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The
purpose of the present article is to outline the high effective properties of
the clinoptilolite matter as a sorbent of toxic substances, especially lead
(Pb), in living organisms. For the first time, the act of zeolite as an
absorbent, in conditions of Pb intoxication in living organism was proved. A
clinoptilolite sorbent was prepared based on natural Bulgarian zeolite and
applied in a 90 day ecotoxicological experiment. The dietary inclusion of the
sorbent reduced the lead concentrations in the Pb exposed and clinoptilolite-supplemented
mice by 84%, 89%, 91%, 77% and 88% in carcass, liver, kidneys, bones and feces,
respectively. Significant improvement of the chromosome aberrations, mitotic
index and erythrocyte morphology and erythropoiesis and body weight was
established in the clinoptilolite-treated animals.
A
mathematical model for lead concentration was elaborated and thus, the
coefficient (η) of Pb absorption by gastrointestinal mucosa in the supplemented
mice was found. It is: η=3.53% (versus η=15% in non-supplemented ones).
For the
first time a mathematical model for the mitotic index was proposed. It shows
that the animals’ recovery goes in parallel with the Pb bioaccumulation and the
susceptibility of the mouse’s organism to Pb load decreases, while the recovery
rate of the genetic apparatus increases with the time.
Keywords: Zeolite, Clinoptilolite,
Lead intoxication, Chromosome aberration, Mitotic index, Mathematical models,
Absorption coefficient
INTRODUCTION
Zeolites are a
large, widespread family of minerals. Their physical and chemical properties
make them suitable means for different areas of applications [1,2]. Because of
their specific structure, they are excellent absorbents and thus-an excellent
means against chronic heavy metals intoxication and particularly lead
intoxication. These minerals are intensively used for augmentation of animal
production, growth performance, feed utilization [3] and prevention of certain
farm animal diseases [4] as well as for the protection of the environment
[5-7]. Zeolites are applied in biotechnology and medicine [8]. They are very useful
in processes of the removal of ammonium [9].
Clinoptilolite
is a mineral from the zeolite group. It is known as one of the most excellent
toxin absorbents. It is the most widely used zeolite in animal studies due to
its structural stability under high temperatures and acidity.
For the first
time we proved the effect of zeolite, as absorbent, in conditions of Pb
intoxication of a living organism. A modified clinoptilolite sorbent, named
“KLS-10-MA”, water modification (made in Bulgaria, by Dr. Nikolay Popov), was
applied, as a food supplement, in a 90-day ecotoxicological experiment with
laboratory mice inbred line ICR.
Here, the
morphophysiological and cytogenetical parameters, indicting the physiological
status of a living organism, are designated as “biomarkers”.
One of the most
important of them is the chromosomal aberration frequency (CAF) [10,11]. Along
the increase of CAF, the decrease of the proliferation capacity of the cells,
indicated by mitotic index (MI), is used as indicator (biomarker) of different
disorders in the genetic apparatus.
Especially
useful are CAF and MI for the assessment of the environmental contamination
degree [12-14].
A mathematical
model of the kinetics of lead bioaccumulation in bones in the exposed and
exposed-supplemented animals was constructed (it is presented in the respective
section). For the first time a mathematical model, presenting the common trends
of MI change in conditions of a chronic intoxication was elaborated.
Because of
their specific, well-defined pore structure [2], zeolites are extensively used
in primarily three fields of application: adsorption, catalysis and ion
exchange. In addition, natural zeolites because of their lower cost are used in
bulk mineral applications. Zeolites are already in use in the food industry.
According to EMFEMA [15], zeolites allow better performance of intestinal micro
flora.
The mineral
clinoptilolite (K, Na, Ca, Mg (AlSi3O8).5H2O)
belongs to Clinoptilolite-Heulandite group [16]. Clinoptilolite is a Heulandite
which is reach of silica (silicon dioxide) SiO2. (Heulandite is
named in honor of the English collector of minerals John Henry Heuland). The
name “clinoptilolite” is derived from the Greek words klino (κλίνω; “oblique”),
ptylon (φτερών; “feather”) and lithos (λίθος; “stone”). Really the texture of
the clinoptilolite patterns resembles in some degree a feather. Clinoptilolite
is a natural zeolite comprising a micro porous arrangement of silica and
alumina tetrahedra.
Clinoptilolites
are minerals with specific crystalline lattice-hydrated aluminosilicates of
alkali and alkaline earth cations, consisting of three-dimensional frameworks
of SiO44- and AlO45-tetrahedra linked through the shared
oxygen atoms [4]. They have a relatively open structure with a total pore
volume of approximately 35%.
The great
benefit of the clinoptilolite as absorbent and particularly perfect heavy
metal-trap is founded on the character of its structure. The Si-block in
crystalline unit is neutral, while the Al-block is negative, thus it charges
the mineral’s lattice negatively. The existence of Na, K and/or Ca cations
determines the neutrality of the minerals. These cations easily exchange in
solutions with cations of certain metals, such as Pb, Cd, Hg, etc. [17-19]
established that clinoptilolite exhibited the highest capability in absorbing
Pb2+ ion in complex solution with pH value of 1.2 at 37°C, achieving
the capacity of 7 mg/g, two times more than that by other zeolites and six
times over that by activated carbon.
The effects of clinoptilolites in animals appear to be related to their
high cation-exchange capacity [20], which affects tissue uptake and utilization
of NH4+, Pb2+, Cd2+, Cu2+, Cs+
and other cations [18,21].
Clinoptilolites support the immune activity [22-24]. They appear to be
stable in the gastrointestinal tract [25] and as unique selective absorbents,
they could absorb toxins, heavy metals and free radicals from the body and
excretes [4,26]. Therefore, the harmful lead ions (Pb2+) could be
trapped in the stomach. It is believed that the high affinity of clinoptilolite
for Pb would significantly reduce the amount of dietary Pb available for
absorption by the intestinal mucosa [19].
Clinoptilolites of high quality occur in Bulgaria. These minerals have
the most widespread distribution in the eastern and north-eastern Rhodopes
Mountain (Rhodopes Mountain is situated in southwestern Bulgaria [27-29].
Clinoptilolite is practically a non-toxic substance [14]. A lack of
toxic effects of clinoptilolite has been reported by other authors also [22,30-32].
MATERIALS AND
METHODS
To explore in
detail the positive effect of the clinoptilolite sorbent as absorbent of toxic
substances, we conducted an experiment with laboratory mice, inbred ICR line.
