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The role of transforming growth factor-β1 and oxidative stress in

podoconiosis pathogenesis

Article  in  British Journal of Dermatology · February 2010

DOI: 10.1111/j.1365-2133.2010.09652.x · Source: PubMed

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C L I N I C A L A N D L A B O R A T O R Y I N V E S TI G A T I O N S British Journal of Dermatology

The role of transforming growth factor-b1 and oxidative

stress in podoconiosis pathogenesis
S. Addisu, T.H. El-Metwally, G. Davey,* Y. Worku and M.A. Titheradge
Department of Biochemistry, Faculty of Medicine, *School of Public Health, Addis Ababa University, PO Box 26905 ⁄1000, Addis Ababa, Ethiopia
Department of Biochemistry, School of Life Sciences, University of Sussex, Brighton, U.K.

Summary

Correspondence Background Podoconiosis (endemic nonfilarial elephantiasis) occurs in susceptible

Gail Davey. individuals who go barefoot in regions of irritant volcanic soil. Silicate particles
E-mail: gailinaddis@hotmail.com absorbed via the skin are thought to induce an inflammatory process and a con-
sequent endolymphangitis of the lower leg lymphatics.
Accepted for publication
1 September 2009
Objectives To establish which oxidative stress biomarkers play a part in the inflam-
matory process, and to test whether transforming growth factor (TGF)-b1 also
Key words has a pathogenetic role.
oxidative stress, pathogenesis, podoconiosis, Patients and methods We enrolled 50 patients with early clinical stage disease, 43
transforming growth factor-b1 patients with advanced stage disease and 35 local healthy controls. Oxidative
stress biomarkers included serum total peroxides (TP), total antioxidant capacity
Conflicts of interest
None declared. (TAC), total nitrate plus nitrite (TN), malondialdehyde (MDA) and total superox-
ide dismutase (SOD) activity. The oxidative stress index (OSI) was also deter-
DOI 10.1111/j.1365-2133.2010.09652.x mined. Serum total TGF-b1 was assayed using sandwich enzyme-linked
immunosorbent assay.
Results Compared with healthy controls, patients with early stage disease showed
significantly higher mean levels of TP (P < 0Æ001), MDA (P < 0Æ05) and OSI
(P < 0Æ01); and significantly lower mean concentrations of SOD (P < 0Æ001) and
TGF-b1 (P < 0Æ001). Mean levels of TGF-b1 were even lower among patients
with advanced stage disease (P < 0Æ001). Mean TAC levels were significantly
lower among patients with advanced disease than either other group
(P < 0Æ001).
Conclusions This is the first study, to our knowledge, to attempt to elucidate the
molecular pathogenetic events in podoconiosis. We conclude that TGF-b1 may
have a pathogenetic role, with oxidative stress playing a minor role in the early
stages of disease.

Podoconiosis is a form of noninfectious elephantiasis caused
by long-term exposure of bare feet to irritant clay soils of vol-
canic origin. It is found in several countries in tropical Africa,
where such soils coexist with high altitude, high seasonal rain-
fall and low income. The pathogenesis of podoconiosis is not
yet fully elucidated, but at present, most evidence suggests an
important role for mineral particles on a background of
genetic susceptibility.1
Silicate particles entering through the skin are thought to be
taken up by macrophages in the lower limb lymphatics, caus-
ing subendothelial oedema, endolymphangitis, and eventually
collagenization and obliteration of the lymphatic lumen.2 The
compromised lymphatic system causes localized fluid retention
and lymphoedema. In other forms of chronic lymphoedema,
the impairment of lymphocyte and Langerhans cell trafficking
from skin to regional lymph nodes leads to inefficient clear-
ance of foreign antigens, and provides the substrate for
chronic inflammatory changes.3,4 Adipocytes, keratinocytes
and fibroblasts accumulate, transforming the initially soft
swollen tissue into a hard fibrotic mass and stiff, thickened
hyperkeratotic skin (Fig. 1).5,6
Most models of silica-induced damage relate to the lungs,
where phagocytosis of soil particles activates macrophages to
release chemokines, cytokines, reactive oxygen species (ROS)
and other mediators that sustain inflammation.7,8 In vitro, oxi-
dative stress-related changes of gene expression and cell sig-
nalling pathways are among the underlying mechanisms of
ultrafine particle damage.9

