TABLE OF CONTENTS
CHAPTER ONE
1.0 INTRODUCTION
AND LITERATURE REVIEW
1.1
INTRODUTION
1.2 LITERATURE
REVIEW
1.2.1 CADMIUM
1.2.1.2 HISTORY
1.2.1.3 USE OF CADMIUM
1.2.1.4 ROLE OF
CADMIUM IN OXIDATIVE STRESS INDUCTION
1.2.1.5 EXPOSURE AND METABOLISM
1.2.1.6 RENAL EFFECTS.
1.2.1.7 TOXICITY AND DISORDERS CAUSED BY CADMIUM
EXPOSURE
1.2.1.8
DIAGNOSIS, TREATMENT AND PREVENTION OF
CADMIUM POISONING
1.2.1.9
MAGNESIUM
1.2.1.10 CHEMISTRY
AND BIOCHEMISTRY OF MAGNESIUM
1.2.1.11 Physiology of Mg
1.2.1.12 CLINICAL
ASPECTS OF Mg
1.2.1.13 MAGNESIUM AND CADMIUM INTERACTION
1.2.1.14
Biochemical parameter
CHAPTER
TWO
2
.0 MATERIALS
AND METHODS
2.1 MATERIAL
2.1.1 EXPERIMENTAL ANIMALS
2.1.2 Chemical and Reagent
2.1.3 EQUIPMENT
2.1.4 ANIMAL FEED
2.2 METHODS
2.2.1 FEED
PREPARATION
2.2.1.1 CADMIUM TAINTED WATER
2.2.2. TREATMENT OF ANIMALS
2.2.2.1 ANIMAL SACRIFICE
2.2.2.1a. PREPARATION OF SERA SAMPLES
2.2.2.1b Preparation of tissue homogenate
2.2.3. BIOCHEMICAL ANALYSIS
2.2.3.1. CREATININE ASSAY
2.2.3.1a. PRINCIPLE
2.2.3.1b. PROCEDURE
2.2.3.1c CONCENTRATION DETERMINATION
2.2.3.2. BLOOD-UREA
ASSAY
2.2.3.2a. PRINCIPLE
2.2.3.2b. PROCEDURE
2.2.3.2c CONCENTRATION DETERMINATION
2.2.3.3. TISSUE
ALKALINE PHOSPHATASE (ALP) ASSAY
2.2.3.3a. PRINCIPLE
2.2.3.3b
PROCEDURE
2.2.3.3c.
CONCENTRATION DETERMINATION
2.2.4 STATISTICAL
ANALYSIS
CHAPTER
THREE
3.0 RESULT
S
3.1
RESULTS OF CREATININE ANALYSIS.
3.2
RESULT OF SERUM UREA NITROGEN.
3.3 RESULT OF KIDNEY ALKALINE PHOSPHATASE
ASSAY
CHAPTER FOUR
DISCUSSION
CONCLUSION
REFERENCES
APPENDIX
1: BODY WEIGHTS OF WISTAR ALBINO RATS
AT DAY ZERO.
APPENDIX
2: BODY WEIGHTS OF WISTAR ALBINO RATS
AFTER ONE WEEK.
APPENDIX
3: BODY WEIGHTS OF WISTAR ALBINO RATS
AFTER TWO WEEKS.
APPENDIX 4: BODY WEIGHTS OF WISTAR ALBINO RATS AFTER THREE WEEKS.
APPENDIX 5: ABSORBANCE VALUES AT 492nm OBTAINED FOR SERUMCREATININE
OF WISTAR ALBNO RATS
APPENDIX 6: ABSORBANCE VALUES AT 546nm OBTAINED FOR BLOOD UERA NITROGEN OF
WISTAR ALBNO RATS
APPENDIX 7: ABSORBANCE VALUES AT 405nm OBTAINED FOR TISSUE ALKALINE
PHOSPHATASE OF WISTAR ALBNO RATS
APPENDIX
7: CALCULATED VALUES FOR SERUM CREATININE, BLOOD UERA NITROGEN
CONCENTRATION AND ALKALINE PHOSPHATASE ACTIVITY.
APPENDIX
8: REAGENT COMPOSITION OF SERUM
CREATININE
APPENDIX
9: REAGENT COMPOSITION OF BLOOD UREA
NITROGEN
APPENDIX
10: REAGENT COMPOSITION OF TISSUE
ALKALINEPHOSPHATASE (ALP)
APPENDIX
11: STATISTCAL ANALYSIS ON SERUM
CREATININE CONCERATION
APPENDIX 12: STATISTCAL ANALYSIS ON BLOOD UREA NIYROGEN CONCERATION
APPENDIX 13: STOCK PREPARATION OF CADMIUM
APPENDIX
14: STATISTCAL ANALYSIS ON TISSUES
ALKALINE PHOSPHATASE ACTIVITY
CHAPTER ONE
1.0 INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUTION
Heavy metals include both non-toxic and toxic
elements. Iron (Fe), Cobalt (Co) Copper (Cu) Manganese (Mn) Molybdenum (Mo) and
Zinc (Zc) Magnesium(Mg) are the trace elements and are required in a very
minute amount, whereas other metals are non-essential, toxic to animals and
even fatal when accumulated these metals includes; Mercury (Hg), Arsenic (As),
Lead (Pb) Plutonium (Pu), Vanadium (v), Tungsten (w) and Cadmium (Cd), (Deevikaet al., 2012).
