Global Journal of Medical Research: J
Dentistry & Otolaryngology
Volume 20 Issue 8 Version 1.0 Year 2020
Type: Double Blind Peer Reviewed International Research Journal
Publisher: Global Journals Inc. (USA)
Online ISSN: 2249-4618 & Print ISSN: 0975-5888
Oral Submucous Fibrosis- God’s Furry or Age Old Habits?
By Dr. Samiha Bari & Dr. Rashmi Metgud
Abstract- Introduction: OSMF is a potentially malignant disorder predominant in people of Asian
descent. Copper plays important role in pathogenesis of OSMF. Lysyl oxidase is a copper
activated enzyme critical for collagen cross linking. The uptake of copper into the epithelial cells
occurs probably by a non-energy dependent diffusion. In Indians, the practice of drinking water
stored in copper vessels for health benefits is being followed since ages.
Aims and Objective: The present study was conducted to study the effect of consuming water
stored in copper vessels in predisposing an individual to OSMF by evaluating cytological
smears.
Materials and Methods: Cytological smears were prepared, stained with Rhodanine stain and
evaluated for the following groups.
Keywords: copper vessel, water, OSMF, rhodanine.
GJMR-J Classification: NLMC Code: WU 113
OralSubmucousFibrosisGodsFurryorAgeOldHabits
Strictly as per the compliance and regulations of:
© 2020. Dr. Samiha Bari & Dr. Rashmi Metgud. This is a research/review paper, distributed under the terms of the Creative
Commons Attribution-Noncommercial 3.0 Unported License http://creativecommons.org/licenses/by-nc/3.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Oral Submucous Fibrosis- God’s Furry or Age
Old Habits?
Dr. Samiha Bari α & Dr. Rashmi Metgud σ
Aims and Objective: The present study was conducted to
study the effect of consuming water stored in copper vessels
in predisposing an individual to OSMF by evaluating
cytological smears.
Materials and Methods: Cytological smears were prepared,
stained with Rhodanine stain and evaluated for the following
groups.
Group 1- Control group (n=10), individuals with no chewing
habit and normal oral mucosa.
Group 2- Study group (n=10), individuals consuming water
stored in copper vessel without areca nut consumption:
a) n=5, Individuals consuming vessel stored water throughout
the day
b) n=5, Individuals consuming vessel stored water once a day
Group 3- Clinically diagnosed OSMF (n=10) with arecanut
consumption history.
Statisical Analysis: Z test was performed to compare the
staining intensity amongst OSMF patients and copper vessel
water consuming subjects.
Results: The numbers of squames revealing presence of
copper granules were higher in smears of Copper vessel water
consuming subjects than OSMF.
Conclusion: In India, copper is used as a cost effective and
traditional method of disinfecting water. This age old habit is
still being practiced and inadvertent use of this habit might
have led to the predisposition of Indians to OSMF.
Keywords: copper vessel, water, OSMF, rhodanine.
Corresponding Author α: Post Graduate, Department Of Oral and
Maxillofacial Pathology, Pacific Dental College and Hospital, Udaipur.
e-mail: samiha.bari@gmail.com
Author σ: Professor and Head of the Department, Department Of Oral
and Maxillofacial Pathology, Pacific Dental College and Hospital,
Udaipur. e-mail: rashmi_metgud@rediffmail.com
T
Introduction
he exact etiology of OSMF is not well understood.
Multiple factors such as genetic, autoimmune,
nutritional and environmental are being studied.
Among the environmental agents different oral habits
like intake of spicy food, chewing of betel nut, betel quid
and other preparations are included. Amongst these
arecanut is confirmed as the main etiological factor.[1]
These factors are known to have either a direct effect or
indirect effect by mediating the immune system which is
compromised in OSMF.[1]
Pathogenesis is believed to involve juxtaepithelial inflammatory reaction and fibrosis in the oral
mucosa, probably due to an increased cross linking of
collagen through an increase in lysyl oxidase activity.
Fibrosis, or the building up of collagen, results from the
effects of areca nut, which increases collagen
production (e.g., stimulated by arecoline, an alkaloid)
and decreases collagen degradation.[2],[3]
Copper, present in arecanut plays an important
role in pathogenesis of OSMF. The enzyme lysyl oxidase
is a copper activated enzyme critical for collagen cross
linking. Copper is absorbed into the epithelial cells
occurs probably by a non-energy dependent diffusion.
