Human and Animal Stratum Corneum As a Partial Model for the 15 Steps of Percutaneous Absorption: Emphasizing Decontamination, Part I

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© Springer Nature Switzerland AG 2020
H. Zhu, H. I. Maibach (eds.)Skin Decontaminationhttps://doi.org/10.1007/978-3-030-24009-7_1



1. Isolated Human and Animal Stratum Corneum As a Partial Model for the 15 Steps of Percutaneous Absorption: Emphasizing Decontamination, Part I



Xiaoying Hui1   and Howard I. Maibach1


(1)
Department of Dermatology, University of California San Francisco, San Francisco, CA, USA

 



 

Xiaoying Hui

Email: Xiaoying.Hui@UCSF.edu


Keywords

Stratum corneumNatural moisturizing factorChemical warfare agentsRougier method


Introduction: 15 Steps of Percutaneous Absorption


The stratum corneum (SC) of humans and animals holds key insights to the development of an efficient protective barrier against contamination and to devising effective decontamination interventions. SC constitutes the main barrier to the absorption of molecules while participating in the homeostasis of the organism particularly by limiting outward movement of water.


Current understanding no longer considers the SC of the skin barrier as dead “brick and mortar”, but rather a dynamic, living defense system. For instance, the acid mantle, a fine film with a slightly acidic pH on the surface of the skin, plays an integral role in making the skin less permeable to water and polar compounds and is formed in higher amounts with increased activity of enzyme phospholipase A2 [20, 57]. Additionally, acute perturbation of SC by organic solvents, detergents, or tape stripping resulting in lipid removal initiates a sequence of biological responses including epidermal lipid synthesis acceleration to rapidly restore the skin lipid content and barrier function [4].


Upon skin exposure, many chemical agents are locked into the SC within minutes [6]. For examples, many organic solvents including methyl chloroform, perchloroethylene and trichloroethylene can be found in expired air of humans subsequent hand exposures to contaminated water within as little as 6 min, proving rapid systemic absorption [32–34, 54]. The SC acts as a “reservoir” for topically applied molecules, and even rapid washing with water post-dermal exposure frequently fails to remove most chemicals [10]. Importantly, Rougier et al. [40, 41] discovered that SC sampling in vivo in humans and rats via tape stripping 30 minutes post exposure accurately predicts and quantifies chemical penetration for up to 4 days with linear-type correlation. While percutaneous penetration is often considered a simplistic one-step diffusion process, it consists of at least 15 steps [51] related to absorption and excretion kinetics, extraction, vehicle characteristics, wash effects, etc. (Table 1.1). Examination of the chemical partitioning/diffusion of a wide array of compounds with differing physicochemical properties showed minimally differing lag times through the SC. Therefore, different compounds have approximately the same diffusion coefficients with regard to their percutaneous absorption in vivo. Thus, for a given thickness of SC and a specific anatomical site, the penetration flux value of a substance depends mainly on its SC/vehicle partition coefficient [42, 43].


Table 1.1

15 Steps related to the process of percutaneous absorptiona
























































Number

Factors determining percutaneous absorption

1

Release from vehicleb


 Varies with solubility in vehicle, concentration, pH, etc.


2

Kinetics of skin penetrationb


 Influenced by anatomical site, degree of occlusion, intrinsic skin condition, animal age, concentration of dosing solution, surface area dosed, frequency of dosing, post absorption, etc.


3

Excretion kineticsb


4

Tissue disposition


5

Substantivity to skinb


6

Wash effectsb


 Wash resistance


 Wash enhancement


7

Rub effectsb


 Rub resistance


 Rub enhancement


8

Transfer––skin, clothing + inanimate surfaceb


9

Exfoliationb


10

Volatilityb


11

Binding––all layersb


12

Anatomic pathways


13

Lateral spreadb


14

Vascular perfusionb


15

Cutaneous metabolismb



aModified with permission from Wester and Maibach [51]


bMetric developed


The abovementioned observations offer us new insights for improving decontamination techniques. The recommended emergency treatment of many chemical sprays and splashes to the skin consist of rinsing or washing with water and/or soap. However, many topically applied chemicals are not easily removed by water washing. Skin decontamination is the primary required intervention for chemical, biological, and radiological exposures and involves immediate removal of the contaminant via the most effective manner. Such removal techniques include physical removal of the contaminant, solvating or emulsifying the contaminant in a liquid vehicle, transferring the contaminant to another medium through absorption/adsorption of the chemical, chemical alteration of the contaminant, and the use of friction to dislodge the contaminant.


