Protective Effects of Lignite Extract Supplement on Intestinal Barrier Function in Glyphosate-Mediated Tight Junction Injury

Abstract

Background: Disruption of tight junctions is associated with various inflammatory diseases, including inflammatory bowel syndrome. Tight junctions form a protective barrier throughout the intestinal tract that is central to health. Glyphosate is known to inhibit tight junction function. The aim of this study was to assess the effect of a soil-derived, lignite extract dietary supplement in protecting against glyphosate’s effect on the tight junctions in cell culture.
Methods: Small bowel (IEC-6) and colon epithelium (Caco-2) cells were incubated until a stable Transepithelial Electrical Resistance (TEER) was measured. A control and the lignite extract supplement (20% concentration) were placed on the cells and left overnight. These cells were then treated with and without glyphosate at 10 mg/ml. TEER was measured at 30 min. The expression of the tight junctions was determined by immunofluorescent microscopy of the ZO-1 tight junction element before and after overnight incubation, and following glyphosate exposure.
Results: TEER increased with the lignite extract on IEC-6 (95%) and Caco-2 (35%) compared to the control. Glyphosate dramatically decreased the TEER in both IEC-6 (80%) and Caco-2 (76%) cells. The lignite extract blocked the glyphosatemediated decrease in TEER.
Conclusion: The lignite extract supplement blocked a glyphosate-dependent decrease in TEER in both cell lines. If found efficacious in clinical trials, RESTORE could prove helpful in combating intestinal sensitivities caused by glyphosatemediated tight junction disruption.

Full text

Journal of Clinical Nutrition & Dietetics
ISSN 2472-1921
2017
Vol. 3 No. 1: 1
iMedPub Journals
ht
t
p://www.
i
m
e
dpub.
c
om
Research Article
DOI: 10.4172/2472-1921.100035
1
© Under License of Creative Commons Attribution 3.0 License | This article is available in: http://clinical-nutrition.imedpub.com/archive.php
John J Gildea1,
David A Roberts2 and
Zachary Bush3
1 Department of Pathology, University of
Virginia, Charloesville, Virginia
2 Chief Public Health Ocer, Biomic
Sciences, LLC, Charloesville, Virginia
3 Director of Clinical Aairs, Revoluon
Health Center, Charloesville, Virginia
Corresponding author: Zachary Bush
 zmbush@gmail.com
Director of Clinical Aairs, Revoluon Health
Center, Charloesville, Virginia.
Tel: 434-321-5257
Citation: Gildea JJ, Roberts DA, Bush
Z. Protecve Eects of Lignite Extract
Supplement on Intesnal Barrier Funcon in
Glyphosate-Mediated Tight Juncon Injury. J
Clin Nutr Diet. 2017, 3:1.
Received: December 12, 2016; Accepted: January 09, 2017; Published: January 16,
2017
Abstract
Background: Disrupon of ght juncons is associated with various inammatory
diseases, including inammatory bowel syndrome. Tight juncons form a
protecve barrier throughout the intesnal tract that is central to health.
Glyphosate is known to inhibit ght juncon funcon. The aim of this study was to
assess the eect of a soil-derived, lignite extract dietary supplement in protecng
against glyphosate’s eect on the ght juncons in cell culture.
Methods: Small bowel (IEC-6) and colon epithelium (Caco-2) cells were incubated
unl a stable Transepithelial Electrical Resistance (TEER) was measured. A control
and the lignite extract supplement (20% concentraon) were placed on the cells
and le overnight. These cells were then treated with and without glyphosate at
10 mg/ml. TEER was measured at 30 min. The expression of the ght juncons was
determined by immunouorescent microscopy of the ZO-1 ght juncon element
before and aer overnight incubaon, and following glyphosate exposure.
Results: TEER increased with the lignite extract on IEC-6 (95%) and Caco-2 (35%)
compared to the control. Glyphosate dramacally decreased the TEER in both
IEC-6 (80%) and Caco-2 (76%) cells. The lignite extract blocked the glyphosate-
mediated decrease in TEER.
Conclusion: The lignite extract supplement blocked a glyphosate-dependent
decrease in TEER in both cell lines. If found ecacious in clinical trials, RESTORE
could prove helpful in combang intesnal sensivies caused by glyphosate-
mediated ght juncon disrupon.
