Niger. J. Physiol. Sci.

26(December 2011) 151 – 160

www.njps.com.ng

Biochemical and histologic presentations of female wistar rats

administered with different doses of paracetamol/methionine

1*
Iyanda A.A. and 2Adeniyi F.A.A.

Department of Chemical Pathology, 1College of Health Sciences, Ladoke Akintola University of Technology,
Osogbo. 2College of Medicine, University of Ibadan, Ibadan, Nigeria

Summary: This study was carried out to compare the hepatoprotective effect of methionine on paracetamol treated rats at
both the peaks of toxicity and absorption. Female Wistar rats were divided into 17 groups consisting of eight rats per group
and treated with different doses of paracetamol/methionine (5:1). Each control rat received 5 ml of physiologic saline. The
study was terminated at two different end points –the 4th & 16th hours. Results show that rats administered with toxic doses
(1000 mg/kg; 3000 mg/kg; 5000 mg/kg BW) of paracetamol exhibited significant increases (p<0.05) in the levels of ALT,
AST, γ- GT compared with controls. These increases were much higher at the 16 th than 4th hour but serum total protein,
albumin and globulin were significantly decreased (p<0.05) by the end of the 16th hour. Histology results of rats in the
3000 & 5000 mg/kg (by the end of the 16th hour) confirmed hepatic damage; light microscopic evaluation of liver showed
remarkable centrilobular necrosis. Moreover, the presence of mononuclear cells in liver section of rats intoxicated with
APAP (5000 mg/kg) suggests a possible involvement of inflammatory process which resulted in regurgitation of bilirubin
leading to its elevated level as well as increase activity of ALP. The hepatoprotective effect of methionine, on the other
hand, was demonstrated in these rats at the 4th & 16th hours, and both results were comparable and therefore not
significantly different (p>0.05) but elevation in GGT level still persisted. In conclusion, data obtained from this study
suggest that these agents may be capable of inducing GGT, although further study is required to establish a possible
relationship between methionine and this enzyme in some other animal species.

Keywords: Paracetamol, Toxicity, Absorption, Liver biochemistry, Liver histology

©Physiological Society of Nigeria

*Address for correspondence: lapeiyanda@yahoo.com

Manuscript Accepted: August, 2011

INTRODUCTION benzoquinoneimine (NAPQI) by cytochrome P450

(Mitchell et al., 1973; Jollow et al., 1973).
Paracetamol (acetaminophen), an over-the-counter Glutathione binds to and detoxifies NAPQI but at
drug, is widely used for the treatment of mild pain toxic level hepatic GSH depletion occurs when
and pyrexia. Because of its over-the-counter status, NAPQI formation exceeds the available supply of
cases of both accidental and intentional paracetamol GSH. The undetoxified NAPQI eventually binds to
(APAP) overdose are numerous, with as many as critical cellular macromolecules (Park et al., 2005)
26,000 subjects being hospitalized each year in the e.g. cellular proteins (Nelson and Bruschi, 2003;
United States of America (Nourjah et al., 2006). As a Bulera et al., 1996), resulting in impairment in
result of this high rate of abuse, paracetamol has been mitochondrial respiration (Meyers et al., 1988;
described by Larson et al. (2005) as the most Burcham and Harman, 1991), opening of the
common cause of drug induced liver failure in the mitochondrial permeability transition pore
United States. At overdose level, paracetamol causes (Masubuchi et al., 2005), elevation of the oxidative
hepatic centrilobular necrosis (McJunkin et al., 1976) stress (Bajt et al., 2004) as well as hepatic necrosis.
which has been linked to excessive generation of N- Kon et al. (2004) have also observed that
acetyl-p-benzoquinoneimine (NAPQI). Paracetamol mitochondria may be the primary targets in
(APAP) is oxidatively transformed to N-acetyl-p- acetaminophen (APAP) toxicity with particular
 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

