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Ecotoxicology and Environmental Safety 78 (2012) 195205

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Ecotoxicology and Environmental Safety


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

Do toxic heavy metals affect antioxidant defense mechanisms in humans?


Monika Wieloch a,1, Piotr Kaminski a,b,n, Anna Ossowska a,1, Beata Koim-Puchowska a,
Tomasz Stuczynski c,2, Magdalena Kuligowska-Prusinska d,3, Grazyna Dymek d,1,3,
Aneta Mankowska d,1,3, Grazyna Odrowaz-Sypniewska d,3
a

Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Department of Ecology and Environmental Protection, Sk!odowska-Curie St. 9, PL 85-094 Bydgoszcz, Poland
ra, Faculty of Biological Sciences, Institute of Biology and Environmental Protection, Department of Biotechnology, Prof. Szafran St. 1, PL 65-516
University of Zielona Go
ra, Poland
Zielona Go
c
Institute of Soil and Plant CultivationGovernment Scientic Institute, Department of Soil Structure, Czartoryskich St. 8, PL 24-100 Pu!awy, Poland
d
Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz, Department of Laboratory Diagnostics, Sk!odowska-Curie St. 9, PL 85-094 Bydgoszcz, Poland
b

a r t i c l e i n f o

abstract

Article history:
Received 30 May 2011
Received in revised form
2 November 2011
Accepted 16 November 2011
Available online 12 December 2011

The aim of this study was to prove whether anthropogenic pollution affects antioxidant defense
mechanisms such as superoxide dismutase (SOD) and catalase (CAT) activity, ferritin (FRT) concentration and total antioxidant status (TAS) in human serum. The study area involves polluted and salted
environment (Kujawy region; northern-middle Poland) and Tuchola Forestry (unpolluted control area).
We investigated 79 blood samples of volunteers from polluted area and 82 from the control in 2008 and
2009. Lead, cadmium and iron concentrations were measured in whole blood by the ICPMS method.
SOD and CAT activities were measured in serum using SOD and CAT Assay Kits by the standardized
colorimetric method. Serum TAS was measured spectrophotometrically by the modied Benzie and
Strain (1996) method and FRT concentrationby the immunonefelometric method. Pb and Cd levels
and SOD activity were higher in volunteers from polluted area as compared with those from the control
(0.0236 mg l  1 vs. 0.014 mg l  1; 0.0008 mg l  1 vs. 0.0005 mg l  1; 0.137 U ml  1 vs. 0.055 U ml  1,
respectively). Fe level, CAT activity and TAS were lower in serum of volunteers from polluted area
(0.442 g l  1 vs. 0.476 g l  1; 3.336 nmol min  1 ml  1 vs. 6.017 nmol min  1 ml  1; 0.731 Trolox-equivalents vs. 0.936 Trolox-equivalents, respectively), whilst differences in FRT concentration were not
signicant (66.109 mg l  1 vs. 37.667 mg l  1, p 0.3972). Positive correlations between Pb (r 0.206),
Cd (r 0.602) and SOD in the inhabitants of polluted area, and between Cd and SOD in the control
(r 0.639) were shown. In volunteers from both studied environments TASFRT (polluted: r 0.625 vs.
control: r 0.837) and FeFRT (polluted area: r 0.831 vs. control: r 0.407) correlations, and PbFRT
(r 0.360) and PbTAS (r 0.283) in the control were stated.
The higher lead and cadmium concentrations in blood cause an increase of SOD activity. It suggests
that this is one of the defense mechanisms of an organism against oxidative stress caused by
environmental factors, whilst non-enzymatic mechanisms marked by TAS are the main antioxidant
defense system in relation with Pb concentration in humans from unpolluted area. Simultaneously, the
higher CAT activity and TAS can indicate that these mechanisms play a key role in the antioxidant
protection in non-stressed environments.
& 2011 Elsevier Inc. All rights reserved.

Keywords:
Anthropogenic pollution
Human serum
Environmental stress
Superoxide dismutase
Catalase
Ferritin
Total antioxidant status

n
Corresponding author at: Nicolaus Copernicus University, Collegium Medicum in
Bydgoszcz, Department of Ecology and Environmental Protection, Sk"odowska-Curie
St. 9, PL 85-094 Bydgoszcz, Poland. Fax: 48 52 585 38 07, 48 68 3287875.
E-mail addresses: monicjus@wp.pl (M. Wieloch), piotr.kaminski@cm.umk.pl,
p.kaminski@wnb.uz.zgora.pl (P. Kaminski),
anna.ossowska@cm.umk.pl (A. Ossowska),
beata.koim@cm.umk.pl (B. Koim-Puchowska), ts@iung.pulawy.pl (T. Stuczynski),
magdalenakuligowska@wp.pl (M. Kuligowska-Prusinska),
grazyna.dymek@op.pl (G. Dymek), anetha7@poczta.onet.pl (A. Mankowska),
odes@cm.umk.pl, grazynaodes@interia.pl (G. Odrowaz-Sypniewska).
1
Fax: 48 68 328 78 75.
2
Fax: 48 81 886 45 47.
3
Fax: 48 52 585 36 03.

0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2011.11.017

1. Introduction
Reactive oxygen species (ROS) takes part in pathogenesis of
many diseases correlated with environmental factors. The relationships between ROS in humans and the morbidity on many diseases
(cancers, rheumatoid arthritis, arteriosclerosis, diabetes, diseases of
central nervous and alimentary system, hematids, etc.) have been
proved (Nohl and Stolze, 1998; Olinski and Jurgowiak, 1999; Schulz
et al., 1999; Siems et al., 2000; Beisswenger et al., 2001; Buonocore
et al., 2001; Darlington and Stone, 2001; Kawanishi et al., 2001;
Sayre et al., 2001).

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M. Wieloch et al. / Ecotoxicology and Environmental Safety 78 (2012) 195205