The experiment covered 90 days.
EXPERIMENTAL ANIMALS
Albino male mice, about 6-8 weeks of age,
inbred ICR strain, were selected. The animals were arranged in four groups each
of 60 mice:
1. Group 1
(Control) animals fed with conventional food for small rodents and water
2. Group 2
animals fed with conventional food+clinosorbent KLS-10-MA (as a powder) and
water
3. Group 3
animals fed with conventional food and water+Pb (NO3)2
4. Group 4
animals fed with conventional food+KLS-10-MA sorbent and water+Pb(NO3)2
(Group 2 was a second control in order to
prove eventual toxicity of the clinoptilolite sorbent)
The mice were bred in vivarium and housed in
individually ventilated cages. The physical size of the cages was in accordance
with European standards. The bedding material was obtained from an ISO 2000
accredited supplier. Mice were acclimatized for a 7 day period before starting
the experiment. A standard temperature of between 19-23°C, a humidity of 45-60%
and a 12 h light/night cycle were kept all the time. The food was in the form
of pellets and not withheld at any time during the experiment. All mice were
allowed access to food and water ad libitum. The water, food and bedding
material were daily inspected and changed when necessary. The animals were
neither medicated nor vaccinated.
CLINOPTILOLITE
For the preparation of the modified
clinoptilolite sorbent KLS-10-MA, natural clinoptilolite obtained from the
deposit in the village Golobradovo (situated in the region of East Rhodops in
South Bulgaria) was used. The sorbent was prepared by Nikolay Popov through a
treatment of natural clinoptilolite (zeolite containing 83.5% clinoptilolite)
[33].
The modification KLS-10-MA could be
considered as a successful sorbent for detoxification purposes. The lead
resorption through the intestinal mucosa is prevented in a great extent.
STUDY DESIGN
The mice were chronically exposed to lead
(Pb) in the form of aqueous solution of Pb(NO3)2, diluted
in the drinking water. The modified clinoptilolite sorbent KLS-10-MA, in the
form of powder mixed with the meal was used as antidote. The bioaccumulations
of Pb and the detoxification effect of KLS-10-MA in carcass, organs and
tissues, chromosomal aberrations and mitotic index, erythrocyte morphology and
proliferation and body weight gain of the laboratory mice were studied.
The exposure of the mice to the lead was
performed as the mice were treated with 0.05 N solution of lead nitrate diluted
1:10 in the drinking water. The clinoptilolite sorbent KLS-10-MA in the form of
powder was mechanically mixed to a 12.5% concentration with conventional
granulated forage for small rodents. Two variants of the feeding stuff [14]
were prepared:
1. Standard (conventional) food [12]
2. Standard food mixed with 12.5% sorbent
KLS-10-MA.
The concentrations of Pb in the whole body,
liver, kidney, bones and feces and the percentages aberrant mitoses, mitotic
indices, blood samples and body weight of the control and experimental animals
were determined on days 15, 40, 60 and 90 [34] from the beginning of the
experiment. At each time point a subset of 8 mice from the four groups were
used.
To determine the Pb concentration, after the
removal of the alimentary tract, the tissues and some internal organs were oven
dried at 60°C to a constant weight. The dried tissues were dissolved in a
mixture of concentrated nitric-perhloric acid (4:1) [35]. The concentrations of
Pb and the element composition of the two food variants were determined in a
certified laboratory by atomic emission spectrometry with inductively coupled plasma
(ICP AES) on a GFAAS-Varian instrument. The detection limits were 0.002 mg/l
for Mn; 0.004 mg/l for Cd; 0.005 mg/l for Zn; 0.03 mg/l
for Pb; 0.04 mg/l for Fe; 0.5 mg/l for Ca, K, Mg
and Na.
STATISTICAL ANALYSIS
The statistical analysis was done
using the SPSS package for Windows, version 15.0. Differences were considered
to be significant when p values were lower than 0.05 (p<0.05). Firstly, the
data was processed according to Kolmogorov-Smirnov test for normality in each
group. All groups showed normal distributions. Secondly, the data was analyzed
by analysis of variance and subsequent Tukey high statistical difference test
(Tukey HSD test) and Dunnet test, for estimating individual differences.
MATHEMATICAL MODEL
FOR LEAD BIOACCUMULATION
This model helps calculate some kinetic
parameters characterizing the Pb-accumulation and the degree of the sorption
effect of the investigated mineral sorbent.
There are not enough studies that suggest
quantitative approach. Physiologically based kinetic models (PBKM) for arsenic,
chromium, mercury and lead have been proposed with any degree of completeness
[36]. A simple mathematical model for Pb concentration in the body of mice, fed
with a contaminated diet, was proposed. Series of PBKM for bone seeking
elements have been developed [36]. Distribution and bioavailability of lead,
chromium and uranium have been considered [37]. For the first time a
mathematical model, describing the time courses of cadmium and zinc concentrations
in liver and kidney of laboratory mice Mus musculus alba was proposed and it
well explains the observed peculiarities of Cd bioaccumulation pattern in liver
and kidney in conditions of very high exposure to heavy metal mixture [34].
In the present mathematical model for the
kinetics of the lead bioaccumulation in mice’s bones, three compartments of Pb
movement are considered: gastrointestinal tract, blood and bones (Figure 1). It was supposed that Pb is evenly
distributed into these compartments. Entering in the gastrointestinal tract, Pb
moves then to the blood and afterwards to the bones.
One can
assume that Pb is distributed evenly into compartments, which allows the use of
differential equation for its kinetics. After entering in gastrointestinal
tract, Pb moves to blood and then to bones. Thus, the
following system of ordinary differential equations takes place:
dx/dt = 1a1x – a2x (1)
dy /dt
= a1x – a3x – a4x (2)
dz /dt
= a3x (3)
Under initial conditions,
t0=0, x (t0)=x0=A, y(t0)=0, z(t0)=z0 (4)
where t
is time [days]; x, y and z are the concentrations [mg/kg] of Pb at a given time t in the
gastrointestinal tract, the blood and the bones, respectively; t0 is the moment when the
experiment starts; a1 ([a1]=[day-1]) and a3 ([a3]=[day-1]) are the rate constants of Pb
accumulation in blood and bones, respectively; a2 ([a2]=[day-1])
and a4 ([a4]=[day-1]) are the rate constants of Pb excretion through the feces
and the urine, respectively; dx/dt, dy/dt and dz/dt are the rates of the change in Pb levels with the time in the
three compartments, respectively.
