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Journal of Applied Sciences and Environmental Management
World Bank assisted National Agricultural Research Project (NARP) - University of Port Harcourt
ISSN: 1119-8362
Vol. 5, Num. 1, 2001, pp. 5-8
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Journal of Applied Sciences & Environmental Management,
Vol. 5, No. 1, June, 2001, pp. 5-8
Effect of a Short Time Post Carbon
Tetrachloride Treatment Interval on Rat Plasma Enzyme Levels and Percentage
Mortality
OBI, F.O* +; OMOGBAI, L.A.; ORIAFO, O.S.J.;
OVAT, O.D.
Department of Biochemistry, Faculty of Science, University
of Benin, P.M.B. 1154, Benin City, Edo state, Nigeria
*Corresponding Author
+Present address: Department of Biochemistry, University of Port Harcourt,
P.M.B. 5323, Port Harcourt River State Nigeria.
Code Number: ja01001
ABSTRACT
The effect of a short time (3 hours) post carbon tetrachloride
treatment interval on rat plasma enzyme levels and percentage mortality have
been examined. Relative to their corresponding activities in the plasma
of carbon tetrachloride-free rats the activities of plasma L-aspartate aminotransferase,
L-alanine aminotransferase and alkaline phosphatase of carbon tetrachloride
treated rats were statistically significantly (P<0.05) increased when
the rats were sacrificed 3 hours post exposure. During this period no mortality
occurred. These results indicate that plasma enzyme levels can still be
used as indices of carbon tetrachloride-induced tissue damage within a short
exposure time rather than a longer post-exposure interval, which carries
the risk of an unacceptably high rate of mortality. @ JASEM
Naturally carbon tetrachloride
(CCl4) was believed to be found in the troposphere by solar-induced
photochemical reactions of chlorinated alkanes (Singh et al., 1975)
but this does not appear to be the major source of environmental CCl4. It
has been detected in volcanic emission gases (Isidorov et al., 1990). Several
studies have shown that global atmospheric levels of CCl4 can
be attributed to anthropogenic sources alone (Singh, et al., 1976). CCl4 can
be produced directly by chlorination of methane, methanol, carbon disulphide,
propane, 1,2- dichloroethane and higher hydrocarbons and indirectly as a
by-product during the manufacture of other products and compounds and during
wood pulp bleaching (US EPA, 1984a).
Carbon tetrachloride like a number of other chemicals can cause cell or
tissue necrosis. Tissue damage or death leads to the leakage of the enzyme
produced in the affected tissue(s) into the bloodstream (Jaeger et al., 1975;
Magos et al., 1982; Siegers et al., 1985). Hence serum or plasma enzyme
levels have be used as indices for monitoring chemically induced tissue damages
(Ngaha et al., 1989; Lin and Wang, 1986). Following the administration of
such chemicals to experimental animals a time interval of 12 48 hours is
often allowed to elapse before the surviving animals are sacrificed and the
plasma/serum enzyme activities analysed (De Zwart et al., 1997).
We adopted this long time interval in a number of studies on the effect
of anthocyanin on CCl4 induced liver damage (Obi and Ozoemena,
1998; Obi et al., 1998; Obi and Okokoro, unpublished results) and in our
experience this was usually accompanied by high mortality rate. Therefore,
the purpose of this investigation was to find out whether a short post CCl4 treatment
interval could lead to higher survival rate and reasonable increase in plasma/serum
enzyme levels that could still allow the effect of this chemical agent to
be satisfactorily monitored.
MATERIALS AND METHODS
Animals: Ten white
albino rats (Wistar strain) used for this experiment were obtained from NIMR, Lagos, Nigeria. They
were maintained after purchase for 7 days on rat chow and water ad libitum before
the commencement of the experiment.
Chemicals: Absolute ethanol
and carbon tetrachloride were the products of BDH Chemical Company Ltd (Poole, England)
and Hopkins and Williams respectively. Other materials include corn oil
(Mazola produced for CPC, UK) and rat chow (Pfizer, Nigeria, Plc).
Treatment of animals: The
rats were divided into two experimental groups of 5 rats each. Rats in-group
I (normal control) were given 50% aqueous ethanol by gavage (2.5 ml/kg body
weight) followed by subcutaneous injection of corn oil (1.5 ml/kg body weight). Rats
in group II were given 50% aqueous ethanol followed by 1.5 ml/kg weight of
a 1:1 (v/v mixture of carbon tetrachloride and corn oil via the same routes
described for group I rats.
Preparation of plasma: Three
hours after the last treatment given to each group of rats, each rat was
anaesthetised in chloroform saturated chamber. While under anaesthesia the
thoracic region was opened to expose the heart. Blood was obtained by cardiac
puncture by means of a 5 ml hypodermic syringe and needle and placed in ice-cold
heparinized bottles. The blood was centrifuged at 5000 rpm (Spinette Damon
/ IEC bench top centrifuge) for 5 min. The plasma samples were collected
and left at 20oC until required.
Plasma protein and enzyme assay: Total
plasma protein was quantified by using biuret reagent. L-aspartate aminotransferase
(L-AST) and L-alanine aminotransferase (L-ALT) activities in the plasma were
determined at 37oC by colorimetric method of Reitman and Frankel
(1957). The activities of alkaline phosphates were determined by the colorimetric
method of Plummer (1978) using phenolphthalein monophosphate as substrate. The
enzymes were assayed using reagents obtained from enzyme assay kits (QCA
Laboratories, Spain).
Statistical analysis: The
mean values of the control and test rats plasma activities of a given enzyme
were compared using Students t-test (Elzey, 1971). The significance level
was set at P<0.05.
