Environmental Toxicology and Pharmacology 60 (2018) 100–109

Contents lists available at ScienceDirect

Environmental Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/etap

Elevated heavy metals levels in cognitively impaired patients from Pakistan T

a b b a,⁎
Ghazala Iqbal , Wahid Zada , Abdul Mannan , Touqeer Ahmed
a
Neurobiology Laboratory, Department of Healthcare Biotechnology, Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology,
Sector H-12, Islamabad, 44000, Pakistan
b
Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad, 22060, K.P.K, Pakistan

A R T I C LE I N FO A B S T R A C T

Keywords: The deficit in the visuospatial skills, thinking, learning and memory is termed as cognitive impairment. Human
Copper exposure to heavy metals is a potential risk factor for developing cognitive impairment. This study aimed to
Lead investigate the possible association between the concentration of heavy metals and the extent of cognitive im-
Aluminum pairment. Blood samples were subjected to microwave assisted acid digestion and evaluated for metals levels
Zinc
using atomic absorption spectrophotometry. We measured the levels of Cu, Pb, Al, Zn, Cd and Mn in 183 patients
Cadmium
diagnosed with cognitive impairment; mild (n = 72), moderate (n = 86) and severe (n = 25) and compared
Manganese
Oxidative stress them to age-matched healthy controls (n = 90). Results showed that all the aforementioned elements were
Mini mental state examination significantly higher in cognitively impaired patients and increasing concentration was strongly correlated with
Age increase in severity of the disease. The correlation study has shown that among the studied metals, Al and Cu are
Metal accumulation strongly associated with the cognitive impairment. This suggests the need for decrease in metal exposure to
humans from environment, food and industries.

1. Introduction uptake mechanism exits to provide essential trace element (such as Zn

Cognition impairment is the diminished ability of an individual to
know the world (Folstein et al., 1985). Cognitive impairment is a
condition in which an individual’s cognitive functions such as re-
membering, language, attention and visuospatial abilities are impaired
and affect their everyday life (Giagulli et al., 2016). This leads to de-
creased activity and deficit in learning and memory; consequently in-
creased risk of disability and health care cost (Plassman et al., 2008).
The cognitive abilities are influenced by various factors, such as; ge-
netics, environment, diet, aging and life style (Deary et al., 2009). The
deficit in cognitive functions are increasing globally and it is predicted
to increase proportionately more in developing countries (Mathers
et al., 2008). During the process of aging, there is a decline in health
and cognitive functions, which is usually proportionally related to the
increased level of oxidative stress and abnormality in protein ag-
gregation (Mattson and Magnus, 2006). Along with this if the in-
dividuals are exposed to various toxins which are responsible for
causing oxidative stress, the susceptibility of worsening the cognitive
functions is significantly increased (Meramat et al., 2017).
The exposure of humans to heavy metals from occupation and en-
vironment is a potential risk factor for cognitive impairment (Afridi
et al., 2011). The metals cannot be synthesized or destroyed by the
human body. Therefore, an efficient absorption, transport and cellular
and Cu) to the body (Clemens, 2006). This transport mechanism is used
by all the heavy metals to enter and get accumulated in the body. These
accumulated heavy metals have the capacity to generate reactive
oxygen species (superoxide and nitric oxide) in biological system and
lead to oxidative stress. This results in increased production of reactive
oxygen species which overwhelms body antioxidant potential and in-
duces various proteins modification and DNA damage, resulting in
various diseases including neurological disorders (Jomova and Valko,
2011). Oxidative stress impairs the function of endothelial cells and
promotes penetration of macrophages to parenchyma of brain, conse-
quently the nutrients availability to brain cells are decreased and in-
itiate the inflammatory process (Markesbery, 1997). Studies have
shown that elevated levels of metals are associated with various dis-
orders enlisted in Table 1.
In Pakistan, we are exposed to heavy metals mainly from environ-
mental and dietary sources as mentioned in Table 1. A relevant study by
Azizullah et al., has shown very high levels of heavy metals (Zn, Cu,
Mn, Cd, Pb, Ni and Hg) in drinking water throughout in the Pakistan.
These levels are significantly higher than those set by WHO (Azizullah
et al., 2011). Textile, pharmaceuticals and fertilizer industries are the
main source of metal contamination in water of Pakistan (Nasrullah
et al., 2006). The crops irrigated with metal (Cu, Zn, Mn. Pb, Co and Ni)
contaminated water is another main source of human exposure to

⁎
Corresponding author.
E-mail address: touqeer.ahmed@asab.nust.edu.pk (T. Ahmed).

https://doi.org/10.1016/j.etap.2018.04.011
Received 16 January 2018; Received in revised form 10 April 2018; Accepted 11 April 2018
Available online 16 April 2018
1382-6689/ © 2018 Elsevier B.V. All rights reserved.
G. Iqbal et al. Environmental Toxicology and Pharmacology 60 (2018) 100–109

heavy metals in Pakistan (Hussain et al., 2013). Hence, these metals

and the oxidative stress produced by these metals are the main culprits
Safe intake quantities of
metal in drinking water
(Organization, 2008) for various diseases including cognitive impairment in developing
countries (Olness, 2003), like Pakistan.
Studies have revealed that elevated levels of metals are associated
with dementia, neurotoxicity, amyloid plaques formation, and

0.005 ppm
0.05 ppm
0.1 ppm

0.4 ppm
Alzheimer’s disease (mentioned in Table 1). However, there is very
1 ppm

5 ppm
limited data available on metal exposure and its effects on cognitive
impairment, especially, from Pakistan where people are highly exposed
Permissible limits of metals in

to metals. Therefore this study was designed to evaluate the effect of

body (Organization, 2008;

metal accumulation with severity of cognitive impairment (measured

through MMSE scores).

0.26 mg/l; 0.06 mg/kg

0.1 mg/l; 0.5 μg/kg

The determination of elements in the human tissue, is the most

Singh et al., 2011)

0.1 mg/l; 25 μg/kg

0.06 mg/l; 7 μg/kg

15 mg/l; 1 mg/kg

common and important application for clinical screening and diag-

nostic procedures. Blood sample may be conveniently used to study
0.1 μg/kg

bio-indicator for this purpose (Kazi et al., 2008c). The evaluation of

trace elements in blood samples is frequently used traditional ap-
proach, however any change in physiological or environmental con-
ditions may result in fluctuation of these elements in the blood (Afridi
Parkinson’s disease (Hirsch et al., 1991), dementia

Motor neuron disease such as Amyotrophic lateral

Alzheimer’s disease (Cuajungco and Fagét, 2003).
Motor neuron diseases (Weihl and Lopate, 2006),

Alzheimer’s disease (Crapper McLachlan, 1986),

and Schneider, 2003; Needleman and Gatsonis,

attention deficit hyperactivity disorder (Lidsky
Neurotoxicity, risk of mild mental retardation,

et al., 2008).
Based on toxicity of these metals and limited data showing the re-
Parkinson’s disease (Powers et al., 2003),
Alzheimer’s disease (Syme et al., 2004).

lationship of these metals with cognitive impairment, we decided to

neurotoxicity (Mergler et al., 1998).

accurately measure the trace element concentration in “blood samples”

Nervous system related disorders

and to measure their correlation with “cognitive impairment”.

sclerosis (Bar-Sela et al., 2001).
1990), lead encephalopathy.

