J. Soc.Cosmet.Chem.24, 473-492
¸1972 Societyof CosmeticChemists
of GreatBritain
The effectof soapupon certain
aspects
of skinbiochemistry
C. PROTTEY, P. J. HARTOP and T. F. M. FERGUSON*
Presentedon the !2th April 1972 in Oxford, at the Symposium
on 'Skin--Environmentalresponses
andprotection',organized
by the Society of Cosmetic Chemists of Great Britain.
Synopsis--Ratshave beenTOPICALLY TREATED with SOAP solutionsand distinctmorphologicalchangesin the skinhavebeenobservedhistologically.Samplesof the treatedtissueshave
beenCULTURED in vitroin the presenceof specificprecursorsof DNA and LIPIDS, when it
was seen that DNA and PHOSPHOLIPID metabolism was stimulated in mildly irritated
cases,but in more severelyirritated tissues,which had receivedgreatlyexaggeratedsoaptreatments compared with routine testsfor irritancy or normal user conditions, both of these biochemicalreactionswere greatly diminished.TRIGLYCERIDE synthesis,on the other hand,
appearedto increaseas the degreeof irritation responseincreased.The mostirritant component
of the soapwas SODIUM LAURATE, and this compoundsimilarlycausedthe most marked
biochemical
alterations.
Theenhanced
triglyceride
synth.esis,
in skinwhichwasseverely
damaged
from exaggeratedsoap treatment,more than replacedthe inhibited phospholipidsynthesis:
indeed,severelyirritated skin was seento synthesizegreatlyincreasedamountsof radioactive
lipids compared with the water-treated controls.
INTRODUCTION
When the surfaceof the skin is repeatedlytreatedwith soapsolutions,an irritationresponse
is oftenseenin the tissue,dependent
generally
uponthefrequency
anddurationof treatment,theformulationof the soap,
and the concentration
of the solutions.The irritationresponse
whichmay
developofteninvolvesmarkedchanges
in the morphologyof the skin,the
rate of keratinformation,andin the overallphysicalpropertiesof the skin.
*Unilever ResearchLaboratory, Colworth House, Sharnbrook,Bedford.
473
474
JOURNALOF THE SOCIETYOF COSMETICCHEMISTS
This paper describescertain biochemicalparameterswhich are seento be
alteredby soap-induced
irritation of rat skin,with particularemphasison
glycerolipidmetabolism.
The soapformulationusedhere is very mild under typical user conditions.The drasticmorphologicaland metabolicchangesdescribedare due
to grosslyexaggeratedtreatments,and in no way would arisefrom normal
usagein cleansinghuman skin.
MATERIALS AND METHODS
Soap treatment
The soapwas a conventionalformulation derivedfrom coconutoil and
tallow. 8•o solutionswere preparedby homogenizingpowderedsoap in
distilledwater (6øhardness),and the pH adjustedto 9.8. The solutionswere
maintainedat 37øduringtreatmentof the animals,whichhad beenclipped
closelyon the dorsalsurfacewith electricclippersprior to the initial treatment. The solution(0.2 ml) was applied to the shavedarea and gently
massagedin, and then left for 7.5 min, after which the treatment was repeated.Excessof soapwasthen removedfrom the animalsby rinsingwith
distilledwater and wiping with tissues.Control animalsreceivedsimilar
treatment,exceptthat distilled water replacedthe soap solutions.The
frequencyof soaptreatmentin eachexperiment(eithertwice--or four times
daily) is quotedat the relevantpoint in the text.
Sodiumsoapsof individualfatty acidswerepreparedby neutralization
of the free acids with sufficientNaOH solution to give final solutionsof
0.25Mat pH 9.8.
Animals
All rats were 3-week-oldfemalesof the Colworth/Wistaralbino strain,
fed Spital diet and water ad libitumthroughouttreatments.Guinea-pigs
were 6-week-oldalbinosex-Porton, of either sex.
Radioactive substrates
These were purchasedfrom the RadiochemicalCentre, Amersham,
Bucks.The specificactivity of each addedto tissueculturewere 5.0 I•Ci
ml4 mediafor 2-x4C-thymidine,
2-x4C-sodium
acetateand U-x•C-glycerol,
EFFECTOF SOAP UPON CERTAIN ASPECTSOF SKIN BIOCHEMISTRY 475
and 50.0[tCiml-• for a2P-orthophosphate.
No carriermaterialwasaddedto
the radioactive substrates.
Other methods
The methodsusedin this studyfor skin and cell culture,extractionof
radioactivity
from tissue,chromatography
of lipidsand isolationof epidermalcellshaveall beenfully described(1).
RESULTS
Histologicalexaminationof rat skin after topicalsoaptreatment
It wasfound generallyafter merelyone or two treatmentswith soap
solutionsthat the animalsshowedvisible signsof an irritation response.
Thiswasseenasaninitialerythema,whichwouldpersistoftenwith oedema,
but in moreseverely
affectedskin(asthe numberof treatments
increased)
scalingof the stratumcorneumand fissuringof the wholeepidermiswas
common.Samples
of the treatedareasof skinwereexcised
afterkillingthe
animalsandpreparedroutinelyfor histology(1), for assessment
of irritation. Microscopic
featuresobserved
includeda generalthickeningof the
stratumcorneum,togetherwith compactingof the squamous
matrix.