Ideally, a decontaminant effectively and rapidly removes the contaminant of interest, easily removes itself without undesirable residue, does not cause enhancement or wash-in effect, and does not damage the skin. Additionally, it is readily available, affordable, and easily disposed of [7]. In the first part of this review, we explore the many steps involved in percutaneous penetration, the laboratory techniques that provided the background for this knowledge, and the insights advancing our knowledge of chemical exposure risk assessment, exposure prevention and barrier methods, and postexposure decontamination.


Stratum Corneum


Stratum Corneum and Barrier Function


SC is the outermost layer of the five layers of the epidermis and is largely responsible for barrier function . The biological and chemical activity of SC is both intricate and complex. As SC is vital to maintain healthy skin, a comprehensive understanding of its structure and function is essential [9] (Table 1.2).


Table 1.2

Stratum corneum structure and function





































































Name

Location—composition

Function

Corneocyte

Cornified envelope: outer surface of the corneocyte


Keratin filaments;


γ-glutamyl isopeptide bonds

Mechanical barrier (impact and shear resistance)


Resiliency of stratum corneum

 

Cytosol: filaggrin proteolytic product; glycerol

Hydration

 

Cytosol: cis-urocanic acid (histidase activity)

Electromagnetic radiation barrier

 

Cytosol: cytokine activation;


Proteolytic activation of pro IL-1α/β

Initiation of inflammation


Extracellular matrix

Lamellar bilayers: ceramides, cholesterol, nonessential fatty acids, proper ratio

Permeability

 

Corneodesmosomes


Intercelluar DSG1/DSC1 homodimers

Cohesion (integrity)/desquamation

 

Lamellar bilayers: antimicrobial peptides, FFA, sph

Antimicrobial barrier (innate immunity)a

 

Extracellular lacunae: hydrophilic products of corneodesmosomes

Toxic chemical/antigen exclusion

 

Lamellar bilayers: cholesterol, FFA, secreted vit. E, redox gradient

Antioxidant

 

Lamellar bilayers: barrier lipids

Psychosensory interface


Natural moisturizing factors

Within SC

Water holding capacity of SC


pH and calcium gradients

Within SC and all through epidermis

Provides differentiation signals and lamellar granule secretion signals


Specialized enzymes (lipases, glycosidase, proteases)

Within lamellar granules and all through epidermis

Processing and maturation of SC lipids, desquamation


Melanin granules and “dust”

Produced by melanocytes of basal layer, melanin “dust” in SC

UV protection of skin



aThis lipid bilayer is analogous to ‘mortar’ and is essential in maintaining the barrier properties of the skin [28, 36].


The structure of SC is synonymous with that of “brick and mortar.” The bricks are corneocytes, protein complexes of tiny keratin threads in an organized matrix, and keratin is capable of holding large amounts of water between the fibers. SC contains approximately 12–16 layers of corneocytes, each with a mean thickness of 1 micrometer that varies with age, anatomical location, and UV radiation exposure [27].


Corneocytes are surrounded by crystalline lamellar lipid regions. Lamellar bodies are formed in the keratinocytes of the stratum spinosum and stratum granulosum layers, and as keratinocytes mature and ascend, enzymes degrade the lamellar body envelopes, releasing free fatty acids and ceramides that fuse to form a continuous lamellar lipid bilayer. This lipid bilayer is analogous to a “mortar” and is essential in maintaining the barrier properties of the skin [28, 36].


Each corneocyte is surrounded by an insoluble protein envelope primarily composed of two proteins, loricrin and involucrin, that have extensive structural links with one another. Cell envelopes are either “rigid” or “fragile” depending on the envelope’s interaction with the lamellar bilayer [28, 36].


Attached to the cell envelope is a layer of ceramide lipids that repel and trap water. This assembly prevents the absorption of water into the lower layers of the epidermis.


The corneocytes are held together by corneodesmosomes that function as “rivets” and are the primary structures that must be degraded for desquamation [28, 36].


Natural moisturizing factor (NMF), a collection of water-soluble compounds specific to the SC, represents approximately 20–30% of the dry weight of the corneocyte. NMF components absorb water molecules from the atmosphere to maintain SC hydration, but their water solubility allows them to easily leach from the cells upon water contact. Repeated contact with water can dehydrate the skin; however, the lipid layer surrounding the corneocyte helps to form a seal to inhibit NMF loss [37].


The stratum basale consists of a single layer of columnar epidermal stem cells attached to the basal lamina via hemidesmosomes. The stratum spinosum just above is rich with progressively enlarging lamellar bodies with ongoing keratin synthesis and lipogenesis. The stratum granulosum is next with mature lamellar bodies capable of differentiating into corneocytes. Here, the intracellular organelles undergo self-destruction, and the packaged lipid in the lamellar granules (LG) is released to the intercellular space. SC forms the outermost seal with 18–21 cell layers of dead corneocytes and lipids that is 20–40 micrometers thick in humans.


