\documentclass[twocolumn,twoside]{article}
\makeatletter\if@twocolumn\PassOptionsToPackage{switch}{lineno}\else\fi\makeatother

\usepackage{amsfonts,amssymb,amsbsy,latexsym,amsmath,tabulary,graphicx,times,xcolor}

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% Following additional macros are required to function some 
% functions which are not available in the class used.
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
\usepackage{url,multirow,morefloats,floatflt,cancel,tfrupee}
\makeatletter


\AtBeginDocument{\@ifpackageloaded{textcomp}{}{\usepackage{textcomp}}}
\makeatother
\usepackage{colortbl}
\usepackage{xcolor}
\usepackage{pifont}
\usepackage[nointegrals]{wasysym}
\urlstyle{rm}
\makeatletter

%%%For Table column width calculation.
\def\mcWidth#1{\csname TY@F#1\endcsname+\tabcolsep}

%%Hacking center and right align for table
\def\cAlignHack{\rightskip\@flushglue\leftskip\@flushglue\parindent\z@\parfillskip\z@skip}
\def\rAlignHack{\rightskip\z@skip\leftskip\@flushglue \parindent\z@\parfillskip\z@skip}

%Etal definition in references
\@ifundefined{etal}{\def\etal{\textit{et~al}}}{}


%\if@twocolumn\usepackage{dblfloatfix}\fi
\usepackage{ifxetex}
\ifxetex\else\if@twocolumn\@ifpackageloaded{stfloats}{}{\usepackage{dblfloatfix}}\fi\fi

\AtBeginDocument{
\expandafter\ifx\csname eqalign\endcsname\relax
\def\eqalign#1{\null\vcenter{\def\\{\cr}\openup\jot\m@th
  \ialign{\strut$\displaystyle{##}$\hfil&$\displaystyle{{}##}$\hfil
      \crcr#1\crcr}}\,}
\fi
}

%For fixing hardfail when unicode letters appear inside table with endfloat
\AtBeginDocument{%
  \@ifpackageloaded{endfloat}%
   {\renewcommand\efloat@iwrite[1]{\immediate\expandafter\protected@write\csname efloat@post#1\endcsname{}}}{\newif\ifefloat@tables}%
}%

\def\BreakURLText#1{\@tfor\brk@tempa:=#1\do{\brk@tempa\hskip0pt}}
\let\lt=<
\let\gt=>
\def\processVert{\ifmmode|\else\textbar\fi}
\let\processvert\processVert

\@ifundefined{subparagraph}{
\def\subparagraph{\@startsection{paragraph}{5}{2\parindent}{0ex plus 0.1ex minus 0.1ex}%
{0ex}{\normalfont\small\itshape}}%
}{}

% These are now gobbled, so won't appear in the PDF.
\newcommand\role[1]{\unskip}
\newcommand\aucollab[1]{\unskip}
  
\@ifundefined{tsGraphicsScaleX}{\gdef\tsGraphicsScaleX{1}}{}
\@ifundefined{tsGraphicsScaleY}{\gdef\tsGraphicsScaleY{.9}}{}
% To automatically resize figures to fit inside the text area
\def\checkGraphicsWidth{\ifdim\Gin@nat@width>\linewidth
	\tsGraphicsScaleX\linewidth\else\Gin@nat@width\fi}

\def\checkGraphicsHeight{\ifdim\Gin@nat@height>.9\textheight
	\tsGraphicsScaleY\textheight\else\Gin@nat@height\fi}

\def\fixFloatSize#1{}%\@ifundefined{processdelayedfloats}{\setbox0=\hbox{\includegraphics{#1}}\ifnum\wd0<\columnwidth\relax\renewenvironment{figure*}{\begin{figure}}{\end{figure}}\fi}{}}
\let\ts@includegraphics\includegraphics

\def\inlinegraphic[#1]#2{{\edef\@tempa{#1}\edef\baseline@shift{\ifx\@tempa\@empty0\else#1\fi}\edef\tempZ{\the\numexpr(\numexpr(\baseline@shift*\f@size/100))}\protect\raisebox{\tempZ pt}{\ts@includegraphics{#2}}}}

%\renewcommand{\includegraphics}[1]{\ts@includegraphics[width=\checkGraphicsWidth]{#1}}
\AtBeginDocument{\def\includegraphics{\@ifnextchar[{\ts@includegraphics}{\ts@includegraphics[width=\checkGraphicsWidth,height=\checkGraphicsHeight,keepaspectratio]}}}

\DeclareMathAlphabet{\mathpzc}{OT1}{pzc}{m}{it}

\def\URL#1#2{\@ifundefined{href}{#2}{\href{#1}{#2}}}

%%For url break
\def\UrlOrds{\do\*\do\-\do\~\do\'\do\"\do\-}%
\g@addto@macro{\UrlBreaks}{\UrlOrds}



\edef\fntEncoding{\f@encoding}
\def\EUoneEnc{EU1}
\makeatother
\def\floatpagefraction{0.8} 
\def\dblfloatpagefraction{0.8}
\def\style#1#2{#2}
\def\xxxguillemotleft{\fontencoding{T1}\selectfont\guillemotleft}
\def\xxxguillemotright{\fontencoding{T1}\selectfont\guillemotright}

