doi: 10.3389/fphar.2017.00107

Lead Induced Hepato-renal Damage

in Male Albino Rats and Effects of
Activated Charcoal
Samuel J. Offor 1 , Herbert O. C. Mbagwu 1 and Orish E. Orisakwe 2*
1
Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria, 2 Department of
Experimental Pharmacology and Toxicology, Faculty of Pharmacy, University of Port Harcourt, Port Harcourt, Nigeria

Lead is a multi-organ toxicant implicated in various cancers, diseases of the hepatic,

renal, and reproductive systems etc. In search of cheap and readily available antidote
this study has investigated the role of activated charcoal in chronic lead exposure in
albino rats. Eighteen mature male albino rats were used, divided into three groups
of six rats per group. Group 1 (control rats) received deionised water (10 ml/kg),
group 2 was given lead acetate solution 60 mg/kg and group 3 rats were given lead
acetate (60 mg/kg) followed by Activated charcoal, AC (1000 mg/kg) by oral gavage
daily for 28 days. Rats in group 2 showed significant increases in serum Aspartate
aminotransferase, Alkaline phosphatase, Alanine aminotransferase, urea, bilirubin, total
cholesterol, triglycerides, Low Density Lipoprotein, Very Low Density Lipoproteins, Total
Edited by: White Blood Cell Counts, Malondialdehyde, Interleukin-6, and decreases in Packed
Chiranjib Chakraborty,
Cell Volume, hemoglobin concentration, Red blood cell count, total proteins, albumins,
Galgotias University, India
superoxide dismutase, glutathione peroxidase and total glutathione. Co-administration
Reviewed by:
Eric Robinet, of AC significantly decreased these biomarkers with the exception of the sperm
Institut Hospitalo-Universitaire parameters. Histopathology of liver and kidney also confirmed the protective effective
de Strasbourg, France
Bashir M. Rezk,
of AC against lead induced hepato-renal damage. AC may be beneficial in chronic lead
Southern University at New induced liver and kidney damage.
Orleans, USA
Keywords: chronic lead toxicity, activated charcoal, hepato-renal damage, biomarkers, public health
*Correspondence:
Orish E. Orisakwe
orishebere@gmail.com
INTRODUCTION
Specialty section:
This article was submitted to Lead constitute an integral source of poisoning to the ecosystem. It primarily affects the central
Experimental Pharmacology and Drug nervous system, hematopoietic, hepatic and renal system, producing serious disorders (Kalia and
Discovery, Flora, 2005). Lead exposure also causes anemia, immunotoxicity and toxicity to the reproductive
a section of the journal organs (WHO, 2016). Excessive dietary intake of lead has been linked with cancers of stomach,
Frontiers in Pharmacology
small intestine, large intestine, ovary, kidney, lungs, myeloma, all lymphomas, and all leukemia
Received: 16 October 2016 (Reddy et al., 2004). Studies in general populations have identified a positive association of lead
Accepted: 21 February 2017 exposure with coronary artery disease (CAD), stroke mortality, and peripheral arterial disease
Published: 14 March 2017
(Lustberg and Silbergel, 2002; Prozialeck et al., 2008). Lead has also been reported of causing
Citation: impairment of the quality of semen in the male reproductive system (Eibensteiner et al., 2005;
Offor SJ, Mbagwu HOC and Telisman et al., 2007). There is no known safe blood lead concentration. Even blood lead
Orisakwe OE (2017) Lead Induced
concentration as low as 5 µg/dL, once thought to be a safe level, may result in decreased intelligence
Hepato-renal Damage in Male Albino
Rats and Effects of Activated
in children, behavioral difficulties and learning problems (WHO, 2016).
Charcoal. Front. Pharmacol. 8:107. Developing nations are particularly of high risk to lead poisoning and carry the highest burden
doi: 10.3389/fphar.2017.00107 of this hazard. In Nigeria, a suspected case of lead poisoning which occurred at Unguwan Magiro