Therefore it demands a highly sensitive technique. Atomic absorption

spectrometry is the extensively used method due to its selectivity and
(Larson et al., 1992).

sensitivity, and numerous reports justify this statement (Afridi et al.,

2011; Kazi et al., 2008a,c). The primary goal of studies was to measure
the metal concentration in blood samples and find out if association
exits with cognitive impairment. Moreover, we also evaluated if metal
accumulation was also related with age or not. It is the first study to
document the association between the concentrations of metals in
whole blood with the age in Pakistani subjects.
Anemia (Lozoff et al., 2000), diabetes mellitus (Kazi

metabolism, a possible higher risk of kidney stones

Kidney damage, disturbed phosphorus and calcium

(Nordberg, 2004), bone damage (Kazantzis, 1979),

Developmental disabilities (Lidsky and Schneider,
Cancer, cardiovascular disease (Kok et al., 1988),
et al., 1991), cancer, cardiovascular disease (Kok
Diseases associated with elevated level of metals

Bone disorder (Baxter et al., 1953), hepatitis (Li

pulmonary dysfunction (Mazzoli-Rocha et al.,

Ischemic heart disease (Costello et al., 2014),

2. Materials and methods

chronic kidney disease (Kurella et al., 2004),

2.1. Compliance with ethical standards

All procedures performed in this study, involving human partici-

cancer (Waalkes et al., 1988).

pants, were in accordance with ethical standard of Internal Review

Board (IRB); letter number IRB-67, Atta-ur-Rahman School of Applied
Biosciences, National University of Sciences and Technology and with
diabetes mellitus.

the 1964 Helsinki declaration. Informed consent was obtained from all
Disease associated with elevated level of metals and their permissible limits.

et al., 2008b).
et al., 1988).

individual participants included in the study.

2010).

2003)

2.2. Assessment of cognitive function

Neuropsychiatric evaluation of all subjects was performed by heath

water treatment plants, antacids (Keith et al., 2008)

Contaminated food, drinking-water, the lead-glazed

Color pigment in paint, in the fabrication of nickel-

products anticorrosive agent, a neutron-absorber in

Diet, drinking water (Bouchard et al., 2011), metal
Beverages cans and foil, antiperspirants, cosmetics,

nuclear power plants, tobacco smoking(Orlowski

care provider. The inclusions criteria of subject are listed in Table 2. All
Food (Galdes and Vallee, 1983), zinc fumes, soil

material for food storage, lead-containing paint,

cadmium batteries, fertilizers, stabilizer in PVC

273 subjects were interviewed to obtain detailed information through

laden dust in industrial areas (Finley, 2004).

an approved questionnaire that covers the demographic data, medical

history and occupation. The cognitive function of each subject was
assessed using mini mental state examination (MMSE). The MMSE also
Fish, meat (Wong et al., 2001)

known as Folstein test, is a 30-point questionnaire test that is used

(Fine et al., 1997), Refineries.

extensively in research and clinical setting to screen cognitive impair-

ment (Folstein et al., 1975). All subjects were then classified into mild,
and Piotrowski, 2003).
Environmental sources

moderate and severe cognitive impairment, according to their MMSE

score as listed in Table 3.

2.3. Measurement of metal levels

petrol.

The analysis of heavy metals was carried out by means of a double-

beam Perkin Elmer atomic absorption spectrometer model 700 (Perkin
Manganese
Aluminum

Cadmium

Elmer, USA) equipped with a flame burner and graphite furnace, a

Copper
Table 1

Metal

Lead
Zinc

pyrocoated graphite tube with an integrated platform and an auto-

sampler AS-800 (Perkin Elmer). The instrumental parameters are

101
G. Iqbal et al.
shown in Table 4. The stock standard solution was prepared according
to instructions provided in manual of the instrument. The straight line
was obtained while plotting the absorbance of standard vs. concentra-
tion. Different standard dilutions were used for each metals and the
absorbance of test samples were only measured after getting a straight
line graph. If the concentration in test sample was high, then it was
further diluted. The standard dilutions for Zn were 0.5 mg/L, 1 mg/L,
1.5 mg/L; Pb were 2 mg/L, 4 mg/L, 6 mg/L; Cu, Al, Mn and Cd were
1 mg/L, 2 mg/L, 3 mg/L. A KENTAX (KNM-20/BW) domestic micro-
wave oven (maximum heating power of 700 W) was used for digestion
of the biological samples.
2.4. Reagents and glasswares
Concentrated nitric acid (65%) and hydrogen peroxide (30%) were
obtained from Merck (Darmstadt, Germany). Working standard solu-
tions of Al, Cu, Pb, Cd, Mn and Zn were prepared prior to their use by
stepwise dilution of certified standard solutions. Before use, all glass-
ware and plastic materials were soaked for 24 h in 5 M nitric acid,
washed and finally rinsed with pure water and dried. Double distilled
and syringe filtered (0.2 μm) water was used throughout the work.
2.5. Blood sample collection and pretreatment
Before the sample collection, all healthy and cognitively impaired
patients were informed about the study and all agreed to participate.
Venous blood samples (3 mL) were collected in EDTA K3 vacutainer
tubes (Bio Tube, Inc.), and the whole blood sample was thoroughly
mixed and transferred to a storage boxes at −4 °C until further pro-
cessed for acid digestion.
2.6. Microwave-assisted acid digestion of blood samples for metal detection
For digestion of blood samples, 0.5 ml of heparinized human whole
blood samples of each patient and control subjects were directly taken
into separate, properly labeled test tube. The mixture of concentrated
HNO3–H2O2 (2:1, v/v) was freshly prepared and three milliliters of this
mixture was added in each test tube, thoroughly mixed and kept at
room temperature for 10 min. This mixture was then heated in micro-
wave oven at 500 W. Microwave digestion was carried out to shorten
digestion time. Complete digestion of blood samples required 1–2 min.
After cooling, the digested samples were filtered through Whatman 42
filter paper and brought to a required volume with pure water (Afridi
et al., 2011; Kazi et al., 2007, 2008b; Olmedo et al., 2010). After fil-
tration, the samples were ready for the measurement of metal by the
atomic absorption spectrometer.
2.7. Statistical analysis
Data were expressed as mean ± standard error of mean (SEM) and
n = number of subjects. Statistical analysis of results was performed
Table 2
Inclusion and exclusion criteria.
Inclusion Criteria
Objective evidence (from physical and neurological examination and laboratory tests usually
(MMSE) Mini mental state examination).
The presence of a disorder in cognitive function for most of the time for at least two weeks, as
reported by the individual or a reliable informer
History of cerebral disease, damage or dysfunction, or of systemic physical disorder known to
cause cerebral dysfunction, including hormonal disturbances and non-psychoactive drug
effects
Either gender
Individuals with age ≥50 years.
Irrespective of Race and origin
102
Environmental Toxicology and Pharmacology 60 (2018) 100–109
Table 3
Classification of subjects according to MMSE score.
Groups MMSE Score(Crum et al., Number of
1993; Sayed et al., 2014) subjects
Age-matched healthy Control 25–30 90
Mild Cognitive Impairment 21–24 72
(Mild CI)
Moderate Cognitive Impairment 10–20 86
(Moderate CI)
Severe Cognitive Impairment 0–9 25
(Severe CI)
using “Graph Pad Prism” software and taken as significant only if the p
value was less than 0.05. One-way ANOVA was applied using
BonferroniPost-hoc test. Correlation analysis of “metal concentration”
with “MMSE scores” and “metal concentration” with “age” was per-
formed using Pearson’s correlation test using Graph Pad Prism.
3. Results
3.1. Demographic data
The demographic data obtained from all the groups i.e. age-matched
healthy control, mild CI, moderate CI and severe CI is summarized in
Table 5. Among the studied groups the percentage of female was high in
severe cognitively impaired group. It was 60% in case of severe CI,
59.30% in moderate CI, 54.20% in mild CI and 46.6% in age-matched
healthy controls; while the percentage of male was 40%, 40.70%,
45.8% and 53.4% respectively. The income status is categorized into 3
groups i.e. lower, middle and upper according to the dollars earned per
person per day (Durr-e-Nayab, 2011). Lower income status subjects
were; severe CI 64%, Moderate CI 53%, Mild CI 25% and age-matched
healthy control 12.5%; in case of middle income status these were 32%,
24.4%, 57%, 30% respectively; and in case of upper class income status
these were 4%, 22.6%, 18%, 57.3% respectively.
The educations level is categorized according to the years of edu-
cation, in to 4 groups i.e. 0–5 years, 6–10 years. 11–14 years and 14+
years of education. Among the 0–5 years groups subjects were, severe
CI 68%, moderate CI 50%, mild CI 48.6% and age-matched healthy
control 28.9%, while in case of 6-10 years group,32%, 45.3%, 44.4%
and 54.4%, in case of 11-14 years group 0%, 3.5%, 4.2%, 5.6% and in
case of 14+ years, 0%, 1.2%, 2.8% and 11.1% respectively.
The occupation of the mild CI, moderate CI, severe CI and age
matched healthy control is mentioned in Table 6. The groups are fur-
ther divided into male and female and their occupation is mentioned.
3.2. Copper concentration in blood samples
The levels of Cu in all the biological samples of mild, moderate and
severe cognitive impairment patients were higher as compared to the
Exclusion Criteria
Suggestive evidence for an alternative causation of the mental disorder, e.g. a
highly loaded family history for a clinically similar or related disorders
Known genetic neurologic disorder or history of psychiatric illness
Systemic physical disorder
Obvious kidney disease (Kidney failure)
Subject carrier of blood transmitted infections
Known liver disease (Hepatitis, jaundice)
Patients having cancer/ tumors
G. Iqbal et al. Environmental Toxicology and Pharmacology 60 (2018) 100–109