attention on the mitochondrial permeability the experiment. On the day of the experiment,
transition. Involvement of other generated reactive paracetamol (APAP) obtained from Sigma-Aldrich
oxygen species such as nitric oxide and superoxide Chemicals (St. Louis, MO) was dissolved in
anion cannot also be discounted in paracetamol- physiologic saline obtained from Unique
induced hepatocyte death (Hinson et al., 2004). Apart Pharmaceuticals, Sango-Ota, Ogun State. Each
from the hepatocytes, Lawson et al. (2000); Gardner control rat received 5 ml of saline while each APAP-
et al. (2003); Liu et al. (1994) have suggested the treated rat or APAP/methionine treated-rat received
contribution of non- parenchymal cells such as appropriate amount of the drug dissolved in saline.
Kupffer cells, Natural killer cells, and neutrophils that The APAP or APAP/methionine (ratio of 5:1,
secrete cytokines and chemokines during according to the study of Neuvonen et al., 1985)
acetaminophen-induced liver injury. doses employed for the study were 350mg\kg
Results obtained from studies of Henderson et al. (subtoxic dose, Abraham, 2004), 1000, 3000, 5000
(2000) and Dahlin et al. (1984) have also indicated mg\kg (toxic doses, Abraham, 2004; Trumper et al.,
that liver toxicity is initiated by P450-mediated 1992, Grypioti, 2005) body weight paracetamol.
reactions that convert APAP to the reactive The experiment was terminated at two-end
electrophile, N-acetyl-p-benzoquinone imine periods, the 4th hour, the peak of absorption (Lewis &
(NAPQI), leading to glutathione depletion and Paloucek, 1991; Albert et al., 1974; Zarro, 1987) and
covalent binding (Henderson et al., 2000). This the 16th hour, the peak of toxicity (Trumper et al.,
invariably results in mitochondria, cell membranes 1992). The peak of paracetamol toxicity has been
and nuclei damage. Disruption of cell death- and reported to be between the 16th and the 24th hours.
survival-related signalling pathways causes extensive Route of administration was by gastric gavage. At the
necrosis and apoptosis (Lauterburg and Mitchell, end of both the 4th and the 16th hours, blood was
1982). Apart from CYP2E1, the major isoform in collected by retro-orbital bleeding. The liver from
biotransformation of paracetamol, other P450s such each animal was promptly removed and preserved in
as CYP1A2, CYP2A6 and CYP3A, have been formalin. Serum was separated immediately through
identified as APAP-metabolizing enzymes. The centrifugation at 3000 r.p.m. for the determination of
involvement of generated Reactive Oxygen Species liver enzymes (alanine amino transferase, aspartate
(ROS), such as superoxide anion (O 2-•), hydrogen amino transferase, alkaline phosphatase, γ-glutamyl
peroxide (H2O2) and hydroxyl radical (HO•), reactive transferase), bilirubin, total protein and albumin. All
nitrogen species (RNS), such as nitric oxide and experimental protocols complied with Institutional
peroxynitrite (ONOO-), and peroxidation reaction Ethical Committee guidelines as well as
products have been highlighted in mechanisms internationally accepted principles for laboratory
associated with APAP-induced hepatotoxicity (James animal use and care as found in US guidelines (NIH
et al., 2003a; James et al., 2003b; Reid et al., 2005; publication\85-23, revised in 1985).
Bessems and Vermeulen, 2001).
A number of antidotes have been suggested for the Analytical Methods
treatment of APAP-induced liver damage, one of Creatinine was estimated by the Jaffé reaction while
which is methionine. The aim of this study is to the level of urea was also measured by the diacetyl
determine the hepatoprotective effect of methionine monoxime oxidase method. Activities of alanine
on female Wistar rats concurrently administered with aminotransferase & aspartate aminotransferase (AST
toxic doses of paracetamol and methionine in the & ALT) were obtained by the method of Bergmeyer
ratio of 5: 1, using liver enzymes (AST, ALT, ALP, et al; 1978, alkaline phosphatase (ALP) was
GGT); total protein, albumin and liver histology as measured by method of Mc Comb and Bowers
indices of study and to compare the effect at the peak (1972). On the other hand, bilirubin and albumin
of absorption with that of peak of toxicity. were determined using modified Jendrassik-Groff
(Koch and Doumas, 1982) and standard
MATERIALS AND METHODS bromocresol method respectively. Total proteins
were measured using Biuret’s method (Kingsley,
Animals/Experimental design 1982). Hitachi® 902 Automated machine supplied
Female Wistar rats weighing between 250-350 g by Roche Diagnostic, Germany was used for these
obtained from the animal house of the Department of estimations.
Veterinary Physiology, University of Ibadan were
utilized for this study. They were kept in cages and Tissue histology
maintained on standard diet and supplied water ad The hepatic tissues were processed using paraffin
libitum. The rats were divided into 17 groups embedding method; sections of five micron thickness
consisting of eight rats per group. A two-week were processed in a microtome. Slides were
acclimation period was allowed before initiation of examined under the microscope to detect histologic
Hepatic presentation of female rats dosed with paracetamol/methionine 152
 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

changes subsequent to staining with haematoxylin All the hepatic indices used to assess hepatic
and eosin (H & E). damage in rats administered with
paracetamol/methionine show non-significant
Statistical Analysis difference (p>0.05) at all levels of exposure and at
Results are expressed as mean ± standard deviation both end-points compared with control as shown in
(SD). Level of significant difference between each of Table 3 except for 1000 mg/kg. Table 4 also shows
APAP or APAP/methionine treated group and control non-significant difference (p>0.05) between serum
was determined using Student ‘s T test, Analysis Of activities of hepatic enzymes in rats administered
Variance (ANOVA) was used to determine with paracetamol/methionine compared with control.
significant difference between the three groups The histologic presentations are shown in Figures 1-5
(APAP, APAP/methionine, control) at each exposure below. Figure 1 shows the photomicrographs of
level. SPSS package version 15 was used for this liver sections of rats exposed to paracetamol at the
purpose. The 0.05 level of probability was used as the end of the 4th hour, some of the presentations include
criterion of significance for experimental groups. periportal fatty infiltration, extensive vacuolar
degeneration and central portal congestion. Figure 2
RESULTS on the other hand shows histologic presentations of
paracetamol/methionine-administered rats also at the
Results of this study are shown in Tables 1-4 and 4th hour; with nonvisible lesion at 350 mg/kg and
Figures 1-5 below. These results revealed that rats diffuse hepatic degeneration at toxic levels. Figure 3
administered with paracetamol showed significant shows among other features hepatic necrosis and
increases (p<0.05) in the serum levels of bilirubin cellular infiltration by mononuclear cells for rats
compared with controls, especially at toxic levels of exposed to toxic doses of paracetamol whereas Figure
exposure at the two end -points i.e. 4th & 16th hours 4 shows that even at the end of the 16th hour rats
except for 1000mg/kg when bilirubin was not exposed to paracetamol/methionine manifested
significantly different (p>0.05) at the 4th hour. Total absence of necrosis but featured diffuse hepatic
protein, albumin and globulin are significantly vacuolar degeneration. The control rats on the other
decreased (p<0.05) at 3000 mg/kg and 5000 mg/kg hand, manifested non-visible lesion as shown in
by the end of the 4th and 16th hours, although total Figure 5.
protein was also significantly decreased (p<0.05) at Using ANOVA, inter-group comparisons between
1000 mg/kg by the end of the 16th hour as shown in the controls, 4th & 16th hour-groups show a marked
Table 1. In Table 2 significant increases in the difference between the degrees of hepatic damage in
activities of hepatic enzymes were noted in paracetamol-exposed group, with the 16th hour group
paracetamol administered rats at both end -points revealing a greater degree of hepatic damage than the
compared with control especially at toxic levels of 4th hour. The hepatic enzymes are significantly
exposure of 1000 mg/kg; 3000 mg/kg & 5000 mg/kg higher (p<0.05) in the 16th hour group compared with
BW levels, although at sub-toxic level, such increases the 4th hour group and control, an observation which
were also noted at the end of the 16th hour. was supported by the histology results.

