zeolites versus lead toxicity jbb.1000209

7/26/2019 Zeolites Versus Lead Toxicity Jbb.1000209

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Volume 7(1): 012-029 (2015) - 12J Bioequiv AvailabISSN: 0975-0851 JBB, an open access journal

Open Access

Beltcheva et al., J Bioequiv Availab 2015, 7:1

http://dx.doi.org/10.4172/jbb.1000209

Research Article Open Access

Bioequivalence & Bioavailability

Zeolites versus Lead ToxicityBeltcheva M1*, Metcheva R1, Topashka-Ancheva M1, Popov N3, Teodorova S2, Heredia-Rojas JA4, Rodrguez-de la Fuente AO4andRodrguez-Flores LE5

1Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1, Tzar Osvoboditel Blvd., 1000 Soa, Bulgaria2Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, 72, Tzarigradsko shaussee Blvd., 1784 Soa, Bulgaria3Mineralagro LTD, 53, Cherni vrah Blvd., 1407 Soa, Bulgaria4Departamento de Ciencias Exactas y Desarrollo Humano, Facultad de Ciencias Biolgicas, Universidad Autnoma de Nuevo Len, Av. Pedro de Alba y Manuel L.

Barragn s/n Cd. Universitaria, C.P. 66451, San Nicols de los Garza, Nuevo Len, Mxico5Departamento de Patologa, Facultad de Medicina, Universidad Autnoma de Nuevo Len, Av. Francisco I. Madero pte. s/n y Av Gonzalitos, Col. Mitras Centro. C.P.

66460, Monterrey, Nuevo Len, Mxico.

Keywords: Bioaccumulation; Intoxication; Clinoptilolite sorbents

Introduction

Zeolites-natural and modified because o their specific structure,are excellent adsorber and thus can diminish the harmul effect oheavy metals. Clinoptilolites, due to its structural stability under hightemperatures and acidity, are the most widely used zeolites in animalstudies. Te important research data are presented, indicating thepositive influence o the in-eed inclusion o clinoptilolite on animalhealth.

Ecotoxicological experiment with laboratory inbred ICR line micewas carried out, covering 90 days. Te mice were chronically exposedto lead (Pb) in the orm o aqueous solution o Pb(NO

3)

2, diluted in

the drinking water, and treated with modified clinoptilolite sorbentKLS-10-MA. Tey were allocated into our groups: group 1, (control):animals ed with conventional ood or small rodents and water; group2: animals ed with conventional ood + clinosorbent KLS-10-MAand water; group 3: animals ed with conventional ood and water +Pb(NO

3)

2; and group 4: animals ed with conventional ood + KLS-10-

MA and water + Pb(NO3)

2. Group 2 was a second control in order to

prove eventual toxicity o the sorbent and influence o clinoptiloliteon growth perormance. o assess the detoxification effect o theclinoptilolite sorbent, the ollowing bioindicator characteristics

were chosen: Pb concentrations in carcass, organs, tissues, and eces,chromosomal aberrations requency, mitotic index, erythrocytemorphology, erythropoesis, and body weight gain o the mice.

Te modified clinoptilolite KLS-10-MA (water modification) wasprepared on the base o natural Bulgarian clinoptilolite rom the depositin East Rhodops, South Bulgaria. As a powder, it was mechanicallymixed at 12.5% concentration with the conventional orage or smallrodents and was administrated as eed supplement or the mice romgroups 2 and 4. A significant reduction o Pb bioaccumulation in theexposed and supplemented mice (group 4) was observed: the Pb levelsin the mice rom group 4 were 84%, 89%, 91%, 77%, and 88% loweror carcass, liver, kidney, bones, and eces, respectively, comparedto the Pb levels in the mice rom group 3. Essential improvement

o the cytogenetical, hematological, and physiological parameterswas noted. On day 90 the ollowing relations were calculated: Pb-exposed and clinoptilolite-supplemented mice exhibited 2.3-old lowerchromosome aberrations requency, 2.5-old higher mitotic index, 1.5-

Abstract

The workers in metal industry as well as the people inhabiting industrially polluted regions are Health hazardgroups regarding the development of chronic heavy metal intoxication. Different means could be used to correct, atleast partly, the consequences of such intoxication. However, the best substances for treatment are those that can

prevent the metals entering in the blood. In case, metals enter in the organism through the digestive tract, zeolitesare the most suitable means for trapping of metal ions. The substantial role of the clinoptilolite as a factor essentiallyreducing Pb bioaccumulation is considered in an experiment with small mammals chronically exposed to lead.

As a feed additive, clinoptilolites have been used so far in poultry and livestock to positively inuence fecesconsistency, reduce diarrhea, bound mycotoxins and aatoxins, and allow better performance of intestinal microora.The present work is the rst study of the effect of clinoptilolite, used as a food supplement, in conditions of Pbintoxication.

Modied clinoptilolite KLS-10-MA was prepared and applied as food-additive in laboratory inbred ICR line mice,chosen as experimental animals. In the experiment the degree of the positive effect of this sorbent in the reduction ofPb bioaccumulation was explored. Evidences that clinoptilolite is practically non-toxic substance were presented. Amathematical model of Pb bioaccumulation in exposed and exposed-supplemented animals was proposed.

Such investigations are important for human and veterinary medicine, pharmacy and for the explanation of somebiological and chemical problems. The authors hope that the obtained results could help in further efforts to createdrugs based on clinoptilolite sorbents. An application of such drugs could be of great importance for human and

animals in regions that are industrially polluted with heavy metals, and particularly with Pb, in order to protect theorganisms as well as the quality of the environment.

*Corresponding author:Beltcheva M, Institute of Biodiversity and EcosystemResearch, Bulgarian Academy of Sciences, 1, Tzar Osvoboditel Blvd., 1000 Soa,Bulgaria, Tel: 35929883709; E-mail: [email protected]

Received November 10, 2014; Accepted November 29, 2014; PublishedDecember 02, 2014

Citation:Beltcheva M, Metcheva R, Topashka-Ancheva M, Popov N, TeodorovaS, et al. (2015) Zeolites versus Lead Toxicity. J Bioequiv Availab 7: 012-029.doi:10.4172/jbb.1000209

Copyright: 2015 Beltcheva M, et al.This is an open-access article distributedunder the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided theoriginal author and source are credited.
http://dx.doi.org/10.4172/jbb.1000209http://dx.doi.org/10.4172/jbb.1000209


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Citation: Beltcheva M, Metcheva R, Topashka-Ancheva M, Popov N, Teodorova S, et al.(2015) Zeolites versus Lead Toxicity. J Bioequiv Availab 7:012-029. doi:10.4172/jbb.1000209

J Bioequiv Availab

ISSN: 0975-0851 JBB, an open access journalVolume 7(1): 012-029 (2015) - 13

old higher percentage normal erythrocytes, and 1.3-old higher bodyweight compared to Pb-exposed and unsupplemented animals.

Te obtained results support the suggestion o other authors thatclinoptilolite is quite effective in gastric juice medium and could besuitable or wide application in mammals under conditions o Pbpoisoning.

No data were ound about applications o zeolites as eed additivein animals exposed to heavy metals. Te proposed exploratory researchabout detoxification capacity o the clinoptilolite is the first step in thisdirection.

Adequate mathematical model was constructed to explain themain regularities in Pb bioaccumulation in the bones. Based on theexperimental data the kinetic parameters o the Pb accumulationand lead excretion in mouses organism were fitted. A value ogastrointestinal Pb resorption coefficient in KLS-10-MA-supplemented

mice = 3.53% was calculated versus normal value = 15%.

Te results o the presented study encourage the urther efforts orthe finding o reliable drug based on the tested clinoptilolite substancein cases o chronic lead intoxication in animals and human.

Heavy metals intoxication is a serious social problem. It directlyaffects humans or animals health and due to the withdrawal o illpersons rom the active work in companies or processing o nonerrousmetals also the production affects. Heavy metals in the orm o aerosolsand dust, especially in industrial areas all into the water, soil, air, andplants. It creates unavorable environmental situation.

In order to minimize risks to lie, health and working capacity o thepopulation, it is necessary to find methods o reducing harmul effect

o heavy metals. Such agents may be sought in three major directions:

1) Decreasing the harmul emissions rom the actories by usingspecial purification filters.

2) Use o appropriate materials with specific properties andstrongly expressed absorptive capacity to cleanse indoor air pollutionin industrial companies, wastewater treatment, soil and ertilizers.

3) Use o suitable substances with strongly expressed absorptivecapacity or internal application in humans and animals in order toneutralize heavy metals that ell within the organism to minimizingtheir resorption rom the gastrointestinal tract.

One o the best means suitable in cases 2) and 3) are the zeolites.Series o ecotoxicological experiments and careul studies haveconfirmed their high efficacy as adsorbers o waste products [1-3].Clinoptilolites are ofen utilized to draw heavy metals rom solutions[4-6] and to puriy deleterious emissions [7]. Diets manipulated byzeolites have the potentiality to reduce both the excess o N and P inswine manure, and to minimize the negative effects o odor and othergaseous emissions such as NH

3 and H

2S rom the swine waste [8,9].

As a eed additive, clinoptilolites have been used so ar in poultry andlivestock in order to positively influence eces consistency, reducediarrhea, bound mycotoxins and aflatoxins, allow better perormanceo intestinal microflora [10]. No data were ound about applicationso zeolites as eed additive in animals exposed to heavy metals. Tisexploratory research was the first step in this direction. Clinoptilolitewas used as ood supplement in laboratory mice, in conditions o

chronic Pb-exposure. An effect o essentially diminished Pb toxicity inexperimental animals was established.