Table 4
Atomic absorption spectrometer parameters for heavy metal analysis.
Parameters Aluminum Zinc Lead Copper Cadmium Manganese

Lamp current 10.0 mA 5.0 mA 5.0 mA 3.0 mA 4.0 mA 6.0 mA

Slit width 0.2 nm 0.2 nm 0.2 nm 0.2 nm 0.2 nm 0.2 nm
Wave length 309.3 nm 213 nm 405.8 nm 327.4 nm 228.8 nm 285.2 nm
Sensitivity 0.55 μg/ml 0.008 μg/ml 0.06 μg/ml 0.025 μg/ml 0.5 μg/ml 0.3 μg/ml

age-matched healthy control subjects. The level of Cu in severe cogni-
tive impairment (2.37 ± 0.12 mg/L) was higher (p < 0.001) from
mild (1.41 ± 0.10 mg/L) and moderate cognitive impairment
(1.97 ± 0.11 mg/L). There was significantly(p < 0.001) decreased Cu
level in age-matched healthy control (0.7 ± 0.06 mg/L) relative to
mild cognitive impairment (Fig. 1a).
3.3. Aluminum concentration in blood samples
The high concentration of Al was associated with the decrease in
MMSE score. The level of Al in blood samples were significantly
(p < 0.001) higher in severe cognitive impairment subjects
(127.8 ± 4.83 μg/L) when compared to mild (63.37 ± 3.52 μg/L) and
moderate (92.4 ± 3.57 μg/L) cognitive impairment groups (Fig. 1b).
Al concentration was significantly (p < 0.001) low in age-matched
healthy control group (47.30 ± 2.76 μg/L) when compared to cogni-
tively impaired groups.
3.6. Manganese concentration in blood samples
Mn levels were significantly (p < 0.001) higher in severe
(92.08 ± 6.8 μg/L) and moderate (77.8 ± 2.4 μg/L) cognitively im-
paired group as compared to the age-matched healthy control group
(52.8 ± 2.8 μg/L). The mild (64.97 ± 3.76 μg/L; p < 0.05) cogni-
tively impaired group was also having significantly elevated levels of
Mn compared to the age-matched healthy control group (Fig. 1e).
3.7. Cadmium concentration in blood samples
Cd concentration in mild (3.87 ± 0.25 μg/L; p < 0.05), moderate
(4.86 ± 0.27 μg/L; p < 0.001) and severe (6.11 ± 0.38 μg/L;
p < 0.001) cognitively impaired groups were significantly high when
compared to the age-matched healthy control (2.85 ± 0.19 μg/L)
group (Fig. 1f).
3.8. Correlation analysis of metal levels with MMSE

3.4. Zinc concentration in blood samples
The concentration of Zn was significantly high in the moderate
(6.4 ± 0.26 mg/L; p < 0.01) and severe (7.92 ± 0.32 mg/L;
p < 0.001) cognitive impairment groups when compared to the age-
matched healthy control group (4.95 ± 0.33 mg/L). There was no
significant difference in concentration of Zn among age-matched
healthy control and mild cognitively impaired group (5.98 ± 0.24 mg/
L) but the increasing trend was there (Fig. 1c).
In order to examine the association of different metals concentra-
tions on the extent of cognitive impairment, correlation analysis was
performed. Pearson’s correlation test revealed the negative correlation
between the metal concentration and MMSE score. The maximum
correlation was observed with Al (r = −0.638; p < 0.001) followed by
Cu (r = −0.610; p < 0.001), Pb (r = −0.554; p < 0.001), Cd
(r = −0.418; p < 0.001), Mn (r = −0.417; p < 0.001) and Zn
(r = −0.329; p < 0.001) respectively (Fig. 2a–f).
3.9. Correlation analysis of metal levels with age

3.5. Lead concentration in blood samples
The concentration of Pb was significantly (p < 0.001) high in se-
vere (274.1 ± 11.92 μg/L) cognitively impaired group as compared to
the age-matched healthy control group (157.6 ± 7.76 μg/L). Moderate
(229.2 ± 6.93 μg/L; p < 0.001) and mild (188.7 ± 6.23 μg/L;
p < 0.05) cognitively impaired groups had significantly higher con-
centration of Pb, relative to the age-matched healthy control group
(Fig. 1d).
Correlation analysis was done in order to examine the relationship
of different metals concentrations with the age. Pearson’s correlation
test was performed and it showed the correlation between the metal
concentration and age in years of all the subjects studied in all the
groups. Among the studied metals, the maximum correlation was ob-
served with Al (r = 0.187; p < 0.01) followed by Pb (r = 0.148;
p = p < 0.05) and Cd (r = 0.14; p < 0.05). There was no correlation
observed with the concentration of Cu (r = −0.025), Zn(r = 0.111)
and Mn (r = 0.106) with the age (Fig. 3a–f).

