The Possible Role of Interstitial Stem Cells Potential for Correcting Histopathological Changes in Liver and Kidney of Rats after Copper Toxicity
O.A. Abu- Zinadah
The aim of this study is to show the potential of interstitial stem cells or their precursors to be involved in the regeneration of the lost tissue mass in liver and kidneys after copper toxicity clearance. The present experiment was designed using the local albino Sprague Dawely rats. The animals were sacrificed during and after toxication period as well as after non-toxication periods. Copper accumulates in animal tissues until reaching the toxic level, at which time the histopathological changes then develops. The belief is that the genetic defects are responsible for disease and copper toxicosis is a cause rather than the result of tissue disease but it is not the primary cause. The tissue alterations, vacoulation, degeneration and necrosis, decreased after stopping copper toxicity and the organs excrete the excess copper by certain ways. This clearance enhances the interstitial stem cells or their precursors to incorporate in regeneration of the lost tissue mass. Moreover, to some extents, the bone marrow stem cells may introduce some plasticity to regenerate some of this lost tissue mass. The results support the opinion that the interstitial stem cells or even bone marrow stem cells are incorporated into regeneration of the lost tissues after toxication and clearance.
The relationship between dietary copper exposure and body copper nutritional
status is complex because copper homeostasis allows a wide range of copper intake
without resulting on adverse effects. Hematopoietic stem cells possess an extraordinary
developmental renewing capacity. It has been reported that hemopoietic stem
cells generate the brain, liver and skeletal muscles (Ferrai
et al., 1998; Gussoni et al., 1999;
Theise et al., 2000; Lagasse
et al., 2000; De Haan, 2002; Rodrigo
et al., 2009). On the other hand, muscle and neural stem cells have
been reported to generate blood cells (Jackson and Goodell,
1999; Bjornson et al., 1999). In this respect,
the use of bone marrow stem cells transplantation to correct disease in an intoxicated
mouse (a murine model for Wilsons disease) was evaluated. The transplantation
of bone marrow cells partially corrects the metabolic phenotype (Allen
et al., 2004; Rodrigo et al., 2009;
Christopher, 2010). As we know copper storage disease
is known as Wilsons disease in which copper concentration is increased up to
50 times than the normal value (Crawford et al.,
1985). Copper accumulates in the liver until reaching toxic levels, at which
time pathological changes then develop. The belief is that a genetic defect
is responsible for the disease (Crawford et al.,
1985). It is suggested that copper toxicosis is a cause rather than a result
of liver disease, but not the primary cause (Franklin and
Saunders, 1988). An autosomal recessive copper toxicosis gene has been linked
to the copper metal metabolism disorder (Brewer et al.,
Bhunya and Jena (1996) studied the clastogenic potential
of copper sulphate in chicks. They found an increase in micronucleolus counts
in both bone marrow and peripheral blood erythrocytes in time- and dose-response.
This observation reveals the genotoxic potential of copper sulphate in chick
in vivo. Similar observation was also recorded by Richards
(1999) who revealed some of the distinct changes occurring in the metabolism
of zinc, copper, iron in both maternal and fetal tissues fluids during gestation
in the pig. Misezta (1990) studied the effects of copper
as copper sulphate in the bone marrow cells of rats in vitro cultures,
loosely arranged macrophages, fibroblasts and adipocytes which appeared as early
as the control tissue culture. All these observations reflect the different
copper mode of actions which includes its accumulation in the cells in greater
level than the cellular needs; of these effects is the remodulation of stem
cells due to the geno- or cyto-toxicity of copper. This suggestion is supported
by Rodriguez et al. (2002) who speculated that
copper plays a functional role which stimulates the mesenchymal stem cells differentiation.
Tissue regeneration entails complex interaction between multiple signals and
material platforms. Orchestrating of these signals may greatly enhance the regeneration
of lost tissue mass. Vascular endothelial growth factors and lost cells precursor
capable of responding of these cues and forming the lost tissues. Huang
et al. (2005) demonstrated that the bone formation is a coordinated
process involving various biological factors. Also, Goldschmidt
et al. (2003) found that bone marrow stem cells transplant will lead
to maximum tumour reduction and development by modulation to form bone marrow
cells. The aim of this study is to show the potential of interstitial stem cells
or their precursors to be involved in the regeneration of the lost tissue mass
in liver and kidneys after copper toxicity clearance.
MATERIALS AND METHODS
Eighteen immature local albinos Sprague Dawely rats weighing about 65±3.5 g (Vide No. 288, 2010) were obtained from the animal house, King Fahd for medical researches, King Abdulaziz University, Jeddah. The animals were housed in stainless steel cages under room temperature and air conditioning. They were fed commercial diet, vegetables, crushed wheat and corn. They were left for a week to be acclimatized. Then, they were divided into a control group (6 animals) and a treated group (12 animals). A dose of 4 mg Cu SO4/kg body weight was force-fed daily by stomach tube to the rats of the second group for three weeks. After each week one rat of the control group and two rats of the treated group were sacrificed. Liver and kidneys were removed, fixed in 10% neutral formalin, embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Then, the rest of the experimental animals (3 control and 6 treated) were left without copper treatment for other three weeks and after each week one control animal and two animals from the previously treated groups were sacrificed. Also, liver and kidneys were dissected out and treated as mentioned before.