Lead is one o the most popular environmental pollutants resulting

rom anthropogenic activities. It predominates as a toxic agent inindustry. Lead along with cadmium and mercury is the most toxicand highly deleterious heavy metal. It causes alterations in growth

and behavior, renal unction deficits, hypertension, osteoporosis, lead-induced anemia, etc. in human and animal organisms. [11-13]. Leadis also a significant genotoxic agent [14-16]. Tereore, it is o greatimportance to take pains or possible reduction o lead toxicity.

Clinoptilolites are minerals with specific crystalline structure, whichmakes them a perect heavy metals trap [17-19]. Besides, they are themost abundant natural zeolites, occurring in volcanic and sedimentaryrocks [1,7] and this act acilitates their use [20] established thatclinoptilolite retains its structural stability under high temperaturesand acidity. According to there experiments clinoptilolite exhibitedthe highest capability in adsorbing Pb2+ ion in complex solution withpH value o 1.2, at 37 oC, achieving the capacity o 7 mg/g, two timesmore than that by other zeolites and six times over that by activated

carbon. Te authors suggest that a significant part o lead (Pb2+)intakeis possible to be neutralized in the stomach by a clinoptilolite sorbent.Tus, clinoptilolite, which reveal a unique selective adsorption, couldbe considered as a reliable means to diminish Pb intoxication.

When clinoptilolite is applied, as a ood supplement, the process oions exchange occurs in the gastrointestinal tract and thus the resorptiono toxic metals through the intestinal mucosa could be prevented in agreat extent. Te present inormation is targeted to explore the degreeo the positive effect o a modified clinoptilolite sorbent KLS-10-MA(water modification). Te sorbent was administrated as eed-additivein laboratory inbred ICR line mice in condition o chronic poisoningby Pb. Clinoptilolite is practically a non-toxic substance [21]. Tisstatement was confirmed also by present results.

Laboratory inbred ICR line mice, were chosen as experimentalanimals. Small mammals, and especially rodents, are preerred asbioindicators in ecotoxicological experiments due to their basicposition in ood chain, ast reaching o maturity, high total metabolismand specific biological reactions (substantial increase o chromosomeaberrations requency, changes o hematological indices etc.).

In the present study bioaccumulation o Pb in carcass, organsand tissues, as well as chromosomal aberrations and mitotic index,erythrocyte morphology and prolieration, and body weight gain o themice were chosen as suitable bioindicator characteristics to assess thedetoxification effect o the modified clinoptilolite sorbent KLS-10-MAadministrated as a eed supplement in the mice chronically exposed

to Pb.Te mathematical model o Pb bioaccumulation in bones, in

exposed-unsupplemented and exposed-supplemented mice, alloweddetermining the coefficient o absorption o Pb by gastrointestinalmucosa in the supplemented mice and some kinetic parameters othe dynamics o Pb bioaccumulation both in unsupplemented andsupplemented animals. Te quantitative investigations o the impact oKLS-10-MA on cell and physiological parameters during a chronic Pbexposure o small mammals could help in urther explorations to usethis sorbent against chronic metal poisoning.

Other aspect is also in the scope. Many authors report the positiverole o zeolites in livestock breading [22-26]. Tus, one o the aimso the present work is to prove whether a significant change in body

weight would be observed in mice supplemented with clinoptilolite.For this reason, and in order to examine the reaction o mammalsorganism to the sorbent, an additional control was used. Tese were


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healthy animals non-exposed to Pb and ed with conventional oragemixed with 12.5% KLS-10-MA.

Zeolite structure and the use of zeolites as excellent adsorberof waste products and heavy metals

Zeolites are an excellent trapper o waste products and heavymetals because o their chemical composition and specific latticestructure. Tese minerals are crystalline, hydrated aluminosilicates oalkali and alkaline earth cations (Na, K and/or Ca cations). Zeoliteshave an infinite, open structure [2]. Tey consist o threedimensionalrameworks o SiO

44 and AlO

45 tetrahedra linked through the shared

oxygen atoms. Both natural and synthetic zeolites are porous materials,able to adsorb molecules o appropriate cross-sectional diameter andto exchange their constituent cations without major change o theirstructure. Tus, zeolites appear to posses two important properties:adsorption property and ion-exchange property [17]. Te exploitation

o these properties underlies the use o zeolites in a wide range oindustrial and agricultural applications and particularly in animalnutrition since mid-1960s [2].

Te most important o zeolites are: analcime, chabazite,clinoptilolite, erionite, aujasite, errierite, heulandite, laumonitte,mordenite, phillipsite.

With large size deposits and wide geographic distribution,clinoptilolites are considered as most abundant members o the 48minerals in the zeolite group [18,27]. Clinoptilolite usually occursin volcanic and sedimentary rocks [1,7]. Due to its abundance, widedissemination and ion exchange characteristics, clinoptilolite mineralsare among the most ofen used zeolites.

As zeolite, clinoptilolite is crystalline, hydrated aluminosilicateso alkali and alkaline earth cations, consisting o three-dimensionalrameworks o SiO

44and AlO

45tetrahedra linked through the shared

oxygen atoms. Te molar Si/Al ratio in clinoptilolites is above 4 [28,29].Clinoptilolites have a relatively open structure with a total pore volumeo approximately 35% [30], and chemical ormula (K, Na, Ca, Mg).(AlSi

43O

8).5H

2O [2]. Te structure o clinoptilolites is characterized by

large intersecting open channels o 10- and 8-member tetrahedral rings[20]. Such structure ensures the clinoptilolite capacity o to absorb andaccumulate heavy metals. Clinoptilolites are ofen utilized to reducethem rom solutions [3,4,6,31].

Why are clinoptilolites a perect heavy metal-trap? Te Si-blockis neutral, while the Al-block in crystalline unit is negative, thus it

charges the minerals lattice negatively. Te existence o Na, K and/orCa cations determines the neutrality o the minerals. Tese cations areexchanged in solutions with cations o certain metals, such as Pb, Cd,Hg, etc [18,19].

In order to meet increasingly stringent Environmental QualityCriteria (EQC) clinoptilolites are widely applied or heavy metalsremoving rom polluted streams and wastewater [4,19,32-37] andor the elimination o gas pollutants in confinement acilities [7,10].Besides, this mineral has widespread applications in agriculture [1,2].According to [38], zeolites also allow better perormance o intestinalmicroflora.

Te effects o clinoptilolites in animals appear to be related totheir high cation-exchange capacity, which affects tissue uptake and

utilization o NH4+, Pb2+, Cd2+, Cu2+, Cs+, and other cations [6,19].Clinoptilolites have shown antioxydative and anticancer effects [39-41]. Tey support the immune activity [20,42,43] note that clinoptilolite

with low toxicity and high adsorption capacity toward Pb2+ in a strongacid solution has a potentiality or application in the lie sciences.Clinoptilolites appears to be stable in the gastrointestinal tract [44], and

as unique selective adsorbers, they could adsorb toxins, heavy metals,and ree radicals rom the body and excretes [17,45]. It is believed thatthe high affinity o clinoptilolite to Pb would significantly reduce theamount o dietary Pb available or absorption by the intestinal mucosa[20]. Present results confirmed this suggestion. Te lead ions could betrapped by clinoptilolites in the stomach because the obtained Pbconcentrations in exposed and clinoptilolite-supplemented animalswere much lower compared with those in exposed unsupplementedones. Te sorbent KLS-10-MA (water modification) appeared as highreliable means or detoxification o animal organisms chronicallypoisoned by lead.

Preparation of modified natural clinoptilolite for theexperiment

In Bulgaria, clinoptilolite are o most widespread distribution in thenorth-eastern Rhodopa Mountain. For the preparation o the modifiedclinoptilolite KLS-10-MA (water modification) natural clinoptilolite

rom the deposit in the village Golobradovo was used. Golobradovois situated in the region o East Rhodops, South Bulgaria (Figure 1).Te chemical compositions o this clinoptilolite and the modifiedclinoptilolite KLS-10-MA are presented in able 1. Te values oexchangeable alkaline and alkaline earth cations are displayed in able2.

Te modified clinoptilolite was prepared through a treatment onatural clinoptilolite (zeolite containing 82% clinoptilolite). Te naturalclinoptilolite was heat-treated at 240-250oC and then chemically-

mechanically activated with 10% alkaline salt, addition o 25-weight %distilled water, and 4 hours processing in ball-crusher (wet activation).Chemical composition o the clinoptilolite sorbent KLS10-MA wasdetermined by common analytical method or silicate materials.Cations exchange capacity was determined according to the methodo [46].

Clinoptilolites o high quality occur in Bulgaria [47-49]. able 1shows that the molar ratio Si/Al was 5.97 in natural clinoptilolite and6.22 in modified KLS-10-MA based on this clinoptilolite. A rather

Figure 1: A map of the distribution of natural clinoptilolites in Bulgaria. Thearrow indicates Golobradovo deposit.


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minor ratio (6.02) has the purified natural clinoptilolite used or thetreatment o diarrhea in the orm o the drug Enterex, approved bythe [48]. A value o 5.98 has the Greek natural clinoptilolite successullyused or treatment o solutions containing Pb2+, Cu2+and Zn2+[18].

Te ratio SiO2/Al

2O

3 in zeolites is a marker o great importance

to determine their acid stability. A greater SiO2/Al

2O

3ratio correlates

with higher acid stability [45]. Tus, the specific structure and highSiO

2/Al

2O

3 ratio in clinoptilolite makes it the preerable sorbent in

different acid solutions, including gastric juice [20] investigatingNa-clinoptilolite established that it maintained structure stability insolution with pH value o 1.2 [20], while [50] indicated a prematurecollapse o zeolites NaY and NaA in such strong acid solution [5].

