Open Access Research Journal
www.pradec.eu
Medical and Health Science Journal, MHSJ
ISSN: 1804-1884 (Print) 1805-5014 (Online) Volume 13, 2012, pp.99-104
PREVENTIVE ACTIVITY OF ASCORBIC ACID ON LEAD ACETATE INDUCED
CEREBELLAR DAMAGED IN ADULT WISTAR RATS
Among the environmental contaminants, lead is one of the most
hazardous to living matter. In mammals, the main target is the
central nervous system, particularly in the young. Ascorbic acid is
an antioxidant which is a substance that may protect your cells
against the effects of free radicals.
This study investigated Experiment the protective effect of
ascorbic acid on the cerebellum of adult Wistar rats following oral
administration of different doses of Lead acetate. Thirty adult
Wistar rats of average weight of 215g were used in this study. The
animals were divided into five (5) groups of six animals per group
and were administered different doses of lead acetate (60mg/kg
bwt of 1/10th LD50 and 30mg/kg bwt of 1/20th of LD50) and
ascorbic acid (4.3mg/kg bwt) orally over a period of three (3)
weeks.
Group 1 (control) was administered distilled water and Group 2
and 3 were administered 30mg/kg and 60mg/kg of Lead acetate
respectively while Group 4 and 5 were given co-administration of
30mg/kg of Lead acetate+4.3mg/kg of ascorbic acid and
60gm/kg of Lead acetate+4.3mg/kg of ascorbic acid respectively.
Histopathologically, Lead acetate induced cellular damage in the
cerebellum of adult Wistar rats and it was also observed that
ascorbic acid prevents or minimize lead-induced cellular damage
in the cerebellum of adult Wistar rats.
Keywords:
UDC:
SUNDAY ABRAHAM MUSA,
ILIYASU, MUSA OMONIYE,
WILSON OLIVER HAMMAN,
AUGUSTINE OSELOKA IBEGBU,
UDUAK EMMANUEL UMANA
Department of Human Anatomy,
Faculty of Medicine, Ahmadu Bello
University, Zaria, Nigeria
Lead acetate, ascorbic acid, cellular, cerebellar damage
574:543.9+613.632:612.015
Introduction
Human and animal populations throughout the world are exposed on daily basis to low
levels of environmental contaminants (Bolognesi and Morasso, 2000). The human race is
burdened by an increasing load of environmental contaminants, affecting not just health
and behavior, but finally survival of the species itself. Among these chemicals, lead (Pb) is
one of the most hazardous to living matter. The metal is primarily found in leaded
gasoline, (Srianujata, 1997; Tong, Von Schirnding, and Prapamontol, 2000). Automobile
emissions have been an important source of lead exposure for urban residents, particularly
in areas with congested traffic. The main source of adult human exposure is food, which is
believed to account for over 60% of blood levels; air inhalation accounts for
approximately 30% and water of 10% (John, Cheryl, Richard, and Christine, 1991).
Lead is highly toxic and can interrupt the body’s neurological, biological and cognitive
function. Children are particularly susceptible, and according to the World Health
Organization, high levels of lead exposure can cause brain, liver, nerve, and stomach
damage, as well as permanent intellectual and developmental disabilities (Elombah and
HRW, 2012).
Lead poisoning is rarely fatal, but medical workers in Nigeria reported that the lead
concentration in Zamfara State ore is so toxic that in 2010 in seven villages the mortality
rate was estimated as high as 40 percent among children who showed symptoms of lead
poisoning (Elombah and HRW, 2012). In a study conducted in Jos, Nigeria, risk factors
© 2012 Prague Development Center
- 99 -
Medical and Health Science Journal / MHSJ / ISSN: 1804-1884 (Print) 1805-5014 (Online)
such as the use of lead-containing eye cosmetics, chipping paint in the home and
proximity to battery smelters and gasoline sellers have been identified as the major sources
of high blood lead levels amongst the inhabitants (Wright, Thacher, Pfitzner, Fischer, and
Pettifor, 2005).
