K.N. SULOCHANA et al.

Indian Journal of Pharmacology 2002; 34: 428-431 SHORT COMMUNICATION

Correspondence: S. Ramakrishnan
e-mail: drsr@sankaranethralaya.org
EFFECT OF CIGARETTE SMOKING ON CATARACT: ANTIOXIDANT ENZYMES
AND CONSTITUENT MINERALS IN THE LENS AND BLOOD OF HUMANS
K.N. SULOCHANA, R. PUNITHAM, S. RAMAKRISHNAN
Biochemistry Research Department, Sankara Nethralaya, Vision Research Foundation,
18, College Road, Chennai - 60.
Manuscript Received: 1.2.2002 Revised: 29.4.2002 Accepted: 7.7.2002
Objective: To find out the mechanism of cataract in smokers by investigating the antioxidant enzymes
and their constituent minerals in the lens and blood.
Methods: The study was carried out on 53 patients who underwent cataract surgery in Sankara
Nethralaya, India. The study population comprised of chronic smokers (27 males) and non-smokers
(26 males) with cataract. The determination of copper, zinc and selenium was carried out in their lens
materials and blood samples using inductively coupled plasma Atomic Emission Spectrometer.
Glutathione, glutathione peroxidase, superoxide dismutase were estimated by standard
spectrophotometeric methods in samples of blood and lens of all the subjects, while ceruloplasmin
was estimated in blood only.
Results: The significant finding of the study was a decrease in the activity of superoxide dismutase
and content of zinc in blood as well as in the lenses of cigarette smokers when compared to
nonsmokers. A significant decrease in the levels of glutathione and glutathione peroxidase activity
was found in lens, but not in blood of smokers when compared with nonsmokers. However, there was
no difference in the levels of ceruloplasmin in blood and copper and selenium in both blood as well as
lens samples of both the groups.
Conclusion: Decreased levels of superoxide dismutase in smokers may be due to depletion of zinc
in their blood and lens. Decreased levels of superoxide dismutase, glutathione and glutathione
peroxidase in lens of smokers suggest an oxidative insult to lens as the mechanism of cataract in
them.
Oxidative stress tobacco smokers trace elements nuclear cataract
ABSTRACT
KEY WORDS
INTRODUCTION
The association between cigarette smoking and nu-
clear cataract has been established by a series of
epidemiological studies both in the United Kingdom
and the United States
1
. Oxidative stress brought about
by Reactive Oxygen Species (ROS) present in smoke
has been implicated in the pathogenesis of cataract.
Oxidative stress could also be brought about by trace
elements like iron (Fe) and copper (Cu) through
glycoxidation
2
and production of super oxide anion
(O
2
l l
) and hydroxyl (OH
l l
) free radical which can oxi-
dise membrane proteins and lipids of lens. Secondly
a phenomenal accumulation of cadmium (Cd) in the
lens and blood of smokers with cataract has been re-
ported for the first time by the authors
3
. Rats when
exposed to tobacco smoke were found to accumulate
Fe and Cu along with Cd in their lenses
4
. Tobacco
leaves contain significant amounts of Cd which is ab-
sorbed into the body when a person smokers or chews
tobacco
3
. This Cd can replace the bivalent metals like
Zn, Cu and manganese (Mn) from superoxide
dismutase (SOD) a powerful antioxidant. It has also
been reported that Cd decreases the bio availability of
selenium (Se)
5
and this, in turn, may affect the activity
of glutathione peroxidase (GPx) another antioxidant
enzyme which requires glutathione (GSH) as a sub-
strate. Thus, there is a close collaboration in biochemi-
cal function between antioxidant enzymes, their sub-
strates and trace elements. Hence it was considered
CATARACT IN SMOKERS BY OXIDATIVE STRESS
desirable to investigate the levels of SOD, GPx, GSH
and the concerned minerals Zn, Cu and Se in the blood
and the cataractous lens and ceruloplasmin in blood
of people habituated to cigarette-smoking and com-
pare the results with those of nonsmokers.
