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Reversal of perfluorooctanesulfonate- caused immunotoxicity by glucan-resveratrol-vitamin C combination

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Perfluorinated compounds (PFCs) perfluoroctanic acid (PFOA) and perfluooctane sulphonic acid (PFOS) are environmentally widespread bioaccumulative chemicals known to induce profound effects on the immune system. In this study, two types of PFC, i.e., PFOS and PFOA, were administered orally (daily) a dose of 20 mg/kg/d. After exposure, all mice exhibited significant immunosuppressive effects upon both cellular (phagocytosis and NK cell activity) and humoral (antibody response) branches of their immune responses. The mice were then fed with a 4 mg/kg/d dose of a combination of resveratrol-glucan-vitamin C (RVB 300). The results showed that treatment with PFCs and RVB 300 resulted in significantly lower level of immunotoxic effects from PFCs. These outcomes suggest to us that RVB 300 can potentially be successfully used as a natural remedy against immunotoxicities induced by low-level exposure(s) to perfluorinated compounds.
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1 23
Oriental Pharmacy and Experimental
Medicine
ISSN 1598-2386
Volume 13
Number 1
Orient Pharm Exp Med (2013) 13:77-84
DOI 10.1007/s13596-013-0105-7
Reversal of perfluorooctanesulfonate-
induced immunotoxicity by a glucan-
resveratrol-vitamin C combination
V.Vetvicka & J.Vetvickova
1 23
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RESEARCH ARTICLE
Reversal of perfluorooctanesulfonate-induced
immunotoxicity by a glucan-resveratrol-vitamin C
combination
V. Vetvicka &J. Vetvickova
Received: 21 August 2012 /Accepted: 10 January 2013 / Published online: 29 January 2013
#Institute of Oriental Medicine, Kyung Hee University 2013
Abstract Perfluorinated compounds (PFCs) perfluoroctanic
acid (PFOA) and perfluooctane sulphonic acid (PFOS) are
environmentally widespread bioaccumulative chemicals
known to induce profound effects on the immune system. In
this study, two types of PFC, i.e., PFOS and PFOA, were
administered orally (daily) a dose of 20 mg/kg/d. After expo-
sure, all mice exhibited significant immunosuppressive effects
upon both cellular (phagocytosis and NK cell activity) and
humoral (antibody response) branches of their immune
responses. The mice were then fed with a 4 mg/kg/d dose of
a combination of resveratrol-glucan-vitamin C (RVB 300).
The results showed that treatment with PFCs and RVB 300
resulted in significantly lower level of immunotoxic effects
from PFCs. These outcomes suggest to us that RVB 300 can
potentially be successfully used as a natural remedy against
immunotoxicities induced by low-level exposure(s) to per-
fluorinated compounds.
Keywords Glucan .Immunotoxicity .Phagocytosis .
Antibodies .PFOS .PFOA
Introduction
Perfluorinated hydrocarbons (PFC) are used in thousands of
products. Two major classes are perfluorinated carboxylates
like perfluoroctanic acid (PFOA) and perfluoroctane sulfo-
nates such as perfluooctane sulfonic acid (PFOS). These com-
pounds have an extremely long lifetime in the environment
and are detectable in the blood of both animals and humans
(Lau et al. 2007). A study of people with high exposure to
perfluorinated compounds (as food contaminants) indicated
that there was a significant reduction in humoral immune
responses to immunizations (Grandjean et al. 2012). Animal
studies noted severe suppression of both humoral and cellular
immunity caused by exposure to these agents (Peden-Adams
et al. 2007). Among the reactions suppressed by chronic
exposure to this class of agents were: natural killer (NK) cell
activity; lymphocyte proliferation and antibody responses
(Dong et al. 2009; Brieger et al. 2011); release of tumor
necrosis factor (TNF)-α(Brieger et al. 2011); and, secretion
of interleukins (IL)-6 and 10 (Corsini et al. 2012). Wang et
al. (2011) reported induction of thymic and splenic hyper-
trophy. Qazi et al. (2009) indicated there was an exposure-
related decrease in levels circulating neutrophils. For a full
review of the immunotoxicologic effects of PFC compounds,
the reader is directed to De Witt et al. (2012).
