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\begin{document}


\def\RunningAuthor{Vaishali  S Pawar and Ajit Sontakke}
\firstPage{2942}
\articleType{Original Article}
\receivedDate{14 Feb 2020}
\acceptedDate{17 Mar 2020}
\revisedDate{14 Mar 2020}
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\title{Impact of tobacco use on oxidative stress in cataract}
\author{Vaishali~~S~Pawar, 
            Ajit~Sontakke\textsuperscript{*}~\\[5pt]{Department of Biochemistry, Krishna Institute of Medical Sciences, Karad - 415539, Maharashtra, India}}

\begin{abstract}
Even today, age related cataract is prominent cause of visual deterioration. Cataract is associated with various risk factors and tobacco is one of the modifiable risk factors. Association between smoking and increased oxidative stress among cataract patients has been shown by some studies. But studies regarding smokeless tobacco are not done. This study was intended to estimate antioxidant enzyme Superoxide Dismutase (SOD) and MalonDiAldehyde (MDA) which is a lipid peroxidation product in tobacco user's Age Related Cataract (ARC) subjects and compare them with tobacco nonuser age related cataract (ARC) subjects. Out of 120 subjects, 60 were tobacco nonuser age related cataract subjects and 60 tobacco users were age related cataract subjects. 60 tobacco user cataract subjects subdivided into 5 different groups as tobacco smokers, tobacco chewers, tobacco mishri users, tobacco chewers as well as mishri users and smokers as well as smokeless tobacco users. Erythrocytic SOD and serum MDA was estimated. The study showed that in tobacco user group age of cataract patients was significantly less than tobacco nonuser cataract patients (P{\textless}0.05). Levels of Erythrocytic SOD were  significantly low and levels of serum MDA  were significantly elevated in tobacco user cataract patients than tobacco non-user cataract patients (P{\textless}0.001). In tobacco user group, nuclear cataract was significantly high (P{\textless}0.05). On comparison of subtypes of cataract in tobacco user subgroup, no significant difference was found. From these results, we may conclude that tobacco use intensifies the oxidative stress that is already present in age related cataract and this may accelerate the progression of ARC mainly nuclear cataract. 
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Age related cataract,\newline Malondialdehyde,\newline Oxidative stress,\newline Superoxide Dismutase,\newline tobacco users
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Ajit~Sontakke~\\ Phone:\ 9423867394~\\  Email:\ ajitsontakke712@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
In current situation, tobacco abuse is a substantial public health problem around the world and is a preventable cause of many diseases like chronic obstructive pulmonary disease, heart related diseases, cancers, reproductive outcomes, oral diseases, including cataract \unskip~\citep{716035:16980158}. In India, scenario of tobacco consumption is far worse, as it is prevalent in all socio-economic and ethnic groups in urban as well as remote villages.

Tobacco use is mainly in two forms: smoking and smokeless tobacco products \unskip~\citep{716035:16980152}. India is one of the topmost producer and second largest consumer of tobacco in the world with 229 million tobacco users, mainly in the form of smokeless tobacco (SLT) \unskip~\citep{716035:16980152}. \ensuremath{^{}}

Although the basic mechanism implicated in the pathogenesis of diseases associated with tobacco use are not precisely known, free radical induced damage has been suggested to play an influential role \unskip~\citep{716035:16980140}.\ensuremath{^{}} Out of 5,000 compounds present in tobacco, most are well known sources of free radicals \unskip~\citep{716035:16980157}. When tobacco enters in blood circulation, it causes alterations in cells ensuing increased production of free radicals. 

\ensuremath{^{}}Senile or age-related cataracts contribute to more than 80\% of all cataracts. There is no conclusive preventive means or medical treatment for cataract and is conventionally treated with surgery. In the last two decades, world has witnessed tremendous advancement in cataract surgery from intra-capsular cataract extraction to femtosecond laser cataract surgery; still cataract remains the predominant cause of visual deterioration. According to Andhra Pradesh Eye Disease Study (APEDS), in India around 44\% of the total blindness is due to cataract \unskip~\citep{716035:16980138}. \ensuremath{^{}}