Table 5
Demographic data of subjects included in study.
Age-matched healthy Control Mild CI Moderate CI Severe CI P-value

Percent Male 53.4 45.80 40.70 40

Percent Female 46.6 54.20 59.30 60

Age ( ± SEM) 58.1 ± 0.69 61.7 ± 1.071 65.1 ± 1.18 79.68 ± 1.4 < 0.0001

Percent Income status

Lower 12.2 25 53 64
Middle 30 57 24.4 32
Higher 57.3 18 22.6 4

Education in years (percent)

0–5 28.9 48.6 50 68
6–10 54.4 44.4 45.3 32
11–14 5.6 4.2 3.5 0
14+ 11.1 2.8 1.2 0

103
G. Iqbal et al. Environmental Toxicology and Pharmacology 60 (2018) 100–109

Table 6
: Occupation of the subjects included in the study.
Groups No of Male Occupation No of Female Occupation

Age-matched healthy control 48 8 teachers; 11 Govt. servant; 27 private; business; 2 labor 42 6 Home tuitions; 14 Tailor; 22 House wife

Mild 33 12 Govt. servant; 21 private business 39 1 lady health worker; 2 maid; 36 house wife

Moderate 51 3 school teachers; 21 private business; 12 mason; 13 labor; 2 Tailor 35 4 maid; 31 house wife

Severe 10 2 driver; 2 mason; 6 labor 15 2 maid; 13 house wife

3.10. Metal levels in sub-fractions of the control group according to the age
The average age of control subjects and the average age of mild,
moderate and severe cognitive impairment groups was different,
therefore, we wanted to check the effect on metal accumulation with
increasing age. The control group was sub-fractioned to 50–59 years,
60–69 years and 70–79 years; to evaluate whether metal accumulation
is related to the age or not. There was no significant difference observed
in the metal concentration among the sub-fractions of age-matched
healthy control. Only the Cu concentration was decreasing with the
increase in age. All the other metals showed no change (there was no
increase) as shown in Fig. 4a–f.
4. Discussion
This population based study explored the association between the
levels of metals in blood to the extent of cognitive impairment. The
heavy metals exposure causes oxidative stress in brain tissue and is a
risk factor for cognitive impairment (Deibel et al., 1996; Huang et al.,
2004; Praticò et al., 2002; Smorgon et al., 2004).There are several
evidences which highlight the role of oxidative stress mechanisms and
free radical damage in pathology of cognitive impairment (Solfrizzi
et al., 2006). In this study, we determine the Cu, Pb, Al, Zn, Cd and Mn
levels in acid digested blood samples from the patients of cognitive
impairment and compared them with the age-matched healthy control
subjects. The degree of cognitive impairment was measured by MMSE
score. MMSE score measures all five dimensions of cognition which are
specified by the diagnostic and statistical manual (DSM-iv) that in-
cludes; memory impairment (both short and long term), aphasia (de-
terioration of language function), apraxia (impaired ability to execute
motor activities), agnosia (inability to recognize objects) and dis-
turbances in executive functioning.
Despite the unavailability of accurate data about the prevalence of
cognitive impairment in Pakistan, there are various studies that in-
dicate that dementia and mental disorders are increasing in developing
countries like Pakistan, either due to poverty or illiteracy (Ferri et al.,
2006; Patel and Kleinman, 2003; Prince et al., 2003). Cognitive im-
pairment is classified as mild, moderate and severe cognitive

Fig. 1. Bar graph showing the metals concentration in blood samples of age-matched healthy control, mild cognitive impairment, moderate cognitive impairment
and severe cognitively impaired subjects. (a) Cu concentration in blood samples. (b) Al level in blood samples.(c) Zn level in blood samples. (d) Pb level in blood
samples. (e) Mn level in blood samples and (f) Cd blood level comparison among groups. *p < 0.05, **p < 0.01, ***p < 0.001 compared with age-matched
healthy control group; # p < 0.05, ##p < 0.01, ###p < 0.001 comparison with mild cognitive impaired group; n is sample size.