Numerous factors influence the bioavailability and
subsequent absorption of copper by the oral mucosa. In
Indians, the practice of drinking water stored in copper
vessels for health benefits is being followed since ages.
The present study was being conducted to study the
effect of this age old habit as a cause for predisposing
Indian population to OSMF.
II.
Materials and Methods
Exfoliative cytology was performed using
cytobrush from buccal mucosal site. The participants
were asked to rinse their mouth before smears were
prepared. The prepared smears were transferred on the
glass slides and were fixed using alcohol based fixative.
The slide was stained for light microscopy with
Rhodanine (Lindquist method 1969)[4].
Additional smears were prepared from control
group. The smears were kept overnight in
supersaturated copper sulphate solution and were used
as positive controls.
The following groups were taken:
Group 1: Control group (n=10) i.e. individuals with no
chewing habit and normal oral mucosa.
© 2020 Global Journals
2020
I.
Year
disorder predominant in people of Asian descent. Copper
plays important role in pathogenesis of OSMF. Lysyl oxidase is
a copper activated enzyme critical for collagen cross linking.
The uptake of copper into the epithelial cells occurs probably
by a non-energy dependent diffusion. In Indians, the practice
of drinking water stored in copper vessels for health benefits is
being followed since ages.
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Abstract- Introduction: OSMF is a potentially malignant
Oral Submucous Fibrosis- God’s Furry or Age Old Habits?
Year
2020
Group 2: Study group (n=10) consisting of individuals
consuming water stored in copper vessel without any
history of areca nut consumption. It was further divided
into two sub groups depending on the duration of
consuming water stored in copper vessel
a) n=5, Individuals consuming vessel stored water
throughout the day
b) n=5, Individuals consuming water stored in vessel
for overnight
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Group 3: Subjects clinically diagnosed as Oral
Submucous Fibrosis (n=10)
The whole smear was evaluated in a zig zag
manner under 400x and 1000 cells per slide were
evaluated for the presence of staining. For interobserver
variability 3 persons randomly observed the slides and
divided them into three groups depending on staining
intensity as no stain, intermediate staining and intense
staining.
III.
Statistical Analysis
Z test was performed to compare the staining
intensity amongst OSMF patients and copper vessel
water consuming subjects. Also a comparison between
the staining intensity of subjects consuming water
stored in copper vessel throughout the day and subjects
consuming water stored overnight (once a day) was
done.
IV.
Results
People consuming copper vessel stored water
revealed staining comparable to that of OSMF patients.
The majority of squames in both the groups revealed
presence of copper granules. The results obtained were
found to be statistically significant with a p value of 0.02.
(Table 1)
Table 1: Comparison between subjects consuming Copper vessel stored water and OSMF
Groups
Mean
sd
Copper vessel
677.1
283.92
OSMF
628
226.9
p value
0.02(S)
z test was performed and p < 0.05 was considered to be significant
People consuming copper vessel stored water
throughout the day revealed dark red staining in majority
of squames than the ones consuming water once a day.
The results obtained were statistically highly significant
with a p value < 0.0006 (Table 2).
Table 2: Comparison between copper vessel stored water consumed throughout the day and consumed once a day
Groups
Mean
sd
A (consumed throughout
the day)
848.2
506
B (once a day)
138.236
298.46
p value
0.0006
z test was performed and the results were found to be highly significant
V.
Discussion
An epidemiological survey done have shown an
increase in number in India from 250000 cases reported
in 1980 to 2 million cases in 1993.[5] The epidemiological
assessment of the prevalence of OSF among Indian
villagers, based on baseline data, recorded a
prevalence of 0.2% (n 10,071) in Gujarat, 0.4% (n
10,287) in Kerala, 0.04% (n 10,169) in Andhra Pradesh,
and 0.07% (n 20,388) in Bihar. The prevalence among
101,761 villagers in the state of Maharashtra (central
India) was 0.03%.[2] A study of Moradabad district
(Nigam et al, 2014)[6] showed a prevalence rate of 6.3%
while in a study of rural Jaipur population (Rohit Sharma
et al, 2012)[7] showed prevalence rate of 3.39%. The
variation can be due to difference in the availability of
different products.
