Critical Review on Zeolite Clinoptilolite Safety and Medical Applications in vivo

Abstract

Unique and outstanding physical and chemical properties of zeolite materials make them extremely useful in a variety of applications including agronomy, ecology, manufacturing, and industrial processes. Recently, a more specific application of one naturally occurring zeolite material, clinoptilolite, has been widely studied in veterinary and human medicine. Due to a number of positive effects on health, including detoxification properties, the usage of clinoptilolite-based products in vivo has increased enormously. However, concerns have been raised in the public about the safety of clinoptilolite materials for in vivo applications. Here, we review the scientific literature on the health effects and safety in medical applications of different clinoptilolite-based materials and propose some comprehensive, scientifically-based hypotheses on possible biological mechanisms underlying the observed effects on the health and body homeostasis. We focus on the safety of the clinoptilolite material and the positive medical effects related to detoxification, immune response, and the general health status.

Full text

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REVIEW
published: 27 November 2018
doi: 10.3389/fphar.2018.01350
Edited by:
Lei Xi,
Virginia Commonwealth University,
United States
Reviewed by:
Panagiotis Dimitrios Katsoulos,
Aristotle University of Thessaloniki,
Greece
Stephane Nizet,
GLOCK Health, Science and
Research G.m.b.H., Austria
*Correspondence:
Sandra Kraljevi ´
c Paveli ´
c
sandrakp@biotech.uniri.hr
Specialty section:
This article was submitted to
Translational Pharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 20 June 2018
Accepted: 02 November 2018
Published: 27 November 2018
Citation:
Kraljevi ´
c Paveli ´
c S, Simovi ´
c
Medica J, Gumbarevi ´
c D, Filoševi ´
c A,
Pržulj N and Paveli ´
c K (2018) Critical
Review on Zeolite Clinoptilolite Safety
and Medical Applications in vivo.
Front. Pharmacol. 9:1350.
doi: 10.3389/fphar.2018.01350
Critical Review on Zeolite
Clinoptilolite Safety and Medical
Applications in vivo
Sandra Kraljevi ´
c Paveli ´
c1*, Jasmina Simovi ´
c Medica2, Darko Gumbarevi ´
c1,
Ana Filoševi ´
c1, Nataša Pržulj3and Krešimir Paveli ´
c1,4
1Department of Biotechnology, Centre for High-Throughput Technologies, University of Rijeka, Rijeka, Croatia,
2General Hospital Pula, Pula, Croatia, 3Computer Science Department, University College London, London,
United Kingdom, 4Juraj Dobrila University of Pula, Pula, Croatia
Unique and outstanding physical and chemical properties of zeolite materials make them
extremely useful in a variety of applications including agronomy, ecology, manufacturing,
and industrial processes. Recently, a more specific application of one naturally
occurring zeolite material, clinoptilolite, has been widely studied in veterinary and
human medicine. Due to a number of positive effects on health, including detoxification
properties, the usage of clinoptilolite-based products in vivo has increased enormously.
However, concerns have been raised in the public about the safety of clinoptilolite
materials for in vivo applications. Here, we review the scientific literature on the health
effects and safety in medical applications of different clinoptilolite-based materials and
propose some comprehensive, scientifically-based hypotheses on possible biological
mechanisms underlying the observed effects on the health and body homeostasis. We
focus on the safety of the clinoptilolite material and the positive medical effects related
to detoxification, immune response, and the general health status.
Keywords: zeolite, clinoptilolite, toxicology, immunostimulation, antioxidant properties
CHEMICAL PROPERTIES AND BIOLOGICAL APPLICATION OF
NATURAL ZEOLITE CLINOPTILOLITE
Zeolites possess unique and outstanding physical and chemical properties. These characteristics
make them very useful in a variety of applications including agronomy, ecology, certain
manufacturing, industrial processes, medicine, and cosmetics. Recently, the application of a specific
natural zeolite material, clinoptilolite, has been documented in veterinary and human medicine.
Abbreviations: 5-HT(1A) and 5-HT(1B), serotonergic 5-hydroxytryptamine receptors in the brain; 3H-8-OH-DPAT,
3[H]8-hydroxy-2-(di-n-propylamino)tetralin; Al, aluminum; Al3+, aluminum(III)-cation; ALT, aspartate aminotransferase;
AST, alanine aminotransferase; Ba, barium; Ca, calcium; Co, cobalt; CO2, carbon dioxide; Cd, cadmium; Cr, chromium;
Cs, caesium; Cu, copper; EDTA, ethylenediaminetetraacetic acid; EFSA, The European Food Safety Authority; EGF-R,
epidermal growth factor receptor; Fe, iron; GALT, gut-associated lymphoid tissue; GGT, gamma-glutamyl transferase;
GSH, glutathione; HEU, clinoptilolite; Hg, mercury; H2S, hydrogen sulfide; H2O2, hydrogen peroxide; K, potassium; Mn,
manganese; MALT, mucosa-associated lymphoid tissue; MDA, malondialdehyde; Na, sodium; NfkB, nuclear factor kB; Ni,
nickel; O•−
2, superoxide anion; OECD, Organization for Economic Cooperation and Development; ·OH, hydroxyl radical;
Pb, lead; (PKB)/Akt, protein kinase B/Akt kinase; PMA, micronized clinoptilolite material; Prx, peroxiredoxin; ROS, reactive
oxygen species; Si, silicon; SOD, superoxide dismutase; Sr, strontium; TCLP/EPA/RCRA, Toxicity Characteristic Leaching
Procedure/Environmental protection agency/Resource Conservation and Recovery Act; Trx, thioredoxin; Zn, zinc.
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FIGURE 1 | The generally accepted and studied clinoptilolite effects on the
human body in vivo. Observed clinically relevant effects on organs and
systems for different clinoptilolite materials in vivo are due to major clinoptilolite
properties: detoxification, antioxidant effect, release of trace elements, and
positive influence on the microbiota status in the intestine as described in
Table 1. These effects were documented in animals and humans for
clinoptilolite material used as supplementation to regular diet in a powdered
form.
Subsequently, the market of clinoptilolite-based products for use
in vivo has been continuously growing (Figure 1) (Paveli´
c and
Hadžija, 2003).
The name ‘zeolite’ originates from the Greek words ‘zeo’(to
boil) and ‘litos’ (a stone). The current nomenclature and
classification of zeolite materials has been provided by the
Structure Commission of the International Zeolite Association
that identifies each material based on their framework with
a three-letter mnemonic code; for instance, natural zeolite
clinoptilolite is denoted as HEU (Baerlocher et al., 2007).
By origin, zeolites can be natural or synthetic materials.
They are aluminosilicate minerals with rigid anionic frameworks
containing well-defined channels and cavities. These cavities
contain metal cations, which are exchangeable, or they may
also host neutral guest molecules that can also be removed
and replaced. The majority of natural zeolites are of volcanic
origin and have a general formula, M2/n:Al2O3:xSiO2:yH2O,
where M stands for the extra-framework cation (Bogdanov
et al., 2009). The mineral structure is based on AlO4and
SiO4tetrahedra, which can share 1, 2, or 3 oxygen atoms, so
there is a wide variety of possible structures as the network is
extended in three dimensions. This unique structural feature
is a basis for their well-known microporous structure. Based
on the pore size and absorption properties, zeolites are among
the most important inorganic cation exchangers and are used
in industrial applications for water and waste water treatment,
catalysis, nuclear waste, agriculture, animal feed additives, and in
biochemical applications (Bogdanov et al., 2009).
The variety of zeolites’ application is indeed a consequence of
their porous structure: pores form negatively charged channels
and cavities, which are occupied with positively charged alkali,
and alkali earth monovalent (i.e., Na+, K+), and divalent (i.e.,
Ca2+) ions, OH-groups or H2O molecules, which can be easily
exchanged by other molecules and cations from the surroundings
(Figure 2). It is logical then, that the final Si/Al ratio in a zeolite
determines the ion exchange capacity and attraction of cations
that come to reside inside the pores and channels (Mumpton,
1999;Canli et al., 2013a).