Table 1:
Serum levels of hepatic indices in paracetamol - exposed Wistar rats- 4 & 16 hours post dosing.
Bilirubin Total protein Albumin Globulin
(µmol\L) (g\dl) (g\dl) (g\dl)
Controls 6.75±2.37 6.03±0.54 3.26±0.34 2.68±0.21
350mg\kg
4 hours 7.25±1.49 5.88±0.57 3.18±0.28 2.58±0.65
16 hours 8.13±1.81 5.58±0.42 3.01±0.09 2.21±0.61
1000mg\kg (F=8.94)ǂ
4 hours 8.36±2.20 5.68±0.41 3.24±0.37 2.48±0.43
16 hours 11.11±1.90* 5.53±0.36* 3.01±0.57 2.52±0.48
3000mg\kg (F=10.58)ǂ (F = 8.63)ǂ (F = 3.31)ǂ (F = 3.16)ǂ
4 hours 10.25±2.43* 5.50±0.39* 3.08±0.35 2.41±0.31
16 hours 13.75±4.03* 5.08±0.44* 2.83±0.34* 2.35±0.29*
5000mg\kg (F = 23.21)ǂ (F = 19.31)ǂ (F = 11.15)ǂ (F = 6.48)ǂ
4 hours 11.75±4.23* 5.31±0.24* 2.93±0.27 2.36±0.34*
16 hours 18.33±2.16* 4.73±0.29* 2.47±0.33* 2.10±0.34*
Results are expressed as mean ± standard deviation. *p <0.05 is significant when each exposure group is compared with
control. ǂ p <0.05 is significant at the dose level, when control, 4th and 16th hours groups are compared using ANOVA.

Hepatic presentation of female rats dosed with paracetamol/methionine 153

 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

Table 2:
Serum activity of hepatic enzymes in paracetamol- exposed Wistar rats- 4 & 16 hours post dosing.
AST (IU\L) ALT (IU\L) GGT (IU\L) ALP (IU\L)
Controls 36.36±10.76 33.75±12.14 40.00±5.78 52.25±15 .21
350mg\kg (F = 48.62)ǂ (F = 28.98)ǂ (F = 67.53)ǂ (F = 41.44)ǂ
4 hours 49.00±9.24 37.75±7.55 42.38±9.29 71.25±1.49*
16 hours 92.38±16.34* 68.63±12.31* 68.0±12.31* 119.88±18.86*
1000mg\kg (F = 215.17)ǂ (F = 36.71)ǂ (F = 10.32)ǂ (F = 52.82)ǂ
4 hours 99.75±12.37 * 61.0±1.81* 83.75±13.29* 115.75±37.59*
16 hours 487.33±80.39* 379.78±153.92* 242.11±33.41* 358.33±101.34*
ǂ ǂ ǂ
3000mg\kg (F = 277.34) (F = 234.75) (F=168.88) (F = 204.92)ǂ
4 hours 498.75±155.40* 315.88±87.83* 211.38±32.47* 198.00±27.12*
16 hours 1872±233.68* 1506±233.86* 432.63±201.15* 743.38±120.74*
5000mg\kg (F =482.99 )ǂ (F = 188.42)ǂ (F = 680 98)ǂ (F = 663.43)ǂ
4 hours 695.0±105* 581.0±121.10* 286.25±62.78* 338.13±66.89*
16 hours 3882±450.95* 2916±539.38* 707.50±199.66* 890.50±19.77*
Data are presented as Mean ± SD, ALT-alanine aminotransferase; AST- aspartate aminotransferase; ALP- alkaline
phosphatase; GGT- γ-glutamyl transferase.*p <0.05 is significant when each exposure group is compared with control. ǂ p
<0.05 significant at the dose level, when control, 4th and 16th hours groups are compared using ANOVA.