Heavy metals with established toxic action to humans
include cadmium (Morrow, 2010 and Hayes, 2007) lead (Eric, 2013 and Patrick,
2006 and mercury (Bojorklund, 1995).Each of these has been studied in isolation
for toxicity (Morrow, 2010; Patrick,2006 and Clarkson, and Magoss. 2006). But
in the ecosystem, be it air, (atmosphere, land and water) where they occur they
do not exist in isolation. They occur in close association with other metal and
non-metallic elemental pollutants. Among the metallic pollutant could be
calcium, copper, zinc, magnesium, manganese, iron and others. Metals are known to
interact with one another. Interaction can bring about two element together
include proximately and could cause out right displacement with one another.
When ingested in food and water they can antagonize each other. When it comes
to intestinal hand and pulmonary absorption it is there conceivable that the presence of other
element can affect the toxic potential of each of the heavy metal that has been
study in isolation.
Egborge (1994) reported that Warri River had and unacceptability
high cadmium level, 0.3mg/l of H2O, was 60-fold above the maximal
allowable level of 0.005mg/l. This report prompted our early studies on the
hepato-, nephro- and goladal toxicity of cadmium in rat expose to this high close
via water and diet. The diet was formulated with fish expose to 0.3mgCd/water
in the ambient water, as protein source and the toxic effects investigated and
reported. (Asagba and Obi, 2000; Asagba and Obi, 2001; Obi and Ilori, 2002;
Asagba and Obi, 2004a; Asagba and Obi, 2004b; Asagba and Obi, 2005). The studies focus on cadmium without taken into
consideration the fact that other metal were also present the river water and
as such were co-consumed by the communities using the river water for
cooking drinking and other domestic
purposes. Hence it is desirable to know if the presence of other metal would
enhance or diminish the toxic potential of cadmium or indeed that of any other
metal such as lead that was mention above. Therefore the aim of this present
studies was to re-examine the toxic potential of cadmium in the presence of
other metals such as magnesium. The
objective set out to achieve were:
v Re-examination
of the kidney toxicity of cadmium using established toxicity index for kidney
such as Creatinine, Blood urea nitrogen and Alkaline phosphatase.
v Re-examine
the status of the parameters in the absence of cadmium but in the presence of
magnesium.
v Re-examine
this parameter in the presence of cadmium and magnesium.
1.2 LITERATURE
REVIEW
1.2.1 CADMIUM
Cadmium (Cd) in its purest form is a
soft silver-white metal that is found naturally in the earth’s crust. It melts
at 3210c and boils at 7670c. The divalent element
has an atomic number of 48, a relative atomic mass of 112.41 and belongs to
group 12, period 5 and block – d of the periodic table. It is insoluble in
water, although its chloride and sulphate salt are freely soluble. The most
common forms of cadmium found in the environment exist in combination with
other elements. For example, cadmium oxide (CdO),(a mixture of cadmium and
oxygen),cadmium chloride (CdCl) (combination of cadmium and chlorine), and
cadmium sulphide (CdS) (a mixture of cadmium and sulphur) are commonly found in
the environment.
1.2.1.2 HISTORY
Cadmium is typically a metal of the
20th century, even though large amount of this by- product of zinc
production have been emitted by non ferrous smelters during the19th
century. Currently, cadmium is mainly used in rechargeable batteries and for
the production of special alloys although emission in the environment have markedly
declined in most industrialized countries, cadmium remains a source of concern
for industrial workers and for populations living in polluted areas, especially
in less developed countries (Sethi and Khandelwal,2006). In the industries,
cadmium is hazardous both by inhalation and ingestion and can cause acute and
chronic intoxications. Cadmium dispersed in the environment can persist in
soils and sediments for decades. When taken up by plants, cadmium concentrates
along the food chain and ultimately accumulates in the body of people eating
contaminated foods. Cadmium is also present in tobacco smoke, further contributing
to human exposure. By far, the most salient toxicological property of cadmium
is its exceptionally long-life in the human body. Once absorbed, cadmium
irreversibly accumulates in the human body, particularly in the kidneys and
other vital organs such as the lungs or the liver. In addition to its
extraordinary cumulative properties, cadmium is also a high toxic metal that
can disrupt a number of biological systems, usually at those that are much
lower than those toxic metals (Norsbergget
al., 2007; Bernard, 2004).