Te most important eature o zeolites, which determines theirwide useulness, is their cation exchangeability [5]. Te aluminum ionis small enough to occupy the position in the center o the tetrahedrono our oxygen atoms, and the isomorphous replacement o Al 3+ orSi4+raises a negative charge in the lattice [19]. Te negatively charged

ramework counter-balanced by positive cations (Na, K and Ca), resultsin a strong electrostatic field on the internal surace. Tese cations can beexchanged to fine-tune the pore size or the adsorption characteristics.All clinoptilolite modifications are based on these principles. Te poresize has an essential unction in the Na+, K+, or Ca++

exchange withcertain cations such as Pb2+, Cd2+, Zn2+etc. in solutions.

Regarding the selectivity o some metal and other cations towardsthe Bulgarian natural clinoptilolite, the ollowing order has beenestablished [47]:

Cs+> K+> NH4

+> Ba2+ > Pb2+> Sr2+ > Se2+ > Na+> Ca2+> Fe3+ > Al3+

> Hg2+ > Mg2+> Li+

Te above relations show that practically, Pb occupies the first

position among the metals encountered in industry and thereore, inthe environment. For this reason the main use o clinoptilolites ormetal adsorption deals on the base o Pb.

Te sorbent KLS-10-MA used in the present experiment is aNa-enriched one [20] established that the structure variations oNa-clinoptilolite in the acidic solution or 24 h would have a minor

influence [20]. Te cation exchange capacity o KLS-10-MA was almost1.4-old higher compared to that o the natural clinoptilolite (able 2).Te exchangeable sodium cations in KLS-10-MA were 5.7-old moreand the total exchangeable cations were 1.9-old more than those inthe natural matter. Results o several authors indicate that Na-enrichedorm o modified clinoptilolite has highest static ion exchange abilitytowards Pb2+, Cd2+, NH

4+etc. [20,49-51]. Tus, the modification KLS-

10-MA could be considered as a successul sorbent or detoxificationpurposes.

Experimental Setting

Animals and treatment

Te eco-toxicological experiment was conducted according to

approved protocols and in compliance with the requirements o theEuropean Convention or the Protection o Vertebrate AnimalsUsed or Experimental and Other Specific Purposes and the currentBulgarian laws and regulations.

Experimental animals were bread in vivarium and housed inindividually ventilated cages. Te physical size o the cages was inaccordance with European standards. Te bedding material wasobtained rom an ISO-2000 accredited supplier. Mice were acclimatizedor a 7 days period beore starting the experiment. A standardtemperature o between 19C and 23C, a humidity o 4560% and a12-hour light/night cycle were kept all the time. Te ood was in theorm o pellets and not withheld at any time during the experiment.All mice were allowed access to ood and water ad libitum. Te water,

ood and bedding material were daily inspected and changed whennecessary. Te animals were neither medicated nor vaccinated.

Te experiment covered 90 days. Only males, about 68 weekso age, laboratory mice, inbred ICR strain, were used. Te exposureto Pb was perormed as the mice were treated with 0.05 N solutiono lead nitrate diluted 1:10 in the drinking water during all theinvestigating period. Te clinosorbent KLS-10-MA in orm o powderwas mechanically mixed to a 12.5% concentration with conventionalgranulated orage or small rodents.

Animals were arranged in our groups each o 60 specimens,as ollows: group 1, (control) animals ed with conventional oodor small rodents and water; group 2 animals ed with conventional

ood + clinosorbent KLS-10-MA and water; group 3 animals ed withconventional ood and water + Pb(NO3)

2; group 4 animals ed with

conventional ood + KLS-10-MA and water + Pb(NO3)

2.

wo variants o the eed mixture were prepared: (1) standard(conventional ood) and 2) standard ood treated with 12.5% sorbentKLS-10-MA. Te elements composition in the variants 1 and 2 arepresented in able 3.

Te concentrations o Pb in the whole body, liver, kidney,bones, and eces o the control and experimental animals, as well ascytogenetical and hematological parameters were determined on days15, 45, 60, and 90 rom the beginning o the experiment.

Elements content analysis

o determine the Pb concentration, afer the removal o thealimentary tract, the tissues and some internal organs were oven dried at600C to a constant weight. Te dried tissues were dissolved in a mixture

Clinoptilolite chemical composition %

Constituent Natural KLS-10-MA

SiO2

69.3 66.09

Al23 11.6 10.62

Fe2O

31.7 0.76

TiO2

0.1 0.12

CaO 2.11 3.03

MgO 2.37 0.33

Na2O 0.98 4.49

K2O 1.59 3.11

I. L. 10.32 11.37

Molar Si/l 5.97 6.22

Table 1:Chemical composition (%) of clinoptilolite samples: natural (Golobradovo,South-East Rhodops Mountain, Bulgaria) and modied (KLS-10-MA).

Samples Natural KLS-10-

Cation exchange capacity 102 139.5

Exchangeable cations

Na+ 23.34 133.14

K+ 19.14 36.08

Ca+ 61.22 24.68

Mg+ 1.05 2.41

Total 104.75 196.31

Table 2:Cation exchange capacity (meq/100g, NH4+) and exchangeable cations

(meq/100g) of clinoptilolite samples from Golobradovo, South-East RhodopsMountain, Bulgaria (natural and modied).


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o concentrated nitricperhloric acid (4:1) [52]. Te concentrationso Pb and the element composition o the two eed variants weredetermined in a certified laboratory by atomic emission spectrometrywith inductively coupled plasma (ICP AES) on a GFAASVarianinstrument.Te detection limits were 0.002 mg/lor Mn; 0.004 mg/l

or Cd; 0.005 mg/lor Zn; 0.03 mg/lor Pb; 0.04 mg/lor Fe; 0.5 mg/lorCa, K, Mg, and Na.

Te statistical analysis was done using the SPSS Package orWindows, version 15.0. Differences were considered to be significantwhenpvalues were lower than 0.05 (p < 0.05). Firstly, the data were

processed according to Kolmogorov-Smirnov test or normality ineach group. All groups showed normal distributions. Secondly, thedata were analyzed by analysis o variance and subsequent ukey highstatistical difference test and Dunnet test, or estimating individualdifferences.

Cytogenetical and hematological analyses

Percentages o aberrant mitoses, mitotic indices, and blood sampleswere determined on days 15, 45, 60 and 90 o exposure. At each timepoint, a subset o eight mice rom the our groups was used.

Te cytogenetical analysis was perormed as described by [53].Mitomycin C (3.5 mg/kg) (Fluka) was used as a positive control. Teother animals were injected with only 0.2 ml 0.9% NaCl.

o perorm bone marrow chromosomal aberration assays, theanimals were injected ip with colchicine at a dose o 40 mg/kg, 1 hbeore isolation o bone marrow cells. Te bone marrow cells wereflushed rom emur with 0.075 M KCl and hypotonized at 37C or20 min. Tereafer, the cells were fixed in methanol-acetic acid (3:1),dropped onto cold slides, and air dried. o examine the chromosomalaberrations, the slides were stained with 5% Giemsa solution (SigmaDiagnostic). At least 50 well-spread metaphases were analyzed peranimal at random.

Te mitotic indices were determined by counting the numbero dividing cells among 1500 cells per animal. Te requencies oabnormalities and the mitotic index were determined or each animal.Te mean SD or each group was calculated, and the data were

statistically evaluated or their significance by analysis o variance usingStudent ttest.

Te hematological analysis was carried out on the same groupso animals using standard clinical methods. Peripheral blood sampleswere collected between 9 and 11 AM rom the orbital sinus [54]. Tepercentages various orm o cells were determined using Giemsa stains.About 150200 cells were counted in each stain.

For the statistical analysis, calculation o the mean SD or eachgroup was perormed, and the data were evaluated or their significanceby analysis o variance using Student ttest.

Mathematical model for lead bioaccumulation in mices bone

Te problems o bioaccumulation o heavy metals and toxic

elements are closely connected to the health and need quantitativeconsideration. A mathematical model or the kinetics o leadaccumulation in the mices bone in conditions without treatment and

treatment with clinoptilolite sorbent is elaborated in the present study.Te model helps calculate some kinetic parameters characterizing thePb bioaccumulation status and the extent o the sorbent effect.

Tere are not enough studies that suggest quantitative approach via mathematical modeling o the processes o metals bioaccumulationin living organisms. Physiologically based kinetic models (PBKM)or arsenic, chromium, mercury and lead have been proposed withany degree o completeness [12]. A simple mathematical model orPb concentration in the body o mice, ed with a contaminated diet,was proposed rom [55]. Series o PBKM or bone seeking elements

[12,56-60], have developed. Distribution and bioavailability o lead,chromium and uranium have been considered [61]. For the first time amathematical model, describing the time courses o cadmium and zincconcentrations in liver and kidney o laboratory mice Mus musculusalba was proposed [62]. It was constructed taking into accountthat these metals induct metallothionein synthesis. Te model wellexplained the observed peculiarities o Cd bioaccumulation pattern inliver and kidney in conditions o very high exposure to heavy metalmixture.

Te present mathematical model is confined to Pb bioaccumulationin mouses bones. Tree compartments o Pb movement areconsidered: gastrointestinal tract, blood and bones (Figure 2). Onecan assume that Pb is distributed evenly into compartments, whichallows the use o differential equation or its kinetics. Afer entering in

gastrointestinal tract, Pb moves to blood and then to bones. Tus, theollowing system o ordinary differential equations takes place:

1 2

dxa x a x

dt= (1)

1 3 4

dya x a y a y

dt= (2)

3

dza y

dt= (3)

under initial conditions:

0 0 0 0 0 00, ,( ) ( ) (, )0t x t x A y t z t z = = = = = (4)where t is time [days]; x, y, and z are the concentrations [mg/

kg] o Pb at a given time tin the gastrointestinal tract, the blood, andthe bones, respectively; t

0is the moment when the experiment starts;

a1 ([a

1] = [day-1]) and a

3 ([a

3] = [day-1]) are the rate constants o Pb

accumulation in blood and bones, respectively; a2([a

2] = [day-1]) and

a4 ([a

4] = [day-1]) are the rate constants o Pb excretion through the

eces and the urine, respectively; dx/dt, dy/dt,and dz/dtare the rateso the change in Pb levels with the time in the three compartments,respectively.

