Journal of Environmental Protection, 2010, 1, 475-479
doi:10.4236/jep.2010.14055 Published Online December 2010 (
Copyright © 2010 SciRes. JEP
Pollution Tolerance of Smoke in the Distribution of
Neurotransmitter Enzyme (Acetylcholine Esterase)
and Total Cholesterol in Tissues of Wistar Rats
Albert C. Achudume, Funso Aina, Bill Onibere
Obafemi Awolowo University, Il-eIfe Institute of Ecology and Environmental Studies, Ile-Ife, Nigeria.
Received June 19th, 2010; revised August 16th, 2010; accepted August 20th, 2010.
This study was designed to assess total animal exposure to non-occupational but environmentally induced smoke
through short-term landfill burning toxicity tests at the biochemical levels. Exposure to municipal land-fill burning us-
ing rat model focused primarily on inhalation exposure. The environmental monitoring consisted of 60 days exposure to
refuse burning by evaluating the level of protein concentrations, neurotransmitter enzyme acetylcholine esterase
(AcHE), and total cholesterol in different tissues of Wistar rats. Protein concentrations tended to decrease in the brain,
liver and kidney and slightly increased in the plasma while acetylcholine esterase decreased in brain and liver and in-
creased in the kidney. The non-depletion in total cholesterol levels in the tissues tended to be due to active mobilization
towards tissue metabolism. The data were sufficient to support risk assessment for human.
Keywords: Municipal Land-Fill, Smoke Pollutants, Acetylcholine Esterase, Total Cholesterol
1. Introduction
The ever increasing population due to rapid industrializa-
tion and technological advancements, traffic congestion
resulting from increase in motor vehicles and urbaniza-
tion contribute to a large extent toxic smoke pollution [1].
The degree of intensity and composition of the pollutants
vary depending on the level of economic development
and industrialization [2]. Owing to frequent power out-
ages experience in Nigeria, a number of households and
commercial establishments use electric generators. The
smoke releases from these generators change the atmos-
pheric composition of air in the immediate environment
and affect the wellbeing of residents [3,4]. Again, the
dependency of indigenes on use of charcoal pot and
sawdust packed stove as energy source are major causes
of indoor/outdoor smoke pollution [5,6].
Inadequate technology still permits waste refuse to be
dumped in land-fills. Some people here-in referred to as
“Human scavengers” manually sort for various reposi-
tory materials. During sorting in the mid of smoke,
scavengers prowl for assorted goods and many more
squat in the vicinity. Waste refuse in land-fill may con-
sist of inorganic and organic matters and other toxic sub-
stances such as disposed asbestos, fabrics and pharma-
ceutical products. Urban dumping most times contains
surprisingly high particulate matter (PM). What is more,
National Agency for Food and Drug Administration and
Control (NAFDAC) burned sizeable drugs in open air [7].
The byproducts of various noxious chemicals including
heavy metals pose a serious threat to the environment,
animals and humans in the surrounding areas [8].
Exposures to particulate matters have long been known
as a serious health threat [9]. New information suggests
that pollutants in smoke are more toxic than previously
thought [6,10]. Particle pollution is a silent killer [3,11].
Short-term exposure to low levels of particulates smoke
pollution may increase the risk of stroke or mini-stroke
symptoms [12] and acute chronic bronchitis [13] and
heart disease [14].
Evaluating such working environment is essential. It is
generally required to identify hazards that are present and
implement controls such as presorting of hazardous ma-
terials, use of personal protective devices and more ef-
fective control of burning refuse. The likelihood of ex-
posure to decomposition products may affect the basal
biochemical mechanisms that may ultimately cause nu-
merous public health problems. In the present study, only
Pollution Tolerance of Smoke in the Distribution of Neurotransmitter Enzyme (Acetylcholine Esterase) and
Total Cholesterol in Tissues of Wistar Rats
Copyright © 2010 SciRes. JEP
the results for neurotransmitter enzyme acetylcholine
esterase and total cholesterol were given.
2. Materials and Methods
Wistar rats, 8 to 10 wks, weighing 190 ± 12 g were pro-
cured from the animal house, Department of Anatomy
and Cell Biology, Obafemi Awolowo University, Ile-Ife,
Nigeria and acclimatized to laboratory conditions (tem-
perature 24 ± 3, humidity 30-70% and 12 hr light, 12
hr dark rhythm) for a week prior to the experiment.
The animals (30 males) were randomized into 3
groups. Group 1 was placed in the center of the smoke
polluted area of the Ojota Municipal Refuse land-fill
bearing in mind the unidirectional smoke wind for 60
days. Group 2 was placed in abandoned refuse land-fill
area, thus served as cham control while the third was
housed two kilometers from refuse land-fill. Twenty-four
hours after the end of the exposure animals were sacri-
ficed by cardiac puncture. Blood was collected in non-
heparinised tubes. The plasma was used for biochemical
analysis. The brain, liver and the kidney were dissected
out, rinsed and separately homogenized in phosphate
buffer 0.1 µg/ml, pH 7.4. The homogenates were used
for biochemical analysis.