MATHEMATICAL MODEL FOR THE MITOTIC INDEX
The
mathematical model, here presented, outlines the common trends in the change of
the mitotic index over time, during a chronic Pb intoxication of small mammals.
This change results from two main processes:
1. Decrease of MI due to the toxic
effect of Pb
2. Increase of MI due to recovery
processes running in the mouse’s organism
In the
unsupplemented animals the recovery process is quite week, so there only a drop
of MI was observed, although this drop was going with a decreasing rate. In
this case the following differential equation is adequate (Group 3):
dm /dt
= -am
Under initial condition,
t0=0, m
(t0)=m0
(6)
where m (%) is the value of MI (the percentage
of cells undergoing mitosis), dm/dt is the rate of change of MI
with the time, a (t) ([a]=[day-1]) is sensitivity (a parameter,
characterizing the cell sensitivity to Pb toxicity, expressed as a “rate
constant” of the diminution of the cells with proliferative activity). This
parameter is presumed as a time dependent variable because the experimental
data clearly shows a decreasing intensity of the cell reaction to Pb bioaccumulation,
probably due to the certain detoxification of the organism on the basis of
liver and kidney activity in the course of the Pb treatment. Thus, the
sensitivity of the bone marrow cells to Pb decreases during the experiment.
Therefore, the parameter a (t) should decrease with the time.
At
simplest a (t) cold is modeled according to the following mechanism:
da /dt
= -ra
Under initial condition,
t0=0, a (t0)=a0 (8)
where r ([r]=[day-1])
could be denoted as reduction parameter (reduction of the sensitivity regarding
Pb toxicity). The solution of the differential equation (7) under initial
condition (8) is:
Taking into account (9), the differential
equation (5) could be written in the form:
dm /dt
= -a0e-rt m
In the supplemented animals the recovery
processes are appreciable and more intensive because the clinoptilolite absorbs
a significant part of Pb in the digestive tract. After day 45, MI significantly
increased. It is known that natural clinoptilolite exerts an immunostimulatory
effect [22]. Our results showed that MI in clinoptilolite-supplemented mice
remains on a visible higher level compared with that in unsupplemented ones (Figure 2).
The following differential equation is
adequate to describe MI behavior in the case of clinoptilolite supplementation
(Group 4):
dm /dt
= -a0e-rt m +
k(t) (11)
Under initial condition,
t0=0, m (t0)=m0 (12)
The
parameter k ([k]=[% day-1]) could be named recovery
rate and it could be considered as an integral characteristics of the
complex of recovery processes developing in animal organism. These processes,
accelerated by clinoptilolite supplementation, run in parallel with the
injuries, caused by Pb intoxication. The experimental data suggest that
parameter k is a variable quantity. It seems reasonable to propose the
following differential equation for the time course of the recovery rate k:
dk /dt
= g - ck
Under initial conditions,
t0=0, k (t0)=k0 (14)
The
parameter g ([g] = [day-2]) is presumed as a
genetically determined parameter and it could be considered as an integral
characteristic of the recovery potential of the genetic apparatus. The
parameter c ([c] = [day-1]) plays a role of a
restriction constant related to the inhibition of the recovery process by the
toxicant.
RESULTS AND
DISCUSSION
Pb concentrations
Lead is significant genotoxic
agent [38]. It is well studied as a factor damaging the chromosome structure in
mammalian cells [12,13,39-41]. Our results are a new contribution to the
previous studies.
The theoretical results (as well
as the experimental points) of the Pb concentrations in bones are displayed in Figure 3. Point “0” of the time axis in
all figures corresponds to the concentration measured in Control group.
The background Pb level in carcass, liver,
kidney, bones and feces of the control mice were 0.22 ± 0.06, 0.5 ± 0.07, 0.44
± 0.08, 9.3 ± 1.3 and 23.6 ± 6.7mg/kg, respectively. On day 90 the Pb
concentrations in the mice from Group 3 in carcass, liver, kidney, bones and
feces were 1467-fold, 133-fold, 1337-fold, 1523-fold and 406-fold higher
compared to those in the mice from Control group. Regarding Group 4, the Pb
concentrations were 237-fold, 17-fold, 125-fold, 357-fold and 51-fold higher
compared to Control group (Group 1). Significant differences (p>0.001) were
established in carcass, kidney, bone and feces between groups 3 and 4 after day
15. The reduction in Pb levels in the exposed and supplemented mice compared to
exposed non-supplemented ones was as follows: 84%, 89%, 91%, 77% and 88% for
carcass, liver, kidney, bones and feces, respectively.
When
denote (Pb90/Pb15) Group 3=R3 and
(Pb90/Pb15) Group 4=R4, the
following relations could be constructed:
(R3/R4)Carcass=2.7;
(R3/R4) Feces=2.19 (15)
(R3/R4)Liver=11.27;
(R3/R4) Kidney=3.77; (R3/R4)
Bone=1.74 (16)
As
seen from (15) and (16), the most value has the relation R3/R4 calculated
about the liver. It is an expected result: The liver is “the purification
plant” of the organism and there the highest toxicant concentration should be
registered.
To
consider more accurately the bioaccumulation differences in the different
tissues resulting from the sorbent supplementation, the following ratios were
calculated for day 90:
Group 3
PbBone/PbLiver=19; PbBone/PbKidney=2.5;
PbKidney/PbLiver=7.6
(17)
Group 4: PbBone/PbLiver=40;
PbBone/PbKidney=6.4; PbKidney/PbLiver=6.3
(18)
The most ratio is this between bone and liver
(Bone/Liver ratio) in Group 4. This fact confirms the significant role of the
clinoptilolite to avert the reaching of high lead concentrations in the soft
tissues. The relatively low value of Bone/Kidney ratio in Group 3 indicates
that the kidneys take much larger part of the Pb load compared to the liver.
Another relation, calculated also for day 90,
could help to estimate the significant decrease of the Pb bioaccumulation in
the conditions of the sorbent supplementation:
Bone PbGroup 3/PbGroup 4=4.3 (19)
Liver PbGroup
3/PbGroup 4=9
(20)
Kidney PbGroup 3/PbGroup
4=11 (21)
The ratios for the liver and especially for
the kidney are rather high compared to the bone-ratio. This fact once more
points out the power of the clinoptilolite sorbent as a means for
detoxification of the soft tissues in conditions of lead bioaccumulation. Also,
the relatively low bone-ratio is in agreement with the fact that Pb is a bone
seeking element.
