Influence of metabolism in skin on dosimetry after topical exposure.

Metabolism of chemicals occurs in skin and therefore should be taken into account when one determines topical exposure dose. Skin metabolism is difficult to measure in vivo because biological specimens may also contain metabolites from other tissues. Metabolism in skin during percutaneous absorption can be studied with viable skin in flow-through diffusion cells. Several compounds metabolized by microsomal enzymes in skin (benzo[a]pyrene and 7-ethoxycoumarin) penetrated human and hairless guinea pig skin predominantly unmetabolized. However, compounds containing a primary amino group (p-aminobenzoic acid, benzocaine, and azo color reduction products) were substrates for acetyltransferase activity in skin and were substantially metabolized during absorption. A physiologically based pharmacokinetic model has been developed with an input equation, allowing modeling after topical exposure. Plasma concentrations in the hairless guinea pig were accurately predicted for the model compound, benzoic acid, from in vitro absorption, metabolism, and other pharmacokinetic parameters.


Introduction
The skin can serve as an important portal of entry of chemicals into the body. When systemic effects of a chemical are estimated after topical exposure, metabolism in skin must also be considered. Numerous studies have reported substantial activity of metabolic enzymes. These enzymes probably have an important role in the activation or elimination of chemicals absorbed into the body. Most enzymes found in other tissues are present in skin. Frequently the activity of these enzymes is less than the activity of the corresponding enzymes in the liver.
Only recently have investigators recognized the importance of skin's metabolic activity during percutaneous absorption. The lack of data is due to the difficulty in measuring skin metabolism in vivo. When body fluids are sampled, the contribution of skin cannot be separated accurately from metabolism in the liver and other organs. The advent of in vitro techniques for measuring absorption and metabolism has enabled investigators to more accurately assess the importance of skin metabolism.
The influence of skin metabolism is determined in part by the area of skin exposure. Skin is the largest organ of the body, with a surface area of 1.8 m2 and a total weight estimated at 2.6 kg (1). Therefore, full body exposure could result in substantial metabolism during absorption, whereas application of the same dose of chemical to a small area of skin might saturate the enzymes, resulting in much less metabolism.
We have developed methods for measuring skin absorption and metabolism with an in vitro flow-through diffusion cell system. Using these procedures, we are investigating the effects of skin metabolism during percutaneous absorption. Results of some of these initial investigations indicate the extent to which skin metabolism can affect topical absorption. A physiologically based pharmacokinetic (PBPK) model has been developed to predict blood and tissue levels after percutaneous absorption of chemicals.

Historical Perspectives Skin Metabolism ofPolycydlic Aromatic Hydrocarbons
Many of the published studies on skin metabolism have dealt with the dermal toxicology of the polycyclic aromatic hydrocarbons (PAHs). These chemicals, which are found frequently in our environment, are potentially toxic to skin and other organs. The effects of these chemicals are relatively easy to study because they cause skin tumors that can be seen on test animals. Because metabolic activation of these compounds is required to produce toxic effects, researchers have focused attention on skin metabolism in toxicological studies.
Aryl hydrocarbon hydroxylase (AHH) is an important enzyme in the metabolism of PAHs. Its activity has been measured in the skin, liver, lung, and other tissues (2). The activity in skin exists primarily in the relatively thin, but metabolically active, epidermal layer (Table 1). AHH activity in liver is more than 10 times that of skin, regardless of whether the activity is expressed as milligrams of tissue protein, grams of tissue, or activity in the whole organ. AHH activity in lung is similar to that found in skin. Induction of enzyme activity by Aroclor occurs extensively in liver, skin, and lung.
The activities of AHH and other major enzymes involved in PAH metabolism in skin were compared with those found in rat liver (Table 2) (3). The specific activities of all five enzymes that were compared were higher in the liver, ranging from 4fold higher activity for NADPHcytochrome c-reductase to 185-fold higher activity for 7-ethoxycoumarin deethylase. Not only microsomal enzymes but also the soluble enzymes (epoxide hydratase and glutathione transferase) had lower activity in the skin.
We compared the activities of glutathione S-epoxide transferase in the liver, skin, and lung, using data from a series of publications by   (Table 3). Using mouse data, we found the liver is more than twice as active as the lung and more than 10 times as active as skin in metabolizing styrene oxide.
Environmental Health Perspectives Rat and mouse liver were equally active in this assay. Glutathione S-epoxide transferase activity in human skin was less than half that found in mouse skin.

Skin Metabolism of Steroids
The metabolism of steroids in skin has been extensively studied. In skin, the endogenous steroids testosterone and estradiol have pharmacological actions on hair growth and the rates of epidermal cell turnover. Anti-inflammatory steroids are probably the most widely used class of drugs for dermatological problems. Researchers are interested in how these drugs are activated, deactivated, and delivered across biological membranes (in the form of prodrugs) during metabolism.
In vitro studies with skin homogenates demonstrated that testosterone is converted to the 5a-hydroxy metabolite exclusively in  (3)(4)(5).
skin, whereas in the liver this reaction results in the formation of both the a and 3 isomers (6). The importance of the skin enzymes in the metabolism of testosterone was further illustrated in the clinical studies of Mauvais-Jarvis et al. (7). Labeled testosterone was administered to four human volunteers by two different routes simultaneously: The 3H isomer was applied topically and the 14C isomer was administered intravenously (iv). The 5a and 5,B metabolites of testosterone were determined in the urine and expressed as the 5ao/5p ratio of androstanediols (primary metabolites) and 17-ketosteroids (secondary metabolites) ( Table 4). The androstanediol data show that the tritiated metabolites from percutaneous absorption resulted in a 3-fold greater ratio of 5a/5p metabolites. These data confirm the earlier in vitro findings of a preference in skin for 5a metabolism of testosterone. Also, metabolism in skin during percutaneous absorption is sufficient to affect the pattern of systemic testosterone metabolites; hence, the different ratios of testosterone metabolites after iv and topical administration.

