Methods development for the in vitro assessment of cutaneous drug

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Methods development for the in vitro assessment of cutaneous drug metabolism

Thohan, Sanjeev, M.S.

The University of Arizona, 1991

U-M-I 300N.ZeebRd. Ann Arbor, MI 48106

Methods Development for the In Vitro Assessment of Cutaneous Drug Metabolism

by

Sanjeev Thohan

Sanjeev Thohan 1991

A Thesis Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE WITH A MAJOR IN TOXICOLOGY In the Graduate College The UNIVERSITY OF ARIZONA

199 1 2 STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interest of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED:,

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

Date Professor and Head of Pharmacology afid Toxicology 3

Dedication

I dedicate this thesis to my parents, Tarsem Lall and Rama Thohan. It is with the constant love and support of my entire family that this research effort was brought to its fruition.

I think Kabir said it very well in couplet VI.8:

What a stone you have become, ever absorbed in intellectual pursuits not a drop of love has touched you.

O Kabir, remember: Without love it is all worthless and dreary. 4 Acknowledgements

I owe a great debt of gratitude to Dr. Inderjit Singh for allowing me, as an undergraduate, to participate in his research efforts at Johns Hopkins. I also am indebted to Dr. Julie Eiseman and Dr. Merrill Egorin at the University of Maryland Cancer Center (UMCC). It was through your guidance and support that I was able to make the right decisions to be where I am. As for the rest of the UMCC staff, thank you all for providing a very productive and pleasant work environment. Thanks for the many enjoyable lunches at the "Market" whenever I came home and keeping in touch with me.

I would like to take this chance to thank my major advisor Dr. I. Glenn Sipes for not only providing me with a positive environment in which to conduct scientific research, but for his critical evaluations, guidance and patience over the duration of this work. I also like thank Dr. Dean E. Carter for his assistance in the direction of this thesis. Dean, your door was always open to me and / will be indebted to you for the many lessons I learned in my discussions with you. Dr. Klaus Brendel, I thank you for the many insightful discussions and assistance with the completion of this work.

Dr. John Barr thanks for your friendship, guidance, tutelage and critical insight into my development as a scientist. John, you have been a friend in every sense of the word. I miss those discussions on philosophy and some of those obtuse questions that came up on sleepless 'Liver Nights'. You always saw the good in all situations and helped me to realize more than I would have done on my own. Thank you my friend.

To my 'partners in crime': Kevin Divine, Timothy Harlan, and Jonathan Patrick Rigsby Francis Monteleone: "You were right, I don't know how I would have made it without you guys." Thanks for always being there and those incredible "Power Science Lunches." Sanjeev Sharma thanks for many an '!impromptu night." Also, Sanjeev you were there to help me to see the good in situations that I thought were bad. Latika, Michele, Nilima and Suresh, thank you for your support from 'home'. Suketu Desai, thanks for your friendship and unique viewpoint. Dr. Shana Azri, thanks for being there on the other end of the phone. Ms. Jeanette O'Hare, thank you for invaluable assistance with art and visual aids throughout this work. Ms. Rita Pedroza, thanks for not only your help and friendship but also for your unique understanding of life. Finalfy, 1 would like to thank Tariok (Kandi) Kandola and Harjinder Gill for their brotherly love and for providing me with a family atmosphere here in the desert. I consider myself to very fortunate to have had advisors and friends such as you in my time here. TABLE OF CONTENTS

List of Figures 10

List of Tables 12

Abstract 14

Introduction .15

1.0 Drag Metabolism - General Introduction 16 1.1 The Mixed Function Oxidase (MFO) System 19 1.1.1 Brief History 19 1.1.2 Components of the MFO system 20 1.1.2.1 Cytochrome P-450 (P-450) "... 20 1.1.2.2 Multiplicity of P-450 20 1.1.2.3 NADPH Cytochrome P-450 reductase (reductase) — 22 1.1.2.4 Cytochrome b5 22 1.1.2.5 Lipid 23 1.1.3 Organization of the MFO System 24 1.1.4 Generalized Reaction Scheme for Microsomal Oxidations T...... 27 1.1.5 Generalized Scheme for Microsomal Reductive - Reactions 30 1.1.6 Rate Limiting Step 31 1.1.7 Induction 33 1.1.7.1 Induction of Rat liver P-450 Isozymes 34 1.2 Conjugation Reactions- Phase n . 38 1.2.1 Cassification of Phase n Reactions 38 1.2.1.1 Glucuronidation 41 1.2.1.2 43

2.0 Skin - General Introduction . 45 2.1 Anatomy 45 2.1.1 Epidermis 46 2.1.2 Dermis 46 2.1.3 Subcutaneous Tissue 47 2.1.4 Cutaneous Appendages 47 6 2.2 Metabolism in Skin 51 2.2.1 Carbohydrate Metabolism 51 2.2.2 Glucose Metabolism 53 _ 2.2.3 53 2.2.4 Protein Metabolism ;.. 54 2.2.5 - Hormone Metabolism 55 2.2.6 Cutaneous Phase I Metabolism 57 2.2.7 Cutaneous Phase II Metabolism 60 2.2.7.1 Glucuronidation 60 2.2.7.2 Sulfation 60 2.2.7.3 Methylation 61

3.0 Purpose 62

4.0 Model Substrates 64 4.1 Alkoxycoumarins 64 4.2 Paranitrophenol Conjugates 65

Materials and Methods 66

1.0 Animals 67

2.0 Human Skin 68

3.0 Chemicals 68

4.0 Animal Pretreatment 68

5.0 Subcellular Fractionation 69 5.1 Skin Microsomal Preparation 70 5.2 Liver Microsomal Preparation 73

6.0 Cutaneous Culture Techniques 73 6.1 Short Term Culture: "Swirly Culture" 73 6.2 Long Term Culture: LTC 74 7 7.0 Assays 75 7.1 Protein Determination 75 7.2 Cytochrome P-450 Content 75 7.3 Cytochrome c Reductase Determination 76 7.3 O-Dealkylase Activity Determination 76 7.3.1 Metabolite Analysis 77 7.3.1.1 Microsomal Incubations 77 7.4 Paranitrophenol (PNP) Deconjugation Assay 80

8.0 Statistical Analysis 80

Results 81

1.0 Protein Determinations 82 1.1 Species Differences in Microsomal Protein Yield 85

2.0 Species Comparison - Microsomal Studies 87 2.1 Cytochrome P-450 Determination 87 2.2 NADPH Cytochrome c Reductase Determination 89 2.3 Alkoxycoumarin Metabolism 95 2.3.1 7-EC Metabolism 99 2.3.2 7-MC Metabolism 100

3.0 The Effects of Aroclor Pretreatment on Rat Microsomal Activities 102 3.1 Microsomal Protein Yield 102 3.2 Cytochrome P-450 Determination 103 3.3 NADPH Cytochrome c Reductase Determination 106 3.4 Alkoxycoumarin Metabolism 109 3.4.1 7-EC Metabolism Ill 3.4.2 7-MC Metabolism Ill 8 4.0 Culture of Rat Skin Explants 113 4.1 Xenobiotic Metabolism by Rat Skin Explants in Swirly Culture ... 115 4.1.1 Phase 17-EC Metabolism: A Comparison of Squares and Strips of Rat Skin 115 4.1.2 Phase I and Phase II Integrated Metabolism by Rat Skin. 116 4.1.3 Phase II conjugation of 7-HC by Rat Skin 119 4.1.4 Bacterial Contribution to the Skin Metabolism of 7-EC .. 121 4.1.5 The Deconjugation of Para-Nitrophenol (PNP) Derivatives by Rat Skin 121 4.2 Xenobiotic Metabolism by Rat Skin Explants in Long term Culture 123 4.2.1 Phase I and Phase II Metabolism of Alkoxycoumarins: A Comparison of Supported and Submerged Incubations 124 4.2.2 Phase II conjugation of 7-HC by rat skin 125

Discussion 127

Protein Determinations 130 Cytochrome P-450 133 Cytochrome c Reductase 136 Alkoxycoumarin Metabolism 139 Rat Skin Explant Metabolism: STC 144 Rat Skin Explant Metabolism: LTC 147

Appendix 149

Literature Cited 150 UST OF FIGURES

Figure 1: A Schematic of the interactions of the two phases of drug metabolism 18

Figure 2: The association of Cytochrome P-450 and the reductase in a membrane ....; 26

Figure 3: A Schematic representation of the metabolism of substrates by Cytochrome P-450 28

Figure 4: A schematic of epidermis and full thickness skin SO

Figure 5: Microsomal Preparations 72

Figure 6: Swirly Culture Incubation 73

Figure 7: Long Term Culture Incubation with agar support . 74

Figure 8: O-Dealkylase Assay 78

Figure 9: A typical dithionite difference spectra of rat liver microsomes .... 87

Figure 10: Protein dependence of reductase activity in skin (A) and liver (B) - microsomal fractions from male F-344 rats 90

Figure 11: Protein dependence of reductase activity in skin (A) and liver (B) microsomal fractions from male New Zealand rabbits. 91

Figure 12: Protein dependence of reductase activity in skin (A) and liver (B) microsomal fractions from male B6C3F1 mice. 92

Figure 13: Protein dependence of reductase activity in skin (A) and liver (B) microsomal fractions from Humans . 93

Figure 14: The metabolism of 7-MC and 7-EC (100 mM) by skin (A) and liver (B) microsomes from male F-344 rat 96

Figure IS: The metabolism of 7-MC and 7-EC (100 mM) by skin (A) and liver (B)microsomes from male New Zealand rabbits 97 10 Figure 16: The metabolism of 7-MC and 7-EC (100 /iM) by skin (A) and liver (B) microsomes from male B6C3F1 mice 98

Figure 17: The metabolism of 7-MC and 7-EC (100 /iM) by liver microsomes from humans 99

Figure 18: The effects of Aroclor pretreatment on the protein dependence of NADPH cytochrome c reductase activity in rat skin (A) and liver (B) microsomes 107

Figure 19: The effects of Aroclor on the metabolism of 7-EC (100 #iM) by skin (A) and liver (B) microsomes from F-344 rats 110

Figure 20: The effects surface area of exposure on the metabolism of 7-EC (100 mM) by untreated rat skin explants from F-344 rats 116

Figure 21: The phase I metabolism of 7-EC (100 /iM) by thin strips of rat skin in Short Term Culture 117

Figure 22: The integrated phase I and phase II metabolism of 7-EC (100 mM) by thin strips of rat skin in Short Term Culture 118

Figure 23: The phase n metabolism of 7-HC (100 /iM) by thin strips of rat skin in Short Term Culture 120

Figure 24: The deconjugation of PNP derivatives by thin strips of rat skin in Short Term Culture 123

Figure 25: The phase I and phase II integrated metabolism of 7-EC (100 jucM) by rat skin in Long Term Culture 125

Figure 26: The phase n metabolism of 7-HC (100 juM) by rat skin in Long Term Culture 126 11 UST OF TABLES

Table 1 Oxidative Reactions Catalyzed by Cytochrome P-450 29

Table 2 Reductive Reactions Catalyzed by Cytochrome P-450 31

Table 3 Immunological estimation of the various forms of rat liver Cytochrome P-450 after treatment with various inducing agents 37

Table 4 Survey of Conjugation Reactions 40

Table 5 Glucuronidation reactions and generalized structures 42

Table 6 activities and acceptor molecules 44

Table 7 Carbohydrate Metabolism in the Skin 52

Table 8 Cutaneous Endogenous and Xenobiotic Phase I Metabolism 58

Table 9 Lowiy Protein Assay 82

Table 10 Bio-Rad Protein Assay 83

Table 11 Microsomal Protein Yields in Rat, Rabbit, Mouse and Human 85

Table 12 Species Differences in Skin and Liver Cytochrome P-450 content 88 12 Table 13 Species Differences in Skin and Liver Reductase Activity 94

Table 14 Species Differences in Alkoxycoumarin Metabolism by Skin and Liver 101

Table 15 Microsomal Protein Yields in Control and Aroclor Pretreated F-344 rats .. 102

Table 16 The Effects of Aroclor Pretreatment on Rat Microsomal P-450 Contents. .. 105

Table 17 The Effects of Aroclor Pretreatment on Skin and Liver NADPH Cytochrome c Reductase Activity 108

Table 18 Aroclor Effects on Alkoxycoumarin Metabolism by Skin and Liver Microsomes from F-344 rats 112 13 Abstract

The major objective of this work was development of procedures for the quantification of cutaneous microsomal cytochrome P-450 mediated metabolism using

model substrates. The Fischer-344 rat was the species of choice for this developmental work. The versatility of this method was demonstrated through its application to a variety of species including man. An extension of the rat microsomal work entailed the use of inducing agents, specifically, the Aroclor series of polychlorinated biphenyls. The effects of these inducing agents were demonstrated and characterized in skin and liver with respect to microsomal marker and metabolism. In addition to the microsomal studies, the use of rat skin explants in culture, provided a simple system for the characterization of phase I and phase II coupled metabolism of the model substrates. 14

Introduction 15 1.0 Drug Metabolism - General Introduction

In the course of one's interactions with the environment there is a constant exposure to a myriad of foreign compounds (xenobiotics) in the form of environmental and therapeutic agents. The fate of these xenobiotics is determined largely by the enzymatic metabolism (biotransformation) to pharmacologically inactive or active metabolites (Williams, 1981).