Therewasalsoevidence
of parakeratosis.
Concurrently
therewasa thickening of the Malpighianlayer, oftenwith a lossof definitionof the stratum
granulosumcells.The cellsof thestratumbasalewerearrangedmoreirregularly than normally.In the upperregionsof the dermislargeinfluxesof
leucocytes
were obvious.In casesof more severeirritation, leucocyteinfiltrationinto the Malpighianlayerwasseen,oftenwith leucocyteexudates
within the stratumcorneum.In animalsreceiving12 soaptreatments,for
example,discontinuities
of theMalpighianlayercouldbeseen,togetherwith
dilatation of microcapillariesin the upper dermis.Someof thesefeatures
may be observedin Fig. la, which showsskinfrom an animalreceiving12
soap treatments.For comparison,Fig. lb showsskin from a control rat
which received12 topicaltreatmentswith water.
Variationin irritationresponse
in rats receivingidenticalsoaptreatments
In preliminaryexperiments
it was observedthat in large groupsof rats,
eachreceivingthe samenumberof soaptreatmentsduringsimilarperiods,
476
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
individualirritation responses
were alwaysvaried. Some animalsmight
showwell-developed
responses
asmeasuredhistologically
in termsof alterations in morphologyof the stratum corneum,whereasotherswould show
little, and occasionally,no changesrelativeto control animals. In order to
rationalizethis, all histologicalsamplesfrom soap-treatedanimalswere
subjectively
analysed,and the specificmorphologicalchangesobserved(i.e.
corneumthickening,leucocyteinfiltration, capillary dilatation etc.) were
assessed
under increasingdegreesof severity.*These arbitrary categories
were then given a numerical weightingas follows: very slight reaction
(numericalweighting= 0.25); slightreaction(0.5); fairly distinctreaction
(0.75);quitedistinctreaction(1.0); well-developed
reaction(2.0) andsevere
reaction(3.0). The cumulativescorefor the criteriaof the irritation response
were then compiledfor each specimen.For example,if a sampleof soaptreatedskinshoweda quitedistinctthickeningof the stratumcorneum,but
only a slightthickeningof the Malpighian layer, and very slightleucocyte
accumulationin the upper dermis,the total scoreof irritation would be
1.75 (i.e. 1.0 + 0.5 + 0.25). Generally,control animalsreceivingwater
treatmentsexhibitedvery low total scores(0-2.0) whereassoap-treated
animalswould havecumulativescoresof 3.0 up to maybe 20, depending,
necessarily,on the number and duration of the treatments.
Individualfatty acidsand irritancyof skin
The soapusedin all of thesestudieswasa conventionalmixture of palm
kernel oil and tallow fatty acids and the major fatty acidspresentwere
laurate,palmirateand oleate.Other fatty acids,presentin lower concentrationsincludedstearate,myristateand caprate.In order to test the contribution of individualingredientfatty acidsto the overallirritation response
observed,a group of rats were topicallytreatedwith pure solutionsof certain sodiumsoapsof fatty acidsasfollows.Twenty-sevenrats,in ninegroups
of three, were treatedrespectivelywith sevenindividual sodiumsoapsof
pure fatty acids,at 0.25 M strength,a total of five timesduring 21/: days.
One group receivedfive treatmentswith 8•o completesoap solution,and
one group, receivingfive treatmentswith water, servedas controls.After
treatmentsamples
wereexaminedhistologically
and the irritationresponses
scoredas describedabove.The resultsare showngraphicallyin Fig. 2.
*The authorsacknowledgethe help and criticismgivenby Mr H. L. Jenkins,who also developed
the method for quantitative histologicalassessment
of irritation.
JOURNAL
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-<
Figure1. Histologyof soap-treated
and controlrat skin. (a) Tissueafter 12
treatments with 8•o soap during 3 days. Arrow indicates infiltration of
leucocytes
into epidermis.(b) Control skin (12 treatmentswith water during
3 days).Stainedwith haematoxylinand eosin. x 408.
Facing page 476
EFFECT OF SOAP UPON CERTAIN ASPECTS OF SKIN BIOCHEMISTRY
477
)ically applied soap (five treatments)
Figure 2. The irritancy of sodium soapsof fatty acids. All individual fatty
acid sodium soapswere 0.25 M, pH 9.8. The soap mixture was 8 •o, pH 9.8.
The most irritant fatty acid soap was sodium laurate. The irritation
response
wassoseverethat in manyregionsof the skinstudiedhistologically
there was completenecrosisof the epidermis,thus, an evaluationof the
irritation scorecouldnot be made.The sodiumsoapof decanoicacid also
causeda severeirritation response,followed,in order of magnitude,by the
completesoap solutionused in the above studies,and sodium oleate.
Soapsof the fatty acidsC8:0,Cm0, Cm0 and C•s:0gaveirritation responses
very closeto thoseof the water-treatedcontrols.It was concludedthat the
most irritant componentof the complete soap formulation was sodium
laurate.