BLOOD PARAMETERS (ERYTHROCYTE MORPHOLOGY AND ERYTHROPOIESIS)
Lead influence on
erythropoietic bone marrow cells causes morphological changes: Microcytic and
hypochromic erythrocytes appear. More than 90% of Pb in blood accumulates in
erythrocytes and there are at least two major compartments for Pb in red blood cell:
one associated with the membrane and the other associated with the hemoglobin. The
increased mechanical fragility of cell membrane shortens the erythrocyte
lifespan. Depression of the chemosynthesis by Pb is due to the inhibition of
SH-containing enzymes controlling it in bone marrow [42]. In addition, the
decrease of the hemoglobin level is due to the decrease of the number of
dividing erythroblasts and consequently newly-formed erythrocytes. All these
features indicate a lead-induced anemia. In our experiment, a drop in the
proliferative activity of the bone marrow stem cells and well expressed anemia
were clearly observed in the Pb-exposed mice from Group 3.
The hematological
analysis was carried out on the same groups of animals using standard clinical
methods. Peripheral blood samples were collected between 9 and 11 AM from the
orbital sinus [10]. The percentages of various forms of cells were determined
using Giemsa stains. About 150-200 cells were counted in each stain.
As known, the
hemoglobin level and erythrocyte number are most often investigated. Our
attention here was focused mainly on the changes of the erythrocyte morphology
because of its usefulness as a clear indicator for Pb intoxication.
The data of the
quantitative erythrocyte morphological analysis in our experiment are presented
in Figure 4. Lead bioaccumulation caused statistically significant
reduction of the percentage normal erythrocytes and respectively significant
increase of the percentage pathological erythrocytes in the peripheral blood of
the mice from Group 3, compared to the Control group (p<0.001). The normal
and pathological erythrocytes decreased and increased, respectively, in Group
4, but the normal red blood cells in Group 4 were at significantly higher level
and the pathological ones were at significantly lower level compared with Group
3 (p<0.01). Besides, within Group 4 the normal erythrocytes remained
significantly higher than pathological ones (p<0.001) up to the end of
experiment.
Between
groups 3 and 4 and Control group (Group 1), on day 90, the following ratios
were established regarding the normal and pathological erythrocytes,
respectively:
N1/N3=2, N1/N4=1.35
(22)
P1/P3=0.43, P1/P4=0.53
(23)
Between
groups 3 and 4 the following relations were determined on days 15 and 90:
Normal
Er: N4/N3=1.2
(15), N4/N3=1.5 (90) (24)
Pathological Er: P4/P3=0.7
(15), P4/P3=0.83 (90) (25)
The proportion between the pathological
erythrocytes in Group 4 and Group 3 remained up to the end of the experiment
less than unity, i.e., the quantity of the pathological erythrocytes in Group 4
was less than the same quantity in Group 3.
These results one more time indicate the
importance of the clinoptilolite treatment.
A significant drop in the erythropoiesis rate
was recorded in the mice from Group 3. Statistically significant differences
(p<0.05) were observed on days 15 and 90 between groups 3 and 4. As
expected, the percentages proliferating erythrocytes in Pb-exposed and
supplemented mice (Group 4) were higher than those in Pb-exposed mice without
clinoptilolite supplement (Group 3).
The differences between groups 3 and 4 and
Control group, especially on day 90, were quite significant (p<0.001).
Comparing PE in the control mice to PE in the mice from groups 3 and 4 on day 90
the following interrelation was obtained:
PEl/PE3=10, PE1/PE4=3.2 (26)
The first relation of (26) indicates a
10-fold drop in the number of dividing erythroblasts in Group 3 compared with
Control group. The supplementation with KLS-10-MA reduced the respective ratio
in Group 4 almost by three times.
CHROMOSOME ABERRATIONS FREQUENCY
Chromosome aberrations are a sensitive
endpoint for detecting genotoxic effects induced by various harmful agents,
including heavy metals and toxic chemicals [43]. Chromosome aberration
frequency (CAF) could be used as an effective biomarker (indicator) of genetic
injuries.
The cytogenetical analysis of the samples was
performed as described by Preston et al. [44]. Mitomycin C (3.5 mg/kg) (Fluka)
was used as a positive control. The other animals were injected with only 0.2
mL 0.9% NaCl. Bone marrow chromosomal aberration assays were performed in
groups of animals, each one consisting of eight specimens. The animals were
injected IP with colchicine at a dose of 40 mg/kg, 1 h before isolation of bone
marrow cells. The bone marrow cells were flushed from femur with 0.075 M KCl
and hypotonized at 37°C for 20 min. Thereafter, the cells were fixed in
methanol-acetic acid (3:1), dropped onto cold slides and air dried. To examine
the chromosomal aberrations, the slides were stained with 5% Giemsa solution
(Sigma Diagnostic). At least 50 well-spread metaphases were analyzed per animal
at random [14].
The results are
presented in Figure 5. It is remarkable that no statistically
significant differences were observed between CAF in Group 4 (intoxicated and
supplemented animals) and CAF in Group 2 (healthy and supplemented animals)
except on day 60 (p<0.05). Significant differences (p<0.001) were recorded
between Group 3 and other groups at each time points. The maximum CAF in groups
3 and 4 were observed on day 60, when the following correlations take place:
CAF3/CAF1=4.56; CAF4/CAF1=2; CAF3/CAF4=2.3 (27)
These
results underline once again the essential effect of the clinoptilolite sorbent
in the reducing of the lead intoxication.
The distribution of the different type
chromosome aberrations is displayed in Figure
6. Chromosome breaks and Robertsonian translocations or c/c fusions were
the predominant harmful effects of the mouse’s genetic apparatus. They had
maximal values in Group 3, on day 15: 4.7% and 5%, respectively. This fact
indicates that the chromosome injuries appear and increase rapidly from the
beginning of the lead treatment and demonstrates the great sensitivity of the
genetic apparatus to toxic substances. Fragments were found below 1.5%. Only in
Group 3, on day 60, they showed its highest value (3%).
These results allow make the following
conclusions:
1. The
genetic apparatus of the mouse’s organism is very sensitive and fast reacting.
(This is a well-known fact. Because of that the rodents are the preferable
experimental animals).
2. The
genetic apparatus of the mouse’s organism reacts to toxic substances mainly via
chromosome breaks and Robertsonian translocations.
3. The
treatment with the clinoptilolite sorbent significantly reduces the chromosome
breaks and fragments and to a lesser degree the Robertsonian translocations.