Prolonged exposure to lipophilic xenobiotics may result in their accumulation due to preferential partitioning into cellular membranes. This is of particular importance since excretory fluids are aqueous in nature and therefore lipophilic compounds may be reabsorbed and retained in the system. If these compounds were not effectively removed from the system, they would accumulate and eventually kill the organism (Conney, 1986). The development of biotransformation enzymes in mammalian systems provides the necessary machinery to avert the routine accumulation of lipophilic compounds through interactions with the environment

(Boxenbaum, 1983).

Historically, biotransformation was first demonstrated by Keller (1842) who reported the conversion of ingested benzoic acid to urinary hippuric acid. Twenty five years later Schultzen and Grabe (1867) showed that chlorobenzoic acid derivatives yielded the corresponding hippuric acids in the urine of patients. Towards the end of the 19th century the role of biotransformation as a protective function was 16 presented (Williams 1947, 1959). However, it is now realized that there are a

considerable number of instances where the metabolism of xenobiotics produces a

more toxic agent (bioactivation). These sequence of events may result in tissue

necrosis, carcinogenesis or teratogenesis (Conney, 1986). The biotransformation

enzymes and the reactions that they catalyze have been conveniently divided into two

categories, Phase I and Phase II (Williams, 1959). In essence, the phase I reactions

may introduce or expose a functional group in a compound (e.g. -OH, -SH, -NH2,

and -COOH). The phase II enzymes conjugate these sites with hydrophilic moieties

(e.g. glucuronic acids, sulfates, amino acids, etc.) The overall purpose and effect of

the sequence of biotransformation is the conversion of lipophilic xenobiotics to compounds which are more amenable to excretion from the system. A summary of

the interactions between phase I and phase II may be found in figure 1 (Sipes and

Gandolfi, 1986).

The enzymes involved in biotransformation, both phase I and phase II, may be found in the highest diversity and concentration in the liver. Functionally, this is not surprising since the liver receives most of the chemicals absorbed from the gastrointestinal tract and the splanchnic blood flow. Consequently most of the xenobiotic biotransformation investigations have focused on the liver. Extrahepatic tissues (lung, kidney, intestine, skin and gonads) also participate in the biotransformation of xenobiotics, but they are limited in their activity and substrate specificity (Sipes and Gandolfi, 1986). 17

PHASE i PHASE II

Expose or Add Functional Groups Primary Biosynthetic Secondary XENOBIOTIC Oxidation Conjugation Product Reduction Foreign Hydrolysis Compounds

EXCRETION

LIPOPHILIC •HYDROPHILIC (lonizable)

Figure 1: A Schematic of the interactions of the two phases of drug metabolism

(Sipes and Gandolfi, 1986) 18 1.1 The Mixed Function Oxidase (MFO) System

1.1.1 Brief Histoiy

The most important with regards to phase I metabolism is the

cytochrome P-450 system and its associated reductase. The first description of their

involvement in xenobiotic metabolism was presented by Mueller and Miller (1949).

They reported the reductive cleavage of the azo linkage of the carcinogenic dye 4-

dimethylamino-azobenzene by a microsomal fraction isolated from rat liver

homogenates. In 1953, Mueller and Miller reported the oxidative N-demethylation

of 4-dimethylamino-azobenzene by hepatic microsomes [microsomes are the vesicles

formed by the endoplasmic reticulum upon cellular disruption and homogenation

procedures. These vesicles may be isolated by differential centrifugation and exhibit

properties of the intact membranes (Testa and Jenner, 1976)].

The requirements of molecular oxygen and reducing equivalents (NADPH and

NADH) for optimal activity of microsomal reactions were determined by Mueller

and Miller (1953). The enzymes effecting the transfer of one molecule of oxygen to a substrate yielding hydroxylated products was termed "mixed function oxidase" by

Mason (1957) and "monooxygenase" by Hayashi (1964). This molecular sequence was supported by data obtained by Posner (1961). Posner utilized radiolabeled oxygen in the hydroxylation of acetanilide by rabbit liver microsomes. The presence of the

radiolabel in the products provided the evidence that molecular oxygen rather than 19 cellular water was the oxygen source in the reaction. The generalized reaction is presented below to aid clarification.

02 + RH + NADPH > ROH + NADP+ + HzO

1.1.2 Components of the MFO system

1.1.2.1 Cytochrome P-450 (P-450)

P-450 was first described by Klingenberg (1958), Garfinkel (1958) and Omura and Sato (1962). It receives its name from the spectral characteristics, i.e. the reduction of the heme iron (Fe+2) and combination with carbon monoxide yields a product which absorbs light maximally at 450 nm (Omura and Sato, 1964a). However, treatment of the P-450 with various solubilizing agents will result in the formation of cytochrome P-420 (P-420) which is the catalytically inactive form of P-450 (Omura and Sato 1964a, 1964b Imai and Sato, 1967).

1.122 Multiplicity of P-450

The reactions mediated by cytochrome P-450 were thought of as being catalyzed by one multifunctional enzyme possessing an extremely broad substrate specificity. However, evidence from studies of the rates of different oxidations known to be effected by this system are suggestive of the multiple isozyme nature of this system. Axelrod (1956) first postulated the existence of more than one drug metabolizing system based upon a series of experiments involving the species 20 comparison of the N-demethylation of several narcotic compounds. Soon thereafter,

Conney, et.al, (1959) demonstrated that there were not only different enzymes in different species, but that there were different enzymes in the liver of one animal

which were capable of catalyzing the same reaction. It was not until 1965 that it P-

450 was clearly shown to be the terminal oxidase in the hepatic drug metabolizing system (Cooper et.al, 1965; Omura and Sato, 1965). This led to great efforts to better

understand the role of P450 in drug metabolism and investigate mechanistically the nature of this enzyme.

The broad substrate specificity of this enzyme system is attributable to the many known isozymic forms and a considerable overlap of the specificities of the substrates handled by these isozymes (Guengerich, 1987). These isozymes may be regulated by endogenous effectors, developmental factors, tissue specific expression and chemical modifiers - xenobiotics (Adesnick and Atchison, 1986). The total number of isozymes in not yet known, but purification and metabolism studies using reconstituted systems has identified no fewer than twenty isoforms in rabbits, rats and mice (Guengerich, 1987). From recent investigations, it was found that the number of P-450 genes far exceeds the distinct forms of P-450 that have been isolated to date

(Adesnick and Atchison, 1986). This finding supports the proposition by Nebert

(1979) that the multiplicity of the P-450 system may resemble that of the immunoglobulin, i.e there may be millions of possible forms. 21 11-2-3 NADPH Cytochrome P-450 reductase

Horecker (1950) described the purification of the reductase from pig liver.It was termed NADPH cytochrome c reductase due to its ability to reduce cytochrome c in vitro. It was not until 1962 that the microsomal localization of this enzyme was reported (Williams and Kamin, 1962, and Phillips and Langdon, 1962). The role of this flavoprotein in vivo was not elucidated until microsomal metabolism inhibition studies were performed with an antibody against the trypsin solubilized reductase

(Kuriyama et. al, 1969 and Omura, 1969). This enzyme is present in amounts one- tenth to one-thirtieth of the P-450 concentration (Sipes and Gaiidolfi, 1986)

1.1.2.4 Cytochrome fr.

Cytochrome b5 is an intermediate electron carrier in the microsomal NADH dependant stearyl-Co-A desaturase system (Oshino et.aL, 1966; Shimakatae/.ai, 1972 and Strittmatter et.aL, 1974). Hildebrant and Estabrook (1971) concluded that cytochrome b5 is a part of the MFO system to explain the earlier observations of

Conney et.aL, (1953) that the addition of NADH had a synergistic effect on the reduction of P-450. In reconstitution studies, the addition of cytochrome b5 was found to enhance the metabolism of certain substrates (Imai and Sato, 1977; Sugiyama et. al, 1982; Bosterling and Trudell, 1982). In addition to these studies, cytochrome bs and cytochrome b5 reductase produced a stimulation of the hydroxylation activity

(Taniguchi et.aL, 1984), 22 1.12.SMM

Lu and Coon (1968) found that microsomes were separable into three distinct

fractions of which one was a heat stable, organic extractable lipid factor. They also

determined that the lipid factor was needed in conjunction with P-450 and reductase

for the maximal activity of the drug metabolizing system. Phosphatidyl choline was

determined to be the active component of the lipid factor necessary for the electron

transfer between NADPH and P-450 (Strobel et.ai, 1980).

Singer (1974) proposed that membranes are composed of a lipid bilayer into

which proteins are imbedded. These proteins and lipids are laterally mobile in the

membrane, but are constrained by the fluidity of the membrane. One of the

important properties of bilayer membranes is that of phase separation equilibria

(Shimshick and Mc Connel, 1973). Different lipid components may not be randomly

distributed in the membrane, but could occur in areas that are separate from the

bulk phase and exist in a different physical state. This physical state determines the

arrangement of the enzymes in the matrix as well as their conformational status.

Receptor binding of a substance may initiate a phase separation, e.g. calcium (Oshini

and Ito, 1973) and (Oldfield and Chapman, 1972) have been shown to induce phase changes. Structural changes in the bulk of the membrane or in the lipoprotein composition of the microenvironment of the P-450 reductase complex

may be induced by substrate binding and alter the phase equilibria in either direction

(Yong, et. al, 1970). 23 1.1.3 Organization of the MFO System

The characteristics of the monooxygenase system found in microsomes is determined partially by the interactions between the lipids and the proteins in the membrane. Microsomal membranes contain a mixture of phospholipids with a large portion of the fatty acyl residues (Dallner and Ernster, 1968). Cholesterol, which accounts for 8 % of the lipid content, is an important factor in determining the packing of the membrane and thus related to its fluidity (Gander and Mannering,

1982).

The spatial arrangement of the monooxygenase system as predicted by De

Pierre and Ernster (1980) would suggest that the reductase is anchored by a small segment of the protein while the bulk of the protein is located on the cytoplasmic side of the endoplasmic reticulum. The studies of Thomas, et.al, (1977) also suggested this spatial arrangement. P-450 and the phase n enzymes appear to be more deeply immersed in the membrane. Figure 2 presents a schematic of the association of P-450, its reductase and the membrane matrix. (Nebert, 1981 in Sipes and Gandolfi, 1986).