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Offor et al. Activated Charcoal on Lead Induced Hepato-renal Damage

and Unguwan Kawo communities in Rafi Local Government Area Animal Husbandry
of Niger state in which 48 cases mostly children, (with BLL Eighteen mature male albino Wistar rats, weighing 145–170 g
between 171.5–224 µg/dL) including 14 deaths were reported obtained from the University of Uyo Animal house, were
in May 15, 2015 (WHO, 2015). In March, 2010, Medecins acclimatized for 2 weeks, maintained under controlled conditions
Sans Frontieres, MSF/Doctors without Borders, an international, of temperature (23 ± 2◦ C) and humidity (50 ± 5%) and a 12-h
independent, medical humanitarian organization was alerted to light–dark cycle, were used for the experiment. The animals were
a high number of child fatalities in Zamfara state, northern housed in sanitized polypropylene cages containing sterile paddy
Nigeria. An estimated 400 children died. Laboratory testing later husk as bedding. The bedding of the cages was changed daily
confirmed high levels of lead in the blood of the surviving and the cages were cleaned as well. They had free access to
children. The root cause of the lead poisoning crisis was unsafe standard rat pellet diet and water ad libitum. The procedures were
mining and ore processing (Medecins Sans Frontieres [MSF], performed according to the guidelines on the use of animals and
2012). Artisanal gold mining as well as agriculture are the approved by the Institutional Animal Ethical Committee of the
predominant occupations in the affected communities. Lead University of Uyo.
poisoning from Lead-acid battery recycling was also reported
in Dakar, Senegal (WHO, 2010). The current drug treatment Experimental Design
of lead poisoning is Chelation Therapy with drugs such as The animals were divided into three groups of six rats per group
Dimercaprol, Ethylenediaminetetraacetic acid (EDTA), Succimer as follows: The animals were treated as follows;
and D-penicillamine (Cuprimine) (Kessel and O’Connor, 2001).
Activated charcoal is a light, finely divided, black fluffy powder Group 1 – Normal control received deionised water 10 ml/kg
prepared by pyrolysis of carbonaceous material such as wood, by oral gavage daily for 28 days in addition to standard feed
coconut shells, or petroleum and oxidation using steam or and water.
air at high temperature (600–900◦ C) (Orisakwe, 1994; Cooney, Group 2 – Lead acetate solution 60 mg/kg by oral gavage daily
1995; Olson, 2010; Vaziri et al., 2013). It adsorbs a wide range for 28 days addition to standard feed and water (Cheong and
of substances and organisms (Cooney, 1995). According to Roh, 2006).
Cooney (1995), the adsorption of most metals including lead to Group 3 – Lead acetate (60 mg/kg) (Cheong and Roh, 2006),
activated charcoal is poor and consequently it is seldom used in followed by Activated charcoal AC (1000 mg/kg) by oral gavage
management of lead poisoning. While AC is mainly associated daily for 28 days (American Society for the Prevention of
with treatment of poisoning substances, it has other important Cruelty to Animals [ASPCA], 2015). In all animals received
roles in the treatment of patients with chronic kidney disease activated charcoal 90 min after administration of lead (Olson,
which enhances the outcome of renal dialysis (Alkhatib and Al 2010).
Zailaey, 2015).
Many antidotes are biological products and their cost,
methods of production, potential for eliciting immunogenic Necropsy
responses, the time needed to generate them, and stability Treatments continued for 28 days. Blood was collected
issues contribute to their limited availability and effectiveness. by retro orbital sinus puncture (Stone, 1954). Each blood
These factors exacerbate a world-wide challenge for providing sample was divided into two portions. The first one was
treatment (Weisman et al., 2015). In resource poor nations of sub mixed well with the anticoagulant, dipotassium EDTA
Saharan Africa with rampant lead poisoning, the cost of chelation by shaking and used for hematological screening. The
therapy is considered prohibitive. There is a need therefore to second portion (without anticoagulant) was kept at room
explore readily available and natural antidotes in the management temperature for 30 min to clot. Afterward, the clotted blood
of lead poisoning. Hitherto there is sparse information on the sample was centrifuged at 3,000 rpm for 10 min. The clear
in vivo adsorptive capacity of activated charcoal on lead (Cheong serum supernatant was then carefully aspirated and stored
and Roh, 2006). The present study seeks to add to the fund in a clean sample bottle for the determination of some
of knowledge for the clarification of the usefulness of activated biochemical parameters. Rats were sacrificed under ether
charcoal in the management of the hepato-renal complications of anesthesia (Okolo et al., 2016) and concussion stunning
chronic lead exposure since available data so far have focused on involving manually applied trauma on the head (AVMA,
acute lead exposure. 2013); the kidney and liver were excised, weighed, rinsed in
saline, and preserved in 10% formalin for histopathological
study.
MATERIALS AND METHODS
Hematological Screening
Materials Total White Blood Cell Counts (TWBC), Packed Cell
Chemicals Volume (PCV), Red blood cell (RBC) count, Lymphocytes
Lead acetate trihydrate (May & Baker, England), Activated % (L%) and Neutrophils % (N%) were determined using the
Charcoal, AC (Merck KGaA, Darmstadt, Germany). Lead acetate Hemocytometer method (Thrall and Weiser, 2002). Hemoglobin
salt was dissolved in deionised water, while AC was dispersed in (Hb) concentration was determined by the Cyanmethemoglobin
deionised water to form a suspension. method (Higgins et al., 2008).