Effect of topicalsoaptreatmenton DNA synthesisin rat skin
Twelve rats were dividedinto four groupsof three animals.Group 1,
the controls,weretopicallytreatedwith water,receivingfour treatmentsper
day for a total of threedays;Group 2 weretopicallytreatedwith 85/osoap
solution,receivinga total of four treatmentsduring1 day; Group 3 received
eight soap treatmentsduring 2 days and Group 4 receiveda total of 12
treatmentsduring 3 days.After completionof respectivetopical treatments
the animals of each group were killed and the degreeof irritation of the
treatedareasexaminedhistologically.
478
JOURNALOF THE SOCIETYOF COSMETICCHEMISTS
By meansof an electrokeratotome
portionsof the treatedareasof each
treatedrat were excisedand were approximately200 pm thick, containing
the whole of the epidermis,togetherwith a smallamountof the upperdermis, but no sebaceous
glands.Thesefragmentswere culturedfor 24 h in the
presenceof 2-•4C-thymidine.After culture, total DNA was extractedand
the amount determined, together with total DNA radioactivity. DNA
specificradioactivitieswere calculatedand are shownin Table I.
Table I
Metabolismof DNA in rat epidermisafter topical soaptreatment
DNA specificactivity
Animal
Treatment
1
(dpm [zg-x DNA)
Average
308
2
12
3
151
196
129
4
189
5
6
8
7
154
176
173
193
8
4
9
141
160
174
10
Controls
92
11
12
(12 treatmentswith
distilled water only)
35
83
70
Each culturecontainedtwo fragmentsof epidermis,measuringapproximately 5 x 8 mm, in the presenceof 5.0 •tCi 2-•4C-thymidine.
In the three groupsof soap-treatedrats the specificacitivity of the
epidermalDNA was consistentlyhigher than the water-treatedcontrols,
indicativeof an increasedrate of DNA synthesis.
Also, the stimulatedDNA
activitieswere roughlyproportionalto the extent of soaptreatment,and,
consequently,the degreeof irritation observed.
Effectof topicalsoaptreatmentuponlipid synthesis
in rat skin
Certain lipids play an integralrole in the normal functionof skin (2),
and in order to investigatethe effectof topical soaptreatmenton skin
lipogenesis,
epidermalsections
from a soap-treated
rat wereculturedin the
presenceof 2-x•C-sodiumacetate.The animal in this experimentreceived
EFFECT OF SOAP UPON CERTAIN ASPECTS OF SKIN BIOCHEMISTRY
479
14 soap treatmentsduring 7 consecutivedays,the control rat receivinga
similar number of applicationsof water. After culture, total radioactive
lipidswereextractedand analysedby thin-layerchromatography,the results
of' which are shown in Table H.
Table II
Lipid synthesis
from x4C-sodium
acetatein soap-treated
rat epidermis
% of total lipid radioactivity
Lipid class
Control
Soap treated
Phospholipids
32.7
18.7
SteroIs
35.7
17.5
Free fatty acids
Triglycerides
Sterol/wax esters
Squalene
6.6
15.9
2.7
0.4
9.4
40.0
5.3
2.4
6.0
6.8
Others
Each culturecontainedtwo epidermalfragments,measuringapproximately5 x 8 mm in the presence
of 5.0 [tCi 2-•C-sodiumacetate.Lipids
wereseparatedby thin-layerchromatography
(ref. 1).
After culture,total lipidswereextractedfrom the culturedskinsamples
whenit wasfoundthat 5.35/0of the initial radioactivityhad beenincorporatedinto the lipids of controltissues,and 3.3•o into thoseof soap-treated
skin.It wasseenthat thepercentage
of labelincorporated
intophospholipid
decreased
after soaptreatment(from 32.7•o in the controlto 18.7•o in the
soap-treatedskin),asdid thelabellingof free sterols.This wascounteracted
by a massiveincreasein the proportionof triglyceride(from 15.9•oin the
control skin to 40•o in the soap-treatedskin). This experimentsuggested
thatsoaptreatment
causes
a reduction
in thelabelling
ofphospholipids
ar/d
sterolswhichare membranecomponents
of the skin and an increasein triglyceridesynthesis,
whichare not thoughtto be integralmembranelipids.
BecauseTable H suggested
that after soap treatment the balance of
glycerolipid
metabolism
wasdrasticallyaltered,skinfrom animalsreceiving
identicaltreatmentto thepreviousexperiment
wasculturedin thepresence
of U-•C-glycerol. The radioactivelylabelledlipidswereextractedand the
results are summarized
in Table III.
Conditionsasfor TableII, exceptthat culturescontained5.0 •Ci U-•C glycerolinsteadof •C-sodium acetate.
480
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Table III
Lipid synthesisfrom x4C-glycerolin soap-treatedrat epidermis
% of total lipid radioactivity
Lipid class
Phospholipids
Control
Soap treated
55.8
11.7
Sterois/lower
glycerides 17.9
Free Fatty acids
Triglycerides
Sterol/wax esters
Squalene
Others
7.0
1.1
17.9
1.3
0.4
1.1
73.1
2.2
0.4
5.6
4.5
After culture,it wasfoundthat 0.5• of theinitial radioactivityaddedas
glycerolwasincorporatedinto the lipids of the controlskin,and 1.3•o for
the soap-treated
skin.The mostobviousfeatureobserved
wasa decrease
in
the proportionof phospholipid
labelling(by 44•o), with a smallerdecrease
in free sterol radioactivity,whereasthe proportion of triglycerideradioactivityincreasedfrom 1.9 to 73.1•o. Also, the presenceof radioactivity
in free fatty acids,free and esterifiedsterolsand squaleneindicatedthat a
portionof the x4C-glycerol
substratehad beenmetabolizedby the skin in
cultureto •4C-acetateunits, which were then resynthesized
into complex
lipids.Thesedataclearlycorroborated
the findingsof TableII thatfollowingtopicalsoaptreatmentthepatternof cutaneous
lipogenesis
wasaltered.