\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Age, gender and group wise distribution of cataract patients.}} }
\label{tw-23d9084b4787}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.22490000000000002\linewidth-2\tabcolsep}p{\dimexpr.24299999999999997\linewidth-2\tabcolsep}p{\dimexpr.1529\linewidth-2\tabcolsep}p{\dimexpr.15009999999999998\linewidth-2\tabcolsep}p{\dimexpr.2291\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{Group}} & {\multirow{2}{*}{No. of participants}} & \multicolumn{2}{p{\dimexpr(.30299999999999997\linewidth-2\tabcolsep)}}{Gender} & {\multirow{2}{*}{Age in years \mbox{}\protect\newline Mean \ensuremath{\pm}SD}}\\
\rowcolor{kwdboxcolor} &  & Male & Female  & \\
\tblmidrule 
Tobacco nonuser (G1) &
  60 &
  29 &
  31 &
  52.53 \ensuremath{\pm}5.50\\
Tobacco user (G2) &
  60 &
  48 &
  12 &
  50.25\ensuremath{\pm}5.69 \\
Total &
  100 &
   &
   &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Comparison of SOD \& MDA between the study groups}} }
\label{tw-d48600093ae0}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.3611\linewidth-2\tabcolsep}p{\dimexpr.3491\linewidth-2\tabcolsep}p{\dimexpr.2898\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Study groups & E.SOD (U/ml of whole blood)  & S. MDA (nmol/ml) \\
\rowcolor{kwdboxcolor} & Mean\ensuremath{\pm}SD  & Mean\ensuremath{\pm}SD \\
\tblmidrule 
G1 &
  326.56\ensuremath{\pm}34.82 &
  2.33\ensuremath{\pm}0.36\\
G2a &
  271.08\ensuremath{\pm}20.02 &
  2.82\ensuremath{\pm}0.12\\
G2b &
  273.08\ensuremath{\pm}19.54 &
  2.79\ensuremath{\pm}0.19 \\
G2c &
  296.41\ensuremath{\pm}19.41 &
  2.55\ensuremath{\pm}0.19 \\
G2d &
  273.83\ensuremath{\pm}21.45 &
  2.73\ensuremath{\pm}0.30 \\
G2e &
  265.25\ensuremath{\pm}16.76 &
  2.76\ensuremath{\pm}0.14 \\
ANOVA(F value) &
  20.65  &
  12.10 \\
P Value &
  {\textless}0.001 &
  {\textless}0.001\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Comparison of subtypes of cataract in study groups}} }
\label{tw-39b6913a952b}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.276\linewidth-2\tabcolsep}p{\dimexpr.26729999999999997\linewidth-2\tabcolsep}p{\dimexpr.2482\linewidth-2\tabcolsep}p{\dimexpr.2085\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Type of cataract & \multicolumn{3}{p{\dimexpr(.724\linewidth-2\tabcolsep)}}{Study groups}\\
\rowcolor{kwdboxcolor} & Tobacco nonuser group(G1) & Tobacco user group (G2) & Total\\
\tblmidrule 
PSC &
  18(48.6\%) &
  19(51.4\%) &
  37(100\%)\\
Cortical  &
  6(46.2\%) &
  7(53.8\%) &
  13(100\%)\\
Nuclear &
  6(25.0\%) &
  18(75.0\%) &
  24(100\%)\\
Mixed &
  30(65.2\%) &
  16(34.8\%) &
  46(100\%)\\
Total &
  60(50\%) &
  60(50\%) &
  120(100\%)\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textbf{Comparison of subtypes of cataract in tobacco user group}} }
\label{tw-b0e2767c7790}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.139\linewidth-2\tabcolsep}p{\dimexpr.14099999999999998\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.14\linewidth-2\tabcolsep}p{\dimexpr.16\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{Type of cataract}} & \multicolumn{5}{p{\dimexpr(.701\linewidth-2\tabcolsep)}}{Study groups} & \\
\rowcolor{kwdboxcolor} & G2a & G2b & G2c & G2d & G2e & Total\\
\tblmidrule 
PSC &
  3(15.8\%) &
  4(21.1\%) &
  3(15.8\%) &
  3(15.8\%) &
  6(31.6\%) &
  19(100\%)\\
Cortical &
  1(14.3\%) &
  2(28.6\%) &
  2(28.6\%) &
  0(0\%) &
  2(28.6\%) &
  7(100\%)\\
Nuclear &
  4(22.2\%) &
  4(22.2\%) &
  4(22.2\%) &
  4(22.2\%) &
  2(11.1\%) &
  18(100\%)\\
Mixed &
  4(25\%) &
  2(12.5\%) &
  3(18.8\%) &
  5(31.2\%) &
  2(12.5\%) &
  16(100\%)\\
Total &
  12(20\%) &
  12(20\%) &
  12(20\%) &
  12(20\%) &
  12(20\%) &
  60(100\%)\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\ensuremath{^{}}The etiology of cataract is multi-factorial. The common cause of cataract is the normal aging process. The progression of age related cataract is a slow as well as complex process; the exact mechanism has not been clearly defined \unskip~\citep{716035:16980145}.\ensuremath{^{}} There is increasing biochemical evidence that oxidative stress has been concerned with development of age related cataract \unskip~\citep{716035:16980156}.\ensuremath{^{}}