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998 Journal Compilation  2010 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp998–1003

Fig 1. A 15-year-old patient with stage 3 disease, before treatment;
this patient was not one of the patients approached for this study.
In addition to oxidative stress-related mechanisms there is
some evidence for a role of transforming growth factor
(TGF)-b1 in mediating ultrafine particle damage. The effects
of TGF-b1 are diverse and include the modulation of cell pro-
liferation and differentiation, angiogenesis, inflammatory or
immune responsiveness, and induction of extracellular matrix
production.10 TGF-b1 has been implicated in the pathogenesis
associated with lung silica exposure where its expression
co-localizes with silicotic granulomas in rodents and
humans.11–13
TGF-b1 and oxidative stress mediators are both clearly
linked with ultrafine silica damage in tissues other than the
lower leg lymphatics. This study was designed to explore
serum oxidative stress status and TGF-b1 levels in early and
advanced stage podoconiosis, and to compare them with those
found in healthy individuals in the same community.
Patients and methods
Study subjects and sampling
Patients were adults over 18 years of age attending podocon-
iosis clinic sites in Areka and Gununo villages, Wolaita Zone,
Southern Ethiopia for the first time. All participants had lived
in the area for at least 18 years. Clinical examination of
long-term residents in an endemic area such as Wolaita Zone
has been shown to have very high predictive value for podo-
coniosis.14 Patients with any other disease likely to affect
oxidative status (including tuberculosis, malaria and leish-
maniasis) were excluded. Controls were healthy individuals
from the same villages who were free of symptoms and
signs. Sample size calculation using information on likely
means and standard deviations15 showed that including 50
individuals in each group would enable a difference of at
least 20% in biomarker level to be demonstrated at the 95%
significance level. Disease was staged according to the Tekola
staging system, which is based on the proximal extent of
 2010 The Authors
Journal Compilation  2010 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp998–1003
Podoconiosis and TGF-b1, S. Addisu et al. 999
swelling, dermal nodules, ridges and bands (Table 1).16
Stages 1–3 were considered early and stages 4 and 5 were
considered advanced disease. Each potential participant was
offered information about the study and signed or thumb-
printed a consent form. The study was approved by the
Research and Publication Committee of the Faculty of Medi-
cine, Addis Ababa University, Addis Ababa, Ethiopia. Fasting
morning sera from ~6 mL of blood were separated, aliquot-
ted and frozen at )80 C.
Laboratory investigations
All assays were performed in the Department of Biochemis-
try, Addis Ababa University. Serum levels of malondialdehyde
(MDA), total peroxides (TP) and nitrate plus nitrite (TN)
formation were measured because markers of oxidative stress
and generation of superoxides and other free radicals such as
these have previously been implicated in silica particle-
induced inflammation7,8 and also lymphatic filiarisis due to
Wuchereria bancrofti infection.15 Similarly, serum levels of
superoxide dismutase (SOD) activity and total antioxidant
capacity (TAC) were determined to evaluate the involvement
of any reduction in the defences against oxidative stress dur-
ing the course of the disease and also to determine the oxi-
dative stress index (OSI), an important indicator of the
redox balance between oxidation and antioxidation in many
disease states.17 The oxidative stress markers, SOD activity
and antioxidant capacity were correlated with the OSI to
determine the importance of each of these parameters in the
development of the disease and also with total TGF-b1, a
possible mediator.11–13
Table 1 Tekola staging system16
Stage 1 Swelling reversible overnight
The swelling is not present when the patient first
gets up in the morning
Stage 2 Below-knee swelling that is not completely reversible
overnight; if present, knobs or bumps are below the
ankle ONLY
Persistent swelling that does not reach above the
knee; if knobs or bumps are seen or felt, they are
only present below the ankle, NOT above the ankle
Stage 3 Below-knee swelling that is not completely reversible
overnight; knobs or bumps present above the ankle
Persistent swelling that does not reach above the
knee; knobs or bumps can be seen or felt above the
ankle as well as below
Stage 4 Above-knee swelling that is not completely reversible
overnight; knobs or bumps present at any location
Persistent swelling that is present above the knee;
knobs or bumps can be seen or felt at any place on
the foot or leg
Stage 5 Joint fixation; swelling at any place in the foot or leg
The ankle or toe joints becomes fixed and difficult to
flex or dorsiflex; this may be accompanied by
apparent shortening of the toes
1000 Podoconiosis and TGF-b1, S. Addisu et al.