Macroscopic Observation of animals and organs: Liver and kidneys did
not show any defects after the first week of copper toxication; no decolourization
or hemorrhage were observed except the vomiting of some animals after treatment.
At the end of the third week decolouriztion of the all organs was observed.
After stopping the copper doses for three weeks all organs restored their normal
colours and shapes.
Microscopic examination: Liver sections displayed vacuolations around
the central vein. This vacoulation was followed by degeneration of cell masses
and finally some necrotic cells were observed (Fig. 1A, B).
Kidneys tissues showed signs of degeneration during copper toxicosis. Some
uriniferous and collecting tubules were vacuolated and degenerated. The glomeruli
were also shrunken and the Bowmans capsules showed some dilatation; these changes
were increased by time (Fig. 2A, B).
||(A and B) Liver sections showing vacuolar degeneration around
the central vein (arrow) and some necrotic cells (head arrow) after the
third week of toxication, (C): A liver section shows normal structure after
the third week of clearance (H and E) X300
||(A) A kidney section with severe vacuolation of the uriniferous
tubules (arrow) and necrotic cells (head arrow). (B) A kidney section shows
certain dilatation (arrow) and degeneration of Bowmans capsule (head
arrow). (C) A kidney section shows normal appearance (H and E), X300
After stopping copper toxicities for one week these alterations were slightly
decreased in both liver and kidneys in all animals left for three weeks without
treatment. Liver and kidneys almost showed the normal structure and almost all
the pathological alterations disappeared (Fig. 1C, 2C).
Diminishing of some degenerative alterations after stopping copper toxicities
observed in this study may be due to the clearance of copper toxicities; also
regeneration of lost cell masses may be due incorporation of some stem cells
to regenerate such lost cell masses. However, the modulation done by copper
toxicities to certain cells may enhance them to incorporate or to share partly
in regeneration of the lost tissues. The potential of bone marrow stem cells
to correct liver dysfunction in the mouse model of Wilsons disease was studied
by Allen et al. (2004) who found a direct evidence
of functionality and disease correction following liver repopulation. This repopulation
was succeeded after bone marrow transplantation which partially corrects the
metabolic phenotype in mouse which comes in agreement with our results. Histological
examination of liver of cats showed chronic hepatitis and cirrhosis associated
with massive accumulation of copper in hepatocytes and macrophages particularly
in fibrotic areas between the regenerative nodules. These criteria together
with the pattern of hepatic copper storage and the associated inflammation and
fibrosis, strongly suggest a primary copper storage disorder (Meertens
et al., 2005).
In fact, in normal digestion, food is moved through the digestive tract by
rhythmic contractions called peristalsis. When animal suffers from a digestive
motility disorder, these contractions are abnormal. This disorder can be due
to one of two causes: a problem within the muscle itself followed by disturbance
in the intracellular lining or a problem with the nerves or hormones that control
the muscles contractions (Bowen, 2002) which support
our observations and may demonstrate the reason of vomiting of some experimental
animals after treatment.
On the other hand, the partial recovery of liver and kidney degenerated cells
may be due to the fact that such organs get rid of excess copper accumulated
during toxication by certain ways. Data which obtained here was supported by
Thornburg et al. (1986) who concluded that copper concentration in
the liver actually decreases after necrosis and thus repair occurs. The cells
that die during necrosis are the same ones that are first to accumulate copper.
As the cells die, copper is released into the blood and excreted in urine as
the hepatocytes are regenerated and scar tissue is formed.
The renewal of hepatocytes may be due to corporation of certain growth factors
or the interstitial stem cells with some plasticity generate the hepatocytes
(Ferrai et al., 1998; Gussoni
et al., 1999; Theise et al., 2000;
Lagasse et al., 2000; Gillson,
It was observed that the cytotoxicity of certain compounds, copper compound
included, to the renal epithelial cells is due to the generation of reactive
oxygen precise and peroxidation or may depend upon depletion of cellular glutathione
GSH (Nakanishi et al., 2005). So, by excretion
of excess copper, this is followed by depletion of reactive oxygen precise and
peroxidation as well as increase of glutathione (GSH) levels may play a role
in regeneration of the altered areas in liver and kidneys (Nakanishi
et al., 2005). Tyrosine kinase receptors play an important role in
proliferation and differentiation of cells (Visser et
al., 1996) including hemopoietic growth factor receptors (Rosnet
et al., 1993).
Moreover, Galhardi et al. (2004) investigated
the effect of copper intake on lipid profile oxidative stress and tissues damage.
It was concluded that copper renal toxicity was associated with oxidative stress
and reduction of at least one of the antioxidant enzymes. Present results were
come in agreement with the opinion stated that after clearing of copper toxicosis
bone marrow cells may contribute to regeneration of damaged glomerular endothelial
cells and show some plasticity and can differentiate into a wide range of specialized
cells and promoting angiogenesis in regions of progressive glomerular lesions
(Ikarashi et al., 2006).
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