Variationin glycerolipidmetabolism
as a functionof the degreeof
irritation response
We found routinelythat groupsof animalsreceivingidenticalnumbers
of soaptreatmentsexhibitedvariedirritation responses.
In order to seeif
the changesin glycerolipidmetabolismdescribedabovevaried with the
irritationresponse
developed,the followingexperimentwasperformed.A
groupof eightrats receivedsoaptreatmentstwice daily for 7 consecutive
days:two controlanimalsweretreatedwith water. At the end of the treatmentperiodthe animalswerekilled and samplesof the treatedareaswere
examinedhistologically.Epidermal sectionswere also preparedwith a
keratotome and cultured in the presenceof •4C-glycerol.After culture,
radioactivelipids were extractedand analysedby thin-layerchromatographyas alreadydescribed.In eachcaseit wasnoticedthat wheneverthe
irritation response
had developedtherewerealsochanges
in therelativepro-
EFFECT OF SOAP UPON CERTAIN ASPECTS OF SKIN BIOCHEMISTRY
481
portionsof radioactivephospholipids
and triglycerides.
Theseproportions
were plotted againstthe respectivecumulativeirritation scoresobtained
[•romhistologicalanalysis,and the resultsare shownin Fig. 3.
I00
-
-
•
•
Phospholipids
5o
_
-
\
I
I
I
I
I
I
I
I
I
I
I
2
3
4
5
6
7
8
9
I0
Scored assessment
,I
II
I
12
of irritation
Figure 3. Relationship between assesseddegree of irritation and the types
of glycerolipidssynthesized.Tissueswere culturedfor 24 h in the presenceof
5.0 IsCi U-x4C-glycerol.Open symbols,control; closedsymbols,treated.
It was seen that as the scoredassessment
of irritancy increased,the
distributionof radioactivitybetweenphospholipid
and triglyceridealtered.
In controlanimalsapproximately705/oof the total lipid radioactivitywas
found in phospholipids,
and only about 105/oin triglyceride.In the most
severelyirritated skin, however(mean scoreof 11.25) thesevalueswere
reversed.Another significantfinding was the wide spreadof scores,indicating large variationsin irritation responsewithin a group of animals
receivingidenticalsoaptreatments.
From the above data it was concludedthat in skin following irritation
from topical soap treatmenttwo distinct biochemicalfeaturesoccurred,
both of which were dependentupon the degreeof irritation. Thesewere:
(a) enhancedDNA synthesis(Table I), and (b), changesin glycerolipid
metabolism(TablesH and III).
Absoluterates of DNA metabolismand lipid synthesisin
soap-irritatedskin
The experimentsabove showingbiochemicalchangesin soap-treated
culturedskin couldnot be compareddirectly,sinceindividualamountsof
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JOURNAL OF THE SOCIETYOF COSMETICCHEMISTS
the cultured tissueswere not accuratelyknown. In order to achievestandardizationof radioactivitydata, total DNA wasmeasuredin all tissuesand
labellingparameterswerethen expressed
relativeto tissueDNA levels.For
thispurposeit wasassumedthat total DNA wasrepresentative
of the total
tissuecell nuclei, and, accordingly,the absoluteamount of tissuetaken.
Total weightsof cultured tissueswere never consideredsincelarge errors
were alwaysincurredin weighingsmallfragmentsof tissue.
A soap-treatmentexperimentwas performedon both rats and guinea-
pigs,to deducewhetherthe effectsdemonstrated
werespecies
specific
for the
rat, or moregeneralphenomenaassociated
with the responseof mammalian
skin to soapirritation. The animalsreceivedfour treatmentswith soapper
day, one of eachspeciesbeingtreatedfor 3 consecutivedays,one eachfor
2 days and one each for 1 day (giving, respectively,12, 8 and 4 soap
treatments).Control animalsof eachspeciesreceived12 topicaltreatments
with water during 3 days. At the end of the treatmentperiod histological
sampleswere prepared for assessment
of irritancy, and 200 gm thick
epidermalsectionswere culturedwith either •4C-thymidineor •C-glycerol.
DNA specificactivitieswere measuredin the thymidine experiment(in
dpm incorporatedgg-XDNA) and lipid specificincorporationscalculated
from the glycerolexperiment(expressed
as dpm incorporatedinto lipid
gg-XDNA). The distributionof radioactivitywithin the variouslipid classes
wasmeasuredby thin-layerchromatography.The resultsare summarized
in Table IV.