Keywords: Lignite; Transepithelial Electrical Resistance (TEER); Caco-2; IEC-6;
Glyphosate; Tight juncons; Terrahydrite; Intesnal permeability
Abbreviaons: TEER: Transepithelial Electrical Resistance; ZO-1: Zonula Occludens
1; GALT: Gastrointesnal-Associated Lymphoid Tissue
Protecve Eects of Lignite Extract
Supplement on Intesnal Barrier Funcon in
Glyphosate-Mediated Tight Juncon Injury
Introducon
Intesnal barrier permeability refers to a dysfuncon in ght
juncons whereby foreign substances (e.g. potenal angens,
inorganic compounds, and pathogenic organisms) are allowed
unregulated passage from the apical side to the basolateral
side of these epithelial cells. Tight juncons are found in all
epithelial and endothelial systems such as the nasosinuses,
digesve tract, renal tubules, and blood-brain barrier. These ght
juncons serve as rewalls, regulang the absorpon of water
and macronutrients, and parcipate in immune system response.
Tight juncons are comprised of dozens of proteins that aid in this
funcon, including occludin proteins, the Junconal Adhesion
Molecules (JAM) family of proteins, and the claudin family of
proteins, all of which span the paracellular space. Occludin
proteins are responsible for intercellular signaling within the ght
juncons, while the JAM proteins aid in tracking of immune
cells to injured or inamed ssues [1]. The claudin proteins help

2017
Vol. 3 No. 1: 1
Journal of Clinical Nutrition & Dietetics
ISSN 2472-1921
This article is available in: http://clinical-nutrition.imedpub.com/archive.php
2
determine the permeability properes of epithelial cells, thus
controlling the ow of molecules in the intercellular space [2].
Perhaps the most well-known ght juncon proteins are the
Zonula Occludens (ZO) proteins, which act as scaolding in the
plasma membrane and provide a cytoplasmic structural anchor
for the paracellular protein complexes [3]. Tight juncons also
serve as a primary barrier or channel through which solutes and
ions are selecvely transported through the intercellular space.
This transport is by passive diusion, yet is highly regulated,
discriminang on three characteriscs of the material in
transport: electrical conductance, size, and charge selecvity [4].
Tight juncons have also been found to be important in cellular
dierenaon, proliferaon, and communicaon [4,5].
With ght juncons being crical to proper cellular funconing,
knowing how ght juncons are disrupted is important to
understanding disease processes such as vascular and neural
inammatory diseases and epithelial cancers (which comprise
90% of all cancers). The disrupon of properly funconing ght
juncons can occur with exposure to various toxins. The body is
exposed to environmental toxins through the air we breathe by
inhalaon, through contact with skin by absorpon, and through
ingeson of food and drink. With a clinically signicant level of
toxins in processed foods, this study focuses on the epithelial
ssue of the small and large intesnes aer toxins are ingested.
Gut permeability caused by ght juncon dysregulaon is
increasingly understood to iniate the pathogenesis of a variety
of inammatory diseases, including ulcerave colis and Crohn’s
Disease [6-11]. The inammatory foundaon of these condions
points to the chronic acvaon of the immune system in the
Gastrointesnal-Associated Lymphoid Tissue (GALT) through
exposure from ght juncon dysregulaon.
Glyphosate is a nonselecve, broad-spectrum herbicide
commonly used to kill weeds and grasses that compete with
crops, or to kill and desiccate crops prematurely to accelerate
harvest. Glyphosate is a glycine amino acid that is chemically
modied with a phosphate group resulng in its categorizaon
as an organophosphate. It funcons as a compeve inhibitor
of 5-Enolpyruvylshikimate-3-Phosphate (EPSP) synthase, an
enzyme produced by plants and microorganisms that is necessary
for the producon of the aromac amino acids (tryptophan,
phenylalanine, and tyrosine) which are necessary constuents of
crical plant and human protein structures including hormones
and cell membrane components. With these amino acids
inhibited, protein synthesis is arrested, leading to retarded plant
growth and eventually death. Glyphosate’s high solubility in water
*Mann D. Glyphosate now the most-used agricultural chemical ever.
In: News Week. Feb 2, 2016. hp://www.newsweek.com/glyphosate-
now-most-used-agricultural-chemical-ever-422419. Accessed 14 Aug
2016.
† Honeycu Z. Glyphosate tesng full report: Finds in American mothers’
breast milk, urine and water. 2014. hp://www.momsacrossamerica.
com/glyphosate_tesng_results. Accessed 2 Aug 2016.