RESULTS AND DISCUSSION
The percentage mortality and
survival of rats in our earlier studies involving long post CCl4 interval
and that of the present study of 3 hours post CCl4 are presented
in Table 1. Plasma L-AST, L-ALT and alkaline phosphatase activities in the
presence and absence of CCl4 are presented in Table 2. A significant
increase (P<0.05) in the plasma activities of the three enzymes was observed
in the CCl4 treated rats when compared with that of the CCl4 free
(control) rats.
Table 1: Percentage
mortality and survival of rats exposed to CCl4
Expt. #
|
Initial No. of rat
|
Final No of rats
|
Post CCl4 interval (h)
|
% Mortality
|
% Survival
|
Ia*
|
10
|
5
|
18
|
50.0
|
50.0
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IIa*
|
20
|
14
|
18
|
30.0
|
70.0
|
IIIa**
|
16
|
10
|
24
|
37.5
|
62.5
|
IVb**
|
5
|
5
|
3
|
0.0
|
100.0
|
aI, II & III
are previous experiments. {Obi and Ozoemena, 1998 (I); Obi et. al, 1998
(II); Obi and Okokoro-unpublised results (III)}
bIV, present study
*CCl4 administered by intraperitoneal injection (i.p)
**CCl4 administered by subcutaneous injection (s.i)
Table 2: Effect
of CCl4 on plasma enzyme levels 3 hours post CCl4
Group #
|
Treatment
|
Plasma Enzyme Activities
U/L/mean mg protein
Mean + SD (n)a
|
|
|
L AST
|
L ALT
|
AP
|
I(Control)
|
-CCl4
|
2.13 + 0.10(5)
|
7.22 + 0.22(5)
|
1.02 + 0.47(5)
|
II(Test)
|
+CCl4
|
2.51 + 0.14(4)b,
c
|
9.50 + 0.34(4)b
|
1.62 + 0.58(4)b
|
aAP Alkaline phosphatase; n - number of samples
analysed.
bValues statistically significantly higher than the
corresponding control value (P<0.05).
cOne of the samples in this group was lost post-sacrifice. Hence
n = 4.
When CCl4 is administered
to rats orally, intraperitoneally or subcutaneously it is normal practice
to allow 12 48 hours to elapse before tissue and blood analyses (De Zwart
et al., 1997; Reinke et al., 1988). Within this period maximum levels of
serum/plasma L-alanine aminotransferase and L-aspartate aminotransferase
(Teschke et al., 1983; Nakata et al., 1985) and glutamate dehydrogenase (Teschke
et al., 1983) activities have been demonstrated. In our earlier studies
in which a good number of rats died before the expiration of 24 hours the
doses of CCl4 used were in the range of 0.5 1.5ml CCl4/kg
body weight. This dose range is not outrageous. Other investigators have
used 1.5 ml (Teschke et al., 1983) 2.5 ml (Dingell and Heimberg 1968; Moore
et al., 1976) 1.25 ml (Larson and Plaa 1965) and 2.0 ml (Marchand et al.,
(1970) CCl4/kg body weight orally. Dose of 3 ml/kg body weight
has been used (Hase et al., 1996) subcutaneously which is considerably higher
than our dose. Most of these reports are silent on the issue of mortality
of the experimental rat, which in a number of cases, are the same stain of
rat we used. However since our doses are well within the dose range used
by others we are inclined to attribute the death to the long post CCl4 interval
instead of overdose.
Following intraperitoneal or subcutaneous injection of CCl4 the
rats that did not survive started dying barely 4 hours post exposure. Based
on this fact we decided to allow only 3 hours post exposure interval. This
time period as reported by others is sufficient for an oral dose to reach
peak levels in blood, liver, kidney, brain and muscle (Watanabe et al.,
1986; Teschke et al., 1983). As the data presented in Table 1 reveals
this time interval ensured that the rats remained alive until they were sacrificed. The
three enzymes whose activities were analysed are frequently used for assessing
liver injury (Ngaha et al., 1989; Lin and Wang, 1986; Teschke et
al., 1983; Nakata et al., 1985; Magos et al., 1982; Jaeger et
al., 1975; Siegers et al., 1985). Theoretically when an agent damages
an organ the enzymes it elaborates leak into the bloodstream leading to increased
serum or plasma activities of the biomarker enzymes. In order to demonstrate
this change unequivocally sufficient post-exposure time, 12 48 hours, is
allowed (De Zwart et al., 1997; Reinke et al., 1988). In this
study a clear and significant margin was demonstrated between the activities
of the enzyme in the plasma of CCl4 free rats and CCl4 treated
ones. The increased enzyme activities in the plasma of CCl4 treated
rats suggests that the toxicant was able to reach the liver and induce detectable
damage within 3 hours. It may, therefore, be worthwhile to consider and
adopt a short post CCl4 exposure time which not only allows
a high survival rate but a reasonable increase in serum/plasma enzyme levels
for assessing the toxic potency of this chemical.
A number of substances increase the biotransformation of CCl4 and
potentiates its intoxication. Among the substances are high fat diet and
ethanol (Strubelt, 1984; McCay et al., 1984; Reinke et al.,
1988). Ethanol in particular is thought to be responsible for the induction
of the cytochrome P450 isoenzyme involved in CCl4 bioconversion
to metabolites that initiate lipid peroxidation and attendant tissue damage. Therefore
in this study we treated both group of rats with 50% aqueous ethanol simply
to achieve reasonable damage within 3 hours of CCl4 exposure.
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Copyright 2001 - Journal of Applied Sciences & Environmental
Management
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