The involvement of the nervous system in lead toxicity is well known. Encephalopathy
with Seizures and coma is one of the most striking and serious complication of lead
poisoning (Martin, Paul, and Thomas, 1970). Cerebellar dysfunction may occur in
association with exposure to a wide variety of toxins including heavy metals (such as
mercury, lead, thallium, and manganese), drugs and solvents. These toxins may adversely
affect the cerebellum directly or as part of a more generalized encephalopathy. Practically,
all toxins in high dose can cause neurologic sign and symptoms (Fredericks, 2011).
Permanent abnormalities are induced only by sustained use of exposure of these chemicals
in a greatly excessive dosage and the most sensitive elements of the cerebellar cortex to
these chemicals are the Purkinje cells (Maurice and James, 1972).
The toxic effects of lead are treated with by chelation therapy which also depletes the
body store of essential cations (Ruff, Markowitz, and Bifur, 1996). Therefore there is need
to look for an alternative solution to lead poisoning.
Ascorbic acid is an antioxidant. Antioxidants are substances that may protect our cells
against the effects of free radicals. It is a water-soluble vitamin that is found intra- and
extracellularly as ascorbate (Chihuailaf, Contreras, and Wittwer, 2002). It is a natural
antioxidant that prevents the increased production of Free Radicals induced by oxidative
damage to lipids and lipoproteins in various cellular compartments and tissues (Sies, Stahi,
and Sundqwist, 1992). It has been shown to react directly with superoxide (Nishikimi,
1975; Hemila, Roberts, and Wikstrom, 1985), hydroxyl radicals (Bielski, 1982) and singlet
oxygen (Bodannes and Chan, 1999). It is generally regarded as a primary first-line
protective agent that repairs or nullifies Free Radicals by donating a single electron,
followed by a proton to yield a chemically dehydroascorbic acid (Carr, Zhu, and Frei,
2000; Halliwell, 2001). Ascorbic acid plays a significant role in the toxicity inversion of
lead by forming inert complexes and inhibiting its toxicity on the dopaminergique neurons
(Satija and Vij, 1995).
In this present study we aimed to evaluate the protective effect of ascorbic acid on the
cerebellum of adult Wistar rats following oral administration of Lead acetate.
Materials and methods
Purchase of Chemicals: 500g of Lead acetate of 99% to 103% purity, manufactured by BDH
Chemicals Ltd England and with the product No 29021 was purchased from a reputable
Chemical supplier, Cardinal Scientific, No.11/12 Samaru/Sokoto Road, Opposite
Longman Nig. Plc. The chemical was taken to the Department of Chemistry Ahmadu
Bello University Zaria for identification and it was confirmed to be Lead acetate. Ascorbic
acid (Vitamin C) tablets manufactured by a reputable Pharmaceutical Company Cinamon
Drugs Ltd. Plot C9 Emene Industrial layout, Emene Enugu, Enugu State, were purchased
from a licensed Pharmaceutical Store, Samaru, Zaria, Nigeria.
Experimental Animals: Thirty (30) healthy adult Wistar rats (14-16 weeks old) of average
weight of 215g reared in the Animal House of the Department of Human Anatomy,
Ahmadu Bello University, Zaria, Nigeria, were used for this study. The rats were
acclimatized to experimental condition for a period of two weeks in the same Department
and fed with rat chow and clean tap water.
© 2012 Prague Development Center
- 100 -
Medical and Health Science Journal / MHSJ / ISSN: 1804-1884 (Print) 1805-5014 (Online)
Experimental design: Based on the reported oral LD50 of Lead acetate for Wistar rats by
Sujatha et al., 2011, we administered 1/10th of LD50 (60mg of lead acetate/kg body
weight) and 1/20th of LD50 (30mg of lead acetate/kg body weight) in this study.
Route and duration of administration: Lead acetate and ascorbic acid were administered orally
by gastric gavage once per day for three weeks.
TABLE 1. SHOWING THE GROUPING AND THE TREATMENT OF THE ANIMALS
Groups
1
2
3
4
5
No of animals
6
6
6
6
6
Treatment (mg/kg body weight)
Distilled water (control)
30mg/kg Lead acetate
60mg/kg Lead acetate
30mg/kg Lead acetate + 4.3mg/kg Ascorbic acid
60mg/kg Lead acetate + 4.3mg/kg Ascorbic acid
Animals Sacrifice: the weights of the animal were taken with a laboratory weighing balance.