MATERIALS AND METHODS
Twenty seven smokers and 26 nonsmokers partici-
pated in the study. The subjects were all males. This
is because smokers among females in this part of
India are rare. The maximum age limit was restricted
to 58 years, as aging itself could cause accumula-
tion of minerals and depletion of antioxidants. Since
cataract is a multifactorial disease the minimum age
limit was fixed at 40 years, as causes for cataract in
younger age group may be different. Smokers included
in the study were habituated to smoking atleast 10
cigarettes a day for more than 10 years. Individuals
with diabetes mellitus, hypertension, glaucoma, his-
tory of alcoholism were excluded as these are con-
sidered as other risk factors for cataract. The sub-
jects of the study were selected from community oph-
thalmic services wing of Sankara Nethralaya, India
and they were duly informed about the nature of the
study. With their consent, a questionnaire linked to
this study along with their personal details about their
addresses, clinical history, number of cigarettes
smoked per day were obtained. Necessary clinical
parameters were assessed by a physician and then
cataract was defined on the basis of slit lamp exami-
nation by an ophthalmologist. Patients with nuclear
cataract with and without posterior and anterior
subcapsular cataract were only included for the study.
People with cortical cataract were excluded from the
study. The study was approved by Institutional Re-
search and Ethics Committee of Vision Research
Foundation and all experiments pertaining to human
subjects strictly adhered to tenets of Helsinki decla-
ration.
Blood taken by venepuncture before surgery and the
intact cataractous lenses extracted by surgery were
used immediately for the analysis.
Analysis of minerals: A volume of 2 ml of whole
blood was digested with 3 ml of concentrated nitric
acid and perchloric acid mixture (5:1) by heating. The
residue was made up to 2 ml with five times distilled
water and a drop of concentrated HNO
3
was added to
prevent adsorption of minerals to the wall of the con-
tainer. Half the lens material was dried between the
folds of the filter paper, weighed, digested and made
up as the case of blood. The solutions were analyzed
for Cu, Zn and Se by Inductively Coupled Plasma
Atomic Emission Spectrometer (ICPAES) model 3410
ICP with mini torch spectrometer system (Perkin El
mer, CA, USA). Standards from Sigma chemical com-
pany, USA were used for calibration. The mineral analy-
sis was carried out at Regional Sophisticated Instru-
mentation Center, Indian Institute of Technology,
Chennai. Results were expressed as g/g of Hb in
blood, g/mg of protein in lens for antioxidant param-
eters. The levels of minerals were expressed as mg/
dl in blood and g/gm protein in lens.
Analysis of antioxidants: The remaining portion of
the lens was used for the determination of SOD
6
, glu-
tathione peroxidase
7
and GSH
8
. The blood samples
were also subjected to these analyses and also
ceruloplasmin estimation by following the standard
protocols described earlier
9
. Protein estimation was
done by the method of Lowry et al. using bovine se-
rum albumin as standard
10
.
Statistical analyses of results were done by compar-
ing smokers and nonsmokers using Mann Whitney
U test. Normal donor eye ball lenses obtained within
6 h of death from both men and women (non-smok-
ers) were used as control to get reference values for
the biochemical parameters studied. Values of GSH,
GPx and SOD in blood from healthy volunteers were
used as control reference value. The controls were
not subjected to statistical analysis and they were used
only for reference. P values <0.05 were considered
significant. The values were expressed as mean SD.
RESULTS
The levels of trace minerals Cu, Zn and Se in blood
and lens samples are given in Table 1. The levels of
Zn in blood and lens of cigarette smokers were de-
creased when compared to nonsmokers. This differ-
ence was statistically significant in both lens and
blood. However there was no difference in the levels
Cu and Se in blood and lens of smokers when com-
pared to nonsmokers.
Levels of SOD, GPx and GSH in blood and lens are
given in Table 2. Important finding was the highly
significant decrease in the activity of SOD in smok-
ers (group 1) in blood as well as in their lenses when
compared with nonsmokers (group 2). (P<0.001 for
429
K.N. SULOCHANA et al.
Table 1. Levels of trace elements in lens and blood from control, smokers and nonsmokers.