1.2.1.3
USE OF CADMIUM
Cadmium metals have a specific
property that makes it suitable for a wide variety of industrial applications. These
include excellent corrosion resistance, low melting temperature, high
ductility, high thermal and electrical conductivity (National resources Canada,
2007). It is used and traded globally as a metal and as a component in 6
classes of products, where it impacts distinct performance averages. According
to the US geological survey, the principal use of cadmium where: nickel-cadmium
(Ni-Cd) batteries, 83%, pigments 8%; coating and platting 7%; stabilizer for
plastics, 1.2%; and others (including non ferrous alloys, semi conductors and
photovoltaic devices). 0.8% (USGS, 2008).Cadmium is also present as an impurity
in the non ferrous metals (Zinc, Lead and copper), iron and steel, fossil fuels
(coal, oil, gas, peat and wood), cement and phosphate fertilizers. In these
products the presence of cadmium generally does not affect performance; rather,
it is regarded as an environmental concern (international cadmium association,
2011). Cadmium is also produced from recycled materials (such as Ni-Cd
batteries and manufacturing scrap) and some residues (e.g. cadmium containing
dusts from electrical and furnaces) or intermediate products. Recycling
accounts for approximately 10- 15% 0f the production of cadmium in developed
countries (National Resources Canada, 2007).
The primary use of cadmium in the
form of cadmium hydroxide is the electrode Ni-Cd batteries because of their
performance characteristics (e.g. higher cycle lives, excellent low- and high –
temperature performance), Ni-Cd batteries are used extensively in the
rail-roads and air craft industries (for starting an emergency power), and in
consumer products (e.g. cordless power tools telephones, portable computer,
camcorders, portable household appliances and toys) (ATSDR, 2008; USG, 2008).
Cadmium sulphide compounds (e.g. cadmium sulphide, cadmium sulphoselenide and
cadmium lithopone) are used as pigments in a wide variety of applications,
including engineering plastics, glass, glazes, ceramics, rubber, enamels,
artists colours and fireworks. Ranging in colour from yellow to deep-red
maroon, cadmium pigments have good covering power, and are highly resistant to
a wide range of atmospheric and environmental conditions (e.g. the presence of
hydrogen sulphide or sulphur dioxide. Light, high temperature and pressure)
(Herron, 2001; ATSDR,2008; International Cadmium Association, 2011).
Cadmium and cadmium alloys are used
as engineered or electroplated coatings on iron, steel, aluminum, and other
non-ferrous metals. They are particularly suitable for industrial applications
requiring, a high degree of safety or durability (e.g. aerospace industry, industrial
fastener selectrical parts automotive systems military equipment, and marine
/offshore installations) because they demonstrate good corrosion resistance in
alkaline or salt solutions, have a low coefficient of friction and good
conductive properties, and are readily solderable (UNEP, 2008; International Cadmium
Association, 2011).Cadmium salts of organic acid (generally Cadmium Laurate or
Cadmium Stearate, used in combination with barium sulphate) were widely used in
the past as heat and light stabilizers for flexible polyvinyl chloride and
other plastics (Herron, 2001; UNEP, 2008). Small quantities of cadmium are used
in various alloys to improve their thermal and electrical conductivity, to
increase the mechanical properties of the base alloy (e.g. strength, durability,
extrudability, hardness, wear resistance, tensile and fatigue strength), or to
lower the melting point. The metals most commonly alloyed with cadmium include
copper, zinc, and lead, tin, silver and other precious metals. Other minor use
of cadmium includes telluride and cadmium sulphidein solar cells. And other
semi-conducting cadmium compounds in a Varity of electronic applications
(Morrow, 2011; UNEP, 2008; International Association, 2011).
1.2.1.4
ROLE OF CADMIUM IN OXIDATIVE STRESS INDUCTION
The first
evidence of increased lipid peroxidation (LPO) in mice hepatocytes co-cultured
with Cd was given by Muller in 1987. The author described Cd-induced production
of reactive oxygen species (ROS) through interaction with critical subcellular
sites such as mitochondria, peroxisomes and microsomes that resulted in the
generation of free radicals and LPO in subcellular membranous structures.
Production of ROS has been reported later in a variety of cell culture systems,
as well as in intact animals via all routes of exposure (Hartet al.,
1999; Amaraet al., 2008). They also found early signs of oxidative stress in
the liver of mice exposed to a single oral Cd dose (20 mg kg-1 b. w. in the
form of CdCl2) through increased LPO level, expressed as
malondialdehyde (MDA) after 6 h, 12 h, and 24 h (Djukić-Ćosićet
al., 2008). Since Cd has no redox activity, it may
enhance ROS production by suppressing free-radical scavengers such as
glutathione (GSH) and by inhibiting detoxifying enzymes such as superoxide
dismutase, catalase, and GSH peroxidase, and/or through other indirect
mechanisms (Valko et al., 2005). The
ways in which Cd can induce the formation of reactive species are summarised in
Figure 1. Available data confirm that the formation of free radicals such as
superoxide ion, hydrogen peroxide, and hydroxyl radicals involves depletion of
GSH and changes in the activity of antioxidant enzymes (Liuet al.,2009). Our recent findings
(Djukić-Ćosićet al., 2007) showed that an
acute oral Cd dose (20 mg Cd kg-1 b. w.) significantly decreased the
glutathione (GSH) content in mice liver 4 h, 6 h, and 12 h after Cd
administration and increased GSH in the kidney after 12 h, 24 h, and 48 h, but
did not cause significant GSH changes in the testis. A two weeks oral Cd
exposure (at dose of 10 mg kg-1 b. w. of Cd given as aqueous solution of CdCl2)
lowered renal levels and increased liver and testicular levels of GSH. These results,
together with related findings of other authors, show that the effect of Cd on
GSH tissue levels varies with animal species, dose, route, and duration of
exposure. In general, acute exposure to metals decreases GSH levels due to the
formation of metal-GSH complexes and/or consumption by the GSH peroxidaseunder
oxidative stress induced by metals.