\newif\ifmultipleabstract\multipleabstractfalse%
\newenvironment{typesetAbstractGroup}{}{}%

%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

\usepackage[authoryear]{natbib}



\newif\ifnokeywords\nokeywordsfalse

\makeatletter\input{size10-pointfive.clo}\makeatother%
\definecolor{kwdboxcolor}{RGB}{242,242,242}
\usepackage[hidelinks,colorlinks=true,allcolors=blue]{hyperref}
\linespread{1}                 
\def\floatpagefraction{0.8}

\usepackage[paperheight=11.69in,paperwidth=8.26in,top=1in,bottom=1in,left=1in,right=.75in,headsep=24pt]{geometry}

\usepackage{multirow-custom}

\makeatletter

\def\hlinewd#1{%
  \noalign{\ifnum0=`}\fi\hrule \@height #1%
  \futurelet\reserved@a\@xhline}
\def\tbltoprule{\hlinewd{1pt}\\[-14pt]}
\def\tblbottomrule{\noalign{\vspace*{6pt}}\hline\noalign{\vspace*{2pt}}}
\def\tblmidrule{\hline\noalign{\vspace*{2pt}}}



\let\@articleType\@empty
\let\@journalDoi\@empty
\let\@journalVolume\@empty
\let\@journalIssue\@empty
\let\@crossMarkLink\@empty
\let\@receivedDate\@empty
\let\@acceptedDate\@empty
\let\@revisedDate\@empty
\let\@publishedDate\@empty
\let\@copyrightYear\@empty
\let\@firstPage\@empty
\def\articleType#1{\gdef\@articleType{#1}}
\def\journalDoi#1{\gdef\@journalDoi{#1}}
\def\crossMarkLink#1{\gdef\@crossMarkLink{#1}}
\def\receivedDate#1{\gdef\@receivedDate{#1}}
\def\acceptedDate#1{\gdef\@acceptedDate{#1}}
\def\revisedDate#1{\gdef\@revisedDate{#1}}
\def\publishedDate#1{\gdef\@publishedDate{#1}}
\def\copyrightYear#1{\gdef\@copyrightYear{#1}}
\def\journalVolume#1{\gdef\@journalVolume{#1}}
\def\journalIssue#1{\gdef\@journalIssue{#1}}
\def\firstPage#1{\gdef\@firstPage{#1}}
\let\@issuelogo\@empty%
\let\@issuelogoex\@empty%
\def\issuelogo#1{\gdef\@issuelogo{#1}}
\def\issuelogoex#1{\gdef\@issuelogoex{#1}}


\def\author#1{%
  \gdef\@author{%
    \hskip-\dimexpr(\tabcolsep)\hskip5pt%
    \parbox{\dimexpr\textwidth-1pt}%
    {\fontsize{11}{13}\selectfont\raggedright #1}%
  }%
}


\usepackage{pharmascope-abs}
\usepackage{caption}
\usepackage{lastpage}
\usepackage{fancyhdr}
\usepackage[noindentafter,explicit]{titlesec}
\usepackage{fontspec}

\setmainfont[%
BoldFont=cambriab.ttf,%
ItalicFont=CAMBRIAI.TTF,%
BoldItalicFont=CAMBRIAZ.TTF]{Cambria.ttf}
\lefthyphenmin = 3

\def\title#1{%
  \gdef\@title{%
      \vspace*{-40pt}%
       \ifx\@articleType\@empty\else{\fontsize{10}{12}\scshape\selectfont\hspace{8pt}\@articleType\hfill\mbox{}\par\vspace{2pt}}\fi%
       \minipage{\linewidth}
         \hrulefill\\[-0.7pt]%%{\includegraphics[width=75pt,height=50pt]{ijrps_logo.png}}\hfill
         \mbox{~}\hspace{5pt}\parbox{.1\linewidth}{\IfFileExists{images/\@issuelogo.\@issuelogoex}{\includegraphics[width=79pt,height=61pt]{images/\@issuelogo.\@issuelogoex}}{\includegraphics[width=75pt,height=50pt]{ijrps_logo.png}}}\hfill
         \fcolorbox{kwdboxcolor}{kwdboxcolor}{\parbox{.792\linewidth}{%
          \begin{center}\fontsize{17}{17}\selectfont\scshape\vskip-7pt International Journal of Research in Pharmaceutical Sciences\hfill\end{center}%
          \vspace*{-10pt}\hspace*{4pt}{\fontsize{8}{9}\selectfont Published by JK Welfare \& Pharmascope Foundation\hfill  Journal Home Page: \href{www.ijrps.com}{\color{blue}\underline{\smash{www.ijrps.com}}}}\hspace*{4pt}\mbox{}}}%
       \par\vspace*{-1pt}\rule{\linewidth}{1.3pt}%
       \endminipage%
    \par\vspace*{9.2pt}\parbox{.98\linewidth}{\linespread{.9}\raggedright\fontsize{14}{17}\selectfont #1}%
    \vspace*{-8pt}%
  }
}