The animals were then anaesthetized through chloroform inhalation. Incision was made
through the skin and muscle of the skull. The skull was opened through the mid sagittal
suture and the cerebellum was removed and fixed in Bouin’s fluid and the tissues were
process for histological study using bench model Automatic Tissue Processor, available in
the histology laboratory of Human Anatomy Department, Ahmadu Bello University,
Zaria. The processed tissues were embedded in paraffin and stained in Haematoxylin and
Eosin and viewed under a light power microscope and photomicrographs were taken
using digital optical eyepiece.
Results
Photomicrographs
FIGURE1. PHOTOMICROGRAPH OF GROUP 1 (CONTROL), FIGURE 2. PHOTOMICROGRAPH OF GROUP 2 (30MG/KG
BW OF LEAD ACETATE), TRANSVERSE SECTION OF
TRANSVERSE SECTION OF CEREBELLUM. SHOWING THE
MOLECULAR LAYER (ML), PURKINJE LAYER (PL) AND CEREBELLUM. SHOWING THE MOLECULAR LAYER (ML),
GRANULAR LAYER (GL). H & E, MG X 250
PURKINJE LAYER (PL) AND GRANULAR LAYER (GL).
H & E STAIN X 250
© 2012 Prague Development Center
- 101 -
Medical and Health Science Journal / MHSJ / ISSN: 1804-1884 (Print) 1805-5014 (Online)
FIGURE 3. PHOTOMICROGRAPH OF GROUP 3 (60MG/KG FIGURE 4. PHOTOMICROGRAPH OF GROUP 4 (30MG/KG
BW OF LEAD ACETATE), TRANSVERSE SECTION OF
BW OF LEAD ACETATE + 4.3MG/KG ASCORBIC ACID),
CEREBELLUM. SHOWING THE MOLECULAR LAYER (ML), TRANSVERSE SECTION OF CEREBELLUM. SHOWING THE
PURKINJE LAYER (PL) AND GRANULAR LAYER (GL). H & MOLECULAR LAYER (ML), PURKINJE LAYER (PL) AND
E, MG X 250
GRANULAR LAYER (GL). H & E, MG X 100
FIGURE 5. PHOTOMICROGRAPH OF GROUP 5 (60MG/KG
BW OF LEAD ACETATE + 4.3MG/KG ASCORBIC ACID),
TRANSVERSE SECTION OF CEREBELLUM. SHOWING THE
MOLECULAR LAYER (ML), PURKINJE LAYER (PL) AND
GRANULAR LAYER (GL). H & E STAIN, MG X 250
Histopathological studies
No histopathological changes were observes in control group 1, and all the layers of the
cerebellar cortex were intact as seen in Figures 1 above. In experimental group 2 which
was administered 30mg/kg BW of Lead acetate; there was cellular degeneration of cells in
the Purkinje layers. In experimental group 3 administered 60mg/kg BW of Lead acetate,
there was more pronounced cellular damaged of the Purkinje and granular layers than in
group two. While in the co-administration groups 4 and 5 there were lesser cellular
damage of Purkinje and granular layers in these groups when compare with group two and
three. In group 5 there was more protection than group 4. The molecular layer seems not
to be affected at all in group 5; this is due the protective of ascorbic acid.