MDA, a stable end-product of lipid peroxidation, was

assayed colorimetrically using 2-thiobarbituric acid (TBA).18
Total SOD activity was assayed using a commercial SOD deter-
mination kit (Superoxide dismutase assay kit, catalogue no.
19160; Fluka, Buchs, Switzerland), based on the ability of
SOD to inhibit the reduction of water-soluble WST-1 [2-(4-
iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetra-
zolium, monosodium salt] by O
2 :19
TP were measured as H2O2 equivalents by the oxidation of
xylenol orange into a purple-coloured product by ammonium
ferrous sulphate.20 TAC was measured as Trolox (6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents, a
water-soluble a-tocopherol analogue. The reaction is based
upon the antioxidant-induced bleaching of the ABTS•+ radical
cation (prepared by the reduction of 10 mmol L)1 2,2¢-azin-
obis-3-ethylbenzothiazoline-6-sulfonate by 2 mmol L)1 H2O2
in 30 mmol L)1 acetate buffer, pH 3Æ6) in 400 mmol L)1
acetate buffer, pH 5Æ8.17
OSI was calculated as a ratio of TP content in lmol L)1 to
TAC in mmol L)1 multiplied by 100 of each sample.17 TN
concentration was determined after reduction of NO3 into
NO2 using formate–nitrate reductase and determination of the
NO2 using Griess reagent.21–23 Total TGF-b1 was measured
by sandwich enzyme-linked immunosorbent assay using a
commercial kit (Transforming Growth Factor-b1 Multispecies
ELISA kit, Catalogue no. KAC1688; Invitrogen Corporation,
Carlsbad, CA, U.S.A.). The concentration of TGF-b1 in the
serum was calculated from a standard calibration curve (0Æ0–
2000 pg mL)1).
Statistical analysis
Data were analysed using an Excel data sheet and Prism 3.0
statistical package (GraphPad Software, Inc., San Diego, CA,
U.S.A.). Data are expressed as mean ± SEM. Results were anal-
ysed statistically using column statistics and one-way ANOVA
with Newman–Keuls Multiple Comparison Test as a post test.
Within each group, the correlation among the investigated
parameters was tested by the nonparametric Spearman’s analy-
sis. The level of significance was set at P < 0Æ05.
Results
Fifty patients with early stage podoconiosis (aged 19–
65 years; mean 38Æ9 ± SEM 1Æ8); 43 with advanced disease
(aged 23–72 years; mean 43Æ5 ± SEM 1Æ6) and 35 healthy
local control individuals free from symptoms or signs of the
disease (aged 22–56 years; mean ± SEM 37Æ3 ± 1Æ6) took part
in the study. The average levels of MDA, SOD, TP, TAC, OSI,
TN and TGF-b1 in the different groups are summarized in
Table 2 and Figure 2.
Mean levels of parameters
Mean TP concentration among patients with early stage disease
was significantly higher than that among controls or patients
with advanced disease (P < 0Æ001). The same pattern was seen
in relation to OSI. Mean MDA was significantly higher among
patients with early stage disease compared with the healthy
controls. Mean total SOD activity was significantly lower in
patients with early disease than either healthy controls or
patients with advanced disease. Mean TAC was significantly
lower (P < 0Æ001) in patients with advanced disease than
among controls or patients with early stage disease. No signifi-
cant differences between groups were seen in relation to mean
TN concentration. Mean serum total TGF-b1 levels were 50%
lower in patients with both early and late stage disease com-
pared with healthy controls (P < 0Æ001).
Correlations between parameters
In healthy controls, only TP was significantly positively corre-
lated with OSI (r = 0Æ952; P < 0Æ001), while TN was signifi-
cantly positively correlated with TGF-b1 (r = 0Æ313;
P < 0Æ05). In patients with early stage disease, OSI was signifi-
cantly negatively correlated with TGF-b1 (r = )0Æ298;
P < 0Æ05) and TAC (r = )0Æ437; P < 0Æ01), but positively
correlated with TP (r = 0Æ785; P < 0Æ001); TN was positively
correlated with SOD (r = 0Æ266; P < 0Æ05); TGF-b1 was nega-
tively correlated with MDA (r = )0Æ409; P < 0Æ01) and TP
(r = )0Æ260; P < 0Æ05); and SOD was positively correlated