Figure 1 Pathways of Cd-induced generation of reactive
oxygen species (adapted from ref. (Valko et al., 2005).By DanijelaĐukić-Ćosić).
Cadmium impairs enzyme activity of antioxidative defence system
(superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase,
GSH-Px; glutathione-S-transferase, GST; gluathionereductase, GR) and of the
non-enzymatic component glutathione, GSSG and GSH Cadmium also
elevates the levels of Fenton metals (Fe + 3+ Cu 2+),
which can break down hydrogen peroxide, H202 to a
reactive hydroxyl radical, OH.
(Casalinoet al.,
1997) have proposed yet another mechanism of Cd-induced ROS production via iron
Cadmium may displace Fe from various cytoplasmic and membrane proteins and
consequently, increased concentration of ionic Fe stimulates ROS production in
tissue. This is in agreement with the recent findings, which clearly show that
acute and subacute Cd exposures change Fe content in the liver of mice (Djukić-Ćosićet
al., 2008). The results of this study also indicate that
Cd affects MDA levels in a time-dependent manner. The observed MDA levels
positively correlated with Fe in the liver. Oral exposure to a single dose of
Cd (20 mg kg-1 b. w.) significantly increased LPO in the liver at 6 h, 12 h,
and 24 h, and liver Fe after 4 h and 6 h up to 110 % of control level. However,
in mice orally exposed to Cd for two weeks (10 mgkg-1 b. w. in form of CdCl2
in aqueous solution), we found liver Fe reduced by 80 % and MDA by 74 %.There
are several other proposed mechanisms of Cd-induced oxidative stress. One of
these is Cd induced inflammation in the liver. Kupffer cells are reactivated in
response to Cd overload and produce inflammatory mediators such as IL-1β,
TNF-α, IL-6,and IL-8, which in turn stimulate generation of free radicals in
the liver (Yamanoet al., 2000). It has also
been suggested that mitochondria are an important target of Cdtoxicity (Belyaevaet al.,
2008).When it comes to ROS production, the results of long-term
exposure to low levels of Cd depend on experimental conditions such as dose,
time intervals of oxidative stress evaluation, and animal species studied. It
seems that ROS production has an important role in chronic Cd nephrotoxicity (Shaikh et al.,
1999), immunotoxicity (Ramirez and Gimenez, 2003).and
carcinogenesis (Waisberget al., 2003). On the other
hand, chronic exposure to Cd often elevates tissue GSH, without elevating
tissue LPO levels (Amaraet al.,2008;Shaikh et al., 1999). This is confirmed by our own results (Djukić-Ćosićet
al., 2008), which showed initial increase in liver LPO
and Fe levels 24 h after a single oral dose, which dropped after repeated Cd dosing.
Prolonged Cd exposure through drinking water also caused a two-phase ROS
response. At the beginning, ROS production rose, only to drop back to normal after
eight weeks of exposure (Thijssenet al., 2007). It has
been shown that ROS production does not play an essential role in chronic
Cd-induced malignant transformation in rat liver cells (Diwan et al.,2005).
1.2.1.5
EXPOSURE AND METABOLISM
Workers are mainly exposed to cadmium
via fume or dust inhalation in the workplace through the respiratory route.
Cadmium can also be orally introduced by consuming contaminated food or water through
the gastrointestinal track (GLT). About 50-80% of absorbed cadmium accumulates
in the liver and kidneys, where the accumulation ratio depends on the
administration route; non oral (such as inhalation, subcutaneous, intra-peritoneal
or intravenous) exposure to cadmium initially results in liver accumulation,
followed by a subsequent decrease and shift to the kidneys, whereas oral
exposure to cadmium results in accumulation in the kidneys as well as the liver
(Ohtaand Cherian,1991; Ohtaet al., 2000).