Besides metal cations and water resident in zeolites’ cavities
and pores, other molecules and cationic groups may be
accommodated as well, such as, for instance, ammonia,
and nitrate ions, all of which are bound to different
zeolites at different affinity levels (Gaikwad and Warade,
2014). For example, selectivity alignments of the zeolite
clinoptilolite cation exchange have been given as Ba2+>Cu2+,
Zn2+>Cd2+, Sr2+>Co2+by Blanchard et al. (1984), as
Pb2+>Cd2+>Cs+>Cu2+>Co2+>Cr3+>Zn2+>Ni2+>
Hg2+by Zamzow et al. (1990), or as Co2+>Cu2+>Zn2+>
Mn2+by Erdem et al. (2004).
The mineral assemblies of the most common zeolite
occurrences in nature are clinoptilolite- and mordenite-
containing tuffs, in which the zeolite clinoptilolite and mordenite
content is high (80% and over). It may appear with the aluminum
phyllosilicate clay smectite (bentonite) and accompanying phases
present in lower percentages cristoballite, calcite, feldspar,
and quartz. However, other types of zeolites (e.g., phillipsite,
chabazite) and clay minerals may dominate the mineral tuff
assemblage, and properties of such materials may vary in the
widest sense with respect to the final mineral content (Cejka,
2005).
The widely tested zeolite suitable for medical applications
in vivo is the clinoptilolite tuff but the mordenite tuff was also
studied by Selvam et al. (2014) So far the word ‘zeolite’ has been
used in the literature for different types of zeolites, tuffs, and
clays. For example, both clinoptilolite and clay materials may be
used for ion-exchange reactions. Still, their structural properties
and toxicology profiles may be different (Maisanaba et al., 2015).
The structure of mineral clays is, for instance, organized in layers
(sheets), while clinoptilolite has tetrahedra arranged in a way that
they form large amounts of pore space in the crystals. Different
physical-chemical properties between clinoptilolite and clays,
e.g., kaolinite were documented accordingly in the literature
(Ghiara et al., 1999;Miranda-Trevino and Coles, 2003;Payra and
Dutta, 2003;Hecht, 2005;Svoboda and Šulcová, 2008;Bibi, 2012;
Dimowa et al., 2013;Jurki´
c et al., 2013). For example, the kaolinite
structure may change during the ion-exchange processes due
to the displacement of H+ions or due to the swelling of the
structure as a consequence of Pb, Zn, or Cd cations absorption,
which is opposite to the clinoptilolite constancy during ion-
exchange process (Miranda-Trevino and Coles, 2003).
Clinoptilolite shares a high structural similarity with the
zeolite heulandite (they are isostructural) and it is distinguished
from helaundite by a higher silicon to aluminum ratio in favor to
silicon, where Si / Al >4.0 and (Na +K) >(Ca +Sr +Ba). The
thermal behavior of clinoptilolite and heulandite is also different.
The clinoptilolite structure is still not destroyed after 12 h of
heating at 750◦C, whereas the heulandite structure is destroyed
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FIGURE 2 | A simplified schematic of the clinoptilolite structure: linked SiO4tetrahedra and pores with metal cations available for ion-exchange with environmental
cations (e.g., caesium, Cs+) that are consequently trapped into the clinoptilolite pores. As naturally occurring clinoptilolite comes with pre-loaded cations (e.g.,
calcium, Ca2+), ion-exchange may occur depending on the ion-exchange capacity and cation affinity of the material, as well as on physical properties of the
surrounding environment. In the herein presented simplified example, Cs+enters in the zeolite pores instead of Ca2+(adapted from
http://www.chemtube3d.com/solidstate/SS-Z-Clinoptilolite.htm Creative Commons Attribution-Non-commercial-Share Alike 2.0 UK: England and Wales License).
A detailed explanation of the clinoptilolite structure is given in the Database of Zeolite Structures (http://europe.iza-structure.org/IZA-SC/ftc_table.php).
after 12 h at 450◦C (Ghiara et al., 1999). This structural stability
is an essential element for in vivo applications.
For instance, a synthetic material known as Zeolite A,
used widely for ion-exchange in industrial processes, has the
framework composition with a high Al content and the molar
ratio of Si/Al of almost 1. This is indeed the highest aluminum
content possible in tetrahedral alumosilicate frameworks (Payra
and Dutta, 2003). In Zeolite A, the Al-framework is balanced
out by the maximum number of cation exchange sites; it has
high cation contents and superior exchange capacities. However,
it is not appropriate for in vivo applications since, similar to
other low-silica zeolites, zeolite A is unstable in acids. In contrast,
zeolites with higher silica content, such as clinoptilolite, are stable
in acids (Payra and Dutta, 2003).
We present a comprehensive review of clinoptilolite
applications in veterinary and human medicine. We consider all
of the above clinoptilolite properties and propose its mechanisms
of action in vivo (summarized in Table 1) and propose some
comprehensive, scientifically-based hypotheses on possible
biological mechanisms underlying observed effects on the health
and body homeostasis.
USE OF CLINOPTILOLITE IN
VETERINARY AND HUMAN MEDICINE
Studies performed in the last decades showed a high potency of
clinoptilolite in diverse medical applications in vitro and in vivo
(Jurki´
c et al., 2013). A large number of documented positive
clinoptilolite medical effects were attributed to basic clinoptilolite
material properties, in particular, to reversible ion-exchange and
adsorption capacity (Mumpton, 1999;Paveli´
c et al., 2001a;Jurki´
c
et al., 2013). This central clinoptilolite characteristic related to
elimination of toxic agents, which may be seen as a support to
the ‘body homeostasis,’ could be widely exploited in a number of
medical applications. For instance, a high affinity of clinoptilolite
toward ammonia was proven in different systems for elimination
of ammonia from water (Demir et al., 2002;Sprynskyy et al.,
2005;Zabochnichka-´
Swiatek and Malinska, 2010). This is why
clinoptilolite has widely been used for years in animal production
as an additive to animal feed, or for the removal of ammonia in
animal manure (Auerbach et al., 2003). This ammonia affinity is
an interesting feature for medical applications in humans as well.
For example, detrimental roles of the end-products of protein
TABLE 1 | Documented properties and effects of clinoptilolite relevant for biomedical applications and effects in animals and humans.
Clinoptilolite properties Clinoptilolite effects
Cation exchange capacity (Mumpton, 1999;Paveli ´
c et al., 2001a;Paveli ´
c and
Hadžija, 2003)
Detoxicant, mineral donor (EFSA Panel on Additives and Products or
Substances used in Animal Feed, 2013;Jurki ´
c et al., 2013;Exley, 2016;
Kraljevi ´
c Paveli ´
c et al., 2017)
Molecular sieve (size and shape selectivity) (Mumpton, 1999;Paveli ´
c and
Hadžija, 2003)
Impact on the intestine status (Yao et al., 2016)
Selective adsorption of water (Kotova et al., 2016) Immunomodulation (Ivkovic et al., 2004;Montinaro et al., 2013)
Removal of ammonia ions and uremic toxins (urea, uric acid, creatinine,
p-cresol, indoxyl sulfate) (Demir et al., 2002;Auerbach et al., 2003;Sprynskyy
et al., 2005;Joughehdoust and Manafi, 2008;Zabochnichka- ´
Swiatek and
Malinska, 2010)
Effect on pathogens and microbiota (Zarkovic et al., 2003;Saribeyoglu, 2011;
Prasai et al., 2016)
Reversible binding of small molecules (Paveli ´
c and Hadžija, 2003) Enzyme mimetics, metaloenzyme mimicry (Herron, 1989)
Biosensors (Soldatkin et al., 2015) Antitumor adjuvant (Wesley, 1996;Krewski et al., 2009)
Drug carrier/delivery (Hayakawa et al., 2000;Bonferoni et al., 2007) Vaccine adjuvant (Garces, 1999)
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fermentation, such as ammonia, have been recognized on the
colonic microbiota and epithelial health, in particular on the
colonocytes life span and function (Hughes and Magee, 2000;Yao
et al., 2016;Hamid Said, 2018).