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Offor et al. Activated Charcoal on Lead Induced Hepato-renal Damage

Biochemical Analysis in accordance with manufacturer’s recommended protocols

Total serum bilirubin (Doumas et al., 1973), serum total proteins (RayBiotech, Inc. USA and Assaypro LLC, USA).
(Lubran, 1978), serum Albumins (Doumas and Peters, 1997),
serum Globulin (calculated by subtracting the quantity of Statistical Analysis
albumins from that of total proteins), serum total cholesterol Results were expressed as mean ± standard deviation, SD.
(Allain et al., 1974), High Density Lipoprotein (HDLs) (Albers Statistical analysis was carried out with one way analysis of
et al., 1978), Low Density Lipoprotein (LDL) and Very Low variance (ANOVA) followed by Tukey’s HSD post hoc test. Values
Density Lipoproteins (VLDL) (Friedewald et al., 1972; Warnick of p < 0.05 were considered to be significant.
et al., 1990), serum triglycerides (Bucolo and David, 1973), serum
creatinine (Blass et al., 1974) and serum urea (Fawcett and Scott,
1960) and Searcy et al. (1967) were determined. The following RESULTS
liver enzymes were also assayed: Alanine Aminotransferase
(ALT) and Aspartate aminotransferase (AST) (Reitman and Effect of Activated Charcoal on
Frankel, 1957) and Alkaline phosphatase (ALP) (Babson et al., Hematological Parameters
1966). Treatment of rats with lead acetate (Group 2) caused significant
(p < 0.05) decreased in PCV, Hb concentration and RBC
Antioxidants Study and Assessment of Lipid count when compared with normal control. These decreased
Peroxidation/Oxidative Stress parameters were increased significantly (p < 0.05) in group 3
Determination of Antioxidant Levels Namely animals which were given Activated charcoal after lead acetate
(i) Superoxide dismutase (SOD) in whole blood using treatment. Rats in group 2 (given lead acetate only) also had
Superoxide Dismutase kit in accordance with significant increase (p < 0.05) in total white blood count (WBC)
manufacturer’s recommended protocols (Fortress when compared to rats in the normal control group (Group
Diagnostics Limited, UK). 1), while the total WBC in group 3 animals was significantly
(ii) Glutathione peroxidase (GSH-PX) in whole blood (p < 0.05) decreased. There was no effect on lymphocyte and
using Glutathione peroxidase kit in accordance with neutrophil percentages (Table 1).
manufacturer’s recommended protocols (Fortress
Diagnostics Limited, UK). Effect of Activated Charcoal on
(iii) Plasma Total Glutathione using RayBio Glutathione R

Biochemical Parameters
Colorimetric Detection Kit in accordance with Table 2 show the effect of activated charcoal on the serum
manufacturer’s recommended protocols (RayBiotech, levels of AST, ALP, and ALT in lead acetate-treated male albino
Inc. USA) Wistar rats. Treatment of rats with lead acetate (group 2) caused
(iv) Measurement of Malondialdehyde (MDA), a prototype significant (p < 0.05) increase in the following liver enzymes
of the thiobarbituric reactive substances (TBARS) as a when compared with the normal control group (group 1): AST,
biomarker of lipid peroxidation and oxidative stress using ALP, and ALT. These enzymes decreased significantly (p < 0.05)
the modified thiobarbituric acid method (Todorova et al., in group 3 animals which were given activated charcoal after lead
2005). acetate treatment (Table 2).
The effect of activated charcoal on serum total proteins,
Determination of Serum Levels of Pro-Inflammatory Albumins, Globulins, Urea, Creatinine, and Bilirubin in lead
Cytokines acetate-treated male albino Wistar rats is shown on Table 3. There
Serum levels of pro-inflammatory cytokines (Tumor necrosis was significant (p < 0.05) decrease in serum total proteins and
factor-alpha, TNF-α) and Interleukin-6 (IL-6) were determined albumins in rats in group 2 compared to rats in group 1. These
using rat ELISA (Enzyme-linked immunosorbent assay) kits two parameters increased significantly (p < 0.05) in group 3

TABLE 1 | Effect of Activated charcoal on the hematological parameters of lead acetate-treated male albino Wistar rats.