The topography of the phase I enzymes involved in biotransformation makes it likely that the organization and mobility of these enzymes is related to their activity (Stier, 1976). The constituent molecules (lipid and protein) known to diffuse laterally may also form aggregate patches to form concentrated sub regions of enzyme (Demel, et.aL, 1973). This aggregative process would serve to favor 24 circumstances that would result in an increased substrate P-450 interaction (Adam and Delbruck, 1968). In addition, the immobilization on the membrane of two enzymes catalyzing a sequence of reactions would enhance product formation. 25

Figure 2: The association of Cytochrome P-450 and the reductase in a membrane matrix. (Sipes and Gandolfi, 1986). 26 1.1.4 Generalized Reaction Scheme for Microsomal Oxidations

In the many substrates (endogenous and exogenous) that are metabolized by the MFO enzyme system there appears to be a common sequence of events (See

Figure 3 for schematic representation):

1. Association of the substrate (RH) to oxidized P-450.

2. Reduction of the reductase by NADPH.

3. Reduction of the P-450 substrate complex by the reductase.

4. Addition of oxygen to the reduced P-450 substrate complex.

5. Reduction of the oxygenated reduced P-450 substrate complex by another electron, probably from the reduced reductase.

6. Decomposition of the oxygenated reduced P-450 substrate complex to yield a hydroxylated product, oxidized P-450 and water.

This complex system of reactions may be conceptually simplified and characterized as different types of hydroxylation reactions. However, simplification underrates the substrate diversity of these oxidative reactions that have been studied in microsomes (Brodie, et.al, 1958, Gillette, 1963, 1966). These reactions include: aliphatic and aromatic hydroxylations; epoxidation; N-, O-dealkylations; deamination,

N-, S- oxidation; desulfiiration; denitrification; denitrosation and dehalogenation.

Examples of these reactions, substrates and products may be found in Table 1 (Yang and Lu, 1987). 27

P-450 RH RH (xenobiotic) e- NADPH—^NADPH- - Cytochrome P-450 Reductase

RH

RH HjO KOH e e~ NAPH " • NADH- Cytochrome bs--'( Cytochrome P-450 2H b5 Reductase RH

Figure 3: A Schematic representation of the metabolism of substrates by Cytochrome P-450. 28 Table 1 Oxidative Reactions Catalyzed by Cytochrome P-450

Reaction Example of Substrate(s)

Aliphatic Hydroxylatwn Fatty acids, n-alkanes, cyclohexane, hexobarbital, and testosterone.

Aromatic oxidation Halogenated benzenes, biphenyls, and pofycyclic aromatic hydrocarbons.

Alkene epoxidation Aflatoxin Benzo(a)-pyrene- 7,8- dihydrodiol; Aldrin.

N-Dealkylation Benzp he famine, Aminopyrine and Ethylmorphine.

Oxidative deamination Amphetamine.

O-Dealkylation 7- ethoxycoum arin and Phenacetin.

N-Oxidation 2-Acetylaminofluorene, Phenacetin and Phenteramine.

Oxidative desulfuration Parathion and Carbon Sulfide.

Sulfoxidation Chlorpromazine

Oxidative dehalogenation Dibromoethane and Chloroform

Oxidative denazification 2-Nitropropane

Oxidative denitrosation N-dinitrosodimethylamine

Table X Adapted from Yang and Lu (1987). 29 1.1.5 Generalized Scheme for Microsomal Reductive Reactions

The implied function of drug metabolizing systems as oxidative reactions has been perpetuated by nomenclature such as "mixed function oxidase" and

"monooxygenase." However, the P-450 system is also responsible for catalyzing reductive biotransformation. Gillette, et.aL (1968) proposed an electron transfer scheme which would explain reductive and oxidative metabolism of compounds by the P-450 system.

Studies from azo dye reductions resulted in the proposal of two mechanisms whereby the reductive metabolism would proceed: (1) a carbon monoxide (CO) and solubilization sensitive pathway requiring P-450. (2) a CO insensitive pathway which required NADPH cytochrome P-450 reductase and was insensitive to detergent solubilization (Gillette et.aL, 1968).

Reductive biotransformation proceeds readily in environments of low oxygen tension. During the sequence of reactions, electrons transferred from the P-450 system are used to reduce the substrate P-450 complex and not molecular oxygen as discussed earlier. In the presence of oxygen, reductive metabolism does not proceed due to competition for electrons by molecular oxygen. These reactions and examples of the substrates and products are presented in Table 2 (Yang and Lu, 1987). 30 Table 2 Reductive Reactions Catalyzed by Cytochrome P-450

Type of Reaction Example of Substrate(s)

Nitro Reduction p-Nitrobenzoic Acid and Nitrobenzene.

Azo Reduction Prontosil

Tertiary Amine N-oxide Imipramine N-oxide reduction

Arene Oxide reduction Benzo(a)-pyrene 4,5-oxide

Reductive Dehalogenation Carbon tetrachloride

Chromate reduction Chromate (VI)

Table 2 adapted from Yang and Lu (1987)

1.1.6 Rate Limiting Step

Taking into consideration the structural and topographical facets of this enzyme system, the overall rate of the reaction is governed by the concentration of the P-450- substrate complex. The formation of this complex is dependent not only on the P-450 31 and substrate, but also the various states of the reduced reductase. As mentioned previously, these enzymes are in a dynamically fluid matrix which also is a determinant of interactions.

Designation of a rate limiting step in a multi step pathway, in principle, can be accomplished by quantification of the accumulation of an intermediate in the process analyzed. Attempts to assign a rate limiting step to the P-450 system has been fraught with many difficulties (Mannering, 1981 and Bjorkhem, 1982). Some of these difficulties include:

a) Intermediates formed in the P-450 catalyzed reactions are often low in concentration and otherwise difficult to isolate.

b) There is a heterogeneity with respect to the isozymes of the terminal oxidase, P-450. The substrate diversity is such that an association between a specific isozyme and a substrate has been difficult.

c) The diversity of substrates metabolized may have influences on a variety of the intermediate steps, which would make it difficult to make general conclusions.

d) The presence of endogenous substrates, such as cholesterol and fatty acids, in the reconstituted systems may compete for reactions that are under study.

e) The concentration of the P-450-substrate complex is limited to the amount of P-450 that is present in the membrane, i.e there is a 10 to 30 fold excess of the reductase present.

f) These are all membrane bound systems that are mobile within the structural confines of the membrane and thus may not follow the principles set for soluble systems. 32 However, the following events in the sequence of reactions are not considered to be rate limiting:

a) The binding of the substrate to the oxidized form of P-450 (Shenkman, 1968).

b) The reduction of the reductase by NADPH (Iyanaga and Mason, 1973).

c) The binding of oxygen to the reduced P-450-substrate complex.

1.1.7 Induction

The term enzyme induction refers, generally, to de novo protein synthesis which results in the enhanced expression of an enzyme at a greater than normal level. However, alternative mechanisms involving the reduced degradation of the protein, enzyme stabilization, or activation of existing enzyme may also effect the increase in the enzyme amount and concomitant function (Gelboin, 1971). The amount of microsomal P-450 in the liver and extrahepatic tissues may be increased by the in vivo administration of modifying agents which have been classified a inducing agents (Yang and Lu, 1987).

Inducing agents have been categorized on the basis of a) the number of enzymatic reactions affected; and b) the type of cytochrome P-450 (isozyme) that is elaborated in the response to the inducing agent. There exist three classes of inducing agents which may be classified using the above criteria. They are: 33 1. Barbiturate (Phenobarbital, etc.) type in which P-450 content and other associated components of the system are increased.

2. Polycyclic Aromatic Hydrocarbon (3-Methylcholanthrene, Aroclor series of compounds, etc.) type in which P-448, a spectrally distinct P-450 isozyme, content is increased.

3. Steroid Hormones (Glucocorticoids, 16-a carbonitrile [PCN]) which may effect increases in P-450 by increased synthesis or the stabilization of an emyme.

1.1.7.1 Induction of Rat Liver P-450 Isozymes

The use of different inducing agents to manipulate the biochemical and

biophysical properties of the MFO system has allowed for the identification and

characterization of a number of the multiple forms of cytochrome P-450. The total

number of distinct isozymes elaborated in the various tissues of the body are not

known; however, in rat liver nearly a dozen isozymes have been identified and

characterized (Adesnick and Atchison, 1986).

The spectral properties of the various forms of P-450 elaborated by inducing

agent pretreatment range from 447nm to 452 nm. Spectral characteristics of the

microsomal suspensions represent a composite of the different forms of the isozymes

present in the suspension. These observations have been confirmed by the analysis

of the purified isozymes with respect to their spectral characteristics (Lu and West,

1980).

The barbiturate type inducing agents (i.e, phenobarbital) effect the elaboration 34 of four types of P-450 isozymes known as PB types -b, -c, -d and /PCNe. Of the

different forms isolated, types b and d are the forms which are highly induced

(Dannan, et.al, 1982). The absorbance maxima for these forms range from 450 - 451

nm. These PB types of P-450 exhibit substrate specificity towards substrates such as

Benzphetamine, N,N-dimethylaniline, parathion and N,N-dimethylphenteramine

(Guengerich, 1977; Kamataki, et.aL, 1976; Miwa, et.aL, 1978; Miwa, et.aL, 1980; Ryan,

et.aL, 1979;and West, et.aL, 1979).

The 3-Methylcholanthrene (3-MC) type inducing agents lead to a high level

of activity associated with the P-45O-0NF-B and -/JNF/ISFG isozymes. These

isozymes produce absorbance maxima at 447 to 448 nm as a reduced-CO complex.

The 3-MC type isozymes have been shown to catalyze the metabolism of a variety

of aromatic hydrocarbons, 7-ethoxycoumarin, 7-ethoxyresorufin, and zoxazolamine

(Lu and West, 1980 and Guengerich et.aL, 1982).

The Aroclor series of compounds, mixtures of polychlorinated biphenyls.have

been found to posses both 3-MC and PB type inducing properties (Alvares and

Kappas, 1977 and Parkinson, et.aL, 1980 a,b). These isozymes produce a characteristic absorption maxima at 448 - 449 nm and have been demonstrated to posses catalytic activity of both the 3-MC and PB type substrates (Alvares and Kappas, 1977). It is due to the potency and broad spectrum of induction that this inducing agent was employed in the work presented in this thesis.

The administration of PCN resulted in the induction of a PCN type of P-450 35 isozyme which has an absorbance maxima at 450 mn (Elsourbagy and Guzelian,

1980). This isozyme was also found to be inducible with synthetic and endogenous (Schuetz, et.aL, 1984a,b).

A comparison of the various isozymes and their inducing abilities is presented in Table 3. The results presented are from the work of Guengerich etal, (1982) and

Dannan et.aL, (1983). Contents presented are nmol P-450/mg microsomal protein as determined by immunologically. 36 Table 3 Immunological estimation of the various forms of rat liver Cytochrome P-450 after treatment with various inducing agents

None PB 3MC PB+3MC PCN Aroclor 1254 Form of P-450 UT-A 0.53 0.30 0.22 0.14 0.33 0.27 PB-B 0.01 1.58 0.01 0.76 0.10 1.29 /3NF-B 0.02 0.10 1.52 1.08 0.06 1.45 PB-C 0.41 0.88 033 0.85 0.31 0.36 PB-D 0.08 2.00 0.06 1.35 0.09 1.46 PB/PCN-E 0.38 0.76 0.24 0.65 1.32 0.77 UT-F 0.11 0.09 0.10 0.09 0.08 0.15 0NF/ISF-G <0.02 <0.02 0.35 0.56 <0.03 1.23

All values presented are in nmol/mg protein.

Abbreviations: PB, phenobarbital; 3-MC, 3-Methylcholanthrene; PCN, Pregnenolone 16-a carbonitrile; 0NFt p napthoflavone; UT, untreated. - 37 1.2 Conjugation Reactions* Phase II

The group of synthetic reactions involving the addition of an endogenous

molecule and a xenobiotic (or its metabolite) are known as conjugation or "phase II"

reactions. Phase II reactions occur at suitable functional groups which are either part

of the molecule or a result of phase I metabolism. In addition to these functional groups present, chemically unstable species such as epoxides, arene oxides and carbonium ions may also be susceptible to phase II reactions (Caldwell, 1980).

