j. Soc.Cosmet.
Chem.,34, 1-11(January/February
1983)
Theeffectof zincpyrithione
onhumanskincellsin vitro
GENJI IMOKAWA andKIKUHIKO OKAMOTO, Kao Soap
Company,
Ltd., TochigiResearch
Laboratories,
2606Akabane,
Ichikai-Machi Haga-Gun,Tochigi,.Japan.
Received
February
5, 1982.
Synopsis
A highlyactiveantidandruff
agent,zincpyrithione
wasinvestigated
on humanskincells(JTC-17)to test
the hypothesis
thatlikeselenium
sulfide,it mayalsohaveanantimetabolic
actionon epidermis,
actingon
dandruff
byreducing
increased
epidermal
turnover.
It hasbeenfoundfromtheuptakeof 3H-thymidine
intocellsthatzincpyrithione
addedat levelsof 0.25-1.0/xg/mlis effectivein suppressing
reversibly
the
DNA synthesis
of humanskincellsin vitrowithouta comparable
inhibitoryeffecton RNA andprotein
synthesis,
asshownby therespective
uptakes
of 3H-uridine
and3H-leucine.
Its homologues,
sodium
pyrithione
andomadinedisulfide,
at levelsof 0.25-1.0/xg/mlalsoexhibita similarreversible
inhibitionof
DNA synthesis.
On longtermcultureof humanskincells15-27%growthinhibitionwasobserved
with 0.2
/xg/mlof zincpyrithione.
Analysis
usingsynchronized
cellsrevealed
thatzincpyrithionecanact on all
periods
of DNA synthesis
to suppress
it. Thesefindingssupport
theideathatzincpyrithione
mayhavean
antidandruff
effectby its antimetabolic
actionto the skin,ratherthanits antiyeast
action.
INTRODUCTION
Previousstudiesto test the relationship
betweenmicroorganisms
and dandruffhave
revealed
thata decreased
numberof Pityrosporum
ovaledoesnotcontribute
primarily
to
thereductionin dandruffcausedby a highlyactiveantidandruff
agent,zincpyrithione
(ZPT) (1).Similarresultshavebeendescribed
usingselenium
sulfideasanantidandruff
agent(2).Thus,scalporganisms,
especially
P. ovalewhoseincrease
hasbeenconsidered
asa pathogenic
factorfor dandruff
(3),playnoprimaryimportant
rolein production
of
dandruff.
Thesefindingsled usto furtherinvestigate
thepossibility
thatZPT mayhave
an influenceon the scalpskinin a differentmannerfrom the way by whichZPT
suppresses
P. ovale.The amountof dandruffis directlyrelatedto cellularreproduction
in the basalcellsof the skin.In the presentstudy,the effectof ZPT andits derivatives
on variouscellularmetabolisms,especiallyDNA synthesis,
was investigatedon
culturedskincellsto testthe possibility
that the reproductive
activityby mammalian
epitheliamightalsobe suppressed
by ZPT.
2
.JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
MATERIALS
AND
METHODS
MATERIALS
EaglesMinimalEssential
Medium(EMEM) andtrypsin(0.25%)
werepurchased
from
Grand Island BiologicalCompany.Fetal calf serum(FCS) was obtainedfrom
Microbiological
Associates.
PlasticPetridisheswerepurchased
from Becton,DickinsonandCo.3H-Thymidine
(3H-TdR,specific
activity2Ci/mM),3H-Uridine
(3H-UR,
specific
activity2-5 Ci/mM), and3H-Leucine
(specific
activity0.5-1 Ci/mM) were
obtainedfromNew EnglandNuclear.Zinc pyrithione
(ZPT), Omadinedisulfide(DS)
and sodiumpyrithione(SPT) were obtainedfrom Olin JapanInc. IrgasanDP-300
(DP-300)was purchased
from Ciba Geigy Company.Amethopterin
and all other
chemicals
werepurchased
fromSigmaChemicalCo.
CELLS
AND
CELL
CULTURE
TheJTC-17humanskincell line (Figure1), whichwasderivedfrom a humanmale
bearingan XX sex chromosome
constitution(4, 5), was culturedin EMEM
Figure 1. Phasecontrast
microscopy
ofJTC-17humanskincells.Notethatmorphological
appearance
of
the cellshasepithelialproperties.
x 300
supplemented
with 10%fetalcalfserum,4 mM glutamine,100unit/mlpenicillinand
100/•g/ml streptomycin
at 37øCwith a 5%CO2/95%air atmosphere.