Treatment Packed cell Hemoglobin, Hb Red blood cell count, Total WBC count Lymphocyte Neutrophil
volume, PCV (%) concentration (g/dl) RBC (106 /µL) (103 /µL) (%) (%)

Group 1: Deionised water 40.67 ± 1.63 16.31 ± 0.72 7.89 ± 0.18 19.24 ± 4.85 71.00 ± 3.35 26.83 ± 3.55
(10 ml/kg)
Group 2: Lead acetate 32.83 ± 1.29a 12.57 ± 0.66a 4.41 ± 1.05a 30.66 ± 3.40a 77.00 ± 6.42 26.33 ± 1.51
(60 mg/kg)
Group 3: Lead acetate 38.75 ± 0.42b 15.47 ± 0.42b 7.45 ± 0.52b 18.12 ± 4.70b 69.00 ± 559 26.83 ± 2.93
(60 mg/kg) + AC
(1000 mg/kg)

Data were expressed as mean ± SD. a Significantly different when compared to the control group (p < 0.05). b Significantly different when compared to the lead acetate-
treated group (p < 0.05) (n = 6).

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Offor et al. Activated Charcoal on Lead Induced Hepato-renal Damage

animals given activated charcoal after lead acetate treatment. The compared to those in the normal control group (group 1). This
serum level of globulin was not significantly affected (Table 3). biomarker was significantly (p < 0.05) decreased in group 3. Rats
There was a significant increase (p < 0.05) in the following in group 2 showed significant (p < 0.05) increase in the pro-
parameters: urea and bilirubin in group 2 animals compared inflammatory cytokine, IL-6 in comparison with normal control
to that of rats in the normal control group (group 1). These (group 1). Level of this cytokine was significantly (p < 0.05)
parameters decreased significantly (p < 0.05) in group 3 animals. decreased in group 3. However, there was no effect on the serum
However, there was no significant effect (p > 0.05) on creatinine level of another pro-inflammatory cytokine, TNF-α.
level.
On the lipid profiles, rats in group 2 showed significant Histopathology of the Kidney and liver:
(p < 0.05) increase in total cholesterol, triglycerides, LDL and Liver
VLDL, compared to those in the control group. These parameters The histological photomicrograph of the liver tissue stained with
were significantly (p < 0.05) reduced in group 3 animals except H&E techniques of Group 1 – control that received deionised
for VLDL. There was no significant effect (p > 0.05) on HDL water 10 ml/kg by oral gavage daily for 28 days is shown on
values (Table 4). Figures 1A,B. Central veins with normal intact hepatocytes
Table 5 shows the effect of activated charcoal on surrounding it (1A Mag × 160). Figures 1C,D show the
some antioxidant, lipid peroxidation parameters and histological photomicrograph of the liver tissue stained with H&E
pro-inflammatory cytokines of Lead acetate-treated male techniques of Group 2 – Lead acetate 60 mg/kg daily for 28 days.
albino Wistar rats. Liver samples from group 2 (lead acetate-treated group) were
Treatment of rats with lead acetate (group 2) caused observed to have massive cellular (neutrophils and lymphocytes)
significant (p < 0.05) decrease in the following antioxidant infiltrations around the peri-portal areas, some hepatocyte
enzymes/parameters: GSH-PX, SOD, and Total Glutathione necrosis and there was also vacuolations/vacuolar degeneration
when compared with the normal control group (group 1). These of peripheral hepatocytes (Figures 1C,D). Figures 1E,F,G show
parameters were significantly (p < 0.05) increased in group 3 the Histological photomicrograph of the liver tissue stained with
animals. Also, the value of the biomarker of lipid peroxidation, H&E techniques of Group 3 – Lead acetate (60 mg/kg) followed
MDA was significantly (p < 0.05) increased in group 2 rats, by AC (1000 mg/kg) daily for 28 days. There was no vacuolar

TABLE 2 | Effect of activated charcoal on the serum levels of Aspartate aminotransferase (AST), Alkaline phosphatase (ALP) and Alanine
aminotransferase (ALT) in Lead acetate-treated male albino Wistar rats.

Treatment AST (U/L) (U/L) ALP (U/L) ALT (U/L)

Group 1: Deionised water (10 ml/kg) 55.77 ± 0.88 180.79 ± 0.17 17.67 ± 2.05
Group 2: Lead acetate (60 mg/kg) 76.77 ± 9.20a 211.71 ± 9.63a 35.53 ± 2.82a
Group 3: Lead acetate (60 mg/kg) + AC (1000 mg/kg) 53.82 ± 5.93b 183.31 ± 15.07b 19.39 ± 5.00b

Data were expressed as mean ± SD. a Significantly different when compared to the control group (P < 0.05). b Significantly different when compared to the lead acetate-
treated group (P < 0.05) (n = 6).