12.1 Classification of Phase II Reactions

The enzymes responsible for phase n reactions are found both in the cytoplasm and intracellular membranes. As a rule, these reactions require high energy intermediates and specific enzymes to transfer the conjugate moiety. The existence of multiple forms of the enzymes has greatly complicated the study of conjugation reactions (Aitio, 1978). However it is known that these reactions proceed rapidly and yield polar metabolites which are much less lipid soluble that the parent compounds. This process is responsible for the deactivation and excretion of a great number of xenobiotics. For this reason, conjugation reactions have been (and often still are) considered to be detoxification reactions. However, the presence of specific hydrolytic enzymes such as: fi glucuronidase, , deacteylase, and O- and N- demethylases in the body has given support to the idea that phase II may not 38 represent solely a detoxification mechanism. Conjugates formed may be transported to sites distant from their metabolism site and acted upon by the hydrolytic enzymes.

This could result in the release of a toxic metabolite at a site distant from that of its metabolism (Caldwell, 1980; Kadlubar, et.aL, 1978; Kauffman, 1987). As may be seen from Table 4 there are a considerable number of phase II reactions to be considered.

However, for this work, the discussion has been limited to glucuronidation and sulfation reactions. 39 Table 4 Survey of Conjugation Reactions

Conjugation Reaction Functional Groups High Energy Intermedi­ ate

Glucuronidation •OH, -COOH, -NH3 Uridine diphosphate -SH Glucuronic Acid (UDPGA).

Sulfation -OH, -NH2 Adenosine-3'-phosphate 5'•phosph osulfate (PAPS).

Glucoside -OH; -COOHi "NH2 Uridine diphosphate Glucose (UDPG).

Amino Acid -COOH thioesters

Methylation -OH, -NH2 S-Adenosyl methionine (SAM); 5-methyltetra- hyrofolic acid.

Acetylation -OH, -NHZ Acetyl Coenzyme A (Acetyl CoA)

Glutathione ArylHalide, arene oxide, Arene oxides, epoxides epoxides, carbonium (mercapturic acid ions

synthesis) 40 1.2.1.1 Glucumidgtim

Glucuronidation is a major conjugative pathway in many species over a range of tissues, and accounts for most of the detoxicatoiy material in the urine and bile

(Smith and Williams, 1966; Smith, 1968). Glucuronidation is catalyzed by a family of

UDP glucuronyl which mediate the transfer of a d-glucuronic acid moiety to a functional group on a molecule of endogenous or xenobiotic origin. Glucuronic acid is readily available from glucose or glycogen stores and is less likely to become a limiting factor such as with amino acids or sulfates (Dutton, 1980).

The major portion of glucuronidation activity has been localized to the endoplasmic reticulum (microsomal membranes)(Gram, et.aL, 1968). However, activity has also been reported in the nuclear envelope (Gorski and Kasper, 1977), mitochondria (Beaufay, et.al., 1974), Golgi apparatus (von Bahr, et.aL, 1972; Nyqvist and Morre, 1971) and plasma membranes (von Bahr, et.al., 1972). The versatility of this enzyme family may be better appreciated from the wide variety of substrates handled (Dutton, 1980) (See Table 5).

Glucuronide conjugates may be cleaved specifically be enzymes known as B- glucuronidases, which are known to be quite ubiquitous in animals, plants and bacteria (Wakabyashi, 1970; Levy and Conchie, 1966). Hydrolysis of glucuronide conjugates may alter the pharmacokinetics of a material in the body significantly ( e.g. enterohepatic recirculation of morphine (Woods, 1954). Commercial availability of these enzymes has aided the characterization of glucuronide conjugates. Table 5 Glucuronidation reactions and generalized structures

Group Structure

Linkage through O; Aryl-OH AR.O.GA

Aryl or alkyl enolic •CH=CO.GA

Alkyl-OH (primary, secondary or ter­ CO.GA tiary)

Acyl-OH (aryl or akyl) C.COO.GA

Hydroxylamic -N.O.GA

Linkage through S; Thiolic -S.GA

Carbodithiolic -C.S.S.GA

Linkage Through N: Amino (aryl) Ar.NH.GA

Uriedo (carbamate) -NH.CO.NH.GA

1-Thiouredo -NH.CS.NH.GA

Sulfonimido -SO2N.GA

Heterocyclic =N.GA

Linkage through C: C.GA

Table 5 adapted from Dutton (1980) 42 12.12 Sulfation

The are a group of enzymes which catalyze the formation of sulfate esters with either adenosine 5'-sulfatophosphate (APS) or adenosine 3'- phosphate 5'-sulfatophosphate (PAPS) as the sulfate donor. The sulfotransferases are localized to the cytosol; however, their presence in the "soluble fraction" of a homogenate does not exclude the potential for being membrane bound. The supply of inorganic sulfate which is required for sulfation is obtained mainly from the food under normal non-fasting conditions. Under fasting conditions, inorganic sulfate may be supplied from the catabolism of proteins and other highly sulfated macro- molecules (Mulder, 1980).

Sulfation usually implies a detoxification and elimination from the body of a sulfated conjugate by either urinary or biliary excretion. However, steroid sulfates may be a storage form of these compounds (Mulder, 1980). The sulfation of steroids may also confer an altered metabolism profile, relative to glucuronidated and unconjugated steroids (Kornel and Miyabo, 1975; Matsui and Hakozaki, 1977; Holler, et.aL, 1977). The sulfate conjugates are also more unstable than the glucuronide conjugates of the same substrate because the sulfate moiety is a better leaving group than the glucuronide moiety (Maher, et.aL, 1968; Irving, 1971). The sulfation of a compound may result in increased reactivity with cellular macromolecules, i.e. the involvement of the N-O-sulfate conjugate in the carcinogenic action of N-Acetyl-2- aminofluorene (King and Phillips, 1968; De Baun, et.aL, 1968). 43 As with the glucuronides, there exist specific which act to

deconjugate sulfate esters (Dodgson and Spencer, 1957; Roy, 1971; Dodgson and

Spencer, 1970; Nicholls and Roy, 1971). The commercially available preparations contain residual ^-glucuronidase activity, which must be selectively inhibited to yield a specific sulfatase enzyme for metabolite analysis. Table 6 provides a brief overview of the variety of sulfations effected by the sulfotransferases (Mulder, 1980).

Table 6 Sulfotransferase activities and acceptor molecules

Activity Typical Acceptors)

Phenol sulfotransferase 4-Nitrophenol, tyrosine deriva­ tives,biogenic amines Steroid sulfotransferases: sulfotransferase Estrone Androstenolone sulfotransferase Dehydroepiandrosterone sulfotransferase Bile-salt sulfotransferase Lithocholate Etiocholanolone sulfotransferase 3-Hydroxy-5 B Steroids Testosterone sulfotransferase Testosterone Cardeolide sulfotransferase Digitoxigertin Sterol sulfotransferase Scymnol

Alcohol sulfotransferase Aliphatic alcohols Ascorbate sulfotransferase Ascorbic acid Calciferol sulfotransferase Calciferol Luciferin sulfotransferase Luciferin N-hydroxyarylamine sulfotransferase N-Hydroxyphenacetin Arylamine sulfotransferase 2'Napthylamine

Table 6 adapted from Mulder (1980) 44 2.0 Skin - General Introduction

The skin, one of the largest organs of the body, is in constant contact with the environment and is subsequently under a barrage of varied environmental insults.

Traditionally, the skin has been considered to be a protective wrapping functioning as a passive, inert barrier between the inner and outer environments. However, it must be noted that the skin is an organ system and thus comprised of many integral parts functioning as one. It provides not only protection, but also sensory information, temperature and water regulation, and biotransformation (bioactivation) of cutaneously exposed compounds (Hsia, 1971; Pannatier, et.ai, 1978; Mukhtar and

Khan, 1989).

2.1 Anatomy

Structurally, the skin is a diverse organ consisting of many specialized cellular structures in a complex matrix. The three major divisions, exemplified in fig 4, are the epidermis, dermis and subcutaneous tissue (hypodermis). The epidermis is the topmost layer and itself is further subdivided into component layers as seen in fig 4. 45 2.1.1 Epidermis

The epidermis is completely avascular and regularly renews itself via cell division in the stratum basale. The daughter cells are pushed up into the stratum spinosum which comprises most of the mechanical bulk of the whole epidermis.

Occasional mitoses may occur in the deeper layers which lend support to the stratum basale growth and development. The stratum granulosum is comprised of cells from the previous layers in which metabolic changes have occurred. These changes include the gradual extrusion of the nucleus and the increased synthesis of keratin and lipids.

Keratin is a high molecular weight protein which imparts strength and pliability to the cells as they proceed to the surface. Lipids and other materials are secreted as the cement between the adjacent cells to form the familiar barrier function of the epidermis. The stratum lucidum is a layer of densely compacted keratinized cells which overlays the stratum granulosum. The topmost layer is the stratum corneum

(horny layer) and is comprised of the overlapping keratinized cells from the germinal layers beneath. This layer serves as the mechanical and chemical barrier between the inner and outer environments (Montagna, 1962).

2.12 Dermis

Underlying the epidermis is the gel like structure of the dermis which is composed of collagen, elastin and acid mucopolysaccharides (ground substance). The dermis is in intimate contact with the germinal layers of the epidermis at contact points called dermal papillae. These papillae provide an interdigitation of the two 46 membranes and provide added structural integrity to the structure as a whole. The

interwoven matrix of the dermis from its components provides a great resilience to

trauma and thus also imparts an added degree of difficulty in separation/fraction-

ation for study. The papillae are nourished by a capillary network that is embedded

within the matrix. Nutrients and waste products are exchanged between the epidermis

and the blood dependent upon diffusion; also, the dermis may function as a reservoir

of nutrients for the growing epidermis above (Johnson and Fusaro, 1972). The blood supply of the skin was determined to be between 0 and 2.5 ml/min/100 g of tissue

(Champion, 1970). However, it was determined that a blood flow rate of 0.1

ml/min/100 g tissue would be adequate for the metabolic needs of the skin (Burton,

1961). Fig 4 illustrates the relative sizes and components of the two layers. Within

the support matrix are present cutaneous appendages: hair follicles and associated

muscles; sweat and sebaceous glands; cutaneous nerves, and a vascular supply.

2.1.3 Subcutaneous Tissue

The subcutaneous tissue provides added support to the upper layers in

absorbing trauma and providing a cushioning for the deeper structures such as vascular supply, bone, and muscle. The thickness of this structure is location dependent.

2.1.4 Cutaneous Appendages

The hairs and nails are composed of keratin and are basically dead tissue of epidlxmal origin. Hair follicles, sebaceous glands, and sweat glands are also of epidermal origin, but are living structures (Hsia, 1971).

Hair follicles develop from invaginations of the epidermis during the second month of fetal development. Hair follicles develop at fixed distances from one another and no new follicles develop after birth. The hair follicle is usually slanted at an angle and extends into the dermis which contains a rich nutrient supply. One or more sebaceous glands are usually associated with each hair follicle which are collectively called the pilosebaceous unit (Hsia, 1971). The bulb-like root of the hair follicle is one of the most metabolically active in the body (Rongone, 1987).

Differential density of hair follicles at varied locations are due to the variable increases in the surface area of the skin (Szabo, 1958).

Sebaceous glands synthesize and secrete lipids which may be found on the surface of the skin. Secretions are pushed out onto the surface of the skin via piliary canals that are associated with the pilosebaceous unit. The size and turnover of these glands is hormonally dependent and particularly sensitive to (Strauss and

Pochi, 1963). Epstein and Epstein (1966) used autoradiographic techniques and demonstrated that radioactivity was present in the germinal layers 40 min after the administration of the radiolabel into sebaceous glands. They also found that differentiation of these cells began to occur at approximately 14 days.