\setlength{\parindent}{0pt}
\setlength{\parskip}{0.4pc plus 1pt minus 1pt}

\def\abbrvJournalTitle{Int. J. Res. Pharm. Sci.}

\fancypagestyle{headings}{%
  \renewcommand{\headrulewidth}{0pt}%
  \renewcommand{\footrulewidth}{0.3pt}
  \fancyhf{}%
  \fancyhead[R]{%
    \fontsize{9.12}{11}\selectfont\RunningAuthor,\ \abbrvJournalTitle,\ \ifx\@journalVolume\@empty X\else\@journalVolume\fi%
    \ifx\@journalIssue\@empty\else(\@journalIssue)\fi%
    ,\ \ifx\@firstPage\@empty 1\else\@firstPage\fi-\pageref*{LastPage}%
  }%
  \fancyfoot[LO,RE]{\fontsize{9.12}{11}\selectfont\textcopyright\ International Journal of Research in Pharmaceutical Sciences}%
  \fancyfoot[RO,LE]{\fontsize{9.12}{11}\selectfont\thepage}
}\pagestyle{headings}

                                                         
\fancypagestyle{plain}{%
  \renewcommand{\headrulewidth}{0pt}%
  \renewcommand{\footrulewidth}{0.3pt}%
  \fancyhf{}%
  \fancyhead[R]{%
    \fontsize{9.12}{11}\selectfont\RunningAuthor,\ \abbrvJournalTitle,\ \ifx\@journalVolume\@empty X\else\@journalVolume\fi%
    \ifx\@journalIssue\@empty\else(\@journalIssue)\fi%
    ,\ \ifx\@firstPage\@empty 1\else\@firstPage\fi-\pageref*{LastPage}%
  }%
  \fancyfoot[LO,RE]{\fontsize{9.12}{11}\selectfont\textcopyright\ International Journal of Research in Pharmaceutical Sciences}%
  \fancyfoot[RO,LE]{\fontsize{9.12}{11}\selectfont\thepage}
  \ifx\@firstPage\@empty\else\setcounter{page}{\@firstPage}\fi
}


\def\NormalBaseline{\def\baselinestretch{1.1}}
\usepackage{textcase}
\setcounter{secnumdepth}{0}

\titleformat{\section}[block]{\bfseries\boldmath\NormalBaseline\filright\fontsize{10.5}{13}\selectfont}
{\thesection}
{6pt}
{\MakeTextUppercase{#1}}
[]
\titleformat{\subsection}[block]{\bfseries\boldmath\NormalBaseline\filright\fontsize{10.5}{12}\selectfont}
{\thesubsection}
{6pt}
{#1}
[]
\titleformat{\subsubsection}[block]{\NormalBaseline\filright\fontsize{10.5}{12}\selectfont}
{\thesubsubsection}
{6pt}
{#1}
[]
\titleformat{\paragraph}[block]{\NormalBaseline\filright\fontsize{10.5}{10}\selectfont}
{\theparagraph}
{6pt}
{#1}
[]
\titleformat{\subparagraph}[block]{\NormalBaseline\filright\fontsize{10.5}{12}\selectfont}
{\thesubparagraph}
{6pt}
{#1}
[]

\titlespacing{\section}{0pt}{.5\baselineskip}{.5\baselineskip}  
\titlespacing{\subsection}{0pt}{.5\baselineskip}{.5\baselineskip}  
\titlespacing{\subsubsection}{0pt}{.5\baselineskip}{.5\baselineskip}  
\titlespacing{\paragraph}{0pt}{.5\baselineskip}{.5\baselineskip}  
\titlespacing{\subparagraph}{0pt}{.5\baselineskip}{.5\baselineskip}

\captionsetup[figure]{skip=1.4pt,font=bf,labelsep=colon,justification=raggedright,singlelinecheck=false}
\captionsetup[table]{skip=1.4pt,font=bf,labelsep=colon,justification=raggedright,singlelinecheck=false}

\def\bibyear#1{#1}
%\def\bibjtitle#1{#1}   %%Publisher request
\def\bibauand{}
\setlength\bibsep{3pt}
\setlength\bibhang{8pt}

\makeatother
\date{}


\usepackage[flushleft]{threeparttablex}

\usepackage{float}

\begin{document}


\def\RunningAuthor{Murugesan Sudha and Thangarasu Vetrichelvan }
\firstPage{1379}
\articleType{Original Article}
\receivedDate{26 Jan 2021}
\acceptedDate{01 Mar 2021}
\revisedDate{24 Feb 2021}
\journalVolume{2021, 12}
\journalIssue{2}
\journalDoi{ijrps.v12i2.4694}
\copyrightYear{2021}
\def\authorCount{2}
\def\affCount{2}