Table 3:
Serum levels of hepatic indices in paracetamol/methionine exposed Wistar rats- 4 & 16 hours post dosing.
Bilirubin Total protein Albumin (g\dl) Globulin (g\dl)
(µmol\L) (g\dl)
Controls 6.75±2.37 6.03±0.54 3.26±0.34 2.68±0.21
350mg\kg
4 hours 6.63±3.25 6.36±0.86 3.39±0.43 2.98±1.01
16 hours 6.25±2.38 5.85±0.58 3.14±0.29 2.58±0.33
1000mg\kg
4 hours 7.25±3.45 5.90±0.48 3.19±0.36 2.71±0.55
16 hours 7.50±4.07 5.25±0.35* 2.80±0.40* 2.44±0.33
3000mg\kg
4 hours 6.86±2.75 5.95±0.67 3.31±0.53 2.64±0.46
16 hours 7.75±3.06 5.85±0.42 3.31±0.37 2.63±0.72
5000mg\kg
4 hours 7.25±3.45 6.12±0.63 3.36±0.54 2.76±0.97
16 hours 6.88±3.94 5.89±0.58 3.10±0.28 2.69±0.46
Results are expressed as mean ± standard deviation; *p <0.05 is significant when each exposure group is compared with
control.

Table 4:
Serum levels of hepatic enzymes in paracetamol/methionine - exposed Wistar rats- 4 hours post dosing.
AST (IU\L) ALT (IU\L) GGT (IU\L) ALP (IU\L)
Controls 36.36±10.76 33.75±12.14 40.00±5.78 52.25±15 .21
350mg\kg (F = 31.31) ǂ
4 hours 32.38±17.97 29.38±13.54 40.75±8.43 52.38±14.46
6 hours 38.75±9.36 31.25±11.59 51.50±8.50* 55.63±23.14
1000mg\kg (F = 28.51) ǂ
4 hours 32.00±8.94 31.50±12.42 43.13±9.89 51.50±20.12
16 hours 33.88±11.87 29.88±17.45 59.00±14.10* 51.00±13.06
3000mg\kg (F = 22.81) ǂ
4 hours 34.25±17.52 30.25±11.79 49.13±3.40* 48.75±16.61
16 hours 32.75±13.63 30.25±10.95 67.25±14.54* 57.25±18.92
5000mg\kg (F = 18.86) ǂ
4 hours 34.25±17.52 26.13±11.96 54.75±15.52* 54.25±22.00
16 hours 37.88±12.64 29.50±12.56 71.00±14.31* 55.63±13.91
Results are expressed as mean ± standard deviation, ALT-alanine aminotransferase; AST- aspartate aminotransferase;
ALP- alkaline phosphatase; GGT- γ-glutamyl transferase. *p <0.05 is significant when each exposure group is compared

Hepatic presentation of female rats dosed with paracetamol/methionine 154

 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

with control. ǂ p <0.05 is significant at the dose level, when control, 4th and 16th hours groups are compared using
ANOVA.
toxicity, the 16th hour post-administration than at the
4th hour, the peak of absorption.
Specifically, results show that administration of
paracetamol (APAP) at toxic doses of 1000, 3000,
5000 mg\kg caused significant increases (p<0.05) in

The inter-group comparisons between the control, 4th

& 16th hour-groups of paracetamol/methionine treated
rats on the other hand, show non-significant
difference (p>0.05) in the levels of the hepatic
enzymes.
aminotransferase (AST), aspartate aminotransferase
DISCUSSION (ALT), γ- glutamyl transferase (GGT), and alkaline
Serum levels of hepatic enzymes e.g. ALT, AST are phosphatase (ALP) at both the 4th and the 16th hour,
very sensitive markers usually employed in the with the 16th hour results being more significantly
diagnosis of liver diseases. When the hepatocellular (p<0.05) higher than the 4th hour. Both AST and ALT
plasma membrane is damaged, the enzymes normally are sensitive indicators of hepatic function with the
present in the cytosol are released into the blood serum activity being raised as early as the 4th hour;
stream. They are usually quantified to assess the type Ilic et al. (2010) have even revealed that elevation in
and extent of liver injury (Sallie et al., 1991). All the activity of AST & ALT can occur as early as 25 min
indices used to assess hepatic damage were post-paracetamol administration. These increases are
significantly increased (p<0.05) in paracetamol an indication of the hepatotoxic effect of paracetamol
exposed rats compared with controls; with these (Sallie et al., 1991).
increases being more prominent at the peak of
Hepatic presentation of female rats dosed with paracetamol/methionine 155
 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

The higher level of AST compared to ALT is a & Ebong, 2007; Balamurugan et al., 2008) have
confirmation of the involvement of not only the indicated that at huge doses of APAP and after many
cytosol but also of the mitochondria; AST occurs in hours of exposure, increase in ALP serum activity
both compartments. Decreased plasma membrane may occur. Rajesh & Latha (2004) have also
Ca2+-ATPase activity and impaired mitochondrial identified that ALP increase may be the result of
sequestration of Ca2+ which lead to influx of defective excretion of bile by the liver cells due to
extracellular Ca2+ has been suggested by Tsokos- hepatic injury.
Kuhn et al. (1988); and Tirmenstein & Nelson, The significant increase (p<0.05) in the activity of
(1989). In addition, large-scale calcium cycling by serum ALT, AST, ALP and GGT in paracetamol
mitochondria resulting in oxidative stress and cell treated rats were also accompanied by significant
death has been reported as some other complications