Sweat glands are simple tubular glands which open to the surface through the ridges of the skin. The secretory parts of the gland are located in the dermis in a very twisted and coiled manner around blood vessels. These glands are divided into two 48 categories, eccrine and apocrine. The eccrine sweat glands are located all over the body and aid in the dissipation of body heat via secretion of water. The apocrine glands are larger than the eccrine glands and do not open to the surface of the skin.

The apocrine sweat reaches the surface via the piliary canals in a manner similar to sebaceous gland secretions. Apocrine secretions are odorless ,but upon interactions with bacteria that are present on the skin surface characteristic odors may be noticed

(Rongone, 1987). * Stratum corfttum • Stratum lucrum

Stratum nrnoaum

Stratum otiaia

Hair anatt

Amctor Smmi oora plli ffMJSCI* \

Duetol swmt gland Stratum comeum <^|5=8jj ^ Eoidarmis Stratum geimmaovum Paoillaiy layer of aermis ; > Dermis Oarmai * papula ^atcuiaf layer' 3t daimia V Subcutaneous t ttssua

/ M Sweat gtana Adipos* tiuua Connectiva tistu* Haif bulb

Figure 4: A schematic of epidermis and full thickness skin. 50 2.2 Metabolism in Skin

2.2.1 Carbohydrate Metabolism

Enzymes of aerobic and anaerobic glycolysis were found to be present in skin; as were the enzymes for carbohydrate metabolism (Cruickshank et.al, 1957;

Griesemer and Gould, 1954; Rippa and Vignali, 1965; Haplrin and Ohkawara, 1966a,

1966b, 1966c; Mier and Cotton, 1970). Techniques for the study of these enzymes has included tissue slices of whole skin (epidermis and dermis), epidermal and dermal homogenates, and differential centrifugation techniques. See table 7 for details. 51 Table 7 Carbohydrate Metabolism in the Skin

Enzvmefs) Investigated Investigator (s)

Citric Acid Cycle Halprin and Chow, 1961

Hexose monophosphate shunt Freinkel, 1960 Pomerantz and Asbomsin, 1961

Glucose-6-phosphogluconic dehydroge­ Rippa and Vignali, 1965 nase Halprin and Ohkawara, 1966a, 1966b Frienkel, 1960 Jacobson and Davidson, 1962.

Glycogen synthesis: Halprin and Ohkawara, 1966a

UDPG pyrophosphorylase, glycogen synthetase, hexokinase, phosphogluco- mutase, glucose-6-phosphate dehydro­ genase, phosphorylase, and UDPG dehy­ drogenase 52 2.22 Glucose Metabolism

The utilization of glucose by the skin is quantitatively different from that in liver and muscle. The major product of glucose metabolism in the skin is that of lactate production via the Embden-Meyerhof pathway. Frienkel (1960) demonstrated that glucose was metabolized by the hexose monophosphate shunt in skin rather than the tricarboxylic acid cycle. This is contrary to the liver where pyruvic acid is formed and subsequently converted to acetyl CoA. Although systems are present in the skin for carbohydrate metabolism they were found to be highly inefficient (less than 2 % of the glucose in skin entered the tricarboxylic acid cycle for ATP synthesis) (

Freinkel, 1960). Further evidence for support of this observation was provided from the studies of Adachi and Uno (1969). They found that greater than 90% of the glucose utilization by hair follicles resulted in the production of lactate, indicating the prominence of the Embden-Meyerhof pathway.

223 Lipid Metabolism

The surface lipids that are found abundant in skin are from either epidermal origin or in the form of sebum from the sebaceous glands of the skin. Character­ ization of the lipids from human skin were found to include: triglycerides, waxes and waxy esters, free fatty acids, squalene, sterols and sterol esters (Lewis and Hayward,

1971; Felger, 1969). Studies by Kellum (1967) of lipids extracted from sebaceous glands of the scalp were found to be squalene, wax esters, and triglycerides. No free fatty acids were detected which led to the suggestion that the free fatty acids in 53 surface lipids are bacterial hydrolytic products of the sebum.

Investigations of the effects of microorganisms on the lipid content of the skin were carried out by Puhvel, et.aL, (1975). Sterilized human sebaceous glands were cultured with Propionibacterium acnes, P. granulosum and Staphylococcus epidermidis subgroup II. The bacteria were found to hydrolyze the sebaceous triglycerides followed by the esterification of sebaceous cholesterol to cholesteroyi esters. Reductions in free fatty acid/fatty esters ratios were noted by Pablo and

Fulton (1975) after treatment with clindamycin for 1 week. These studies point out the importance of considering bacterial modifications of different substrates.

2.2.4 Protein Metabolism

Specific amino acids were found to be differentially utilized-in specific areas of the epidermis. Glycine, leucine and alanine were found to be incorporated in the basal layer cells. Arginine, histidine, and methionine were found to be incorporated in the granular layers of neonatal rats (Fukuyama et.aL, 1965; Fukuyama and Epstein,

1968). The comparison of the incorporation of amino acids by liver, hair root cell and epidermis was investigated by Freeberg (1972). The result from the liver indicated that leucine, alanine, proline, threonine, and serine were most actively incorporated and that methionine and histidine were at lower levels of incorporation.

Citrulline and ornithine were essentially not incorporated in the liver system. In the hair root preparations, cysteine was the most actively incorporated followed by arginine, serine and proline. Glycine had a low incorporation into hair cell 54 preparations. Leucine and valine were most actively incorporated followed by glutamic acid and serine in the epidermal preparations. Cysteine had a low rate of incorporation in the epidermal system examined.

2.2.5 Steroid - Hormone Metabolism

Development of secondary sex characteristics in the skin was observed to be dependent (for a review see Strauss and Pochi, 1963). A variety of steroid metabolism reactions have been classified in skin such as: alcohol oxidation, acyclic carbon hydroxylation, carbonyi reduction, carbon-carbon double bond reduction and certain hydrolytic reactions. The salient features of these reactions will be presented in this section.

The activity of 17 0-hydroxysteroid dehydrogenase is responsible for the metabolism of alicyclic alcohol groups found in some steroids. The metabolism of hydrocortisone to by human skin samples was observed by Hsia et.al.

(1971). The skin was also demonstrated to oxidize to estrone (Weinstein, et.aL, 1968). In addition to these studies, Wotiz et.aL, (1956) found that testosterone was metabolized to seven metabolites by human skin.

Studies performed by Faredin et.aL, (1969) involving the metabolism of dehydroepiandrosterone (DHA) revealed some interesting comparisons between the cutaneous and hepatic metabolism of this steroid. Human abdominal skin produced

60 % 7 a-hydroxydehydroepianrosterone, 16% 7 /3-hydroxy-dehydroepianrosterone, and 16 % of the keto-hydroxydehydroepianrosterone. Hepatic enzymes produced only 55 the 7 a-hydroxylated metabolite of DHA.

Carbonyl reduction of groups on steroids is also performed by skin as exemplified by the reduction of the 7- keto-DHA mentioned earlier. Additionally, the reduction of the 20-oxo group (carbonyl) to a secondary alcohol in the case of hydrocortisone (Hsia et.aL, 1971) and progesterone (Frost et.aL, 1969).

Carbon-carbon double bond reduction is present in skin, but was found to be more stereospecific than that present in the liver. Cutaneous biotransformation yields only the a-epimer, whereas hepatic biotransformation yields both the a and (3 epimers. An example of this is the activity of the 5 -a-reductase towards testosterone to produce 5 a-dihydrotestosterone and progesterone to produce 5 -a-pregane (Frost, et.aL, 1969)

The specificity of cutaneous esterases was demonstrated by Cheung et.al.,

(1985). They found that the 21 ester of betamethasone-21-valerate was more susceptible to hydrolysis by esterases than the 17 ester of betamethasone-17-valerate.

Using in vitro flux chambers, Valia et.aL, (1985) demonstrated that estradiol esters were metabolized to estradiol with a rank order which paralleled the diffusion through the skin preparation. A result which may suggest that there is a correlation between the lipid solubility, residence time in the skin and metabolism by the cutaneous esterases. 56 2.2.6 Cutaneous Phase I Metabolism

The skin is far from the static, passive barrier that has come to be its

classification. In addition to the endogenous metabolism, there is also evidence for

the metabolism of xenobiotics to innocuous and reactive metabolites by the skin.

Many of the studies conducted have been in neonatal rats, mice and some studies

with human tissues. The summary of the reactions studied, based on a literature

review are presented in table 6. Central to these reactions is the mixed function

oxidase (MFO) enzyme system which is localized in the endoplasmic reticulum. This

system is composed of a family of hemoproteins classified as cytochrome P-450

isozymes (P-450). The P-450 and its associated NADPH Cytochrome P-450 reductase

(reductase) form a highly versatile system with an extremely broad substrate specificity for both endogenous (i.e. steroids, fatty acids, bile acids, etc.) and xenobiotic compounds. Table 8 Cutaneous Endogenous and Xenobiotie Phase I Metabolism

Reaction Tvpefs^ Investigator^

Oxidation

17 /3-hydroxysteroid dehydrogenase Hsia, et.aL, 1965 Gomez and Hsia, 1968 Weinstein et.aL, 1968

Aiyl Hydrocarbon Hydroxylase Faredin, et.aL, 1969 Fox, et.aL, 1975 Di Giovanni, et.aL, 1977 Pannatier, et.aL, 1981b Kao, et.aL, 1985

O-dealkylase Pohl, et.aL, 1976 Pannatier, et.aL, 1981a Goerz et.aL, 1981

Monoamine Oxidase Hakanson and Moller, 1963a

DOPA Decarboxylase Hakanson and Moller, 1963b

Aniline Hydroxylase Pohl, et.aL, 1976

Adenosine Deaminase Yu, et.aL, 1980

Table 8 adapted from Martin, et.aL (1987) Table 8 (continued) Cutaneous Endogenous and Xenobiotic Phase I Metabolism

Reaction Tvpefs^ Investigator^

Reduction

Ketoreductase Weinstein, et.aL, 1968 Frost, et.aL, 1969 Hsia, et.aL, 1969 Segal, eLai, 1975

5 o-reductase Hsia, et.aL, 1965 Frost, et.aL, 1969

Hvdrolvsis

Epoxide Hydratase Bentley, et.aL, 1976

Esterases Findlay, 1955 Tauber and Toda, 1976 Rawlins, et.aL, 1979 Bodor, et.aL, 1980 O'Neill and Carless, 1980 Nacht, et.aL, 1981 Pannatier, et.aL, 1981b Loftsson, et.aL, 1982 Sloan and Bodor, 1982 Baines,et.aL, 1984 Jofaanssen, et.aL, 1986

Table 8 adapted from Martin, et.aL (1987) 59 22.1 Cutaneous Phase II Metabolism

2.2.7.1 Glucurortidation

The possibility of the formation of glucuronides by the skin was first suggested by

Harper (1958) and Zini (1952). This was confirmed by Stevenson and Dutton (1960)

using 2-aminophenol with strips and homogenates of shaved skin from guinea pig and

mouse. They also found a four fold enhancement of the glucuronidation activity

towards 2-aminophenol by pretreatment (painting) with 3,4,-benzopyrene in hairless

mice (Stevenson and Dutton, 1962).

Using cultures and homogenates of whole human skin, Rugstad and Dybing

(1975) were able to demonstrate the glucuronide conjugates of para-nitrophenol and

para-aminophenol.

Moloney, et.aL, (1982) demonstrated the presence of UDP-glucuronyl

transferase activity in microsomes that were isolated from hairless mice and rats.

Using 1-napthol as a model substrate, they found that glucuronidation activity of the was 50 % of that found in similar preparations from liver.

22.72 Sulfation

Berliner et.aL (1968) demonstrated the formation of water soluble steroids by human skin. They incubated human abdominal wall skin for five days with DHA and isolated two sulfated metabolites. These metabolites were determined to be DHA- sulfate and A 5-androstene-3 13-11 /3-diol sulfate. 60 22.13 Methvlation

The presence of catechol-O-methyl transferase (COMT) in cutaneous tissue of rat, rabbit, mouse and human was demonstrated by Hakanson and Moller (1963).