While most of the current literature is focused on the
description of immunotoxic effects of PFCs, there is little
known about the possible blocking of these effects. Studies
showing the strong potential of glucans to help overcome
immunosuppressive effects induced by immuno- modulat-
ing processes such irradiation or chemotherapy (Patchen
and MacVittie 1982; Vetvicka et al. 2007b; Vetvicka and
Vetvickova 2009) led us to the hypothesis evaluated herein.
Specific- ally, this study sought to first compare the type-
s/degrees of immunosuppression caused by either PFOS or
PFOA and to then to evaluate if any suppression could be
reversed by a therapy that utilized a glucan-resveratrol-
Vitamin C combination.
The choice of this particular combination to examine was
based on our own earlier studies demonstrating significant
stimulation of both cellular and humoral branches of the
immune response using a combination of glucan and humic
acid (Vetvicka et al. 2010). Another study showed that both
glucan and resveratrol stimulated phagocytosis, increased
splenic cell expression of CD4, and induced restoration of
V. Vetvicka (*):J. Vetvickova
Department of Pathology, University of Louisville School of
Medicine, 511 S. Floyd, MDR Bldg., Room 224,
Louisville, KY 40202, USA
e-mail: vaclav.vetvicka@louisville.edu
Orient Pharm Exp Med (2013) 13:7784
DOI 10.1007/s13596-013-0105-7
Author's personal copy
splenic population profiles after an experiment- ally-induced
leukopenia (Vetvicka et al. 2007a). When effects of all of
these substances on the expression of some important genes
(i.e., NF-κB2 and Cdc42) in breast cancer cells were mea-
sured, up-regulation of Cdc42 expression was evident only
with the use of both immuno- modulators combined
(Vetvicka et al. 2007b; Vetvicka and Vancikova 2010).
Follow-up studies showed that, compared to each compo-
nent alone, a glucan-resveratrol-Vitamin C combination
caused the strongest inhibition of experimentally-induced
lung and breast cancers (Vetvicka and Vetvickova 2012a).
Materials and methods
Animals
Female BALB/c mice (8-wk-old) were purchased from the
Jackson Laboratory (Bar Harbor, ME). All mice were
housed in a sterile animal facility that was maintained at
22 °C and at a 35 % relative humidity, with a 12-hr light/-
dark cycle. All mice had ad libitum access to standard
mouse chow and filtered water throughout the studies. All
mice were allowed to accli- mate for a minimum of 10 day
prior to any exposure being performed. All mice were
weighed prior to the first dosing and again daily to assure
that dosing volumes were constant over the course of the
treatments. All animal work was done according to a
University of Louisville IACUC protocol and with the ap-
proval of that committee IACUC #04050.
Reagents
RVB 300 mixture containing resveratrol, yeast-derived glu-
can #300, and Vitamin C (at a ratio of 2:2:1 [w/w/w]) was
purchased from Restart Your Life (Lexington, SC). RPMI
1640 medium, Freunds adjuvant, tri-nitrophenol (TNP),
bovine γ-globulin, MTT ([3-(4,5-dimethyl- thiazol-2-yl)-
2,5-diphenyltetrazolium bromide]), bovine serum albumin
(BSA), Concanavalin A (ConA), lipopolysaccharide (LPS),
sodium dodecyl sulfate, perfluorooctanesulfonate (PFOS;
85 % purity), and perfluorooctanoic acid (PFOA; 96 %
purity) were all purchasedfromSigma(St.Louis,
MO). Fetal calf serum (FCS) was obtained from
Hyclone Laboratories (Logan, UT).
Treatment
Each treatment group in these studies contained five mice.
Some mice received an oral (non-sedated gavage) dosing of
PFOS or PFOA in phosphate-buffered saline (PBS; pH 7.4) at a
dose of 20 mg/kg/day. Control mice received PBS only. Some
mice (with and without any PFC treatments) also received 4 mg
RVB 300/kg/d by oral gavage; these treatments occurred with
the PFC or PBS instillation on each day. All treatments were for
7 day (except the antibody production) and all experiments
were repeated three times. For all of the protocols outlined
below (except those pertaining to antibody formation), 1 day
after the final treatment, all mice were euthanized by CO
2
asphyxiation and their blood/organs (i.e., spleen, thymus) were
aseptically removed for analyses. Blood was isolated from the
orbital vein into 5 ml plastic tubes (Fisher, Pittsburgh, PA) and
then placed at room temperature to allow for clotting. Serum
was subsequently isolated and stored; the cells were then used
in some of the protocols outlined below. The organs were blot
dried, trimmed of extraneous tissue matter, and weighed; each
value was normalized to body weight and reported as somatic
index (i.e., 100 × organ weight/body weight).