TABLE 3 | Effect of activated charcoal on serum total proteins, albumins, globulins, urea, creatinine, and bilirubin in lead acetate-treated male albino
Wistar rats.

Treatment Total proteins Albumins Globulins Urea Creatinine Bilirubin

(g/dl) (g/dl) (g/dl) (mg/dl) (mg/dl) (mg/dl)

Group 1: Deionised water (10 ml/kg) 8.55 ± 0.34 4.28 ± 0.38 4.27 ± 0.35 17.92 ± 2.83 0.59 ± 0.08 0.27 ± 0.11
Group 2: Lead acetate (60 mg/kg) 6.61 ± 0.16a 2.90 ± 0.17a 3.71 ± 0.25 29.00 ± 3.23a 0.64 ± 0.05 0.97 ± 0.31a
Group 3: Lead acetate (60 mg/kg) + 7.76 ± 0.32ab 3.90 ± 0.14b 3.84 ± 0.38 18.91 ± 1.47b 0.56 ± 0.05 0.35 ± 0.10b
AC (1000 mg/kg)

Data were expressed as mean ± SD. a Significantly different when compared to the control group (P < 0.05). b Significantly different when compared to the lead
acetate-treated group (p < 0.05) (n = 6).

TABLE 4 | Effect of activated charcoal on lipid profiles of lead acetate-treated male albino Wistar rats.

Treatment Total cholesterol (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl) Triglyceride (mg/dl)

Group 1: Deionised water (10 ml/kg) 53.29 ± 5.57 22.16 ± 6.22 26.30 ± 9.21 4.94 ± 0.47 24.17 ± 2.84
Group 2: Lead acetate (60 mg/kg) 78.51 ± 13.66a 22.62 ± 5.00 40.07 ± 4.92a 22.64 ± 12.64a 113.20 ± 63.17a
Group 3: Lead acetate (60 mg/kg) + AC (1000 mg/kg) 55.03 ± 5.74b 20.56 ± 3.30 26.06 ± 1.51b 10.69 ± 4.55 45.70 ± 20.39b

Data were expressed as mean ± SD. a Significantly different when compared to the control group (P < 0.05). a Significantly different when compared to the lead
acetate-treated group (P < 0.05) (n = 6).

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Offor et al. Activated Charcoal on Lead Induced Hepato-renal Damage

TABLE 5 | Effect of activated charcoal on some antioxidant, lipid peroxidation parameters and pro-inflammatory cytokines of lead acetate-treated male
albino Wistar rats.

Treatment Malondialdehyde Glutathione Superoxide Total glutathione Interleukin-6 Tumor necrosis

(µmol/L of peroxidase (U/L of dismutase (U/ml of (ng/µL) (IL-6) factor-alpha
plasma) blood) blood) (TNF-α)

Group 1: Deionised 1.58 ± 0.09 482.85 ± 53.43 144.80 ± 7.00 1.11 ± 0.03 74.41 ± 5.45 0.01 ± 0.00
water (10 ml/kg)
Group 2: Lead acetate 1.90 ± 0.17a 247.18 ± 70.40a 122.39 ± 4.63a 0.56 ± 0.31a 113.58 ± 13.46a 0.00 ± 0.00
(60 mg/kg)
Group 3: Lead acetate 1.62 ± 0.14b 327.65 ± 96.32 142.13 ± 8.82b 1.20 ± 0.08b 72.68+11.68b 0.00 ± 0.00
(60 mg/kg) + AC
(1000 mg/kg)

Data were expressed as mean ± SD. a Significantly different when compared to the control group (P < 0.05). b Significantly different when compared to the lead
acetate-treated group (P < 0.05) (n = 6).