The increase of studies focused on the impact of chemical


elements on human health and oxidant stress in humans has been
observed recently. However, the researches include mostly individuals exposed to the impact of occupational factors, causing
changes in the defense mechanisms of an organism against
oxidant stress. Among examined elements especially toxic heavy
metals are harmful for heath. Simultaneously, the environmental
exposure upon these factors is rather not considered in detail.
Simultaneously, the investigations describing particular impact of
environmental stress on the antioxidant barrier of an organism
are poor (Sbrana et al., 1990; Tang et al., 1990; Anwar and Gabal,
1991; Senft et al., 1992; Topaktas et al., 2002; Kobal et al., 2004).
Lead and cadmium are important occupational and environ
mental pollutants (Nriagu, 1978; Ewers and Schlipkoter,
1991).
The extraction, smelt, lead purication and production of products
containing lead can cause the emission of this metal to the
environment. Lead exposure occurs during the production of
batteries, cables and wires, in the chemical industry and the beds,
printer0 s type production, from covers to protect from the radioactive radiation and manufacture of dyes and insecticides (KabataPendias and Pendias, 1984; Kabata-Pendias and Mukherjee, 2007).
Lead cumulates in soils and dusts and because it does not decay,
this is an essential source of exposure. The consumption of plants
farmed in the polluted areas causes the health hazard (Senczuk,
2002; Kaniuczak, 2004). Cadmium emission to the environment
follows mostly from smelters of zinc, nickel and other non-ferrous
metals, such as lead and copper (60% of all anthropogenic sources),
as well as a result of coal combustion. Environmental pollution
with cadmium as a result of industrial activity has often a local
character (to about 40 km). The easy movement of this toxic
element in the trophic chain causes the health hazard. Moreover,
it is more toxic than lead (Bonda et al., 2007). The inuence of lead
on the ferritin concentration got conrmed with many investigations. These studies include the groups of workers exposed to lead
and suggest a direct inuence of iron depletion on lead poisoning
(Osman et al., 1998; Kim et al., 2002, 2003; Wright et al., 2003;
Kwong et al., 2004).
In the areas studied by us there is a sustainable process of
penetration into the soil and groundwater with chemicals contained in them, especially cadmium and lead. These metals are
derived from both the nearby soda plants, and glass works, and
municipal waste treatment plants as well, especially from municipal landlls, active and inactive. Additionally, the investigations
were carried out in salted Kujawy Region (northern-middle
Poland) in Inowroc"aw Region of Ecological Danger IRED (salinity,
acidication, disturbed Ca, Mg, Na and Fe management, the
impact of toxic metals). Thus here it should be taken into account
that these environments were of various degrees of degradation
where sodium manufacture, wastes utilization, very polluted
neighborhoods and industrial manufacture areas with anthropogenic impact are here a constant source of toxic metals, especially
cadmium and lead. The separate and independent sources of
these metals are here landll and agriculture itself, areas immediately adjacent, i.e. surrounded districts with dumping grounds
of wastes and agricultural cultivation (agrocenoses). Overall
environmental effects of these highly unfavorable biogeochemical
conditions here are signicantly strengthened by a rich network
of groundwater, streams, ood waters, stagnant waters and local
so-called dystrophic ponds. This favors the leaching of organic
matter, both autochthonous and allochtonous, inside the basin
(Wilkon-Michalska, 1971; Ciesla and Dabkowska-Naskret, 1984;
Czerwinski et al., 1984; Czerwinski, 1996; Rytelewski et al., 1993;
Borsuk and Stachowiak, 1994; Niklewska et al., 2000). At the
same time the signicant effect of iron in the areas we examined
should be noted. This element is there in large concentrations
in the soil, groundwater in the network, trophic chains, etc.

The destabilization of iron compounds economy in these areas


is reected in the form of numerous small ferruginous water
tanks, the so-called meshes that are a source of iron leaching
into the environment.
In addition to natural biogeochemical local processes the
anthropogenic impact is here signicant. The development of
saline sodium industry and therefore the activity of industrial
giants of this region, leads to imbalance of the environment.
Contaminations associated with activity of agricultural production include surface and underground waters, atmospheric air and
soil (Rytelewski et al., 1993; Niklewska et al., 2000). The salts of
sodium and chlorine combine here with compounds of calcium
and potassium, and then are directed to the settlers, the so-called
white seas, which are becoming a source of serious instability of
the environment. Another source of contamination is spray dried
lime sodiumcalcium settlers, and damage to wells and pipelines
brine (Czerwinski et al., 1984, 1996; Rytelewski et al., 1993).
Furthermore, the increase in salinity also causes failures of
various devices, such as various hydrological equipment, which
results in saline waste entering the emergency ditches watercourses, which leads to formation of saline spills contaminating
surface water and arable land (Rytelewski et al., 1993). In
enlarging the white seas also plays a role in geomorphology of
land, which inuences the direction of water ow and salt
deposits conducive to this process (Wilkon-Michalska, 1971).
Human industrial and rural activities in the studied Kujawy
region led to secondary soil salinity in the region, namely the
formation of soils known as anthropologically saline. The result of
this process is to change the properties of soils, which are a result
of accumulation of sodium and toxic metals, mainly Cd and Pb, in
the complex of absorbent dry-up during drought and form a hard,
difcult to grow rock. However, in the state of soil moisture they
are boggy, impervious and windproof (Rytelewski et al., 1993).
ROS produced by toxic metals exposure causes tissue damage
by a variety of mechanisms (Yetkin-Ay et al., 2007). However, the
broad defense mechanisms of an organism exist, targeting to
control physiological level of ROS in the organism (Rukgauer
et al., 2001; Armutcu et al., 2004; Gacko et al., 2006; uszczewski
et al., 2007). Antioxidant enzymes activity determines the rst
line of protection and their charge is the limitation of ROS
formation. The mechanism of these antioxidants activity is well
recognized and described by many researchers (Harrison and
Arosio, 1996; Orino et al., 2001; Rukgauer et al., 2001; Armutcu
et al., 2004; Gacko et al., 2006; uszczewski et al., 2007).
Antioxidant enzymes are very important for aerobic organisms
exposed to oxygen. Superoxide dismutase metabolizes reactive
oxygen species to hydrogen peroxide. Catalase converts hydrogen
peroxide to water and oxygen (Bowler et al., 1992). Thus the
inuence of heavy metals on the antioxidant enzymes activity
became conrmed scientically.
It should be emphasized that superoxide dismutase and
catalase are among the so-called triad of enzyme. Based on the
evaluation of changes in the activity of these enzymes we can
create the antioxidant status of an organism and the environment,
which is complemented by non-enzymatic mechanisms for performance analysis (Fandos et al., 2009; Mansego et al., 2011).
Most research conrms that SOD, CAT and non-enzymatic antioxidants can be indicated as the main antioxidant defense
mechanisms in humans (Sadowska-Krepa and Poprzecki, 2005;
Corrales et al., 2011; Mansego et al., 2011; Skolmowska and
Kmiec, 2011); e.g., the application of CAT and SOD in daily
practice encounters many difculties connected with their loss
of activity and with their degradation. These enzymes practically
do not penetrate biological membranes, which limits or even
makes impossible their protective activity directed against ROS.
However, the penetration of the membranous barrier becomes