KINETICS OF THE LEAD
BIOACCUMULATION
For the equation (3) under conditions (4),
the following analytical solution was obtained:
z(t)
= z0 + Aa1a3 ( (1/b1b2)–
(1/b1(b2 – b1)) e -b1t + 1/ b2(b2-b1)
* e -b2t (8)
where
b1=a1+a2 and b2=a3+a4.
The
solution z (t) represents the process
of Pb bioaccumulation in the bones of the mice. The graphical time course of
bone Pb is presented in Figure 4.
The initial condition z (t0)=z0 corresponds to the bone Pb
concentration in the control group, z0=1
mg/kg.
The
concentration of Pb in the drinking water of the experimental animals was 620 mg/L≈
620 mg/kg. The daily water consumption per animal was about 7 mL/day and
therefore, the daily Pb dose per animal could be approximately B=4.34 mg/day. Extrapolating over the
experiment and taking into account the value of gastrointestinal resorption
coefficient η=15% it is
calculated that in Group 3 the entire quantity of Pb absorbed by the
gastrointestinal mucosa and entering into the blood during the experiment might
be AGroup3=58.6 mg. Coefficient η (0<η< 1)
is a dimensionless coefficient indicating what fraction of ingested metal dose
resorbs in the digestive tract. Converted to concentration in mg/kg and taking
into account that the mean mouse body weight during the experiment was about 30
g, we obtained AGroup3=x (t0)=1953 mg/kg.
The parameters were fitted by minimization of 2 by the use of an iterative
Gauss-Newton procedure [45,46]. Thus, the following values were found-for Group
3: a1=0.022 day-1, a2=0.001
day-1, a3=0.099 day-1 and
a4=0.004 day-1; for Group 4: AGroup4 =x
(t0)=459.6 mg/kg, a1=0.022 day-1,
a2=0.002 day-1, a3=0.099
day-1 and a4=0.004 day-1.
The
model shows that the rate constants of Pb excretion by the feces a2
in Group 4 are twofold higher than that in Group 3: a2
Group4 =2a2 Group 3. This result corresponds to the
real situation, because as a ballast matter, the clinoptilolite sorbent
accelerates the intestine passage.
The
mathematical model of Pb kinetics is in good agreement with the experimental
data for Pb concentration in bones. This model allows determining the
coefficient of Pb gastrointestinal absorption (absorption coefficient) in the experimental animals from Group
4, using the formula:
η = A Group4 P / 1000BT (29)
where AGroup 4=x(t0)Group 4 is the concentration of lead absorbed in the digestive tract and entering the blood extrapolating over the experiment (for the mice of Group 4), P is the mean mouse body weight during the experiment, B is the daily dose of Pb per animal and T is the duration of the experiment. The value η=3.53% obtained in clinoptilolite supplemented mice (Group 4) is 4.25 fold lower compared with η=15% in non-supplemented animals. A reduction of 76% occurred! In fact, this is a significant result: KLS-10-MA diminished the Pb absorption in gastrointestinal tract of mammals’ organism more than four times! This result allows prognosticate an excellent perspective for the application of the clinoptilolite sorbent in mammals.
KINETICS
OF THE MITOTIC INDEX
The
mitotic indices (%) were determined by counting the number of dividing cells
among 1500 cells per animal. The frequencies of abnormalities and the mitotic
index were determined for each animal. The mean SD for each group was
calculated and the data was statistically evaluated for their significance by
analysis of variance using student t test.
A trend
of continuous decrease of the mitotic index during the experiment was
established in the exposed-non-supplemented mice. Contrariwise, in the
exposed-supplemented mice, the level of the mitotic index decreased up to day
45 but then steadily increased (p<0.001)
(Figure 7).
The
following comparisons were made and respective ratios calculated regarding the
mitotic indices (M) (the time points are given in brackets):
M1/M3=1.8 (15) M1/M3=2.5
(45) M1/M3=2.7 (60) M1/M3=2.9
(90) (30)
M1/M4=1.4 (15) M1/M4=1.5
(45) M1/M4=1.3 (60) M1/M4=1.2
(90)
(31)
M4/M3=1.3 (15) M4/M3=1.6
(45) M4/M3=2.1
(60) M4/M3=2.5 (90) (32)
The mitotic indices in the mice from Group 4
in all points of the observation were close to those in Control group. The
relations M4/M3 steadily increased during
the experiment and this indicates the determinant
role of the clinoptilolite sorbent in the adsorption of the toxicant and
therefore in the improvement of the genetical status in the mice from Group 4.
In the
unsupplemented animals the recovery process is quite week, so there only a drop
of MI was observed, although this drop was going with a decreasing rate. In
this case the following differential equation is adequate (Group 3):
dm /dt
= -a(t)m
Under initial condition:
t0 =0, m (t0)=m0 (34)
where m (%) is the value of MI (the percentage of cells undergoing mitosis), dm/dt is the rate of change of MI with the time, a (t) ([a]=[day-1]) is sensitivity (a parameter, characterizing the cell sensitivity to Pb toxicity, expressed as a “rate constant” of the diminution of the cells with proliferative activity). This parameter is presumed as a time dependent variable because the experimental data clearly show a decreasing intensity of the cell reaction to Pb bioaccumulation, probably due to the certain detoxification of the organism on the basis of liver and kidney activity in the course of the Pb treatment. Thus, the sensitivity of the bone marrow cells to Pb decreases during the experiment. Therefore, the parameter a (t) should decrease with the time. For a (t), under initial condition: t0 =0, a (t0)=a0, we found:
a(t) = a0e-rt (35)
Taking into account of (35), the differential
equation (28) could be written in the form:
dm /dt = -a0e-rt m (38)
The following differential equation is
adequate to describe MI behavior in the case of clinoptilolite supplementation
(Group 4):
dm /dt
= -a0e-rt m +
k(t) (39)
Under initial condition:
t0 = 0, m (t0)=m0 (40)
The
parameter k ([k]=[% day-1]) could be named recovery rate and it could
be considered as an integral characteristics of the complex of recovery
processes developing in animal organism. These processes, accelerated by
clinoptilolite supplementation, run in parallel with the injuries, caused by Pb
intoxication. The experimental data suggest that parameter k is a variable quantity.