degeneration of peripheral hepatocytes (intact hepatocytes) and to the aforementioned limitations. As a further justification for
mild cellular infiltration with few lymphocytes around the portal this research, there is currently sparse information on the in vivo
areas efficacy of AC in the treatment of lead poisoning.
In this study, treatment of rats with lead acetate caused
Kidney significant increase in the activity of serum AST, ALT, ALP,
Figure 2 shows the histological photomicrograph of the kidney bilirubin and urea, while the levels of albumin and total proteins
stained with H&E techniques of Group 1 – control that received were decreased. Similar results were reported by Azoz and Raafat
deionised water 10 ml/kg by oral gavage daily for 28 days (2012), Ibrahim et al. (2012), and Azab (2014). These parameters
(Figures 2A,B). There was normal dilated tubules and glomeruli were, however, reversed by treatment with activated charcoal
at the periphery of the cortex. Group 2 – Lead acetate 60 mg/kg in this study. Increasing levels of AST and ALT in the lead
daily for 28 days –Degeneration and necrosis of the renal acetate-treated rats signify damage to the structural integrity
parenchymal cells with massive inflammatory cells infiltration of the liver. It is mainly due to the leakage of these enzymes
(2C Mag × 160). The kidney of rats in Group 2 – Lead acetate from the liver cytosol into the blood stream (Concepcion et al.,
60 mg/kg daily for 28 days showed areas of degeneration and 1993). Releasing of AST and AST from the cell cytosol can
necrosis of the renal cortical parenchymal cells with massive occur as secondary changes to cellular necrosis (Gaskill et al.,
lymphocytic cellular infiltration, with vacuolation/vacuolar 2005). The high AST and ALT activities are accompanied by high
degeneration of the peripheral interstitial cells of the cortex liver microsomal membrane fluidity, free radical generation and
(Figures 2C,D).Tissue sections from the kidneys of rats in Group alteration in the liver tissue (Ibrahim et al., 2012). Elevated level
3 – Lead acetate (60 mg/kg) followed by AC (1000 mg/kg) daily of ALP suggests biliary damage or an obstruction of the biliary
for 28 days had varied areas of normal and degenerated cortical tree, which disrupts the flow of blood to the liver (Farida et al.,
tubules, with mild cellular infiltration of the renal parenchyma 2012). The decrease in serum levels of these enzymes may be
(Figures 2E,F). due to the prevention of their leakage from the liver cytosol by
activated charcoal, probably due to reduction in blood lead level.
The increase of bilirubin values in rats treated with lead acetate
DISCUSSION in this study may be due to excessive heme destruction and
blockage of biliary tract resulting in inhibition of the conjugation
Lead is one of the most toxic heavy metals of great public health reaction and release of unconjugated bilirubin from damaged
significance. Exposure to low-level heavy metals such as lead hepatocytes (Ali et al., 2010). Bilirubin has a protective role
may contribute much more toward the causation of chronic against oxidative damage of cell membrane induced by metal
diseases (diabetes, renal disease, cancer, male infertility etc.) and (Noriega et al., 2003).
impaired functioning than previously thought (Orisakwe, 2014). Lead acetate administration in this study caused significant
Chelators, the currently available antidote for the treatment of increase in serum urea level, but only a slight increase in serum
lead poisoning have many adverse effects such as being painful, creatinine level. The serum urea was significantly decreased
hepatotoxicity, gastrointestinal symptoms, among others. In with the administration of activated charcoal in this study but
addition to difficulties in the administration of some chelators, showed only a slight decrease in the value of creatinine after
they are also expensive and not readily available. Also, chelation 28 days. This is in agreement with the work of Cheong and Roh
therapy is recommended by the CDC to be used only in extreme (2006), whose data showed a significant decrease in the value of
cases where the blood lead level is over 45 ug/dL. It is generally blood urea nitrogen after 1 week of administration of activated
not used in cases where levels are under 25 ug/dl and it neither charcoal, while the serum level of creatinine was only significantly
removes all of the lead from the body nor does it undo damage reduced by activated charcoal after 72 h. This study confirms the
already done to organ. Activated charcoal is thus investigated in conclusion by Cheong and Roh (2006) that activated charcoal
this study as a possible alternative to the classical antidotes owing may protect the lead-induced toxicity on kidney.

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Offor et al. Activated Charcoal on Lead Induced Hepato-renal Damage