It wasseenthat in both rats and guinea-pigsthe biochemicalresponses
to soapirritation were quite similar.The irritancyscoresfor the guinea-pig
werehigherthan the rat, probablyreflectinga greatersusceptibility
to the
methodof treatment,and it was seenthat therewasa correspondingmassive
increasein total lipid radioactivityafter 12 treatments.Analysisof the
labelledlipidsshoweda similarincreasein the proportionof triglycerides
comparedwith phospholipids,
as was seenwith the rat. In both species
the
specificactivity of DNA increasedwith numberof soaptreatmentsand in
the animalstreated 12 times,this beganto decline.In the rat, total lipid
labellingfollowed this trend too. In both speciesstudied,soaptreatment
generallycausedincreasesin specificincorporationinto lipid and also in
the proportionof triglyceridelabelledtoo. When thesetwo valueswere
combined(columnA x columnC), to calculatespecificincorporations
into
triglycerides(expressed
in dpm incorporatedinto triglyceridegg-XDNA)
it was found that there were increasesin this parameter with treatment,
reflectinga greatersynthesis
of thislipid class.Thesemassiveincreases
were
EFFECT
OFSOAP
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OFSKIN
BIOCHEMISTRY
483
484
JOURNALOF THE SOCIETYOF COSMETICCHEMISTS
not seenfor phospholipidspecificincorporations,however(columnA x
columnB).
Phosphorus
metabolismin soaptreatedskin
The previous experiment (Table IV) showed that whereasin soaptreatedskin therewasa generalstimulationof DNA labellingand massive
increases
in triglyceridesynthesis,
the phospholipids
did not follow a clear
trend.This wasfurtherexaminedby usinga•P-orthophosphate
asa substrate
for both DNA and lipids.Duplicatepairsof rats weretreated(four treatmentsdaily) suchthat individualpairsreceivedtotalsof eight,six,four and
two treatmentsrespectively.Control animalsreceivedeight treatmentswith
water. At the end of treatment200 [tm thick epidermalsectionswere cultured with •P-orthophosphatefor 24 h, after which total DNA was extractedand the specificactivitymeasured(in dpmincorporatedpg-• DNA).
Total phospholipidswereextractedand the specificincorporation(in dpm
incorporatedpg-• DNA) measured.Thesedata are shownin Fig. 4.
It was seenthat DNA specificactivity wasmaximal after only two soap
treatments,whenrat skin exhibitsa very low irritationresponse
generally.
However, as the frequencyof soap treatmentincreasedthe level of incorporationinto DNA wasseento fall, and, after eightsoaptreatments,when
the irritationresponse
is generallywell developed,DNA labellingwasvery
_
I
I
2
I
I
I
4
6
8
_
Number of soap treatments
(Each point represents the mean of two values)
Figure 4. Metabolism of a:P-orthophosphateby soap-treatedand control
rat skin cultured in vitro. Tissueswere cultured for 24 h in the presenceof
50.0 •tCi ::P-orthophosphateafter completionof treatmentsin vivo. x--x,
DNA; &--&, phospholipids.
EFFECT OF SOAP UPON CERTAIN ASPECTS OF SKIN BIOCHEMISTRY
485
low indeed.The incorporationof a•P-orthophosphate
into total phospholipidsfolloweda similartrend,namely,a slightstimulationin labellingafter
two treatments,which rapidly declinedas soaptreatmentsincreased,with
virtually no phospholipids
beingsynthesized
in the rats treatedeighttimes.
Theseresultscorresponded
with thoseshownin Table IV for the •4Cthymidineand •4C-glycerolexperimentsexceptthat in the presentexperiment there was a far greater inhibition of phosphorusmetabolismafter
eight soap treatmentscompared with the metabolism of the carbonlabelled substrates.This suggested
that the animalsusedin the a•P-orthophosphateexperimentrespondedmore to the soap treatment than in
previous experiments.Such variations in irritation responsecannot be
explained.
The individual phospholipidtypes labelled during the cultureswere
separatedby thin-layerchromatographyand the controlpattern of activity
wascomparedwith that from soap-treatedskin.There wereno changesin
the overallpatternof phospholipids
labelled,however,the generalproportions of radioactivitybeing: phosphatidylcholine,
60•o; phosphatidylethanolamine,145/o;sphingomyelin,10•o; lysophosphatidylcholine,
105/o
and phosphatidic
acid, 2.5•o. Unidentifiedmaterialaccountedfor 3.55/0.
Effectof topicallyappliedsodiumlaurateon lipid and DNA
metabolism in rat skin
In orderto establishwhetherthe irritant componentof the soap,sodium
laurate, also causedthe biochemicalchangesobservedin soap-irritated
skin, the following experimentwas performed.One rat was treated four
timesduring1 daywith 0.25M sodiumlauratesolution,anotherwastreated
eighttimesduring2 days,and a third, controlanimal,wastreatedeight
timeswith water.Samplesof epidermiswerethen culturedin the presence
of x*C-thymidine
or x•C-glycerol
andsamples
of wholeskinwereexamined
histologically.
Total x'C-labelledlipids were extractedand examinedby thin-layer
chromatography,
the labellingpatternsbeingshownin Table V.
The resultsshowa dramaticreversalin the proportionof labelledphospholipidsand triglycerides.
In the water-controlepidermis,greaterthan
80•o of the glycerolincorporatedinto lipidswaslocatedin phospholipids
(principallyphosphatidylcholine),
whereasin the skinirritatedby sodium
laurate,triglyceridecontained805/0of the radioactivity,and phospholipids
lessthan 20•o.