Phagocytosis
The phagocytic activity of peripheral blood cells (phagocytes)
in the blood samples iso- lated above was assessed using a
technique described in Vetvicka et al. (1982,1988). Briefly,
peripheral blood cells (0.1 ml) were combined with 50 μlofa
suspension of 2-hydroxyethyl methacrylate particles (HEMA;
5×10
8
/ml) and then incubated 60 min at 37 °C, with intermit-
tent gentle shaking. Thereafter, the samples were prepared as
smears on glass slides that were, in turn, treated with Wright
stain. Cells were then examined using a CK 30 microscope
(Olympus, Pittsburgh, PA); cells with three or more HEMA
particles were considered as positive for phagocytic activity.
At least 200 cells in 60 high-power fields were examined per
slide; five slides/animal were analyzed.
Lymphocyte proliferation assay
Splenocytes were prepared from each harvested organ using a
standard protocol (Fernandez-Botran and Vetvicka 1995). Only
preparations containing cells with viability >95 % were used in
the experiments. Ultimately, isolated splenocytes were washed
three times in RPMI 1640 medium supplemented with 10 %
FCS and re-suspended at 5 ×10
6
cells/ml. An aliquot (100 μl)
of each suspension was added to triplicate wells of a 96-well
plate that contained either ConA or LPS (each at 10 μg/ml, final
concentration after cell addition) or medium (negative control).
The plates were then incubated for 48 h at 37 °C in a humidified
5%CO
2
incubator. Thereafter, 10 μl of MTT (5 mg/ml) was
added to each well, and the plates were incubated a further 4 h
before 100 μl of 20 % sodium dodecyl sulfate (w/v) solution
was added to each well. After incubation overnight at 37 °C, the
plates were placed in an ELISA reader (Tecan, Research
Triangle Park, NC) and the absorbance values in each well
monitored at 570 nm (OD). The proliferation index was then
calculated as: (OD
ConA
-stimulated cells/OD non-stimulated
cells) or (OD
LPS
-stimulated cells/OD non-stimulated cells).
78 V. Vetvicka, J. Vetvickova
Author's personal copy
Ex vivo cytotoxicity assay
Spleen cells from the above preparations were also used for
assessments of ex vivo cytotoxic activity. YAC-1 cells
(obtained from ATCC; Manassas, VA) were grown in
RPMI 1640 supplemented with 10 % FCS (without anti-
biotics) and then labeled as outlined in Vetvicka et al.
(2007a). Splenocytes (10
6
/ml stocks) were then placed as
100 μl aliquots into V-shaped 96-well microplates and in-
cubated with 50 μl of the labeled YAC-1 target cells, at an
effector- target ratio of 64:1. After gently centrifuging at 250
× g for 5 min, the plates were incubated for 4 h at 37 °C. The
cytotoxic activity of the cells was then determined using a
CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega,
Madison, WI), following manufacturer instruc- tions.
Briefly, 10 μl of lysis solution was added to select wells (i.e.,
those for determination of maximum killing potential) 45 min
before the end of the incubation period. The plates were then
centrifuged at 250 × g for 5 min and 50 μl of culture superna-
tant from each well was then trans- ferred to corresponding
wells of a flat-bottom 96-well microplate. Kit-provided reac-
tion sub- strate (50 μl) was then added to each well, and the
plate was covered and incubated (in dark) for 30 min at room
temperature. The optical density (at 492 nm) in each well was
then determined using the ELISA plate-reader. Specific cell-
mediated cytotoxicity was ultimately calculated via the formu-
la: Percent-specific killing (% cytotoxicity) =100 × [(OD
exp
OD
spont
)/(OD
max
OD
spont
)], where spontaneous release was
obtained from target cells incubated with medium alone and
maximum release was obtained from target cells lysed with the
solution provided in the kit.
Splenic and thymic cellularity
To determine cellularity in two key immune system organs in
the mice, spleen and thymuses were suspended in RPMI 1640
and single cell suspensions were prepared using a steel mesh.
The suspension was then washed twice with cold medium and
any erythrocytes present were lysed with a 0.74 % (w/v)
ammonium chloride lysing buffer. After washing via
centrifuga- tion, the final cells present in the pellet were re-
suspended in medium and counted in a hemo- cytometer.