The excessive production of ammonia, but also of other
gaseous products, including CO2and H2S, may occur as a
consequence of protein-rich or imbalanced diets, or in diverse
pathogeneses where excessive protein fermentation occurs,
including irritable bowel syndrome, ulcerative colitis, and
colorectal carcinogenesis (Hughes and Magee, 2000;Yao et al.,
2016). Clinoptilolite has a high affinity toward ammonium and
may prove useful in these cases as an adjuvant to the standard
therapy (Yao et al., 2016). From this perspective, clinoptilolite
was evaluated in a recent trial performed on aerobically trained
subjects (Lamprecht et al., 2015). In this study, endurance-
trained subjects were recruited and supplemented with a
clinoptilolite/dolomite/maca-based product (Panaceo SportR
).
Athletes, indeed, often report intestinal symptoms including
nausea, stomach and intestinal cramps, vomiting, and diarrhea.
These symptoms may be a consequence of typical athletes’ diets
with high protein content, as in such circumstances excessive
protein fermentation may occur and is accompanied by higher
ammonia release in the intestine as well. These subjects also
have increased intestinal wall permeability. A well-known and
complex relationship between exercise and oxidative stress
depends on many diverse factors. For instance, regular moderate
exercise increases the resistance against oxidative stress, while
acute and vigorous exercise can generate free radicals in excess.
Consequences of exercise at exhaustion levels include increased
number of leukocytes due to the damage of muscle fibers
and connective tissue (Morillas-Ruiz and Hernández-Sánchez,
2015) as well as elevated lipid-peroxidation marker MDA in the
plasma (Pingitore et al., 2015). It is, therefore, not surprising
that a number of professional athletes show gastrointestinal
symptoms, which may result in medical problems, infections,
and autoimmune disease (Waterman and Kapur, 2012;Oliveira
et al., 2014). Interestingly, the supplementation with Panaceo
Sport positively influenced the intestinal wall integrity, which was
witnessed through decreased concentrations of the tight junction
modulator zonulin, a marker of increased intestinal permeability
(Lamprecht et al., 2015).
Other studies on detoxification properties of clinoptilolite
materials in vivo performed so far have mainly been done
on animals and they provide strong evidence on alleviating
effects during exposure to different toxicants upon clinoptilolite
supplementation. For instance, a prolonged consumption of
water with increased nitrate levels by dairy cattle is known
to impair protein metabolism and glucose utilization. In these
cows, dietary administration of clinoptilolite alleviated the nitrate
burden to the body and reduced the negative systemic effects
of nitrates (Katsoulos et al., 2015). Similarly, a dietary mixture
containing 3% of a clinoptilolite-based product showed an
increase in the nitrogen excretion in feces and a decrease in
the nitrogen excretion in urine in growing pigs. Importantly, no
effects on the protein retention values were observed and the
protein deposition was not altered (Poulsen and Oksbjerg, 1995;
Laurino and Palmieri, 2015).
Moreover, clinoptilolite incorporated into the diet may be
effective in fighting mycotoxins by direct absorption. Affinity
toward aflatoxins, zearalenone, ochratoxin, and the T2 toxin was
proven in vitro in the presence of aminoacids and vitamins,
where the latter were not absorbed by the clinoptilolite material
(Tomasevic-Canovic et al., 1996). The specificity for aflatoxin
M1 was also shown in vivo, and the dietary administration of
clinoptilolite, especially of the material with the smallest particle
size at the rate of 200 g per cow per a day, effectively reduced milk
aflatoxin M1 concentration in dairy cattle (Katsoulos et al., 2016).
It is important to note that the supplementation with
clinoptilolite in dairy cows may have additional benefits, such
as the reduction of parturient paresis. A study by Katsoulos
et al. (2005a), for instance, showed that the clinoptilolite
supplementation reduced its incidence and did not affect
serum concentrations of total calcium, phosphate, magnesium,
potassium, and sodium. This veterinary application showed that
mineral levels in the blood were not affected by clinoptilolite
supplementation which may be relevant for human applications
as well. Indeed, the demand for healthier food products and
a balanced diet is being increasingly recognized as a central
paradigm for the preservation of the body’s homeostasis and
health. Moreover, it is widely known that the contamination
of poultry by food-borne pathogens is considered a major
problem in the poultry industry. This is why antibiotics are
standardly used in poultry meat production. Such a wide use of
antibiotics in poultry, but also in the production of other meat,
has recently been accepted as a major cause for development of
antibiotic-resistant bacteria (Aminov and Mackie, 2007). New,
natural possibilities for improvement of animal health in meat
production have therefore been widely discussed (Diaz-Sanchez
et al., 2015) and clinoptilolite may be a natural alternative.
For instance, clinoptilolite has been tested as a possible
supplementation to broilers feed as an alternative to antibiotics
for: (1) the control of the total flora at broiler farms, where
clinoptilolite supplementation showed a positive effect on the
total flora, a parameter often used in the evaluation of the
gastrointestinal health status in poultry (Mallek et al., 2012),
as well as on the performance of production and organoleptic
parameters, especially on the increase of omega-3 fatty acid
levels in eggs (Mallek et al., 2012); (2) the improvement of
the antioxidant capacity in broilers where the supplementation
of clinoptilolite materials increased the activities of glutathione
peroxidase, catalase, total SOD, and the total antioxidant capacity
(Wu Y. et al., 2013); (3) the reduction of mycotoxin effects on
broilers health, where the number of aflatoxin-affected broilers,
or the number of severe lesions in the liver of chickens,
was reduced in the clinoptilolite-supplemented group (Ortatatli
and O˘
guz, 2001). All these documented effects are due to
the clinoptilolite capacity to adsorb harmful substances in the
gastrointestinal tract that are not confined only to micotoxins and
ammonia but include heavy metals and organic compounds as
well.
Indeed, different studies have shown that clinoptilolite
materials provide direct detoxifying performance in vivo. For
instance, in lead-intoxicated mice, a clinoptilolite sorbent KLS-
10-MA decreased the lead accumulation in the intestine by
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more than 70% (Beltcheva et al., 2012, 2015). Moreover, in rats
exposed to organophosphate poisoning, zeolite tuff containing
61% of clinoptilolite and added 5 min prior to intoxication at
dosage 1 g/kg, proved efficient in the restoration of cholinesterase
activity in the brain, liver, spleen, femoral muscle, heart,
stomach, duodenum, colon, and erythrocytes of intoxicated
animals (Mojzis et al., 1994). Two possible ways of binding
organophosphates may be envisaged. One is the esterification
reaction of the free OH moiety and the carboxyl functional group
of the acid. The second option is through adsorption by forming
a dipol–dipol interaction between the polar channel and/or the
zeolite surface and fluorine, or on the acid. It can generally
be stated that clinoptilolite loaded with potential toxicants in
the intestine is then excreted along with toxicants (EFSA Panel
on Additives and Products or Substances used in Animal Feed,
2013).
It seems that this detoxifying effect may have additional
systemic effects. The role of clinoptilolite has been recognized
in medical applications, where its usage in zootechnology
and veterinary medicine has provided strong evidence on
improvement of pets’ fitness and efficiency in the removal of
numerous harmful substances from the organism, including
radioactive elements, mycotoxins, and poisons (Laurino
and Palmieri, 2015). In addition, EDTA and clinoptilolite
supplementation exerted a protective effect on the brain tissue of
mice intoxicated with lead by inducing antioxidant mechanisms
and greater activity levels of catalase, SOD, glutathione
peroxidase, and glutathione (Basha et al., 2013). Moreover,
a study in humans showed the ability of tribomechanically
micronized clinoptilolite to decrease the absorption of ingested
ethanol by reducing blood alcohol levels at a dose of 5 g (Federico
et al., 2015). If the clinoptilolite-containing product dosage
is lower or if it is not administered at the time of alcohol
consumption, this effect may not be visible as shown by Gandy
et al. (2015) where clinoptilolite still proved highly efficient in the
reduction of veisalgia symptoms and signs up to 40–50%.