© 2020 Global Journals
Hypersensitivity to chili or betel quid is
explained as a common factor in OSMF development.
However, its development is very rare in people of
Mexico and South America even though their intake of
chilies is equal to or even exceeds from people of India
or south East Asia. Thus a genetic perspective is
considered vital to explain this condition. Almost all
carcinogenic agents, whether physical (radiation),
chemical, or infectious (viruses), act as mutagens. They
change the structure of the genetic material, producing
point mutations, deletions, insertions, or rearrangement.
Copper increases absorption of lysyl oxidase in oral
submucous fibrosis patients that causes cross linking of
collagen and makes it resistant to digestion by
collagenase enzyme. Thus it could be stated that an
increase in lysyl oxidase activity leads to accumulation
of collagen[8].
© 2020 Global Journals
Year
for the identification of abnormal tissue copper levels.
They observed that a minimum of 60 g of copper per
gram of tissue has to be present for cytochemical
identification of copper using Rhodamine staining
technique.[14]
Copper is widely used in household plumbing
materials. It is also enters the water (“leaches”) through
contact with the plumbing. Copper leaches into water
through corrosion. Copper can leach into water primarily
from pipes, but fixtures and faucets (brass), and fittings
can also be a source. The amount of copper in water
also depends on the types and amounts of minerals in
the water, how long the water stays in the pipes, the
amount of wear in the pipes, the water’s acidity and its
temperature. Safety of leached copper does not appear
to be an issue since studies have shown that the current
WHO guideline of 2 mg Cu/L is safe.[15,16]
Arakeri G et al (2014)[17] conducted a study to
evaluate that OSMF was significantly associated with a
raised concentration of copper in drinking water. The
study was carried out in a heterogeneous population in
Hyderabad-Karnataka, India, a region with a high
incidence of the condition. They evaluated 3 groups,
each of 100 patients: those with OSMF who chewed
gutkha, those who chewed gutkha but did not
have OSMF, and healthy controls who did not chew
gutkha. The difference between the groups in the mean
concentration of copper in water measured by atomic
absorption spectrometry was significant (p<0.001).
There were also significant differences between the
groups in mean concentrations of serum copper,
salivary copper, and ceruloplasmin (p<0.001). The
results confirm that copper in drinking water contributes
to the pathogenesis of OSMF, but ingestion of copper is
unlikely to be the sole cause.
In the present study a comparison between
normal subjects, copper vessel stored water consuming
and OSMF subjects showed difference in the no of
squames showing positive staining. The normal subjects
showed no staining (Fig1). Subjects consuming copper
vessel stored water throughout the day and those
consuming water stored overnight showed difference in
the staining intensity (Fig 2, 3). People consuming
copper vessel stored water throughout the day showed
majority of squames showing dark red staining than the
ones consuming water once a day(Fig4). The OSMF
patients also showed positive staining but the staining
was comparable with that of copper vessel subjects (Fig
5) as the OSMF patients taken were of clinically
diagnosed Stage 2 patients.
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According to Rajendran et al 2001[9] OSMF is
believed to be a localized lesion of fibrosis in that part of
the oral mucosa that had localized contact of copper,
however visceral organ fibrosis was not evident. The
bioavailability of copper and its absorption in the oral
mucosa is influenced by numerous factors. Some of
these include presence of amino acids, dietary fats,
carbohydrates, mineral elements, pH of oral
environment.
However the exact mechanism through which
copper is absorbed by the cells of the oral mucosa is
not properly known. In some literature role of membrane
bound copper transporting adenosine triphosphates is
evident and mentioned at cellular level.[10]
It is evident that copper binding sites form an
extended polypeptide chain at the amino terminus of the
transmembrane domain that regulates its absorption.
The reason for accumulation of copper by cells are
explained by the extracellular presence of tripeptide
glycyl-L-histidyl-Llysine (GHL), where the first two
residues of the GHL molecule are involved in the binding
of copper, whereas the side chain of lysine may be
involved in the recognition of receptors that function in
the uptake of copper into cells. This tripeptide may be
liberated within the lamina propria of areca chewers
during the initial inflammatory phase of OSMF.[11]
Interleukin -1 beta which has been shown to potentiate
collagen synthesis in vitro is another important regulator
of fibrosis that participates in the mediation of OSMF.