© 2012 Prague Development Center
- 102 -
Medical and Health Science Journal / MHSJ / ISSN: 1804-1884 (Print) 1805-5014 (Online)
Discussion
In this study, cellular damage of the cerebellar layers of adult Wistar rats was induced by
different dosages of lead acetate as showed above in Photomicrographs. This observation
agrees with the reports of Maurice and James (1972), that stated that many heavy metals
such as lead, mercury, etc and other organic compounds have the capacity to damage
nervous system and the most sensitive elements of the cerebellar cortex to these chemicals
are the purkinje cells in the middle purkinje layer; The report was also supported by the
work of Fredericks, 2011. Our study further confirmed these two findings. Cerebellar
dysfunctions may occur in association with exposure to a wide variety of toxins including
heavy metals and Martin et al., 1970: the involvement of the nervous system is well
known. Encephalopathy with Seizures and coma is one of the most striking and serious
complication of lead poisoning as earlier documented. Even at low BPb levels, lead
exposure has been associated with deficits in the early developmental years. Canfield et
al.’s study (2003) on the effects of low level lead poisoning (<10 µg/dL) on cognitive
functioning in children and infants, found that IQ declined 7.4 points as average blood
lead concentrations increased from 1 µg/dL to 10 µg/dL, and then declined 4.6 points for
every 10 µg/dL increase after that. Lead exposure has also been linked to behavioral
problems. In his landmark study, Needleman et al., (1979) measured dentine Pb levels in
312 first-and second-grade students (mean age 7.3 years), and administered
neurobehavioral tests. The results showed that behavioral and performance problems in
primary school children were more prevalent with increasing dentine (teeth) lead levels.
These behavioral problems included hyperactivity, distractibility, impulsivity,
disorganization, non-persistence, inability to follow simple instructions and overall poor
functioning. Lead elicits it´s activity through the generation of free radicals which are very
harmful to the cells in the living system. Ascorbic acid is generally regarded as a primary
first-line protective agent that repairs or nullifies Free Radicals by donating a single
electron, followed by a proton to yield a chemically dehydroascorbic acid (Carr et al.,
2000; Halliwell, 2001). Ascorbic acid plays a significant role in the toxicity inversion of
lead by forming inert complexes and inhibiting its toxicity on the dopaminergique neurons
(Satija and Vij, 1995). The scavenging activities of ascorbic acid was demonstrated in these
present study. The groups co-administered with ascorbic acid show some degree of
protection to the cells of the cerebellum.
In conclusion, it is also established in this study that administration of ascorbic acid has a
clear improvement in preventing the degeneration of brain cells when compare with rats
exposed to lead acetate. Lead exposure induced a significant degeneration in the
cerebellum of adult Wistar rats and the need to prevent the neurotoxicity induced by lead
acetate using ascorbic acids has been established. And the preventive effect of ascorbic
acid was dose dependent. The damage that lead poisoning can have on the
neuropsychological functioning of the central nervous system can be devastating. It can
affect anybody, at any age, although children are most often the worst affected, as their
cognitive development can be disrupted. Now we are beginning to realize that there are
consequences beyond what was once thought the neurological endpoint for this harmful
substance, and hopefully we will one day fully understand the mechanisms by which lead
destroys the mind. It is also established in this study that administration of ascorbic acid in
groups four and five have a clear improvement in preventing cerebellar degeneration
when compare with rats exposed to lead acetate only (group two and three). Our study
further validate the antioxidant activity of ascorbic acid as earlier reported by other
studies.
Recommendations
From our findings in the study we are recommending that the preventive activity of
ascorbic and other antioxidants on Lead acetate induced cerebellar degeneration should
further be investigated on a large sample size of experimental animals. The use of Lead
should be minimize or avoided, especially in materials such as petroleum fuel. Also
analysis of the components of natural ores should be carried out before extraction;
consumers of lead containing products such as paints, house dusts, pesticides, and some
industrial products such as glazes, colorings for ceramics, hose and pipes should always be
conscious of the percentage of lead in them and people in urban residents particularly in
© 2012 Prague Development Center
- 103 -
Medical and Health Science Journal / MHSJ / ISSN: 1804-1884 (Print) 1805-5014 (Online)
area with congested traffic and those using products containing Lead should eat food rich
in ascorbic acid and other antioxidants.