Viability of the final populations were assessed via
trypan blue exclusion; only suspensions with a viabili-
ty>95 % were analyzed.
Cells were stained with monoclonal antibodies in 12×75-
mm glass tubes using standard techniques (Fernandez-Botran
and Vetvicka 1995). For these analyses, fluorescein isothio-
cyanate (FITC)-labeled anti-CD4, -CD8, and -CD49b anti-
bodies (BD Pharmingen, San Jose, CA) were employed.
Briefly, aliqouts of 5×10
5
cells were combined with 10 μlof
FITC-labeled anti- bodies (120 μg/ml in PBS) and placed on
ice (in dark) for 30 min. Thereafter, the cells were diluted with
ice-cold PBS, centrifuged to remove non-adherent antibody
materials, and then re-suspended in PBS containing 1 % BSA
and 10 mM sodium azide. Flow cytometry was then per-
formed using a FACScan (Becton Dickinson, San Jose, CA)
system. For each sample, a minimum of 10,000 events were
analyzed using Flowjo software (Tree Star, Ashland, OR).
Antibody formation assay
Formation of antibodies was evaluated using ovalbumin
(OVA) as antigen. Mice were injected twice (2 week apart,
IP) with 0.1 mg of ovalbumin (in 100 μl volume) and the
serum was collected 7 day after the second injection. Total
level of specific antibodies against OVA was detected using
an ELISA assay. As positive control, a combination of OVA
and Freunds adjuvant (with no co-treatments with any
PFCs) was used. All test groups here received daily admin-
istration of the PFCs (alone or in combination with RVB) or
PBS for the entire 3-wk period of OVA exposures.
Measurement of IgM antibodies
Serum levels of IgM antibodies directed specifically against
TNP hapten was measured by ELISA in 96-well plates with
wells that had been coated with 100 μlTNP(10μg/ml) conju-
gated to bovine γ-globulin by incubation overnight, followed
by blocking with 1 % (w/v) BSA. Briefly, 100 μl of test serum
was added to each well and the plate incubated for 120 min at
37 °C. After washing with PBS-Tween to remove non-adherent
proteins, goat anti-mouse IgM conju- gated with alkaline phos-
phatase (1:2000, Sigma) was added and the plate incubated a
further 120 min at 37 °C. After another round of washing to
remove the detection antibody, p-nitrophenyl phosphate sub-
strate (diluted according to manufacturer instruction; Sigma)
was added and the absorbance in each well was measured
using the ELISA plate-reader. Wells containing standard- ized
amounts of mouse anti-TNP (IgM) antibody (BD Biosciences,
San Jose, CA) were analyzed in parallel to generate standard
curves from which levels of anti-TNP IgM in each test sample
could then be extrapolated. All test groups here received daily
administration of the PFCs (alone or in combination) or PBS
for the entire 3-wk period of antigen exposures.
Statistics
All experiments were repeated three times with at least 5
mice/group (15 mice total). Initially, a one-way analysis of
variance (ANOVA) was used to determine if there were
signifi- cant differences between groups for a given mea-
sured endpoint. If significance was noted, a Studentst-test
was then used to compare all treatment groups to the con-
trols. Significance in each analysis was assigned when a p-
value0.05 was obtained.
Reversal of perfluorooctanesulfonate-induced immunotoxicity 79
Author's personal copy
Results
Natural trans-resveratrol with a purity of 99.62 % (HPLC)
was used in these experimens. Vitamin C was pharmaceuti-
cal grade, 1,3/1,6-D-β-glucan #300 was originally from
Transfer Point and has a purity over 85 % (Vetvicka et al.
2007b; Vetvicka and Vetvickova 2012a). At the beginning
of the experiment, there were no significant differences in
body weights among the mice in the various groups (Fig. 1).
However, from Day 4 and thereafter, there was a significant
deterioration in body weights in mice in the PFOS and
PFOA groups. Simultaneous addition of PFCs and RVB
300 produced no changes in body weight. On the last day
of the treatment (i.e., Day 8), body mass was significantly
decreased and liver mass significantly elevated in both the
PFOS and PFOA groups (Table 1). Treatment with RVB
300 returned the body mass to normal levels and signifi-
cantly improved the liver mass as well.