Filaggrin, a key protein in the hydration and formation of the SC barrier , acts as a source of hygroscopic amino acids and their derivatives including NMF [28, 36, 37].


Stratum Corneum Hydration


Normal skin has a consistent water content of 5–15%, independent of variations in environmental humidity. The superficial SC plays a dual barrier role of minimizing transepidermal water loss (TEWL) and preventing the entrance of external molecules. Hydration fluctuations alter SC permeability, and percutaneous absorption may be enhanced with increasing SC water content. Water is an endogenous skin constituent with minimal irritants or toxic exposure effects that are quickly reversible. Skin hydration can be increased easily with an occlusive vehicle or more elegantly with vehicles containing specific NMFs or polymer patch delivery systems [61].


High SC water content is essential in sustaining its flexibility, and its water holding capacity correlates directly with its protein and lipid domains and water-soluble substances [18].


Table 1.3 shows the water holding capacities and lipid content of normal powdered versus delipidized powdered SC (the protein fraction) as determined by the amount of [3H]-water (μg equivalent) per milligram (mg) powdered SC after equilibration [17]. No statistical differences (p > 0.05) were observed in the capacities of normal powdered SC, delipidized powdered SC, or the combination of delipidized powdered SC plus its lipid content. Powdered SC can absorb up to 49% of its dry weight in water, a value consistent in the literature. Middleton [26] found that the amount of water bound to guinea-pig footpad SC intact, in small pieces, or powdered was 40%, 40%, and 43% of dry corneum weight, respectively. Leveque and Rasseneur [21] demonstrated that human SC absorbs up to 50% of its dry weight in water.


Table 1.3

Lipid content and water holding capacity of powdered human stratum corneuma















































































Stratum corneum source

Lipid content


(% dry wt)

Water uptake (μg/mg dry powdered SC)


Normal SC

Delipidized SC


Lipid

Protein

Total

1

2.38

495.85

26.44

452.40

478.84


2

2.21

452.49

39.26

364.96

404.22


3

2.39

585.62

23.09

498.40

521.49


4

2.69

554.27

40.05

492.31

532.36


5

2.08

490.04

49.86

363.30

413.16


6

2.01

381.61

14.82

324.18

339.00


Mean

2.29

493.31

32.26

415.92

448.18


SD

0.25

72.66

12.97

74.50

75.47



aModified with permission from Hui et al. [18]. The powdered SC absorbs up to 49% by weight of dry untreated, which is consistent with literature reports


Powdered SC not in an intact sheet exhibits lower water retention capacity. Powdering ruptures the corneocyte walls, allowing water to extract the hydrophilic NMFs, a process normally requiring a solvent. The results obtained suggest the protein domain of the powdered SC plays an important role in water absorption. Furthermore, depletion of the powdered SC lipid content did not significantly affect water retention (p > 0.05, [17]).


Additionally, if powdered SC is pretreated with water and ethanol or the dosing vehicle contains an ethanol concentration of 40% (v/v) or higher, the water retention capacity of the powdered SC can be reduced significantly (p < 0.05) when compared to untreated powdered SC or when vehicles with lower concentrations of ethanol are used (<40%, [17]). The data suggest that NMF is removed by the ethanol solution during processing.


Regional Variation in Stratum Corneum


Percutaneous absorption in both humans and animals varies depending on the anatomical location to which the chemical is applied. This implies a regional deviation of the barrier structure. Because the effect of any chemical applied to the skin is limited by the amount absorbed, estimation of the human health hazard effects from the environmental contaminant exposure illustrates the need to understand this variation. For example, after skin exposure to pesticide residue, estimation of prognostic factors and treatment doses requires the pesticide mass absorbed, and accuracy of this measure is crucial.


We review regional variation in skin absorption and its application to assess human risk. Risk assessment is achieved by determining skin absorption and multiplying it by the skin area involved. Accuracy improves by factoring in clothing and anatomical regions [50].


Feldmann and Maibach [11] explored the potential for regional variation in percutaneous absorption of hydrocortisone and/or parathion in men in vivo. The anatomical site absorbing the highest proportion of steroid was the scrotum, providing insight to the causation of scrotal cancer in chimney sweeps. Absorption was lowest for the sole of the foot and higher for the head and face.


