Arsenic contamination 1.1 Overview Arsenic contamination of groundwater is natural and found in high concentrations in deeper levels of groundwater. It is a high-profile problem due to the use of deep tubewells for water supply in the Ganges Delta, causing serious arsenic poisoning to large numbers of people. A 2007 study found that over 137 million people in more than 70 countries are probably affected by arsenic poisoning of drinking water. Arsenic contamination of the groundwater in Bangladesh is a serious problem. Prior to the 1970s, Bangladesh had one of the highest infant mortality rates in the world. Ineffective water purification and sewage systems as well as periodic monsoons and flooding exacerbated these problems. As a solution, UNICEF and the World Bank advocated the use of wells to tap into deeper groundwater. Millions of wells were constructed as a result. Because of this action, infant mortality and diarrheal illness were reduced by fifty percent. However, with over 8 million wells constructed, approximately one in five of these wells
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Arsenic contamination
1.1 Overview
Arsenic contamination of groundwater is natural and found in high
concentrations in deeper levels of groundwater. It is a high-profile problem due
to the use of deep tubewells for water supply in the Ganges Delta, causing
serious arsenic poisoning to large numbers of people. A 2007 study found that
over 137 million people in more than 70 countries are probably affected by
arsenic poisoning of drinking water.
Arsenic contamination of the groundwater in Bangladesh is a serious problem.
Prior to the 1970s, Bangladesh had one of the highest infant mortality rates in
the world. Ineffective water purification and sewage systems as well as periodic
monsoons and flooding exacerbated these problems. As a solution, UNICEF
and the World Bank advocated the use of wells to tap into deeper groundwater.
Millions of wells were constructed as a result. Because of this action, infant
mortality and diarrheal illness were reduced by fifty percent. However, with
over 8 million wells constructed, approximately one in five of these wells is
now contaminated with arsenic above the government's drinking water standard.
In the Ganges Delta, the affected wells are typically more than 20m and less
than 100m deep. Groundwater closer to the surface typically has spent a shorter
time in the ground, therefore likely absorbing a lower concentration of arsenic;
water deeper than 100m is exposed too much older sediments which have
already been depleted of arsenic.
The crisis came to international attention in 1995. The study conducted in
Bangladesh involved the analysis of thousands of water samples as well as hair,
nail, and urine samples. They found 900 villages with arsenic above the
government limit.
The acceptable level as defined by WHO for maximum concentrations of
arsenic in safe drinking water is 0.01 mg/L. The Bangladesh government's
standard is at a slightly higher rate, at 0.05 mg/L being considered safe. In
Bangladesh, 27% of shallow tube-wells have been shown to have high levels of
arsenic (above 0.05mg/l). It has been estimated that 35 - 77 million of the total
population of 125 million of Bangladesh are at risk of drinking contaminated
water (WHO bulletin, volume 78, page 1096). Approximately 1 in 100 people
who drink water containing 0.05 mg arsenic per liter or more for a long period
may eventually die from arsenic related cancers.
Arsenic is a well-recognized human carcinogen. Its exposure was shown to
depress the antioxidant defense system leading to oxidative damage to cellular
macromolecules including DNA, proteins, lipids(Shi et al. 2004), wreak havoc
in biological system by tissue damage, altering biochemical compounds and
corroding cell membranes (Wiseman & Halliwell 1996). Though liver appears
to be the main target of arsenic (Ferrini et al. 1997 & Runge et al. 2004),
kidney, and spleen are also vulnerable to arsenic toxicity (Siewicki, T.C. 2008).
Inorganic arsenic acts as a tumor promoter through reactive oxygen
species (ROS) generation in mammalian cells resulting in oxidative stress
(Garcia-Chavez, 2003) and carcinogenesis in man (Ahmad et al. 2000).