In addition, clinoptilolite has interesting antioxidant,
hemostatic, and anti-diarrheic properties that may be exploited
in human medicine, especially as adjuvants to standard therapies
(Paveli´
c and Hadžija, 2003). However, the number of clinical
studies with clinoptilolite materials on humans is still low, and
the previously described immunomodulatory, anticancer, and
antioxidant effects of clinoptilolite in vivo should be studied in
more detail.
Even though the efficacy and potential of clinoptilolite
materials in medicine seems high, questions have been raised
on to possible clinoptilolite effects on physiologically relevant
elements, i.e., micronutrients and trace elements, or effects on
important processes in the organism. The results published thus
far show that clinoptilolite does not affect the homeostasis of trace
elements and micronutrients, but acts rather selectively on heavy-
metals and toxicants. For instance, clinoptilolite-treated dairy
goats showed no changes in serum concentrations of fat-soluble
vitamins, macro-elements, and trace elements, or activities of
hepatic enzymes. In addition, clinoptilolite supplementation
improved milk fat percentage and milk hygiene (Katsoulos et al.,
2009). No effects of clinoptilolite on physiological mineral levels
have been observed in cows (Katsoulos et al., 2005a;Valpoti´
c
et al., 2017).
ZEOLITES EFFECTS ON OXIDATIVE
STRESS AND IMMUNE SYSTEM
In aerobic organisms, production of small quantities of ROS,
including peroxides, superoxides, hydroxyl radicals, and singlet
oxygen, occurs continuously (Hayyan et al., 2016). A controlled
production of ROS is indeed essential to the body’s homeostasis
(Covarrubias, 2008), while an excessive production of ROS is
known to cause damage to the DNA, proteins, and lipids (Gulam
and Ahsan, 2006). Some ROS are produced endogenously,
while others are derived exogenously, such as those formed
by ionizing radiation. The endogenous sources of ROS are the
mitochondria, cytochrome P450 metabolism, peroxisomes, and
inflammatory cell activation (Inoue et al., 2003). For example,
mitochondria-produced ROS are the superoxide anion (O•−
2),
hydrogen peroxide (H2O2), and the hydroxyl radical (·OH).
Other routes and factors may induce ROS in the organism as
well, such as ROS produced through the activity of xanthine
oxidase, in reactions of hypoxanthine to xanthine and xanthine
to uric acid conversions, where molecular oxygen is reduced to
superoxide anion, followed by a generation of hydrogen peroxide
(Valko et al., 2004). It is understood that homeostasis in normal
cells includes a balance between ROS production and antioxidant
defense activity. Indeed, antioxidant mechanisms in the human
body, which are the main regulators of ROS levels, are based on
enzyme and non-enzyme systems. Enzyme systems rely mainly
on SOD, catalase, peroxiredoxin (Prx), thioredoxins (Trx),
and glutathione (GSH) enzymes’ activity, while non-enzymatic
systems comprise flavonoids, vitamin A, vitamin C, vitamin E,
and melatonin (Rahman, 2007). In addition to these antioxidant
systems inherent to the body, other exogenous antioxidants are
important in the regulation of constant body’s ROS homeostasis
as well. For example, dietary compounds are highly important
for elimination of excessive ROS caused by external stimuli and
include, for instance, carotenoids, tocopherols, bioflavonoids,
anthocyanins, and phenolic acid (Smilin Bell Aseervatham et al.,
2013). When ROS production exceeds antioxidant capacity, we
usually perceive the process as “oxidative stress” that leads
to organic damage. Increased oxidative damage to cells and
tissues and the modulation of the ROS-regulated signaling
pathways have recently been acknowledged in the pathogenesis
of a wide number of diseases, including obesity, atherosclerosis,
heart failure, uremic cardiomyopathy, kidney pathologies,
hypertension, neurological disease, and cancer (Chen et al.,
2016;Miranda-Díaz et al., 2016;Patel, 2016;Srikanthan et al.,
2016;Ding et al., 2017). It should be noted that for a proper
functioning of the body, antioxidant defenses, co-factors, or
molecules that activate enzymes by binding to their catalytic sites
are also required. In case of antioxidant enzymes, these co-factors
may include the coenzyme Q10, vitamins B1 and B2, carnitine,
selenium, and often transition metals Cu, Mn, Fe, and Zn
(Khalid, 2007). Recently, a preliminary efficacy study performed
on patients with dyslipidemia has also shown a positive effect of
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clinoptilolite supplementation on lowering the total lipid count
and LDL (low density lipoproteins), which may also be indirectly
correlated with its general antioxidative effect (Cutovic et al.,
2017).
Due to a certain amount of pre-loaded elements, it is possible
to assume that clinoptilolite may positively affect the body’s
metal homeostasis, including either the levels or the availability
of some physiological metal ions that are pre-loaded in the
material, on signal pathways responsible for the production of
endogenous antioxidant enzymes. Still, no direct data supports
these assumptions that may partially explain the observed effects
on the oxidative stress defense mechanisms, which are visible
as activation or restoration of activity and levels of natural
antioxidant enzymes. Still, this effect should be evaluated along
with factors such as, for example, the applied daily dosage,
health status, or lifestyle. For example, in the study of Lamprecht
et al. (2015), the daily dosage of 1.85 g clinoptilolite material
supplementation did not show an effect on the measured redox
markers in the blood of healthy athletes. Furthermore, interesting
effects of clinoptilolite supplementation were documented in
animals as well. In hepatectomized rats, for instance, common
oxidative stress markers are induced upon trauma, including
MDA in the plasma and liver tissue. When hepatectomized rats
were supplemented with a micronized clinoptilolite preparation,
‘Froximun,’ MDA levels were significantly lower, while liver
tissue antioxidant mechanisms were strengthened, as witnessed
by a significantly higher activity of Cu-Zn SOD and GSH
(Saribeyoglu, 2011). Also, in chicken, daily supplementation with
a natural clinoptilolite, or a modified clinoptilolite, efficiently
improved the antioxidant capacity by increasing the antioxidant
enzyme activities in intestine mucosa and decreasing the free
radical NO content and inducible nitric oxide synthase activity
in the serum. Moreover, upon prolonged supplementation
in chicken, both tested clinoptilolite materials increased the
activities of glutathione peroxidase, catalase, total SOD, and
the total antioxidant capacity (Wu Q.J. et al., 2013). Similarly,
in doxorubicin treated mice, micronized clinoptilolite proved
efficient in counteracting lipid peroxidation in the liver (Zarkovic
et al., 2003).
An interesting effect of clinoptilolite was observed in
fluoride-intoxicated rats (Madhusudhan et al., 2009). Fluoride
is neurotoxic upon penetration through the blood–brain
barrier during gestation and post-gestation periods. As a
consequence of fluoride-intoxication, inhibition of antioxidant
enzymes occurred in pups along with lipid peroxidation.
Upon supplementation of pups with clinoptilolite, oxidative
damage was restored and levels of GSH-Prx were substantially
ameliorated in the cerebral cortex and medulla oblongata. Similar
results were, however, observed in animals supplemented with
vitamins E and C as well (Madhusudhan et al., 2009). In
line with these results, it should also be hypothesized that
clinoptilolite might have the potential to combat acute fluoride-
intoxication in animals, as well as in humans. In the gastric
juice, fluoride anions are converted into hydrofluoride acid. Such
a weak hydrofluoride acid may form hydrogen bonds with the
clinoptilolite framework and be eliminated from the body in the
stool.
We believe that exact mechanisms of clinoptilolite effects on
systemic restoration of homeostasis and increased antioxidant
capacity are still not fully understood, as these effects are in
our opinion probably connected both to general detoxifying
effects occurring in the intestine, to immunomodulatory effects,
or even to the release of physiologically-relevant cations from the
clinoptilolite framework during the ion exchange process, e.g.,
Ca, Mn, Zn, and Mg, which are then readily available to the
organism and the antioxidant mechanism. Similar indirect effects
of clinoptilolite on the antioxidant mechanisms in the body were
also observed in different pathologies and disease models. For
instance, tribomechanically-micronized zeolite increased SOD
activity in a transgenic mouse model of the Alzheimer disease
in the hippocampus and cortex, while it concomitantly reduced
Aβ(x-42) amyloid beta levels in the hippocampus (Montinaro
et al., 2013). Moreover, zinc-bearing clinoptilolite proved to exert
a protective effect on the performance and gut health of broilers
against S. pullorum infection as well as to improve the SOD
activity of ileal mucosa and reduced MDA contents of jejunual
and ileal mucosa (Wang, 2012).