of toxicity (Thomas & Reed, 1988). Disturbed Ca2+

homeostasis is likely to activate Ca 2+-dependent
catabolic processes such as phospholipid degradation,
protein degradation, disruption of the cytoskeleton increases (p<0.05) in the level of bilirubin in the 16
and DNA fragmentation (Ray et al., 1990; Orrenius et hour-exposure group; the liver cells are the main site
al., 1991), thereby resulting in, among other things, of detoxification and excretion of bilirubin.
hepatocellular membrane damage. Hepatocellular Furthermore, the hepatotoxic nature of
plasma membrane damage causes leakage and paracetamol was confirmed by the results of total
subsequent release of these enzymes into the protein and albumin. Both were significantly
extracellular compartment. Although ALP is not a decreased (p<0.05) especially at 1000, 3000, 5000
sensitive indicator of hepatic damage, studies (Ekam mg\kg levels, these decreases were more evident at
Hepatic presentation of female rats dosed with paracetamol/methionine 156
 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

known to play critical role as cellular effectors of

nonspecific host defense and which are also potent in
their role as secretory cells, producing an array of
mediators (e.g. pro-inflammatory and cytotoxic
cytokines and growth factors, bioactive lipids,
hydrolytic enzymes, and reactive oxygen and
nitrogen intermediates) can also not be discounted,
most of these mediators have also been implicated in
tissue injury, with a consequent rise in liver enzymes
(Laskin & Laskin, 2001).
Such increases in the activities of these enzymes
have been reported by Rajesh et al. (2009) to reflect
also in urine as a spill-over effect. da Silva et al.
(2006) equally observed such reflection; that urinary
activity of GGT, ALP and LDH enzymes were
significantly higher (P<0.05) 24 hours after drug
administration compared to the activity of controls.
the 16th hour – which Trumper et al. (1992) have Moreover, Kocaoğlu et al. (1997) indicated that
described as the peak of toxicity- compared with the twenty-four hours after a single dose of 900 mg/kg
4th hour results as well as the controls. Most of the paracetamol (APAP) administered to rats
plasma proteins are synthesized by the hepatocytes, intraperitoneally. Urinary GGT activity in the APAP
the abnormality of which resulted in lower serum administered group was significantly higher than in
level. the control group, histological examination of the
Stocker et al. (1987) have indicated that increased kidneys under light microscopy also showed tissue
serum bilirubin in APAP treated groups can be linked damage.
to regurgitation of bile due to obstruction within the Our results of altered activity of liver enzymes as
liver as a result of damage or inflammation caused by well as alteration in the levels of albumin and total
paracetamol whereas, Sallie et al. (1991) have protein in the serum of paracetamol (APAP) treated
identified that regurgitation of bile resulted in the rats are in agreement with the observation of
increase of ALP activity in rats they administered Balamurugan et al. (2008). These workers observed a
with APAP but Moss and Butterworth (1974) have reduction in serum total protein and an increase in
pointed out that elevated level of serum ALP may be serum ALP, AST, ALT, bilirubin and liver
due to the increased synthesis in presence of thiobarbituric acid reactive substances (TBARS) due
increasing biliary pressure. In the methionine treated to liver injury in the paracetamol-administered rats.
paracetamol intoxicated rats, we observed that the The significant decrease (p<0.05) in the levels of total
levels of bilirubin and ALP were not significantly protein and albumin by the end of the 16th hour of
different (p>0.05) compared with controls, an exposure could not entirely have been as a result of
indication that the liver normal activity and integrity decreased synthesis although the liver is the site of
was preserved by action of methionine. production of a number of these proteins; only 4 % of
The significant increases (p<0.05) in the activities albumin (which constitute about 50% of all serum
of ALT and AST in this study are in agreement with a proteins) is turned over each day (Crook, 2006).
number of other studies; Kuvandik et al. (2008) who Depletion in levels of albumin and total protein is
also used adult female Wistar Albino rats equally more of a common presentation of chronic liver
observed significant elevations in serum AST and diseases where albumin: globulin ratio is always
ALT activities in the APAP treated group. Light decreased (Murray, 2006). Paracetamol
microscopic evaluation of their rats’ livers showed (acetaminophen) is also nephrotoxic at overdose level
that there were remarkable centrilobular (zone III) and loss through the urine is another probable cause
hepatic necrosis and mild to moderate sinusoidal of low serum protein level. Being also a negative
congestion in the APAP group, just like our study acute phase protein, the depletion in albumin can also
where histologic presentation varied from periportal be linked to acute phase response especially as liver
fatty infiltration, extensive vacuolar degeneration and histology confirmed infiltration by mononuclear
central venous congestion (at lower doses) to severe cells.
portal congestion, focally extensive, centrilobular The hepatoprotective effects of methionine also
hepatic necrosis and fatty infiltration. There was also reflected in results of biochemical indices used to
mild cellular infiltration by mononuclear cells (at assess hepatic damage. They were not significantly
5000 mg\kg). The involvement of macrophages different (p>0.05) in the APAP\methionine exposed
Hepatic presentation of female rats dosed with paracetamol/methionine 157
 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