486
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Table V
Glycerolipidmetabolismin rat epidermisafter topical treatmentwith sodiumlaurate
•o of total radioactivelipids*
Group
Phospholipids
Triglycerides
Control
82.1
Sodium laurate']'(4 treatments)
22.0
17.9
78.0
Sodium
laurate
(8 treatments)
13.9
86.1
*Epidermisculturedwith 5.0 •Ci U-x•C-glycerol
(aftertreatmentwith soap).
'•0.25 M sodium laurate in water, pH of solution = 9.8.
In this experimentthe specificactivitiesof the tissueDNA were also
measured:lipid radioactivitieswere also expressed
relative to total tissue
DNA levels, so that direct comparisonsbetween control and treated
animalsmay be made.Thesedata are shownin Fig. 5.
Certain featuressimilar to the data abovewere seen,namely as in the
experimentof TableIV, as the numberof topical applicationsof soapincreased,the distributionand specificactivity of triglyceridesincreased,
whereasthe distributionand specificactivity of phospholipidsfell. Other
featureswere not so clear, however.TablesI and IV, and Fig. 4 showthat
DNA specificactivity roseas soaptreatmentincreased,and fell sometimes
in severeresponsesof tissue.Here, however,in the skins treated with
Number
Figure 5.
I
4
of treotments
8
Glycerolipid and DNA labelling in rat skin in vitro after topical
treatment in vivo with 0.25 M sodium laurate solution. DNA
cultures were for
24 h in the presenceof 5.0 I•Ci 2-x4C-thymidine.
Glycerolipidcultureswerefor
24 h in the presenceof 5.0 IxCi U-x4C-glycerol.l--l,
Total glycerolipids;
ß --- O, triglycerides;x -- x, DNA; lm&, phospholipids.
EFFECT OF SOAP UPON CERTAIN ASPECTS OF SKIN BIOCHEMISTRY
487
sodiumlaurate, DNA metabolismwas almost completelyinhibited. Similarly, total lipid specificactivitiesfell as treatment with sodium laurate
increased,
whereaswith increasing
soaptreatment(TableIV) lipid specific
activitiesrose.Theseanomaliesare probablyexplainedby referenceto the
histologicalsamplesof the laurate-treatedskins,where there was evidence
of verysevereirritation. In the animaltreatedfour timeswith sodiumlaurate
therewassomelossof the stratumcorneumin someplacesand thickening
and compactingin other places,and containingmuch nuclear debris. In
the Malpighianlayer the stratumgranulosumwas generallyabsent,with
somelossof the stratumspinosum
andthe basallayer.The restof the epidermiswas disruptedand disorganized.In the dermisthere were slight
increases
in leucocytes
and capillarydilatation.In the skin of the rat treated
eighttimeswith sodiumIauratetherewasnecrosis
overthewholelengthof
the sectionsexamined,with completelossof a definedepidermis.Under
conditions
whereviableepidermalcellswereabsentit wouldnot be surprising to find lossof anabolicbiochemical
processes,
suchas DNA synthesis
and membraneformation.This,however,doesnot explainwhy formation
of triglycerideincreasedso markedlyat the sametime.
Thesiteof increased
lipogenesis
in skintreatedtopicallywithsoap
All metabolismstudieswere performedon 200 [tm thick keratotome
sections,includingthe wholeof the epidermis.When sectionsare excisedin
thismannerthe tissueremainingcontainsthe lowerdermis,completewith
sebaceous
glandsandhair follicles.In preliminaryexperiments
thesedermal
sampleswerealsoexcisedfrom soap-treatedskin and culturedwith radioactive precursors.In no cases,however, were alterations in DNA and
glycerolipid
metabolism
observed
aftersoaptreatment,
although
corresponding sectionsof the overlyingepidermisshowedsuchchanges.It was thus
concludedthat the specificbiochemical
changes
alreadydescribed
wereconfinedexclusively
to theepidermis
of thesoaptreatedskin,theregionwhere
the major morphologicalchangeswere seentoo.
Sinceit wasthe Malpighianlayerwhichshowedmostmarkedchanges
as the irritationresponse
developed,
it was attractiveto suggest
that the
observed
biochemical
changes
alsooccurred
in thisregiontoo. Thispossibility wasexaminedin the followingexperiment.
A groupof four ratsreceiveda total of 10 soaptreatments
during3 consecutive
days.Four control
grouprats weresimilarlytreatedwith water. The treatedareaswereexcised
and epidermalsectionscut and pooledwith thosefrom the other animals
488
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
of the treatmentgroup. Epidermalcellswere harvestedfrom the pooled
sectionsby mild trypsinization(1). These were then cultured with •4Cglycerol.Samplesof the epidermalsectionswere also cultured.After cell
and tissuecultures,lipid radioactivitiesand total DNA levelsweremeasured.
Radioactivelipidswereanalysedby thin-layerchromatography,
the results
of which are shown in Table VL
Table VI
Glycerolipidmetabolismin whole epidermisand isolatedepidermalcellsof soap-treated
rat skin*
Group
Tissue
Lipid
specific
incorporation
Lipid labellingpattern
(% of total lipid radioactivity)
(dpmpig-x DNA) PhospholipidsTriglycerides Diglycerides
Control
Whole
1 702
85.6
11.8
2.7
747
76.3
17.4
6.3
1 847
74.9
25.0
0.0
1 029
48.7
48.2
3.1
epidermis
Isolated
cells
Soap
treated,'
Whole
epidermis
Isolated
cells
*Tissuesculturedwith 5.0 •Ci U-x4C-glycerol.