Finally, for m (t) one can write:
dm /dt = -a0e-rt m + k0e-ct + g/c (1 - e-ct ) (41)
The solution (Figure 7) of the equation (39) for the unsupplemented mice (Group 3) under initial condition (40) is:
m(t) = m0
e –(a0/r (1-e-rt)) (45)
The initial condition is m (t0)=m0=12%
The
equation (44), for the time course of MI in the mice from Group 4, cannot be
solved analytically and a numerical solution was obtained. It is presented in Figure 8.
In the observed
mitotic indices no statistically significant differences were established (p>0.1) between Control group and
Group 2. Significant differences (p<0.05-p<0.001) were found between Group
3 and Group 4, between Control group and Group 4 (p<0.002) and especially between Control group and Group 3 (p<0.001). On day 90, MI in Group 4
was almost 2.5-fold higher compared to that in Group 3 (Figures 2 and 7). On the same day MI in Control group was almost
3-fold higher than that in Group 3 and only 1.2-fold higher than that in Group
4. A continuous decrease of MI during the experiment was established in the
mice from Group 3. Contrariwise, in the exposed and supplemented mice from
Group 4, MI decreased up to day 45 but then steadily increased.
BODY
WEIGHT
The body weight is an important biological
marker for the estimation of the vital status of the living organisms in
different injuries, including metal intoxication. Each intoxication causes damages
on the organs and tissues in the animal
organism and reflects particularly
on the growth and biomass.
Many
researches have proved that the dietary inclusion of zeolites, particularly clinoptilolites, as a feed additive
in animal’s nutrition improves body mass, average daily gain and/or feed
conversion ratios in pigs, ruminants, sheeps, poultry, broilers [31,47,48].
Apart from the positive effects on animal’s performance, dietary
supplementation of zeolites appears to represent an efficacious, complementary,
supportive strategy in the prevention of certain diseases and the improvement
of animal’s health status.
The
changes with time of mean body weight of the animals are presented in Figure 2.
CONCLUSION
The
inclusion of the modified clinoptilolite sorbent KLS-10-MA (prepared, based of
natural Bulgarian zeolite) as a food supplement significantly decreased Pb
concentrations in the body and organs of the experimental animals, loaded with
lead. This is due to the fact that the clinoptilolite sorbent, as a reliable
absorbent, trapped in the stomach a great part of lead and thus limited the Pb
entering the blood. In the sorbent-supplemented animals the bioaccumulation
coefficients is η=Pb90/Pb15
=3.53% [49-55]. (Pb15 and Pb90 are the respective
lead concentrations on days 15 and 90). (This value is about 4 times lower
compared to the known coefficient for Pb absorption in non-treated animals:
15%). In the supplemented mice the lead concentrations in carcass, liver,
kidneys, bones and feces decreased by 84%, 89%, 91%, 77% and 88% respectively,
compared to those in the unsupplemented ones [56-60].
The
structure of the chromosomes and red blood cells as well as the mitotic index
and erythropoiesis, also the body mass, were significantly improved in the
group of the intoxicated mice, supplemented with KLS-10-MA. These mice appeared
active and healthy [60-63].
The
mathematical model for the lead bioaccumulation clearly shows that the recovery
processes in the animals run in parallel with the Pb bioaccumulation and that
the susceptibility of the mouse’s organism to Pb load decreases and the
recovery rate of the genetic apparatus increases during the experiment [64].
The
mathematical model for the mitotic index allowed made an exact quantitative
evaluation on the positive effect of the clinoptilolite treatment. The theoretical
results could also help in order to a more precise determination of the doses
of the clinoptilolite supplement. The model confirms the authors’ hypothesis
that the sensitivity of the bone marrow cells to the toxicant and the recovery
rate of the genetic apparatus remain not constant with the time. Really, the
sensitivity decreased and the recovery rate increased with the time due to the
stimulated defense reaction of the organism. The model could predict the state
of cell proliferation in each time point of the observation and allows a
quantitative evaluation of the positive effect of the treatment. Such models
could help in the toxicological investigations [65-67].
This
study demonstrates a great benefit of the clinoptilolite as a food additive. We
have every reason to conclude that the used clinoptilolite sorbent KLS-10-MA is
quite effective in gastric juice medium and could be excellent food additive
for a wide application in mammals, under conditions of Pb intoxication and
perhaps in a wide range of influences of toxic substances. Besides, the
clinoptilolite sorbent exerts a significantly favorable effect on the mammal
organisms. In fact zeolites reveal “magical” features!
Clinoptilolite
substances could be valuable in agriculture and livestock as well as in human
medicine in cases of chronic lead intoxication. The present work gives ground
to create a new effective drug based on the clinoptilolite sorbent for
supplementation of animals in regions that are industrially polluted with heavy
metals and particularly with lead, in order to protect the animals’ health and
the quality of the environment.
ACKNOWLEDGEMENT
The
authors express their most heartfelt thanks to Dr. Tsvetelina Gerasimova from
IBER BAS, Dr. Jose Antonio Heredia Rojas, Dr. Abraham Octavio Rodrigues de la
Fuente and Laura Ernestina Rodrigues Flores from UANL, Mexico for their assistance
in conducting the search.
1.
Dogliotti G, Malavazos AE, Giacometti S, Solimene
U, Fanelli M, et al. (2012) Natural zeolites chabazite/phillipsite/analcime
increase blood levels of antioxidant enzymes. J Clin Biochem Nutr 50: 195-198.
2.
Eyde DT (2016) Zeolites. Min Eng 68: 79-83.
3.
Shariatmadari F (2008) The application of zeolite
in poultry production. World Poultry Sci J 64: 76-84.
4.
Papaioannou D, Katsoulos PD, Panousis N, Karatzias
H (2005) The role of natural and synthetic zeolites as feed additives on the
prevention and/or the treatment of certain farm animal diseases: A review.
Microporous Mesoporous Mater 84: 161-170.
5.
Gorimbo J, Taenzana B, Muleja AA, Kuvarega AT,
Jewell LL (2018) Adsorption of cadmium, nickel and lead ions: Equilibrium,
kinetic and selectivity studies on modified clinoptilolites from the USA and
RSA. Environ Sci Pollut Res Int 25: 30962-30978.
6.
Kanyilmaz M, Tekelioğlu N, Sevgili H, Uysal R,
Aksoy A (2015) Effects of dietary zeolite (clinoptilolite) levels on growth
performance, feed utilization and waste excretions by gilthead sea bream juveniles
(Sparus aurata). Ani Feed Sci Technol 200: 66-75.
7.