104
G. Iqbal et al.
Fig. 2. Correlation graph is between the metal concentration in blood of studied samples and MMSE score. (a) Correlation of Cu concentration with MMSE scores. (b)
Correlation of Al concentration with MMSE scores. (c) Correlation of Zn concentration with MMSE scores. (d) Correlation of Pb concentration with MMSE scores. (e)
Correlation of Mn concentration with MMSE scores and (f) Correlation of Cd concentration with MMSE scores.
impairment based on MMSE scores. In mild cognitive impairment, the
learning and memory is compromised to a greater extend as can be
expected at certain age. However the moderate and severe cognitive
impairment is much severe condition and patient sometimes need
nursing care (Crum et al., 1993; Sayed et al., 2014). There are various
risk factors which are associated with the cognitive impairment and
metals may be a suspect risk factor for decreased level of cognition. A
population based survey in French men has shown that metals and pH
of drinking water has been related to cognitive impairment in elderly
(Jacqmin et al., 1994). In Pakistan, heavy metals are extensively found
in environment and humans are exposed to these trace metals from
many sources, such as contaminated, water, air, soil and food (Jomova
and Valko, 2011). Numerous studies point out that these metals act as
catalysts in the oxidative reactions at cellular level in biological func-
tions (Deibel et al., 1996; Patra et al., 2001; Valko et al., 2005). Metals
induced oxidative stress results in high yield of reactive oxygen species
in neuronal cells and this might be responsible for the neurotoxic effects
of these metals (Barnham et al., 2004; Ercal et al., 2001; Iqbal et al.,
2016; Squitti et al., 2011). The oxidative stress act as an early signal to
mediate apoptosis of neurons and cerebral endothelial cells of blood
brain barrier (Bush, 2000; Greenlund et al., 1995). This damage of
blood brain barrier makes it more vulnerable to neurotoxicants (Oteiza
et al., 2004), further worsening the situation and leading to mental
disorders. We found that as the concentration of these metals in blood is
increasing, the cognitive impairment shifts from mild to moderate and
then severe. This is strongly proven by our correlation studies between
metal concentrations with MMSE score. Among the examined metals, Al
and Cu are found to be more associated with the extent of cognitive
105
Environmental Toxicology and Pharmacology 60 (2018) 100–109
impairment as compared to Pb, Mn, Cd and Zn. The negative correla-
tion between metal concentration and MMSE score depict that as the
metal concentration is increasing, there is decreasing trend of MMSE
score. The correlation analysis are supporting the literature that Al le-
vels are strongly associated with cognitive impairment and can leads
towards dementia (Rondeau et al., 2000). We found that the con-
centration of Cu, Zn and Mn in blood is not associated with the increase
in age. On the other hand, the correlation among the age and Al, Cd and
Pb concentration depicted that with the increase in age these metals
were also increasing. But the association between metal concentration
and age is weaker than the relationship of metal concentration with the
MMSE score. Hence, based on the strong association of metals with
MMSE score, we concluding that elevated metals concentration is a risk
factor for the cognitive impairment and its progression.
Our study demonstrates that individuals with high concentration of
Al in blood have greater cognitive impairment. The strongest correla-
tion of Al concentration was shown with the MMSE score. Previous
studies also reports that Al from environmental source leads to the
dementia and cognitive decline (Solfrizzi et al., 2006). Al has been
shown to convert amyloid beta alpha-helix conformation to beta-ple-
ated sheets thereby causing aggregation of amyloid protein and for-
mation of amyloid plaques(Exley et al., 1993) and neuronal death, thus
leading to cognitive impairment (Exley et al., 1993).Our study has
shown that with increase in age, the Al concentration is also increasing,
and it is consistent with the previous finding reporting that Al accu-
mulate in age dependent manner in aorta and cerebral artery (Minami
et al., 1996). But further studies are needed for detailed elaboration and
underlying mechanism exploration.
G. Iqbal et al.
Fig. 3. Correlation graph showing the relationship between metal concentrations in blood samples of all the subjects and their age in years. (a) Correlation of Cu
concentration with age in years. (b) Correlation of Al concentration with age in years. (c) Correlation of Zn concentration with age in years. (d) Correlation of Pb
concentration with age in years. (e) Correlation of Mn concentration with age in years and (f) Correlation of Cd concentration with age in years.
In our study, Cu was at second number for its association with
MMSE. Squitti et al, have also reported that Cu concentration can help
to discriminate the cognitively impaired subjects from the control
subjects (Squitti et al., 2011). Cu act as a cofactor for cytochrome C
oxidase and superoxide dismutase enzymes which is involved in redox
reactions (Valko et al., 2005). Cu induces neurotoxicity by two me-
chanisms, first, Cu directly catalyzes the formation of reactive oxygen
species (Prousek, 2007), and, secondly high level of Cu decrease glu-
tathione level, which is a powerful antioxidant in cell. Decreased glu-
tathione level further aggravates oxidative stress in cell(Mattie and
Freedman, 2004). Cu accumulation in blood is independent of the age
as documented in the results. Wills et al., found that Cu and Zn con-
centration build up in retina of eye with age(Wills et al., 2008), but
there is no data available about Cu accumulation in the whole blood
with age.
Blood Pb concentration can affect various physiological functions
(Kim et al., 1996)and early life exposure to Pb leads to the formation of
amyloid plaques (Wu et al., 2008). Previous data reported that there is
higher possibility of cognitive impairment in individuals who are more
exposed to environmental Pb (Rogan et al., 2001). Our results con-
firmed that Pb at high concentration in blood is associated with im-
paired cognitive functions as confirmed from correlation studies and
the patients with severe cognitive impairment showed the highest blood
concentration of Pb as compared to age-matched healthy control sub-
jects.
There is no known physiological function of Cd and it is absorbed
and transported by the system acquired by other essential metals
(Satarug et al., 2011) from the environment and diet. Cd is readily
106
Environmental Toxicology and Pharmacology 60 (2018) 100–109
absorbed by respiratory tract as compared to digestive tract and smo-
kers are more prone to Cd toxicity (Elinder et al., 1983). Cd up-reg-
ulates glutamate dehydrogenase and enhances excitotoxicity of gluta-
mate as well as reduces superoxide dismutase level. All these factors
result in impairment of blood brain barrier and ultimately cognitive
functions (Bar-Sela et al., 2013). Our results indicate that severity of the
cognitive impairment is significantly increased as the concentration of
Cd in blood is elevated. These results are consistent with the previously