The highest concentration of cadmium
(10-100ppm) are found in internal organs of mammals, mainly in the kidneys and
liver as well as in some species of fish, muscles and oysters; especially when
caught in polluted seas. Tobacco smoking
is an important additional source of exposure to cadmium. Absorption by oral
route varies around 5%, but can be increased up to 15% in subjects with low
iron stores. When exposure is by inhalation, it is estimated that between 10%
and 50% of cadmium is absorbed, depending on the particle size and the
solubility of cadmium compounds. In the case of cadmium in tobacco smoke
(mainly in the form of CdO), an average of 10% of Cd is absorbed. Absorption of
cadmium through the skin is negligible (Nordberg et al., 2007; Bernard and Lauwerys,1986).Regardless the route of
exposure, cadmium is efficiently retained in the organism and remains
accumulated throughout life. The cadmium body burden, negligible at birth,
increases continuously during life until approximately the age of about 60-70
years from which cadmium body burden levels-off and can even decrease cadmium
concentrates in the liver and even more in the kidneys; which can contain up to 50% of the total body burden of cadmium in
subjects with low environmental exposure. Accumulation of cadmium in kidney is
due to the ability of this tissue to synthesize matallothionein (a
cadmium-inducible protein that protect the cell by tightly binding the toxic Cd2+
ion). The stimulation of metallothionein by zinc probably explains the
protection effect of this essential element towards cadmium toxicity. Because
of its small size, metallothionein is rapidly cleared from plasma by glomerular
filtration before being taken up by the proximal tubular cells. This glomerular
filtration pathway is at the origin of the selective accumulation of cadmium in
proximal tubular cells and thus in the renal cortex where this segment of the
nephron is located. Cadmium does not easily cross the placental or haemato-
encephalic barriers, explaining its very low toxicity to the foetus and the
central nervous system as compared with other heavy metals. Cadmium is mainly
eliminated via the urine. The amount of cadmium excreted daily in urine is
however very low, representing somewhat 0.005-0.01% 0f the total body burden.
1.2.1.6 RENAL EFFECTS.
There is now a consensus among
scientists to say that in chronic cadmium poisoning, the kidney (which is the main
storage organ of cadmium) is the critical target i.e. the first organ to
display signs of toxicity (Nordberget al.,
2007; Bernard and lauwerys, 1986; Bernardet
al., 1992). Cadmium nephropathy has been described in industrial workers
exposure mainly by inhalation, and in the general population exposed through
contaminated foods. The various studies conducted on human populations and
experimental animals have demonstrated that cadmium exerts its renal toxicity
in a strictly dose- dependent manner, the adverse effects occurring only when
the cadmium concentration in the renal cortex reaches a critical threshold. The
total concentration of cadmium in the renal cortex from which renal effects are
likely to occur has been estimated at 150-200ppm (μg/g wet weight of renal
cortex), both in human subjects and in experimental animals (Bernard and
Lauwerys, 1986; Bernard et al.,
1992). As most renal cadmium is bound to metallothionein, the form of cadmium
responsible for renal damage is the highly toxic Cd2+ion that avidly
reacts with cellular components. The critical concentration of free cadmium has
been estimated at about 2ppm (Bernardet
al., 1987).The earliest manifestation of cadmium –induced renal damage
considered as critical consists in an increased urinary excretion of micro
proteins (molecular weight <40 KD). Among the proteins, β2-microglobulin,
retinol-binding proteins and α 1- microglobulin have been the most validated
for the routine screening of tubular proteinuria. The increased loss of these
proteins in urine is a reflection of the decreased tubular reabsorption
capacity. In health, these proteins are almost completely reabsorbed by the
proximal tubular cells meaning that a minute decrease of their fractional
reabsorption drastically increases their urinary excretion. A modest increase
in the urinary excretion of these proteins, as found at the early stage of
cadmium nephropathy (in the range of 300-1000 μg/g creatinine for
retinol-binding protein), is unlikely to compromise the renal function (Bernard,
2004). Such as small increase might even be reversible after removal from
cadmium exposure. By contrast, when the urinary excretion of these proteins is
increased by more than one order of magnitude. Tubular dysfunction caused by
cadmium become irreversible and may be associated with a lower glomerular filtration
rate(GFR) and an accelerated decline of the GFR with ageing (Bernard,2004).
Other solutes excreted in greater amount in the urine of subjects with cadmium nephropathy
include total protein, albumin, amino acids, enzymes (e.g. N-acetyl-β–D
glucosaminidase), tubular antigens, glucose calcium and phosphate. The
disturbances of calcium may lead to bone demineration the formation of kidney
stones and bone fractures.
1.2.1.7 TOXICITY
AND DISORDERS CAUSED BY CADMIUM EXPOSURE
Long-term exposure to cadmium leads to
kidney damage and renal tubular dysfunction as assessed by increased urinary
excretion of low molecular weight (MW) proteins such as α 1- microglolin and β-
microgloblin (Kjellstron et al.,1977;
Nogawa et al., 1984). Individual
expose to high levels of cadmium develop severe renal dysfunction and bone
damage characterized by osteoporosis. Osteomalacia bone mineral loss (Goyer et al., 1994; Zhu et al., 2004) and anaemia (Horiguchi et al., 1996). One mechanism of bone damage is depletion of the
bone calcium pooldue to disrupted vitamin D metabolism in the kidneys,
resulting from renal tubular dysfunction caused by cadmium and the continuous
urinary excretion of cadmium and phosphorus (Akibaet al., 1980; Nogawa et al.,
1987). Cellular cadmium uptake is thought to be mediated by calcium channel c
(Lopezet al., 1989). Thus cadmium
competition for cellular calcium uptake also explains the calcium deficiency in
individuals exposed to cadmium olfactory toxicity (Gobba, 2006), male
infertility (Queirozet al., 2006). Hypertension
and cardiovascular diseases (Glauseret al.,
1976) are also associated with cadmium poisoning.