group compared with controls, except GGT which hepatocytes: protection by N-acetyl Cysteine.
was slightly higher than controls at 3000 & 5000 Toxicol. Sci. 80:343–349.
mg\kg. The cause of mild increase in GGT activity Balamurugan, M., Parthasarathi, K., Ranganathan,
without hepatic dysfunction is difficult to identify L.S. and Cooper, E.L. (2008). Hypothetical mode
especially as APAP has not been identified as a GGT of action of earthworm extract with
inducers, though the ability of APAP to induce some hepatoprotective and antioxidant properties. J
CYP enzymes has been recognized. Crook, (2006) Zhejiang Univ. Sci B. 9(2): 141–147.
has recognized that mild increases in GGT activity Bergmeyer, H.U., Scheibe, P. and Wahlefeld, A.W.
are sometimes difficult to interpret. (1978). Methods of aspartate aminotransferase and
Liver histology revealed absence of hepatic alanine amino transferase.Clin.Chem.24:58-73.
necrosis but changes in liver architecture were Bessems, J.G.M. and Vermeulen, N.P.E. (2001).
observed only at the highest level of exposure i.e. Paracetamol (acetaminophen)-induced toxicity:
5000 mg/kg. These changes can be linked to the molecular and biochemical mechanisms,
massive influx of methionine to the liver because analogues and protective approaches. Crit. Rev.
earlier on Hardwick et al. (1970) on postmortem Toxicol. 31:55-138.
examination had discovered that liver of rats exposed Bulera, S.J., Cohen, S.D. and Khairallah, E.A.
to high doses of methionine was found to be fatty; (1996). Acetaminophen-arylated proteins are
lipid being concentrated in the periportal hepatocytes detected in hepatic subcellular fractions and
with a little present around the central veins. Just like numerous extra hepatic tissue in CD-1 and
this study hepatic lipid appeared as early as 16 h into C57Bl/6 mice. Toxicology.109:85–99.
their experiment, after a total dose of 584 mg/kg. No Burcham, P.C. and Harman, A.W. (1991).
hepatic lipid was found in their control animals as Acetaminophen Toxicity in Site-Specific
well as ours. Mitochondrial Damage in Isolated Mouse
Contrary to the previous reports that APAP causes Hepatocytes. J. Biol. Chem. 255:5049–5054.
damage to plasma membrane of liver cells leading to Crook, A.M. (2006). Clinical Chemistry and
significantly higher levels of AST and ALT, the Metabolic Medicine. seventh ed. London Edward
results of this study revealed the presence of Arnold (Publishers) pp 277.
mononuclear cells in liver histology of rats Dahlin, D.C., Miwa, G.T., Lu, A.Y.H. and Nelson,
intoxicated with APAP suggests a possible S.D. (1984). N-Acetyl-p-benzoquinone imine; a
involvement of inflammatory process which resulted cytochrome P-450-mediated oxidation product of
in regurgitation of bilirubin leading to its elevated acetaminophen. Proc. Nat. Acad. Sci. U S A.
level as well as increased activity of alkaline 81:1327-1331.
phosphatase. In addition, the hepatoprotective ability Ekam, V.S. and Ebong, P.E. (2007). Serum protein
of methionine was demonstrated in female Wistar rats and enzyme levels in rats following administration
administered even with toxic doses, not just few of antioxidant vitamins during caffeinated and
hours after exposure but at the 16th hour of exposure. non-caffeinated paracetamol induced
GGT was also observed to be raised in hepatotoxicity. Niger. J Physiol. Sci. 22(1-2):65-8.
hepatoprotected rats. Since the ability of methionine Gardner, C.R., Laskin, J.D., Dambach, D.M., Chiu,
to induce GGT has not earlier been reported, further H., Durham, S.K., Zhou, P., Bruno, M., Gerecke,
study may be necessary to determine a possible D.R., Gordon, M.K. and Laskin, D.L. (2003).
relationship between methionine and this enzyme in Exaggerated hepatotoxicity of acetaminophen in
some other animal species. mice lacking tumor necrosis factor receptor-1.
Potential role of inflammatory mediators. Toxicol.
REFERENCES Appl. Pharmacol. 192:119–130.
Grypioti, A.D., Theocharis, S.E., Papadimas, G.K.,
Abraham , P. (2004). Increased plasma biotinidase Demopoulos, C.A., Papapoulos-Daifoti, Z.,
activity in rats with paracetamol-induced acute Basayiannis, A.C. and Mykoniatis MG. (2005).
liver injury. Clin. Chim. Acta 349(1-2):61-5. Platelet-activating factor (PAF) involvement in
Albert, K.S., Sedman AJ and Wagner JG. (1974). acetaminophen-induced liver toxicity and
Pharmacokinetics of orally administered regeneration. Arch. Toxicol. 79(8):466-74.
acetaminophen in man. J.Pharmacokinet. Hardwick, D.F., Applegarth, D.A., Cockcroft, D.M.,
Biopharm. 1974;2:381-393. Ross, P.M. and Calder, R.J. (1970). Pathogenesis
Bajt, M.L., Knight, T.R., LeMaster, J.L. and of methionine-induced toxicity. Metabolism. 19:
Jaeschke, H. (2004). Acetaminophen induced 381-391.
oxidant stress and cell injury in cultured mouse Henderson, C.J., Wolf, C.R., Kitteringham, N.,
Powell, H., Otto, D. and Park, B.K. (2000).
Hepatic presentation of female rats dosed with paracetamol/methionine 158
 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