References
Bielski, B., 1982. “Chemistry of ascorbic acid radicals,” in: Seiob, P. and Tolbert, B. (eds.), Ascorbic acid:
Chemistry, metabolism and uses, Washington DC: American Chem. Soc., pp.81-100
Bodannes, R., Chann, P., 1979. “Ascorbic acid as a scavenger of singlet oxygen,” FFBS Letter, Vol.105,
pp.195-96
Bolognesi, C., Morasso, G., 2000. “Genotoxicity of pesticides: Potential risk for consumers,” Trends in Food
Science and Technology, Vol.11(4-5), pp.182-87
Canfield, R., Henderson, C., Slechta, D., Cox, C., Jusko, T. and Lanphear, B., 2003. “Intellectual impairment in
children with blood lead concentrations below 10µg per decilitre,” The New England Journal of Medicine,
Vol.348(16), pp.1517-526
Carr, A., Zhu, B., Frei, B., 2000. “Potential antiatherogenic mechanisms of ascorbate and alpha-tocopherol,”
Circulation Research, Vol.87, pp.349-54
Chihuailaf, R., Contreras, P., Wittwer, F., 2002. “Pathogenesis of oxidative stress: Consequences and
evaluation in animal health,” Vet Mex., Vol.33(3), pp.265-83
Elombah. com and HRW. (2012). Nigeria’s Child Lead Poisoning Crisis is ‘worst in modern history’ HRW.
Fredericks, CM., 2011. “Disorders of the cerebellum and its connections,” in: Saladin LK, Fredericks CM,
Pathophysiology of the motor systems: principles and clinical presentations, Philadelphia: F.A. Davis,
pp.445-66
Halliwell, B., 2001. “Role of free radicals in the neurodegenerative diseases: Therapeutic implications for
antioxidant treatment,” Drugs Ageing, Vol.18(9), pp.685-716
Hemila, H., Roberts, P., Wikstrom, M., 1985. “Activated polymorphonuclear leucocytes consume vitamin C,”
FEBS Letter, Vol.178, pp.25-30
John, H., Cheryl, H., Richard, S., Christine, S., 1991. Toxics: A to Z - A guide to everyday pollution hazards,
Berkeley: University of California Press
Martin, R., Paul, M., Thomas, W., 1970. An Appraisal of Rodent models of lead encephalopathy. In
neurotoxicology, Vol.1 edited by Leon, R. and Hirotsugu, S., New York: Raven press
Maurice, V. and James, A., 1972. The nutritional and metabolic diseases of the cerebellum. Clinical and
pathological aspects. In the cerebellum in health and diseases, edited by: William, S. and William, D.,
London: Adam Hilger, pp.412-49
Needleman, H., Schell, A., Bellinger, D., Leviton, A., Allred, E., 1990. “The long-term effects of
exposure to low doses of lead in childhood: An 11-year follow-up report,” The New England Journal of
Medicine, Vol.322(2), pp.83-88
Nishikimi, M., 1975. “Oxidation of ascorbic acid with superoxide anion generated by thexanthine-xanthine
oxidase system,” Biochemistry and Biophysics Research Communication, Vol.63, pp.463-68
Ruff, H., Markowitz, M., Bifur, P., 1996. “Relationships among blood lead levels, iron deficiency, and
cognitive development in two-year-old children,” Envoron. Health Perspect., Vol.104(2), pp.180-85
Satija, N., Vij, A., 1995. “Preventive action of zinc against lead toxicity,” India Journal of Physiol Pharmacol,
Vol.39, pp.377-82
Sies, H., Stahi, K., Sundqwist, A., 1992. “Antioxidant functions of vitamins vitamin E and C, betacarotene,
and other carotenoids,” Annals of New York Academy of Science, Vol.669, pp.7-20
Srianujata, S., 1997. “Lead, the toxic mental to stay with human,” Journal of Toxicological Sciences, Vol.23,
pp.237-40
Sujatha, K., Srilatha, C., Anjaneyulu, Y., Amaravathi, P., 2011. “Lead acetate induced neurotoxicity in wistar
albino rats: A pathological, immunological, and ultrastructural studies,” Journal of pharma and bio science
Vol.2, pp.459-62
Tong, S., Von Schirnding, Y., Prapamontol, T., 2000. “Environmental lead exposure: A public health problem
of global dimensions,” Bulletin WHO, Vol.78(9), pp.1068-1077
Wright, N., Thacher, T., Pfitzner, M., Fischer, P., Pettifor, J., 2005. “Causes of lead toxicity in a Nigerian city,”
Archives of Disease in Childhood, Vol.90(3), pp.262-66
© 2012 Prague Development Center
- 104 -