\def\journalTitle{International Journal of Research in Pharmaceutical Sciences}

\title{Genoprotective effect of D-Pinitol isolated from aerial parts of Soybean plants against Doxorubicin-induced genotoxicity evaluated by \textit{in vitro} comet assay in Vero cell lines}
\author{Murugesan~Sudha\textsuperscript{*}\textsuperscript{1}, 
            Thangarasu~Vetrichelvan\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Department of Pharmacology, Adhiparasakthi College of Pharmacy, Melmaruvathur, Tamil Nadu, India}~\\{\textsuperscript{2}Department of Pharmaceutical Analysis, Adhiparasakthi College of Pharmacy, Melmaruvathur, Tamil Nadu, India}}

\begin{abstract}
Doxorubicin is a chemotherapeutic agent with a genotoxic effect on normal cells at its therapeutic dose itself. D-Pinitol is an abundantly available carbohydrate in Soybean plants and has been proven for antioxidant and anti-inflammatory activities. Our investigation was examined by \textit{in vitro} comet assay to explore the genoprotective effect of D-Pinitol in normal cells against Doxorubicin-induced genotoxicity in Vero cell lines. \textit{In vitro} comet assay treatment groups were: Vero Cell lines with culture medium (control group), Doxorubicin (0.15\ensuremath{\mu }g/ml), D-Pinitol (0.05\ensuremath{\times}10\ensuremath{^{3}} mM, 0.125\ensuremath{\times}10\ensuremath{^{3}} mM, and 0.25\ensuremath{\times}10\ensuremath{^{3}} mM) alone, and pretreatment with D-Pinitol (0.05\ensuremath{\times}10\ensuremath{^{3}} mM, 0.125\ensuremath{\times}10\ensuremath{^{3}} mM, and 0.25\ensuremath{\times}10\ensuremath{^{3}} mM) before Doxorubicin (0.15\ensuremath{\mu }g/ml) treatment. When compared to the control group, D-Pinitol alone treated groups showed no significant changes in the percentage of DNA damage. For the evaluation of the genoprotective effect of D-Pinitol, the \% DNA damage in the D-Pinitol, and Doxorubicin simultaneously treated groups were compared to the Doxorubicin alone treated group. The results showed that Doxorubicin-induced genotoxic effect in Vero cell lines was significantly reduced by D-Pinitol in a dose-dependent manner by reducing DNA damage. Our findings confirmed that D-Pinitol had no genotoxic effect and it showed a genoprotective effect against Doxorubicin-induced genotoxicity.  
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Genotoxicity,\newline Doxorubicin,\newline D-Pinitol,\newline In vitro Comet assay
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Murugesan~Sudha~\\ Phone:\ 09944640202~\\  Email:\ kaviyasudha7@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
        \textsc{DOI:}\ \href{https://doi.org/10.26452/\@journalDoi}{\textcolor{blue}{\underline{\smash{https://doi.org/10.26452/\@journalDoi}}}}\par%
        \vspace*{-11pt}\hrulefill\\{\fontsize{9.12}{10.12}\selectfont Production and Hosted by}\par{IJRPS |~\href{www.ijrps.com}{www.ijrps.com}}\par%
        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
        \vspace*{-11pt}\rule{\linewidth}{1.2pt}
        \makeatother
        
    
\section{Introduction}
Doxorubicin (DOX) is a potent antibiotic of anthracycline class in anticancer drugs. Although DOX is very useful for treating various types of human cancers, having severe side effects at its therapeutic dose is one of the most undesired outcomes of using it. Hence, management of its side effects is essential to achieve patient's treatment, tolerability, and overall quality of life \unskip~\citep{1026090:21586086}. Previous reports suggested that oxidative stress and free radicals production are the main reasons for DOX-induced cardiotoxicity, cytotoxicity, and genotoxicity \unskip~\citep{1026090:21586104}. Apart from these side effects, DOX also induces severe inflammatory responses in various organs, including the kidney, liver, blood vessels, and intestine. The previous reports also showed an increase in pro-inflammatory cytokine levels after DOX administration \unskip~\citep{1026090:21586097,1026090:21586085}. Since DOX has an important role in cancer treatment, it is very crucial to minimize its toxic effects on normal cells. This reduction in DOX-induced toxicity to normal cells can be attained by simultaneous administration of free radical scavenging agents, antioxidants and anti-inflammatory agents. Decreasing DOX's toxicity by attenuating oxidative stress and pro-inflammatory mediators is a forthcoming therapeutic approach against DOX-induced toxicity \unskip~\citep{1026090:21586104}.

Hence, the compounds that can scavenge free radicals and reduce the levels of pro-inflammatory mediators will safeguard the normal cells from DNA damage when they subjected to the genotoxic agents \unskip~\citep{1026090:21586101}. D-Pinitol has been identified as a major carbohydrate present in \textit{Glycine max} L. Merr. \unskip~\citep{1026090:21586095,1026090:21586096}. It has been reported for its free radical scavenging capacity \unskip~\citep{1026090:21586098}, antioxidant activity \unskip~\citep{1026090:21586099,1026090:21586087}, anti-inflammatory activity \unskip~\citep{1026090:21586103,1026090:21586100}, hepatoprotective effect \unskip~\citep{1026090:21586088,1026090:21586092}, and Cardioprotective effect \unskip~\citep{1026090:21586090}. These therapeutic effects of D-Pinitol may be useful in the prevention of the genotoxic effect of DOX on normal cells. This research's main goal was to evaluate the genoprotective effect of D-Pinitol in the Vero cell line from the DNA damage induced by DOX through \textit{in} \textit{vitro} comet assay.  The \textit{in vitro} Comet assay presents a visual technique to analyze DNA damage in the cells when treated with chemicals inducing toxic effects \unskip~\citep{1026090:21586094}. Hence this assay is an advantageous, fast, and effective\textit{ in vitro} method for studying the genotoxicity of chemical agents \unskip~\citep{1026090:21586093,1026090:21586084}. 
    