Exponentially
growingcellswereharvested
andplatedat a celldensity
of 105cell/cm
2 in Falcon
plasticculturedishes
(35mmdiameter)
containing
three15mmdiameter
glasscoverslips
perdish.Mediumwasreplaced
threetimesperweekfor growthexperiments.
Forthe
examinationof the effectof drugson the synthesis
of DNA, RNA, and protein,1/•Ci
permlof 3H-thymidine,
uridine,
andleucine
respectively
wereaddedto thecultures
for
a desired
periodof time.Afterincubation,
cellswerewashed
3 timeswithcoldHank's
buffer solution. Fixation was done with two successive ten minute treatments with 5%
EFFECT
OF
ZINC
PYRITHIONE
ON
SKIN
CELLS
3
coldtrichloroacetic
acid(TCA), followedby successive
treatments
with 70%,90%and
100%EtOH for dehydration.The TCA insolublefractionwas dissolvedin 0.Sml of
Soluene©-350
(Packard)and mixedwith 10 ml of toluenebasedscintillationfluid.The
radioactivity
wascountedin a liquidscintillation
spectrometer.
Cellcountswerecarried
out using a hemocytometer
after trypsinization
of cells adheringto the glass
coverslips.
KERATINOCYTE
CULTURE
Primaryculturesof keratinocytes
wereinitiatedfromthe earsof guineapigs(Hartley
strain)according
to themethodof Christophers
(6).Theearskinwasfloatedovernight
on0.25%
trypsinin Hank'sbuffersolution
(pH 7.2)at 4øC.Theepidermis
wasseparated
from the skin,andthe epidermalcellswereremovedby verygentlescraping
with a
scalpelandfilteredthroughcottoninto a tubecontaining
the medium.At 72 hr after
seeding
at105cell/cm
2,incorporation
experiments
werecarried
outinthesame
manner
as described above.
SYNCHRONOUS
CULTURE
To obtainsynchronized
cellsin S-phase,a doubletreatmentwith excessTdR and
amethopterin
wasusedaccording
to themethodof Ebinaetal. (7).Briefly,
JTC-17cells
werefirstsynchronized
withexcess
TdR (2raM)for 24hr.Afterreplacement
withfresh
medium
for10hr,medium
containing
10-6Mamethopterin
and5 x 10-5Madenosine
wasaddedfor 16 hr. Then the cultureswerewashedrapidlywith Hank'ssolution3
timesandreceivedfreshmediumfor startingDNA synthesis
in earlyS phase.
RESULTS
INHIBITORY
EFFECT
OF
ZPT
ON
DNA
SYNTHESIS
Figure2Ashows
theeffectof ZPT ontheincorporation
of 3H-TdRintoJTC-17
cells.
Resultsindicatedthat ZPT added at the level of 0.25-1.0•g/ml is effectivein
suppressing
the DNA synthesis
of the cell by approximately
20-90%.This inhibitory
effectat 1.0•g/ml wasmaximalaftera 2 hr period(Figure3), andwasfoundto be
diminished
within 3 to 6 hr afterremovalof ZPT from the culturemedium(Figure
2B).
A similarlyactiveantidandruffagent,DS also exhibitsa similardose dependent
inhibitionof DNA synthesis
with a reversible
effect(Figure2). However,an active
antimicrobialagent,DP-300,whichhasbeenfoundto haveno antidandruffeffectin
vivo(1), showsno significant
inhibitionevenat the levelof 2.0/xg/ml, althoughit
demonstrates
reversible
inhibitionat highdosesof 5/xg/ml and 10/xg/ml(TableI).
Table I also shows that ZPT and DS but not DP-300 have a similar reversible anti-DNA
synthesizing
effecton keratinocytes
fromthe guineapig ear.Sodiumpyrithione,
a Zn
lackingderivativeof ZPT, wasalsohighlyeffectivein suppressing
the DNA synthesis
of theJTC-17cellswith a similardosedependence
(TableI). Observation
of cultures
with phasecontrastmicroscopy
demonstrated
that therewasno lethalor cytotoxic
appearance
occurringat leastwithin8 hr of ourexperiments.
4
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
(A)
[ (B)
I•]
[]
ZPT
DS
1.0•og ml
Control 0.25 0.5
1.0/tg/ml
Control
0
3
hourafter
6
removal
Figure2. Effects
ofZPTandDSon3H-TdR
incorporation
intoJTC-17
cells
(A)andtheirreversibility
(B).Cells
were
seeded
atapproximately
2 x 10• cells/15
mmdiameter
glass
coverslip.