Plasma 0.4ml aliquot was incubated in a mixture of
5:5-dithiobisnitrobenzoic acid (DTNB), 100 µl, and ace-
tylcholine chloride 20 µl (AchCl) as substrate. The sub-
strate decomposition was measured after 2 min stabiliza-
tion. Each tissue homogenate was treated as above fol-
lowing the methods of Kuhnen [15]. Absorbance was
read at 412 nm.
Catalase activity (CAT) in the organs was estimated
by the methods given by Aebi [16] with slight modifica-
tion. 0.2 gm of fresh tissue was homogenized in 2 ml of
extraction buffer under cold conditions. The homogenate
was centrifuged at 10,000 rpm for 20 min at 40. The
supernatant was used for quick assay. Catalase activity
was determined by observing the disappearance of H2O2
by measuring the spectrophotometer. Reaction, carried
out in the total volume of H2O2, was allowed to run for
3min. Activity was calculated by using extinction coeffi-
cient () 0.036 mM-1 Cm-1.
Superoxide dismutase (SOD) activity was measured
by the method described by Dhindsa [17]. 0.2 g of liver
was homogenized in 2.0 ml of extracting buffer in a pre-
cooled mortar and pestle. The homogenate was centri-
fuged at 15,000 rpm at 4 and supernatant was stored at
4. And supernatant was assayed by its ability to inhibit
photochemical reduction of nitroblue tetrazolium. The
test tubes containing assay mixture (1.5 reaction buffer,
0.2 ml of methionine, 0.1 ml enzyme extract with equal
amount of NaCO3, NBT solution, riboflavin, EDTA and
1.0 ml DDW) were incubated in light under 15 W inflo-
rescent lamps for 15min, illuminated and non-illuminated
reactions without supernatant served as calibration stan-
dard. Absorbance was read at 560nm wavelength. One
unit of enzyme activity was defined as the quantity of
enzyme that reduced the absorbance reading of samples
to 50% in comparison with blank.
Glutathione reductase (GR) activity was determined
by the methods of Foyer and Halliwell [18]. 0.5 g of
fresh liver was ground in 2 ml of extraction buffer and
centrifuged at 10,000 rpm for 10 mins. The supernatant
was collected and used for assay. GR activity was deter-
mined by monitoring the glutathione-dependent oxida-
tion of NADP at 340 nm wavelength on spectropho-
tometer. 1ml reaction mixture containing NADP, GSSG
and enzyme extract was allowed to run for 3 min at 25.
Corrections were made for any GSSG oxidation in the
absence of NADP. Activity was calculated by using ex-
tinction coefficient () 6.2 mM-1 Cm-1, and expressed in
enzyme units (mg protein)-1. One unit of enzyme is the
amount necessary to decompose 1 µmol of NADP per
min at 25.
Total cholesterol was estimated by modification of the
methods of Abell et al. [19]. Samples of each tissue (0.4
ml) were added to 2.0 ml of alcoholic potassium hydrox-
ide. The mixture was incubated at 37 for 1hr. Five ml
of petroleum ether was added to the mixture after cooling
to room temperature followed by distilled water (2.0 ml).
The mixture was centrifuged at low speed. Aliquot
(4.0ml) of the petroleum ether layers was vaporized by
heating to 60 and thermostated to 25 allowing 10
min for the temperature to stabilize. After 30 s, 60 ml of
Leiberman-Burchard reagent was added. The mixture
was allowed to stabilize to room temperature, the ab-
sorbance (A) was read at 620 nm.
The experimental data was analyzed statistically, mean
and standard error (S.E.) was calculated. Three-ways
ANOVA was used to assess the enzyme activity and
cholesterol level in the tissues. Level of significance was
set at 0.05%.