Table 2 Laboratory results in sera of patients

Mean ± SEM Control Early stage Advanced stage with podoconiosis (early vs. advanced stage)
and disease-free controls
MDA, lmol L)1 0Æ377 ± 0Æ016 0Æ443 ± 0Æ018*-C, ns-A 0Æ415 ± 0Æ020ns-C
SOD, U mL)1 16Æ0 ± 0Æ3 14Æ7 ± 0Æ1***C, **A 15Æ5 ± 0Æ2ns-C
TP, lmol L)1 26Æ9 ± 0Æ946 31Æ3 ± 0Æ818***C ⁄ A 26Æ4 ± 0Æ6ns-C
TAC, mmol L)1 Trolox 1Æ62 ± 0Æ01 1Æ62 ± 0Æ03ns-C 1Æ49 ± 0Æ02***C ⁄ E
equivalents
OSI 1Æ66 ± 0Æ06 1Æ96 ± 0Æ06***C, *A 1Æ77 ± 0Æ04ns-C
TN, lmol L)1 62Æ8 ± 2Æ0 61Æ1 ± 1Æ3ns-C ⁄ A 59Æ3 ± 1Æ6ns-C
TGF-b1, ng mL)1 28Æ9 ± 1Æ9 15Æ6 ± 1Æ0***C, ns-A 14Æ1 ± 1Æ2***C

MDA, malondialdehyde; OSI, oxidative stress index; SOD, total superoxide dismutase activ-
ity; TAC, total antioxidant capacity; TGF, transforming growth factor; TN, total nitrate plus
nitrites; TP, total peroxides; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid; C, vs. healthy controls; A, vs. advanced stage; E, vs. early stage; ns, nonsignificant.
ANOVA P-value: *, 0Æ05; **, 0Æ01; ***, 0Æ001.

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Journal Compilation  2010 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp998–1003