Arsenicosis is the effect of arsenic poisoning, usually over a long period such as
from 5 to 20 years. Long-term exposure to arsenic contaminated water causes a
wide range of adverse health effects, including skin lesions like raindrop
pigmentation, hyper pigmentation, keratosis of skin; anemia, vascular diseases,
conjunctivitis in the eyes, neuropathy, lung diseases and non-melanocytic
cancer of skin and different internal organs. But absorption of arsenic through
the skin is minimal and thus hand-washing, bathing, laundry, etc. with water
containing arsenic do not pose human health risks.
Intermittent incidents of arsenic contamination in groundwater can arise both
naturally and industrially. The natural occurrence of arsenic in groundwater is
directly related to the arsenic complexes present in soils. Arsenic can liberate
from these complexes under some circumstances. Since arsenic in soils is
highly mobile, once it is liberated, it results in possible groundwater
contamination
Arsenic poisoning is treated by some thiol containing chelating agents. They
can be administered either alone or in combination with antioxidants. But the
clinical applications of these chelators cause side effects include
1.3 Bangladesh Perspective of Arsenic Contamination
Groundwater arsenic contamination in Bangladesh is reported to be the biggest
arsenic calamity in the world in terms of the affected population. The
Government of Bangladesh has addressed it as a national disaster. Arsenic
contamination of groundwater in Bangladesh was first detected in 1993(Khan et
al. 19997). Recent studies in Bangladesh indicate that the groundwater is
severely contaminated with arsenic above the maximum permissible limit of
drinking water. In 1996, altogether 400 measurements were conducted in
Bangladesh. Arsenic concentrations in about half of the measurements were
above the maximum permissible level of 0.05 mg/l in Bangladesh. In 1998,
British Geological Survey (BGS) collected 2022 water samples from 41 arsenic-
affected districts. Laboratory tests revealed that 35% of these water samples
were found to have arsenic concentrations above 0.05 mg/l.
The experts from Bangladesh Council for Scientific and Industrial Research
(BCSIR) have been found the highest level of arsenic contamination, 14 mg/l of
shallow tube-well water in Pabna (Flora et al. 2004). The recent statistics on
arsenic contamination indicate that 59 out of 64 districts of Bangladesh have
been affected by arsenic contamination. Approximately, arsenic has
contaminated the ground water in 85% of the total area of Bangladesh and about
75 million people are at risk (Flora et al. 2005). It has been estimated that at
least 1.2 million people are exposed to arsenic poisoning. The reported number
of patients seriously affected by arsenic in drinking water has now risen to
8500(Nandi et al. 2005). As the people are getting arsenic also from food chain
such as rice, fish and vegetables, the problem is growing more severe. The
current statistics of arsenic calamity given in Table 1 present the severity of
arsenic contamination in Bangladesh.
Table 1: Statistics of Arsenic Calamity in Bangladesh (Flora et al. 2005)
Total Number of Districts in Bangladesh 64Total Area of Bangladesh 148,393 km2Total Population of Bangladesh 125 millionWHO Arsenic Drinking Water Standard 0.01 mg/l
Bangladesh Arsenic Drinking Water Standard 0.05 mg/lNumber of Districts Surveyed for Arsenic Contamination 64Number of Districts Having Arsenic above 0.05 mg/l inGroundwater
59
Area of Affected 59 Districts 126,134 km2Population at Risk 75 millionPotentially Exposed Population 24 millionNumber of Patients Suffering from Arsenicosis 8,500Total Number of Tube-wells in Bangladesh 4 millionTotal Number of Affected Tube-wells 1.12 million
1.4 Mechanism of arsenic contamination
The large-scale withdrawal of groundwater has caused rapid diffusion of
oxygen within the pore spaces of sediments as well as an increase in dissolved
oxygen in the upper part of groundwater (Figure 1). The newly introduced
oxygen oxidizes the arseno- pyrite and forms hydrated iron arsenate compound
known as pitticite in presence of water. This is very soft and water-soluble
compound. The light pressures of tube-well water break the pitticite layer into
fine particles and make it readily soluble in water. Then it seeps like drops of
tea from the teabag and percolates from the subsoil into the water table. Hence,
when the tube-well is in operation, it comes out with the extracted water. This
mechanism is portrayed in Figure 2.