In Chapter 9 the results rom this mathematical model, applied tothe dynamics o Pb bioaccumulation in mouses bones, are presented,compared with the experimental data, and discussed.

Lead toxic effects on animals and humans

Te distribution, accumulation and transer o heavy metals,

Elements Na K Ca Mg Fe Zn Cu Mn Pb

Standard food 885 45 8164 245 7575 227 2319 92 412.5 78 76.99 15.4 20.9 1.4 101.9 10.2 2.25 0.3

Food + 12.5%

KLS-10-MA 1924 96 9397 282 8415 252 2205 88 378.6 72 75.22 14.9 15.4 0.9 121.2 11.9 2.76 0.3

Table 3:Quantities of some elements (mg/kg) in the two variants feed mixture of ICR mice.


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especially zinc, copper, lead, and cadmium in living organisms have

been studied in detail [63] established that zinc does not accumulatewith continued exposure, because body content is modulated byhomeostatic mechanisms. Copper distribution has been estimated inhuman tissues [64], and transport mechanisms and serum levels ocopper have been well studied [65]. Te main storage sites o cadmiumare liver and kidney [11,66-69]. Cadmium accumulation, as well asthe decreasing cadmium kidney/liver ratio as the total body burden ocadmium increases, has been studied [62,66-70]. Te lead absorptionin mammalian organism is affected by the specific exposure pathwayand dietary actors; thus, the absorption rates o lead in mammals may

vary rom 10 to 50% or inhalation and rom 2 to 20% or ingestion[71]. Te ollowing order o Pb bioaccumulation was established inmammals: bone > kidneys > liver > brain > muscle [71,72]. Kidneys areknown as the usually way or Pb excretion [73].

Te toxic action o heavy metals has also been studied. Mechanismso lead toxicity have been studied at the cellular and subcellularlevels [74,75] evaluated the carcinogenic activity o cadmium [73]have published on the distribution, lethal doses, toxicokinetics, andtoxicity mechanism o metals and other inorganic compounds. Also,[11] has presented a detailed view on the toxicokinetics, toxicity andpathological effects o the metals.

Lead, as ubiquitous environmental pollutant, is o particularinterest as a toxicant and a contaminant. It is widely distributed andrepresents a significant toxicological and ecotoxicological risk actor.Its distribution is closely connected with human activities. Becauseo that chronic Pb intoxication in workers engaged in the respective

activities is a serious health and social problem.Lead has long been recognized as a potential hazard to human

health [74,76-78]. A significant ecotoxicological risk or a wildpopulation, associated with high Pb tissues concentration has beenestimated [79]. Lead affects a wide range o physiological systemsand organs, including the central and peripheral nervous system, thecardiovascular system etc [71]. Lead produces an excessive amount oreactive oxygen species resulting in oxidative stress, thereby resultingin hypertension [13] and chronic kidney disease [80]. Lead has alsomultiple hematological effects as well as genotoxic effects [76].

Lead encephalopathy may occur in children with high exposureto Pb. Symptoms o Pb encephalopathy begin with lethargy, vomiting,irritability, loss o appetite, and dizziness, progressing to obvious

ataxia, a reduced level o consciousness which may progress to comaand death. Peripheral neuropathy is a classic maniestation o Pbtoxicity, particularly the ootdrop and wristdrop that characterizes the

house painter and other workers with excessive occupational exposureto Pb [11].

Lead may affect blood pressure by altering sensitivity o vascularsmooth muscle to vasoactive stimuli via the ollowing mechanism:Pb alters calcium-activated unctions o vascular smooth muscle cellsincluding contractility by decreasing Na+/K+-APase activity andstimulation o the Na/Ca pump. Lead acts also indirectly by alteringneuroendocrine input to vascular smooth muscle [11].

Lead has a harmul effect on bones. It is known that the total bodyburden o lead is divided into two pools, which have different rateso turnover. Te largest and kinetically slowest pool is the skeleton,with a hal-lie o more than 20 years, and a much more labile (about20 days) is the sof tissue pool [11,73]. Lead occurs in bone in muchhigher concentration than in other organs [60,72,73,81] because it is astrongly bone-seeking element [82,83]. Our data support this concept[62]. Well-defined age dependence was ound among total bone leadturnover, bone ormation rate, and Pb accumulation in bone [59].[84] note that hair and epidermal compartments (nails etc.) can alsoaccumulate Pb. Much higher concentrations o lead have been observedin the shells than in the sof tissues o some marine shellfish andcrustaceans [85]. Obviously, these acts are connected to the behavioro lead as bone-seeking (or more generally, as calcium-ormations-seeking) element. Te accumulation o lead in metabolically inactivetissues, mainly in bone, may be considered to a certain extent aprotective mechanism or organisms. But OFlaherty and coauthorsshowed that lead sequestered in bone can become bioavailable insome conditions, related to physiological states or aging [12,86]. Asubstantial transplacental transer o endogenous Pb rom maternalbone during pregnancy was observed in primates [86] modeled the

process o osteoporotic bone loss in aging as a result rom the return obiologically significant amounts o Pb to the blood. In agreement withexperimental observations, an increase o blood Pb, previously storedin bone, was modeled afer menopause. Te model predicts a blood Pbconcentration o 17.6 g/dl in a 50-year-old US woman and 18.5 g/dlin a 60-year-old US woman in the period 1976-1978. Lead adverselyinfluences bone development through disruption o mineralizationduring growth [82]. Te trends or the accumulation o Pb in corticalbone and the release o Pb rom bone stores are well predicted by theOFlaherty model o Pb kinetics and model results are consistent withthe hypothesis that a polymorphism in the delta-aminolevulinatedehydratase enzyme modifies the kinetics o Pb in humans [87].

Lead nephropathy is one o the oldest recognized health effectso Pb. Lead nephropathy appears as acute (reversible) or chronic(reversible) [11].

Acute Pb nephropathy is limited to unctional and morphologicchanges in proximal tubular cells. Te unctional changes arethought to be related to a Pb effect on mitochondrial respiration andphosphorilation. A characteristic microscopic change is the ormationo a lead-protein complex, which appears in renal tubular cells asinclusion bodies. Formation o such a complex was also ound tooccur in renal tubular cells by [88,89]. Also, the primary renal toxicityo cadmium affects proximal renal tubular unction and is maniestedby increased Cd in the urine. It was supposed [62] that the increasedurinary leak (the polyuric stage o nephropathy [90] is the reason orthe essential change o the pattern o the Cd kidney/liver ratio (Cd

kidney/

Cdliver

). Tere is a strong relation between the intake o Cd in the diet

and the levels o this element in the kidney and liver, both respondingby accumulating and storing the cadmium [91]. Usually in human Cdconcentration in kidney is greater than in liver. Cd

kidney/Cd

liver is well

Gastrointestinal GIT Blood Bones

Tract (GIT) Mucosa

x(t) y(t) z(t)

a1 a3

GI Pb Pb Blood Pb Bone Pb

a2 a4

Feces Urine

Figure 2: Scheme of the compartments, pathways, and rate constants. x(t),y(t),and z(t) are the Pb concentrations [mg/kg] (varied with the time t) in thegastrointestinal tract, blood, and bones, respectively.


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above one (about 5) [11]. Te same is valid also or rodents [68,91].However, an essential decrease, even to inversion (well below one) inthis ratio has been observed in the conditions o very high Cd exposure

[62,91]. Tus, the ratio Cdkidney/Cdliverdecreases with the increase o Cdcontamination degree.

Lead may produce a chronic intersticial nephropathy, mostcommonly with blood Pb levels greater than 60 g/dL [92]. However,depressed glomerular filtration rates have been reported in a groupo lead-exposed workers whose blood Pb levels were as low as 40 g/dL [92]. Te risk o death rom renal disease increases with increasingduration o employment [92]. It was reported that o 4519 battery plantworkers and 2300 lead production workers there was excess mortalityrom chronic nephritis [92] have established that Pb nephropathy,characterized unctionally by depression o effective renal plasma flow,glomerular filtration rate, and maximum glucose reabsorption rate,is associated with prolonged occupational exposure to Pb. Tere is a

relationship between chronic Pb exposure and gouty nephropathy.Gout patients with renal disease have a greater chelate-provoked Pbexcretion then do renal patients without gout. Lead reduces uric acidexcretion. Elevated blood uric acid has been demonstrated in rats withchronic Pb nephropathy [93].

Severe Pb-exposure effects on health are typically related alsoto blood pathology. Reticulocytosis, microcytosis, hypochromy,basophilic erythrocytic granulation and hemoglobin decrease indicate alead-induced anemia, a well-known symptom o chronic Pb poisoning.More than 90% o Pb in blood is in red blood cells (RBC) and there areat least two major compartments or Pb in RBC: one associated with themembrane and the other with hemoglobin [11]. Te anemia is causedby two basic deects: shortened erythrocyte liespan and impairment o

heme synthesis. Shortened liespan o the RBC is thought to be due toincreased mechanical ragility o the cell membrane [11]. Te shortenedliespan o RBC results in reticulocyte production as a compensatoryreaction. Te basophilic stippling occurred in the reticulocytes resultsrom inhibition o the enzyme pyrimidine-5-nucleosidase (Py-5-N)[94]. Tere is an inverse relationship between Py-5-N inhibition andblood Pb concentration. Te depression o the heme synthesis is dueto the inhibition o SH-containing enzymes by Pb [73]. Besides, ironin the orm o apoerritin and erruginous micelles may accumulatein mitochondria o bone marrow reticulocytes in conditions oPb poisoning Failure to insert iron into protoporphyrin results indepressed heme ormation [11].