M. Wieloch et al. / Ecotoxicology and Environmental Safety 78 (2012) 195205

possible after the location of the enzymes in lipid structures. One


of the technologies of stabilization of the activity of enzymes
consists in closing them in aliposome structure. The incorporation
of the enzymes into a closed, such as liposome, structure or into
their lipid bilayer screens and simultaneously protects them
against degradation that they would undergo during use in the
unshaded form (Skolmowska and Kmiec, 2011).
Occupational exposure studies suggest that the inuence of
lead on the antioxidant enzymes activity is relative to the doses
and time of exposure (Monteiro et al., 1985; Sugawara et al.,
1991; Aydinn et al., 2004; Han et al., 2005; Patil et al., 2006).
Simultaneously, it has been proved that lead can both decrease
and increase the activities of SOD and CAT. The increase is
observed at lower levels of Pb-exposure, whilst decrease has been
shown at higher exposure over a long period of time (Patrick,
2006). Also repeated exposure of cell line on lead caused signicant increase in SOD activities (Zaree Mohmodabady et al.,
2006). The increase of SOD activity and simultaneously lack of
essential changes in CAT in Cd exposed HF2FF (human skinhead,
the broblast cell line) was shown in the experimental conditions,
whilst in the research on laboratory animals the inhibition of SOD
and the increase of CAT activities were stated (Caisova and Eybl,
1988; 1993; Zaree Mohmodabady et al., 2006).
Important effects of stress proteins in the above relationships
have also been stated; e.g., ferritin as a protein binding and
storing ferric ions in readily available, but non-toxic form, can
also be considered as a defense mechanism (Florianczyk, 2008).
Moreover, it is well known that iron is required for normal cell
growth and proliferation, but it is potentially harmful in excess
and it can catalyze the formation of ROS in the Fenton reaction.
Ferritin plays a central role in the maintenance of intracellular
iron balance because of its importance in the antioxidant
mechanisms (Harrison and Arosio, 1996; Orino et al., 2001). There
are also several experiments in which a role of FRT as a protectant
against oxygen free radical-mediated damage has been proved.
The exposure of endothelial cells to hemin was observed to
induce ferritin synthesis and concordantly reduce cytotoxic
response of these cells to toxic doses of H2O2 (Balla et al.,
1992). Simultaneously, FRT synthesis is signicantly stimulated
in the liver of rats subjected to oxidative stress by treatment with
phorone, a glutathione-depleting drug that increases intracellular
levels of ROS (Cairo et al., 1995).
The other research showed that UV irradiation, which produces oxygen free radicals, induces ferritin protein (Vile and
Tyrrell, 1993). These ndings are supported by studies in which
human skin cells were treated with ultraviolet A radiation in vivo.
The increased concentration of FRT in human skin following acute
UV radiation could afford increased protection against subsequent
oxidative stress (Applegate et al., 1998). On the other hand, some
experiments suggest that superoxide can mobilize iron from
ferritin and promote oxidative damage (Biemond et al., 1986).
Thus the role of FRT in the oxidative damage is still unclear.
Gender of the volunteers might also have some impact on the
results. Among participants of research, females dominated
(79.75% and 63.41% in polluted and control area, respectively;
Table 1, this paper). Simultaneously we have not stated signicant
correlations between changes in antioxidants and gender of
volunteers from study environments. The results of Bolzan et al.
(1997) study suggest higher levels of SOD and CAT in females
than males. However other authors did not detect any sex-related
differences in the antioxidants activity (Winterbourn et al., 1975;
Ceballos-Picot et al., 1992). It suggests that gender is not probably
one of the factors affecting antioxidant mechanisms formation.
Antioxidant system of the organism includes also non-enzymatic antioxidants, such as uric acid, a-tocopherol, ascorbic
acid, bilirubin, albumin and many other active substances. The

197

Table 1
Age and gender structure (Ffemales, Mmales) of volunteers from Mogilno
neighborhood (polluted area) and Tuchola Forestry (unpolluted control area).
There was no signicant difference in age structure in examined environments
(chi-square Pearson test: p 0.088109).
Environment

Age (years): Age (years): Age (years):


2130
3140
4150

Polluted area 20
Control area 31

37
26

22
25

63
52

16
30

79
82

methods assessing the total antioxidant status in human serum


have been developed because of the difculties of measuring
particular antioxidant components. Among these the ferric reducing ability of plasma or serum (FRAP) assay by the method of
Benzie and Strain (1996) is signicant. The FRAP assay is simple
and relatively inexpensive, because it measures the ferric-toferrous iron reduction in the presence of antioxidants, whilst
TAS is expressed as one-electron equivalent of Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid). The percentage
proportion of main antioxidants in total antioxidant status measured by FRAP is uric acid (61.7%), ascorbic acid (10.1%), albumin
(7.26), a-tocopherol (5.84%), bilirubin (4.34%) and others (10.76%;
Cao and Prior, 1998). Experimental studies on animals showed
signicant decrease in TAS in the rats treated with 1% of aqueous
solution of lead acetate for 9 months (Marchlewicz et al., 2006).
Metallothioneins (Mt) serve a protective function against
various factors causing oxidative stress. The increase in the
synthesis of Mt was found as a result of the increased concentration of hydroxyl radicals, anion-superoxide radicals and organic
radicals. Continued increases in the Cd exposure concentrations
resulted in declines in the levels of MT mRNA. ROS production
decreased in a concentration-dependent fashion at CdCl2 concentrations. At these high Cd levels, the concomitant decrease in MT
mRNA inducibility and suppression of ROS production were
probably manifestations of the general cytotoxicity of Cd. ROS
suppression can result in reduced resistance to infectious agents,
suggesting that Cd is immunotoxic (Sato and Bremner, 1993;
Roesijadi et al., 1977). The principal roles of MT in the detoxication of heavy metals and regulation of the metabolism of essential
trace metals are generally accepted. However, there is increasing
evidence that it can act as a free radical scavenger. It can thus be
concluded that there is evidence supporting such a physiological
role and induction of MT synthesis by oxidative stress, possible
mediators for this induction, and the radical scavenging capability
of MT in tissues and cells. The relationship between MT and other
antioxidant defense systems and the medical implications of the
free radical scavenging properties of MT are of great importance
for biochemical dependences in the case of environmental stressors activity (Sato and Bremner, 1993). Studies by Takahashi et al.
(2004) report that endogenous ROS accumulate more in MT /
cells with the progress of the cell cycle than in MT / cells
owing to the absence of the antioxidant; MT and the MT / cells
are arrested at the G2M phase, resulting in slower proliferation
than in the case of MT / cells. Exogenous ROS also retard
proliferation more obviously in MT / cells than in MT /
cells. Based on these studies, it is suggested that MT plays a role
as a specic antioxidant against endogenous ROS generated in the
cell cycle, especially in the nucleus (Takahashi et al., 2004).
The aim of this study was to prove whether lead, cadmium and
iron, as anthropogenic pollutants, affect the defense mechanisms
such as antioxidant enzymes (SOD, CAT) activity, ferritin (FRT)
concentration and total antioxidant status (TAS) in humans
against oxidant stress. This research determines, which defense
mechanisms play the main role in the protection of humans

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M. Wieloch et al. / Ecotoxicology and Environmental Safety 78 (2012) 195205

against oxidative stress caused by environmental factors, i.e. lead,


cadmium and iron impacts, and what kinds of relationships
among different antioxidant mechanisms exist in serum of
humans resident in polluted areas as compared with the control.