Increased resistance to acetaminophen (2005). Acetaminophen sets record in the United

hepatotoxicity in mice lacking glutathione S- States: Number 1 analgesic and number 1 cause of
transferase Pi. Proc. Nat. Acad. Sci. USA acute liver failure. Hepatology. 42:1364–1372.
97:12741-12745. Laskin, D.L. and Laskin, J. D. (2001). Role of
Hinson, J.A., Reid, A.B., McCullough, S.S. and macrophages and inflammatory mediators in
James, L.P. (2004). Acetaminophen-induced chemical induced toxicity. Toxicology. 160 (1 –
hepatotoxicity: role of metabolic activation, 3): 111 – 8.
reactive oxygen/nitrogen species, and Lauterburg, B.H. and Mitchell, J.R. (1982). Toxic
mitochondrial permeability transition. Drug doses of acetaminophen suppress hepatic
Metab. Rev. 36:805–822. glutathione synthesis in rats. Hepatology 2:8-12.
Ilic, S, D.D., Zarkovic, K., Kolenc, D., Coric, M., Lawson, J.A., Farhood, A., Hopper, R.D., Bajt, M.L.
Brcic, L., Klicek, R., Radic, B., Sever, M., Djuzel, and Jaeschke, H. (2000). The hepatic
V., Ivica, M., Boban Blagaic, A., Zoricic, Z., inflammatory response after acetaminophen
Anic, T., Zoricic, I., Djidic, S., Romic, Z., overdose: role of neutrophils. Toxicol. Sci. 54:
Seiwerth, S. and Sikiric, P. (2010) High 509-516.
hepatotoxic dose of paracetamol produces Lewis, R.K. and Paloucek FP. (1991). Assessment
generalized convulsions and brain damage in rats. and treatment of acetaminophen overdose. Clin.
A counteraction with the stable gastric Pharm. 10:765-774.
pentadecapeptide BPC 157 (PL 14736). J. Physiol. Liu, M.L., Mars, W.M., Zarnegar, R. and
Pharmacol. 2010 Apr;61(2):241-50. Michalopoulos, G.K. (1994). Uptake and
Jollow, D.J., Mitchell, J.R., Potter, W.Z., Davis, D.C., distribution of hepatocyte growth factor in normal
Gillette, J.R. and Brodie, B.B. (1973). and regenerating adult rat liver. Am. J. Pathol.
Acetaminophen-induced hepatic necrosis. II. Role 144: 129-140.
of covalent binding in vivo. J. Pharmacol. Exp. Masubuchi, Y., Sudua, C. and Horie, T. (2005).
Ther. 187:195–202. Involvement of mitochondrial permeability
James, L.P.., Mayeux, P.R. and Hinson, J.A. (2003a). transition in acetaminophen induced liver injury in
Acetaminophen-induced hepatotoxicity. Drug mice. J. Hepatology. 42:110–116.
Metab. Dispos. 31:1499-1506. Mc Comb, R.B. and Bowers, G.N.Jr. (1972). A study
James, LP, McCullough, S.S., Lamps, L.W. and of optimum buffer conditions for measuring
Hinson, J.A. (2003b). Effect of N-acetylcysteine alkaline phosphatase activity in human serum.
on acetaminophen toxicity in mice; relationship to Clin. Chem.. 18:97.
reactive nitrogen and cytokine formation. McJunkin, B., Barwick, K.W., Little, W.C. and
Toxicol. Sci. 75:458-467. Winfield, J.B. (1976). Fatal massive hepatic
Kingsley, C.R. (1982). The indirect biuret method for necrosis following acetaminophen overdose.
determination of serum proteins. J.Lab.Clin.Med. JAMA. 236:1874–1875.
28:1093-1103. Meyers, L.L., Beierschmitt, W.P., Khairallah, E.A.
Kocaoğlu, S., Karan, A., Berkan, T. and Basdemir, G. and Cohen, S.D. (1988). Acetaminophen induced
(1997). Acute acetaminophen nephrotoxicity and inhibition of hepatic mitochondrial respiration in
urinary gamma-glutamyl transferase activity in mice. Toxicol. Appl. Pharmacol. 93:378–387.
rats. Drug Meatbol. Drug Interact. 14(1):47-54. Mitchell, J.R., Jollow, D.J., Potter, W.Z., Gillette,
Koch, T.R. and Doumas, B.T. (1982). Bilirubin: J.R. and Brodie, B.B. (1973). Acetaminophen-
Total and conjugated, modified Jendrassik- Grof induced hepatic necrosis. IV. Protective role of
method. Am. Ass. Clin. Chem. 113. glutathione. J. Pharmacol. Exp. Ther. 187:211–
Kon, K., Kim, J.S., Jaeschke, H. and Lemasters, J.J. 217.
(2004). Mitochondrial permeability transition in Moss, D.W. and Butterworth, P.J. (1974)
acetaminophen-induced necrosis and apoptosis of Enzymology and Medicine. London: Pitman
cultured mouse hepatocytes. Hepatology 40: Medical; p. 139
1170-1179. Murray, R.K. (2006). Metabolism of xenobiotics. In:
Kuvandik, G., Duru, M., Nacar, A., Yonden, Z., Harper’s illustrated Biochemistry. The McGraw-
Helvaci, R., Koc, A., Kozlu, T., Kaya, H. and Hill companies pp 633-640.
Sogüt, S. (2008). Effects of erdosteine on Nelson, S.D. and Bruschi, S.A. (2003). Mechanisms
acetaminophen-induced hepatotoxicity in rats. of Acetaminophen induced liver disease. In:
Toxicol. Pathol. 36(5):714-9. Kaplowitz N, DeLeve LD, editors. Drug Induced
Larson, A.M., Polson, J., Fontana, R.J., Davern, T.J., Liver Disease. Marcel Dekker; NY: pp. 287–325.
Lalani, E., Hynan, L.S., Reisch, J.S., Schiodt, Neuvonen P.J, Tokola O., Toivonen M.L. and Simell
F.V., Ostapowicz, G., Shaki, A.O. and Lee, W.M. O. (1985) Methionine in paracetamol tablets, a
Hepatic presentation of female rats dosed with paracetamol/methionine 159
 Niger. J. Physiol. Sci. 26 (2011): Iyanda and Adeniyi