Lead bioaccumulation caused essential reduction o the percentagenormal erythrocytes and respectively significant increase o thepercentage pathological erythrocytes in the peripheral blood [95]. Tisfinding confirms the study o [94] that has been shown that Pb causesmorphological changes in erythropoietic bone marrow cells, leadingto production o microcytic and hypochromic erythrocytes. Significantdecreases in RBC and mean corpuscular hemoglobin and significantincrease in the requency o micronucleated polychromatic bonemarrow erythrocytes have been ound in Algerian mice (Mus spretus)exposed to Pb [96].

In addition, lead is a genotoxic actor. It damages the chromosomalstructure in mammalian cells [15,16,97-101]. Mutagenicity andcarcinogenicity o Pb have been reported by [102] as well as by [103].At toxic doses, lead acetate and lead nitrate have induced DNAbreaks determined by nick translation [104]. Lead essentially reduces

the prolierative activity o the bone marrow cells [105]. [99] notedthat lead and cadmium cause DNA strand breaks and chromosomalaberrations (CA) only afer treatment with high, toxic doses, however,

both metals exert pronounced indirect genotoxic effects, which maybe due to an interaction with DNA repair processes. Te inhibition oDNA repair could play a role in the accumulation and stability o DNA

damage, resulting in initiation o carcinogenic process.

Chromosomal aberrations (CA) are changes in normalchromosome structure or number. Te structural CA are two maintypes: breaks and exchanges. Te numerical CA are aneuploidy andpolyploidy. CA can be ormed also spontaneously e.g. rom double-strand breaks, generated by cellular events such as topoisomeraseaction, DNA replication, transposable elements, and ragile sites, andin excision repair o oxidative DNA damage [106]. Te comparison ochromosome aberration requency (CAF) in spontaneous and agent-induced cases allows assessing the extent o harm o the respectiveagent.

Te numerous and various adverse health effects caused byexposure to lead have motivated an earnest quest to find an effectiveagent that could, albeit partially, prevent the harmul influence o leadon humans and animals. Zeolites have proven to be such suitable agent.

Toxicity test for clinoptilolite

o calculate the mean Lethal Dose (LD50

) o any toxicant, theKarber-method was used as the most punctual method, allowingdetermination o reliability interval. Tis method requires an equalnumber o animals in each group. LD

50is calculated by the ormula:

( )( )50 100 : . :LD LD z d m=

where m= const is the number o animals in a given group; z(z1,

z2, etc.) is the number o dead animals rom two neighboring groups

divided in two; d (d1, d

2, etc.) is the dose in mg (mL) between two

neighboring doses:

1 1 2 2 3 3 4 4 5 5. . . . . .z d z d z d z d z d z d= + + + +

Te test was conducted in order to establish the Lethal Dose (LD50

)

o the clinoptilolite sorbent administrated to the mice. Te data o theinvestigation on chromosome aberrations, some blood parameters and

body weight gain suggested that the used clinoptilolite sorbent waspractically non-toxic. A lack o toxic effects o clinoptilolite has been

also demonstrated by other authors: [2,45,107-109].

Te LD50

test and its variants were ofen undertaken because

o legislative requirements. Tese tests are considered obsolete andnowadays they are rarely but still perormed in many countries.

Internationally accepted alternative tests have been developed. Tey

use ewer animals and absence o evident toxicity, rather than death,as their criterion. Tey have been accepted by most regulators as validalternatives to LD

50 testing. Nonetheless, LD

50 values are still quoted

and enquiries received by the Royal Society o Chemistry suggest thatthere is still a need or inormation about their meaning [110]. Tis was

the reason the examination o KLS-10-MA or toxicity to be reportedbased on LD

50test.

Te experimental results indicated that all animals rom group 2

(healthy and clinoptilolite-supplemented mice) survived up to the endo experiment, gained in body weight, and exhibited a good activity

and lie condition. No symptoms o an increased toxicity regardingthe physiological status were recorded during the experiment.

Unavorable pharmacological effects were not established. In addition,

an unusual behavior was not observed. Moreover, the Pb-exposedand supplemented mice demonstrated a significant improvement othe vital parameters. Tereore, due to the lack o any acute toxicity


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at the applied dose o the clinoptilolite sorbent, it was impossible todetermine LD

50o this substance and the sorbent KLS-10-MA was

assumed practically nontoxic. However, it is important to note that an

increase o sorbent concentration would be wrong because it wouldworsen the nutrition quality o the animal ood.

Lead bioaccumulation in clinoptilolite unsupplemented andclinoptilolite supplemented mice.

Experimental data and model results:Te comparative analysiso lead bioaccumulation between clinoptilolite unsupplemented andclinoptilolite supplemented experimental animals showed substantialdifferences in these groups (able 4, Figures 36). Tis act confirmsthe useulness o the clinoptilolite sorbent as a reliable means versuslead intoxication. Below the experimental and modeling results o theinvestigation is presented and discussed.

Te concentration o Pb [mg/kg] in the whole body, liver, and

kidneys measured in mice rom groups 3 and 4 are presented in Figure3. Te concentration o Pb in bones and eces are displayed in Figures4 and 5, respectively. Point 0 o the time axis in all o these figurescorresponds to the concentration measured in the control group.

Te highest Pb concentrations were established in eces, ollowedby those in bones in the mice rom group 3. Te background Pb levelin carcass, liver, kidney, bones, and eces o the control mice were0.22 0.06, 0.51 0.07, 0.44 0.08, 0.99 0.15, and 23.6 6.7 mg/kg, respectively. On day 90 the Pb concentrations in carcass, liver,kidney, bones, and eces o the mice rom group 3 were 1440-old, 134-old, 1355-old, 1518-old, and 406-old higher compared to those inthe control group. In group 4 these Pb concentrations were 237-old,17-old, 125-old, 357-old, and 51-old higher compared to those in

the control group. Significant differences (p> 0.001) were establishedin carcass, kidney, bone, and eces between groups 3 and 4 afer day45. Te reduction in Pb levels in the exposed and supplemented micecompared to exposed unsupplemented ones was as ollows: 84%,89%, 91%, 77%, and 88% or carcass, liver, kidney, bones, and eces,respectively.

Te progressive accumulation o Pb in the bones is due to theinert character o bone tissue making the reverse resorption almostimpossible. Te obtained data confirm this state (Figs. 3 and 4). On day90, Pb

Bone/Pb

Carcass= 5 in group 3 and Pb

Bone/Pb

Carcass= 6.9 in group 4.

Significant differences (p


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0 10 20 30 40 50 60 70 80 90

-50

0

50

100

150

200

250

300

350

400

450

500

550

600

650

Carcass, group 3

Liver, group 3Kidneys, group 3Carcass, group 4Liver, group 4Kidneys, group 4

Le

adconcentration[mg/kg]

Time [days]

Figure 3: Lead concentrations in carcass, liver and kidneys of ICR mice from groups 3 and 4during the ecotoxicological experiment. Time point 0corresponds to the concentrations in the control group.

0 20 40 60 80 100

0

200

400

600

800

1000

1200

1400

1600

1800

Group 3Group 4

Leadconcentrationinbones[mg/kg]

Time [days]

Figure 4: Lead concentration in bones of ICR micefrom groups 3 and 4duringthe ecotoxicological experiment model solutions and experimental points.Time point 0 corresponds to the concentrations in the control group.

0 20 40 60 80 100

0

2000

4000

6000

8000

10000

Group 3

Group 4

Feces

leadconcentration[mg/kg]

Time [days]

Figure 5: Lead concentration in feces of ICR mice from groups 3 and 4during the ecotoxicologicval experiment. Time point 0 corresponds to theconcentrations in the control group.

Body Liver Kidneys Bones Excreta

0

1

2

3

4

5

6

7

8

Group 3Group 4

Pb

90/

Pb

15

Samples

Figure 6: The Pb90/Pb15 ratio for carcass, liver, kidney, bones, and feces,indicating the signicant reduction of Pb bioaccumulation in group 4 versusgroup 3 due to the clinoptilolite sorbent supplement.


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J Bioequiv Availab


tissues, and eces in the exposed and supplemented animals (group4). Te reduction o 88% o Pb content in the eces o the micerom this group compared to the mice rom group 3 indicates that

clinoptilolite supplementation can also prevent the contamination othe environment.

Te results o the mathematical model are in a good agreement withthe experimental data or Pb concentration in bones (Figure 4) andallow determining some parameters o lead-bioaccumulation kinetics.

For the equation (3) under conditions (4) the ollowing analyticalsolution was obtained:

1 23

0 1

1 2 1 2 1 2 2 1

1 1 1( ) ( )

( ) ( )

b t b t z t z Aa a e e

b b b b b b b b

= + +

(9)

where b1= a

1+ a

2and b

2= a

3+ a

4.