\section{Materials and Methods}
\textbf{Materials Required}

Doxorubicin HCL (TCI chemicals, India), D-Pinitol isolated from aerial parts of Soybean (\textit{Glycine max} L. Merr.,) plants, DMEM medium (Gibco, USA), Fetal Bovine Serum (Gibco, USA), Antibiotic solution (Gibco, USA),  Ethidium Bromide (Merck, India), Low melting agarose (Merck, India), Normal agarose (Merck, India), Phosphate Buffer Saline Solution (Himedia, India) and Olympus BX 50 microscope (Olympus Optical Co., Germany).

\textbf{Methodology \textit{- In vitro} Comet Assay}

\textbf{Cell line and cell culture}

Vero cell lines (African green monkey kidney cells) were cultured in liquid Dulbecco's Modified Eagle's medium (DMEM) enriched with 10 percentage Fetal Bovine Serum (FBS), 100 u/ml penicillin and 100 \ensuremath{\mu}g/ml streptomycin, and maintained under an atmosphere of 5 percentage CO\ensuremath{_{2}} stored at 37\ensuremath{^{o}}C.\textbf{\space }


\begin{table*}[!htbp]
\caption{\boldmath {Treatment protocol } }
\label{tw-705b6f5b65b0}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.13670000000000002\linewidth-2\tabcolsep}p{\dimexpr.348\linewidth-2\tabcolsep}p{\dimexpr.5153\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Groups & Labeled & Treatment\\
\tblmidrule 
 I~&
  Control &
  Vero Cell lines with culture medium  \\
 II &
   Positive Control &
  Doxorubicin 0.15\ensuremath{\mu }g/ml\\
III &
   &
  D-Pinitol 0.05\ensuremath{\times}10\ensuremath{^{3}} mM\\
IV &
   &
  D-Pinitol 0.125\ensuremath{\times}10\ensuremath{^{3}} mM\\
V &
   &
  D-Pinitol 0.25\ensuremath{\times}10\ensuremath{^{3}} mM\\
VI &
  Tests &
  Doxorubicin 0.15\ensuremath{\mu }g/ml + D-Pinitol 0.05\ensuremath{\times}10\ensuremath{^{3}} mM\\
VII &
   &
  Doxorubicin 0.15\ensuremath{\mu }g/ml+D-Pinitol 0.125\ensuremath{\times}10\ensuremath{^{3}} mM\\
VIII &
   &
  Doxorubicin 0.15\ensuremath{\mu }g/ml +D-Pinitol 0.25\ensuremath{\times}10\ensuremath{^{3}} mM\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Selection of doses of Doxorubicin and D-Pinitol were based on the articles Abd-ElAziz et al., and Al-Ashaal et al., respectively \unskip~\citep{1026090:21610815,1026090:21610814}.}
\end{tablenotes}
\end{threeparttable}

\end{table*}
Briefly, Vero cells were incubated at 37\ensuremath{^\circ}C for 24 h in a humidified 5\% CO\ensuremath{_{2}} incubator after they were seeded (density of 10,000 cells/well) in a six-well plate. After the wells were washed with sterile Phosphate Buffer Saline Solution (PBS), the cell lines were treated with D-Pinitol and DOX as per the treatment protocol given in Table~\ref{tw-705b6f5b65b0}. Trypsinization was done before harvesting of cells in a 1.5 ml tube. The microscopic slides were coated first with 200 \ensuremath{\mu }l of 0.75 \% normal melting agarose (the first layer) and 100 \ensuremath{\mu }l of 0.5\% low melting agarose (the second layer). Next, the slides were distributed with 20 \ensuremath{\mu }l cell suspensions in 60 \ensuremath{\mu }l of 0.5\% low melting agarose (the third layer). Then incubation of slides was done in cell lysis buffer (2.5 M NaCl, 0.2 M NaOH, 100 mM Na\ensuremath{_{2}}EDTA, 10 mM Tris{\textendash}HCl, 1\% Triton X-100 and 10\% dimethyl sulfoxide, pH =10.0) for overnight at 4\ensuremath{^\circ}C. After that, the slides were immersed in double- distilled water three times, followed by 20 min incubation with unwinding solution (3M NaOH). The slides were subsequently placed in a horizontal gel electrophoresis tank containing electrophoresis solution (1 mM Na\ensuremath{_{2}}EDTA and 300 mM NaOH, pH =13). The electrophoresis was conducted at 25 V (1 V/cm, 300 mA) for 25 min. The incubation of slides was done in neutralization buffer (0.4 M Tris{\textendash}HCl, pH = 7.5) for 10 min followed by immersion in ultrapure water for three times and then air-dried. The staining of cells was done with 50 \ensuremath{\mu }l of ethidium bromide (5 mg/L) and then the cells were observed under a fluorescent microscope. 15 minutes after staining, the comets of each group were examined on the microscope at 100 X magnification. In order to minimize extra DNA damage, all steps in the procedure were carried out in dim light. The percentage of DNA damage events was calculated by manual counting. Comets were visually scored and classified into five classes, a) Class 1 - no damage, {\textless}5\%; b) Class 2 - low level damage, 5{\textendash}20\%; c) Class 3 - medium level damage, 20{\textendash}40\%; d) Class 4 - high level damage, 40{\textendash}95\%; e) Class 5 - total damage, {\textgreater}95\% (Figure~\ref{f-3e9c55e89370})based on the level of DNA migration \unskip~\citep{1026090:21586084}. The experiment was performed in triplicate for each group and, the analysis was performed on100 cells for each experiment \unskip~\citep{1026090:21586091,1026090:21586105}.