FIGURE 1 | (A) Histological photomicrograph of the liver tissue stained with H&E techniques of Group 1 – control that received deionised water 10 ml/kg by oral
gavage daily for 28 days – Central veins with normal intact hepatocytes surrounding it (1A Mag × 160). (B) Histological photomicrograph of the liver tissue stained
with H&E techniques of Group 1 – control that received deionised water 10 ml/kg by oral gavage daily for 28 days – Intact hepatocytes on the periphery (no
vacuolations – 1B Mag × 640). (C) Histological photomicrograph of the liver tissue stained with H&E techniques of Group 2 – Lead acetate 60 mg/kg daily for
28 days – Massive cellular infiltration around the portal areas with some hepatocyte necrosis (1C Mag × 160). (D) Histological photomicrograph of the liver tissue
stained with H&E techniques of Group 2 – Lead acetate 60 mg/kg daily for 28 days – Vacuolation or vacuolar degeneration of peripheral hepatocytes (1D
Mag × 640). (E) Histological photomicrograph of the liver tissue stained with H&E techniques of Group 3 – Lead acetate (60 mg/kg) followed by AC (1000 mg/kg)
daily for 28 days – Mild cellular infiltration around the portal areas (1E Mag × 160). (F) Histological photomicrograph of the liver stained with H&E techniques of
Group 3 – Lead acetate (60 mg/kg) followed by AC (1000 mg/kg) daily for 28 days – Mild cellular infiltration with few lymphocytes around the portal areas (1F
Mag × 640). (G) Histological photomicrograph of the liver stained with H&E techniques of Group 3 – Lead acetate (60 mg/kg) followed by AC (1000 mg/kg) daily for
28 days – Intact hepatocyte.

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Offor et al. Activated Charcoal on Lead Induced Hepato-renal Damage

FIGURE 2 | (A) Histological photomicrograph of the kidney stained with H&E techniques of Group 1 – control that received deionised water 10 ml/kg by oral gavage
daily for 28 days – normal dilated tubules and glomeruli at the periphery of the cortex (2A Mag × 160). (B) Histological photomicrograph of the kidney stained with
H&E techniques of Group 1 – control that received deionised water 10 ml/kg by oral gavage daily for 28 days – normal renal cortical tubules and glomeruli (2B
Mag × 640). (C) Histological photomicrograph of the kidney tissue stained with H&E techniques of Group 2 – Lead acetate 60 mg/kg daily for 28 days –
Degeneration and necrosis of the renal parenchymal cells with massive inflammatory cells infiltration (2C Mag × 160). (D) Histological photomicrograph of the kidney
tissue stained with H&E techniques of Group 2 – Lead acetate 60 mg/kg daily for 28 days – Massive lymphocytic cellular infiltration, with degeneration and necrosis
(2D Mag × 640). (E) Histological photomicrograph of the kidney stained with H&E techniques of Group 3 – Lead acetate (60 mg/kg) followed by AC (1000 mg/kg)
daily for 28 days –showing normal medullary tubules (2E Mag × 160). (F) Histological photomicrograph of the kidney stained with H&E techniques of Group 3 – Lead
acetate (60 mg/kg) followed by AC (1000 mg/kg) daily for 28 days – some degenerated tubules and mild cellular infiltration of the renal parenchyma(2F Mag × 640).

Charcoal in various forms administered with low protein diets was able to significantly increase these parameters. Decrease in
has been reported to control effectively some uremic symptoms serum total protein may be due to both hepatic and renal damage
in patients with different stages of renal disease, and this is induced by lead (Ahmed and Shalaby, 1999), or may be due to
achieved through the binding of urea and other urinary toxins binding of lead to plasma proteins, where it causes alteration in
to charcoal, and its excretion with feces, creating a concentration a high number of enzymes and can also disturb protein synthesis
gradient for continued diffusion of these toxins (Ash, 2009). in hepatocytes (Goering, 1993). Decreasing of serum total protein
Simultaneous treatment of rats with adenine and AC (20% w/w in values may be attributed to a decrease in hepatic DNA and RNA
the feeds for 28 days) has been shown to produce a broad, dose- induced by lead intoxication or due to decreased utilization of
dependent, nephroprotective action in adenine-induced chronic free amino acids for protein synthesis (Moussa and Bashandy,
Renal failure (Ali et al., 2014). Also in this study, lead acetate 2008).
caused a significant decrease in the values of serum total proteins In our study, administration of lead acetate was found to
and Albumins. Administration of activated charcoal in this study cause elevation of Total cholesterol, triglycerides, LDL and VLDL.

