Cytotoxic  Effects  of  Electronic  Cigare3es  on  16HBE  Human  Bronchial  Epithelial  Cells  In  Vitro  

INTRODUCTION  Promoted  as  a  means  of  reducing  smoking,  Electronic  cigare5es  (ECs)  have  been  the  subject  of  much  interest  contribu=ng  to  its  open  considera=on  as  a  safer  smoking  alterna=ve.  Recently  approved  by  the  Britain’s  medicine  regulator  for  this  purpose,  sales  are  expected  to  grow  significantly  in  the  next  few  years,  where  prescrip=on  through  the  NHS  could  become  readily  available1.  However,  healthcare  prac==oners,  remain  uncertain  of  the  safety  and  efficacy  of  electronic  cigare5es  as  a  consequence  of   limited  evidence,   inconsistencies  in  results,  methodologies  and  absence  of   long-­‐term  con=nuous  studies.  Besides  chemical  evalua=ons2,   limited  studies  have  performed   in  vitro  on  the  airway  epithelial;  therefore  no  definite  conclusions  can  be  drawn  on  the  poten=al  cytotoxic  effects  and  safety  of  ECs.  Thus,   in  order  to  compare  cellular  reac=ons   induced  of  E.liquid  and  it’s  aerosol,  the  current  project  aimed  to   implement  a  realis=c  simula=on  of  E.C  use.    We  developed  an  in  vitro  cytotoxicity  model,  analyzing  a  high  nico=nic  content  (18mg/ml)  ice  mint  flavor,  Bri=sh  e-­‐liquid,  in  order  to  evaluate  the  cytotoxic  poten=al,  with  and  without  pH  adjustments,  in  addi=on  to  cellular  levels  of  poten=al  pro-­‐inflammatory  cytokine  release  IL-­‐6  and  TER  of    airway  epithelial  cells  16HBE.    

RESULTS  

       Figure   1:   DisrupGon   of   16HBE   cell   lines   following   exposure   to  different   treatments,   (Vape   and   E.liquid   at   1.25%   v/v   and   a  control)  for  different  exposure  duraGons  (4  and  26  hours).      16HBE  cells  on  inserts  were  challenged  apically  with  E.C  and  Vape  at   1.25   %   v/v   concentra?ons.   An   untreated   control   was   also  analyzed.  TER  (Ω  cm2)  was  measured  before  cell  treatment  (t=0)  and   at   4h   and   26h   respec?vely.   Data   calculated   as   a   %   mean  change   from  pre-­‐treatment   reading  ±SD,  4   replicates,  3   repeats.  *represents  significant  difference  in  measured  TER  with  respect  to  the  control  group;  p<  0.05;  2  way  ANOVA  Tukey.  

       Figure   2:   Change   in   expression   release   of   IL-­‐6   by   16HBE   in  response  to  24  hour  exposure  to  “Vape”  and  E.liquid    (0.306%-­‐  5%  v/v)  or  posiGve  control.     Il-­‐6   release   was   assed   using   Human   Il-­‐6   Elisa   set.   Absorbance  was   measured   at   450nm,   represented   as   mean   values   to  respec?ve   treatments   ±SD   of   4   replicates.   IL-­‐6   expression   was  significantly  different  for  vape  and  E.liquid  (p<0.001);  and  E.liquid  from  control  (p<0.05),  remarkably  at  5%  v/v  for  E.liquid  (p<0.05)  represented  by  *;  Kruskal-­‐wallis  and  post  hoc  Mann-­‐Whitney.  

Figure   3:   Cytotoxic   screening   following   exposure   of   a)   “Vape”   and   E.liquid   on   16HBE,   b)   pH   treatment   and   their   respecGve  controls    a)  Cytotoxicity,  measured  from  LDH  ac?vity  of  16HBE  aber  24  hours  of  exposure  to  treatments  at  0.306-­‐5%  v/v  concentra?ons.  

Data  is  presented  as  mean  values  ±SD  of  10  replicates  for  each  treatment,  18  controls.  b)   Cytotoxic  assessed  from  LDH  ac?vity  post  24  hour  exposure  to  pH  treatment    adjusted  to  7.3  from  8.23  (Vape)  8.53  (E.liquid)  

revealing  strong  alkaline  proper=es,  physiologically  incompa=ble  with  cellular  environment  and  func=on.  .  Experiments  were  conducted  in  4  replicates;  error  ±SD  (Standard  Devia?on).  

13815280/MENDES  

Disrup.on  of  Epithelial  Barrier  Func.on    

a)   b)  

CONCLUSIONS    à   Cellular   events   occurring   post   treatment   of  

E.liquid   and   Vape   include   increase   in  cytotoxicity  and    =ght  junc=on  degrada=on  in  a  dose/=me  rela=onship  respec=vely  (Figure  1  and  3)  

 à  The   release   of   IL-­‐6   is   independent   of   dose,  

and   further   suppressed   at   5%,   presumably  due  to  mass  cell  death.  (Figure  2)  

 à  Cellular   cytotoxicity   is   found   to   be  

sta=s=cally     higher   in   E.liquid   compared   to  Vape,  where  4  readings  out  of  10  were  above  moderate  range  cytotoxicity  (70%)  according  to  -­‐ISO  10993-­‐5  protocol.3  

 à  There  is  a  significant  effect  of  pH  contribu=ng  

towards  the  cytotoxicity  of  our  cell  model.  

 

à  These   finding   are   in   agreement   to   several  studies,   however   pH   unrecognized   issue  must   be   further   exploited   in   order   to  d e t e rm i n e   t h e   p o t e n = a l   h e a l t h  consequences  in  a  long-­‐term  E.  cigare5e  use.  