2. Study area
Studies were carried out in Mogilno (521400 11.2000 N;
521370 4.6400 S; 181110 50.2100 E; 171560 35.1400 W, central Poland) neighborhood (polluted and salted region) and in Tuchola Forestry
(531420 40.6800 N; 531320 51.0900 S; 171590 3.0600 E; 171470 45.8100 W, N
Poland), which serves as a control (unpolluted areas). Mogilno
district is situated on the south-west of KuyavianPomeranian
province and sits next to Inowroc"aw Region of Ecological Danger
(Fig. 1). Since heavy metals from industrial plants get through to the
atmosphere, the pollution involves not only areas directly adjoining
but also neighbor districts (Senczuk, 2002; Kaniuczak, 2004).
The control areas were chosen on the basis of earlier research
carried by Ossowska et al. (2009), in which metals concentrations in
blood of humans resident in the Tuchola district were measured.
Tuchola Forestry and landscape parks district are situated in the
northern part of the province (Fig. 1) and most of this territory is
determined by forests and forest-lands. This area serves as a control
also for the reason of lack of heavy industry.
3. Material and methods
3.1. Human population
The study material included blood samples of volunteers from polluted
Mogilno neighborhood and unpolluted Tuchola Forestry. The number of humans
studied in 2008 and 2009 was 79 and 82 for Mogilno and Tuchola Forestry,
respectively. The volunteers were at the age below 60 years because of depletion
of antioxidants (Sulochana et al., 2002). The age structures (Table 1) of volunteers
from polluted (Mogilno neighborhood) and control (Tuchola Forestry) were
similar. The volunteers divisions depending on the nicotinic habits from polluted
and the control were also similar (Table 2).
Blood samples were collected in tubes to receive blood serum for antioxidant
enzymes activity, ferritin and total antioxidant status detection (test-glasses with
accelerator of blood coagulating in the vacuum-system for biochemical research
on the serum) and to the tubes with heparin for elements concentration analysis.

Fig. 1. Study area: polluted and salted environments in the neighborhood of


Mogilno district situated in the south-west of KuyavianPomeranian province and
adjacent to the Inowroc"aw Region of Ecological Danger (central Poland).

Table 2
Volunteers depending on smoking cigarettes in Mogilno neighborhood (polluted
area) and Tuchola Forestry (unpolluted area). There were no signicant differences
in numbers of smokers and non-smokers in examined environments (chi-square
Pearson test: p 0.301307). NP1: non-smokers since 1 year; NP5: non-smokers
since 5 years; NP10: non-smokers since 10 years; NP: non-smokers all life; P1:
smokers since 1 year; P5: smokers since 5 years; P10: smokers since 10 years;
P15: smokers since 15 years; P20: smokers since 20 years; N: total number of
smokers and non-smokers in polluted and unpolluted environments.
Environment

NP1

NP5

NP10

NP

P1

P5

P10

P15

P20

Polluted area
Control area

3
2

3
3

5
11

47
43

58
59

2
1

3
4

1
4

5
3

10
11

21
23

Blood samples in tubes for receiving blood serum were centrifuged at 2000g for
15 min at 4 1C for SOD and CAT activity measurements serum aliquots were stored
at  80 1C until analysis. All of the above procedures were adopted in accordance
with the methodology specied by Johansson and Borg (1988), Wheeler et al.
(1990), Liu (1996), Sherwood et al. (1998), Andrews (1999), Cook (1999), Kricka
(1999), Van den Bosch et al. (2001)and Maier and Chan (2002).

3.2. Chemical elements concentration


Lead, cadmium and iron concentrations were measured by the ICPMS
method. A volume of 2 ml of blood as placed in glass tubes calibrated to the
capacity 25 ml. Samples were vaporized to dry in the DIGIPREP block at the
temperature of 180200 1C; then they were mineralized in the stove at the
temperature of 480 1C for 14 h. After mineralization samples were treated with
3 ml of pure nitric acid and were then placed in DIGIPREP block, bringing the
temperature to 200 1C (1 h). Then the temperature was increased to 250 1C (2 h).
After chilling, solutions after mineralization were supplemented with redistilled
water up to 5 ml. Samples were mixed and placed in polythene test-glasses. The
solutions were analyzed for lead concentration by inductively coupled plasma
mass spectrometry (ICPMS), i.e. AGILENT 7500CE plasma ICPMS spectrophotometer from Agilent Technologies Inc. (Palo Alto, CA, USA; Nixon et al., 1986). The
results were interpreted comparatively with standard well-known concentration,
i.e. Pb, Cd, Fe levels were made in relation to the analysis of reference materials.
Parallelly measurements were taken in the blind trials. The results were given in
mg l  1 (Pb, Cd) or g l  1 (Fe).

3.3. Serum antioxidant enzymes activity


3.3.1. Superoxide dismutase activity
Superoxide dismutase activity was measured by standard in serum using SOD
Assay Kit (Cayman Chemical Catalog No. 706002). All of the procedures were
adopted in accordance with the methodology specied by Liu (1996) and by Maier
and Chan (2002). This method is performed on a 96-well plate, and uses a
tetrazolium salt for detection of superoxide dismutase radicals generated by
xanthine oxidase and hypoxanthine. One unit of superoxide dismutase is dened
as the amount of enzyme needed to exhibit 50% dismutation of superoxide radical.
In brief, to 10 ml of samples and standards, 200 ml of the radical detector was
added. Reaction was initiated by adding 20 ml of xanthine oxidase. The absorbance
was read at 450 nm after 20 min incubation at room temperature on a shaker
using a plate reader. The results were interpreted comparatively with standard
well-known concentration. The results were given in U ml  1.

3.3.2. Catalase activity


Catalase activity was measured in serum using CAT Assay Kit (Cayman
Chemical Catalog No. 707002), according to manufacturers instructions. All of
the procedures were adopted in accordance with the methodology specied by
Johansson and Borg (1988) and by Wheeler et al. (1990). This method is performed
on a 96-well plate, and uses colorimetric measurement of formaldehyde produced
in the reaction of CAT with methanol in the presence of H2O2; 4-amino-3hydrazino-5-mercapto-1,2,4-triazole was used as a chromogen to colorimetrically
measure formaldehyde. One unit of CAT is dened as the amount of enzyme that
will cause the formation of 1.0 nmol of formaldehyde per minute at 25 1C. In brief,
to 20 ml of samples, standards and positive control, 100 ml of Assay Buffer and
30 ml of methanol were added. Reaction was initiated by adding 20 ml of hydrogen
peroxide. After 20 min of incubation at room temperature on the shaker, 30 ml of
potassium hydroxide was added to terminate the reaction and then 30 ml of
chromogen was added. After 10 min of incubation at room temperature on the
shaker, 10 ml of potassium periodate was added, and after 5 min incubation at
room temperature on the shaker the absorbance was read at 540 nm using a plate
reader. The results were interpreted comparatively with standard well-known
concentration and were given in nmol min  1 ml  1.