They incubated norepinephrine from the various species anaerobically and were able to isolate normetanephrine as a metabolite. They also found that the reactions was inhibited by pyrogallo, which is a specific COMT inhibitor. Barnshad (1969) compared the activities of COMT in human epidermis and dermis. He found that the

COMT activity was higher in the epidermis than the dermis or the whole skin. 61 3.0 Purpose

The purpose of this work was to develop procedures for the quantification of microsomal P-450 mediated metabolism in full thickness skin (epidermis and dermis intact). To this end, microsomal fractions were isolated from skin and liver. The presence of MFO system in these fractions was determined initially by the presence of cytochrome P-450 (spectrophotometrically quantified) or NADPH cytochrome P-

450 reductase (activity assayed by the reduction of cytochrome c). The activity of the

MFO system was quantified by using well characterized (model) substrates. O- dealkylase activities in skin and liver microsomal preparations were compared.

Implementation of this developed procedure also allowed for the comparison between four species (adult male Fischer-344 (F-344) rats, male New Zealand rabbits, B6C3F-1 male mice and humans). The determination of the liver microsomal activities towards substrates tested provide not only a comparison parameter for activities, but also provide an index for the relative contribution of the skin microsomal activity.

The inducibility of the MFO system using various compounds has been well documented (see section 1.1.7.1). The broad spectrum induction of hepatic P-450 by

» the pretreatment with Aroclor isomers has been extensively studied. This situation has provided a characterization of the isozyme profile and related substrate specificities. Studies were undertaken to characterize the effects Aroclor 1221 and 62 1254 (A1221 and A1254, respectively) pretreatment on the activity of rat P-450 (skin and liver) towards the model substrates.

Furthermore, simple procedures for the assessment of integrated phase I and phase II biotransformation ability of rat skin were developed. These systems allowed for the quantification of the capacity of full thickness skin to metabolize the model substrates. 63 4.0 Model Substrates

To satisfy the major objective of this work, i.e. the assessment of biotransfor­

mation ability of the skin, model substrates were chosen. Model substrates are those

substrates for which the major routes of metabolism are known. Additionally, these

substrates must provide a sensitive and reliable index for the assessment of

biotransformation (phase I and phase II).

4.1 Alkoxycoumarins

Alkoxycoumarin molecules have been utilized as a measure of the hepatic

MFO system in a variety of in vitro techniques (Kremers, et.aL, 1981; Matsubara, et.aL, 1983; Belinski et.aL, 1983; Gugler and Jensen, 1985). The O-demethylation of

7-methoxycoumarin (7-MC) and the O-deethylation of 7-ethoxycoumarin (7-EC) both yield a highly fluorescent molecule, 7-hydroxycoumarin (7-HC). The nature of the

product, 7-HC, provides a sensitive measure of the P-450 related activity (Ullrich and

Weber, 1972; Ullrich, et.aL, 1973).

In cellular preparations, the O-dealkylated product, 7-HC may be further metabolized by phase II conjugation enzymes. The major conjugates formed from the

metabolism of 7-HC are glucuronides and sulfates. These two pathways represent quantitatively the most important routes of phase II metabolism. The use of specific 64 hydrolytic enzymes allows for the quantification of the phase II metabolites. Thus, using 7-EC, 7-MC and 7-HC will allow for the quantification of the three quantita­ tively most important enzymes of biotransformation, i.e. P-450, glucuronyltransferase and sulfotransferase.

4.2 Paranitrophenol Conjugates

The use of the glucuronide and sulfate conjugates of paranitrophenol provided a simple and sensitive index for the assessment of the deconjugation capabilities of rat skin. The production of paranitrophenol (PNP) from the conjugates by endogenous ^-glucuronidases and arylsufatases was determined spectrophotometrical- ly. The product PNP, in alkaline pH, is a bright yellow color with an absorption maxima at 400 nm. Materials and Methods 1.0 Animals

Adult male, Fischer-344 rats (weighing 180 - 220 g) and male B6C3F-1 mice,

weighing 17-20g were purchased from Harlan Sprague Dawley (Indianapolis, IN.)

Male New Zealand White Rabbits weighing 2.5 Kg were purchased from the Blue

Ribbon Ranch (Tucson, AZ.). All animals were acclimated for at least seven days

prior to use. Animals were housed in wire bottom hanging cages in animal housing

areas within the Department of Animal Resources at the University of Arizona.

Animals were exposed to a 12 hour white fluorescent light/ 12 hour dark light cycle and room temperature was maintained at 22°C. The animals were allowed standard laboratory chow and water ad libitum. Maintenance of the food, water and cage cleaning was

performed by the facility management personnel according to standard operating

procedures.

Rats and mice were terminated by cervical dislocation and rabbits were terminated by captive bolt pistol. The livers were removed immediately and placed in ice cold isotonic KCL The dorsum of the animal was shaved with Oster small animal clippers (Milwaukee, WL), dissected from the body and placed in ice cold

0.9% saline. 67 2.0 Human Skin

Human full thickness skin was procured from the department of Pathology in the College of Medicine at the University of Arizona. Abdominal cadaver skin samples (4 x 30 cm sections) were obtained from cadavers less than 24 hours after death. Tissues were stored and transported in Krebs buffer pH 7.4 on ice until use.

3.0 Chemicals

Cytochrome c (type III from horse heart), 7-ethoxycoumarin (7-EC), 7- hydroxycoumarin (7-HC), p-nitrophenol, p-nitrophenyl-/3-D-glucuronide and p- nitrophenyl sulfate, NADP, NADPH, Glucose-6-phosphate (G-6-P), Glucose-6- phosphate dehydrogenase (G-6-PD), Butylated Hydroxytoluene (BHT), Ovalbumin,

Bovine serum albumin, Agar, Hthylenediaminetetraacetic acid (EDTA), Trizma base and Trizma Hydrochloride (Tris and Tris-HCl, respectively) were purchased from the

Sigma Chemical Company (St. Louis, MO.) 7-methoxycoumarin (7-MC) was purchased from the Aldrich Chemical Company (Milwaukee, WI.). 4-(2-Hydroxy- ethyl)-l-piperazine-ethanesulfonic acid (HEPES) was purchased from Boehringer

Manheim Biochemicals (Indianapolis, IN). Penicillin-streptomycin and fungizone were purchased from Gibco Laboratories (Long Island, NY). Aroclor 1221 and 1254 mixtures (A1221 and A1254, respectively) were obtained from Monsanto (St. Louis,

MO.). All other chemicals and solvents were of the highest purity commercially available. 68 4.0 Animal Pretreatment

For the induction studies, A1221 or A1254 were administered to F-344 male rats as a single intraperitoneal injection (500 mg/Kg in corn oil). Dosing solutions of Aroclor mixtures 1221 and 1254 were prepared in corn oil at a concentration of

250 mg/ml. Animals were fasted overnight prior to a single intraperitoneal injection of 500 mg/Kg of treatment or the vehicle solutions (Parkinson, et.al, 1983). Control and treatment groups were allowed food and water ad libitum until termination 4 days later. Rats were terminated in the manner described earlier.

5.0 Subcellular Fractionation

Established methods used for the study of tissues such as the liver were found to be ineffective in the study of the various sources of skin (full thickness epidermis and dermis). Conventional mincing and homogenization techniques created many problems which severely impeded the study of these tissues. Quick freezing in liquid nitrogen and subsequent pulverization by mechanical means provided a reproducible method for the homogenization of cutaneous tissues. 69 5.1 Skin Microsomal Preparation

Pelts and human abdominal skin areas were cleaned of subcutaneous tissue and adherent material by abrasion with cotton gauze. These cleaned pelts were then immersed in ice cold 0.9% saline until use. Full thickness pelts (epidermis and dermis intact) were then sectioned into approximately 0.2 x 2.0 cm strips on a glass plate (4°C) and immediately immersed in liquid nitrogen. Frozen strips (1-4 rat pelts/pool) were then placed in a prechilled Waring Blendor equipped with a stainless steel chamber. A powder of the tissue was achieved by high speed pulse of the blendor (10-15 seconds in duration); care was taken to ensure that the tissue did not thaw during pulverization. The resulting powder was then resuspended at 20 % w/v in a pH 7.4 buffer containing 0.05 M Tris-HCl, 0.1 M KC1, 0.001 M EDTA and

2.0 x 10'5 M BHT (Homogenization buffer). This mixture was then subjected to three

30 second pulses of a Polytron Homogenizer equipped with an ST-10 generator. The resulting mixture was centrifuged at 10,000 x g for 20 min at 4°C. The supernatant or homogenate fraction was then centrifuged at 100,000 x g for 60 min at 4°C to yield the microsomal pellet and the supernate containing the cytosol. The microsomal pellet was then resuspended in a pH 7.4 buffer containing 0.01 M Tris-HCl, 0.001 M

EDTA, 20 % Glycerol (v/v) and 1 x 10 "* M Phenyl Methyl Sulfonyl Fluoride

(Resuspension buffer). Subcellular fractions were stored at -20°C until use. 70 5.2 Liver Microsomal Preparation

Individual or pooled (3-4 animals/pool) liver tissues from the animals were utilized for comparison subcellular studies. Minced livers were placed in homogeni- zation buffer at 20% w/v. These were then subjected to three 10 second pulses of a

Polytron Homogenizer equipped with an ST-10 generator. The homogenates were the exposed to three passes in a ground glass homogenizer to ensure uniform particle size. Homogenates were centrifuged as described in the above method. Identical buffers and centrifugal conditions were employed for subcellular isolation. The resulting pellet post 100,000 x g centrifugation was then resuspended in Resuspension buffer and stored at -20°C with the related fractions. 71

Figure 5: Microsomal Preparations

Liver Skin a. mince into small pieces a. section into strips b. resuspend at 20% weight/volume b. Immerse in liquid nitrogen until frozen. c. Pulverize into a fine powder. d. Resuspend at 20% weight/volume

Polytron Homogenize (setting 6)

Centrifuge the resulting homogenate (10,000 x g 20 min at PC)

Pellet' 'Supemate (discard) /(S-9 fraction) Centrifuge the S-9 fraction (100,000 xgfor60 min at PC)

Pellet Supemate 72 6.0 Cutaneous Culture Techniques

6.1 Short Term Culture: "Swirly Culture"

Swirly Culture studies were performed on pelts obtained in the same manner as above. Pelts were cut into either 2.0 x 2.0 cm or 0.2 x 2.0 cm sections and coded

as to their location. Areas equivalent to 2 x 2 cm were then incubated in Ehrlen-

meyer flasks containing substrate (100 /aM 7-EC) dissolved in Krebs-Henseleit buffer supplemented with Hepes, pH 7.4 (10 ml final volume). The flasks were then

incubated in an air controlled bath at 37°C rotating at 250 - 300 rpm for the length

of the incubation. This speed was necessary to prevent the tissue from settling to the

bottom, or adhering to the side of the vessel. Flasks were loosely covered to prevent

media evaporation over the extended time periods. Reactions were terminated by the

removal of the tissue. 1 ml aliquots of media were utilized for metabolite analysis.

Figure 6: Swirly Culture Incubation 73 62 Long Term Culture: LTC

LTC studies were performed with full thickness skin using a modification of the method described by Trowell (1958). Rats were terminated and the pelts prepared as described above. Pelts were sectioned into 2.0 x 2.0 cm pieces using ethanol washed razor blades on a glass plate (4°C) and placed in ice cold 0.9% saline until use (approximately 10 min). Sections were placed in the center of a sterile petri dish containing either an agar support block or no support (submerged incubation).