‡ Rohrich JD. The truth about toxic wheat. In: Hungton Post. 2014.
hp://www.hungtonpost.com/jenny-dewey-rohrich/the-truth-about-
toxic-wheat_b_6180498.html. Accessed 14 Aug 2016.
and ability to be absorbed by plants is another reason for its
herbicidal ecacy, and its rapid penetraon into the ecosystem.
Hundreds of millions of pounds of glyphosate are applied on U.S.
farms, and more than 1.2 billion pounds are used worldwide each
year*. The volume of usage of this herbicide has led to reports
of signicant levels of glyphosate in water, air, rainfall and even
human breast milk†,‡.
Healthy soil contains numerous minerals, nutrients and other
chemicals released during the bacteria and fungi digesve
processes. This is very similar to the human intesnal ecosystem.
In both soil and intesnal environments, the chronic use of
glyphosate since its debut in 1976 has resulted in subopmal
nutrient availability and delivery to plants and the animals and
humans that consume those plants.
With the decrement in the nutrional value of our food, clinicians
worldwide have begun to turn to fossilized soil (lignite) extracts
for mineral supplementaon for human nutrion as the nutrient
density in the soils decrease due to modern agricultural pracces.
Shilajit, fulvic acids, and humic acids are naturally-oxidized lignite
extracts that have been ulized as mineral supplements in Asia
for hundreds of years. Due to their oxidave nature and frequent
contaminaon, their use in the clinical seng has been limited.
Signicant toxicity introduced from both the mining and the
manufacturing of these mineral supplements have been seen in
numerous commercial sources [12,13].
RESTORE is the inial lignite-derived mineral supplement in an
alkaline liquid form that carries trace minerals and amino acids
and that delivers a stabilized family of carbon-based redox
molecules as the acve ingredient.
This study aimed to examine how lignite extract inuenced the
ght juncons of the intesnal barrier system in the presence
and absence of glyphosate, measuring the membrane integrity
via Transepithelial Electrical Resistance (TEER) of polarized
epithelial membranes of normal small bowel epithelium cells
(IEC-6) and colon epithelial cells (Caco-2) [14-16]. Because
glyphosate has been determined to cause intesnal permeability,
this study examined the protecve eects this lignite extract had
on the intesnal epithelial cell model systems in combang this
common toxic herbicide [17].
Methods
Cell culture
Rat ileum epithelial (IEC-6) cell lines and human colorectal
carcinoma (Caco-2) cell lines were obtained from the American
Type Culture Collecon (Manassas, VA, USA; ATCC catalogue
CRL-1592 [IEC-6 cells]; ATCC catalog HTB-37 [Caco-2 cells]). Their
respecve specic media was used to propagate both cell lines
according to manufacturer protocols.
Exposure of cells to glyphosate
Glyphosate (Sigma-Aldrich, St. Louis, MO, USA) that was freshly
dissolved at a concentraon of 10 mg/ml was introduced to the
apical membrane and TEER was determined at 30-min.

2017
Vol. 3 No. 1: 1
Journal of Clinical Nutrition & Dietetics
ISSN 2472-1921
3
© Under License of Creative Commons Attribution 3.0 License
Exposure of cells to lignite extract
We introduced the cell membranes to the lignite extract
supplement (20% concentraon) and puried water. RESTORE
itself is comprised of the 150 mg of Terrahydrite™ lignite extract
at an alkaline pH (over 7.0) and therefore cannot be considered
humic acid or fulvic acid. The supplement also contains puried
water, as well as a proprietary blend of the following ingredients,
comprising less than 1500 ppm: (inorganic compound) chloride,
sodium, lithium, calcium, phosphorus, sulfur, bromide, potassium,
iron, anmony, zinc, copper, gold, magnesium, (organic
compounds) alanine, glycine, hisdine, isoleucine, methionine,
threonine, and valine.
Transepithelial electrical resistance
Transepithelial electrical resistance (TEER) is widely accepted
as an indicator of ght juncon permeability [17-20]. TEER
was measured in both IEC-6 and Caco-2 cells seeded in 24-
well transwell plates (1-micron pore size, Becton Dickinson).