 Oxidative stress in two different ways can trigger cataractogenesis: directly by Reactive Oxygen Species (ROS) and indirectly by reactive products of lipid peroxidation (LPO) \unskip~\citep{716035:16980144}. ROS are required for physiological functions nominally, but they become toxic to the cells at high levels.  They are simply produced during normal metabolic activities in the body. They can also be formed by external agents such as drug metabolism, air pollutants, electromagnetic radiation, particulate radiation or tobacco use \unskip~\citep{716035:16980141}. 

  These ROS are efficient in commencing and exalting oxidative damage by disabling endogenous antioxidant defense systems. The defense system consists of enzymes such as superoxide dismutase, catalase and glutathione peroxidase and non-enzyme molecules (uric acid, ascorbic acid, reduced glutathione etc.) which keep the ROS within essential limit. Smoking accelerates production of ROS which may exceed the capacity of defense system or due to inadequacy of antioxidants it may culminate in the oxidant vs. antioxidant imbalance and results in oxidative stress \unskip~\citep{716035:16980148}.   

Attack of oxidizing ROS generated by cigarette smoke on the polyunsaturated fatty acids of plasma membranes leads to lipid peroxidation (LPO) and it is substantially detrimental because it proceeds as a never ending chain reaction. Structural transformation of the plasma membrane occurs due to LPO with the liberation of the cell contents and the organelle and the loss of the essential fatty acids, with generation of cytosolic aldehyde and peroxide products. Malondialdehye (MDA) is chief end product of LPO \unskip~\citep{716035:16980155}. Extended accumulation of ROS and lipid peroxidation products also damage the lens crystalline which aggregate and precipitates, forming lens opacities \unskip~\citep{716035:16980153}.\ensuremath{^{}}\ensuremath{^{}}In almost all cells accessible to oxygen, one imperative antioxidant enzyme Superoxide dismutase (SOD) is present, with the most important function to catalyze the dis-mutation of two superoxide molecules into oxygen and hydrogen peroxide \unskip~\citep{716035:16980147}. Glutathione peroxidase and catalase enzymes further detoxify H\ensuremath{_{2}}O\ensuremath{_{2}}. Thus the cell membrane is protected from the damage by ROS.\ensuremath{^{}}

  Smoking is well studied and most prevailing form of tobacco use. Generally detrimental effects of tobacco are only linked with smoking and consequences of smokeless tobacco are overlooked. Only few studies were done for smokeless tobacco use, cataract and oxidative stress. Therefore, the purpose of this study was to estimate antioxidant enzyme Superoxide Dismutase (SOD)  and Malondialdehyde (MDA) in tobacco user Age Related Cataract (ARC) subjects and compare them with tobacco nonuser age related cataract (ARC) subjects.
    
\section{Materials and Methods}
\textbf{Study design}

This was a cross sectional study, done in Ophthalmology OPD and Biochemistry department, KIMS, Karad, Maharashtra from January 2016 to December 2017.

\textbf{Ethics}

Ethical clearance was taken from Institutional Ethics Committee. 

\textbf{Methodology}

Based on the previous study done by Sulochana, K.N., et al., 2002 calculation of sample size was done as follows: Mean difference in blood SOD level was 1072 units/gm of Hb (3106\ensuremath{\pm} 257 units/gm of Hb vs 2034 \ensuremath{\pm} 152 units/gm of Hb) among nonsmokers and smokers cataract patients. According to formula n= (SD1\ensuremath{^{2}} + SD2\ensuremath{^{2 }} )( Z~\ensuremath{_{1-\ensuremath{\alpha\ }/2}} + Z~\ensuremath{_{1-\ensuremath{\beta\ }\ }} )\ensuremath{^{2}} /d\ensuremath{^{2}} , with permissible error 10\%, confidence interval 95\% and power 80\% sample size came around minimum 10 in each group .\textit{}

The study comprised of two groups with total 120 subjects between age group 40- 65 years. First group comprised of 60 tobacco nonuser cataract patients and second group comprised of 60 tobacco user cataract patients. Group 2 subdivided into 5 groups. 