M. Wieloch et al. / Ecotoxicology and Environmental Safety 78 (2012) 195205

3.3.3. Serum total antioxidative status


Serum total antioxidative status was measured spectrophotometrically by the
modied Benzie and Strain (1996) method. Solution containing ions Fe3 (FeCl6)
was added to portion of serum. Antioxidants being found in serum reduce Fe3 to
Fe2 . A colored complex is created in the presence of 2,4,6 tripirydylo-S-triazine
(TPTZ). The increase of absorbance of TPTZFe2 complex is proportional to the
quantity of antioxidants present in the sample. In brief, to 20 ml of samples, 580 ml
of solution containing acetate buffer, FeCl6  6H2O and 2,4,6-tris(2-pyridyl)-1,3,5triazine was added. After 20 min etc incubation at room temperature the
absorbance was read at 593 nm. The results were interpreted comparatively with
Trolox standard well known concentration. The results were given in Troloxequivalents (Bartosz, 2006).
3.3.4. Serum ferritin concentration
Ferritin concentration was measured by the immunonefelometric method in the
BN system. All of the procedures were adopted in accordance with the methodology
specied by Andrews (1999), Cook (1999), Sherwood et al. (1998) and Van den Bosch
et al. (2001). This method uses particles of polystyrene coated with specic
antibodies of human ferritin, which reduce the aggregation during mixing with
samples containing human FRT. These aggregates cause the split of light beam
passing through the sample. The intensity of diffused light is proportional to the
concentration of FRT in the sample. The results were interpreted comparatively with
standard well-known concentration and were given in mg l  1.

4. Statistical analyses
By the central terminal statement for the number of cases
above 50, to nd whether essential differences in Pb, Cd, Fe and
FRT concentrations, and SOD and CAT activities and TAS exist, the
z test was used, and a value of p o0.05 was considered to be
signicant. To show what kinds of interactions between studied
parameters exist, the multiple regression analysis was used
(Stanisz, 2006). Arithmetic means with standard deviations,
minimum and maximum values of analyzed parameters and p
value for z test for comparison were described in the tables,
while the correlations were demonstrated on scattering graphs
(3 W for three parameters with the surface tted method of least
squares, 2 W for two parameters with lineal or multinomial
match). The results were presented as arithmetic means7SD
for volunteers from studied environments.
This study was undertaken following the Guidelines of the
European Union Council and the current laws in Poland, according
to the Ethical Commission No. 05/2005. The work required a
permit from the Local Committee for Bioethical Research of
Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz
(No. KB/153/2008).

5. Results
Lead, cadmium and iron concentrations in blood, and antioxidant enzymes activity and total antioxidant status in serum were
found to be different in volunteers from studied areas. We have
stated signicant higher lead and cadmium concentrations in blood
samples of volunteers from polluted area as compared with
those from the control (0.0236 mg l  1 vs. 0.014 mg l  1 and
0.0008 mg l  1 vs. 0.0005 mg l  1, respectively), but a lower iron
concentration (0.442 g l  1 vs. 0.476 g l  1; Table 3). The activity of
SOD in serum of volunteers from polluted area was over two times
higher than in serum of volunteers from unpolluted (0.137 U ml  1
vs. 0.055 U ml  1), whilst CAT activity in serum of volunteers from
polluted area was lower than in serum of those from the control
(3.336 nmol min  1 ml  1 vs. 6.017 nmol min  1 ml  1; Table 3).
Simultaneously, we have demonstrated no signicant differences
in FRT concentration (66.109 mg l  1 vs. 37.667 mg l  1, p0.3972)
and signicant lower TAS in serum of volunteers from polluted area
compared with those from the control (0.731 Trolox-equivalents vs.
0.936 Trolox-equivalents, respectively); Table 3. The interactions
between analyzed parameters were examined and we stated

199

Table 3
Superoxide dismutase (SOD) and catalase (CAT) activities, lead (Pb), cadmium
(Cd), iron (Fe) and ferritin (FRT) concentrations, and total antioxidant status (TAS)
in serum of volunteers from Mogilno neighborhood (polluted area) and Tuchola
Forestry (unpolluted control area); p for z test of comparisons, Nnumber,
xarithmetic mean, SDstandard deviation, min. and max.minimum and
maximum values, respectively.
Parameter

Polluted area

Control area

Polluted vs.
control area

SOD [U ml  1]
N
x 7 SD
min.max.
Median

79
0.137 7 0.077
0.0470.346
0.106

82
0.055 70.018
0.0110.100
0.057

p 0.0000

CAT [nmol min  1 ml  1]


N
79
x 7 SD
3.336 7 0.659
min.max.
1.3264.463
Median
3.408

82
6.017 7 3.000
2.10114.476
4.587

p 0.0000

FRT [mg l  1]
N
x 7 SD
min.max.
Median

82
37.6677 22.710
9.20093.200
31.200

p 0.3972

TAS [Trolox-equivalents]
N
79
x 7 SD
0.731 7 0.174
min.max.
0.2171.135
Median
0.695

82
0.936 7 0.180
0.5231.304
0.924

p 0.0000

Pb [mg l  1]
N
x 7 SD
min.max.
Median

79
0.0236 7 0.027
0.0100.079
0.020

82
0.014 70.007
0.0030.038
0.013

p 0.0000

Cd [mg l  1]
N
x 7 SD
min.max.
Median

79
0.0008 7 0.0005
0.00040.0035
0.0006

82
0.0005 70.0003
0.00020.0014
0.0005

p 0.0000

Fe [g l  1]
N
x 7 SD
min.max.
Median

79
0.442 7 0.077
0.2360.854
0.438

82
0.476 7 0.071
0.2380.884
0.479

p 0.0028

79
66.109 7 39.944
20.800161.000
50.900

Table 4
Parameters of multiple regression for the dependence between superoxide
dismutase (SOD) activity in serum and lead (Pb) and cadmium (Cd) concentrations
in the blood of volunteers from polluted area, involving partial correlation
coefcient and semipartial correlation coefcient, p for F test and value of
F test (model of multiple regression essentiality) and model equation (N 79).

Element

Partial
correlation

Semipartial
correlation

Cd
Pb

0.833225
0.256971

0.602094
0.206850

0.0001

11.418

SOD 50.262Cd 0.891Pb 0.054

positive correlations between lead (semipartial correlation:


r0.207) and cadmium (semipartial correlation: r0.602) concentrations in blood and SOD activity in serum of volunteers from
polluted area (Table 4, Fig. 2). However, only positive correlations
between Cd level in blood and SOD activity in serum of volunteers
from unpolluted area (r 0.637; Fig. 3) were noted. Ferritin concentrations in serum were correlated with iron in both studied
environments (polluted area: r 0.831 vs. control: semipartial
correlation: r 0.407) and with lead in the control (semipartial
correlation: r0.360; Table 5, Figs. 4 and 5). Lead concentration in

200

M. Wieloch et al. / Ecotoxicology and Environmental Safety 78 (2012) 195205

Fig. 4. Correlation between iron (Fe) concentration in blood and ferritin (FRT)
concentration in serum of volunteers from polluted area.