Carcinogenesis caused by cadmium exposure
represents another concern. Many experimental and epidemiological studies have
examined cancers of various organs such as the liver, kidneys, lung, prostate,
breast, brain and nervous system, testis and hematopoietic system (Glauseret al., 1976). However, many human
epidemiological surveillance investigations have not identified a relationship
between cadmium exposure and cancer risk, although experiment animal studies
have clearly demonstrated an association. Therefore, although the International
Agency for research on cancer (IARC) has classified cadmium as carcinogenic in
1993, debate about this association has continued (Arisawaet al., 2007).
Metallothionein is the most important
factor regulating the biological effects of cadmium. Treating mice with low
doses of heavy metals (such as cadmium or zinc), induces metallothionein and
obviously reduces the toxicity of subsequently administered lethal doses of
cadmium (Wobb, 1979). Transgenic mice that constantly over-express
metallothionein genes are also cadmium tolerant (Palmiteret al., 1993). In contrast, knockout mice with defective metallothionein
genes are more sensitive to cadmium toxicity than wild type mice (Liuet al., 2000). The findings of many
similar studies support the notion that metallothionein is the main cellular
determinants of the sensitivity of mammals and cultured mammalian cells of
cadmium.
1.2.1.8 DIAGNOSIS, TREATMENT AND PREVENTION OF
CADMIUM POISONING
Diagnosis of chronic cadmium poisoning
basically relies on the screening of proximal tubular dysfunction and the
assessment of the cumulative exposure to cadmium using environment or
biological indicators. The earliest manifestations of cadmium nephropathy
consist in an increased urinary excretion of micro proteins (tubular
proteinuria). The most commonly micro protein are β2-microgloblin, retinol-binding
protein and α – microgloblin. These proteins are usually measured in untimed
urine samples and the β2- microgloblin test presents the drawback that β2-microgloblin
is unstable in urine samples with a PH above 5.6, which necessitates to collect
a new urine sample in above 20-30% of the cases. α-globlin is very stable in
urine but because of its larger size, it is less specific of tubular damage and
slightly less sensitive than the two other micro proteins. Retinol-bindind
presents advantage of being stable, specific and as sensitive as β2-
microglobin .when tubular dysfunction is at an early stage, there is a
possibility of some reversal at least when the cadmium body burden is not too
high (e.g cadmium in urine below 20 μg/g creatinine).
Otherwise, cadmium-induce micro proteinuiria
is irreversible and predictive of a faster decline of the GFR with ageing. There
are no efficient treatments for chronic cadmium poisoning. Even after cessation
of exposure, renal dysfunction and pulmonary impairment may progress. The only
possible intervention is removal from the emphasis must be placed on primary
prevention in order to maintain the levels of cadmium in the environment or in
the food chain as low as possible (Bernard, 2008).
1.2.1.9
MAGNESIUM
Magnesium (Mg) is the main intracellular
earth metal cation with a free
concentration in the cytosol around 0.5 mmol/ l (Shilset al., 1994; Quamme, 1997; Grubbs
and Maguire,1987; Williams, 1970;Flatman,
1991 ). It is evident that Mg, whose
gradient over the plasma membrane is slight, and whose free extracellular
concentration (ionized Mg) is about 0.7 mmol/ l, at most can play the
complementary role of a more long-term regulatory element (Shilset al., 1994; Grubbs and Maguire, 1987; Williams, 1970 ). Nevertheless, with
the recent developments in analytical methods and instrumentation for measuring
both ionized and cytosolic free Mg concentrations it has been possible to gain
a better insight into the physiology of Mg.
1.2.1.10 CHEMISTRY
AND BIOCHEMISTRY OF MAGNESIUM
In order to understand the behaviour of
Mg, it is useful to recall some basic facts about it. Mg is a smaller ion that attracts water
molecules more avidly. Thus in practice, the ion is quite large (Williams,
1970; Jung and Brierley, 1994). Its six coordination bonds also have more rigid
coordination distances and directions than the more flexible Ca with its six to
eight coordination bonds (Williams, 1970). In contrast to Ca, Mg binds to
neutral nitrogen groups such as amino-groups and imidazol in addition to oxygen
especially in acidic groups, while calcium binds to oxygen in multidentate
anions (Williams, 1970). As a result, magnesium binding to protein and other
molecules generally is weaker than that of calcium, and it is more difficult
for it to reach and adapt to more deeply-situated binding sites in proteins
(Carafoli, 1987).and to pass through narrow channels in biological membranes.
This may also be the reason for the difficulty in finding probes that are
highly Mg-specific. Mg is a cofactor in hundreds of enzymatic reactions (Grubbsand
Maguire, 1987; Wackerand Parisi, 1968; Romani and Scarpa, 1992).And is especially
important for those enzymes that use nucleotides as cofactors or substrates.