With regard to cellularity, 7 days of exposure to PFOA
caused only an insignificant decrease in this value in the
spleen, but a strong (i.e., >50 %) decrease in this parameter
in the thymus. Exposure to PFOS resulted in a significant
decrease in cellularity in both organs. Co-treatment with
RVB 300 either returned the number of cells to normal
levels (spleen and PFOS + RVB 300 in thymus) or at least
significantly increased the number of cells in comparison to
levels in PFOA-RVB 300 in thymus (Fig. 2). In light of this
finding of altered cellularity in the spleen, any changes in
CD4, CD8 and CD49b sub-populations were also evaluated
(Fig. 3). Whereas there were no changes in CD4 or CD8
sub-population levels after any of the various test treat-
ments, RVB 300 alone caused significant increase in levels
of NK (CD49b
+
) cells.
To evaluate the effects of the PFOS and PFO - and the
possible mitigation of same by the test agents - on lympho-
cyte proliferation, mitogen-induced proliferation of B- and
T-lympho- cytes was evaluated. Figure 4illustrate how each
PFC significantly suppressed proliferation of T-
lymphocytes. PFOA exposure also inhibited proliferation
of B-lymphocytes. In both cases, addition of RVB 300
returned cell proliferation activities to normal levels.
Effects of each PFC on phagocytosis (here, by peripheral
blood neutrophils) were also evaluated using a well-
established model of synthetic microspheres. Data in
Fig. 5reflect the fact that on one hand, both PFCs signifi-
cantly inhibited phagocytosis. On the other hand, the stim-
ulation of phagocytic activity caused by RVB 300 was
strong enough not only return the reducedphagocytosis
to control values, but to a level even significantly higher
than that noted with cells from the PBS control hosts.
Potential effects on cellular immunity were also evaluat-
ed by monitoring NK activity in cells harvested from the
exposed hosts. The results of these studies showed almost
the same results as with phagocytosis. Both PFOA and
PFOS significantly inhibited NK cell activity and co-
treatment with RVB 300 not only restoredactivity, but
increased it to levels above those of cells from the PBS-
treated control mice (Fig. 6).
To assess effects on humoral immunity, an experimental
model of mice immunized with ovalbumin (OVA) was
employed. Mice were injected twice (14 days apart) with
OVA and the serum was collected 7 days after the second
14
16
18
20
22
24
26
28
0123567
PBS PFOA PFOS RVB PFOA+RVB PFOS+RVB
Body weight (g)
DAYS
Fig. 1 Body weight changes in mice treated with the test substances.
Body weights were measured daily. Values shown are mean ± SD
Table 1 Body mass change and organ mass change after PFOA, PFOS, and RVB 300 treatments
Body mass change
a
Spleen mass
b
Thymus mass
b
Liver mass
b
PBS 1.74± 0.24 0.44± 0.03 0.29 ±0.01 6.12± 0.10
PFOA
*
1.11± 0.20 0.42± 0.03 0.30±0.01
*
8.11± 0.11
PFOS
*
1.02± 0.23 0.40± 0.02 0.27 ±0.02
*
9.73± 0.14
RVB 300 1.75± 0.27 0.45± 0.04 0.28± 0.01 6.13± 0.12
PFOA + RVB 300
**
1.60± 0.22 0.44± 0.05 0.29 ±0.02
**
6.77± 0.12
PFOS + RVB 300
***
1.71± 0.20 0.43± 0.04 0.30 ±0.01
***
6.40± 0.11
a
Final mass (g) start mass (g)
b
Calculated as: [organ mass (g)/body mass (g)]×100
*
Significantly different at
*
p<0.05 from PBS values,
**
p<0.05 from PFOA alone,
***
p<0.05 from from PFOS alone
80 V. Vetvicka, J. Vetvickova
Author's personal copy
injection. In these studies, mice were being concur- rently
injected with the various test materials over the entire 3-wk
study period. The results indicate that both PFC caused
significantly decreased antibody formation and that RVB
300 co- treatment helped to significantly mitigate only the
reduction in antibody formation induced by PFOA (Fig. 7);
RVB 300 led to an increase of 48 % vs. that from PFOS
alone, but this change was not significant. These results
were further confirmed by evaluation of the IgM formation
using immunization with a different antigen (i.e., TNP).
Once again, both PFOA and PFOS inhibited IgM formation
and RVB 300 co-treatment more than doubled this produc-
tion (Fig. 8).
Discussion
In the last decade, considerable attention has been paid to
PFOS and related compounds, due to their presence in the
environment and subsequently animal and human systems.