Copper Zinc Selenium
Groups
(Lens) (Blood) (Lens) (Blood) (Lens) (Blood)
g/gm of g/dl g/gm of g/dl g/gm of protein g/dl
protein protein protein
Group I 2.10 0.45 89 33 0.90 0.15 530 26 0.27 0.18 10.5 0.9
Smokers
n = 27
Group II 1.72 0.46 84 20 1.46 0.4* 610 48 0.27 0.15 13.0 1.1
Nonsmokers (1.59 0.8) (86 36) -- (606 56) (0.31 0.16) (12.4 3.2)
n = 26
Values in parentheses are the data from donor eyeballs/blood of both men and women aged 1-58 years and served as control for
reference purpose. P<0.05 when compared to group I.
Table 2. Levels of antioxidants in lenses and blood from control, smokers and nonsmokers.
GSH GPx SOD Ceruloplasmin
Groups
(Lens) (Blood) (Lens) (Blood) (Lens) (Blood) (Blood)
g/mg mg/gm of Hb mol of GSH mol of units/mg units/gm of Hb mg/dl
protein consumed/mg of GSH protein
protein consumed /
gm of Hb
Group I 1.9 1.5 2.56 1.19 1.2 0.67 29.6 3.3 0.29 0.23 2034 152 27.1 1.73
Smokers
n = 27
Group II 3.55 2.1* 2. 19 0.89* 2.5 0.8* 33.5 4.7 1.5 0.79* 3106 257** 27.2 2.13
Nonsmokers (5.6 2.8) (3.29 1.06) -- (34.5 8) (2.9 1.028) (3173 845) (18 - 45)
n = 26
Values in parentheses are the data from donor eyeballs/blood of both men and women aged 1-58 years and served as control for
reference purpose. *P<0.05; **P<0.001 when compared to group I.
GSH-glutathione; GPx-glutathione peroxidase; SOD-superoxide dismutase.
blood and < 0.05 for lens). Significantly low levels of
GSH were found in lens of smokers (1.9 g/mg
protein) when compared to nonsmokers (3.55 g/mg
protein). However, there was no difference in blood
levels of GSH between the two groups. GPx activity
in lens but not in blood was also significantly de-
creased in smokers.
DISCUSSION
Earlier studies have indicated that increased oxidative
stress and depletion of antioxidants particularly in vi-
tamin C, E and Beta carotene in plasma could be a
possible reason for cataract
11
. The present study re-
lates the effect of tobacco on lenticullar antioxidant
enzymes, concerned substrates and trace elements.
Significant reduction in the levels of GSH and GPx is
seen in the lenses removed from smokers, indicating
the importance of GSH metabolism in lens. Loss of
GSH and protein thiol and increased hydrogen perox-
ide in lenses is considered a major cause for the on-
set of cataractogenesis. However the results of re-
ports of such studies are mixed
12,13
. Present study
gives support to the view that smokers may have
decreased levels GSH and GPx in their lenses when
compared with those of nonsmokers indicating a defi-
nite insult to lens in smokers. Though the lenses af-
fected had decreased GPx and GSH, blood levels
were not altered.
430
CATARACT IN SMOKERS BY OXIDATIVE STRESS
In the present study a statistically significant decrease
in the levels of SOD both in blood and in the lens of
people who smoke has been established. It is reported
that the activity of SOD decreased in human lenses
with senile cataract as the cataract advances
14
. This
decrease in the level of SOD was correlated with the
decrease of Zn in smokers. SOD is a first order anti-
oxidant enzyme which requires Zn for its activity. De-
pletion of SOD activity may be primarily due to inad-
equate availability of Zn in blood and lens. This has
been confirmed by our recent clinical study, which in-
dicated that supplementation of Zn can help in increas-
ing erythrocyte SOD in smokers
15
. Decrease in Zn may
also be due to the competition with Cd as the latter is
reported to accumulate in lens of smokers. Surpris-
ingly Cu in the lens and blood
16
and copper containing
enzyme ceruloplasmin in blood were not altered.