Fig. 2. Correlation between lead (Pb) and cadmium (Cd) concentrations in


blood and superoxide dismutase (SOD) activity in serum of volunteers from
polluted area.

Fig. 3. Correlation between cadmium (Cd) concentrations in blood and superoxide


dismutase (SOD) activity in serum of volunteers from unpolluted area.

Fig. 5. Correlation between iron (Fe) and lead (Pb) concentrations in blood and
ferritin (FRT) concentration in serum of volunteers from unpolluted area.

Table 5
Parameters of multiple regression for dependence between ferritin (FRT) in serum
and iron (Fe) and lead (Pb) concentrations in the blood of volunteers from control
area, involving partial correlation coefcient and semipartial correlation coefcient, p for F test and value of F test (model of multiple regression essentiality)
and model equation (N 82).

Element

Partial
correlation

Semipartial
correlation

Fe
Pb

0.462010
0.418763

0.406606
0.359933

0.0000

16.678

FRT 0.485Fe 3139.408Pb  47.295

blood was also positively correlated with TAS (r 0.283) in volunteers from unpolluted area (Fig. 7). Positive correlations between
FRT concentration and TAS in serum in volunteers from both
environments were also stated (polluted: r0.625 vs. control:
r0.837; Fig. 6).

Fig. 6. Correlation between ferritin (FRT) concentration and total antioxidant


status (TAS) in serum of volunteers from polluted and unpolluted area.

M. Wieloch et al. / Ecotoxicology and Environmental Safety 78 (2012) 195205

We can nd a higher lead and cadmium danger for the people


inhabiting polluted area, which results from signicantly higher
toxic metals concentration in blood of volunteers from Mogilno
neighborhood. Simultaneously, positive correlations between Pb in
blood and FRT, TAS and SOD activity, and Cd in blood and SOD
activity and Fe in blood and FRT suggested direct inuence of these
elements upon the effectiveness of these defense mechanisms. It is
supported by results of other research, which showed the inuence
of toxic metals on antioxidant defense elements of the organism
(Aydinn et al., 2004; Han et al., 2005; Kwong et al., 2004;
Marchlewicz et al., 2006; Osman et al., 1998; Patil et al., 2006).

6. Discussion
Our research allows proof of the effect of actual lead, cadmium
and iron exposure, marked by these elements level in blood, upon
defense antioxidant mechanisms in humans (Tables 3 and 5).
Environmental factors are essential either for human health or for
the condition of any living organism, because the increase of
concentration of toxic metals plays signicant role in disorders
involving macro- and microelements homeostasis in the organism. It causes disorders of physiological balance of an organism
and serious danger of many abnormalities or diseases, including
cancer, and weakens defense mechanisms (Nohl and Stolze, 1998;
Olinski and Jurgowiak, 1999; Schulz et al., 1999; Siems et al.,
2000; Beisswenger et al., 2001; Buonocore et al., 2001; Darlington
and Stone, 2001; Kawanishi et al., 2001; Sayre et al., 2001).
There are many antioxidant mechanisms targeting to control
physiological level of reactive oxygen species and to protect
human organism against oxidant damage. We tried to estimate
the impact of environmental stress, which is determined by the
magnitude of lead and cadmium concentrations in blood, on the
antioxidant enzymes activity (CAT, SOD), FRT concentration and
TAS in serum (this study). We can thus evaluate this impact;
however there is lack of similar environmental studies carried on
humans. Nevertheless, many publications concerning occupational exposure on lead and its inuence on the antioxidant
enzymes activity exist. They found the impact of lead upon
antioxidant enzymatic mechanisms in relation to doses and time
of exposure (Monteiro et al., 1985; Sugawara et al., 1991; Aydinn
et al., 2004; Han et al., 2005; Patil et al., 2006). It is supported by
studies in which plasma oxidant and antioxidant statuses, in cement
plant workers who were in the profession over the last 10 years
ranging in age between 29 and 54 years, were measured; e.g., SOD
activity was decreased in cement plant workers compared with
control group (Aydinn et al., 2004). The investigations by Han et al.
(2005) involved measurements of oxidative stress biomarkers in
serum of shipyard welders with 133 yr of exposure history in a
large ship-building industry, full-time employed and used a gas
metal arc-welding process with carbon dioxide as shielding gas. The
measurements of ambient air samples taken outside the welding
helmet had a mean welding fume particulate load (13.21.8 mg/m3)
signicantly higher than the Occupational Safety and Health Administration American Conference of Governmental Industrial Hygienists occupational exposure limits (5 mg/m3), and there was no
signicant difference between SOD activity in serum of exposed
workers and the control group (Han et al., 2005). In the other
research, antioxidant enzymes activity was measured in the erythrocytes from workers occupationally exposed to lead, e.g., there
was signicant decrease in SOD and CAT activities in the erythrocytes from workers exposed to lead compared with those from
control group. When a direct effect of lead on both puried enzymes
was tested, SOD activity was inhibited by about 10% and CAT
activity by about 20% at a lead concentration of 400 mg/dl
(Sugawara et al., 1991). The relation between the exposure time