\textbf{Statistical analysis}

Statistical analysis was performed by One way ANOVA method. Values were expressed as mean and for n = 6. All data were analyzed with The GraphPad Prism 8.0.1 software. A difference at P \textless\ 0.05 was considered statistically significant.
    
\section{Results and Discussion}

\begin{table*}[!htbp]
\caption{\boldmath {Number of different classes of comet events occurred in Vero cell lines} }
\label{tw-d3d837335c8c}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.085\linewidth-2\tabcolsep}p{\dimexpr.106\linewidth-2\tabcolsep}p{\dimexpr.09799999999999999\linewidth-2\tabcolsep}p{\dimexpr.115\linewidth-2\tabcolsep}p{\dimexpr.11800000000000002\linewidth-2\tabcolsep}p{\dimexpr.11379999999999995\linewidth-2\tabcolsep}p{\dimexpr.1231\linewidth-2\tabcolsep}p{\dimexpr.11709999999999999\linewidth-2\tabcolsep}p{\dimexpr.124\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Nature of Comet events & Group  \mbox{}\protect\newline I(Vero Cell lines and culture medium) & Group  \mbox{}\protect\newline II(DOX  \mbox{}\protect\newline 0.15 \mbox{}\protect\newline \ensuremath{\mu }g/ml) & Group  \mbox{}\protect\newline III(D-P \mbox{}\protect\newline  0.05\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}} mM) & Group  \mbox{}\protect\newline IV(D-P  \mbox{}\protect\newline 0.125\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}} mM) & Group  \mbox{}\protect\newline V(D-P  \mbox{}\protect\newline 0.25\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}} mM) & Group  \mbox{}\protect\newline VI(DOX  \mbox{}\protect\newline 0.15\ensuremath{\mu }g/ml +D-P 0.05\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}}mM) & Group  \mbox{}\protect\newline VII(DOX \mbox{}\protect\newline  0.15\ensuremath{\mu }g/ml +D-P 0.125\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}}mM) & Group  \mbox{}\protect\newline VIII(DOX  \mbox{}\protect\newline 0.15\ensuremath{\mu }g/ml +D-P 0.25\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}}mM)\\
\tblmidrule 
Class 1 &
  94.33 \ensuremath{\pm} \mbox{}\protect\newline 1.453 &
  29.67 \ensuremath{\pm} \mbox{}\protect\newline 4.372 &
  94.67 \ensuremath{\pm} \mbox{}\protect\newline 0.882 &
  94 \ensuremath{\pm} \mbox{}\protect\newline 0.577 &
  93.33 \ensuremath{\pm} \mbox{}\protect\newline 0.667 &
  41.33 \ensuremath{\pm} \mbox{}\protect\newline 3.283 &
  53.33 \ensuremath{\pm} \mbox{}\protect\newline 3.528 &
  81 \ensuremath{\pm} \mbox{}\protect\newline 2.082\\
Class 2 &
  5.667 \ensuremath{\pm} \mbox{}\protect\newline 1.453 &
  33 \ensuremath{\pm} \mbox{}\protect\newline 0.577 &
  5.333 \ensuremath{\pm} \mbox{}\protect\newline 0.882 &
  6 \ensuremath{\pm} \mbox{}\protect\newline 0.577 &
  6.667 \ensuremath{\pm} \mbox{}\protect\newline 0.667 &
  25.33 \ensuremath{\pm} \mbox{}\protect\newline 1.764 &
  21 \ensuremath{\pm} \mbox{}\protect\newline 1.155 &
  5 \ensuremath{\pm} \mbox{}\protect\newline 0.577\\
Class 3 &
  0 &
  7.333 \ensuremath{\pm} \mbox{}\protect\newline 0.667 &
  0 &
  0 &
  0 &
  15 \ensuremath{\pm} \mbox{}\protect\newline 1.528 &
  13.33 \ensuremath{\pm} \mbox{}\protect\newline 0.882 &
  11.33 \ensuremath{\pm} \mbox{}\protect\newline 0.882\\
Class 4 &
  0 &
  18.67 \ensuremath{\pm} \mbox{}\protect\newline 2.333 &
  0 &
  0 &
  0 &
  15.33 \ensuremath{\pm} \mbox{}\protect\newline 0.882 &
  10.33 \ensuremath{\pm} \mbox{}\protect\newline 1.202 &
  2 \ensuremath{\pm} \mbox{}\protect\newline 0.577\\
Class 5 &
  0 &
  11.33 \ensuremath{\pm} \mbox{}\protect\newline 1.764 &
  0 &
  0 &
  0 &
  3 \ensuremath{\pm} \mbox{}\protect\newline 0.577 &
  2 \ensuremath{\pm} \mbox{}\protect\newline 0.577 &
  0.667 \ensuremath{\pm} \mbox{}\protect\newline 0.333\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{DOX {\textendash} Doxorubicin;D-P {\textendash} D-Pinitol}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Effect of D-Pinitol on Doxorubicin-induced genotoxicity by \textit{in vitro} comet assay in Vero cell lines} }
\label{tw-aba785835e7d}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.10339999999999998\linewidth-2\tabcolsep}p{\dimexpr.1076\linewidth-2\tabcolsep}p{\dimexpr.10100000000000001\linewidth-2\tabcolsep}p{\dimexpr.113\linewidth-2\tabcolsep}p{\dimexpr.112\linewidth-2\tabcolsep}p{\dimexpr.111\linewidth-2\tabcolsep}p{\dimexpr.11500000000000002\linewidth-2\tabcolsep}p{\dimexpr.117\linewidth-2\tabcolsep}p{\dimexpr.12\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Criterion & Group  \mbox{}\protect\newline I(Vero Cell lines and culture medium) & Group  \mbox{}\protect\newline II(DOX 0.15 \mbox{}\protect\newline \ensuremath{\mu }g/ml) & Group  \mbox{}\protect\newline III(D-P \mbox{}\protect\newline  0.05\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}} mM) & Group  \mbox{}\protect\newline IV(D-P  \mbox{}\protect\newline 0.125\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}} mM) & Group  \mbox{}\protect\newline V(D-P  \mbox{}\protect\newline 0.25\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}}  mM) & Group  \mbox{}\protect\newline VI(DOX  \mbox{}\protect\newline 0.15\ensuremath{\mu }g/ml + D-P  \mbox{}\protect\newline 0.05\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}} mM) & Group  \mbox{}\protect\newline VII(DOX \mbox{}\protect\newline 0.15\ensuremath{\mu }g/ml + D-P  \mbox{}\protect\newline 0.125\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}} mM) & Group  \mbox{}\protect\newline VIII(DOX  \mbox{}\protect\newline 0.15\ensuremath{\mu }g/ml + D-P  \mbox{}\protect\newline 0.25\ensuremath{\times} \mbox{}\protect\newline 10\ensuremath{^{3}} mM)\\