This is because, as a rule, it is not the free nucleotide but its Mg complex
that is the actual cofactor or substrate. This is true for phosphotransferases
and –hydrolases such as AT Pases which are of central importance in the
biochemistry of the cell, particularly in energy metabolism. In addition, Mg is
required for protein and nucleic acid synthesis. There is also experimental
evidence for substitutive effects. Thus, Mg deficiency in the rat produces an
increase in the spermidine content of brain cortex ( Khawaja et al., 1984 ).
1.2.1.11Physiology
of Mg
It has long been known that Mg is
important for normal neurological and muscular function, hypomagnesemia leads
to hyperexcitability due mainly to cellular Ca transport and signalling (Shilset al., 1994; Grubbs and Maguire,1987;
Wacker and Parisi, 1968). The adult body contains approximately21–28 g (about 1
mole) of Mg, muscle and soft tissues accounting for almost half of this and
bone for slightly more than half (Shilset
al., 1994). Only about 1% of Mg is present in the blood plasma and red
cells.
v RENAL Mg EXCRETION
Approximately 75% of the total plasma Mg
is filtered through the glomerular membrane. In contrast to Na and Ca, only 15%
of the filtered Mg is reabsorbed in the proximal tubules, most (50–60%) in the
thick ascending loop of Henle (Shilset al.,
1994;Quamme, 1997). Under normal conditions only 3–5% of the filtered Mg is
excreted in the urine (Quamme, 1997).
As in the mucosa, both the paracellular
pathway and epithelial transport are important in the tubular reabsorption of
Mg which varies extensively with the filtered load. Several drugs, particularly
diuretics, thiazides, cisplatin, gentamycin and cyclosporin cause Mg loss into
the urine by inhibiting the Mg reabsorption in the kidneys (Shilset
al., 1994; Quamme, 1997). The thiazide-sensitive Na –Clsymporter in the
distal convoluted tubule is implicated as being involved since in the rat its amount
correlates closely with dietary Mg intake, plasma ionized Mg and urinary
excretion of Mg (Fanestilet al.,
1999) In analogy with the mucosa, its function could be to provide Cl to a Mg
–2Cl symporter. The mechanism of the paracellular transport of Mg has remained
elusive but now rapid progress is to be expected. A study of a rare genetic
disease with Mg wasting — renal hypomagnesemia with hypercalciuria and
nephrocalcinosis —has identified a mutated gene, paracellin-1, coding
for a protein located in the tight junctions of the thick ascending limb of
Henle.
1.2.1.12 CLINICAL ASPECTS OF Mg
v Mg
deficiency as a risk factor
The important role of Mg in modulating
transport functions and receptors, signal transduction, enzyme activities,
energy metabolism, nucleic acid and protein synthesis as well as protecting
biological membranes makes Mg deficiency a potential health hazard. The
development of Mg deficiency is usually linked either to disturbances in the
intestinal Mg absorption and/or to an increased renal Mg excretion. In gastrointestinal
disorders like intestinal malabsorption, steatorrhea and chronic pancreatic
insufficiency, non-absorbable magnesium-fatty acid soaps may be formed. Factors
increasing renal Mg excretion are discussed earlier. Anorexia, nausea,
vomiting, lethargy and weakness are typical early symptoms of Mg deficiency. If
severe Mg deficiency develops, paresthesia, muscular cramps, irritability,
decreased attention span and mental confusion often occur. The physical signs
of Mg deficiency are largely due to the associated hypocalcemia and hypokalemia.
There is an accumulating body of evidence to suggest that dietary Mg deficiency
plays an important role in the pathogenesis of ischemic heart disease, congestive
heart failure, sudden cardiac death, cardiac arrhythmias, vascular complications
of diabetes mellitus, pre-eclampsia / eclampsia and hypertension (Arsenian,
1993).
1.2.1.13 MAGNESIUM
AND CADMIUM INTERACTION
Limited
experimental data point to beneficial effects of Mg against Cd toxicity. (Boujelbenet al.,2006)
showed that Mg supplementation could reduce both organ Cd accumulation and
Cd-related LPO. The authors found that parental Mg supplementation (in form of
sulphate) was associated with a dose dependent reduction in Cd levels in rat
kidney, liver, and testis and lowered Cd-induced LPO in the liver and kidney.