Te solution z(t) represents the process o Pb bioaccumulation in

the mouses bones. Graphically, the time course o bone Pb is presentedin Figure 4. Te initial condition z(t0) = z

0corresponds to the bone Pb

concentration in the control group, z0= 1 mg/kg. Te concentration o

Pb in the drinking water o the experimental animals was 620 mg/l 620 mg/kg. Te daily water consumption per animal was about 7 ml/day and thereore, the daily Pb dose per animal could be approximatelyB= 4.34 mg/day. Extrapolating over the experiment, and taking intoaccount the value o gastrointestinal resorption coefficient = 15% [73]it was calculated that in group 3 the entire quantity o Pb absorbedby the gastrointestinal mucosa and entering into the blood during theexperiment, might be A

Group3= 58.6 mg. Coefficient (0 = = 1) is

a dimensionless coefficient indicating what raction o ingested metaldose is resorbed by the digestive tract. Converted to concentrationin mg/kg, and taking into account that the mean mouse body weight

during the experiment was about 30 g, the value AGroup3= x(t0) = 1953mg/kg was obtained. Te parameters were fitted by minimization o 2by the use o an iterative Gauss-Newton procedure [111,112]. Tus, theollowing values were ound or group 3: a

1= 0.022 day-1,a

2= 0.001

day-1,a3= 0.099 day-1,anda

4= 0.004 day-1; or group 4: A

Group4= x(t

0)

= 459.6 mg/kg,a1= 0.022day-1,a

2= 0.002 day-1,a

3= 0.099 day-1,anda

4

= 0.004 day-1.

Te model shows that the rate constants o Pb excretion by theecesa

2 Group 4is 2-old higher than a

2 Group 3. Tis is in accord with the real

situation. Te clinoptilolite sorbent accelerates the intestine passage, asa ballast matter.

Tis model allowed determining the coefficient o Pbgastrointestinal absorption in the experimental animals rom group 4,

using the ormula:

hGroup4

1000

A P

BT= (10)

where AGroup4

= x(t0)

Group4 mg/kg, or the mice o group 4, is the

concentration o lead, absorbed in the digestive tract and entering theblood extrapolating over the experiment, P is the mean mouse bodyweight during the experiment, B is the daily dose o Pb per animal,and T is the duration o the experiment. Te absorption coefficient = 3.53% was calculated or the clinoptilolite supplemented mice(group 4).Te value = 3.53% is 4.25-old lower compared with =15% in unsupplemented animals. Tereore, KLS-10-MA diminishesthe Pb absorption more than our times. Te obtained result is veryimportant! It shows a reduction o 76% in Pb quantity entering rom

the digestive tract to the blood o the mouse organism! Tis resultallows prognosticate an excellent perspective or the application o theclinoptilolite sorbent in mammals.

Genotoxic effects in clinoptilolite unsupplemented andclinoptilolite supplemented mice

Chromosomal aberrations, involving gross alterations o geneticmaterial, have been considered as a sensitive endpoint or detectinggenotoxic effects induced by heavy metal and toxic chemicals. Tus,the study o cytogenetical status is considered highly relevant in thehuman context [113,114].

Te chromosome aberration requency (CAF) in the analyzedmetaphases o bone marrow cells o ICR mice in our experiment ispresented in Figure 7. Te percentages aberrant metaphases in the

control group were within the range o spontaneous requencies. Nostatistically significant differences (p < 0.1) were observed betweenCAF in groups 1 and 2 except on day 90 (p< 0.05). No statisticallysignificant differences were observed between CAF in groups 4 and 2except on day 60 (p < 0.05). Significant differences (p< 0.001) were

recorded between group 3 and other groups at each time points. CAFin group 3 (CAF

3) exhibited on average 2-old higher level compared

to group 4 and 3.5-old higher level compared to the control group. Temaximum CAF in groups 3 and 4 were observed on day 60. For day 60the ollowing relations were established:

3 1 4 1 3 4/ 4.56; / 2; / 2.3CAF CAF CAF CAF CAF CAF = = = (1)

Te distribution o the different type chromosome aberrations (CA)is displayed in Fig. 8. Chromosome breaks (CrB) and Robertsoniantranslocations or c/c usions (C) in group 3 had their maximum valueson day 15 (4.7% and 5%, respectively). Chromosome breaks in group 4were almost equal on days 15 and 90, where they had maximum values(1.75% and 1.5%, respectively). Robertsonian translocations in group

4 had maximum value on day 60 and at this time point groups 3 and4 showed equal values (4.5%). In all, ragments (F) were ound below

10 20 30 40 50 60 70 80 90 100

2

4

6

8

10

12

14

16Group 1Group 2Group 3Group 4

Chromosomeaberrations

frequency%

Time [days]

Figure 7: Frequency of chromosome aberrations in ICR mice.


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J Bioequiv Availab


1.5%. Only in group 3 on day 60 F showed a relatively higher value(3%).

Te ollowing ratios were established about chromosome breaks,Robertsonian translocations, and ragments between groups 3 and 4(the time points are given in brackets):

( )

( ) ( )

3 4 3

4 3 4

/ 2.7 15 , /

7.46 45 , / 12 60

CrB CrB CrB

CrB CrB CrB

=

= =

,

( )3 4/ 2 90CrB CrB = (2)

( ) ( )3 4 3 4/ 1.8 15 , / 1.32 90C C C C = = (3)

( )3 4/ 12 60F F = (4)

Te ollowing ratios were calculated regarding the chromatidebreaks in a comparison o the experimental groups with the control

group (group 1):

( ) ( )

( )

3 1 3 1

3 1

/ 4.7 15 , / 3.2 45 ,

/ 3 60

CrB CrB CrB CrB

CrB CrB

= =

=

,

( )3 1/ 3 90CrB CrB = (5)

( ) ( )

( )

4 1 4 1

4 1

/ 1.8 15 , / 0.43 45 ,

/ 0.25 60

CrB CrB CrB CrB

CrB CrB

= =

=

,

( )4 1/ 1.5 90CrB CrB = (6)

o study the prolierative activity o the bone marrow cells duringthe experiment, mitotic indices were determined in ICR mice (Figure

9). Te observed mitotic indices exhibited no statistically significantdifferences (p> 0.1) between the control and second control groups(groups 1 and 2). Significant differences (p< 0.05 p< 0.001) wereound between groups 3 and 4, between the control group and group4 (p< 0.002), and especially between the control group and group 3 (p< 0.001). A trend o continuous decrease o the mitotic index duringthe experiment was established in the exposed-unsupplemented mice.Contrariwise, in the exposed-supplemented mice, the level o themitotic index decreased up to day 45 but then increased steadily. Teollowing comparisons between the groups were made and respectiveratios calculated regarding the mitotic indices (M) (the time points aregiven in brackets):

( ) ( )

( ) ( )

1 3 1 3

1 3 1 3

/ 1.8 15 / 2.5 45

/ 2.7 60 / 2.9 90

M M M M

M M M M

= =

= = (7)

( ) ( )

( ) ( )

1 4 1 4

1 4 1 4

/ 1.4 15 / 1.5 45

/ 1.3 60 / 1.2 90

M M M M

M M M M

= =

= =

(8)

( ) ( )

( ) ( )

4 3 4 3

4 3 4 3

/ 1.3 15 / 1.6 45

/ 2.1 60 / 2.5 90

M M M M

M M M M

= =

= =

(9)

In our experiment, the increased percentage aberrant bone marrowcells in the mice rom group 3 most probably is due to the very highPb concentration in bones (over 1500 mg/kg on day 90), as seen inable 4. Lead concentration in bone marrow is proportional to that inbones [115]. Lead is well studied as a actor damaging the chromosomestructure in mammalian cells [97,99,115].

Structural chromosome aberrations (CA) may be induced mainlyby direct DNA breakage, by replication on a damaged DNA template,

and by inhibition o DNA synthesis [116,117]. Tey can be dividedinto two main classes: chromosome-type aberrations (CSA) involvingboth chromatids o one or multiple chromosomes and chromatid-type

aberrations (CA) involving only one o the two chromatids [117,118].Metaphase analysis provides inormation on the timing o DNA lesionsrelative to DNA replication [119]. CSA result rom double-strandbreaks (DSB), incompletely- or non-repaired, ormed in G

0/G

1phase o

the cell cycle or rom DSB generated beore replication in early S phase.In this case, in metaphase are seen chromosome-type breaks, dicentricand ring chromosomes, ragments. DSB generated in postreplicativeDNA in later S phase and in G

2phase give rise to CA, i.e. chromatid

type breaks and exchanges [118]. CA may arise also, in response tosingle-strand breaks (SSB) induced in early S phase. CA are usuallygenerated by S-phase-dependent clastogens (e.g. chemicals) [120]. SSBresulting rom Pb influence were reported by [101,121]. Chromatidebreaks observed in metaphase would result rom incomplete or ailed

repair [106]. Te high presence o chromatide breaks and Robertsoniantranslocations in the examined laboratory mice suggested an essentialprevalence o CA. Tus, the data o our experiments, compared withthe data o other authors, provide evidence that Pb is mainly S-phase-dependent clastogen.

In Figure 8 it is seen that among the spontaneous CA in mice,centromere-centromeric usions (c/c usions) predominate. Te mousekaryotype is a special one in some extent because o the acrocentricnature o mouses chromosomes. Robertsonian translocations causestructure orms as meta- and submeta-centric chromosomes throughcentric exchanges o two acrocentrics due to the single strands breaksin the minor SA DNA located in the centromeric regions o micechromosomes. Tus, the rate o exchanges between non-homologouschromosomes in the mouse is particularly high [121].

Tere is a lack o data about the behavior and adsorption capacityo zeolites in animal organism as well as about the influence o theseminerals on genetic apparatus [122] have established that erionite fiberscould cause cytogenetic changes similar to those caused by asbestiormmineral dusts but like asbestos minerals do not induce mutations inhuman lymphoblastoid cells.

Our finding (Figures 7 and 8) showed that clinoptilolite did not giverise to injury o chromosomal structure. Te CAF and the percentages

15 45 60 90 15 45 60 90 15 45 60 90

0

1

2

3

4

5

6

7 Group 1Group 2Group 3Group 4

Typeofchromosomeaberrations

Chr Breaks Fragments c/c

Figure 8:Distribution of chromosome aberrations in ICR mice.