Several important papers have discussed the immunosup-
pressive role of these compounds (Peden-Adams et al. 2007;
Dong et al. 2009;Qazietal.2009;Zhengetal.2009;
Brieger et al. 2011; Corsini et al. 2012). Among immune
system-related reactions that have been documented as ad-
versely impacted by oral exposure to PFOS and PFOA are
cell proliferation, antibody formation, inflam- mation, and
cytokine production. Even more attention was focused on
the toxic and immunosup- pressive effects of these com-
pounds after reports surfaced of reduced humoral responses
to routine immunization among children who had been
exposed to PFCs (Grandjean et al. 2012).
0
2
4
6
8
10
Thymus
Spleen
PBS
PFOA PFOS RVB PFOA+RVB PFOS+RVB
Total cellularity (x106)
* *
**
**
*
Fig. 2 Spleen and thymus cellularity after 7 days of exposures to the
test substances. Values shown are mean ± SD.
*
Value significantly
different from control (PBS only) at p0.05.
**
Significant differences
between PFOA and PFOA + RVB group at p0.05
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
T cells B cells
PBS PFOA PFOS RVB PFOA+RVB PFOS+RVB
Proliferation index
*
* *
Fig. 3 Proliferation index of lymphocytes after 7 days of exposures to
the test substances. Unstimulated data were not different between
groups; therefore the data are presented as the proliferation index.
*
Value significantly different from control (PBS only) at p0.05
0
2
4
6
8
10
12
CD4 CD8 CD49b
PBS PFO A PFO S RVB PFOA +RVB PFOS+RV B
% of positive cells
*
Fig. 4 Splenic populations in mice treated for 7 days.
*
Value signifi-
cantly different from control (PBS only) at p0.05
0
10
20
30
40
50
60
PBS PFOA PF OS RVB PFOA+RVB PFOS+RVB
% of positive neutrophils
*
*
* *
*
Fig. 5 Effects of 7 days of exposure to the test substances on phago-
cytosis by peripheral blood cells. Values shown are mean ± SD.
*
Value
significantly different from control (PBS only) at p0.05
Reversal of perfluorooctanesulfonate-induced immunotoxicity 81
Author's personal copy
However, despite all the current knowledge about immuno-
suppression caused by PFCs, no study about the possible
reversal of these effects exists in the literature. This, together
with our previous report showing that simultaneous treatment
of mercury with glucan (which resulted in significantly low-
ered immunotoxic effects of the metal; Vetvicka and
Vetvickova 2009) suggested to us that glucans can be used as
a natural remedy against low-level exposures to immunosup-
pressants. Since those earlier observations, we have determined
that a combination of glucan-resveratrol-Vitamin C had even
superior effects to that derived from the glucan alone (Vetvicka
and Yvin 2004). Among some of those observations, it was
noted that both glucan and resveratrol stimulated phagocytosis
of blood leukocytes, caused increased expression of CD4 on
spleen cells, and potentiated a restoration of splenic normal
status after experimental induction leukopenia (Vetvicka et al.
2007a; Vetvicka and Vetvickova 2012b). Thus, the aim of this
study was to determine if this combination could be used to
overcome the immunosuppressive effects induced by PFCs.
For the current study, a 7-day oral exposure to PFOS or
PFOA - as originally described by Zheng et al. (2009) - was
employed. As expected, decreases in body weight were fol-
lowed by changes in organ mass (Dong et al. 2009). However,
co-treatment with RVB 300 (the glucan- resveratrol-Vitamin
C combination used here) returnedboth weights body and
liver mass to almost control levels. At the level of the primary
immune organs, cellularity in the spleen and thymus was most
impacted by PFOS. Again, co-treatment with RVB 300
returnedthe number of cells to near normal. In all instances,
RVB 300 itself had no effects on cellularity in either organ.
The effects of each PFC on proliferation are less clear.
Some studies documented suppression of ConA-induced T-
lymphocyte proliferative activity (Zheng et al. 2009). Others,
in contrast, showed an insignificant increase in T- and B-
lymphocyte proliferative capacities (Peden-Adams et al.
2007). Here we demonstrated that, whereas the effects of both
compounds on T-lymphocytes were profound, those on B-
lymphocytes suggested a greater degree of resist- ance. No
effects on the expression of surface markers CD4/CD8/CD49b
were noted, suggesting that PFCs do not affect the migration
of lymphocytes into the spleen or change the development and
maturation of thymocytes.