Burmańczuk A, Markiewicz W, Burmańczuk A, Kowalski
C, Roliński Z, et al. (2015) Possible use of natural zeolites in animal
production and environment protection. J Element 4:803-811.
8.
Bacakova L, Vandrovcova M, Kopova I, Jirka I (2018)
Applications of zeolites in biotechnology and medicine: A review. Biomate Sci
6: 974-989.
9.
Ham K, Kim BS, Choi KY (2018) Enhanced ammonium
removal efficiency by ion exchange process of synthetic zeolite after Na+ and
heat pretreatment. Water Sci Technol 788: 1417-1425.
10.
Hoff J (2000) Methods of Blood Collection in the
Mouse. Lab. Animal 29: 10-50.
11.
International Commission for Protection against
Environmental Mutagens and Carcinogens (1983) Report of Committee I. Mutation Research
114: 120-177.
12.
World Health Organization (1985) Environmental
Health Criteria 51, Guide to Short-term Tests for Detecting Mutagenic and
Carcinogenic Chemicals. Geneva: WHO, pp: 100-114.
13.
Topashka-Ancheva M, Metcheva R, Teodorova S (2003)
Bioaccumulation and damaging action of polymetal industrial dust on laboratory
mice Mus musculus alba II. Genetic, cell and metabolic disturbances. Environ
Res 92: 152-160.
14.
Topashka-Ancheva M, Metcheva R, Teodorova S (2003)
A comparative analysis of the heavy metal loading of small mammals in different
regions of Bulgaria II. Chromosomal aberrations and blood pathology. Ecotoxicol
Environ Saf 54: 188-193.
15.
Topashka-Ancheva M, Beltcheva M, Metcheva R, Rojas
JA, Rodriguez-De la Fuente AO, et al. (2012) Modified natural clinoptilolite
detoxifies small mammal’s organism loaded with lead II. Genetic, cell and
physiological effects. Biol Trace Elem Res 147: 206-216.
16.
EMFEMA (2005) International Association of the
European Manufacturers of Major, Trace and Specific Feed Minerals Secretariat.
Wetstraat-Rue de la Loi 223/3B-1040 Brussels, Belgium. Available at:
https://www.emfema.org/minerals
17.
Kostov I (1971) Mineralogy Sofia. Mir Publishing
House.
18.
Stylianou MA, Hadjiconstantinou MP, Inglezakis VJ,
Moustakas KG, Loizidou MD (2007) Use of natural clinoptilolite for the removal
of lead, copper and zinc in fixed bed column. J Hazard Mater 143: 575-581.
19.
Orhan Y, Kocaoba S (2007) Adsorption of toxic
metals by natural and modified clinoptilolite. Annali di Chimica 97: 781-790.
20.
Tao YF, Qiu Y, Fang SY, Liu ZY, Wang Y, et al.
(2010) Trapping the lead ion in multi-component aqueous solutions by natural
clinoptilolite. J Hazard Mater 180: 282-288.
21.
Sprynskyy M, Buszewski B, Terzyk AP, Namieśnik J
(2006) Study of the selection mechanism of heavy metal (Pb2+, Cu2+, Ni2+ and
Cd2+) adsorption on clinoptilolite. J Colloid Interface Sci 304: 21-28.
22.
Argun ME (2008) Use of clinoptilolite for the
removal of nickel ions from water: Kinetics and thermodynamics. J Hazard Mater
150: 587-595.
23.
Pavelić K, Hadzija M, Bedrica L, Pavelić J, Dikić
I, et al. (2002) Natural zeolite clinoptilolite: A new adjuvant in anticancer
therapy. J Mol Med 78: 708-720.
24.
Sverko V, Sobočanec S, Balog T, Colić M, Marotti T
(2004) Natural micronized clinoptilolite and clinoptilolite mixtures with
Urtica dioica L. extract as possible antioxidants. Food Technol Biotechnol 42:
189-192.
25.
Ivkovic S, Deutsch U, Silberbach A, Walraph E,
Mannel M (2004) Dietary supplementation with the tribomechanically activated
zeolite clinoptilolite in immunodeficiency: Effects on the immune system. Adv
Ther 21: 135-147.
26.
Jung BG, Toan NT, Cho SJ, Ko JH, Jung YK (2010)
Dietary aluminosilicate supplement enhances immune activity in mice and
reinforces clearance of porcine circovirus type 2 in experimentally infected
pigs. Vet Microbiol 143: 117-125.
27.
Armbruster T (2001) Clinoptilolite-heulandite:
Applications and basic research. Stud Surf Sci Catal: 135.
28.
Djurova E, Stefanova I, Gradev G (1989) Geological,
mineralogical and ion exchange characteristics of zeolite rocks from Bulgaria.
J Radioanal Nucl Ch 130: 425-432.
29.
Ivanova T, Stoyanov I, Stoilov G, Kostov P,
Sapunova S (1997) Natural Zeolites. pp: 3-11.
30.
Rajnov N, Popov N, Yanev Y, Petrova P, Popova T, et
al. (1997) Geological, mineralogical and technological characteristics of
zeolitized (clinoptilolitized) tuffs deposits in the Eastern Rhodopes,
Bulgaria. Natural Zeolites pp: 263-275.
31.
Mumpton FA, Fishman PH (1977) The application of
natural zeolites in animal science and agriculture. J Ani Sci 45: 1188-1203.
32.
Mumpton FA (1985) Using zeolites in agriculture in
innovative biological technologies for lesser developed countries. Washington
DC: Workshop Proceedings, pp: 127-158.
33.
Mumpton FA (1999) La Roca Magica: Uses of natural
zeolites in agriculture and industry. Proc Natl Acad Sci U S A 96: 3463-3470.
34.
Bish DL, Ming DW (2001) Natural zeolites:
Occurrence, properties, applications. Mineralogical Society of America,
Washington DC.
35.
Teodorova S, Metcheva R, Topashka-Ancheva M (2003)
Bioaccumulation and damaging action of polymetal industrial dust on laboratory
mice Mus musculus alba I. Analysis of Zn, Cu, Pb and Cd disposition and
mathematical model of Zn and Cd bioaccumulations. Environ Res 91: 85-94.
36.
Sawicka-Kapusta K, Gorecki A, Swiergosz R, Juszczak
G, Mielczarek M, et al. (1987) Effect of metabolic rate on the rate of
elimination of high and low concentrations of cadmium and lead in the bank
vole. Ecologia Polska 35: 399-430.
37.