tool to reduce paracetamol toxicity. Int. J.Clin. Oliveira C.S., Filho, J.C., Padiha, R.Z., Reichel,
Pharmacol. Ther. Toxicol. 23(9) 497-500. C.L., Neto, E.J., Oliveria, R.M., D’avila L.C.,
Nourjah, P., Ahmad, S.R., Karwoski, C. and Willy, Kessler, A. and de Oliveira J.R. (2006) Evaluation
M. (2006). Estimates of acetaminophen of renal enzymuria and cellular excretion as
(paracetamol)-associated overdoses in the United marker of acute nephrotoxicity due to an overdose
States. Pharmacoepidemiology and Drug Safety. of paracetamol in Wistar rats. Clin. Chim. Acta.
15:398–405. 373(1-2):88-91.
Orrenius, S., McConkey, D.J. and Nicotera, P. Stocker, R., Yamamoto, Y., McDonagh, A.F., Glazer,
(1991). Role of calcium in toxic and programmed A.N.and Ames, B.N. (1987). Bilirubin is an
cell death. Adv. Exp. Med. Biol. 283: 419-425. antioxidant of possible physiologic importance.
Park, B.K., Kitteringham, N.R., Maggs, J.L., Science. 235(4792):1043–1046.
Pirmohamed, M. and Williams, D.P. (2005). The Thomas, C.E. and Reed, D.J. (1988). Effect of
role of metabolic activation in drug-induced extracellular Ca++ omission on isolated
hepatotoxicity. Annu. Rev. Pharmacol. Toxicol. hepatocytes. II. Loss of mitochondrial membrane
45: 177-202. potential and protection by inhibitors of uniport
Rajesh, M.G. and Latha, M.S. (2004). Preliminary Ca++ transduction. J Pharmacol Exp. Ther.
evaluation of the antihepatotoxic activity of 245:501-507.
Kamilari, a polyherbal formulation. J Tirmenstein, M.A. and Nelson, S.D. (1989).
Ethnopharmacol. 91 : 99-104. Subcellular binding and effects on calcium
Rajesh SV, Rajkapoor B, Kumar RS and Raju K. homeostasis produced by acetaminophen and a
(2009) Effect of Clausena dentata (Willd.) M. non-hepatotoxic regioisomer, 3-
Roem. against paracetamol induced hepatotoxicity hydroxyacetoanilide in mouse liver. J Biol. Chem.
in rats. Pak J Pharm Sci. 2009 Jan;22(1):90-3. 264: 9814-9819.
Ray, S.D., Sorge, C.L., Raucy, J.L. and Corcoran, Trumper, L., Girardi, G. and Elías, M.M. (1992).
G.B. (1990). Early loss of large genomic DNA in Acetaminophen nephrotoxicity in male Wistar
vivo with accumulation of calcium in the nucleus rats. Arch. Toxicol. 66:107-111.
during acetaminophen-induced liver injury. Trumper, L., Monasterolo, L.A. and Elías, M.M.
Toxicol Appl Pharmacol. 106: 346-351. (1996). Nephrotoxicity of acetaminophen in male
Reid, A.B., Kurten, R.C., McCullough, S.S., Brock, Wistar rats: Role of hepatically derived
R.W. and Hinson, J.A. (2005). Mechanisms of metabolites. Arch. Toxicol. 66:107-111.
acetaminophen-induced hepatotoxicity: role of Tsokos-Kuhn, J.O., Hughes, H., Smith, C.V. and
oxidative stress and mitochondrial permeability Mitchell, J.R. (1988). Alkylation of the liver
transition in freshly isolated muse hepatocytes. J. plasma membrane and inhibition of the Ca 2+
Pharmacol. Exp. Ther. 312:509-516. ATPase by acetaminophen. Biochem. Pharmaco.
Sallie, R., Tredgeri, J.M. and Willion, R. (1991) 37: 2125-2131.
Drugs and the liver. Biopharmaceutics and Drug Zarro VJ. (1987). Acetaminophen overdose. Am.
Disposition. 1991;12(4):251–259. Fam. Physician. 1987;35:235-237.
Da Silva Melo, D.A., Saciura V.C., Poloni, J.A.,

Hepatic presentation of female rats dosed with paracetamol/methionine 160