3. Results and Discussion
On the basis of comparative study of the tolerance to
smoke, four major organs were analyzed. The values for
protein concentrations, acetylcholine esterase and total
cholesterol levels obtained in the present study are in
agreement with the characteristics of other studies as
reported by Walker and Mackness [20-22]. The plasma
in addition to the liver and kidney, seems to be that ani-
mals did not show any variation in total cholesterol levels
among the groups except in the brain with a slight de-
crease, however all groups in the control showed higher
Pollution Tolerance of Smoke in the Distribution of Neurotransmitter Enzyme (Acetylcholine Esterase) and
Total Cholesterol in Tissues of Wistar Rats
Copyright © 2010 SciRes. JEP
increases in protein concentrations, total cholesterol and
AcHE activity (Table 1). The sham values were rela-
tively the same as the actual control in all tissues inves-
tigated. Decreased protein concentrations and acetylcho-
line esterase activity in brain and liver were significant at
P < 0.05. There was a noticeable increase in AcHE activ-
ity in the kidney and considerable decrease in protein
concentrations. Consequently, the levels of protein con-
centrations and AcHE activity were recalculated on the
basis of the number of animals and these values were
compared with the original values. It was found that cor-
rection for protein concentrations and AcHE activity did
not in general, change the outcome of the analysis of
The biochemical analysis of the different tissues pro-
vides valuable information on the status and condition of
mammals. Acetylcholine esterase is a biomarker of neu-
ro-toxic syndrome [23]. Other chemicals like allopurinol
[24], doxorubin [25] on oxidative stress, while, nicotine
and muscarine , though, do not inhibit the enzyme choli-
nesterase directly [20], but act on Ach receptor subtypes,
as agonists hence decreased levels are observed and may
be associated with congenital deficiency and liver disease
[26]. The decrease level of protein in the liver may be an
indication of protonuria [14]. Liver is the major site of
protein synthesis and any observable defect could be an
onset of ill health (Table 1) whereas the slight increase
in the plasma suggests residual protein. In this present
study, the decrease activity of AcHE in the brain indi-
cates onset of neuropath as already charted in animals
poisoned by smoke pollution [4,27]. The AcHE reactive
sites are widely distributed in liver and kidney cytoplasm
and nuclei [28], the inhibition of activity of this enzyme
could affect the cell function, metabolism and signal
The non-depletion in total cholesterol may be due to
their active mobilization towards tissue metabolism
[21,22]. It may also be due to the utilization of triglyc-
erides to meet the additional energy requirement under
stress [10,29]. The overall biochemical parameters and
the prevailing environment including quality of ambient
air cannot be ignored; these factors ultimately regulate
the internal atmosphere of organisms. Since all the ani-
mals were subjected to the same environmental condi-
tions, the pollution tolerance of the smoke in the distribu-
tion of AcHE and cholesterol in the tissues may contrib-
ute towards an estimate of the environmental burden of
stress. Short-term exposure to low levels of particulate
smoke pollution may increase the risk of stroke [1,11,12];
and large majority of the public may be exposed to py-
thoria of potentially toxic pollutants. The limitation of
this study may not be unconnected with unidirectional of
Table 1. Mean ± SD of protein concentrations, acetylcholine
esterase activity and total cholesterol in different tissues of
wister rat exposed to smoke pollution.
Treatment Protein concentration
AcHE activity
Total cholesterol
Control0 0.85 ± 0.11 0.81 ± 0.05 0.17 ± 0.09
Plasma 0.88 ± 0.26 0.84 ± 0.04 0.18 ± 0.01
Control¹ 0.16 ± 0.01 0.90 ± 0.13 0.18 ± 0.06
Brain 0.02 ± 0.1* 0.79 ± 0.03* 0.16 ± 0.02
Control² 0.42 ± 0.3 0.83 ± 0.05 0.15 ± 0.08
Liver 0.16 ± 0.02+ 0.75 ± 0.06 0.15 ± 0.07
Control³ 0.31 ± 0.05 0.94 ± 0.00 0.15 ± 0.02
Kidney 0.21 ± 0.08 0.96 ± 0.02 0.15 ± 0.10
*Significantly different from control1 (p< 0 .05); +Significantly different
from control2 (p < 0.05)
smoke particulates. Nobody knows the level of risk that
may be associated with the chemical cocktail of pharma-
ceutics’ burning in refuse that are found in the air. In
order words, open burning of refuse is not safe, meaning
that the real risks to human remain entirely unknown.
Proper environmental management is the key to avoid-
ing all preventable illness which are directly caused by
environmental factors. The environment influences
health in many ways; through exposures to physical,
chemical and biological risk factors in smoke [30]. In
this respect, official regulatory commission should be
established to monitor daily air pollutions and environ-
mental quality’s operations of particulate matter includ-
ing waste land-fill refuse burning, risk management and
health effects of air pollution, in order to have opera-
tional database. Finally, governments at appropriate level,
should, where necessary, enhance institutional capacity
of local government to procure incinerating facilities and
equipment to sustain development and public health. The
semi-aerobic land-fill technology provide four basic
concepts in sustainable development vis-à-vis to provide
indicator system; to evaluate the degree of stabilization;
to manage the land-fill and finally to direct its ultimate
reuse. At present, there are sufficient data to examine any
deterministic explanation. Additionally, land-fill features
associated with illness, will serve as the basis of working
hypothesis. The latter will only be testable through the
collection of further data on land-fill pollutants.
The advances in these studies indicate that smoke can
cause cancer cells to proliferate faster, slow kidney cell
growth and cause inflammation in blood cells [6,12,14,
31,32]. At a time when most African population is suf-
Pollution Tolerance of Smoke in the Distribution of Neurotransmitter Enzyme (Acetylcholine Esterase) and
Total Cholesterol in Tissues of Wistar Rats
Copyright © 2010 SciRes. JEP
fering from premature deaths, it seems outrageous that
health authorities would not be looking closely at this
issue and working on ways to protect the public from
smoke pollution. In conclusion, the root causes of envi-
ronmental degradation can be found in social and eco-
nomic problems such as poverty, inequality of wealth,
the debt burden and unsustainable production and con-
sumption behaviors.
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Pollution Tolerance of Smoke in the Distribution of Neurotransmitter Enzyme (Acetylcholine Esterase) and
Total Cholesterol in Tissues of Wistar Rats
Copyright © 2010 SciRes. JEP
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