Skin Absorption and Metabolism Methods
Skin metabolism can be studied in combination with skin absorption measurements by using in vitro flow-through diffusion cells (8) with a physiologic buffer as the receptor fluid (9). Viability of the skin can be maintained in diffusion cells for at least 24 hr, as assessed by maintenance of glucose utilization and metabolic activity and by histologic evaluation. Hepes-buffered Hanks' balanced salt solution (HHBSS) is effective as a tissue culture medium (minimum essential medium) in maintaining glucose utilization, but phosphate-buffered saline solution results in the loss of glucose metabolism within 12 hr (Figure 1). The simplified formulation of a balanced salt solution may be advantageous during analysis of receptor fluid contents after an absorption and metabolism study. For this reason, we routinely used HHBSS as the receptor fluid in our studies.

Absorption and Metabolism Data
The influence of metabolism on percutaneous absorption has been examined in diffusion cells by using model compounds with different metabolic pathways. Metabolism by P450 enzymes in skin during absorption was assessed in studies with four readily absorbed compounds: 7ethoxycoumarin (10), benzo [a] pyrene (10), testosterone (9), and estradiol (9). The metabolism of 7-ethoxycoumarin to 7hydroxycoumarin was small in humans and several species of rodents ( Figure 2). Only 0.1% of the absorbed dose was metabolized in human skin, whereas 1.2% was metabolized with skin from the Sencar mouse.
Benzo[a]pyrene is metabolized to numerous compounds in skin and other organs. Metabolites formed in rodent skin were quantitated by thin-layer chromatography and identified by comparison with lable 4. Metabolism of radioactive testosterone simultaneously administered intravenously (14C)  Values were determined from urine samples collected for a 3-day period. Modified from Mauvais-Jarvis et al. (7).
Environmental Health Perspectives standards. The aqueous fraction probably contains polar conjugates of benzo[a]pyrene that cannot be extracted into ethyl acetate. When total metabolism is expressed in terms of the absorbed dose, we obtained about 4% (hairless guinea pig) and 8% (Sencar mouse) (Figure 3). Testosterone and estradiol were more completely metabolized than the previous two compounds during skin permeation in the rat (9). Approximately 20% of the absorbed estradiol was converted to estrone. Testosterone metabolites totaled more than 30% of the absorbed material and cochromatographed with standards for 5x-androstane-3,17-diol; 4-androstane-3,17-dione; and 5a-dihydrotestosterone.
Additional biotransformation reactions were observed in hairless guinea pig skin with benzoic acid, p-aminobenzoic acid (PABA), and benzocaine ( Figure 4) (11). A small amount of absorbed benzoic acid (approximately 7%) was converted to the glycine conjugate of benzoic acid (hippuric acid) during percutaneous absorption. Acetylation of primary amino groups can occur extensively in skin. PABA and benzocaine were substantially converted to the acetylated metabolite during absorption. Benzocaine was also a substrate for esterase, but because the conversion of benzocaine to PABA was small, it is a poor substrate for this enzyme. Metabolism of benzocaine was similar in human and hairless guinea pig skin.
The absorption and metabolism of benzocaine were examined in the hairless guinea pig after application in a water-soluble gel (12). A radiotracer dose (2 pg/cm ) was compared with a 20-fold larger dose used to approximate concentrations of benzocaine applied for topical anesthesia. At the low dose, 80% of absorbed benzocaine was metabolized to N-acetylbenzocaine. At the high dose, saturation of skin acetyltransferase limited N-acetylbenzocaine formation, but 47% of the absorbed benzocaine was acetylated.
In preliminary studies, azo colors were observed to undergo azo reduction during Volume 102, Supplement 11, December 1994 I 3 2 6 skin absorption (13). Several azo compounds that are simple derivatives of aniline were synthesized and found to be metabolized to aniline during azo reduction. The aniline was further biotransformed to other compounds with the N-acetylated derivative acetanilide as the most predominate.

PBPK Model-Dennal Input
A PBPK model is being developed for predicting in vivo blood and tissue concentrations of a chemical after topical exposure (14). In vitro skin absorption and metabolism of benzoic acid were measured with viable hairless guinea pig skin in a diffusion cell and described mathematically for input into the PBPK model. Tissue-plasma parti-tion coefficients and Michaelis-Menton metabolic constants (Km, V ax) were determined by in vitro techniques. Predicted blood levels after three different topical doses of benzoic acid agreed closely with in vivo measurements. A PBPK model may be useful for determining the influence of metabolism at the absorption site on the systemic effects of a toxicant.