•-8% of soapformulationin water. pH of solution-----9.8.
In the control rats it was seenthat the specificincorporationinto the
lipids of the whole epidermiswas far higher than that of the isolated
epidermalcells,and suggested
that therewereotherregionsof theepidermal
samplewith significantlipogenicactivity too. The distributionsof •4Cglycerolbetweenvariousglycerolipidswere quite similar, however,with
mostradioactivityfound in phospholipids.
After soaptreatment,however,
certainchangeswereseen.The specific
incorporationinto lipidsof thewhole
epidermisincreased
by lessthan 10•o,whereasin theisolatedepidermalcells
thisparameterincreased
by nearly40•o aftersoaptreatment,andindicated
that the stimulatedlipid synthesis
wasconcentrated
in the Malpighiancells
of the epidermis.Similarly,the distributionof radioactivityamongdifferent
glycerolipids
wasalteredin soap-irritatedtissues.
In the cultured whole epidermalsectionsthe proportion of labelled
phospholipids
droppedfrom 85.6 to 74.9•o of the total lipid radioactivity,
with a concomitant rise in triglyceride radioactivity, a feature already
amply illustratedin the experimentsabove.However,in isolatedcellsof
EFFECT OF SOAP UPON CERTAIN ASPECTS OF SKIN BIOCHEMISTRY
489
the epidermisthesechangeswere far more prominent.The proportion of
labelled phospholipidsdroppedfrom 76.3 to 48.7•o, and labelled triglyceriderosefrom 17.4 to 48.2•o. This amplificationof the soap-induced
changesin lipogenesis
within the isolatedMalpighian cells,comparedwith
whole epidermissuggested,
therefore,that the Malpighian layer was the
site of altered metabolicactivity.
DISCUSSION
The effectsof soapson mammalianskin havebeenamply documented
since the first observationsby Emery and Edwards (3). More recently
Bettley (4) reviewedsoap action in terms of its antibacterialaction, skin
cleansingability, effecton waterpermeabilityand effecton the stabilityof
the acidmantleof skin.Wood and Bettley(5) describedchangesin the levels
of liberatedsulphydrylgroupin humankeratin after denaturingwith soap
solutions.Emery and Edwards (3) showedin an homologousseriesof
fatty acidsthat sodiumlaurate exhibitedthe most frequent irritation on
human skin. Choman (6), in an in vitro test, showedthat sodiumlaurate
causedfar greaterswellingof collagenthan other soaps.Thesefindingshave
beencorroboratedby Fig. 2 wheretopicallyappliedsodiumlauratenot only
causedmuchgreaterirritationthan otherconstituentfatty acidsof the soap,
but also inducedthe most marked changesin biochemistry(Fig. 5). From
this we concludethat both the irritation potentialof the soapand the resultantdegreeof biochemicalalterationsinducedare due principallyto its
lauric acid content.
The findingsof this study may be summarizedas follows. When rats
are treated topically with soap solutionsan irritation responsedevelops
whichis generallyproportionalin severityto the numberand frequencyof
treatments.At the microscopiclevel this irritation is seenas an increasein
the proliferativeprocesses
of the epidermis,seenasa thickenedMalpighian
layer, a thickened,compactedand parakeratoticstratumcorneum,containing nucleardebris,and with invasionof leucocytesfrom the underlying
dermis.In association
with thesemorphologicalchangeswe haveobserved
a stimulatedDNA synthesisand an alteredglycerolipidmetabolism.No
distinctsequenceof thesebiochemicalphenomenahas been established,
althoughfrom Fig. 4 and TableIV it appearsthat DNA metabolismincreasesearly in the responseof the tissueto soaptreatment,and is prerequisitefor the entry of more cells of the basal layer into mitosis. A
featureof the DNA specificactivitieswasthat after reachinga maximum
490
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
they declinedwith increasingtreatments.In Fig. 5 in particular,whereskin
had undergoneseveretreatmentwith sodiumIaurate DNA metabolismwas
virtually zero after eight applications,this probablybeingdue to the complete necrosisof the cellsof the germinativelayer of the epidermis,which
would be unable to perform anabolicreactions.The a•'P-orthophosphate
experimentof Fig. 4 and the xaC-glycerol
experimentof TableIV showthat
in rat skin treated with soap, phospholipidradioactivityalso reacheda
maximum,similarto DNA data. This parallelmay indicatean initial hypertrophy in the epidermalcells,which would precedethe hyperplasia.seen
histologically,at later stagesof the irritation response.
In a similarfashionto DNA labelling,specificincorporationsinto phospholipidalsogenerallydecreased
afterprolonged
treatment.This,again,may
have beendueto generalimpairmentof thetissue'sabilityto supplythecytidine cofactors required for phospholipid synthesisafter more severe
irritation.
The most novel finding, however,was the dramatic increasein triglyceridelabellingin soap-irritatedskin.Thislipid is not a classical
membrane
component,
asarephospholipids
andsterols,butisgenerallyagreedto serve
as an energystore.The proportionof radioactivetriglyceridein the total
labelledlipidsroseastheirritationresponse
developed
(Fig. 2), andoccurred
muchlater in the time scaleof the irritation responsethan the stimulation
of DNA synthesis.