The groups comprised of-

Group 1(G1) {\textendash} 60 subjects who were tobacco nonuser cataract patients.

Group 2(G2) - 60 subjects who were tobacco user cataract patients subdivided as follows

 G2a- 12 subjects who were tobacco smoker cataract patients.

 G2b- 12 subjects who were tobacco chewer cataract patients.

 G2c - 12 subjects who were tobacco mishri user cataract patients.

 G2d- 12 subjects who were dual, tobacco chewer and tobacco mishri user cataract patients.

 G2e - 12 subjects who were dual, tobacco smoker and smokeless tobacco user cataract patients.

\textbf{Inclusion and Exclusion criteria}

\textbf{Inclusion criteria}

Patients diagnosed as having cataract, age 40-65 years with

Group 1 included subjects who never used tobacco, group 2a included smokers, group 2b included tobacco chewers, group 2c included tobacco mishri users, group 2d included tobacco chewers as well as tobacco mishri users and group 2e included tobacco smokers as well as smokeless tobacco users. 

\textbf{Exclusion criteria}


  
  \begin{enumerate}
  \item \relax Patients with congenital /complicated / traumatic/secondary cataract 
  \item \relax  Patients operated for cataract (pseudophakia , surgical aphakia)
  \item \relax Pre-existing ocular diseases (glaucoma, uveitis , corneal opacity) 
  \item \relax Patients with systemic illness like diabetes mellitus, hypertension, liver, cardiac or renal diseases 
  \item \relax Patients with history of any other substance abuse (alcohol, drugs). 
  \end{enumerate}
  Based on groups, inclusion \& exclusion criteria, subjects were chosen from the patients attending ophthalmology OPD by systematic sampling method. Written consent was taken after proper explanation of need of study from all the participants. A proforma was filled to collect the base line data like demographic data, past history and medical history. Details of tobacco use were collected by filling questionnaire. 

Proper systemic and ocular examination was done. Ophthalmic examination included best corrected visual acuity, tonometry, grading of cataractous lens using slit lamp and the Lens Opacities Classification System III (LOCS III) and fundus examination after dilatation of pupil. Significant cataract was indicated by a grading of \ensuremath{\geq }N II/C I/P I~or a combination.

Cataract subgroups such as pure nuclear (N), posterior sub-capsular (PSC) and cortical(C) had isolated cataract without the presence of other types. Combination of nuclear, cortical or PSC cataract was sub grouped as mixed cataract. For analysis, the opacity grade of the worse eye was measured. 

\textbf{Biochemical investigation}

In plain bulb 4 ml and in EDTA bulb 1ml of venous blood sample was collected after overnight fasting with aseptic precautions from all the subjects. Blood was processed and serum was separated by centrifugation in plain bulb in Biochemistry laboratory of KIMSU, Karad. Kei Satoh method \unskip~\citep{716035:16980143} was used for estimation of serum Malondialdehyde. I.N.T. [2(-4-iodophenyl)-3-(-4 nitrophenol)-5- phenyltetrazolium chloride] method by using RANSOD kit supplied by RANDOX Laboratory USA \unskip~\citep{716035:16980151} was used \ensuremath{^{}} for estimation of erythrocytic Superoxide Dismutase.\textit{\space }

\textbf{Statistical analysis}

 To find the significance of study parameters between different groups Chi-square test, unpaired t test and ANOVA were used. IBM SPSS Statistics, version 20 was used for data analysis. P value {\textless}0.05 was considered as statistically significant.
    
\section{Results and Discussion}
A series of epidemiological studies in the United Kingdom and the United States demonstrated significant association between cigarette smoking and nuclear cataract \unskip~\citep{716035:16980139}. In the present study, we tried to find out the impact of tobacco use in smoking as well as smokeless form on oxidative stress in cataract patients.

Gender wise there was significant difference in groups (Chi-square test=13.08, P\textless\ 0.001) as shown in Table~\ref{tw-23d9084b4787}. In tobacco user group males were significantly more than females. Age wise there was significant difference in groups (Chi-square test=2.23, P= 0.027). In tobacco user group age of cataract patients was significantly less than tobacco nonuser cataract patients.

Statistically significant difference was found on comparison of mean values of erythrocytic SOD and serum MDA between the groups using ANOVA test (P{\textless}0.001) as shown in Table~\ref{tw-d48600093ae0}. Levels of serum MDA  were significantly high and levels of erythrocytic SOD were  significantly low in tobacco user cataract patients (G2a, G2b, G2c, G2d, G2e) than tobacco nonuser cataract patients (G1) (P{\textless}0.001). In other tobacco user cataract patients apart from mishri users, serum MDA levels and erythrocytic SOD levels showed no appreciable difference. 