In the testis, the protective effect of Mg was present only during the early
phase of Cd exposure. We investigated the effect of supplemental magnesium in
mice exposed to Cd (Djukić-Ćosić et al.,
2006). While acute oral Cd exposure (20 mg kg-1 b. w.) resulted in a
significant renal Cd increase, pre- treatment with Mg (40 mg kg-1 b. w)
efficiently lowered kidney Cd levels4 h and 6 h after Cd exposure. Similarly,
the Cd content in the kidney was also elevated following two-week Cd exposure
(10 mg kg-1 b. w. per os), and the effect was diminished by about 30 % in mice
pre treated with Mg (20 mg kg-1 b. w. per day). These results provide evidence
that Mg has the ability to protect the kidney from Cd accumulation and point to
the beneficial effects of Mg supplementation against Cd-altered renal Cu and Zn
levels (Djukić-Ćosić, et al., 2006). Further investigation showed
that Mg pre treatment significantly lowered Cd content not only in the kidney
(~30 %), but also in the lungs (50 %), spleen (~30 %), and testis (~30 %),
following two week Cd exposure. Results of our investigation on rabbits exposed
orally to Cd (as aqueous solution of CdCl2 at dose of 10 mg kg-1 b. w. per day)
for four weeks also showed that concomitant Mg supplementation (40 mg kg-1 b.
w. as an aqueous solution of Mg(CH2COO)2 per day given 1 h after Cd
administration) had the beneficial effects against tissue accumulation of Cd.
Excessive intake of Mg reduced blood, kidney, spleen, and bone Cd levels for
about 30 % in respect to rabbits given only Cd. However, we did not observe any
relevant changes in the lungs, heart, liver, pancreas, muscle, and brain. This
suggests that Mg modifies Cd absorption in the gastrointestinal system by
affecting the intercellular leak of Cd from intestinal lumen to portal blood
and thus reduces peripheral blood Cd. The effect of Mg supplementation on renal
Cd retention could be explained by Cd-Mg competition during reabsorption.
Furthermore, increased Mg in the lumen of the distal nephron could disable Cd
uptake by intercellular transport and promote Cd elimination via urine.
1.2.1.14 Biochemical parameter
creatinine
Creatinine
is synthesized primarily in the liver from the methylation of glycocyamine
(guanidino acetate, synthesized in the kidney from the amino acids arginine and
glycine) by S-adenosyl methionine. It is then transported through blood to the
other organs, muscle, and brain, where, through phosphorylation, it becomes the
high-energy compound phosphocreatine.(Shemeshet al., 1985). During the reaction, creatine and phosphocreatine are
catalyzed by creatine kinase, and a spontaneous conversion to creatinine may
occur. (Grosset al., 2005)
Creatinine is removed from the blood chiefly by the
kidneys, primarily by glomerular filtration, but also by proximal tubular
secretion. Little or no tubular reabsorption of creatinine occurs. If the
filtration in the kidney is deficient, creatinine blood levels rise. Therefore,
creatinine levels in blood and urine may be used to calculate the creatinine
clearance (CrCl), which correlates with the glomerular filtration rate (GFR).
Blood creatinine levels may also be used alone to calculate the estimated GFR
(eGFR).
Clinical significance
The GFR is clinically important because it is a
measurement of renal function. However, in cases of severe renal dysfunction,
the CrCl rate will overestimate the GFR because hypersecretion of creatinine by
the proximal tubules will account for a larger fraction of the total creatinine
cleared. (Harita, 2009)Ketoacids, cimetidine, and trimethoprim reduce
creatinine tubular secretion and, therefore, increase the accuracy of the GFR
estimate, in particular in severe renal dysfunction. The plasma level of
creatinine is relative independent of protein ingestion, water intake rate of
urine production and exercise.
Blood urea nitrogen
Blood
urea nitrogen (BUN) is an indication of renal (kidney) health. The
liver produces urea in the urea cycle as a waste product of the digestion of
protein more than 90% of urea is excreted through the kidneys, with remaining
loses through the gastro intestinal tract and the skin. Kidney disease is
associated with urea retention in the blood. Urea clearance underestimates
glomerular filtration rate (GFR) as it is reabsorbed. Measurement of urea can
indicate kidney functional status and in special circumstances, measurement of
urea in dialysis fluids is widely used in assessing the adequacy of renal
replacement therapy.
Urea was assayed based on the method described by Marcellin
Berthelot in 1860.
Clinical significance
The main causes of an increase in BUN are: high
protein diet, decrease in Glomerular Filtration Rate (GFR) (suggestive of renal
failure) and in blood volume (hypovolemia), congestive heart failure,
gastrointestinal hemorrhage, fever and increased catabolism. The main causes of
a decrease in BUN are severe liver disease, anabolic state, and syndrome of
inappropriate antidiuretic hormone.
ALKALINE PHOSPHATASE
Alkaline phosphatase (ALP) is an enzyme found in all
body tissues. There are many different forms of ALP called isoenzymes. The
structure of the enzyme depends on where in the body it is produced. This test
is most often used to test ALP made in the tissues of the liver and bones. The
ALP isoenzyme test is a lab test that measures the amounts of different types
of ALP in the blood.
Clinical significance
When the serum alkaline phosphatase (ALP) test result
is high, it is indicative of the following diseases:
Bone disease, Hepatitis, Hyperparathyroidism, Leukemia,
Liver disease, Lymphoma, Osteoblastic, bone tumors, Osteomalacia, Paget's
disease, Rickets, Sarcoidosis Lower-than-normalserum levels of ALP is indicative
of the following disease: Hypophosphatasia, Malnutrition, Protein deficiency, Wilson's
disease.
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