Figure 2: Mechanism of arsenic contamination in groundwater around a tube-well
1.5 Source of arsenic in ground water
Arsenic ranks 20th in abundance in relation to other elements in the earth’s
crust and high concentrations are found in granite and in many minerals
including copper, lead, zinc, silver and gold. Arsenic naturally accumulates as
both organic and inorganic forms in soil, surface and groundwater (Smith et al.
1998).
The source and method of arsenic entering the groundwater in Bangladesh is a
controversial issue and has yet to be determined. But it is now widely believed
that the high arsenic levels in the groundwater in Bangladesh have a natural
geological source which may be due to abstraction water from quaternary
confined and semi-confined alluvial or deltaic aquifers. A large number of
diverse chemical and biological reactions, i.e. oxidation, reduction, adsorption,
precipitation, methylation and volatilization participate actively in the cycling of
this toxic element in the groundwater. The main process of arsenic
contamination is explained in two main processes, namely oxidation of arsenic
pyrites or ferrous hydroxides and oxy-hydroxide reduction.
Arsenic pyrites or ferrous hydroxides are very arsenic rich minerals which are
generally stable in reducing environment under the water table and normally
concentrated in organic deposits. But for different anthropogenic activities, like
lowering of water table below the organic deposits, accelerate the oxidation
process. When they oxidized and arsenic is released from the minerals. Some
of them are absorbed onto iron hydroxide. But when water table is recharged
and the arsenic adsorbed onto iron hydroxide returns to the reduced
environment under the water table and mixes with water and caused the
poisoning of water. According to this hypothesis, the origin of arsenic rich
groundwater is man-made, which is a recent phenomenon. Moreover, the whole
processes also accelerate by different geological process like weathering,
erosion, sedimentation, use of irrigation and fertilizers.
According to Oxy-hydroxide Reduction hypothesis, the origin of arsenic rich
groundwater is due to a natural process, and it seems that the arsenic in
groundwater has been present for thousands of years without being flushed from
the delta. Arsenic is assumed to be present in alluvial sediments with high
concentrations in sand grains as a coating of iron hydroxide. The sediments
were deposited in valleys eroded in the delta when the stream base level was
lowered due to the drop in sea level during the last glacial advance. The organic
matter deposited with the sediments reduces the arsenic bearing iron hydroxide
and releases arsenic into groundwater. Organic matter deposited in the
sediments reduce the arsenic adsorbed on the oxyhydroxides and releases
arsenic into the groundwater and dissolution occurs during recharge, caused by
microbial oxidation of the organic matter as bacteria dissolves surrounding
Arsenic is toxic substance to human health and toxicity depends on the amount
of arsenic intake, which is classified into acute, sub-acute and chronic toxicity
respectively. It is a silent killer. It is 4 times as poisonous as mercury and its
lethal dose (LD) for human is 125 milligram. Drinking water contamination
causes the last variety of toxicity. Undetectable in its early stages, arsenic
poisoning takes between 8 and 14 years to impact on health, depending on the
amount of arsenic ingested, nutritional status, and immune response of the
individual. Arsenic toxicity is dose dependent, and particularly on the rate of
ingestion of arsenic compounds and their excretion from the body but it also
accumulate into the body and passes slowly out through hair and nail. Most of
the ingested arsenic is excreted from the body through urine, stool, skin, hair,
nail and breath. In excessive intake, some amount of arsenic is accumulated in
tissues and inhibits cellular enzyme activities.