On comparison of subtypes of cataract in study groups, mixed cataract was significantly more in tobacco nonuser group and nuclear cataract was significantly more in tobacco user group(Chi-Square test- 10.36, P=0.016) as shown in Table~\ref{tw-39b6913a952b}.

On comparison of subtypes of cataract in tobacco user subgroup, no significant difference was found (Chi-Square test- 7.08, P=0.85) as shown in Table~\ref{tw-b0e2767c7790}.

In the present study, mean age of tobacco users was found significantly lower than mean age of tobacco nonusers (P=0.027). This shows that cataract occurs at an earlier age in tobacco users than tobacco nonusers. 

Similar findings were seen in the Framingham study done by \unskip~\citep{716035:16980142,716035:16980146}\textit{\space }that cataract develops at an earlier age in cigarette smokers than non-smokers. But according to \unskip~\citep{716035:16980149} mean age ( \ensuremath{\pm} SD) of tobacco users was remarkably higher than that of the non-tobacco users (p=0.001).

Present study showed that levels of serum MDA  were significantly elevated and levels of erythrocytic SOD were  considerably low in tobacco user cataract patients than tobacco nonuser cataract patients. 

\unskip~\citep{716035:16980154} study showed significant decrease in SOD activity in blood and lenses in smokers than non-smokers (P{\textless}0.001 for blood and {\textless}0.05 for lens). Our previous study showed similar results with significantly elevated levels of serum MDA  and significantly low levels of erythrocytic SOD  in tobacco users than tobacco nonusers \unskip~\citep{716035:16980150}.\ensuremath{^{}}

Superoxide dismutase (SOD) is an antioxidant enzyme which requires Zinc (Zn) for its activity and act as a first line of defense in destroying free radicals. Cadmium (Cd) in tobacco has affinity to replace the bivalent metals like (Zn) from SOD. So in tobacco users, there is decrease in Zn leading to inactivation of SOD. Therefore depletion of SOD activity in tobacco users may be primarily due to inadequate availability of Zn in blood and lens \unskip~\citep{716035:16980154}. Polyunsaturated fatty acids present in cell membrane are destroyed by the ROS produced by tobacco and continues as non ending chain reaction leading to loss of membrane integrity with increase in MDA level. 

In other tobacco users cataract patients except mishri users, no significant variation was established between levels of serum MDA and erythrocytic SOD. This indicates that smokers as well as tobacco chewers have same potential to augment oxidative stress in cataract patients \unskip~\citep{716035:16980150}.\ensuremath{^{}}

The present study showed that nuclear cataract was significantly associated with tobacco user group and mixed cataract with tobacco nonuser group. But on comparison of subtypes of cataract in tobacco user subgroup showed no significant difference.

Enhanced risk of age related cataract mainly nuclear cataract with significant association in ever smoking was found by \unskip~\citep{716035:16980159}. Subgroup analysis showed positive association of ever smoking with nuclear cataract and in cohort studies a marginally significant relationship with PSC cataract. 

Also significant association of smoking with pure nuclear cataract (P=0.036) was found in the study by \unskip~\citep{716035:16980149}. However, after adjustment for age and sex it was found not significant. But significant association of smokeless tobacco use with pure nuclear cataract (P=0.001) was found and remained significant even after adjustment for age and sex. 

Significantly higher prevalence of nuclear cataract and cortical cataract was showed by \unskip~\citep{716035:16980146} study, in cigarette smokers who smoked heavily ({\textgreater}14 pack-years of smoking) compared with never smokers after adjustment. 

Our results confirmed that oxidative stress is more in tobacco user cataract patients. This suggests that tobacco is one of the factors which have impact on oxidative stress in cataract. 

\textbf{Limitations }

Confounding factors such as data on time of sunlight exposure, indoor smoke exposure, occupational smoke exposure etc. were not controlled. Since it was a cross-sectional study, we cannot comment about longitudinal relationship. Additional research is suggested to take account of other confounding factors. 
    
\section{Conclusions}
From these results we may conclude that tobacco use intensifies the oxidative stress that is already present in age related cataract and this may accelerate the progression of ARC mainly nuclear cataract. 

\textbf{Conflict of Interest}

None.

\textbf{Funding Support}

None.
\section*{Acknowledgements}We are grateful to KIMS, Deemed to be University, Karad for funding the study. 
    

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