It is also possible that antibacterial and antiviral effects of
clinoptilolite might be in correlation with immunomodulatory
properties. For instance, in long-term supplementation with
clinoptilolite, a decreased prevalence of E. coli carrying certain
antimicrobial resistance and virulence genes was documented
(Jahanbakhsh et al., 2015). An influence of natural clinoptilolite
on E. coli was also documented in another study on broilers
in vivo (Wu Y. et al., 2013). In this study, a beneficial effect
on intestinal parameters was measured, which was hypothesized
to be based on a direct effect on the microbial population in
the intestine. While the total count of E. coli was significantly
reduced, a rise of Lactobacillus acidophilus occurred in parallel
(Zarkovic et al., 2003). Similarly, clinoptilolite supplementation
of EnterexR
, approved by the Cuban Drug Quality Control
Agency, showed to be highly efficient in ameliorating diarrhea
symptoms in several clinical studies on humans with acute
diarrhea of different etiologies. Moreover, in cases where diarrhea
symptoms were removed and the pathogenic agent was identified
upon Enterex treatment antibiotics were additionally used to
completely eliminate pathogenic bacteria from the intestinal
lumen (Rodríguez-Fuentes et al., 1997). Therefore, this observed
antidiarrheal activity may be in correlation with EnterexR

effect on certain pathogenic bacteria count or the microbiota
status in general rather than with a direct antibacterial effect,
which would have to be confirmed by additional studies.
Recently, a positive effect of a potentiated clinoptilolite material
(AbsorbatoxR
) was also shown to reduce symptoms associated
with endoscopically negative gastroesophageal reflux disease and
non-steroidal anti-inflammatory drug-induced gastritis, where it
significantly prevented mucosal erosion severity (Potgieter et al.,
2014).
Similarly, antiviral properties for clinoptilolite in vitro were
shown on the human adenovirus 5, herpes simplex virus
type 1, and the human enteroviruses coxsackievirus B5 and
echovirus 7 (Grce and Paveli´
c, 2005). This effect may probably
be attributed to a direct adhesion of the viral particles on
clinoptilolite in vitro, which then inhibits viral entrance in
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the cells and viral replication. Even though no in vivo studies
on clinoptilolite antiviral activity have been published thus
far, positive immunomodulatory effects have been observed in
patients treated for immunodeficiency disorders. In a study
performed by Ivkovic et al. (2004), a significant increase in
specific immunity cell counts, B lymphocite CD19+, T-helper
cells CD4+, and activated T-lymphocytes HLA-DR+were
observed in subjects treated with tribomechanically micronized
clinoptilolite. This effect was accompanied by significantly
decreased natural immunity NK CD56+cell counts. Again,
standard blood count parameters of patients remained within
normal referent values (Ivkovic et al., 2004).
A hypothesis for the observed clinoptilolite
immunomodulatory effects may be the modulation of body
defense mechanisms toward ROS. Indeed, ROS induces cell and
tissue damage when the inflammation is initiated as a mechanism
for restoration of the body’s homeostasis. Any impairment of
the host immune and inflammatory mechanisms in the long-
term may cause other inflammatory disorders, e.g., chronic
sinusitis, otitis media and osteomyelitis, or microbial overgrowth
syndromes, such as bacterial vaginosis, or inflammatory bowel
disorders. It is plausible, therefore, to assume that such disorders
have the formation of biofilms in common due to the impaired
immunological reaction of the host organism (Pincus, 2005).
Indeed, previous studies have shown a link between the
antioxidative effect and the stimulation of the immune system
(Knight, 2000;Brambilla et al., 2008).
Clinoptilolite’s positive immunomodulatory effects in similar
conditions may be due to the interactions of clinoptilolite
particles in the intestine with microfold cells (M-cells) (Figure 3).
M-cells are found in the GALT of Peyer’s patches, a rich lymphoid
tissue that communicates with intestinal epithelial cells and
the microbiome of the intestine by diverse immunomodulation
processes as well as in the MALT of other parts of the
gastrointestinal tract. These gastrointestinal cells are known to
initiate mucosal immunity responses on the apical membrane
of the M-cells and to allow the transport of microbes
and particles across the epithelial cell layer from the gut
lumen to the lamina propria where interactions with immune
cells occur (Mabbott et al., 2013). While evaluating possible
clinoptilolite immunomodulatory effects in the intestine, it
should be emphasized that M-cells can uptake nano- and
submicro-particles, which can probably induce changes in the
redox homeostasis in a cell (Igarashi, 2015). These changes
in the M-cells then affect the Peyers patches as well. It is
important to note that M-cells apical and basolateral sides, which
communicate with Peyers patches, are polarized (Society for
Mucosal Immunology, 2012) and one may hypothesize that, due
to this particular phenotype, M-cells retain clinoptilolite particles
or silica particles released from the clinoptilolite material (tuff),
which do not enter the blood system (Nizet et al., 2018) and
act locally on this tissue. Contrary to M-cells, other cells in the
intestine cannot perform macropinocytosis and therefore cannot
absorb negatively charged clinoptilolite particles or silica particles
released from the clinoptilolite material (tuff) due to their
rich negatively-charged glycoprotein-polysaccharide covering,
glycocalix (Egberts et al., 1984). Some probiotics’ metabolites,
e.g., from the lactic acid bacteria, exert the same activating
function on Peyers patches as we suggest for clinoptilolite
particles or silica particles released from the clinoptilolite material
(tuff) and improve intestinal wall integrity (Sung et al., 2016).
Therefore, we propose that this clinoptilolite-induced M-cells’
communication with Peyer’s patches, as similarly shown by
Pavelic et al. (2002), increases the immune response either
through particle intake or microbiota effect as recently shown
in dogs supplemented with the zeolite chabazite (Sabbioni et al.,
2016), and in particular, stimulates IgA producing B lymphocytes
(plasma cells), a defensive mechanism of the intestinal tract
against pathogenic bacteria (Round and Mazmanian, 2009).
In the paper by Nizet et al. (2018), however, (Egberts et al.,
1984), no clinoptilolite particles were detected in the selected
sections of the gut tissue. Even though the inspection of
limited histopathological sections in this study cannot rule
out the suggested hypothesis on clinoptilolite particles or
silica particles released from clinoptilolite material (tuff) in
activation of Peyer patches, experimental analysis of the observed
local immunomodulatory effect should be conducted in more
detail. Indeed, the microbiota-clinoptilolite interaction may also
underlie the observed immunomodulatory mechanism as well.
Indeed, a role of IgA was already described in the reduction
of intestinal pro-inflammatory signaling and bacterial epitope
expression as part of the innate immune mechanism that
contributes to balancing antibodies’ negative impact on the
micriobiota status (Round and Mazmanian, 2009). Evidence
was provided on the role of cross-talking between the adaptive
immune system and gut microbiota by selective generation
of immune responses to bacteria that consequently stimulate
the innate system and production of IgA. By means of this
mechanism, the host can detect new bacterial types and ignore
previously encountered bacteria in the intestine (Peterson et al.,
2007). This immunomodulatory effect of clinoptilolite was
speculated to be the so-called ‘silicate superantigen’ response. The
superantigens generally encompass some bacterial exotoxins and
viral products with a potent non-specific immuno-stimulatory
effect on large T-cells fractions. This immunostimulation occurs
upon simultaneous interaction of the superantigen with MHC
class II molecules and T-cell receptors. Superantigens bind to the
variable Vβregion of the T-cell receptor or to CD28 and do not
follow the peptide-binding pattern. An incredibly heterogeneous
T-cell clonal activation occurs upon binding and different
cytokines are produced massively (Proft and Fraser, 2016).