After3hrincubation
withZPTandDS(13tg/ml),
cells
were
washed
three
times
withHank's
buffer
andplaced
infresh
medium.
3H-thymidine
(! 3tCi/ml)
was
added
forthelast! hroftheincubation
period.
3100 -
1.0/tg/ml z P T
J TC-17
050
Cell
-
2
3
4
Incubation
5
6
hour
Figure3. Inhibition
of3H-TdR
uptake
byZPTplotted
against
theincubation
time.Experiments
were
in
triplicate.
EFFECT
OF
ZINC
PYRITHIONE
Table
ON
SKIN
CELLS
5
I
Effect
of ZPT,DSandSPTon3H-TdRIncorporation
intoJTC-17
CellsandGuinea
PigKeratinocytes
and
Its Reversibility
3H-TdR
GuineaPigKeratinocytes
Control
%Inhibition
11854 _+263a
ZPT
1.0/xg/ml
(3hrb)
DS
1.0/xg/mlwash
c
0.5/xg/ml(7 hr)
1.0/xg/ml
(3 hr)
1.0/xg/ml
wash
1,0/xg/ml
(3 hr)
DP-300
(cpm/dish)
4963+ 174
8423+ 168
1380+ 273
5966_+30
11028+ 132
11245+ 414
58.1
28.9
88.4
49.7
7,0
5.1
3H-TdR
(cpm/104
cell)
JTC-17cells
Sodiumpyrithione
1936+ 286
-
(SP T)
Control
0.25/xg/ml
1739-+345
10.2
(3hrb)
0.50/xg/ml
1073_+241
44.8
256+ 67
86.8
1.00/xg/ml
DP-300
(3 hr)
Control
2306 + 234
1.00/xg/ml
2.00/xg/ml
5.00/xg/ml
5.00/xg/ml
wash
10.00/xg/ml
10.00/xg/ml
wash
2178+ 240
2196+ 98
1814_+146
2353+ 716
1302_+354
2513+ 147
5.5
4.8
21.3
-2.0
43.5
-8.9
a)Meanandstandard
deviation
n = 9, b)Incubation
hour.3H-TdRwasincubated
for thelast1 hr.c) 5 hr
afterreplacement
withfreshmedium.
EXAMINATION
USING
SYNCHRONIZED
CELLS
In orderto confirmif ZPT primarilyinfluences
cellularDNA synthesis,
JTC-17cells
synchronized
by excessTdR (2mM), followed by the additionof amethopterin
(10-6 M) andadenosine
(10-' M), wereassayed
forSphase
initiation
afterremoval
of
amethopterinand adenosine.Figure4 showsthat JTC-17 cellssynchronized
at the
beginningof S phaseare releasedinto an originalcell cyclefollowingremovalof
amethopterin,
therebyinducingDNA synthesis
withinaboutan 8 hr period,asshown
bythepulseincorporation
of 3H-TdRfor30min.In thecaseof addition
of ZPT at 1.0
•tg/ml concentration,
the DNA synthesis
startingin early S phasewas almost
completely
suppressed.
Further,differences
in inhibitoryeffectsdepending
on thetime
of additionwereexaminedto determine
the activephasein the cellcyclefor theZPT's
action.Thus,synchronized
cellsofJTC-17wereincubated
with 1 •tCi/ml of 3H-TdR
immediatelyafter or 2.5 hr prior to the additionof ZPT, and the accumulated
incorporation
of 3H-TdRwascompared
in both.Results
(Figure5) showthe same
inhibitorypatternin bothcases,
indicating
that ZPT canactduringall periodsof DNA
synthesis
to suppress
it.
COMPARISON
WITH
OTHER
CELLULAR
SYNTHESES
Comparison
of inhibitoryeffectsusingTdR, UR, andleucineincorporations
(TableII)
revealed
that ZPT andDS havefewerinhibitoryeffectson 3H-URand3H-Leucine
incorporations
compared
to 3H-TdRincorporation.
Despite
abouta 90%inhibition
of
6
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
x5x10 3
3
Control
3Omi
3H TdR
1/tCi
pulse-label
ing
1
Z PT
3
4
1.0,•g/•l
5
6
7
8
hours after removal of Amethopterin
Figure4. Effectof ZPT onsynchronizedJTC-17
cellsat earlySphase
asshownbypulse3H-TdRuptake
for 30 min.ZPT wasaddedat 1.0/•g/ml immediately
afterremovalof amethopterin.
No lethaleffecton
the treatedcellswasobserved
with phasecontrastmicroscopy
during8 hr.