201

and antioxidant enzymes activity has also been conrmed in earlier


studies (Monteiro et al., 1985). They studied antioxidant enzymes
activity in the erythrocytes of lead-exposed and non-exposed workers. Lead-exposed men had been working for at least 1 year in direct
contact with lead in chemical plant producing lead salts or in
electric-cable manufacturing plant, which uses lead-containing
catalysts. SOD activity was signicant higher in lead-exposed workers than in non-exposed controls. The doseresponse relationship
between this enzyme and lead concentrations was roughly linear for
the workers with 440 mg of lead per 100 g of blood (Monteiro et al.,
1985). On the other hand, Patil et al. (2006) demonstrated that SOD
and CAT activities in the erythrocytes were signicantly lower in
lead-exposed workers as compared with controls. In this study SOD
and CAT activities in the erythrocytes of battery manufacturing
workers were measured. The study group included workers occupationally Pb-exposed over a long 15-year-period of time (Patil
et al., 2006). The in vitro studies consisted of the exposure of human
skin broblast cell line to 20 mM of Pb-acetate for 18 h, and repeated
doses three times in a week. In these studies Zaree Mohmodabady
et al. (2006) observed signicant increase in SOD activity in lead
treated cells after 1 week, whilst changes in CAT activity were not
signicant.
Our studies shown signicant relationships between Cd level in
the blood and SOD activity in serum of humans inhabiting polluted
and salted Mogilno district (Table 4, Fig. 2) and Tuchola Forestry
(control; Fig. 3). Thus, we have noticed these relations in humans
with high Cd level and high SOD activity (Mogilno) and in those
with lower Cd concentration and almost three times lower SOD
activity (Table 3). The research described in literature conrms the
carcinogenic effects of Cd. Experiments on rats have shown that,
depending on the exposure of this element, it can cause the cancer
of lung, skin, kidneys or reproductive organs (Waalkes, 2003). The
impact of Cd on the activity of antioxidant enzymes has also been
stated, but these results are not conclusive. The experiments on rats
also showed that single intraperitoneal injection of a solution of
Cd2 and its oral administration may lead to a decrease in SOD
activity (Novelli et al., 1999). However, in vitro studies on cell lines of
HF2FF (human skin broblast cell line) treated with 20 mM CdCl2
solution showed increase of SOD activity under the inuence of Cd
(Zaree Mohmodabady et al., 2006). Similar results were stated in the
case of blood analyses from inhabitants of polluted Kujawy region
(this paper).
The positive correlation between SOD activity and Cd concentration in humans inhabiting the district of Mogilno (Table 4,
Fig. 2) indicates that increase in serum SOD activity in these
individuals may be an important part of bodys response to
environmental stress associated with increased Cd concentration
in blood. Other studies conducted in the Jinzu basin (Toyama,
Japan) found signicantly higher concentrations of Cd in urine of
exposed persons compared with control group (people living in
the area uncontaminated with Cd) and also signicantly lower
SOD activity in their erythrocytes (Uchida et al., 2004). Generally,
we can thus nd various trends of relations between cadmium
and antioxidant mechanisms, which might certify that this metal
is of signicant importance in development of human antioxidative ecophysiological mechanisms. However, perhaps, contamination with cadmium in contaminated environments examined in
this work was so severe that this metal is splashed in these cases,
into the bloodstream in humans, causing bodys response to
residents expressed through changes in activity of SOD. Some
other results reported decreases of SOD and CAT activities resulting from Cd and Pb increases in humans and rats (Monteiro et al.,
1985; Sugawara et al., 1991; Novelli et al., 1999; Patil et al., 2006;
Zaree Mohmodabady et al., 2006). After all, Cd concentration in
humans examined in this paper was not yet large enough to affect
the activity of SOD-limiting.

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M. Wieloch et al. / Ecotoxicology and Environmental Safety 78 (2012) 195205

Taking into account the results of our studies (this work) and
data from abovementioned authors, we can assume that the
changes of SOD activity depend on the condition of an organism
and environmental determinations. Thus our studies (this paper)
show an increase in SOD activity, as an important mechanism of
antioxidant defenses against environmental stress. Our environmental research analyzes the changes in the antioxidant enzymes
activity in humans living under conditions of environmental
stress, which considers the magnitude of Pb and Cd concentrations in blood. On the basis of our studies we can thus conclude
that signicant increase of SOD activity in serum of volunteers
from polluted areas can be one of the defense systems of an
organism against oxidative stress caused by environmental factors (Tables 3 and 4, Figs. 2 and 3). It is also supported by positive
correlation between Pb and Cd concentrations in blood and SOD
activity in serum of volunteers from polluted area, which can be
interpreted as SOD activity increase together with toxic metals
concentrations (Tables 3 and 4; Fig. 2). On the other hand, we
indicated that CAT activity was signicantly higher in serum of
volunteers from unpolluted area (Table 3). We could thus conclude that, as it is connected from our research (this paper), CAT
might be more sensitive upon environmental impact. Simultaneously we should emphasize that the lack of correlation
between CAT activity and Pb or Cd level in serum of volunteers
from studied areas may suggest however that CAT may not meet
the essential role in the protection of an organism against the
toxic metals in the areas studied in this study. Simultaneously,
our environmental investigations showed the increase of TAS and
positive correlation between Pb concentration in blood and TAS in
serum of volunteers from unpolluted areas. This can be interpreted as saying that together with lead height concentration, TAS
in serum also increases (Fig. 7). These results suggest that nonenzymatic antioxidants, marked by total antioxidant status in
serum, play signicant role in the protection against lead blood
increase in volunteers from unpolluted areas.
Analysis of the results of this study shows a positive correlation
between serum FRT and serum iron in the blood of people living in
county of polluted (Fig. 4) and control areas (Table 5, Fig. 5). The
strength of FRTFe correlation was stronger at the Mogilno countys
residents (polluted) than in the controls, where the highest Fe
concentration was stated (Tables 3 and 5, Figs. 4 and 5). In addition,
a positive correlation has been demonstrated in the case of
dependences between FRT and lead in the control (Table 5, Fig. 5).
Furthermore, determination of serum FRT is used as a valuable
parameter, providing both a shortage and excess of iron in the body
(Nielsen et al., 2000). In healthy individuals, approximately 15% of
the total amount of iron ions are associated with this protein, and
65% with hemoglobin (Bauminger and Harrison, 2003). Therefore, it

Fig. 7. Correlation between lead (Pb) concentration in blood and total antioxidant
status (TAS) in serum of volunteers from unpolluted area.

is anticipated that as a result of changes in Fe there are also similar


changes in serum FRT. We also noted positive correlations FRTFe in
humans living in the county of Mogilno and control area (Table 5,
Figs. 4 and 5), which conrm these assumptions.
The research described in the literature also showed changes
in concentration of acute phase proteins, including ferritin,
together with changes of trace elements level, in the course of
most infections as part of the bodys defense strategy (Kocyigit
et al., 1998; Seyrek et al., 2005); e.g., Olthof et al. (2007) have
shown that in the patients with infections and inammations and
without hematological diseases FRTFe relations were very weak,
whilst in the patients without infections and inammations and
with hematological diseases these relations were very strong.
Based on our studies (this paper) and literature data it can be
concluded that the increase in serum FRT causes the response of
an organism to increased iron concentrations in the blood. The
strength of FRTFe compound depends on the factors that cause
infections and inammations, and pathological substrate. The
literature also describes research on the effects of occupational
exposure to dust containing various metals, including iron, in the
body. It related the long-term exposure to low levels of metals in
welding dust. It found higher concentrations of Fe and FRT in
serum of humans occupationally exposed compared with controls
(Lu et al., 2005). The analysis of the results obtained in this study
also showed signicant differences in Fe concentration in the
blood of people living in studied environments and no signicant
differences in FRT concentrations (Table 3), although the growth
of Fe blood level was accompanied by an increase in serum FRT.
The lack of signicant differences in FRT concentration in the
volunteers studied in this paper might result from a wide range of
reference values established for serum ferritin; FRT values were
signicant in humans studied by us, but they were contained in
reference values, yet. These regularities have also been suggested
by other sources (Sherwood et al., 1998; Andrews, 1999; Cook,
1999; Van den Bosch et al., 2001).
The relationships between lead blood concentration and body
iron status have been described in many studies. Experiments
conducted on laboratory animals have shown a higher concentration of Pb in Fe-decient animals compared with controls
(Bradman et al., 2001). We stated a positive correlation between
serum FRT level and lead blood concentration (Table 5, Fig. 5) in
people from Tuchola Forestry (control). Based on the fact that
serum FRT is a commonly used parameter for determining the
balance of iron oxide in the body, we can conclude that lead blood
concentration affects the economy of iron, and is determined by
the changes in serum ferritin in humans inhabiting unpolluted
areas. In the literature there are studies in which the determination of serum FRT is used to assess the status of iron in the body,
which also showed the relationships between the changes in the
protein levels and lead stress in humans; e.g., in studies by Kim
et al. (2002) FRT concentration was measured in groups of
workers with high exposure (29 men in the secondary lead
smelting), low exposure (46 in the manufacturer of inorganic
pigment) and non-exposed group (56 clerical men from another
plant). These studies indicated signicant decrease in FRT level in
high exposed group compared with non-exposed (Kim et al.,
2002). In other experiments the impact of short-term Pb-exposure on FRT was measured. Blood samples were drawn for Pb and
FRT prior to and after 6 weeks of intensive target practice in the
indoor ring ranges. After 6 week period of exposure to lead dust,
there was a signicant increase in the mean blood Pb level, whilst
mean FRT level decreased (Kim et al., 2003; Wright et al., 2003;
Kwong et al., 2004). As it is connected, from our results there is no
signicant correlation between Pb and FRT in polluted area and in
control area it is positive correlation. This might be explained by
the regularities of cadmium inuence upon ferritin concentration