These cells were incubated three days in a row unl a stable
TEER was determined. TEER was accessed using the epithelial
Volt- ohmmeter ed with a planar electrode (World Precision
Instruments, Sarasota, FL, USA). Media with and without the
lignite extract or other addives were added to water used to
make media from powder (DMEM-F12, Invitrogen, Waltham,
MA, USA). These were placed on the cells and incubated at 5%
CO2 in a humidied 37°C incubator for the appropriate amount of
me. Glyphosate (Sigma-Aldrich, St. Louis, MO, USA), was added
to apical media at 10 mg/ml. Measurements were made at the
30-min me point [21].
Zona occludens protein 1 immunouorescence
microscopy
Directly following TEER measurements, both cell culture
systems were simultaneously xed and permeabilized in 4%
paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) 1% Triton
X100 (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 5 min.
Cells were washed and then blocked in two percent bovine serum
albumin (BSA) and incubated with one to twenty diluon of cell
culture supernatant from Hybridoma clone R26.4c producing an-
ZO-1 monoclonal anbody (Developmental Studies Hybridoma
Bank, Iowa City, IA, USA). Alexa 488 labeled donkey an-mouse
IgG (Invitrogen, Waltham, MA, USA) was used at four µg/ml to
make the anbody uorescent. Nuclei were stained with Hoechst
ve µg/ml. Cells were imaged with a Zeiss (Oberkochen, Germany)
Axiovert automated 6D uorescent microscope and 100 × 1.4 NA
plan apochromac objecve.
Stascs
Experiments were performed in four replicates and stascs
included herein are results of all four. Data herein are presented
as mean values ± the standard error from the mean. P-values
were assessed by performing t-tests between groups.
Results
Eect of glyphosate on TEER
Two of the polarized epithelial cell lines, IEC-6 (small bowel) and
Caco-2 (large bowel), both formed an electrical resistant barrier
on transwell inserts. They were therefore measured for changes
in TEER during exposure to glyphosate, lignite extract, and
glyphosate and lignite extract together.
Glyphosate decreased the TEER in IEC-6 cells by 76%, n=4, p<0.001
(Figure 1) aer glyphosate was applied for 30 min (vehicle vs.
Figure 1 Average TEER in IEC-6 (small bowel) cells. Eects of
glyphosate (10 mg/ml) and lignite extract (LE) (20 vol/
vol concentraon in media) on the transepithelial
electrical resistance (TEER) of IEC-6 monolayers.
Data are presented for one experiment, with four
replicates. Results are wrien as mean ± standard error
(*Represents a TEER value that is stascally signicantly
dierent from the TEER of cells with glyphosate and
those with both glyphosate and LE (p<0.001)).
120
100
80
60
40
20
0
Veh LE Glyph LE/Glyph
Glyphosate Insult
* p<0.001, n=4
*
TEER Ohms X cm2
Figure 2 Average TEER in Caco-2 (large bowel) cells. Eects
of glyphosate (10 mg/ml) and lignite extract (20 vol/
vol concentraons in media) on the transepithelial
electrical resistance (TEER) of Caco-2 monolayers. Data
are presented for one experiment, with four replicates.
Results are wrien as mean ± standard error (*Represents
a TEER value that is stascally signicantly dierent
from the TEER of cells with glyphosate and those with
both glyphosate and lignite extract (p<0.001)).
Veh LE Glyph LE/Glyph
Glyphosate Insult
TEER Ohms X cm2
300
250
200
150
100
50
0
* p<0.001, n=4
*

2017
Vol. 3 No. 1: 1
Journal of Clinical Nutrition & Dietetics
ISSN 2472-1921
This article is available in: http://clinical-nutrition.imedpub.com/archive.php
4
glyphosate). The lignite extract supplement increased TEER in
IEC-6 cells by 93%, n=4, p=0.002 (vehicle vs. lignite). Lignite was
shown to prevent the glyphosate-dependent decrease in the
TEER on IEC-6 cells when lignite and glyphosate were placed
together on the cells, as illustrated by there being no stascally
signicant dierence (p=0.414) between the vehicle and lignite/
glyphosate.
Glyphosate decreased the TEER in Caco-2 cells by 80%, n=4,
p<0.001 (Figure 2) aer glyphosate was applied for 30 min (vehicle
vs. glyphosate). The lignite extract supplement increased TEER
in Caco-2 cells by 35%, n=4, p=0.004 (vehicle vs. lignite). Lignite
was shown to prevent the glyphosate-dependent decrease in the
TEER on Caco-2 cells when lignite and glyphosate were placed
together on the cells, as illustrated by there being no stascally
signicant dierence (p=0.736) between the vehicle and lignite/
glyphosate.