70
Total nitrites
55
40
Total peroxides
25
TGF-b1
10 Superoxide dismutase activity
2·5
Oxidative stress index
2·0
1·5 Total antioxidants
1·0
0·5 Malondialdehyde
0·0
Control Early Stage Advanced Stage
Fig 2. Pattern of change in total nitrites, malondialdehyde, total
antioxidants, total peroxides, oxidative stress index, superoxide
dismutase activity and transforming growth factor (TGF)-b1 in
patients with early and advanced stage podoconiosis compared with
disease-free controls. For the mean ± SEM and ANOVA P-value, see
Table 2.
with TN (r = 0Æ335; P < 0Æ05). In patients with advanced
stage disease, OSI was significantly negatively correlated with
TAC (r = )0Æ427; P < 0Æ01) and positively correlated with TP
(r = 0Æ752; P < 0Æ001); TAC was positively correlated with
TN (r = 0Æ349; P < 0Æ05) and TGF-b1 (r = 0Æ358; P < 0Æ05)
but negatively correlated with TP (r = )0Æ267; P < 0Æ05); TP
and TN were positively correlated with MDA (r = 0Æ350;
P < 0Æ05 and r = 0Æ344; P < 0Æ05, respectively). SOD was
positively correlated with TGF-b1 (r = 0Æ345; P < 0Æ05).
Discussion
Clinically, the early stages of podoconiosis are characterized by
lymphoedema, hyperkeratosis and fibrosis. Small particles of
silica, iron and aluminium oxide have been demonstrated in
the phagosomes of dermal tissue macrophages of the foot with
the activated macrophages thought to recruit other inflamma-
tory cells towards the injured region.24 Early events in lung
silicosis include the accumulation of alveolar macrophages and
lymphocytes as well as the release of cytokines25,26 and it is
proposed that silica-induced cellular generation of ROS, proin-
flammatory cytokines and fibrogenic mediators all contribute
to disease development in lung silicosis.27–30
In vitro studies using different cell systems have shown vary-
ing degrees of proinflammatory- and oxidative-stress-related
cellular responses after dosing with laboratory-generated or fil-
ter-collected ambient ultrafine particles9,31–33 and have sug-
 2010 The Authors
Journal Compilation  2010 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp998–1003
Podoconiosis and TGF-b1, S. Addisu et al. 1001
gested that oxidative stress may be important in the
development of podoconiosis pathogenesis. No previous stud-
ies have measured overall biomarkers such as total antioxidant
capacity and total concentration of peroxides as potential
mediators in podoconiosis, although measurements of some
markers of oxidative stress have been made in chronic lym-
phatic filariasis.15
The significant increase in the TP content measured in this
study includes increases in organic (mainly lipid) and inor-
ganic (H2O2) peroxides. This reflects significant generation of
oxygen radicals such as superoxide anion (O
2 ) and hydroxyl
radical (HO·) as has been previously reported in dermal lym-
phoedematous tissues.34 Interestingly both in vivo and in vitro
studies have also shown that nanoparticles of various composi-
tions induce release of different ROS.8,35,36
The increased peroxide concentration may be pathogenetic
as it was significantly increased only in patients with early
stage disease and was not apparent during the advanced stages.
It is possible that pro-oxidant chemicals in the volcanic
soil24,28,35,37 may induce formation of these peroxides. Evi-
dence for this comes from studies implicating a role for silica-
induced cellular generation of ROS, proinflammatory cytokines
and fibrogenic mediators in the development of lung silico-
sis.28–30 Similarly, intense inflammatory changes in the dermis
and the subdermis have been demonstrated in a mouse model
of chronic lymphoedema, where the upregulation of genes
related to inflammation, oxidative stress response, immune
response, complement activation and wound healing with
excessive fibrosis were reported.4,9 The subsequent elimination
of activated macrophages (the major source for these perox-
ides)30,38 might explain the later decline in the generation of
these peroxides during disease progression. The positive cor-
relations of TP level with OSI and MDA in all subjects investi-
gated was rational as TP level is a major determinant of the
OSI.
Alterations in SOD activity may also be important when
combined with alterations in other antioxidants, particularly
catalase and glutathione peroxidase, that dispose of the H2O2
produced by the SOD reaction.29 In the present study, the
reduction of SOD activity correlated with increased levels of
peroxides and MDA, particularly in the early stages of the dis-
ease. Pro-oxidants and lipid peroxidative products are able to
directly inhibit SOD activity39,40 and therefore the increased
serum MDA and TP concentrations may be responsible for the
decreased serum SOD activity in the early stages of the disease.
A similar correlation between plasma MDA concentration and
SOD activity has also been demonstrated in chronic and occult