The superantigen-activated T-lymphocytes provoke the cellular
immune response and also the humoral immune response,
as postulated by Emmer et al. (2014) in multiple sclerosis
pathogenesis as well. Lymphocytes stimulation by silicates, which
also act as superantigens, was already shown for different silicate
materials in in vitro conditions and this mechanism may underlie
immunomodulation activity of clinoptilolite in the intestine
as well (Ueki et al., 1994;Aikoh et al., 1998). Even though
the exact mechanisms remain elusive, one may speculate that
clinoptilolite silica or released silica acts as a superantigen
that promotes the formation of IgA producing plasma cells,
which is dependent on the presence of superantigen-reactive
T cells. A similar superantigen effect was already observed in
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Peyer’s patches during milk-borne mouse mammary tumor virus
infection (Cabrera et al., 2010). To our knowledge, no negative
effects on immune cells or tissue were documented in the
scientific literature so far. Also, we cannot rule out some other
unrecognized immunomodulatory effects of clinoptilolite due to
a direct interaction with human microbiome (Figure 3).
The majority of studies on clinoptilolite were done by using
different, so-called activated materials to increase either the
surface area or to improve the clinoptilolite general adsorption or
the ion-exchange capacity. Activation may be performed either
through chemical treatment, e.g., with an acid, by replacing
stabilizing cations, or through mechanical modifications by
means of different micronization methods, which may all
increase the surface area and change the ion-exchange properties
and adsorption capacity (Abdulkerim, 2012;Akimkhan, 2012;
Canli et al., 2013b). In the paper by Kraljevi´
c Paveli´
c et al.
(2017), it was specifically shown that different micronization
methods change the clinoptilolite tuff properties by affecting
the surface area, pore size, and silicon to aluminum ratio on
the surface of the material. Moreover, hydrochloric acid (HCl)
that is also present in the stomach may change clinoptilolite
physical chemical properties and has been proven to enhance
the clinoptilolite ion-exchange capacity for Cu2+and Co2+
in a synthetic Cu-Co solution at concentrations relevant for
the stomach in vivo (0.1 M) (MambaI et al., 2010). Still,
the clinoptilolite ion-exchange effects in vivo are complex and
cannot be linearly explained as they are not affected only
by the environmental conditions (pH, temperature, etc.) but
also by the material composition and cation affinity properties.
In a recent article, Turkish clinoptilolite was activated with
hydrogen peroxide, which acts as a weak acid, to improve Ni2+
ions removal from aqueous solutions (Çanli and Abali, 2016).
The authors show changes on the clinoptilolite surface upon
activation that resulted in an improved Ni-ions absorption. This
is important, as hydrogen peroxide dissociates into hydrogen
ion H+and hydrogen peroxide radical (HO•
2)−, and during the
acid-activation process H+ions are brought to the negatively-
charged species on the material surface. As a consequence, de-
alumination of the surface occurs, which increases the Si/Al
surface ratio and absorption capacity for metal cations. This is
a well-known process in industrial applications, while for the
in vivo applications it may also hold certain relevance. In vivo,
the acid concentrations of the intestine are substantially lower
than those used in industrial activation process. For instance,
gastric acid in the stomach contains HCl at 0.05–0.1 M. In such an
environment, a certain release of Al species from the clinoptilolite
surface may well be hypothesized even though aluminum
from the clinoptilolite materials does not enter the blood or
accumulate in the body as shown in athletes supplemented
with zeolite-clinoptilolite supplement (Lamprecht et al., 2015) or
healthy rats supplemented with different clinoptilolite materials
(Kraljevi´
c Paveli´
c et al., 2017) where aluminum released into
systemic circulation was observed only in rats supplemented with
synthetic zeolite A. The latter effect was attributed to the zeolite A
lower stability in the acidic pH relevant for the human intestine
in comparison to clinoptilolite materials. In this study, authors
FIGURE 3 | Proposed model of clinoptilolite positive immunomodulatory effect in the intestinal epithelium (denoted with red arrows) through interaction of
clinoptilolite tuff particles with microfold cells (M-cells). Clinoptilolite tuff released particles are denoted by ‘C.’ M-cells are hypothesized to transport luminal
clinoptilolite tuff released particles across the epithelial barrier and present them to immunological cells (e.g., dendritic cells) in the lamina propria and the Peyer’s
patches. The latter are rich in T cells, macrophages, and clinoptilolite- activated IgA secreting B and plasma cells. The single layer of the intestinal epithelium is
protected by mucus containing mucin glycoproteins where immunoglobulin A (IgA) and antimicrobial peptides prevent interaction of microbiota with the cell surface.
Question marks (?) and blue arrows denote still unknown interactions of clinoptilolite with microbiota and microbiota with the lumen and epithelia.
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also proved that clinoptilolite materials were efficient in the
removal of aluminum from aluminum chloride-intoxicated rats
in vivo. These observations may be attributed to the clinoptilolite
stability, the low bioavailability of Al species from water (around
0.1 to 0.4%), and the immediate precipitation of Al-species as
non-soluble forms. Aluminum(III)-cation (Al3+) has a generally
strong affinity for anions which promote its precipitation. The
Al3+in most situations seeks out complexing agents with
oxygen-atom donor sites, such as carboxylate or phosphate
groups, e.g., from food in the intestine. However, it should be
noted that the aqueous coordination chemistry of Al3+, especially
in the living systems, is rather complex due to the Al-complexes’
tendency to hydrolyze and form polynuclear species, which vary
according to the pH condition of the medium (Wesley, 1996;
Krewski et al., 2009). Interestingly, oral aluminum bioavailability
is known to be increased by acidic pH, such as the pH in the
human intestine, but in case of clinoptilolite tuff, it may be
decreased, as this is a silicon-containing compound that releases
certain amounts of dissolved silica (Jurki´
c et al., 2013). Data has
been provided on the ability of silicon-rich mineral water or
silicic acid to remove Al from the human organism (Buffoli et al.,
2013;Davenward et al., 2013), and this Si and Al relation has
been recognized as the main evolutionary mechanism for fighting
ecotoxicity of aluminum in living organisms. Water-soluble silica
forms may thus be acknowledged as important contributors to
fighting aluminum detrimental effects on human and animal
health, especially nowadays when the exposure to bioavailable
free aluminum cation poses a serious problem due to industrial
development (Exley, 2009;Beardmore et al., 2016;Exley, 2016).
In addition, we hypothesize that previously observed data
on antitumor properties of clinoptilolite in vitro may be
due to the activation of clinoptilolite surface by acids. Even
though in the majority of in vitro studies, the cells were
grown in micronized clinoptilolite pre-treated growth media, no
ultracentrifugation was employed, which means that a colloid
system containing finest clinoptilolite particles was used for
experiments (Paveli´
c et al., 2001b;Katic et al., 2006). For instance,
it is well-known that tumor cells have increased hydrogen
peroxide levels that regulate specific signaling pathways and
hydrogen peroxide may modify cysteine residues on antioxidative
enzymes (Lennicke et al., 2015). Enzymes are deactivated during
modification. Clinoptilolite can react with hydrogen peroxide
(Canli and Abali, 2016), similar to other silica particles, and,
in such situations, oxidative stress is induced either through
the breakdown of hydrogen peroxides to hydroxyl radicals or
through the breakdown of hydrogen peroxides and production
of the hydroperoxyl radicals (Rochette and Vergely, 2008).
Therefore, it is possible that the contact between clinoptilolite
and tumor cells with increased hydrogen peroxide concentrations
induces formation of free radicals; therefore, increases in the
oxidative burden occur in tumor cells, which consequently die.
Tumor cells are susceptible to increased oxidative stress and
in our previous experiments, this effect was not visible or was
lower in normal tested fibroblasts in vitro (Katic et al., 2006).