The percutaneous absorption of multiple pesticides in humans has been shown to vary by anatomical region [24]. Importantly, regional variation was confirmed with parathion and malathion, and the higher absorbing sites including the head and face are more likely exposed to pesticides or other environmental contaminants. Parathion shows absorption variability by site with time, and variation is evident early in exposure [52]. Even though it is completed within minutes after exposure, wash with soap and water is not a perfect decontaminant. Using the combined hydrocortisone and pesticide data, Guy and Maibach [12] constructed penetration indices for five anatomical sites (Table 1.4). Wester et al. [53] examined the percutaneous absorption of paraquat in humans and showed that absorptions for the leg (0.29 ± 0.02%), hand (0.23 ± 0.1%), and forearm (0.29 ± 0.1%) were similar. Interestingly, paraquat showed low absorption and negligible regional variation .


Table 1.4

Penetration indices for five anatomical sites assessed using hydrocortisone skin penetration data and pesticides (malathion and parathion) absorption resultsa



































Site

Penetration index based on


Hydrocortisone data

Pesticide data

Genitals

40

12


Arms

1

1


Legs

0.5

1


Trunk

2.5

3


Head

5

4



aModified with permission from Guy and Maibach [12]


To examine how anatomical site affects chemical penetration and the amount remaining in the SC, Rougier et al. [40] applied benzoic acid (BA) to humans and performed tape stripping after 30 minutes. The total BA penetration varied by anatomical site with the forehead being three times more permeable than the back. Using the BA levels in the SC post 30-minute application and then its penetration over 4 days, additional correlations to anatomical sites were made (r = 0.97, p = 0.001). The forearm is commonly believed to be the best location to test for immediate contact irritation; however, Wertz et al. [49] showed the areas of greatest to least irritant response were the neck, the perioral region, the forehead, and the volar.


The face exhibits its own anatomical variation, and Marrakchi and Maibach [25] used noninvasive skin bioengineering technology to establish a map showing the six biophysical parameters : skin blood flow, transepidermal water loss (TEWL), SC hydration (capacitance), temperature, pH, and sebum content of the skin surface. Testing multiple locations on both young and old human volunteers, both age groups had the greatest skin blood flow on the nose, the chin was the most alkaline area, the neck had the highest capacitance value, and the perioral and nasolabial areas exhibited the highest TEWL. The highest skin temperature was on the neck in the young and the nasolabial area in the old. These values provide a framework for disease and intervention-related considerations.


Regional Stratum Corneum Variation: Application to Human Risk Assessment


Chemical warfare agents (CWAs) are easily produced and of significant threat to military forces and the public. Most well-known CWAs are organophosphorus compounds, a number of commonly used pesticides, including parathion. Investigating the in vitro percutaneous absorption of parathion through dry and sweated uniformed and naked human skin, the percentage absorbed through naked skin (1.78 ± 0.41) was greater than that through dry (0.29 ± 0.17; p = 0.000) and sweated uniformed skin (0.65 ± 0.16; p = 0.000). A difference was also noted between sweated and dry uniformed skin (p = 0.007). Applying the uniformed skin absorption to the skin permeability of the CWA VX for the head, neck, arms, and hands shows a 50% mortality within the first hour of exposure for a sweated soldier wearing a uniform. By 8 hours post-trunk exposure in either a dry or sweated uniform, mortality is predicted. 96 hours after, any uniformed region is exposed individually would be lethal [56].


Another example of human regional variation is exhibited by the bioavailability and body burden after permethrin exposure in the uniformed soldier. Permethrin, a toxic pesticide and repellent of potential disease-carrying insects, is imbedded in military uniform material and provided to soldiers in spray cans for protection. Table 1.5 summarizes the predicted permethrin human bioavailability of uniformed and exposed skin at 1.24 mg/kg body burden [56]. Animal studies generated a no observable effect level (NOEL) of approximately 5 mg/kg, thus estimating a fivefold safety margin.


Table 1.5

Permethrin bioavailability and body burden for uniformed soldiera













































































































Human part

Body surface area (cm2)

Permethrin doseb (μg/cm2)

Percutaneous absorptionc

Body region indexd

Uniforme

Total (μg)

Head

1180

4

0.087

X 4

1

1642.56


Neck

420

4

0.087

X 4

1

584.64


Trunk

5690

125

0.087

X 3

0.29

53834.51


Arms

2280

125

0.087

X 1

0.29

7190.55


Hands

840

4

0.087

X 1

1

292.32


Genitals

180

125

0.087

X 12

0.29

6812.10


Legs

5050

125

0.087

X 1

0.29

15926.44


Feet

1120

4

0.087

X 1

1

389.76


Total (μg)

          

86672.88


Total (mg)

          

86.67


Total body burdenf

          