\tblmidrule 
\% DNA Damage &
  5.667\ensuremath{\pm} \mbox{}\protect\newline 1.453 &
  70.33\ensuremath{\pm} \mbox{}\protect\newline 4.372 \mbox{}\protect\newline \ensuremath{^{a}}\ensuremath{^{* }} &
  5.333 \ensuremath{\pm} \mbox{}\protect\newline 0.882 \ensuremath{^{aNS}} &
  6 \ensuremath{\pm} \mbox{}\protect\newline 0.577 \ensuremath{^{aNS}} &
  6.667 \ensuremath{\pm} \mbox{}\protect\newline 0.667 \ensuremath{^{aNS}} \mbox{}\protect\newline  &
  58.67 \ensuremath{\pm} \mbox{}\protect\newline 3.283 \ensuremath{^{a*bNS}} &
  46.67 \ensuremath{\pm} \mbox{}\protect\newline 3.528 \ensuremath{^{a*b\# }} &
  19 \ensuremath{\pm} \mbox{}\protect\newline 2.082 \ensuremath{^{a@b*}}\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{DOX {\textendash} Doxorubicin;D-P {\textendash} D-Pinitol. Mean \ensuremath{\pm} SEM, n=6, where, a- Group II, III, IV, V, VI, VII \&VIII compared with Group I. b- Group VI, VII \& VIII compared with Group II.\ensuremath{^{*}} P \textless\ 0.001, \ensuremath{^{\#}} P \textless\ 0.01 \& \ensuremath{^{@ }} P {\textless}0.05}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\bgroup
\fixFloatSize{images/f9b218b7-0dbf-4e80-bf1b-44c2f1413d2d-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/f9b218b7-0dbf-4e80-bf1b-44c2f1413d2d-upicture1.png}{\includegraphics{images/f9b218b7-0dbf-4e80-bf1b-44c2f1413d2d-upicture1.png}}{}
\makeatother 
\caption{\boldmath {Histogram of number of different Classes of Comet events occurred in Vero cell lines}}
\label{f-ad01c86c9c43}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/7b9078eb-9e1d-4526-ba92-90efe783f12f-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/7b9078eb-9e1d-4526-ba92-90efe783f12f-upicture2.png}{\includegraphics{images/7b9078eb-9e1d-4526-ba92-90efe783f12f-upicture2.png}}{}
\makeatother 
\caption{\boldmath {Histogram of genoprotective effect of D-Pinitol on Doxorubicin-induced genotoxicity by \textit{in vitro }comet assay in Vero cell lines}}
\label{f-5a31ecd36e2c}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/c30e09af-0da2-432f-ba54-b3e10f7a56b0-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/c30e09af-0da2-432f-ba54-b3e10f7a56b0-upicture3.png}{\includegraphics{images/c30e09af-0da2-432f-ba54-b3e10f7a56b0-upicture3.png}}{}
\makeatother 
\caption{\boldmath {Images of Comets visualized by comet assay in Vero cell lines}}
\label{f-9e55ed6bf6a5}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/71d5b1c6-5b8c-46cf-9463-36fdf4b090c7-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/71d5b1c6-5b8c-46cf-9463-36fdf4b090c7-upicture4.png}{\includegraphics{images/71d5b1c6-5b8c-46cf-9463-36fdf4b090c7-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Images of Classes of Comet events}}
\label{f-3e9c55e89370}
\end{figure}
\egroup
We assessed the effect of D-Pinitol for its ability to prevent DOX-induced genotoxicity. The values were represented as the mean number of different classes of comets that occurred in all groups (Table~\ref{tw-d3d837335c8c} and Figure~\ref{f-ad01c86c9c43}) and as the percentage of DNA damage (Table~\ref{tw-aba785835e7d} and Figure~\ref{f-5a31ecd36e2c}). Images of comets that occurred in all groups were shown in Figure~\ref{f-9e55ed6bf6a5}.  The obtained data elucidates that all three doses of D-Pinitol did not express any significant differences in the percentage of DNA damage values in D-Pinitol alone treated groups when compared to the control group. While increased levels of DNA damage was detected in cells treated only with DOX (0.15\ensuremath{\mu }g/ml), a significant reduction in the levels of DNA damage was seen in the Vero cell lines treated simultaneously with DOX (0.15\ensuremath{\mu }g/ml) and three different concentrations of D-Pinitol (0.05\ensuremath{\times}10\ensuremath{^{3}} mM, 0.125\ensuremath{\times}10\ensuremath{^{3}} mM and 0.25\ensuremath{\times}10\ensuremath{^{3}} mM) when compared with DOX alone treated group (Tables~\ref{tw-d3d837335c8c} and~\ref{tw-aba785835e7d} \& Figures~\ref{f-ad01c86c9c43} and~\ref{f-5a31ecd36e2c}). All three doses of D-Pinitol hopefully decreased the induction of DNA damage by DOX. Concentration-response of D-Pinitol indicated that protection against DOX-induced DNA damage was more intense, with an increase in D-Pinitol concentration. The increased DNA damage reduction was observed in the Vero cell lines pretreated with 0.25\ensuremath{\times}10\ensuremath{^{3}} mM concentration of D-Pinitol. Hence, the higher concentration of D-Pinitol showed a more significant protective effect.