The lignite extract’s and glyphosate’s eect on ght juncon
integrity is seen by examining the ZO-1 of the ght juncon
by immunouorescence microscopy in IEC-6 (Figure 3). In the
vehicle or control (VEH) cells, the localizaon of ZO-1 shows
interspersed gaps in ZO-1 between the cells (a). As lignite extract
and cells were incubated together for only two hours, an increase
in abundance of ZO-1 between cells can be seen (b). Glyphosate
clearly decreased the consistency of ZO-1 localizaon between
cells (c), and lignite extract prevented this loss of ZO-1 localizaon
between cells (d).
Discussion
Inially introduced in a 1962 patent, and commercialized in the
herbicide Roundup in 1976, glyphosate is widely used in both
industrial agriculture and residenal sengs. Because of this, it
is widespread in our daily environment and food industry. In this
study, glyphosate, rapidly disrupted the intesnal ght juncon
barrier in both small intesne and colon epithelial membranes.
While the exact mechanism for ght juncon dysregulaon
from glyphosate is unknown, it is plausible that it is similar to
how gluten disrupts ght juncons through the upregulaon
of zonulin and the Zonulin Occludins Toxin (ZOT) pathway.
Glyphosate’s systemic acons are likely unique due to its small
size (molar mass of 169 g/mol) and ability to pass through the
plasma membrane, in comparison to gliadin’s relavely large size
(>30,000 g/mol).
A proposed mechanism by which glyphosate has been shown
to decrease TEER in IEC-6 and Caco-2 cells is by dysregulaon
of ght juncons [17]. Vasiluk et al. have reported glyphosate-
mediated disrupon of intracellular F-acn bers resulng in
disarrangement of the normal cytoplasmic honeycomb, with
nuclei neatly in the middle of the cell, to a more chaoc localizaon
of the nuclei aer exposure to the herbicide [17]. We demonstrate
this phenomenon again with our IEC-6 data above demonstrang
Figure 3 ZO-1 immunouorescence of IEC-6 cells
The cells were grown and then exposed to Hybridoma clone R26.4c to produce an-ZO-1 monoclonal
anbody. Alexa 488 labeled donkey anmouse IgG was used at 4 μg/ml to make the anbody uorescent.
Nuclei were stained with Hoechst 5 μg/ml. Cells were imaged with a Zeiss (Oberkochen, Germany)
Axiovert automated 6 D uorescent microscope and 100 × 1.4 NA plan apochromac objecve. Images
were taken aer exposure to (a) glyphosate (10 mg/ml) aer 2 min, (b) lignite extract (20% concentraon)
overnight then glyphosate aer 2 min, (c) glyphosate aer 10 min, (d) LE then glyphosate aer 10 min, (e)
glyphosate aer 16 min, (f) LE then glyphosate aer 16 min.

2017
Vol. 3 No. 1: 1
Journal of Clinical Nutrition & Dietetics
ISSN 2472-1921
5
© Under License of Creative Commons Attribution 3.0 License
chaoc nuclei distribuon following glyphosate exposure (Figure
3). Indeed, one advantage of this experiment methodology is
that it provides simplicity with which to determine glyphosate’s
impact on ght juncons and lignite’s protecve ecacy against
this glyphosate-mediated eect. But simplicity comes at a cost.
Further research is needed to determine the specic mechanism
of how glyphosate leads to ght juncon dysregulaon.
Few research studies have illustrated improvements in ght
juncon integrity and TEER of polarized epithelial cells. Some
natural substances that have shown such improvement
include quercen, butyrate, L-glutamine, and the probioc
Bidobacterium, and the lignite extract compound studied herein
[19,22-32]. Larazode, a synthec pharmaceucal and zonulin-
inhibitor, has also shown to improve ght juncon formaon [33].
Among these reported compounds, lignite extract is seen to more
quickly improve ght juncon integrity as measured by the TEER
funconal analysis as seen by immunouorescence of zonulin
protein expression. This improvement in ght juncon integrity
occurred within the rst thirty minutes from introducon.
Conclusion
This study demonstrates the capacity of the lignite extract to
block glyphosate-mediated injury in small intesnal and colon
epithelial membranes, even at glyphosate levels 50 mes higher
than allowable in foods like corn and soy. The direct eects of
lignite extract on large and small bowel ssues evaluated in
this study include a rapid increase in ght juncon expression,
indicang a strengthening of intesnal barrier funcon. The
public health ramicaons of these results may be signicant
as intesnal permeability from ght juncon dysregulaon
connues to be recognized as a foundaonal cause of chronic
inammatory condions [34].