1.24 mg/kg



aModified with permission from Wester et al. [56]


bUniform = 125 μg/cm2/spray open skin 4 μg/cm2


c8.7% (0.087) permethrin dose absorbed/


dregion body absorption index


e0.29 is 29% absorption from cloth relative to open skin (index)


fTotal body burden (mg/70 kg soldier)


In conclusion, review of current data shows important trends; however, regional variation of skin absorption has not been fully explored. Absorption disparities in all areas of animal skin have not been completely uncovered, but more importantly, many areas of human skin remain unstudied. The absorption differences of fingers, nails, eyelids, perirectal regions, and upper versus lower extremities need to be deciphered. While current data is sufficient to estimate human risk of percutaneous penetration by exposure region, evidence of absorption from clothing must also be explored for real-life applications. With more comprehensive absorption maps for a broader range of chemical moieties, refining our knowledge of skin penetration within dermatopharmacology and dermatotoxicology would be possible.


Rougier Method: A Quick Stripping Method to Predict Systemic Absorption Following Short-Time Dermal Exposure


Stratum corneum (SC) is the main barrier against dehydration and xenobiotics. A relatively noninvasive technique, adhesive tape stripping, can partially or entirely remove this micrometer-thin skin layer for a more thorough examination. Initially employed by Pinkus [31], this methodology has become common practice to remove SC layers, to reveal susceptible skin for irritant or allergen application, to predictably disrupt the water barrier for efficacy evaluation of barrier restoring compounds, or to obtain cells for mycological culture or to investigate SC quality [2]. Additionally, bioengineering methods including TEWL measurement can be employed post tape stripping to measure changes in water barrier properties [48].


Tape stripping may also assess the uptake of topically administered compounds into the SC. Rougier et al. [40–43] used tape stripping 30 minutes post chemical application to quantify the amount present in the SC. The reservoir capacity of the SC was determined at different anatomical regions and correlated with systemic excretions to predict the total molecular penetration over 4 days in both humans and rats in vivo.


Rougier Method to Determine Regional Variation of Chemicals in Human Stratum Corneum Post Exposure


The rate of molecule absorption depends on the application conditions, partially explaining the wide absorption variations reported for the same substances. The factors significantly affecting the absorption rate of a compound include the animal species, vehicle, dose applied, and length of contact. Contradicting explanations for regional permeability differences are often stated in literature reviews.


Rougier et al. [40] used tape stripping in vivo to explore how anatomical site affected the relationship between total chemical penetration and the amount remaining in the SC post application. Four chemicals were studied, and 1000 nmol of 14C-radiolabeled chemical was applied to 1 cm2 of skin at various anatomical sites. For each molecule and site, total absorption was determined by measuring urine chemical excretion after an initial application on one side of the body. Another application to tape-stripped areas on the contralateral side 48 hours later measured the substance present in the SC at the end of application (30 minutes). The calculated skin permeabilities varied substantially, depending on both the physicochemical nature of the molecule and on the anatomical location. The forehead was always twice as permeable as the arm or abdomen (Table 1.6). Importantly, the Rougier method provides a rapid means to determine flux in vivo in humans.


Table 1.6

Percutaneous absorption of benzoic acid and anatomical sitea


























































Anatomical site

Urinary extraction


0–24 hour (1)

Total penetration in 4 days (2)

Amounts in stratum corneum 30 minutes after application

Predicted penetrations in 4 days


(nmol/cm2)

(nmol/cm2)

(nmol/cm2)

(nmol/cm2)

Back

6.41 ± 0.99

8.55 ± 1.32

6.19 ± 1.27

10.80 ± 2.32


Arm

7.06 ± 0.77

9.41 ± 1.02

5.92 ± 0.62

10.31 ± 1.14


Chest

8.78 ± 0.98

11.70 ± 1.31

8.07 ± 1.19

14.25 ± 2.18


Thigh

9.38 ± 1.07

12.50 ± 1.43

6.80 ± 0.72

11.92 ± 1.31


Abdomen

11.44 ± 1.44

15.26 ± 1.93

8.20 ± 1.24

14.48 ± 2.18


Forehead

20.74 ± 2.71

27.65 ± 3.61

11.91 ± 2.28

21.24 ± 4.16



(2) Values calculated from (1), i.e., (2) = (1)/0.75. Urinary excretion data was expressed in nmol.cm−2 application area


The table gives the amounts of benzoic acid found in the urine over 24 hours and the total amounts having penetrated over a period of 4 days (calculated). Total benzoic acid penetration in relation to the anatomical sites treated is: back < arm < chest < thigh < abdomen < forehead


aModified with permission from Rougier et al. [40]

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