Also, it cannot be ruled out that some clinoptilolite particles
enter into tumor cells in vitro, as tumor cells are inherently
depolarized (Yang and Brackenbury, 2013) and can uptake
particles by endocytosis (Sincai et al., 2007). Recently, a new
hypothesis has been suggested on the use of lipophilic anions
that target cancer cells due to their distinct electrical properties
(Forrest, 2015). As clinoptilolite particles are negatively-charged
polyanions, they might also target cancer cells and induce
additional oxidative stress upon entrance into the cytoplasm
through hydrogen peroxide activation, increased production of
ROS and its consequent depletion within the cell. The depletion
of hydrogen peroxide and the increased ROS production during
hydrogen peroxide reaction with a clinoptilolite surface may
change the redox status of the cell, e.g., through inhibition
of the transcription factor Nrf2. Indeed, in previous in vitro
experiments on tumor cells, clinoptilolite antitumor effects were
attributed to the modulation of the EGF-R, protein kinase B
(PKB)/Akt, and nuclear factor kB (NfkB) signaling. They are
interconnected with ROS and activity of Nrf2 (Paveli´
c et al.,
2001b;Katic et al., 2006). This might be highly relevant for
the survival of cancer cells as Nrf2 bears a proliferative role. In
tumor cells, Nrf2 is usually activated by ROS-induced oncogenes,
such as KRAS and c-MYC (DeNicola, 2011), and inhibition of
its activity may contribute to the apoptosis of tumor cells and
abrogated tumor growth (Ryoo et al., 2016).
CLINOPTILOLITE TOXICOLOGY IN
ANIMALS AND HUMANS
The basic structure of clinoptilolite is considered to be
biologically neutral and non-toxic (Auerbach et al., 2003). EFSA
recently released an expert opinion on the safety of natural zeolite
clinoptilolite in vivo (EFSA Panel on Additives and Products
or Substances used in Animal Feed, 2013). EFSA evaluated and
proved the zeolite-clinoptilolite non-toxicity for animal feed at
doses of 10000 mg/kg. Oral consumption of this type of zeolite,
due to its extreme chemical stability, in EFSA’s opinion, does not
represent a potential risk for in vivo applications (EFSA Panel
on Additives and Products or Substances used in Animal Feed,
2013).
The first comprehensive acute, subchronic, and chronic
toxicology evaluation of a clinoptilolite material in vivo was
performed by Paveli´
c et al. (2001b). In this preclinical toxicology
study, tribomechanically micronized clinoptilolite was evaluated
at the ‘Ruer Boškovi´
c’ Institute in Zagreb, Croatia, according to
the standards and regulations required at the time by the OECD.
In that study, the effects associated with increasing exposure
times were analyzed in three categories: (1) acute toxic responses
up to 1 month in mice and rats, (2) subchronic toxic responses
up to 3 months in mice and rats, and (3) chronic toxic responses
up to 1 year in rats and 6 months in mice. Clinoptilolite was
administered to the animals as a powder supplementing their
usual diet. Toxicity studies were approached by setting a “limit”
test, which means that high doses of the substance were applied
during 15 or more days. Two doses were selected from the
“limit” test, 400 mg/mice/day (3.2 times higher than the dose
specified by the regulatory agency) and 1000 mg/mice/day (8
times higher). Recalculated from human use, they were 10 and 25
times higher than envisaged potential human exposure dosages
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(60 g/75 kg human body weight and 150 g/75 kg human body
weight). The results showed that the “limit” test doses of the
substance did not cause death for mice. Therefore, the “up
and down” test on mice was performed with doses ranging
from 60 to 400 mg/mice/day. Again, no toxicity was observed.
Classical acute, subacute, and chronic tests on rats and mice
were performed as well. Oral (in diet) administration to mice
and rats showed no effects or changes that could be correlated
to tribomechanically micronized clinoptilolite-supplementation.
In addition, earlier in Pond and Yen (1983) published the
first study on the clinoptilolite effects on the reproduction and
progeny growth in rats with or without cadmium presence. They
have shown protective effects of clinoptilolite on hematocrit and
hemoglobin levels as well as on cadmium levels in the liver
of pigs fed with cadmium in the presence of clinoptilolite in
comparison to animals fed only with addition of cadmium to the
diet.
Similarly, in another study performed by the European
Union Cosmetic Ingredient Review Expert Panel, natural
clinoptilolite showed no effects on female rat reproductive
performance and it proved non-genotoxic in the Ames bacterial
test system (Elmore, 2003). Moreover, in an independent
study performed by Martin-Kleiner et al. (2001) effects
of tribomechanically micronized clinoptilolite on the serum
chemistry and hematopoiesis were evaluated in mice. The authors
showed that the ingestion of clinoptilolite was well-tolerated
and substantiated by unchanged body mass in clinoptilolite-
supplemented mice. An increased level of potassium by 20%
was detected in mice receiving the clinoptilolite-rich diet, while
other changes in the serum chemistry were not observed.
Erythrocyte, hemoglobin, and platelet levels in peripheral
blood were not affected by clinoptilolite supplementation
either.
Also, Muck-Seler and Pivac (2003) studied the effects
of tribomechanically micronized and non-micronized
clinoptilolite materials on the serotonergic 5-hydroxytryptamine
receptors 5-HT(1A) and 5-HT(1B) in the brain of non-
tumorous (control) and mammary carcinoma-bearing
female mice. A reduced binding of 3[H]8-hydroxy-2-(di-n-
propylamino)tetralin (3H-8-OH-DPAT) to 5-HT(1A) receptors
in mammary carcinoma-bearing mice was normalized in
animals supplemented by tribomechanically-micronized
clinoptilolite. Also, the administration of clinoptilolite
materials did not affect the binding of 3H—8-OH-DPAT
to the studied receptors during prolonged administration.
The authors speculated that the observed effects in tumor-
bearing mice may be in correlation with the electrolytes
balance, or immune system response to supplementation.
A neuroprotective effect was also documented by Basha et al.
(2013). Safety of the material was also proven by Ivkovic
et al. (2004) where no adverse reactions to tribomechanically
micronized clinoptilolite supplementation were observed in
immunodeficient patients.
Some concerns were raised in public on the possible
lead leakage from the natural clinoptilolite materials into the
intestine. Still, extremely high affinity of clinoptilolite to lead
has been documented previously, where sorption of lead and
cadmium (Cd) on natural clinoptilolite was shown to be
irreversible or very slowly reversible (Hamidpour et al., 2010),
and, in particular, it was shown to be high in an acidic
environment (Peri´
c et al., 2004). These results were obtained in
very simple in vitro models that may not adequately mimic
human digestion. Furthermore, a high capacity of zeolite lead
adsorption occurs in the pH range 3–11 (Payne and Abdel-
Fattah, 2004) and the leaching of lead from lead-preloaded
clinoptilolite occurs mainly in pH under 1, which is not
relevant to conditions in the human body, as shown by
Petrakakis et al. (2007). The authors conducted the study
according to the standard procedures, Toxicity Characteristic
Leaching Procedure/Environmental protection agency/Resource
Conservation and Recovery Act (TCLP/EPA/RCRA) (1311), EPA
Methods 1310, 1320 and DIN 38414-S4, and provided evidence
of the pH being the main factor affecting Pb leaching from
clinoptilolite. Interestingly, in the pH 3 and higher Pb, the
leakage was less than 1%, while at pH 1 the leakage was
observed up to 20% of the initial lead content. Furthermore,
the authors show that the re-adsorption of Pb particles that
leach from the solid material may occur as well; for lead this
process occurred at pH 1.5 and 2. The Pb leaching percentage
may, in the authors’ opinion, be generally correlated with an
increasing initial load but is not affected by the agitation rate
or particle size. Also, previously published results from trials
on animals and human subjects showed a strong clinoptilolite
detoxifying effect and reduction of Pb content in vivo. For
instance, tissue lead concentrations in lead-intoxicated rats
with or without clinoptilolite supplementation clearly show
that Pb concentrations were not increased in animals fed with
clinoptilolite and that the intoxication burden in animals can
be even alleviated by clinoptilolite supplementation (Beltcheva
et al., 2012, 2015;Basha et al., 2013a). Similarly, in the study
by Fokas et al. (2004), clinoptilolite was added to the diet
of growing pigs at 20 g/kg and no significant increase of
Pb concentration in blood and edible tissues was measured.