Bancroftian filariasis.15 The observed normalization of SOD
activity in the late stages of podoconiosis may also be
explained by the reduction of TP and MDA levels in these
individuals.
We observed a late decline in total antioxidant capacity,
suggesting eventual decreased generation of antioxidants after
their consumption to buffer pro-oxidants produced during the
early stages. In the early stages, MDA and TP are increased,
while SOD activity is reduced, resulting in a high OSI at this
1002 Podoconiosis and TGF-b1, S. Addisu et al.
stage. Later, this effect was ameliorated, possibly indicating
that the initial inflammation and oxidative stress subsided dur-
ing the progression of the disease as a result of apoptosis of
inflammatory and skin cells, the major sources of pro-oxi-
dants. A similar scenario has been reported for alveolar macro-
phages exposed to fine particles41 and a hyporesponsive
inflammatory reaction in lymphatic filariasis has also been
suggested to account for the late nonsignificant changes in
MDA and SOD.15 In the late stages of podoconiosis, chronic
damage results in lymphatic obstruction, hyperkeratosis and
oedema of the legs, so reduced exposure to nanoparticles
might explain the lower OSI. A murine model of silicosis has
also indicated early inflammation and oxidative stress that later
subsided into fibrosis.25
A beneficial role for TGF-b1 in skin health and podoconi-
osis may be inferred from its positive correlation with anti-
oxidant biomarkers and negative correlation with pro-oxidant
biomarkers. TGF-b1 is a potent keratinocyte growth inhibitor
that is overexpressed in keratinocytes in certain inflammatory
skin diseases.42 TGF-b1 also stimulates collagen synthesis
in vitro,28 and this appears to be regulated by glutathione in
fibroblasts.43 In the lung, inorganic dusts induce macro-
phage-derived cytokines including TGF-b1;25,28,29 therefore
the decrease in TGF-b1 we observed was unexpected. The
data in Table 2 show a highly significant reduction in serum
total TGF-b1 concentration in both the early and late stage
of podoconiosis compared with the healthy control group. In
contrast to the measurements of TP, MDA and SOD, there
was no significant difference in TGF-b1 concentration
between patients at early and late stages of disease, the
reduction in TGF-b1 being highly significant in both groups.
There are several possible explanations for the reduction in
TGF-b1. Firstly, oxidative stress or cytokines released by soil
components may lead to inhibition of one of the signalling
pathways controlling the expression of TGF-b1.44 Secondly,
chronic exposure to soil particles may lead to inactivation
and apoptotic death of macrophages and keratinocytes, the
major sources of TGF-b1. This would be consistent with the
progressive reduction in the TGF-b1 level in advanced stage
disease. Thirdly, inherited polymorphisms may have led to
reduction in the expression of the TGF-b1 gene. This last
explanation is consistent with the finding that serum TGF-b1
level is already reduced in early stage disease and worsens in
advanced stages. Previous studies have demonstrated that the
concentration of TGF-b1 is predominantly under genetic
control with a heritability estimate of 0Æ54.45 It is therefore
possible that podoconiosis-susceptible individuals are low
TGF-b1 expressors.
This is the first study, to our knowledge, to attempt to elu-
cidate molecular pathogenetic events in podoconiosis. We
have demonstrated a small but significant increase in oxidative
stress reflected as significantly higher TP, MDA and OSI levels
and reduction of SOD activity in patients with early clinical
podoconiosis. This suggests a mild degree of inflammation in
the early pathogenetic process of the disease. However, reduc-
tion in the total antioxidant capacity was noticed only at the
Journal Compilation  2010 British Association of Dermatologists • British Journal of Dermatology 2010 162, pp998–1003
advanced clinical stage of the disease when oxidative stress
levels were normalized. The most significant observation was
the progressive reduction in TGF-b1, an important modulator
cytokine of skin homeostasis. Future investigations should
address possible links between susceptibility to the disease and
the mechanisms responsible for the decrease in TGF-b1,
including polymorphisms affecting the regulatory sequences
of the TGF-b1 gene in patients compared with genetically
related and unrelated local individuals not showing symptoms
of the disease.
Acknowledgments
We wish to thank Meskele Ashine of the Mossy Foot Treat-
ment & Prevention Association for his assistance with field
work, and all the patients and controls who participated.
Funding was provided from the Association of Physicians
‘Links with Developing Countries’ Scheme.
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