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Offor et al. Activated Charcoal on Lead Induced Hepato-renal Damage

These parameters were reduced by treatment with activated and Ibrahim et al. (2012). On the other hand, total WBC
charcoal. The result is in agreement with the report of Azoz was significantly increased. Administration of activated charcoal
and Raafat (2012). The high lipid levels could be due to significantly reversed these parameters. Lead directly affects
either increased synthesis or decreased removal of lipoproteins. the hematopoietic system through restraining the synthesis of
Decreased removal may occur as a result of the alteration of Hb by inhibiting various key enzymes involved in the heme
cell – surface receptors for lipoprotein (Tarugi et al., 1982) or synthesis pathway, particularly the enzyme Aminolevulinic Acid
as a result of the inhibition of hepatic lipoprotein lipase activity Dehydratase (ALAD). It also reduces the life span of circulating
(Chajet et al., 1989). Furthermore, lead has been shown to depress erythrocytes by increasing the fragility of cell membranes. The
the activity of cytochrome P – 450 (Alvares et al., 1975), this can aftermath of these two processes leads to anemia (Guidotti et al.,
limit the biosynthesis of the bile acids, which is the significant 2008; Flora et al., 2012).
route for elimination of cholesterol from the body. Although Administration of lead acetate caused significant increase in
activated charcoal did not show any significant effect on HDL in the pro-inflammatory cytokine, IL-6. However, administration
this study, AC significantly lowered lead induced increase in LDL, of activated charcoal significantly reduced the level of IL-6. AC
triglycerides and total cholesterol implicated in the development could protect from lead acetate-induced toxicity by attenuating
of heart disease. the increased serum IL-6. However, the exact mechanism through
It was observed in this study that levels of total glutathione, which it is achieved requires further investigation. Howell
SOD, GSH-PX were significantly reduced in lead acetate-treated et al. (2013), have reported good removal of the inflammatory
rats. The values of these biomarkers were increased by the cytokines IL-8 (100% removal), IL-6 (80% removal) and TNF-α
protective activity of activated charcoal, which also significantly (51% removal) from blood using nanoporous activated carbon
reduced the value of the lead acetate-induced biomarker of lipid beads. In another experiment, Inoue et al. (2015) concluded that
peroxidation, MDA. This result is in agreement with the report of AC should be efficient for cytokine adsorption. Measurements
Azoz and Raafat (2012). Oxidative stress represents an imbalance of humoural factors such as cytokines and other inflammatory
between the production of free radicals and the biological mediators or markers can provide predictive clinical information
system’s ability to readily detoxify the reactive intermediates plus insights into disease mechanisms (Zhou et al., 2010).
or to repair the resulting damage (Flora, 2011). It has been Taken together activated charcoal seems to be protective
reported as a major mechanism of lead induced toxicity (Flora against hepato-renal damage induced by chronic exposure of
et al., 2012). Under the influence of lead, onset of oxidative lead in the animal model. The elevation of antioxidant enzymes
stress occurs on account of two different pathways operative level, reduction of lipid peroxidation (MDA), modulation of the
simultaneously. First, the generation of reactive oxygen species, pro-inflammatory cytokine, IL-6; and reversal of lead-induced
ROS and second, the antioxidant reserves become depleted alteration in some hematological and biochemical parameters
(Flora, 2002). Apart from targeting the sulfhydryl groups, lead following administration of AC may be as result of decrease
can also replace the zinc ions that serve as important cofactors in blood lead level BLL, Further studies may be necessary to
for these antioxidant enzymes and inactivate them (Flora et al., understand the precise mechanism of action.
2007). Lipid peroxidation, another indicator of oxidative stress
occurs as a result of the action of ROS on lipid membranes. The
generated free radical captures electrons from the lipids present AUTHOR CONTRIBUTIONS
inside the cell membranes and damages the cell.
Treatment of rats with lead acetate in this study caused SO performed bench study, write up and analyses of data. HM
significant reduction in PCV, Hb concentration and RBC count. designed study and OO conceptualized, designed the study and
Our results were in agreement with Azoz and Raafat (2012) write up.

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WHO (2010). Childhood Lead Poisoning. WHO Library Cataloguing-in-Publication Conflict of Interest Statement: The authors declare that the research was
Data. Geneva: World Health Organization. conducted in the absence of any commercial or financial relationships that could
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WHO (2016). Lead Poisoning and Health. Available at: www.who.
int/mediacentre/factsheets/fs379/en/ [accessed 14th July, 2016]. Copyright © 2017 Offor, Mbagwu and Orisakwe. This is an open-access article
Zhou, X., Fragala, M. S., McElhaney, J. E., and Kuchel, G. A. (2010). distributed under the terms of the Creative Commons Attribution License (CC BY).
Conceptual and methodological issues relevant to cytokine and The use, distribution or reproduction in other forums is permitted, provided the
inflammatory marker measurements in clinical research. Curr. Opin. original author(s) or licensor are credited and that the original publication in this
Clin. Nutr. Metab. Care 13, 541–547. doi: 10.1097/MCO.0b013e328 journal is cited, in accordance with accepted academic practice. No use, distribution
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