Tight juncon injury has also been shown to be involved in a
variety of seemingly dierent diseases, from allergies and asthma
to Alzheimer’s disease and Parkinson’s disease. The debate
connues to be ongoing as to whether the rise in the incidence
of these disease is due to an increase in public and praconer
awareness, or due to notable changes in our food system.
This study shows how the consumpon of food products containing
glyphosate may result in intesnal barrier permeability. This
permeability can lead to unregulated angen presentaon to the
gastrointesnal-associated lymphoid ssue which, in turn, can
cause chronic inammatory condions in our populaon. Further
studies are needed to assess potenal clinical applicaons of
lignite extract supplements.
Acknowledgements
We are grateful to Barbara Brand for her helpful comments on
the manuscript.
Funding
This study, including the experiments, analysis, and publicaon
was funded by Biomic Sciences, LLC (Charloesville, VA USA).
Aurthor’s Contribuons
JJG designed and executed the experiments. DAR and ZMB
contributed to the data analysis and wring of the manuscript.
Compeng Interest
JJG, DAR and ZMB are shareholders in Restore, a supplement that
has been shown to help with intesnal permeability in laboratory
cell culture.

2017
Vol. 3 No. 1: 1
Journal of Clinical Nutrition & Dietetics
ISSN 2472-1921
This article is available in: http://clinical-nutrition.imedpub.com/archive.php
6
References
1 Van Itallie CM, Anderson JM (2014) Architecture of ght juncons
and principles of molecular composion. Semin Cell Dev Biol 36:
157-165.
2 Gunzel D, Yu AS (2013) Claudins and the modulaon of ght juncon
permeability. Physiol Rev 93: 525-569.
3 Bauer H, Zweimueller-Mayer J, Steinbacher P, Lametschwandtner A,
Bauer HC (2010) The dual role of zonula occludens (ZO) proteins. J
Biomed Biotechnol 2010: 402593.
4 Anderson JM, Van Itallie CM (2006) Tight juncon channels. In:
Gonzalez-Mariscal L (ed.) Tight juncons. NY, USA: Springer.
5 Fromm M, Schulzke JD (2009) Molecular Structure and Funcon of
the Tight Juncon: From Basic Mechanisms to Clinical Manifestaons.
NY, USA: Wiley-Blackwell.
6 Wang W, Uzzau S, Goldblum SE, Fasano A (2000) Human zonulin, a
potenal modulator of intesnal ght juncons. J Cell Sci 24: 4435-
4440.
7 Fasano A (2012) Zonulin, regulaon of ght juncons, and
autoimmune diseases. Ann N Y Acad Sci 1258: 25-33.
8 Fasano A (2012) Leaky gut and autoimmune diseases. Clin Rev
Allergy Immunol 42: 71-78.
9 Fasano A (2008) Physiological, Pathological, and Therapeuc
implicaons of zonulin-mediated intesnal barrier modulaon. Am
J Pathol 173: 1243-1252.
10 Fasano A (2011) Zonulin and its regulaon of intesnal barrier
funcon: the biological door to inammaon, autoimmunity and
cancer. Physiol Rev 91: 151-175.
11 Samsel A, Sene S (2013) Glyphosate’s suppression of cytochrome
P450 enzymes and amino acid biosynthesis by the gut microbiome:
pathways to modern diseases. Entropy 15: 1416-1463.
12 Rudnev MI, Maliuk VI, Stechenko LA (1993) An electron microscopic
analysis of the smulang and toxic eects of mumie-containing
preparaons. Lik Sprava 12: 63-64.
13 Saper RB, Phillips RS, Sehgal A, Khouri N, Davis RB, et al. (2008) Lead,
mercury, and arsenic in US- and Indian- manufactured Ayurvedic
medicines sold via the Internet. JAMA 300: 915-923.
14 Quaroni A, Wands J, Trelstad RL, Isselbacher KJ (1979) Epithelioid cell
cultures from rat small intesne. Characterizaon by morphologic
and immunologic criteria. J Cell Biol 80: 248- 265.
15 Rousset M, Chevalier G, Rousset JP, Dussaulx E, Zweibaum A (1979)
Presence and cell growth-related variaons of glycogen in human
colorectal adenocarcinoma cell lines in culture. Cancer Res 39: 531-534.