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J Bioequiv Availab


several chromosome aberrations in group 2 exhibited no significant

changes compared to those in the control group (p< 0.1). Te slightincrease o CAF on day 90 was almost within the range o spontaneousrequencies. Tere were no statistically significant differences (p> 0.1) in the mitotic indices o the control mice and healthy micesupplemented with clinoptilolite.

Te excellent quality end effective influence o KLS-10-MA as a Pb-adsorber in the mouses organism is seen rom equations (1) (6) orthe chromosome aberrations and (7) (9) or the mitotic indices. Te

CAFs in group 4 were 22.3-olds lower than those in group 3. Techromatide breaks and ragments in group 4, especially on day 60, were12-old lower than those in group 3. On day 90, the mitotic index ingroup 4 was 2.5-old higher compared to that in group 3. On the sameday the mitotic index in control group was almost 3-olds higher than

that in group 3, and only 1.2-old higher than that in group 4.Tese results perectly conorm to the data o Pb bioaccumulation

(able 1). Lead concentration in kidney gives inormation concerningthe level o blood Pb. On day 90, the mean kidney Pb concentration ingroup 4 was 11-old lower than that in group 3. Respectively, on day 90the Pb concentration in bones o the mice rom group 4 was 4.3-oldlower compared to Pb bone concentration in mice rom group 3. Tismeans that the lead entering into the blood, organs, and bone marrowo the sorbent-supplemented mice was significantly reduced due to the

high ion exchange capacity o clinoptilolite in the animals digestivetract.

Pathology in the erythrocytes and erythropoesis

in clinoptilolite unsupplemented and clinoptilolitesupplemented mice

Lead influence on erythropoietic bone marrow cells causes

morphological changes, which lead to production o microcyticand hypochromic erythrocytes [94]. More than 90% o Pb inblood accumulates in erythrocytes and there are at least two major

compartments or Pb in red blood cell: one associated with themembrane and the other with hemoglobin [11]. Te increasedmechanical ragility o cell membrane shortens the erythrocyteliespan. Depression o the hemosynthesis by Pb is due to the inhibitiono SH-containing enzymes controlling it in bone marrow [11,73]. Inaddition, the decrease o the hemoglobin level is due to the decrease othe number o dividing erythroblasts and consequently newly-ormederythrocytes. All these eatures indicate a lead-induced anemia, a well-known symptom o chronic Pb poisoning. In our experiment, a dropin the prolierative activity o the bone marrow stem cells and well-expressed anemia were clearly observed in the Pb-exposed mice romgroup 3.

Te hemoglobin level and erythrocyte number are most ofen

investigated. Our attention here was ocused mainly on the changeso the erythrocyte morphology because o its useulness as a clearindicator or Pb intoxication.

Te data o the quantitative erythrocyte morphological analysisare presented in Figure 10. Lead bioaccumulation caused statisticallysignificant reduction o the percentage normal erythrocytes andrespectively significant increase o the percentage pathologicalerythrocytes in the peripheral blood o the mice rom group 3,compared to the control group (p< 0.001). Te normal and pathologicalerythrocytes decreased and increased, respectively, also in group 4,but the normal red blood cells were at significantly higher level andthe pathological ones were at significantly lower level compared withgroup 3 (p< 0.01). Besides, within group 4 the normal erythrocytesremained significantly higher than pathological ones (p< 0.001) up to

the end o experiment.

10 20 30 40 50 60 70 80 90 100

2

4

6

8

10

12

14

Group 1Group 2

Group 3Group 4

Mitoticindex%o

Time [days]

Figure 9:Mitotic indices in ICR mice.

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20

25

30

35

40

45

50

55

60

65

70

75

80

85

90Group 1 - normalGroup 1 - pathologicalGroup 3 - normalGroup 3 - pathologicalGroup 4 - normalGroup 4 - pathological

Erythrocyte

%

Time [days]

Figure 10: Percentage of normal erythrocytes and erythrocytes withpathological changes in ICR mice.


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Te ollowing ratios were calculated between the normal (N) andpathological (P) erythrocytes within groups 3 and 4, on days 15 and 90,respectively:

group 3: ( ) ( )3 3 3 3/ 1.9 15 , / 0.7 90N P N P= = (10)

group 4: ( ) ( )4 4 4 4/ 3.1 15 , / 1.3 90N P N P= = (11)

Between groups 3 and 4, and the control group, on day 90, theollowing ratios were established regarding the normal and pathologicalerythrocytes, respectively:

1 3 1 4/ 2, / 1.35N N N N= = (12)

1 3 1 4/ 0.43, / 0.53P P P P= = (13)

Equations (12) and (13) demonstrate a well-expressed toxicologicalstress in the animals exposed to Pb and a reliable detoxificationeffect o the clinoptilolite sorbent taken as a ood supplement. In the

supplemented mice rom group 4 pathological erythrocytes werereduced and the normal ones were enhanced so that their levels camenear to that in the control group.

Between groups 3 and 4 the ollowing ratios were determined:

normal Er: ( ) ( )4 3 4 3/ 1.2 15 , / 1.5 90N N N N= = (14)

pathological Er: ( ) ( )3 4 3 4/ 1.4 15 , / 1.2 90P P P P= = (15)

Equations (14) and (15) confirm the essential effect o the sorbenton the decrease o harmul effect o Pb on cell level.

A significant drop in the erythropoesis rate was recorded in thePb-exposed mice. Te time course o the prolierating erythrocytes(PE) is presented in Figure 11. Statistically significant differences(p < 0.05) were observed on days 15 and 90 between groups 3 and4. Te percentages prolierating erythrocytes in Pb-exposed andsupplemented mice were higher than those in Pb-exposed mice withoutclinoptilolite supplement. Te differences between groups 3 and 4, andcontrol group 1, especially on day 90 were quite significant (p< 0.001).Comparing PE in the control mice to that in mice rom groups 3 and 4on day 90 the ollowing ratios were obtained:

3 1 4/ 10, / 3.2lPE PE PE PE= = (16)

Te ollowing ratios between PE in group 4 and PE in group 3 takenplace:

( )4 3 4 3/ 1.22 15 , / 3.14PE PE PE PE= = (90) (17)

A 10-old drop was observed in the number o dividing

erythroblasts in group 3 compared with the control group (equation(16). Te supplementation with KLS-10-MA reduced the respectiveratio in group 4 almost by three times. Te greatest differences betweengroups 3 and 4 were established on day 90, equation (17). Te resultindicates that the sorbent improved the erythropoietic unction aferday 60.

No many echinocytes were ound and this could be due to therelatively low Pb level in the liver (compared to other tissues); on day90. In group 3 it was 68.518 mg/kg (able 1). Te echinocytes presencemainly in group 3 suggests that the potassium (K) in the mousesorganism is replaced in high degree by Pb. Te lack o echinocytes in

the mice rom group 4 indicates a normal K level. In act, on day 90 themean liver Pb concentration in group 4 was almost 8-old lower than

in group 3 (able 4) and thereore, the amount o replaced K in group4 was insignificant. Moreover, the supplemented mice have receivedadditional K rom the sorbent. Te observed data are in accord with

the finding o [123] who detected a 20% elevation o serum K in healthylaboratory mice receiving a zeolite-rich diet.

Micronuclei in erythrocytes in peripheral blood were observedin the specimens rom group 3 and 4. In group 4 they appearedsporadically mainly beore day 45. Tis act indicates again that themain part o Pb entering in the mouse organism via drinking waterwas captured by the clinoptilolite sorbent and thus the Pb quantityabsorbed by the intestine mucosa and utilized by the organism at celllevel was significantly reduced.

Growth pattern in clinoptilolite unsupplemented andclinoptilolite supplemented mice

Body weight gain in experimental animals is a reliable indicator or

their healthy status. It is an integral characteristic o the physiology o

the organism. In the experiment here presented the body weight gain othe mice was determined at the same time points, in which the samples

or the cytogenetical and hematological analyses were taken: on days

15, 45, 60, and 90.

Te changes in the body weight o the mice are presented in Fig.

12. Statistically significant differences were ound among groups 3 and4 (p < 0.01) on days 15, 45, 60 and 90 (p < 0.01). A sharp decrease

o body weight was established in the Pb-exposed mice rom group 3

afer day 60. On day 90 these mice exhibited a body weight reduced

with 24% compared to the control mice. On day 90 the body weight in

the exposed and supplemented mice was 21% higher compared to that

in the exposed and unsupplemented ones. No statistically significantdifference was ound between group 4 and the control group during

the experiment.

When compared the body weight (W90) on day 90 to the bodyweight (W

15) on day 90 in the mice rom groups 3, 4 and 1 (control) the

ollowing ratios were calculated:

10 20 30 40 50 60 70 80 90 100

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

Group 1Group 2Group 3

Proliferatingerythrocytes%

Time [days]

Figure 11:Percentage of proliferating erythrocytes in ICR mice.


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group 3: 90 15/ 1.07W W = (18)

group 4: 90 15/ 1.28W W = (19)

group 1: 90 15/ 1.3W W = (20)

As seen rom the relations (18), (19), and (20), the decrease o thebody weight gain in the Pb-exposed and sorbent-unsupplemented micewas so strong, that the body weight o the animals on the third montho their growth was almost equal to the body weight at their 15-day age.In contrast, the body weight o Pb-exposed and sorbent-supplementedmice on the third month o their growth was almost equal to the bodyweight in the mice rom the control group.