M. Wieloch et al. / Ecotoxicology and Environmental Safety 78 (2012) 195205

in human from polluted area, compared with control (our results).


These may be also concluded from results obtained by Osman
et al. (1998), who studied children in polluted environment.
Lower Pb concentration in blood, and lower serum FRT were
stated in humans living in the areas with less pollution, as
compared with people living in the areas with a greater degree
of contamination (Hogervorst et al., 2007). However, in the
experiment covering children aged 15 years with different
ethnic backgrounds, higher lead blood concentrations of children
with low FRT level has been stated, compared with those of
standard FRT concentration, amongst blacks population, a group
of Latin Americans and white children (Bradman et al., 2001).
Similar researches on the relationship between serum FRT and
lead in children from industrialized areas were also conducted in
Poland. They demonstrate that children with lower FRT serum
showed a tendency towards higher Pb in blood (Osman et al.,
1998). The research in the present studies shows, however,
that in people living in the control area the increased Pb blood
level causes the increase in serum FRT (r 0.360, p0,000;
Tables 3 and 5, Fig. 5). We can thus conclude that a different
trend of serum FRT changes under the inuence of lead, depending on the environment, may result from differences in the
concentration of Pb in blood, as well as from the age of individuals. Simultaneously, Bradman et al. (2001) showed a strong
correlation between Fe deciency and Pb level in small children
(12 years) and weaker correlation in older children, whilst adults
had no correlation. This means that the results of research
conducted on a group of children should not be generalized for
the entire population. Pb level of the children in large-scale
territories was also more than twice, compared to the level
obtained for adults from unpolluted areas (this paper; Table 3).
Furthermore, the average Pb and Fe concentrations in the blood
and serum FRT were within reference values (Dade Behring Diagnostics Ltd., unpubl.; Goulle et al., 2005; Thermo Fisher Scientic,
2007; Osman et al., 1998, Table 3). Probably in healthy adults from
areas with environmental exposure to lead, and with no Fe deciency,
no evidence of adverse effects of lead on iron status in the body
occurs. Based on the lack of dependences of PbFRT in the people
living in polluted areas, with signicantly high concentration of Pb in
blood (Table 3) and on the ndings by Bradman et al. (2001) we can
suggest that adults living in areas of anthropogenic impact might be
more resistant to the threat of Fe deciency due to increased Pb
concentration in the blood as compared with young groups of people.
We may support this conclusion on the basis of comparison of
abovementioned results and the statements obtained by Osman
et al. (1998), who studied children in polluted areas. This may also
result from the fact that in residents of anthropogenic Kujawy region
the lead exposure is lower compared with large industrial Polish
areas, as evidenced by lower Pb concentrations in the blood of these
people. Perhaps there is a boundary in the blood lead level, beyond
which a disorder of homeostasis occurs and it may be related with
the risk of iron deciency?
On the basis of our results we can explain the inuence of lead
concentration in the blood on FRT level in serum of humans from
examined environments. We stated no signicant decrease of FRT
in the serum of volunteers from control unpolluted area (Table 3).
This may be connected with lead impact and may suggest that
lead pollution can cause changes of antioxidant mechanisms. We
also noted positive correlation between FRT level and TAS in the
serum of volunteers from both environments (Fig. 6). This shows
that these two mechanisms (FRT and TAS) are common and occur
together with FRT increase. Simultaneously, a positive correlation
between Pb blood concentration and serum TAS was also
demonstrated in the control group (Fig. 7). However, studies by
Antonowicz et al. (1998) in 141 healthy male workers of a copper
smelter exposed to the mixture of metals showed signicant

203

negative correlation with blood Pb concentration and TAS. On the


other hand, studies by Han et al. (2005) found positive relations
between lead and TAS in exposed and unexposed volunteers. Thus
lead exposure, which might occur from exposure to welding, can
cause changes in serum biomarkers of oxidative stress that may
be valuable in clinical monitoring of disease development and
assessing. Similar conclusions were obtained by Han et al. (2005).
Thus summarizing our ndings and the results obtained by
abovementioned authors we may conclude that lead exposure
could not damage antioxidant mechanisms in any cases.
It can be concluded from our research that anthropogenic
pollution can bear on antioxidant mechanism efciency. The
inhabitants of polluted regions developed mechanisms protecting
against oxidative stress based on SOD activity increase, whereas
antioxidant defense in relation with lead concentration in
humans of unpolluted areas is based on the non-enzymatic
mechanisms marked by total antioxidant status. We also noted
signicant CAT decrease and FRT increase in serum of humans
from polluted areas, but without any relation to lead or cadmium
blood concentrations.

7. Conclusions
1. Environmental pollution makes for increase of actual lead and
cadmium exposure, which manifests with lead and cadmium
blood concentrations increase.
2. Superoxide dismutase is resistant to the inuence of environmental factors. Thus increase of SOD activity in serum of
inhabitants from polluted areas is one of the defense mechanisms of an organism against oxidative stress caused by
environmental lead and cadmium exposure.
3. The higher catalase activity and total antioxidant status
indicate that these mechanisms play a key role for antioxidant protection in the unpolluted areas.
4. Non-enzymatic mechanisms marked by a total antioxidant
status are the main antioxidant defense in relation with lead
concentration in humans of unpolluted areas.
5. Ferritin and total antioxidant status are related to each other;
thus antioxidant mechanisms can strengthen one other.

Acknowledgments
This work was supported by a grant from funds of the European
Social Fund and State budget within the framework of the Integrated
Program of Operating-Regional Development (the Activity 2.6
Regional Innovative Strategies and knowledge transfer of individual project of the KuyavianPomeranian Province: Scholarships
for graduate students of 2008/2009-ZPORR) and Nicolas Copernicus
University in Torun within the framework of Rector Magnicus
Grant no. 24/2009.
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