16 Sääf AM, Halbleib JM, Chen X, Yuen ST, Leung SY, et al. (2007)
Parallels between global transcriponal programs of polarizing Caco-
2 intesnal epithelial cells in vitro and gene expression programs in
normal colon and colon cancer. Mol Biol Cell 18: 4245-4260.
17 Vasiluk L, Pinto, LJ, Moore MM (2005) Oral bioavailability of
glyphosate: studies using two intesnal cell lines. Environ Toxicol
Chem 24: 153-160.
18 Gildea JJ, Roberts DA, Bush Z (2016) Protecon against gluten-
mediated ght juncon injury with a novel lignite extract supplement.
J Nutr Food Sci 6: 547.
19 Ling X, Linglong P, Weixian D, Hong W (2016) Protecve eects
of Bidobacterium on intesnal barrier funcon in LPS-induced
enterocyte barrier injury of caco-2 monolayers and in rat NED model.
Plos One 11: e0161635.
20 Peng S, Wang SB, Singh D, Zhao PY, Davis K, et al. (2016) Claudin-3
and claudin-19 parally restore nave phenotype to ARPE-19 cells
via eects on ght juncons and gene expression. Exp Eye Res 151:
179-189.
21 Gildea JJ, Seaton JE, Victor KG, Reyes CM, Bigler Wang D, et al. (2014)
Exosomal transfer from human renal proximal tubule cells to distal
tubule and collecng duct cells. Clin Biochem 47: 89-94.
22 Shen L, Weber CR, Raleigh DR, Yu D, Turner JT (2011) Tight Juncon Pore
and Leak Pathways: A Dynamic Duo. Annu Rev Physiol 73: 283-309.
23 Suzuki T, Hara H (2009) Quercen enhances intesnal barrier funcon
through the assembly of zonula occludens-2, occludin, and claudin-1
and the expression of claudin-4 in Caco-2 cells. J Nutr 139: 965-974.
24 Chuenkiyanon S, Pengsuparp T, Jianmongkol S (2010) Protecve
eect of quercen on hydrogen peroxideinduced ght juncon
disrupon. Int J Toxicol 29: 418-424.
25 Amasheh M, Schlichter S, Amasheh S, Mankertz J, Zeitz M, et al.
(2008) Quercen enhances epithelial barrier funcon and increases
claudin-4 expression in Caco-2 cells. J Nutr 138: 1067-1073.
26 Mercado J, Valenzano MC, Jeers C, Sedlak J, Cugliari MK, et al. (2013)
Enhancement of ght junconal barrier funcon by micronutrients:
compound-specic eects on permeability and claudin composion.
PLoS One 8: e78775.
27 Peng L, Li ZR, Green RS, Holzman IR, Lin J (2009) Butyrate enhances
the intesnal barrier by facilitang ght juncon assembly via
acvaon of AMP-acvated protein kinase in Caco-2 cell monolayers.
J Nutr 139: 1619-1625.
28 McGilligan VE, Wallace JM, Heavey PM, Ridley DL, Rowland IR (2007)
The eect of nicone in vitro on the integrity of ght juncons in
Caco-2 cell monolayers. Food Chem Toxicol 45: 1593-1598.
29 Beutheu S, Ghouzali I, Galas L, Décheloe P, Coëer M (2013)
Glutamine and arginine improve permeability and ght juncon
protein expression in methotrexate-treated Caco-2 cells. Clin Nutr
32: 863-869.
30 Anderson JM, Van Itallie CM (2009) Physiology and Funcon of the
Tight Juncon. Cold Spring Harb Perspect Biol 1: a002584.
31 Lerner A, Mahias T (2015) Changes in intesnal ght juncon
permeability associated with industrial food addives explain the rising
incidence of autoimmune disease. Autoimmun Rev 14: 479-489.
32 Turner JT (2009) Intesnal mucosal barrier funcon in health and
disease. Nat Rev Immunol 9: 799-809.
33 Gopalakrishnan S, Tripathi A, Tamiz AP, Alkan SS, Pandey NB (2012)
Larazode acetate promotes ght juncon assembly in epithelial
cells. Pepdes 35: 95-101.
34 Xavier RJ, Podolsky DK (2007) Unravelling the pathogenesis of
inammatory bowel disease. Nature 448: 427-434.