3H-TdRincorporation,
only40%and55%inhibitions
of UR andleucine
incorporations
respectively
wereseenat theconcentration
of 1.0!ag/mlof ZPT andDS.
INHIBITORY
EFFECT
ON
CELL
GROWTH
Additionof ZPT to normalmediumin therangeof 0.04-0.4/xg/mlcaused
JTC-17cells
to havea growthinhibitionin 5 to 14daysafterinitiationof culturein comparison
with
growthin normalmedium(Figure6). Thisgrowthinhibitionvarieddepending
on the
initial concentrations
of the seededcells.Thus,at a low concentration
of 3 x 104
cells/dish,
about27.9%growthinhibitionwasobserved
with0.2/xg/mlof ZPT (Figure
6A) whilea muchlowerinhibitoryeffectof 12.7%wasdemonstrated
at a higherseed
concentration
of 3.2 x 105cells/dish
(Figure6B).In thelattercase,even0.4/xg/mlof
ZPT exhibits
lessgrowthinhibition
thanisseenat the3 x 104cells/dish
concentration.
DISCUSSION
The presentstudyusingculturedhumanskin cellshasdemonstrated
that ZPT hasa
capacityto inhibit reversiblymammalianDNA synthesis
by influencinga cellular
EFFECT
xlO
OF
ZINC
PYRITHIONE
ON
SKIN
CELLS
7
5
j'
2
Z PT(1.O/jg/ml)
3
4
5
6
7
hours after removalof Amethopterin
Figure 5. Effect of ZPT on synchronized
JTC-17 cells at early S phaseas shown by cumulative
incorporation
of 3H-TdR.1.0/ag/mlof ZPT wasaddedto thesynchronized
culture
atthetimesindicated
by arrows.
8
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Table
II
Effects
of ZPTandDSon3H-Thymidine,
Uridine,
andLeucine
Incorporations
intoJTC-17
Cells
3H-TdR
(cpm/104
cell)
ZPT
DS
% Inhibition
Control
2766 _+333a
0.25#g/ml
b
2206_+263
20.3
--
0.50#g/ml
1.0o#g/ml
0.25#g/ml
0.50#g/ml
1.0o#g/ml
1522_+488
275__+96
1991_+185
1412_+555
385+ 30
45.O
90.1
28.0
49.0
86.1
3H-Uridine
(cpm/104
cell)
ZPT
DS
Control
1012 _+232
0.25#g/ml
0.50#g/ml
1.00#g/ml
0.25#g/ml
0.50#g/ml
1.0O#g/ml
858+ 99
1006+ 243
619+ 127
824_+30O
903+ 91
629+ 109
%Inhibition
-
15.8
0.6
38.8
18.0
10.7
37.8
3H-Leucine
(cpm/104
cell)
Control
ZPT
DS
0.25#g/ml
0.50#g/ml
1.0O#g/ml
0.25#g/ml
0.50#g/ml
1.0O•tg/ml
102 +
40
99 + 24
83 _+ 38
46 + 6
82 + 30
70 + 29
48 _+ 18
%Inhibition
-
3.0
18.3
55.2
19.9
31.3
53.4
a)Meanandstandard
deviations
n = 9, b) Incubation
hours:3 hr Radioactive
precusors
wereincubated
for
the last 1 hr.
process
associated
with DNA synthesis,
ratherthanby actingon the RNA andprotein
synthesizing
processes.
Priestley
(8) speculated,
basedon the acutetoxicityof ZPT to
culturedhumanskincellsat the concentrations
of 0.1-0.5/xg/ml,that ZPT's action
againstdandruffisa resultof non-specific
toxicityfor epidermalcells.It is wellknown
that the degreeof celltoxicityasseenin a cell culturesystemvariesgreatlydepending
on cell speciesas well as the initial concentrationof seededcells. Our growth
experiments
with humanskincellshasrevealedthat evenat the ZPT concentration
of
0.4/xg/mlthe cellscouldfinallyreachconfluency.
Ourprevious
microbialstudy(1),togetherwitha similarstudyof Leydenetal. (2),has
revealedthat the suppressive
effectof antimicrobial
agentslike ZPT and selenium
sulfideonP. ovaleisnotresponsible
for thereductionof dandruff.Leydenetal. (2)also
speculated
thatthe actionof selenium
sulfidewhichcontrolsdandruffis the resultof
its antimetaboliceffect on epidermisas revealedby a decreasein labelingindex.