Any interaction of copper with other agents in the nut,
such as arecoline, and mediators of inflammation, such
as cytokines, need further study.
Since ancient times Ayurveda has advocated
the benefits of drinking water from a copper vessel.
Ayurveda states that when you store water in a copper
vessel it has the ability to balance all the three doshas in
our body. Scientifically speaking, when water is stored in
a copper vessel for over eight hours, very small
quantities of copper get dissolved in this water. This
process is called “oligodynamic effect” and has the
ability to destroy a wide range of harmful microbes,
molds, fungi etc. due to the toxic effect it has on living
cells.
Modified Rhodamine stain is a copper specific
stain and histologically demonstrates copper in the
tissues.[12] Comparision of different histochemical
staining methods has shown modified Rhodamine
technique to be the method of choice for the detection
of copper.[13] Copper appears red to orange-red stain
and the nucleus was stained blue.[4] Irons RD et al have
concluded that the Rhodamine method was found to
produce the most reproducible results and a linear
relationship between microscopical evaluation of the
stain and actual tissue copper levels was observed.
They considered that the Rhodamine method is
applicable for the semiquantitative evaluation of tissue
copper and provides a satisfactory screening method
2020
Oral Submucous Fibrosis- God’s Furry or Age Old Habits?
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Oral Submucous Fibrosis- God’s Furry or Age Old Habits?
Fig. 1: Normal mucosa showing no stain
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Fig. 2: Copper vessel water consumed throughout the day
Fig. 3: Copper vessel water consumed once a day
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Oral Submucous Fibrosis- God’s Furry or Age Old Habits?
Year
Fig. 4
Fig. 5: OSMF showing variability in staining
The copper sulphate dipped smears showed red staining and was taken as positive controls (Fig 6).
Fig. 6: Copper Sulphate Dipped smear
In mammals, copper can be absorbed from the
stomach to the distal small intestine. A critical
component of copper gastrointestinal balance involves
enterohepatic circulation. At least one-half of the amount
of copper reaching the small intestine reappears in the
bile as strongly bound compounds, and is lost in the
stool. The distribution of copper throughout the body is
mediated by ceruloplasmin, albumin, and other
quantitatively less important copper binders.[18]
Copper added to cooked foods with high
protein contents, such as chicken liver or chick peas,
was more poorly absorbed by rats than copper supplied
from other vegetable and animal sources.[19]
A study was conducted by Janet R
Hunt(2001)[20] that showed that although copper was
less efficiently absorbed from a vegetarian diet than
from a nonvegetarian diet, the total apparent copper
absorption was greater from the vegetarian diet
because of its greater copper content.
Storing water in copper and silver pots finds
mention in ancient texts of Ayurveda for purification of
water.[21] A study conducted by Sudha et al (2009)[22]
provided laboratory evidence of the antibacterial activity
of copper pot in distilled water.
In India, copper is used as a cost effective and
traditional method of disinfecting water. It is also used
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because of its health benefits mentioned in the Vedas.
This age old habit of consuming copper vessel stored
water is still being practiced. The present study findings
suggest that the inadvertent use of this age old habit
has led to the predisposition of Indians to OSMF.
Moreover majority of the Indian population is vegetarian
in diet that makes higher copper bioavalability as
compared to those having a non-vegetarian diet.
All these factors may be helpful to some extent
in providing an explanation to the fact that why OSMF is
more prevalent in Indian population. Although further
research with a large sample size is needed to prove
this hypothesis.
References Références Referencias
Pillai R, Balaram P, Reddiar KS. Pathogenesis of
oral submucous fibrosis. Relationship to risk factors
associated with oral cancer. Cancer. 1992;
69(8):2011-20.
2. Shieh T-Y, Yang J-F. Collagenase activity in oral
submucous fibrosis. Proceedings of the National
Science Council, Republic of China Part B, Life
sciences. 1992; 16(2):106-10.
3. Yang SF, Hsieh YS, Tsai CH, Chen YJ, Chang YC.
Increased plasminogen activator inhibitor‐1/tissue
type plasminogen activator ratio in oral submucous
fibrosis. Oral diseases. 2007; 13(2):234-8.
4. Bancroft JD, Gamble M. Theory and practice of
histological techniques: Elsevier Health Sciences;
2008.