In this study, however, Pb levels were not discussed in the
context of stored Pb levels in the bones and Pb levels in the
bones were not assessed. This is why definite conclusions on
eventual lead fate in the blood and organism of animals fed
with clinoptilolite supplemented diet in this study cannot, be
conclusive. Moreover, a clinical study comprising 22 human
subjects evaluated the effects of clinoptilolite treatment on
chronic diseases which could be traced back to heavy metal
poisoning. During treatment with activated clinoptilolite from 7
to 30 days in total, both urine and blood serum were collected
and tested for heavy metals and electrolytes. In this study, the
daily intake of activated clinoptilolite suspension was effective
in removal of toxic heavy metals from the body via urine
(Flowers et al., 2009). Urine is, indeed, important in elimination
of lead released from the bones or body compartments, i.e.,
in chelation therapy where upon quenching of lead from
different sites of the body it is expelled through urine (Flora
et al., 2012). The high lead leakage from the material into the
body is theoretically possible but this would eventually happen
from tuff materials with extremely high content of lead where
the theoretical absorption would be subject to many different
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physiological parameters and health conditions. Another clinical
study on human subjects showed detoxifying effectiveness of
clinoptilolite. A total of 102 heavy-metal contaminated men were
investigated and decreased concentrations of harmful metals
(Cd, Pb, Cu, Cr, and Ni) were measured in their hair after
a 30-day supplementation with clinoptilolite. This decrease in
harmful metal concentrations was a result of the clinoptilolite
detoxification function and probable restoration of the body
mineral metabolism homeostasis (Zhakov, 2003). Importantly,
while great danger exists in removing the physiologically
important electrolytes from the serum in a classical detoxification
process, this has not been observed in clinoptilolite trials
both in humans and animals, where no substantial changes in
physiologically relevant trace elements or vitamins were observed
even after long-term administration (Papaioannou et al., 2002;
Katsoulos et al., 2005b;Flowers et al., 2009).
In conclusion, clinoptilolite materials tested in the scientific
literature proved to be generally safe for in vivo applications
even though each material seems to retain its own physical-
chemical characteristics and exerts specific biological effects
that cannot be readily transferable to other materials. Different
particle sizes, surface areas, and cation compositions may
induce different biological effects and exert different levels
of effectiveness. Biological effects and toxicology data should
therefore be carefully evaluated according to the type of
clinoptilolite material or clinoptilolite-based preparations used in
a particular study or application. In this paper, the cited literature
on clinoptilolite effects in vitro and in vivo provides data for
clinoptilolite materials (tuffs) from different sources/continents,
of different purity, chemical composition, and that were prepared
for oral application by use of different milling processing
methods. Moreover, the research goals and experimental designs
were different. This is why no absolute generalization on the
mechanisms of action for clinoptilolite materials (tuffs) may be
done at this point. Still, presented studies provide intriguing data
on positive medical effects for this type of materials, especially
effects on the immune system and detoxification, all substantiated
by so far presented safe profile. In the future, it would be
highly helpful to gather scientific data on the direct relationship
between specific clinoptilolite material properties and sources
with positive or negative effects and mechanisms of action in vivo.
This will fill in the current gaps in research presented so far as
similarly suggested by Colella (2011). Collela also emphasized
the variability and heterogeneity of the clinoptilolite material
used in different applications and studies and suggested to
study in detail the applications and mechanisms of clinoptilolite
materials in light of known and well-established properties or
behaviors.
CONCLUSION
In agreement with the scientific evidence presented in the
literature so far, it can be generally stated that clinoptilolite-
based materials, including the so-called activated materials,
may be regarded as safe for in vivo consumption. A variety
of highly positive effects on animal and human health were
documented thus far for clinoptilolite-based materials. Due
to clinoptilolite’s remarkable ion-exchange and adsorption
properties and consequent detoxifying effects, it has proven
useful in the elimination of a variety of contaminants from the
body or in amelioration of the intestinal status. An indirect
systemic detoxification effect attributed to clinoptilolite-based
material supplementation in the diet of both animals and
humans was documented in other organs as well, e.g., liver.
However, the observed positive systemic mechanisms are still not
completely understood. We hypothesize that they may be at least
partially attributed to the restoration of the human homeostasis
due to local detoxification properties within the intestine, the
release of dissolved silica forms from the clinoptilolite tuff
that enter from the intestine into the blood, as well as to
clinoptilolite’s immunomodulatory effects. The observed local
immunomodulatory effects of clinoptilolite involve the induction
of immune responses through Peyer’s patches and/or possible
positive effects on microbial intestinal populations through still
unknown mechanisms. These local effects may have a systemic
‘echo’ on the whole immune status as well, as observed in some
studies.
Finally, clinoptilolite’s antioxidant effects and restoration
of antioxidant defense mechanisms may also be linked to the
positive general systemic impact. However, conclusive statements
on the exact applications and benefits of clinoptilolite-
based materials in humans should be carefully investigated
and analyzed for each specific clinoptilolite material as
the mechanisms of action may have correlations with the
specific material’s physical and chemical properties. Currently,
different clinoptilolite-containing materials are used in medical
applications worldwide. These materials contain different
percentages of clinoptilolite and different compositions. Also,
clinoptilolite-containing natural tuffs come with small quantities
of other trace elements, and clinoptilolite is always pre-loaded
with various cations. Some of the alkaline ions contained in
the crystal lattice, mainly Na+, Ca2+, Mg2, and K+, may be
readily released during the ion-exchange process. While these
parameters may not be that relevant for agricultural or industrial
applications, veterinary and human applications would require
a higher level of control via a quality control system in the
production, both of the raw material and the final products.
For example, a proper mining process with adequate cleaning,
sieving, de-hydrating, and pre-milling processes, along with
elemental and microbiological examination of the clinoptilolite
materials, might be considered among essential requirements for
ensuring purity and quality (in relation to the high clinoptilolite
content in the tuff) of the final materials for in vivo consumption.
AUTHOR CONTRIBUTIONS
SKP generated the main idea and wrote the manuscript,
generated and shaped presented hypotheses, performed literature
search and analysis, prepared the figures and tables, discussed
and systematized all literature data. JSM prepared parts related
to clinical aspects of clinoptilolite effects in vivo, was involved in
the preparation of the table. DG performed the literature search,
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participated in writing of the manuscript related to oxidative
stress and immune system, and participated in shaping of the
hypothesis of zeolite molecular effects in vivo. AF performed
literature search on physical-chemical properties of clinoptilolite
and wrote parts of the manuscript related to clinoptilolite
chemistry. NP performed a critical review of data and literature,
edited the paper content and its final content. KP performed
literature search related to clinical aspects and toxicology,
discussed clinical aspects, and helped to draft the manuscript.
FUNDING
We greatly acknowledge the support of the University of Rijeka
(Grant Nos. 13.11.1.1.11 and 13.11.1.2.01). NP was funded by
the European Research Council (ERC) Starting Independent
Researcher Grant 278212, the European Research Council
(ERC) Consolidator Grant No. 770827, the Serbian Ministry
of Education and Science Project III44006, the Slovenian
Research Agency (ARRS) Project Grant No. J1-8155, and the
awards to establish the Farr Institute of Health Informatics
Research, London, from the Medical Research Council, Arthritis
Research UK, British Heart Foundation, Cancer Research UK,
Chief Scientist Office, Economic and Social Research Council,
Engineering and Physical Sciences Research Council, National
Institute for Health Research, National Institute for Social
Care and Health Research, and Wellcome Trust (Grant No.
MR/K006584/1).
ACKNOWLEDGMENTS
We acknowledge the project “Research Infrastructure for
Campus-based Laboratories at University of Rijeka,” co-financed
by European Regional Development Fund (ERDF).
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Frontiers in Pharmacology | www.frontiersin.org 14 November 2018 | Volume 9 | Article 1350