O'Flaherty EJ, Inskip MJ, Franklin CA, Durbin PW,
Manton WI, et al. (1998) Evaluation and modification of a physiologically based
model of lead kinetics using data from a sequential isotope study in cynomolgus
monkeys. Toxicol Appl Pharmacol 149: 1-16.
38.
O'Flaherty EJ (1995) Physiologically based models
for bone-seeking elements. V. Lead absorption and disposition in childhood.
Toxicol Appl Pharmacol 131: 297-308.
39.
Johnson FM (1998) The genetic effects of
environmental lead. Mutat Res 410: 123-140.
40.
Nayak BN, Ray M, Persaud TV (1989) Maternal and
fetal chromosomal aberrations in mice following prenatal exposure to
subembryotoxic doses of lead nitrate. Acta Anatomica 135: 185-188.
41.
Hartwig A, Schlepegrell R, Beyersmann D (1990)
Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells.
Mutat Res 241: 75-82.
42.
Demarque D, Jouanny J, Poitevin B, Saint Jean Y
(1995) Pharmacologie et Matiere Medicale Homeopathique. Boiron - C.E.D.H,
France.
43.
Goyer RA (1996) Toxic effects of metals. In
Casarett and Doull’s Toxicology. The Basic Science of Poisons, pp: 691-735.
44.
Mateuca R, Lombaert N, Aka PV, Decordier I,
Kirsch-Volders M (2006) Chromosomal changes: Induction, detection methods and
applicability in human monitoring. Biochimie 88: 1515-1531.
45.
Preston RJ, Dean BJ, Galloway S, Holden H, McFee
AF, et al. (1987) Mammalian in vivo cytogenetic assays. Analysis of chromosome
aberrations in bone marrow cells. Mutat Res 189: 157-165.
46.
Alexandrov L (1971) Regularized computerizing
processes of Newton-Kantarovich type. JVM i MF 2: 31-41.
47.
Alexandrov L, Drenska M, Karadjov D (1984) Program
system REGN for solution of nonlinear system of equations, PRS-165/REGN RSIC.
Oak Ridge, Tennessee.
48.
Shurson GC, Ku PK, Miller ER, Yokoyama MT (1984)
Effects of zeolite A or clinoptilolite in diets of growing swine. J Anim Sci
59: 1536-1545.
49.
Kyriakis SC, Papaioannou DS, Alexopoulos C,
Polizopoulou Z, Tzika ED, et al. (2002) Experimental studies on safety and
efficacy of the dietary use of a clinoptilolite-rich tuff in sows: A review of
recent research in Greece. Microporous Mesoporous Mater 51: 65-74.
50.
Beltcheva M, Metcheva R, Topashka M, Popov N,
Blajev B, et al. (2010) Variant investigation on the influence of clinoptilolite
sorbent KLS-10 MA and lead in conditions of ecotoxicological experiments with
laboratory mice. International Conference of Occurrence, Properties and
Utilization of Natural Zeolite. Sofia, Bulgaria, pp: 45-46.
51.
Beltcheva M, Metcheva R, Topashka-Ancheva M, Popov
N, Teodorova S E, et al. (2014) Zeolites versus Lead Toxicity. J Bioequivalence
Bioavailab 7: 223-240.
52.
Beyersmann D, Hartwig A (2008) Carcinogenic metal
compounds: Recent insight into molecular and cellular mechanisms. Arch Toxicol
82: 493-512.
53.
Cooke JA, Johnson MS (1996) Environmental
contaminants in wildlife: Interpreting tissue concentrations. Lewis Publishers.
54.
Ma WC, Denneman W, Faber J (1991) Hazardous
exposure of ground-living small mammals to cadmium and lead in contaminated
terrestrial ecosystems. Arch Environ Contam Toxicol 20: 266-270.
55.
Ma WC (1996) Environmental contaminants in wildlife
interpreting tissue concentrations. Lewis Publishers, pp: 281-296.
56.
O'Flaherty EJ (1998) A physiologically based
kinetic model for lead in children and adults. Environ Health Perspect 6:
1495-1503.
57.
O'Flaherty EJ (1998) Physiologically based models
of metal kinetics. Crit Rev Toxicol 28: 271-317.
58.
O'Flaherty EJ (2000) Modeling normal aging bone
loss, with consideration of bone loss in osteoporosis. Toxicol Sci 55: 171-188.
59.
Paglia DE, Valentine WN, Dahlgren JG (1975) Effects
of low-level lead exposure on pyrimidine-5-nucleosidase and other erythrocyte
enzymes. J Clin Invest 56: 1164-1169.
60.
Popov N, Popova T, Zlatev AT, Stanchev P, Marinov V
(1992) Ion-exchange and sorption properties of clinoptilolite from Bulgarian
deposits in respect to their fractional and chemical composition. Proceedings
of the Scientific Practical Conference on People Prevention during Disasters
and Accidents 2: 29-30.
61.
Popov N, Jilov G, Popova T (1997) Study of the use
of natural clinoptilolites and their modifications as effective sorbents of Sr
and Cs and heavy metals from water solutions and drinking waters. Fifth
International Conference of Natural Zeolites “Zeolite-97”, September 21-29,
Ischia (Naples), Italy.
62.
Popov N, Popova T, Georgieva L, Tsvetkov TSV,
Balabansky L, et al. (2003) “Clinosorbent-5”A new mineral product as a food
supplement with an anti-radioactive effect in case of emergency and a clinical
estimate of its physiological impact. Proceedings of X Balkan Mineral
Processing Congress, Varna, pp: 615-619.
63.
Roy NK, Rossman TG (1992) Mutagenesis and
co-mutagenesis by lead compounds. Mutat Res 298: 97-103.
64.
Silbergeld EK, Waalkes M, Rice JM (2000) Lead as a
carcinogen: Experimental evidence and mechanisms of action. Am J Ind Med 38:
316-323.
65.
Sindhu RK, Sindhu KK, Roberts CK (2007) Role of
lead in hypertension and chronic kidney disease. Environ Res J 1: 345-359.
66.
Sprynskyy M, Kosobucki P, Kowalkowski T, Buszewski
B (2007) Influence of clinoptilolite rock on chemical speciation of selected
heavy metals in sewage sludge. J Hazard Mater 149: 310-316.
67.
Zlatev AT, Popov N, Stanchev F, Marinov V (1992)
The ability of clinoptilolites from industrial deposits to absorb cesium.
Scientific-Practical Conference in Defense of the Population from Disaster and
Accidents 2: 29-30.
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