The agar support incubations were arranged such that the dermis was in contact with the agar and the incubation buffer. Incubation buffer (20 ml) consisted of Krebs-

Henseleit buffer (pH 7.4) supplemented with 25 mM HEPES, 2% Penicillin-

Streptomycin, Wo fungizone and 100 mM substrate. Appropriate blank and control incubations were also generated. Petri dishes were covered and incubated at 37°C for

72 hours with shaking (60 rpm) in an environmental shaker (Labline-Instruments,

Melrose Park, IL.). Reactions were terminated by the removal of the tissue. 1 ml aliquots of media were utilized for metabolite analysis.

Figure 7: Long Term Culture Incubation with agar support 74 7.0 Assays

7.1 Protein Determination

Protein content was determined colorimetrically by the method of Bradford

(1976) utilizing ovalbumin as a standard. In addition to the routine Bradford protein determination, protein contents were also determined using the method of Lowry et.al., (1951). Bovine serum albumin was used as the reference standard. However, the Bradford method, referred to as the Bio-Rad, was used as the assay for determination of all protein values used in the studies presented.

7.2 Cytochrome P-450 Content

P-450 content was determined by the method of Omura and Sato (1964).

Microsomal samples were diluted (1.0,0.5 or 0.25 mg/ml as deemed necessary) with

"microsomal solubilization buffer," which contained 100 mM potassium phosphate,

20 % glycerol, 0.5 % sodium cholate, 0.4 % Renex and 1 mM EDTA (pH 7.4). The dithionite difference spectra of subsequent dilutions was generated using a Beckman

DU-7HS spectrophotometer (Beckman Instruments, Irvine, CA.). 75 73 Cytochrome c Reductase Determination

Reductase activity was determined using the method of Phillips and Langdon

(1963). Kinetic measurements of the reduction of cytochrome c were measured at 550 nm using a Beckman DU-7HS spectrophotometer.

7.3 O-Dealkylase Activity Determination

The O-dealkylation of 7-MC and 7-EC was quantified fluorimetrically (Ullrich and Weber, 1973 ; Ullrich, et.aL, 1973) by comparison to standard curve using 7-HC

(excitation 370 nm and emission 450 nm) in Glycine:NaOH (pH 10.4).

Detection and quantification of 7-HC (major metabolite) was achieved using a fluorescence HPLC apparatus as a flow cell detection system. Standard curves for

7-HC were determined to be linear in the range of 1.25 to 1000 pmol/ml. The detection system included a Perkin Elmer 650s Fluorescence Detector, Spectra

Physics SP8700 solvent delivery system, Rheodyne sample injector equipped with a

1 ml sample loop and a Spectra Physics SP4100 integrator. 76 73.1 Metabolite Analysis

7.3.1.1 Microsomal Incubations

Microsomal incubation mixtures (1 ml final volume) contained an NADPH generating system (4.6 n moles NADP, 10 n moles G-6-P, 50 n moles MgCl2 and 0.09

units of G-6-PD) microsomal protein ( 1.0 mg/ml skin and 0.5 mg/ml liver final concentrations) and 100 /xM substrate in 0.1 M Tris-HCl buffer pH 7.4. Assays were performed at 37°C in an environmental shaker (Labline Instruments, Melrose Park,

IL.). Reactions were terminated by the addition of 5 ml ice cold hexane. Samples terminated immediately upon the addition of were utilized as reaction blanks and the fluorescence values subtracted from subsequent time points.

Samples from the microsomal and short term culture (also containing 1 ml media:5 ml ice cold hexane) were shaken for 15 min and the hexane, containing unmetabolized substrate, was removed. The remaining aqueous phase was extracted with 5 ml of ether:iso-amyl alcohol (1000:14) to remove the product. Four ml of the organic layer was removed and evaporated over 2 ml of Glycine:NaOH (pH 10.4).

This procedure allowed for the quantification of the 7-HC present in the sample, i.e, the major product from the microsomal incubations and the free 7-HC from the short term culture incubations.

Aliquots of the aqueous incubation media, after organic extraction to remove unmetabolized substrate and free 7-HC, were subjected to specific hydrolytic enzymes to liberate the conjugated 7-HC remaining in the aqueous layer. Glucuronide 77 conjugates were hydrolyzed using ^-glucuronidase (5000 units/ml) at pH 4.0. Sulfate conjugates were hydrolyzed using a crude sulfatase preparation (700 units/ml) containing 17 mM D-saccharic acid 1,4-lactone in Acetate Buffer (pH 4.0). The 7-HC liberated after a 16 hour incubation at 37°C was determined using the above mentioned etheniso-amyl alcohol extraction procedure. Figure 8: O-Dealkylase Assay

Media : Hexane (1:5)

Aqueous Organic Discard

Extract with 5 vol Ether/IAA

Aqueous Ether/IAA : Gly-NaOH (note extract 3x (4:2) with Ether/IAA) Five 7-HC Quantification AEx:370 nm; A.Em:450 nm

Add hydrolytic enzymes and incubate for 16 hours

Extract with 5 vol Ether/IAA

Aqueous (Discard) Ether/IAA: Gly-NaOH (4:2) Conjugate determination AEx:370 nm; AEm:450 nm 79 7.4 Paranitrophenol (PNP) Deconjugation Assay

Short term culture incubations were generated according to method B with

strips of rat skin. Glucuronide and sulfate conjugates of the PNP were added to

incubations to effect a final concentration of 100 juM. Incubations were prepared as

described earlier and terminated by the removal of tissue and alkalinization of the

medium( > pH 10.0). Aliquots were removed and centrifuged at 2500 rpm for 10

min in a Beckman TJ-6 centrifuge (Beckman Instruments, Irvine, CA.). PNP in the supernate was quantified at 400nm by comparison to a standard curve generated in

buffer, pH 10.4. Appropriate blank and control incubations were generated and used

to correct for background interferences.

8.0 Statistical Analysis

Unless specifically indicated, all data presented are the mean ± the standard error of the mean (SEM). Statistical evaluations were carried out using the Student's

t test, p< 0.10 was considered significant (Rosner, 1982). 80

Results 81 1.0 Protein Determinations

The objective of this section was to provide a comparison of two commonly

used protein assay techniques, i.e, the Bradford (Bio-Rad) and Lowiy assays. To this

end, requirements for reagents and standards were met and the protein values

determined. In addition to standard protein solutions of BSA and Ovalbumin, samples of liver and skin microsomal preparations were also included. The determination of protein values was achieved by comparison to the appropriate standard curves.

Table 9: Lowry Protein Assay

Protein ABS Predicted Sample ABS Predicted (Mg) X 660 Protein (jig) # X 660 .. Protein (Mg) nm nm 0.0 0.000 1 0.320 43.13 5.0 0.054 1.81 2 0.368 - 49.69 10.0 0.114 10.96 3 0.472 65.54 20.0 0.223 27.58 4 0.645 91.91 40.0 0.337 44.96 5 0.487 67.83 60.0 0.474 65.85 6 0.756 108.84 80.0 0.549 77.28 7 0.282 36.57 100.0 0.652 92.98 8 0.334 44.50 9 0.201 24.23 10 0.152 16.75 11 0.345 46.18 12 0.247 31.24 82 Table 10: Bio-Rad Protein Assay

Protein ABS Predicted Sample ABS Predicted (Mg) A. 595 Protein (ng) # k 595 Protein (/ig) nm nm 0.0 0.000 1 0.523 40.39 5.0 0.099 2.93 2 0.489 37.39 10.0 0.202 12.03 3 0.630 49.85 20.0 0.336 23.87 4 0.915 75.03 40.0 0.569 44.46 5 0.795 64.43 60.0 0.786 63.63 6 0.984 81.13 80.0 0.975 80.33 7 0.360 25.99 100.0 1.125 93.59 8 0.452 34.12 9 0.272 18.21 10 0.165 8.76 11 0.472 35.89 12 0.321 22.54

The microsomal samples utilized for these determinations were from rabbit

liver (1,2); control rat liver (3,5); A1254 rat liver (4,6); rabbit skin (7,8); control rat

skin (9,11) and A1254 rat skin (10,12). The standard curves were observed to be

^linear over the range examined with correlation coefficients (r values) of 0.998 and

0.993 for Lowry and Bio-Rad, respectively. All of the samples, with the exception of

#6 in the Lowry assay, were within the range of the standard curve limits. This

sample was repeated at various dilutions and the values found to be comparable to 83 those reported here.

As may be calculated from the data, the Bio-Rad assay had a tendency to

underestimate the amount of protein, as compared to the Lowry estimate (significant at p< 0.10). Bio-Rad assay samples were found to be 77% ± 0.03 of those generated

by the Lowry assay. Therefore, in order to compare between the assay techniques the values obtained from the Bio-Rad assay must be multiplied by a factor of 1.3 to attain a Lowiy equivalent protein value. These comparisons were made based upon using BSA and Ovalbumin as standard references in the Lowry and Bio-Rad assays, respectively.

Comparison of BSA and Ovalbumin as standard references in the Lowry assay showed that Ovalbumin standard comparisons generated values were 92 % (± 0.001,

SEM). However, a similar comparison using the Bio-Rad assay showed that BSA standard comparisons generated values were 66 % of those generated for Ovalbumin

(results not shown). The protein results presented in the remainder of this work are those generated from the Bio-Rad assay using ovalbumin as a standard. 84 1.1 Species Differences In Microsomal Protein Yield

Microsomal fractions from liver and skin were prepared as described earlier in the Methods section. The total microsomal protein yield per gram of tissue are presented in table 11. These values were obtained based upon reconstitution volume, protein content and the tissue weight used. Human liver samples (D43-46, see appendix A for explanation of codes) were included in this analysis for comparison and do not correspond to the skin samples used. However, in the other species examined the liver and skin microsomes are from the same animal.

Table 11: Microsomal Protein Yields in Rat, Rabbit, Mouse and Human

Species Over (L)1 Skin (S)1 S/L Ratio F-344 Rat - 33.01 ± 1.49 4.00 ± 0.37 0.12 Male (n=12) New Zealand 23.48 ± 2.55 2.97 ± 0.38 0.13 Rabbit - Male (n=5) B6C3F1 21.09 ± 3.79 2.19 ± 0.24 0.10 Mouse - Male (n=3) Human - 16.28 ± 2.11 0.96 ± 0.14 0.06 Male (n=4)

1 = mg protein/g tissue ± SEM (n) value represents individual animals/human subjects. 85 As may be readily appreciated from the results presented, the liver has a much higher yield of microsomal protein per gram tissue across the species. The rank order of yield, although not different from one another statistically, was maintained, i.e. rat > rabbit > mouse > human. Skin microsomes yielded between 6 an 13 % of the microsomal protein content of the liver. 86 2.0 Species Comparison - Microsomal Studies

2.1 Cytochrome P-450 Determination

P-450 could not be spectrophotometrically quantified in rat, rabbit, mouse or

human skin microsomes. Protein concentrations ranging from 0.125 - 2.0 mg/ml were prepared in the solubilization buffer (see methods 7.2) and the dithionite difference spectra recorded from 400-500 nm. From all the skin preparations examined from the various species, there was no detectable signal at 450 or 420 nm over the background

"noise." However, using the same methods, liver microsomal P-450 contents were quantifiable. A representative spectra, redrawn for clarity, is presented for rat liver microsomes (figure 9).

0.100T

0.075 -•

0.050--

4> 0.025-•

< -0.025 --

-0.050 --

-0.075 --

-0.100 400 410 420 430 440 450 460 470 480 490 500 Wavelength (nm)

Figure 9: A typical dithionite difference spectra of rat liver microsomes 87 A summary of the liver microsomal contents expressed nmoies P-450/mg microsomal protein may be found in table 12. The rat, rabbit and mouse were not statistically different from one another; however, the human was approximately three fold lower than the other species examined. Due to the close proximity of the values for rat, rabbit and mouse P-450 contents, no rank order could be assigned.