When compared the body weights o the animals between thecontrol group, and groups 3 and 4 on day 90, we obtained the ollowingresult:

3 4/ 0.79W W =

(21)

3 1/ 0.76W W = (22)

4 1/ 0.97W W = (23)

Te relations (21), (22), and (23) show the significant differencebetween the group 3, on the one hand, and groups 1 and 4, on the otherhand. Te measurement o the body weight o the experimental animalsonce more confirms the establishment that the used clinoptilolitesorbent KLS-10-MA plays an important role in prevention o a severePb intoxication in mammals.

Influence of clinoptilolite supplementation on the bodyweight gain of healthy animals

Many researchers have proved that the dietary inclusion o zeolitesimproves average daily gain and/or eed conversion in pigs, calves,

sheep and broilers. Zeolites also enhance the reproductive perormance

o sows, increase the milk yield o dairy cows and the egg production

o laying hens. Tey have also beneficial effects on egg weight and the

interior egg characteristics [17]. However, the extent o perormanceenhancement effects is related to the type o the used zeolite, its purity

and physicochemical properties, as well as to the supplementation

level used in the diets. Besides, the particle size o the zeolitic material,

crystallite size and the degree o aggregation, as well as the porosity o

individual particles determine the access o ingested fluids to the zeoliticsurace during passage across gastrointestinal tract and strongly affect

its ion exchange, adsorption and catalytic properties. Apart rom the

positive effects on animals perormance, dietary supplementation o

zeolites appears to represent an efficacious, complementary, supportive

strategy in the prevention o certain diseases and the improvement o

animals health status.

In our study, a weak acceleration o body weight gain in the

conditions o clinoptilolite supplementation was established. A 5%

higher body weight was recorded in the healthy supplemented mice

compared with the control ones. Tus, the mice rom group 2 presented

the highest body weight in contrast to the other groups.

Te positive effect o the clinoptilolite sorbent KLS-10- on

the supplemented mice confirms the numerous data o other authors

who have observed essential positive results, such as toxin elimination,

conversion nutrition improvement, growth increase, mortality

reduction, as well as improvement o common state o animal healthusing clinoptilolites as a eed additive in poultry, pigs and ruminantsnutrition [17].

Studies illustrate that clinoptilolite supplementation to thediet o swine, poultry and ruminants improved body mass and eedconversion ratios [107,124,125]. In addition, milk yields have improved

in dairy herds and the incidence o scours, enteritis and other intestinaldiseases has decreased substantially [107]. Te use o 3%, 5% and 10%clinoptilolite as a eed supplement or Leghorn chickens, indicated thatat all levels, eed efficiency ratios have increased comparing with thecontrol diet. No adverse effects on the health or vitality o the birds havebeen observed [107]. Clinoptilolite at 2% supplement level by weight ina hog diet increased daily eed intake [126,127]. A significant daily gaino newborn lambs was established in the conditions o basal diet with3% clinoptilolite [128]. A greater weight gain has been reported o 17%and 3.6% in young bee cattle and broilers when clinoptilolite at 4.3%and, 0.5%, 1% and 1.5% by weight was added to cattles and chickensregular diet, respectively [129]. In comparison to a regular diet, theclinoptilolite at 5% and 10% levels by weight in hogs diet resulted in a

weight gain o 27% [130-134] and 8% [129], respectively.In the present experiment the modified clinoptilolite KL-10-MA was

mixed at 12.5% concentration with the conventional orage. Tereore,approximately about 9% by weight was added to the mices daily diet.Te data showed a 5% rise (Figure 12) in body weight o the healthysupplemented animals (group 2) compared with the healthy non-supplemented ones (control group). Comparing the obtained resultswith the data o other authors, the optimal quantity o clinoptilolitesupplement ensuring the maximal body weight gain could be specified.Perhaps there should be doses o about 5% by animal weight.

Conclusion

Te chromosome aberrations, lowered mitotic index, pathologically

changed erythrocytes, diminished erythropoesis, and reduced bodyweight gain observed here in the Pb-exposed laboratory mice,demonstrated a well-expressed toxicological stress due to the chronic

10 20 30 40 50 60 70 80 90 100

22

24

26

28

30

32

34

36

38

40

42Group 1Group 2Group 3Group 4

Meanbodyweight[g]

Time [days]

Figure 12:Mean body weight of ICR mice.


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Pb intoxication. Te structure o chromosomes and red blood cells aswell as mitotic index and erythropoesis were significantly improvedby the supplementation o the animals with modified clinoptilolite

sorbent KLS-10-MA, a Na-enriched alkali earth clinoptilolite basedon natural Bulgarian clinoptilolite. Te mice exposed to Pb andsupplemented with KLS-10-MA exhibited a reduction in Pb levels inseveral samples o about 77 90%. Te bioaccumulation coefficientsPb

90/Pb

15 or carcass, liver, kidney, bones, and eces in supplemented

animals were significantly lower compared to those in unsupplementedones. Tus, the obtained results showed that the sorbent KLS-10-MAstrongly decreases the absorption o lead in animals digestive tractand thereore limits Pb quantity entering the blood. Te time course othe body weight in the supplemented animals differed not significantlyrom that in the control mice. Te supplemented animals survived andappeared active and healthy. A weak rise o the body weight gain wasestablished in healthy clinoptilolite-supplemented animals.

Te mathematical model o Pb bioaccumulation in bones canpredict the time course o Pb concentrations in conditions o chronicexposure to Pb with/without sorbent supplementation. Te aim o themodel is to indicate the main tendencies o Pb bioaccumulation inbones, taking into account the basic actors determining this process.Te model allows determining some kinetic parameters, especially; itwas useul to calculate the coefficient o Pb gastrointestinal absorptionin the case o sorbent-supplemented animals. Tis coefficient was 4times lower compared to the established coefficient or Pb absorption.

Te findings allow conclude that the clinoptilolite sorbent KLS-10-MA exerts a significant avorable effect and thus it appears as a reliablemeans or detoxification o human and animal organisms chronicallypoisoned by heavy metals, particularly lead. Tis work gives ground to

create a drug based on the clinoptilolite sorbent or supplementationo animals in regions that are industrially polluted with heavy metals,and particularly with Pb, in order to protect the animals health andthe quality o the environment. Te use o a drug based on this sorbentcould be valuable in agriculture and livestock as well as in humanmedicine in cases o chronic lead intoxication.

References

1. Sheppard RA (1984) Characterization of zeolitic materials in agriculturalresearch. In: Pond WG, Mumption FA (eds) Zeo-Agriculture: Use of NaturalZeolites in Agriculture and Aquaculture. Westview Press, Boulder, Colorado81-90.

2. Mumpton FA (1999) La roca magica: uses of natural zeolites in agriculture andindustry. Proc Natl Acad Sci U S A 96: 3463-3470.

3. Inglezakis VJ, Stylianou MA, Gkantzou D, Loizidou MD (2007) Removal of Pb(II) from aqueous solutions by using clinoptilolite and bentonite as adsorbents.Desalination 210: 248256.

4. Wingenfelder U, Nowack B, Furrer G, Schulin R (2005) Adsorption of Pb andCd by amine-modied zeolite. Water Res 39: 3287-3297.

5. Sprynskyy M, Buszewski B, Terzyk AP, Namienik J (2006) Study of the selection mechanism of heavy metal (Pb2+, Cu2+, Ni2+, and Cd2+) adsorptionon clinoptilolite. J Colloid Interface Sci 304: 21-28.

6. Argun ME (2008) Use of clinoptilolite for the removal of nickel ions from water:kinetics and thermodynamics. J Hazard Mater 150: 587-595.

7. Bernal MP, Lopez-Real JM, Scott KM (1993) Application of natural zeolites forthe reduction of ammonia emissions during the composting of organic wastesin a laboratory composting simulator. Bioresource Technol 43: 35-39

8. Jongbloed AW, Lenis NP (1998) Environmental concerns about animal manure.J Anim Sci 76: 2641-2648.

9. Sutton AL, Kephart KB, Verstegen MW, Canh TT, Hobbs PJ (1999) Potential for reduction of odorous compounds in swine manure through diet modication.J Anim Sci 77: 430-439.

10. Dwairi IM (1998) Conserving toxic ammoniacal nitrogen in manure using naturalzeolite tuff: a comparative study. Bull Environ Contam Toxicol 60: 126-133.

11. Goyer RA (1996) Toxic effects of metals. In: McGraw-Hill (ed) Casarett

and Doulls Toxicology. The Basic Science of Poisons, Fifth (edn). HealthProfessions ivision, New York London Tokyo Toronto 691-735

12. OFlaherty EJ (1998) Physiologically based models of metal kinetics. Crit RevToxicol 28: 271-317.

13. Robles HV, Romo E, Sanchez-Mendoza A, Rios A, Soto V, et al. (2007) Leadexposure effect on angiotensin II renal vasoconstriction. Hum Exp Toxicol 26:499-507.

14. Johnson FM (1998) The genetic effects of environmental lead. Mutat Res 410:123-140.

15. Topashka-Ancheva M, Metcheva R, Teodorova S (2003) Bioaccumulation anddamaging action of polymetal industrial dust on laboratory mice Mus musculusalba II. Genetic, cell and metabolic disturbances. Environ Res 92: 152-160.

16. Valverde M, Trejo C, Rojas E (2001) Is the capacity of lead acetate andcadmium chloride to induce genotoxic damage due to direct DNA-metal

interaction? Mutagenesis 16: 265-270.17. 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. Micropor Mesopor Mat 84: 161-170

18. Stylianou MA, Hadjiconstantinou MP, Inglezakis VJ, Moustakas KG, LoizidouMD (2007) Use of natural clinoptilolite for the removal of lead, copper and zincin xed bed column. J Hazard Mater 143: 575-581.

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