It is also reported that Cd can decrease the bio-
availablity of Se
5
. As such, the levels of Se in blood
and lens of smokers could also decrease. But, our
results showed that there were no changes in the lev-
els of Se in the study groups indicating that decreased
activity of GPx may not be due to lack of Se. It may
be due to decreased availability of GSH needed for
the action of GPx in quenching ROS. Decrease in
the level of lenticular GSH, as one of the reasons for
cataract has been reported earlier. Increase in the
level of GPx in cataract reported by Delcourt C
et al
17
goes counter to imposition of oxidative stress
in smokers for which there is overwhelming evidence.
In conclusion, this study gives more information
favoring oxidative insult to lens in people who are
habituated to cigarette smoking. Zn supplementation
may be tried to combat this problem.
REFERENCES
1. Flaye DE, Sullivan KN, Cullinan TR, Silver JH, Whitelocke
RAF. Cataracts and cigarette smoking the city eye study.
Eye 1989;3:379-84.
2. Darley UV, Halliwell B. Blood radicals: Reactive nitrogen
species, reactive oxygen species, transition metal ions
and the vascular system. Pharm Res 1996;13:649-62.
3. Ramakrishnan S, Sulochana KN, Selvaraj T, Abdulrahim
A, Lakshmi M, Arunagiri K, et al. Smoking of beedies and
cataract: Cadmium and vitamin C in the lens and blood.
Br J Ophthalmol 1995;79:202-6.
4. Avunduk AM, Yardimci S, Avunduk MC, Kurnaz L, Kockar
MC. Determinations of some trace and heavy metals in
rat lenses after tobacco smoke exposure and their rela-
tionships to lens injury. Exp Eye Res 1997;65:417-23.
5. Hankinson SE, Willett WC, Colditz CA, Seddon JM, Resner
B, Speizer FE, et al. A prospective study of cigarette
smoking and risk of cataract surgery in women. JAMA
1992;268:984-8.
6. Misra HP, Fridovich IC. The role of superoxide anion in the
antioxidation of epinephrine and a simple assay for super-
oxide dismutase. J Biol Chem 1972;247:3170-5.
7. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman
DG, Hekstra WG, et al. Selenium biochemical role as a
component of glutathione peroxidase, purification and as-
say. Science 1973;179:588-90.
8. Beutler E, Duron O, Belly BM. Improved method for the
determination of blood glutathione. J Lab Clin Med 1963;
61:882-8.
9. Henry RJ, Chiamori N, Jacobs SL, Senglore N. Determi-
nation of ceruloplasmin oxidase in serum. Proc Soc Exp
Biol Med 1960;104:620-4.
10. Lowry OH, Rosebrough NJ, Farr AL, Randall RN. Protein
measurement with Folin's phenol reagent. J Biol Chem
1951;193:265-73.
11. Shalini VK, Luthra M, Srinivas L, Rao SH, Basti S, Reddy
M, et al. Oxidative damage to the eye lens caused by
cigarette smoke and fuel smoke condensates. Indian J
Biochem Biophy 1994;31:261-6.
12. Xie PY, Kanai A, Nakajima A, Kitahara S, Ohtsu A, Fujii K, et
al. Glutathione and glutathione-related enzymes in human
cataractous lenses. Ophthalmic Res 1991;23:133-40.
13. Ozmen D, Mutaf I, Ozmen B, Mentes J, Bayindir O. Lens
lipid peroxides and glutathione concentrations in diabetic
cataract. Ann Clin Biochem 1997;34:190-2.
14. Fujiwara H, Takigawa Y, Suzuki T, Nakata K. Superoxide
dismutase activity in cataractous lenses. Jpn J Ophthal-
mol 1992;36:273-80.
15. Sulochana KN, Punitham R, Ramakrishnan S. Oral sup-
plementation of zinc promotes erythrocyte superoxide
dismutase activity in chronic cigarette smokers-report on
a pilot clinic trial. Indian J Pharmacol 2001;33:224.
16. Cekic O. Effect of cigarette smoking on copper, lead, and
cadmium accumulation in human lens. Br J Ophthalmol
1998;82:186-8.
17. Delcourt C, Cristol JP, Leger CL, Descomps B, Papoz L.
Associations of antioxidant enzymes with cataract and
age related macular degeneration. The POLA study
pathologies ocularies Liees al'Age. Ophthalmology
1999;106:215-22.
431