Inhalation, ingestion and skin contact are the primary routes of human exposure
to the arsenic. Chronic arsenic ingestion from drinking water is known to cause
skin cancer, and there is substantial evidence that it increases risk for cancers of
the bladder, lung, kidney, liver, colon, and prostate. Recent studies have also
shown that arsenic is associated with a number of non-neoplastic diseases,
including cardiac disease, cerebrovascular disease, pulmonary disease, diabetes
mellitus and diseases of the arteries, arterioles, and capillaries (Engel & Smith
2004). Individuals with chronic Hepatitis B infection, protein deficiency or
malnutrition may be more sensitive to the effects of arsenic (World Health
Organization WHO (1999). Children and older adults may be other groups at
special risk. The Table 1 shows problems and organ of the human body which is
generally affected by arsenicosis. Observable symptom to the arsenic poisoning
can be thickening and discoloration of skin, stomach pain, nausea, vomiting,
diarrhea, numbness in hand and feet, partial paralysis, blindness.
Table 2: Arsenic infection
Organ System ProblemsSkin Symmetric hyperkeratosis of palms and soles, melanosis or
depigmentation, bowen's disease, basal cell carcinoma and squamous cell carcinoma.
Liver Enlargement, Jaundice, cirrhosis, non-cirrhotic portal hypertensionNervous System Peripheral neuropathy, hearing lossCardiovascular System
Acrocyanosis and Raynaud's Phenomenon
Hemopoietic System
Megalobastosis
Respiratory System
Lung Cancer
Endocrine System Diabetes mellitus and goiter1.7 Arsenicosis effects on cell death signaling
Arsenic induces significant amount of DNA damage. For proper maintenance of
physiological functions, these cells carrying defective genetic information have
to be eliminated from the body, which generally occurs via the programmed cell
death or apoptosis. Significant increase in cytochrome-P450 and lipid
peroxidation accompanied with a significant alteration in the activity of many of
the antioxidants was observed, all suggestive of arsenic induced oxidative
stress. Histopathological examination under light and transmission electron
microscope suggested a combination of ongoing necrosis and apoptosis.
Agarose gel electrophoresis of
DNA of hepatocytes resulted in a characteristic ladder pattern. Chronic arsenic
administration induces a specific pattern of apoptosis called post-mitotic
apoptosis. (Somia et. Al, 2006).
Apoptotic cell death generally occurs through transduction of death signals that
cause morphological changes and affect a number of intracellular key effector
molecules stepwise during the whole process. The cell death signal transduction
triggered by arsenic results in aggregation of the membrane rafts together with
glycosyl phosphotidyl insitol (GPI) anchored cell surface proteins and Thy-1
receptors (in T lymphocytes), reduction of mitochondrial membrane potential,
glutathione production and Bcl-2 expression; elevation of supper oxide
production and Bax protein expression; activation of protein tyrosine kinase
(PTK), mitogen activated protein kinase (MAPK) family kinases, caspases and
Akt; inhibition of NF-кB activity and finally fragmentation of the nuclear DNA
(Scholz et. Al, 2005).
1.8 Blood Serum Parameters
Glucose level and other enzymes like lactate dehydrogenase, alkaline phosphatase, serum glutamic pyruvic transaminase etc. present in blood give important information about how the liver functioning and whether a substance affecting it.
1.8.1 Blood Glucose levels
Glucose, a type of sugar used by the body for energy. The body maintains the
blood glucose level at a reference range between about 3.6 and 5.8 mM
(mmol/L, i.e., millimoles/liter), or 64.8 and 104.4 mg/dL (http://www.faqs.org)
The human body naturally tightly regulates blood glucose levels as a part of
metabolic homeostasis. Glucose is transported from the intestines or liver to
body cells via the bloodstream, and is made available for cell absorption via the
hormone insulin, produced by the body primarily in the pancreas.