reported data mentioning that the people who are exposed to Cd have
greater tendency to have poor cognitive functions (Hart et al., 1989).
Further studies are needed to find Cd pathological effects alone and in
combination in old age population.
The values of Mn in mild, moderate and severe cognitively impaired
individuals were higher as compared to age-matched healthy controls
hence highlighting the toxic role of Mn in cognitive pathology. Mn
rapidly crosses blood brain barrier and causes neurotoxicity. Chronic
Mn exposure leads to Mn psychosis (Verhoeven et al., 2011). The use of
Mn containing pesticides is widespread in agriculture, hence increasing
the human exposure to this toxic metal (Lucchini et al., 1995).Ger-
hardsson et al, have previously reported that increased serum Mn level
is directly related to problems in cognitive functions (Gerhardsson
et al., 2008).
Zn and Cu are essential elements for human health but overexposure
can lead to various health complications (Arora et al., 2006; Fosmire,
1990). Zn at higher concentration act as neurotoxin causing demyeli-
nation (Tokuda et al., 2007), oxidative stress and leads to neuronal cell
death at different concentrations (Koh et al., 1996). Previous study has
reported that Zn is involved in the pathogenesis of dementia (Mizuno
G. Iqbal et al.
Fig. 4. The bar graph showing the sub-fractions of the control group according to the age and metal levels. (a) Cu concentration in sub-fractions of age-matched
healthy control. (b) Al concentration in sub-fractions of age-matched healthy control. (c) Zn concentration in sub-fractions of age-matched healthy control. (d) Pb
concentration in sub-fractions of age-matched healthy control. (e) Mn concentration in sub-fractions of age-matched healthy control. (f) Cd concentration in sub-
fractions of age-matched healthy control. *p < 0.05.
and Kawahara, 2013). Our study also showed the association of the
high Zn concentration in serum with the level of cognitive impairment.
There is marked increase in Zn level in individuals with low MMSE
score.
The significantly high levels of all the aforementioned metals were
observed to be associated with decrease in MMSE score. Numerous
studies from Pakistan have shown the relationship of high metal con-
centration in biological samples with various diseases including pa-
ralysis (Afridi et al., 2011), diabetes mellitus (Afridi et al., 2008), lung
cancer (Kazi et al., 2008a) and myocardial infarction (Kazi et al.,
2008c) but limited data is present on cognitive functions. Conclusively,
oxidative stress caused by these metals may be considered as culprit for
development and progression of cognitive dysfunctions. The persistence
of metals in the environment and diet requires a long term approach to
restrict our exposure by environmental management and maintaining
of lower toxic metals level in water and diet. Along with this the
awareness should be provided to the general public about the toxic
effects of metals on nervous system.
5. Conclusion
This study conclusively suggests that metals exposure is associated
with pathology of cognitive impairment and its progression in old age.
There could be multiple mechanisms responsible for the cognitive im-
pairment caused by metal exposure but, the oxidative stress seems to be
the most significant. However single element could not be responsible
for all various types of cellular and molecular events. This suggests the
107
Environmental Toxicology and Pharmacology 60 (2018) 100–109
need for decrease in metal exposure to humans especially Al and Cu
from environment, food and industries.
Declaration of interest
The authors declare that they have no conflict of interest.
Acknowledgments
The authors will like to thank Atta-ur-Rahman School of Applied
Biosciences, National University of Sciences and Technology, Islamabad
and Higher Education Commission (HEC) of Pakistan for sponsoring
this project.
References
Giagulli, A.V., Guastamacchia, E., Licchelli, B., Triggiani, V., 2016. Serum testosterone
and cognitive function in ageing male: updating the evidence. Recent Pat. Endocr.
Metab. Immune Drug Discov. 10, 22–30.
Afridi, H.I., Kazi, T.G., Kazi, A.G., Shah, F., Wadhwa, S.K., Kolachi, N.F., Shah, A.Q., Baig,
J.A., Kazi, N., 2011. Levels of arsenic, cadmium, lead, manganese and zinc in bio-
logical samples of paralysed steel mill workers with related to controls. Biol. Trace
Elem. Res. 144, 164–182.
Afridi, H.I., Kazi, T.G., Kazi, N., Jamali, M.K., Arain, M.B., Jalbani, N., Baig, J.A., Sarfraz,
R.A., 2008. Evaluation of status of toxic metals in biological samples of diabetes
mellitus patients. Diabetes Res. Clin. Pract. 80, 280–288.
Arora, R., Kulshreshtha, S., Mohan, G., Singh, M., Sharma, P., 2006. Estimation of serum
zinc and copper in children with acute diarrhea. Biol. Trace Elem. Res. 114, 121–126.
Azizullah, A., Khattak, M.N.K., Richter, P., Häder, D.-P., 2011. Water pollution in
Pakistan and its impact on public health—a review. Environ. Int. 37, 479–497.
G. Iqbal et al.
Bar-Sela, S., Reingold, S., Richter, E.D., 2001. Amyotrophic lateral sclerosis in a battery-
factory worker exposed to cadmium. Int. J. Occup. Environ. Health 7, 109–112.
Bar-Sela, S., Reingold, S., Richter, E.D., 2013. Amyotrophic lateral sclerosis in a battery-
factory worker exposed to cadmium. Int. J. Occup. Environ. Health 7 (2), 109–112.
Barnham, K.J., Masters, C.L., Bush, A.I., 2004. Neurodegenerative diseases and oxidative
stress. Nat. Rev. Drug Discov. 3, 205–214.
Baxter, J., Van Wyk, J., Follis Jr, R., 1953. A bone disorder associated with copper de-
ficiency. 2. Histological and chemical studies on the bones. Bull. Johns Hopkins Hosp.
93, 25–39.
Bouchard, M.F., Sauvé, S., Barbeau, B., Legrand, M., Brodeur, M.-È., Bouffard, T.,
Limoges, E., Bellinger, D.C., Mergler, D., 2011. Intellectual impairment in school-age
children exposed to manganese from drinking water. Environ. Health Perspect. 119,
138.
Bush, A.I., 2000. Metals and neuroscience. Curr. Opin. Chem. Biol. 4, 184–191.
Clemens, S., 2006. Toxic metal accumulation, responses to exposure and mechanisms of
tolerance in plants. Biochimie 88, 1707–1719.
Costello, S., Brown, D.M., Noth, E.M., Cantley, L., Slade, M.D., Tessier-Sherman, B.,
Hammond, S.K., Eisen, E.A., Cullen, M.R., 2014. Incident ischemic heart disease and
recent occupational exposure to particulate matter in an aluminum cohort. J. Expos.
Sci. Environ. Epidemiol. 24, 82–88.
Crapper McLachlan, D.R., 1986. Aluminum and Alzheimer’s disease. Neurobiol. Aging 7
(6), 525–532.
Crum, R.M., Anthony, J.C., Bassett, S.S., Folstein, M.F., 1993. Population-based norms for
the mini-mental state examination by age and educational level. JAMA 269,
2386–2391.
Cuajungco, M.P., Fagét, K.Y., 2003. Zinc takes the center stage: its paradoxical role in
Alzheimer's disease. Brain Res. Rev. 41, 44–56.
Deary, I.J., Corley, J., Gow, A.J., Harris, S.E., Houlihan, L.M., Marioni, R.E., Penke, L.,
Rafnsson, S.B., Starr, J.M., 2009. Age-associated cognitive decline. Br. Med. Bull. 92,
135–152.
Deibel, M., Ehmann, W., Markesbery, W., 1996. Copper, iron, and zinc imbalances in
severely degenerated brain regions in Alzheimer's disease: possible relation to oxi-
dative stress. J. Neurol. Sci. 143, 137–142.
Durr-e-Nayab, 2011. Estimating the middle class in Pakistan. Pak. Dev. Rev. 1–28.