In one portion of this study, the focus was upon the
effects of the PFCs on cellular immunity. The effects on
0
10
20
30
40
50
60
PBS PFOA PFOS RVB PFOA+RVB PFOS+RVB
%cytotoxicity
**
*
*
*
Fig. 6 Splenic NK cell activity following 7 days exposure to tested
substances. Each value represents the mean ± SD. *Significant differ-
ence at p 0.05 from control (PBS) group
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
OVA+FA PFOA PFOS RVB PFOA+RVB PFOS+RV B
ODat 405nm
*
Fig. 7 Effects of tested substances glucans on formation of antibodies
against ovalbumin. Mice were injected twice (2 weeks apart) with
antigen and the serum was collected 7 days after the final injection.
The level of specific antibodies against ovalbumin was detected using
ELISA. *Value significantly different between control (ovalbumin and
Freund adjuvant) and samples at p0.05
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
50 100 200 400 800
PBS PFOA PFOS RVB PFOA+RVB PFOS+RVB
Dilution of the serum
OD at 405 nm
Fig. 8 Effects of tested substances glucans on formation of IgM anti-
bodies against TNP. Mice were injected twice (2 weeks apart) with
antigen and the serum was collected 7 days after the final injection. The
level of specific antibodies against ovalbumin was detected using
ELISA. *Value significantly different between control (TNP and
Freund adjuvant) and samples at p0.05
82 V. Vetvicka, J. Vetvickova
Author's personal copy
phagocytic activity were assessed using 2-hydroxyethyl
methacrylate particles that have only a slight negative
charge and thus do not non-specifically adhere to the cell
surface and significantly lowers the chance of false negative
outcomes (Vetvicka et al. 1982). The results revealed that
both PFOS and PFOA inhibited phagocytosis to a level
equal to 30 % of normal values. The co-treatment with
RVB 300, well known to strongly stimulate this activity,
restoredphagocytosis by harvested cells to the levels noted
in mice that received RVB 300 alone.
Some previous studies have noted a dose-dependent de-
crease in NK cell activity as a result of PFOS exposure
(Zheng et al. 2009). However, as with the lymphocyte
proliferative capacity analyses, other studies reported no
effects at all (Nelson et al. 1992). This discrepancy some-
times can be ascribed to mouse strain or gender differences
(as female B
6
C
3
F
1
mice were less sensitive than male mice;
Peden-Adams et al. 2007). The results of the present study
indicated that both PFOS and PFOA imparted strong inhib-
itory effects on the activity of NK cells in the hosts. As with
many of the other endpoints analyzed here, the co-treatment
with RVB 300 once again led to a significant restoration of
this immune function.
In order to also assess the impact of each PFC (and any
mitigating effects of the RVC 300) on humoral responses in
the treated hosts, effects of PFOS and PFOA exposure on
antibody responses were analyzed. Using two different anti-
gens (i.e., ovalbumin and TNP), it was observed that both
PFCs (particularly PFOA) imparted strong inhibitory effects
on both total immunoglobulin formation and on IgM for-
mation. Once again, RVB 300 helped to restoresome of
the antibody formation activity; however, with neither PFC
was the combination co-treatment able to fully restore the
antibody formation capacities in the exposed hosts.
Conclusions
Two major conclusions were reached in this study. The first
was that since both PFOS and PFOA significantly sup-
pressed both branches of immune reactions even after short
7-d exposure - when taken together with findings of signif-
icant loss of body weight and hepato- megaly - these com-
pounds clearly present a significant danger to exposed hosts.
Our study, therefore, provides additional support to regula-
tory agencies seeking to eliminate/minimize PFOA and
PFOS from pollutant emissions and other products. The
second is that, for the first time, PFC-induced immunosup-
pression can be at least partly restored/mitigated by oral
admini- stration of a combination of glucan-resveratrol-
vitamin C. From our data, it could be imagined that RVB
300 might be used in the prophylactic treatment of PFC-
based poisonings. Experi- ments evaluating the mechanisms
of the RVB 300-based restoration of immunosuppression
seen here are currently under way. Furthermore, the clinical
importance of this observation deserves further study.
Declaration of interest The authors have no financial interests in
any company mentioned in this study and report no conflicts of
interest. The authors alone are responsible for the content of this
manuscript.
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