   à  The  study  proves  that  E.liquid  and  vape  have  

a   poten=al   to   alter   the   Airway   Epithelial  morphology,   func=on  and  cell  viability,  even  at  low  exposure  strengths,  which  are  possibly  observed   concentra=ons   of   vapor   absorbed  into  the  lungs.    

MATERIALS  AND  METHODS  Materials:  Ice  Mint  flavor  with  full  strength  nico=ne  levels  18mg,  and  a  VG/PG  ra=o  of  65:35   (Liqualites,Bolton,UK)   was   opted   for   this   experiment.   For   the  produc=on  of  extracts,  a  commercially  available  160W  temperature  control  device   (SMOK   x   box   cube   II,   SMOK   Tech,   Shenzhen,   China)   was   used,  consis=ng  of  lithium  ba5ery,  a  triple  coil  Ni200  alloy,  TFV4  atomizer.  (SMOK  Tech)                                                                                  Known  exact  %  concentra=on  of  E.liquid  vape  condensate  used.    Cell  culture  and  Treatment  preparaGon:  16HBE   cells   were   cultured   with   MEM   supplemented   with   10%   FBS   (PAA  Laboratories)   .   Stock   solu=ons   for   E.liquid   and   Vape   were   prepared,   from  which  serial  dilu=ons  were  conducted.  (5%-­‐0.036  %v/v).    For  cytotoxicity  and  cytokine  experiments,  cell  were  seeded  in  96  and  48  well  plates  respec=vely  in  100μL  MEM  +10%  FBS.  For  transepithelium  resistance,  cells  were   seeded   into   12   transwell   inserts   (Corning   Incorporated,  NY,USA)  with  DMEM  ,  Hams  F-­‐12  mix  (1:1)  (GE  HealthcarePAA  Laboratories,  Austria).  pH  stocks  were  adjusted  to  pH    7.3  from  8.23  (Vape)  8.53  (E.liquid).    Transepithelial  resistance  (TER):  Prior  seeding  16HBE  cells  into  the  12    transwell  inserts,  200μl  of  collagen  was  added   onto   each   insert   coa=ng   (Pure   col).   16HBE   cells,   were   then   seeded  into  the  apical  chamber  at  a  seeding  density  of  4.3  x  105  cells/well  in  500μl  of    appropriate  cell   culture  medium  and   further  1500μl  of  cell   culture  medium  was  added  to  basolateral  chamber.    Aner  24  hours  cells  were  subjected   to  air-­‐liquid   interface,   and   used   on   the   7th   day   following   seeding   where   TER  measurements  were  conducted    using  Epithelial  Tissue  Voltohmeter  (EVOM)  and   hand-­‐held   chops=ck-­‐type   electrode   prior   exposure   of   each   variable  (E.liquid,  Vaped  1.25%v/v  and  control)  and  at  4  and  26  hours  post  treatment  respec=vely.      Cytokine  IL-­‐6:  Cells  were   seeded  with   a   density   of   1.5   x   104     cells/well   and   treated  with  Vape,  E.lqiuid  or  posi=ve  control  (vanadyl  sulphate)  in  appropriate  media  for  24  hours  under  standard  condi=ons,  aner  which   IL-­‐6  release  was  measured  using   a   commercially   available   Human   IL-­‐6   ELISA   kit   (BD  OptEIA™,  Biosciences  Pharmingen  ,USA).      LDH  Cytotoxicity  assay:  Cells   were   seeded   with   a   density   of   1.0   x   104   cells/well   in   96-­‐well  microplates   ,in   appropriate   media   overnight.   Medium   was   subs=tuted   by  treatments   or   len   untreated   (control)   for   24   hours   and   successively,  evaluated   using   a   Pierce   LDH   Cytotoxicity   Assay   Kit.   (Thermo   Scien=fic,  Rockford,  USA)  

The  E.C  was  ac=vated  for  2-­‐2.5  sec.  every  30  secs.  for  a  period  of  1  hour.  Successively  the  extracts  from  the  two  collec=ons  flasks  were  combined  together.  

Cytotoxicity  is  pH  dependent  

a)  2  way  Anova;  Kukey  Post  Hoc  b)  Kruskal-­‐wallis,  and  Mann-­‐Whitney  post  hoc.  

*Represents  the  significant  difference  in  cytotoxicity  with  respect  to  control  treatments  p<0.05.    nRepresents  the  significant  difference  between  cytotoxicity  with  respect  to  Treatment  concentra?on  5  %v/v.    p<0.05    ✚Represents  significant  difference  between    cytotoxicity  of  E.liquid  and  Vape    p<0.05.  

✓

References  1-­‐  Nico=ne  without  smoke  Tobacco  harm  reduc=on    A  report  by  the  Tobacco  Advisory  Group  of  the  Royal  College  of  Physicians  (April  2016)  h5ps://www.rcplondon.ac.uk/file/3563/download?token=uV0R0Twz  (accessed  05.05.16)  2-­‐  Famele,  M.,  C.  Ferran=,  C.  Abenavoli,  et  al.  'The  Chemical  Components  of  Electronic  Cigare5e  Cartridges  and  Refill  Fluids:  Review  of  Analy=cal  Methods',  Nico?ne  &  Tobacco  Research,  vol.  17/no.  3,  (2015),  pp.  271-­‐279.  3-­‐ISO  10993:5  Standard.  Biological  Evalua=on  of  Medical  Devices—Part  5:  Tests  for  in  vitro  Cytotoxicity,  2009.  Available  online:  h5p://www.iso.org/iso/home/store/catalogue_tc/  catalogue_detail.htm?csnumber=36406  (accessed  on  14  March  2016).  

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