Elinder, C.G., Kjellström, T., Lind, B., Linnman, L., Piscator, M., Sundstedt, K., 1983.
Cadmium exposure from smoking cigarettes: variations with time and country where
purchased. Environ. Res. 32, 220–227.
Ercal, N., Gurer-Orhan, H., Aykin-Burns, N., 2001. Toxic metals and oxidative stress part
I: mechanisms involved in metal-induced oxidative damage. Curr. Top. Med. Chem.
1, 529–539.
Exley, C., Price, N.C., Kelly, S.M., Birchall, J.D., 1993. An interaction of β‐amyloid with
aluminium in vitro. FEBS Lett. 324, 293–295.
Ferri, C.P., Prince, M., Brayne, C., Brodaty, H., Fratiglioni, L., Ganguli, M., Hall, K.,
Hasegawa, K., Hendrie, H., Huang, Y., 2006. Global prevalence of dementia: a Delphi
consensus study. Lancet 366, 2112–2117.
Fine, J.M., Gordon, T., Chen, L.C., Kinney, P., Falcone, G., Beckett, W.S., 1997. Metal
fume fever: characterization of clinical and plasma IL-6 responses in controlled
human exposures to zinc oxide fume at and below the threshold limit value. J. Occup.
Environ. Med. 39, 722–726.
Finley, J.W., 2004. Does environmental exposure to manganese pose a health risk to
healthy adults? Nutr. Rev. 62, 148.
Folstein, M., Anthony, J.C., Parhad, I., Duffy, B., Gruenberg, E.M., 1985. The meaning of
cognitive impairment in the elderly. J. Am. Geriatr. Soc. 33, 228–235.
Folstein, M.F., Folstein, S.E., McHugh, P.R., 1975. “Mini-mental state”: a practical method
for grading the cognitive state of patients for the clinician. J. Psychiatr. Res. 12,
189–198.
Fosmire, G.J., 1990. Zinc toxicity. Am. J. Clin. Nutr. 51, 225–227.
Galdes, A., Vallee, B.L., 1983. Categories of zinc metalloenzymes. Met. Ions Biol. Syst. 15,
1–54.
Gerhardsson, L., Lundh, T., Minthon, L., Londos, E., 2008. Metal concentrations in plasma
and cerebrospinal fluid in patients with Alzheimer’s disease. Dement. Geriatr. Cogn.
Disord. 25, 508–515.
Greenlund, L.J., Deckwerth, T.L., Johnson, E.M., 1995. Superoxide dismutase delays
neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death.
Neuron 14, 303–315.
Hart, R.P., Rose, C.S., Hamer, R.M., 1989. Neuropsychological effects of occupational
exposure to cadmium. J. Clin. Exp. Neuropsychol. 11, 933–943.
Hirsch, E., Brandel, J.P., Galle, P., Javoy- Agid, F., Agid, Y., 1991. Iron and aluminum
increase in the substantia nigra of patients with Parkinson’s disease: an X‐Ray mi-
croanalysis. J. Neurochem. 56, 446–451.
Huang, X., Moir, R.D., Tanzi, R.E., Bush, A.I., Rogers, J.T., 2004. Redox‐Active metals,
oxidative stress, and Alzheimer’s disease pathology. Ann. N.Y. Acad. Sci. 1012,
153–163.
Hussain, A., Alamzeb, S., Begum, S., 2013. Accumulation of heavy metals in edible parts
of vegetables irrigated with waste water and their daily intake to adults and children,
District Mardan, Pakistan. Food Chem. 136, 1515–1523.
Iqbal, G., Iqbal, A., Mahboob, A., Farhat, S.M., Ahmed, T., 2016. Memory enhancing
effect of black pepper in the AlCl3 induced neurotoxicity mouse model is mediated
through its active component chavicine. Curr. Pharm. Biotechnol. 17, 962–973.
Jacqmin, H., Commenges, D., Letenneur, L., Barberger-Gateau, P., Dartigues, J.-F., 1994.
Components of drinking water and risk of cognitive impairment in the elderly. Am. J.
Epidemiol. 139, 48–57.
Jomova, K., Valko, M., 2011. Advances in metal-induced oxidative stress and human
disease. Toxicology 283, 65–87.
Kazantzis, G., 1979. Renal tubular dysfunction and abnormalities of calcium metabolism
in cadmium workers. Environ. Health Perspect. 28, 155.
108
Environmental Toxicology and Pharmacology 60 (2018) 100–109
Kazi, T., Memon, A., Afridi, H., Jamali, M., Arain, M., Jalbani, N., Sarfraz, R., 2008a.
Determination of cadmium in whole blood and scalp hair samples of Pakistani male
lung cancer patients by electrothermal atomic absorption spectrometer. Sci. Total
Environ. 389, 270–276.
Kazi, T.G., Afridi, H.I., Jamali, M.K., Arain, M.B., Jalbani, N., Syed, N., 2007. Evaluation
of zinc status in whole blood and scalp hair of female cancer patients. Clin. Chim.
Acta 379, 66–70.
Kazi, T.G., Afridi, H.I., Kazi, N., Jamali, M.K., Arain, M.B., Jalbani, N., Kandhro, G.A.,
2008b. Copper, chromium, manganese, iron, nickel, and zinc levels in biological
samples of diabetes mellitus patients. Biol. Trace Elem. Res. 122, 1–18.
Kazi, T.G., Afridi, H.I., Kazi, N., Jamali, M.K., Arain, M.B., Sarfraz, R.A., Jalbani, N.,
Ansari, R., Shah, A.Q., Khandhro, G.A., 2008c. Distribution of zinc, copper and iron in
biological samples of Pakistani myocardial infarction (1st, 2nd and 3rd heart attack)
patients and controls. Clin. Chim. Acta 389, 114–119.
Keith, S., Jones, D., Rosemond, Z., Ingerman, L., Chappell, L., 2008. Toxicological Profile
for Aluminum. Atlanta, GA.
Kim, R., Rotnitzky, A., Sparrow, D., Weiss, S.T., Wager, C., Hu, H., 1996. A longitudinal
study of low-level lead exposure and impairment of renal function: the normative
aging study. JAMA 275, 1177–1181.
Koh, J.-Y., Suh, S.W., Gwag, B.J., He, Y.Y., 1996. The role of zinc in selective neuronal
death after transient global cerebral ischemia. Science 272, 1013.
Kok, F.J., Van Duijin, C.M., Hofman, A., Van Der Voet, G.B., De Wolff, F.A., Paays, C.H.C.,
Valkenburg, H.A., 1988. Serum copper and zinc and the risk of death from cancer and
cardiovascular disease. Am. J. Epidemiol. 128, 352–359.
Kurella, M., Chertow, G.M., Luan, J., Yaffe, K., 2004. Cognitive impairment in chronic
kidney disease. J. Am. Geriatr. Soc. 52, 1863–1869.
Larson, E.B., Kukull, W.A., Katzman, R.L., 1992. Cognitive impairment: dementia and
Alzheimer's disease. Annu. Rev. Public Health 13, 431–449.
Li, Y., Togashi, Y., Sato, S., Emoto, T., Kang, J., Takeichi, N., Kobayashi, H., Kojima, Y.,
Une, Y., Uchino, J., 1991. Spontaneous hepatic copper accumulation in Long-Evans
Cinnamon rats with hereditary hepatitis. A model of Wilson’s disease. J. Clin. Invest.
87, 1858.
Lidsky, T.I., Schneider, J.S., 2003. Lead neurotoxicity in children: basic mechanisms and
clinical correlates. Brain 126, 5–19.
Lozoff, B., Jimenez, E., Hagen, J., Mollen, E., Wolf, A.W., 2000. Poorer behavioral and
developmental outcome more than 10 years after treatment for iron deficiency in
infancy. Pediatrics 105, e51.
Lucchini, R., Selis, L., Folli, D., Apostoli, P., Mutti, A., Vanoni, O., Iregren, A., Alessio, L.,
1995. Neurobehavioral effects of manganese in workers from a ferroalloy plant after
temporary cessation of exposure. Scand. J. Work. Environ. Health 143–149.
Markesbery, W.R., 1997. Oxidative stress hypothesis in Alzheimer’s disease. Free Radic.
Biol. Med. 23, 134–147.
Mathers, C., Fat, D.M., Boerma, J.T., 2008. The Global Burden of Disease: 2004 Update.
World Health Organization.
Mattie, M.D., Freedman, J.H., 2004. Copper-inducible transcription: regulation by metal-
and oxidative stressresponsive pathways. Am. J. Physiol.-Cell Physiol. 286,
C293–C301.
Mattson, M.P., Magnus, T., 2006. Aging and neuronal vulnerability. Nat. Rev. Neurosci. 7,
278–294.
Mazzoli-Rocha, F., dos Santos, A.N., Fernandes, S., Ferreira Normando, V.M., Malm, O.,
Nascimento Saldiva, P.H., Wanderley Picanço-Diniz, D.L., Faffe, D.S., Zin, W.A.,
2010. Pulmonary function and histological impairment in mice after acute exposure
to aluminum dust. Inhal. Toxicol. 22, 861–867.
Meramat, A., Rajab, N.F., Shahar, S., Sharif, R.A., 2017. DNA damage, copper and lead