However,exceptfor thecases
of selenium
sulfide(9)andomadinedisulfide
(10),there
havebeenno reportsto confirmquantitatively
theantimetabolic
actionof antidandruff
agentsunderexperimentally
simplifiedconditions.Our presentstudyhasrevealedthat
the highlyactiveantidandruff
agentsZPT and DS alsohavecomparable
inhibitory
EFFECT
OF ZINC
PYRITHIONE
ON
SKIN
CELLS
9
effectson DNA synthesis
which,as suggested
by the experiments
with sodium
pyrithione,
areattributable
to theirpyrithionemoieties.Thus,pyrithioneis,exceptfor
itsringstructure,verysimilarto hydroxy-2-thiourea
whichalsoexhibitsDNA synthesis
inhibition. Additionally,it has been shown that all of the hydroxyurearelated
compounds
whichformcomplexes
withmetalionsinhibitsynthesis
of DNA in a HeLa
cellsystem
(11).
l0 v
ß
Control
ß 0.04 jtg/ml
1 05
[]
0.08
•
0.12
o
0.20
ZPT
}
Cultu
re
Day
Figure 6a. Seelegendon followingpage.
,
10
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
1 07
I 06
1 o5
I
I
o
5
Cultu
re
Day
Figure 6b. Effectof ZPT on the growthofJTC-17cellsseededin differentconcentrations.
A: Cellswere
initially
cultured
ata 3.0x 104celldensity
withsimultaneous
addition
of ZPT atindicated
concentrations.
B:Cellswereinitiallycultured
at a 3.2 x 105celldensity
withsimultaneous
additionof ZPT at indicated
concentrations.
Arrowrepresents
thetimeof the additionof drugs.
Sinceactualinhibitionof DNA synthesis
by ZPT requirespenetrationof ZPT into
epidermisaswell as sequential
contactwith epidermalbasalcells,whetheror not the
suppression
of dandruffby ZPT couldreasonably
be interpreted
in the termsof the
inhibitionof DNA synthesis
observedhere,remainsto be established
not only with
regardto ZPT'spermeability
into the scalpskinbut alsofor its actualin vivocellular
metabolicaction.Our previousmicrobialstudy(1) has suggested
that of greater
significance
for inductionof clinicaleffectiveness
is the factorof penetration
of ZPT
intothe skin.Thisobservation
is basedon the factthatthepresence
of ZPT molecules
on scalpin concentrations
capableof maintaining
the continuous
suppression
of P.
ovaledoesnot necessarily
resultin reductionof dandruff.In a biologicalestimation
of
actuallyadsorbedZPT moleculesby in vitrominimuminhibitoryconcentration
(MIC)
valuesfor itsanti-yeast
action,thereseems
to be at least10ppmof ZPT presentwithin
theupperlayersof thescalp(1).Furthermore,
surfactants
usedasmainconstituents
of
antidandruffshampoosbecauseof their enhancedsurfaceactivitiesmay accelerate
percutaneous
absorption(12). There is someevidencethat dandruffscalphas a
EFFECT
OF
ZINC
PYRITHIONE
ON
SKIN
CELLS
11
decreased
numberof hornylayersas comparedto normalscalp(13).Therefore,the
clinicaleffectiveness
on dandruffthroughinhibitionof DNA synthesis
by ZPT seems
aidedthroughthe enhancedpermeabilityof dandruffscalp.This also suggests,
as
supported
by clinicalnoneffectiveness
of ZPT on milddandruffsubjects
(14),thatthe
restorationof the normal number of horny cell layers, becauseof decreased
permeability,
maygenerally
diminishthepotencyof ZPT in exertingits inhibitionof
DNA synthesis.
Scalpepidermal
kineticshaverevealed
thatdandruffisa disorder
of hyperproliferation
(15).Therefore,our findingsthat the highlyactiveantidandruff
agentsZPT and DS
haveaninhibitingeffecton DNA synthesis
of mammalian
cells,supportthehypothesis
that the antidandruff
effectby ZPT mayprimarilybe dueto its anti-metabolic
effect
ratherthanits anti-yeast
effect.Sincepyrithioneis a generalinhibitorof membrane
transportprocesses
in fungi (16), it seemslikely that the decreaseof P. ovale
concomitant
with the decrease
in dandruffcausedby ZPT asobservedpreviously
(1),
involvessimilaritiesin cellularmetabolisms
betweenanti-yeastand anti-DNA synthesizingactions,both of whichare relatedto a markeddecrease
in the activitiesof a
varietyof independently
regulatedtransportsystems
withincells.
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