The examination of dietary components with antioxidant and anti-inflammatory activities has gotten much consideration because the essential factors in the generation and progression of numerous chronic diseases were oxidative stress and inflammation \unskip~\citep{1026090:21586089}. D-Pinitol has been shown its beneficial effects in several experimental models of diseases emerging and worsening because of oxidative stress and inflammation. It's antioxidant \unskip~\citep{1026090:21586099,1026090:21586087} and anti-inflammatory properties \unskip~\citep{1026090:21586100,1026090:21586103} were well reported. 

DOX, an anthracycline anticancer agent, can induce reactive oxygen species (ROS) generation with severe inflammatory responses. DNA damage caused by inflammatory Reactive Oxygen Species can inevitably lead to disturbance of genetic stability. Hence the production of free radicals and pro-inflammatory mediators is the primary mechanism responsible for DOX genetic toxicity and DNA damage to normal cells \unskip~\citep{1026090:21586102}. 

The genotoxic evaluation of D-Pinitol by \textit{in vitro} comet assay in Vero cell lines indicated that this compound did not induce any genotoxic effects. This investigation also showed the capacity of D-Pinitol to protect oxidative DNA damage caused by DOX in Vero cell lines. In our results, all concentrations of D-Pinitol showed a genoprotective effect against DOX-induced genotoxicity. The protective effect of D-Pinitol is possibly due to the result of the expression of several molecular pathways such as ROS scavenging effect through its antioxidant capacity and attenuation of pro-inflammatory mediators by its anti-inflammatory property. Genoprotective effect of D-Pinitol was more pronounced at the larger concentration than smaller concentrations.
    
\section{Conclusions}
Our findings showed that D-Pinitol was not genotoxic to Vero cell lines. Our investigation confirmed the genoprotective effect of D-Pinitol against DOX-induced genotoxicity by protecting normal cell lines from DNA damage. 
\section*{Acknowledgement} The authors thank the authorities of Adhiparasakthi College of Pharmacy for providing the necessary facilities. The published work is a part of the Ph.D., Thesis of The Tamilnadu Dr.M.G.R. Medical University.

\textbf{Funding Support}

The authors declare that they have no funding support for this study.

\textbf{Conflict of Interest}

The authors declare that they have no conflict of interest for this study.
    

\bibliographystyle{pharmascope_apa-custom}

\bibliography{\jobname}
\end{document}