The mean normal blood glucose level in humans is about 4 mM (4 mmol/L or
72 mg/dL). However, this level fluctuates throughout the day. Blood sugar
levels outside the normal range may be an indicator of a medical condition. A
persistently high level is referred to as hyperglycemia; low levels are referred to
as hypoglycemia. Diabetes mellitus is characterized by persistent
hyperglycemia from any of several causes, and is the most prominent disease
related to failure of blood sugar regulation. In diabetes mellitus, hyperglycemia
is usually caused by low insulin levels (Diabetes mellitus type 1) and/or by
resistance to insulin at the cellular level (Diabetes mellitus type 2), depending
on the type and state of the disease. Low insulin levels and/or insulin resistance
prevent the body from converting glucose into glycogen (a starch-like source of
energy stored mostly in the liver), which in turn makes it difficult or impossible
to remove excess glucose from the blood.
1.8.2 Lactate Dehydrogenase (LDH)
Lactate dehydrogenase (also called lactic acid dehydrogenase, or LDH) is an
enzyme found in almost all body tissues. It plays an important role in cellular
respiration, the process by which glucose (sugar) from food is converted into
usable energy for our cells.
Although LDH is abundant in tissue cells, blood levels of the enzyme are
normally low. However, when tissues are damaged by injury or disease, they
release more LDH into the bloodstream. Conditions that can cause increased
LDH in the blood include liver disease, heart attack, anemia, muscle trauma,
bone fractures, cancers, and infections such as meningitis, encephalitis, and
HIV.
1.8.3 Alkaline Phosphatase (ALP)
This enzyme works best at an alkaline pH (a pH of 10) and thus the enzyme
itself is inactive in the blood. Alkaline phosphatase acts by splitting off
phosphorus (an acidic mineral) creating an alkaline pH. This enzyme is found in
several body tissues, including the liver. Kids and teens normally have higher
levels of ALP than adults because of bone growth. But ALP levels that are
higher than normal can be a sign of liver diseases or blocked bile ducts.
The primary importance of measuring alkaline phosphatase is to check the
possibility of bone disease or liver disease. Since the mucosal cells that line the
bile system of the liver are the source of alkaline phosphatase, the free flow of
bile through the liver and down into the biliary tract and gallbladder are
responsible for maintaining the proper level of this enzyme in the blood. When
the liver, bile ducts or gallbladder system are not functioning properly or are
blocked, this enzyme is not excreted through the bile and alkaline phosphatase
is released into the blood stream & found in increased concentration.
1.8.4 Serum Glutamic Pyruvic Transaminase (SGPT)
Serum glutamic pyruvic transaminase (SGPT) is one of these enzymes. It's
found in particularly large amounts in the liver and plays an important role in
metabolism, the process that converts food into energy. Normally, ALT is found
inside liver cells. But if the liver is inflamed or injured, ALT is released into the
bloodstream (for example, from viral hepatitis). Measuring blood levels of ALT
can give doctors important information about how well the liver is functioning
and whether a disease, drug, or other problem is affecting it.
Materials and methods
2.1 Sample Collection
Samples for the DNA analysis & measurement of blood serum were collected
from mouse models.
2.1.1 Collection of serum
Blood was collected in eppendorf tubes and kept at room temperature for 10
minutes. After that blood was centrifuged at 3000rpm for 5 minutes at 4C. The
supernatant was taken out using micropipette and collected in fresh eppendorf
tubes. The serum was stored in -78C refrigerator.
2.1.2 Collection of organs for DNA analysis
Part of kidney, liver, and spleen were collected and stored in-78C refrigerator
until samples were prepared for DNA analysis.
2.2 Estimation of blood serum parameters
2.2.1 Determination of serum glucose level
Glucose test is used to determine the amount of glucose in the blood. Serum
glucose concentration was determined using commercially available assay kit
manufactured by Human Diagnostic, Germany according to the manufacturer’s
protocol. In this test glucose oxidase converts glucose in gluconic acid and
peroxidase converts aminoantipyrine into quinoneimine.