Fig. 5 is remarkablein that it showsthe massivetriglyceridesynthesisin sodiumlaurate-treatedskin, in which DNA and
phospholipid
synthesis
had virtuallyceased.In this experiment
the overall
levelof totallipid synthesis
did not diminishgreatlywith soaptreatmentbut
thin-layeranalysesshowedthat the highlevelof phospholipidsynthesis
of
the control was largely replacedby an almost equally high level of triglyceridesynthesis.
There was no obviousstoichiometricrelationshipbetweenthesetwo
lipid types,because
in TableIV, for boththe rat and the guinea-pig
total
lipid specific
incorporation
greatlyexceeded
the controllevels(TableIV).
Fig. 6 showsthe biosynthetic
pathwayfor certainglycerolipids,
and illustratesthat both triglyceridesand phospholipids
sharea commonprecursor,
namely,1,2-diglyceride.
One might arguethat a block in phosphatidylcholinesynthesis
at the CDP-cholineformationstepscouldlead to an accumulationof 1,2-diglyceride,
whichcouldthen be furtheracylatedto triglyceride.
However,this wouldnot explainwhy the total amountof lipid
radioactivityixmole-x of DNA wasconsistently
higherin soap-treated
skin
comparedwith controls.
EFFECT OF SOAP UPON CERTAIN ASPECTS OF SKIN BIOCHEMISTRY
491
Glycerol
I .•--•
AT
P
•'ADP
25-G!ycerophosphate
I •2 AcyI-S-CoA
•'- CoASH
Phosphatidicacid
1,2-Diglyceride
CDP-choline.
/
•
_..Acyl-S-CoA
CMP
'""•• '• CoASH
Phosph•tidylcholine
Triglyceride
Figure 6. The biosynthesisof triglyceridesand phosphatidylcholine.
Some data reported here are consistentwith the findings of others.
Bertalanaffyet al (7) showedthat after injury by incision,the mitotic index
of rat skinwassignificantly
elevated.Thisparameterwasnot measuredhere,
but ratherDNA replication,whichoccursin S-phase,prior to commencement of the mitotic cycle.Similarly,Penneyset al (8) have demonstrated
that in hyperkeratogenicstatesof certain skin diseasesthe labelling of
nucleiwith aH-thymidineincreases
comparedwith normal skin.
In a seriesof papersMezei and co-workerssuggested
that in rabbit skin
after topicalapplicationof certainsyntheticsurfactants
phospholipidlabelling andtotal DNA levelsincreased
(9). Later (10) it wasreportedthat both
phospholipidand RNA levelsincreased,
but DNA levelsremainedconstant,
and they arguedthat increasedamountsof cell membranes(protein and
phospholipid)were being synthesized
to replacethat sloughedoff by the
surfactanteffect.More recently(11), Mezeihasshownthat changes
in RNA
levelsand radioactivitiesat differentstagesof surfactanttreatmentindicated
that cellular disturbancesexist before histologicalsigns of irritation by
surfactantsare manifested,and that DNA changessuggestthat the surfactantshave a generalaction on cell proliferation. In a similar fashionthe
work of Takasu and Aizawa (12) suggests
that skin inflamedby topical
application of toxic chemicalsdemonstratesincreasedmetabolism of
phospholipids.
The findingherethat increasedtriglyceridesynthesis
wasassociated
with
latter stagesof irritation is an extremelyinterestingfacet of the biochemical
response
of injuredepidermiswhichwarrantsfurtherinvestigation.It would
492
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
appearthat the syntheticapparatusfor thislipid is still functioningin tissues
whichno longerincorporatethymidineinto DNA, or glycerolintophospholipids,andthisis situatedin the severely
damagedor degenerate
epidermis,as
shown histologically.It is known that in the liver of rats which have
sustaineddamagefrom dosagewith chloroform, more radioactivefatty
acidsare incorporatedinto triglycerides,
and lessinto phospholipids
than
control(13), and thusthismay be a generalfeatureof degenerating
tissues.
The degreeof morphologicaland biochemicalchangeinducedby soap
treatment essentiallycorrespondedto the number of soap treatments.
Marked inhibitionof DNA and phospholipidsynthesis,
and stimulatedtriglyceridesynthesis,observedin the severelyirritated skin, which had receivedexaggeratednumbersof treatmentscomparedwith normal usage,
thusmay be a feature of damagedtissuesin general,rather than a specific
consequence
of soap.
Our studieson the specificsite of thesebiochemicalalterationsare incomplete,althoughit is probablethat the cells of the basal layer and/or
the differentiatedcells of the stratum spinosumand granulosumare involved,sincetheseregionsof the epidermisare classicallyacceptedasbeing
the most metabolicallyactiveof the epidermis.
(Received:18th January1972)
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O) Emery, B. E. and Edwards,L. D. The pharmacologyof soaps.at.Amer.Pharm.Ass.29 251
(1940).
(4) Bettley,F. R. Someeffectsof soapon the skin. Brit. Med. at.S187 1675(1960).
(5) Wood, D.C. F. and Bettley,F. R. The effectof variousdetergentson humanepidermis.
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(6) Choman, B. R. Determination of the responseof skin to chemicalagentsby an in vitro
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