5. Guta M, Mhaske S. Oral submucous fibrosis:
Current concepts in etioathogenesis. People′ s J Sci
Res. 2008; 1:39-44.
6. Sharma R, Raj SS, Miahra G, Reddy YG, Shenava S,
Narang P. Prevalence of oral Submucous fibrosis in
patients visiting Dental college in Rural area of
Jaipur, Rajasthan. J Indian Aca Oral Med Radiol.
2012; 24(1):1-4.
7. Mathew P, Austin RD, Soma Susan Varghese M.
Estimation and Comparison of Copper Content in
Raw Areca Nuts and Commercial Areca Nut
Products: Implications in Increasing Prevalence of
Oral Submucous Fibrosis (OSMF). Journal of
clinical and diagnostic research: JCDR. 2014;
8(1):247.
8. Trivedy C, Baldwin D, Warnakulasuriya S, Johnson
N, Peters T. Copper content in Areca catechu (betel
nut) products and oral submucous fibrosis. Lancet.
1997; 349(9063):1447.
9. Rajendra
R,
George
B,
Sivakaran
S,
Narendranathan N. Visceral organ involvement is
infrequent in oral submucous fibrosis (OSF). Indian
journal of dental research: official publication of
Indian Society for Dental Research. 2000;12(1):7-20.
10. Trivedy C, Meghji S, Warnakulasuriya K, Johnson
NW, Harris M. Copper stimulates human oral
11.
12.
13.
1.
© 2020 Global Journals
14.
15.
16.
17.
18.
19.
20.
21.
22.
fibroblasts in vitro: a role in the pathogenesis of oral
submucous fibrosis. Journal of oral pathology &
medicine. 2001;30(8):465-70.
Maquart F, Bellon G, Chaqour B, Wegrowski J, Patt
L, Trachy R, et al. In vivo stimulation of connective
tissue accumulation by the tripeptide-copper
complex glycyl-L-histidyl-L-lysine-Cu2+ in rat
experimental
wounds.
Journal
of
Clinical
Investigation. 1993; 92(5):2368.
Jain S, Scheirer P, J, Archer B, Newman S, P,
SherlockS. Histological demonstration of copper
and copper-associated protein in chronic liver
disease. J C lin Pathol.(8):784-90.
TE B. Comparison of three histochemical staining
methods for the detection of copper in white perch
(Morone americana) with abnormal hepatic copper
storage. J Comp Pathol.
Irons R, Schenk E, Lee J. Cytochemical methods for
copper. Semiquantitative screening procedure for
identification of abnormal copper levels in liver.
Archives of pathology & laboratory medicine. 1977;
101(6):298-301.
Pettersson R, Rasmussen F, Oskarsson A. Copper
in drinking water: not a strong risk factor for
diarrhoea
among
young
children.
A
population‐based study from Sweden. Acta
Paediatrica. 2003; 92(4):473-80.
Araya M, Olivares M, Pizarro F, Llanos A, Figueroa
G, Uauy R. Community-based randomized doubleblind study of gastrointestinal effects and copper
exposure in drinking water. Environmental health
perspectives. 2004; 112(10):1068.
Arakeri G, Hunasgi S, Colbert S, Merkx M, Brennan
PA. Role of drinking water copper in pathogenesis
of oral submucous fibrosis: a prospective case
control study. British Journal of Oral and
Maxillofacial Surgery. 2014; 52(6):507-12.
Wapnir RA. Copper absorption and bioavailability.
The American journal of clinical nutrition. 1998;
67(5):1054S-60S.
Johnson PE, Lee D-Y. Copper absorption and
excretion measured by two methods in rats fed
varying concentrations of dietary copper. J Trace
Elements Exp Med. 1988; 1:129-41.
Hunt JR, Vanderpool RA. Apparent copper
absorption from a vegetarian diet. The American
journal of clinical nutrition. 2001; 74(6):803-7.
Sharma P. Susruta Samhita, Vol 1, Sutra Sthana 45,
Verse 13, 418. Varanasi: Chaukamba Visva Bharati.
2004.
VP,
Singh
KO,
Prasad
S,
Sudha
Venkatasubramanian P. Killing of enteric bacteria in
drinking water by a copper device for use in the
home: laboratory evidence. Transactions of the
Royal Society of Tropical Medicine and Hygiene.
2009; 103(8):819-22.