associates with cognitive function among older adults. J. Nutr. Health Aging 21,
539–545.
Mergler, D., Baldwin, M., Belanger, S., Larribe, F., Beuter, A., Bowler, R., Panisset, M.,
Edwards, R., De Geoffroy, A., Sassine, M., 1998. Manganese neurotoxicity, a con-
tinuum of dysfunction: results from a community based study. Neurotoxicology 20,
327–342.
Minami, T., Ichii, M., Tohno, Y., Tohno, S., Utsumi, M., Yamada, M.-o., Okazaki, Y., 1996.
Age-dependent aluminum accumulation in the human aorta and cerebral artery. Biol.
Trace Elem. Res. 55, 199–205.
Mizuno, D., Kawahara, M., 2013. The molecular mechanisms of zinc neurotoxicity and
the pathogenesis of vascular type Senile Dementia. Int. J. Mol. Sci. 14, 22067.
Nasrullah, R.N., Bibi, H., Iqbal, M., Durrani, M.I., 2006. Pollution load in industrial ef-
fluent and ground water of Gadoon Amazai Induatrial Estate (GAIE) Swabi, NWFP. J.
Agric. Biol. Sci. 1, 18–24.
Needleman, H.L., Gatsonis, C.A., 1990. Low-level lead exposure and the IQ of children: a
meta-analysis of modern studies. JAMA 263, 673–678.
Nordberg, G.F., 2004. Cadmium and health in the 21st century–historical remarks and
trends for the future. Biometals 17, 485–489.
Olmedo, P., Pla, A., Hernández, A., López-Guarnido, O., Rodrigo, L., Gil, F., 2010.
Validation of a method to quantify chromium, cadmium, manganese, nickel and lead
in human whole blood, urine, saliva and hair samples by electrothermal atomic ab-
sorption spectrometry. Anal. Chim. Acta 659, 60–67.
Olness, K., 2003. Effects on brain development leading to cognitive impairment: a
worldwide epidemic. J. Dev. Behav. Pediatr. 24, 120–130.
Organization, W.H, 2008. Guidelines for Drinking-Water Quality [Electronic Resource]:
Incorporating 1st and 2nd Addenda, vol. 1 World Health Organization
Recommendations.
Orlowski, C., Piotrowski, J.K., 2003. Biological levels of cadmium and zinc in the small
intestine of non-occupationally exposed human subjects. Hum. Exp. Toxicol. 22.
Oteiza, P.I., Mackenzie, G.G., Verstraeten, S.V., 2004. Metals in neurodegeneration: in-
volvement of oxidants and oxidant-sensitive transcription factors. Mol. Aspects Med.
25, 103–115.
G. Iqbal et al.
Patel, V., Kleinman, A., 2003. Poverty and common mental disorders in developing
countries. Bull. World Health Organ. 81, 609–615.
Patra, R., Swarup, D., Dwivedi, S., 2001. Antioxidant effects of α tocopherol, ascorbic acid
and L-methionine on lead induced oxidative stress to the liver, kidney and brain in
rats. Toxicology 162, 81–88.
Plassman, B.L., Langa, K.M., Fisher, G.G., Heeringa, S.G., Weir, D.R., Ofstedal, M.B.,
Burke, J.R., Hurd, M.D., Potter, G.G., Rodgers, W.L., 2008. Prevalence of cognitive
impairment without dementia in the United States. Ann. Intern. Med. 148, 427–434.
Powers, K., Smith-Weller, T., Franklin, G., Longstreth, W., Swanson, P., Checkoway, H.,
2003. Parkinson’s disease risks associated with dietary iron, manganese, and other
nutrient intakes. Neurology 60, 1761–1766.
Praticò, D., Uryu, K., Sung, S., Tang, S., Trojanowski, J.Q., Lee, V.M.-Y., 2002. Aluminum
modulates brain amyloidosis through oxidative stress in APP transgenic mice. FASEB
J. 16, 1138–1140.
Prince, M., Acosta, D., Chiu, H., Scazufca, M., Varghese, M., Group, D.R., 2003. Dementia
diagnosis in developing countries: a cross-cultural validation study. Lancet 361,
909–917.
Prousek, J., 2007. Fenton chemistry in biology and medicine. Pure Appl. Chem. 79,
2325–2338.
Rogan, W.J., Dietrich, K.N., Ware, J.H., Dockery, D.W., Salganik, M., Radcliffe, J., Jones,
R.L., Ragan, N.B., Chisolm Jr., J.J., Rhoads, G.G., 2001. The effect of chelation
therapy with succimer on neuropsychological development in children exposed to
lead. N. Engl. J. Med. 344, 1421–1426.
Rondeau, V., Commenges, D., Jacqmin-Gadda, H., Dartigues, J.-F., 2000. Relation be-
tween aluminum concentrations in drinking water and Alzheimer's disease: an 8-year
follow-up study. Am. J. Epidemiol. 152, 59–66.
Satarug, S., Garrett, S.H., Sens, M.A., Sens, D.A., 2011. Cadmium, environmental ex-
posure, and health outcomes. Ciencia Saude Coletiva 16, 2587–2602.
Sayed, K., Taha, T.T., Saad, M., Mohsen, L.A., Ibrahiem, M.A., Fadeel, N.A., Sotouhy, A.,
2014. Is mini‐mental score examination scoring a new predictor of uncontrolled
hypertension? J. Clin. Hypertens. 16, 348–353.
Singh, R., Gautam, N., Mishra, A., Gupta, R., 2011. Heavy metals and living systems: an
overview. Indian J. Pharmacol. 43, 246.
Smorgon, C., Mari, E., Atti, A.R., Dalla Nora, E., Zamboni, P.F., Calzoni, F., Passaro, A.,
Fellin, R., 2004. TRACE ELEMENTS AND COGNITIVE IMPAIRMENT: AN ELDERLY
COHORT STUDY. Arch. Gerontol. Geriatr. 38, 393–402.
109
Environmental Toxicology and Pharmacology 60 (2018) 100–109
Solfrizzi, V., Colacicco, A.M., D’Introno, A., Capurso, C., Parigi, A.D., Capurso, S.A.,
Torres, F., Capurso, A., Panza, F., 2006. Macronutrients, aluminium from drinking
water and foods, and other metals in cognitive decline and dementia. J. Alzheimer’s
Dis. 10, 303–330.
Squitti, R., Ghidoni, R., Scrascia, F., Benussi, L., Panetta, V., Pasqualetti, P., Moffa, F.,
Bernardini, S., Ventriglia, M., Binetti, G., 2011. Free copper distinguishes mild cog-
nitive impairment subjects from healthy elderly individuals. J. Alzheimer’s Dis. 23,
239–248.
Syme, C.D., Nadal, R.C., Rigby, S.E., Viles, J.H., 2004. Copper binding to the Amyloid-β
(Aβ) peptide associated with Alzheimer’s disease folding, coordination geometry, ph
dependence, stoichiometry, and affinity of Aβ-(1–28): Insights from a range of
complementary spectroscopic techniques. J. Biol. Chem. 279, 18169–18177.
Tokuda, E., Ono, S.-I., Ishige, K., Naganuma, A., Ito, Y., Suzuki, T., 2007. Metallothionein
proteins expression, copper and zinc concentrations, and lipid peroxidation level in a
rodent model for amyotrophic lateral sclerosis. Toxicology 229, 33–41.
Valko, M., Morris, H., Cronin, M., 2005. Metals, toxicity and oxidative stress. Curr. Med.
Chem. 12, 1161–1208.
Verhoeven, W.M., Egger, J.I., Kuijpers, H.J., 2011. Manganese and acute paranoid psy-
chosis: a case report. J. Med. Case Rep. 5, 1.
Waalkes, M.P., Rehm, S., Riggs, C.W., Bare, R.M., Devor, D.E., Poirier, L.A., Wenk, M.L.,
Henneman, J.R., Balaschak, M.S., 1988. Cadmium carcinogenesis in male Wistar [Crl:
(WI) BR] rats: dose-response analysis of tumor induction in the prostate and testes
and at the injection site. Cancer Res. 48, 4656–4663.
Weihl, C.C., Lopate, G., 2006. Motor neuron disease associated with copper deficiency.
Muscle Nerve 34, 789–793.
Wills, N.K., Sadagopa Ramanujam, V.M., Kalariya, N., Lewis, J.R., van Kuijk, F.J.G.M.,
2008. Copper and zinc distribution in the human retina: relationship to cadmium
accumulation, age, and gender. Exp. Eye Res. 87, 80–88.
Wong, W.Y., Flik, G., Groenen, P.M.W., Swinkels, D.W., Thomas, C.M.G., Copius-
Peereboom, J.H.J., Merkus, H.M.W.M., Steegers-Theunissen, R.P.M., 2001. The im-
pact of calcium, magnesium, zinc, and copper in blood and seminal plasma on semen
parameters in men. Reprod. Toxicol. 15, 131–136.
Wu, J., Basha, M.R., Brock, B., Cox, D.P., Cardozo-Pelaez, F., McPherson, C.A., Harry, J.,
Rice, D.C., Maloney, B., Chen, D., 2008. Alzheimer's disease (AD)-like pathology in
aged monkeys after infantile exposure to environmental metal lead (Pb): evidence for
a developmental origin and environmental link for AD. J. Neurosci. 28, 3–9.