Table 12: Species Differences in Skin and Liver Cytochrome P-450 content

Species Skin1 Liver1 Rat N.D. 0.57 ± 0.08 (n=3) Rabbit N.D. . 0.52 ± 0.10 (n=5) Mouse N.D. 0.60 ± 0.03 (n=3) Human N.D. 0.21 ± 0.02 (n=4)

1 = nmoies P-450/mg protein ± SEM; A. max at 450 nm N.D. = Not Detected (n) values represent individual experiments 88 22 NADPH Cytochrome c Reductase Determination

NADPH cytochrome c reductase (reductase) is a microsomal P-450 system

linked enzyme and was used as a marker enzyme for the isolation of the microsomal

fraction from skin and liver. The subcellular activity of the reductase was found to

be greatest in the resuspended 100,000 x g pellet (microsomes) from skin and liver.

Since the P-450 content could not be determined in the skin microsomal prepara­

tions, the presence and activity of the reductase was taken to indicate the presence

of the P-450 system in the microsomal fraction. Linearity with respect to time was found to extend to 15 minutes; however, a routine 5 minute assay time was used.

Protein dependence was also established for each of the species examined. The

protein dependency of the reductase in the various tissues is presented on the following pages. 1 1 1 1 1 1 1 1— O.OO 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 Protein (mg/ml)

Protein (mg/ml)

Figure 10: Protein dependence of reductase activity in skin (A) and liver (B) microsomal fractions from male F-344 rats. Protein (mg/ml)

Protein (mg/ml)

Figure 11: Protein dependence of reductase activity in skin (A) and liver (B) microsomal fractions from male New Zealand rabbits. Protein (mg/ml)

1.000 1 Protein (mg/ml)

Figure 12: Protein dependence of reductase activity in skin (A) and liver (B) microsomal fractions from male B6C3F1 mice. 92 10 -r

•o(D

>% o tn

c c 0 • —i— 0.000 0.500 t.000 1,500 2.000 Protein (mg/ml)

Protein (mg/ml)

Figure 13: Protein dependence of reductase activity in skin (A) and liver (B) microsomal fractions from Humans. 93 As may be seen in the preceding figures, skin and liver studies of reductase

activities in the various species were found to be linear with respect to protein

content. A comparison of these species may be found in table 13. The rank order for

the skin reductase activities were mouse > rabbit > rat > > human. However, the liver rank order was rat > human > mouse > rabbit. Upon comparison of the

relative activities of skin as compared to liver, the rat was 5 %; rabbit 15%; mouse

16%; and human 3% of the liver reductase activity expressed nmoles cytochrome c

reduced/min/mg protein.

Table 13: Species Differences in Skin and Liver Reductase Activity

Species Skin (S)1 Liver (L) 1 S/L Ratio Rat 6.54 ± 0.72 128.0 ± 12.6 0.05 (n=3) Rabbit 8.44 ± 2.30 56.1 ± 2.0 0.15 (n=5) Mouse 12.97 ± 0.95 82.9 ± 4.96 0.16 (n=3) Human 2.53 ± 0.80 87.3+ 6.42 0.03 (n=4)

1 = nmoles Cytochrome c reduced/min/mg protein ± SEM (n) values represent individual experiments 2.3 Alkoxycoumarin Metabolism

The metabolism of the alkoxycoumarins, 7-MC and 7-EC, was quantified fluorimetrically as described in the methods section. A modification of the method of Greenlee and Poland (1976) was used to reduce the fluorescence background of unmetabolized substrate. A hexane pre-extraction increased the sensitivity of the assay necessary for the detection of extremely low levels of metabolite production.

Linearity with respect to time and protein was determined for skin and liver microsomes from the various species. Time dependency was studied up to 60 min for all preparations; however, time ranges which were linear are presented here. Routine protein concentrations of 1.0 and 0.5 mg/ml microsomal protein for skin and liver, respectively. Time courses for the metabolism of 7-MC and 7-EC are presented on the following pages for rat (figure 14), rabbit (figure 15), mouse (figure 16) and human (figure 17). The values presented for skin microsomal metabolism are in pico- moles (pmoles) 7-HC generated/mg microsomal protein and the liver expressed in nano-moles (nmoles) 7-HC generated/mg microsomal protein. 95

c 150-r '55•w1 o • 7-EC Rat skin CL CP • 7-MC Rat skin s.£ -o(U 100-- a> c 0 u 50 -- 1 I r-. en

15 30 45 GO Time (min.)

C *

«0J o £ c 20 Time (min.)

Figure 14: The metabolism of 7-MC and 7-EC (100 mM) by skin (A) and liver (B) microsomes from male F-344 rats. Values presented are the mean ± SEM (n=3). 96

4C 1000 • •*—>o> o Q_ O 7-EC Rabbit Skin O • 7-MC Rabbit skin £ 750 -- X)QJ

500 -- ca> o o n I 250 -- vto o eQ. 0#- 15 30 45 60 Time (min.)

c '(D 15t "o u(X O 7—EC Rabbit Liver cn • 7—MC Rabbit Liver E B 10-- -a0) 0) a>c O a x

(/) _Q) O Ec

Time (min.)

Figure 15: The metabolism of 7-MC and 7-EC (100 pM) by skin (A) and liver (B) microsomes from male New Zealand rabbits. Values presented are the mean ± SEM (n=5) 97

c 3000 t 3 o A 7-EC Mouse skin Q. 2500- A 7-MC Mouse skin £ \ ~u 2000 - - CL)

O 1500 -- c O O 1000 X r- V) 500-- o Q.£ 30 Time (min.)

c "

Time (min.)

Figure 16: The metabolism of 7-MC and 7-EC (100 mM) by skin (A) and liver (B) microsomes from male B6C3F1 mice. Values presented are the mean ± SEM (n=3). 98

.E*a> 15 T o CLw • 7-EC Human liver C7> • 7-MC Human liver £

10 -- •a

Time (min.) Figure 17: The metabolism of 7-MC and 7-EC (100 jiM) by liver microsomes from humans. Values presented are mean ± SEM (n=5; D20-D26).

2.3.1 7-EC Metabolism

Untreated adult rat and rabbit and mouse skin microsomal O-deethylase activity towards 7-EC was observed to proceed in a time dependent manner (Figures

14-16A). Time dependency was also observed with the corresponding liver microsomal preparations from these animals (Figures 14-16B and 17). Mouse skin microsomal preparations were found to be the most active in the metabolism of 7-

EC, i.e. approximately 16 fold higher than rat and 2.4 fold higher than rabbit skin microsomes. Rabbit skin microsonial incubations produced 7-HC with approximately a 7 fold greater activity than that observed in rat skin incubations. From these results the rank ordering of activity towards 7-EC by skin microsomal preparations was found to be mouse > rabbit > > rat. Human skin microsomal preparations did not

exhibit any metabolism of 7-EC over the 60 minute incubation period as indicated

by the lack of a fluorescent signal over background levels.

The rat liver microsomal activity was determined to be 1.2 fold greater than

that present in mouse liver incubations, 2.6 fold greater than rabbit and 8.5 fold

greater than human. This resulted in a rank ordering different than that from skin

microsomes, i.e, rat > mouse > rabbit > > human. The O-deethylase activities

present in the skin relative to the liver were determined to be 2.0%, 3.5% and 4%

of rat, rabbit and mouse, respectively. No such comparisons were available for

humans since there was no detectable O-deethylase activity in the skin microsomes.

A summary of the rates expressed pmoles 7-HC generated/mg microsomal protein are presented in table 14.

2.3.2 7-MC Metabolism

The only skin microsomal preparation that exhibited O-demethylase activity was from B6C3F1 mice. The skin O-demethylase activity was found to be time and protein dependent. The rate of skin O-demethylation was found to be approximately

2 fold less than that of O-deethylation. Rat, rabbit and human skin microsomes did not exhibit any O-demethylase activity up to 60 min.

The rank order of rates of liver O-demethylase activity was determined to be mouse > rat > rabbit > human. The O-deethylase activity in rat, rabbit and mouse 100 liver were significantly greater than the O-demethyiase activity in these fractions (p <

0.10). A similar difference was observed between skin O-deethylase and O- demethylase activities from mouse. In contrast to other species examined, the human liver microsomal O-demethylase was found to be significantly higher than liver O deethylase activity (p< 0.10). A summary of the rates of O-demethylase activity are presented in table 14.

Table 14: Species Differences in Alkoxycoumarin Metabolism by Skin and Liver

O-Demethylase Activity1 O-Deethylase Activity1

Species Skin Liver Skin Liver Rat N.D. 376 ± 27 2.0 ± 0.8 990 ± 150 (n=3) Rabbit N.D. 237 ± 13 13.4 ± 1.5 380 ± 36 (n=5) Mouse 17.2 ± 0.9 647 ± 2 31.5 821 ± 51 (n=3) Human N.D. 198 ± 35 N.D. 116+ 17 (n=5)'

1 pmol 7-HC/min/mg protein N.D. = Not Detected (n) values represent independent experiments * The liver and skin are not from the same donor; therefore n=5 for the liver and n=4 for the skin samples. 101 3.0 The Effects of Aroclor Pretreatment on Rat Microsomal Activities

3.1 Microsomal Protein Yield

Microsomal fractions from liver and skin from Aroclor pretreated rats were prepared as described earlier in the Methods section. The total microsomal protein yield per gram of tissue are presented in table 15. These values were obtained based upon reconstitution volume, protein content and the tissue weight used. The liver and skin microsomes are from the same animal and n values represent individual animals from 3 independent experiments.

Table 15: Microsomal Protein Yields in Control and Aroclor Pretreated F-344 rats

Treatment Skin (S) 1 Liver (L) 1 S/L Ratio Corn Oil Vehicle 4.00 ± 0.37 33.01 ± 1.49 0.12 (n=12) Aroclor 1221 3.73 ± 0.15 41.47 ± 6.06 0.09 (n=3) Aroclor 1254 4.16 ± 0.22 41.95 ± 2.18 0.10 (n=10)

1 = mg protein/g tissue ± SEM (n) values represent individual animals from 3 indepen­ dent experiments. 102 The Aroclor pretreatment of F-344 rats was not observed to modulate the protein content and yield in skin microsomes. The Skin microsomal protein yields were determined to be 12%, 9% and 10% of the corresponding liver protein yields from control, A1221 and A1254 treated rats, respectively. No statistical difference was found upon comparison of the treatment groups with control. Similarly, the liver microsomal protein yield per gram tissue were also not significantly different upon comparison to controls. Increases, although not significantly different, in liver protein content were apparent upon expression of results as microsomal protein per gram of liver.

3.2 Cytochrome P-450 Determination

P-450 could not be spectrophotometrically quantified in skin microsomal preparations from control, A1221 and A1254 pretreated F-344 rats. Protein concentrations ranging from 0.125 - 2.0 mg/ml were prepared in the solubilization buffer (see methods) and the dithionite difference spectra recorded from 400-500 nm.

In the skin preparations examined from the various treatment groups, there was no detectable signal at 450 or 420 nm over the background "noise." However, using the same methods, liver microsomal P-450 contents were quantifiable. 103 A summary of the effects of Aroclor mixtures on the P-450 content of rat liver

microsomes from (3 independent experiments, 3-4 animals/treatment group) is

presented in table 16. A1221 pretreatment was found to increase the liver P-450 content (pmoles P-450/mg protein) by 1.6 fold over controls and effected a shift of the maximal absorbance to 449 nm (see table 16). A1254 pretreatment produced a more dramatic increase in the P-450 content (4.5 fold) and shifted the absorbance

maxima to 448 nm (see table 16). If these same results are expressed per gram of tissue, a greater difference is noted in the P-450 contents. The mean values as expressed nmoles P-450/g liver were determined to be 18.82, 37.74, and 107.81 for control, A1221 and A1254, respectively. Given that Aroclor pretreatment increased the liver weight also, the magnitude of the difference would be further enhanced using this data manipulation. However, for the sake of brevity and clarity these numbers are not presented. Table 16: The Effects of Aroclor Pretreatment on Rat Microsomal P-450 Contents.

Treatment Skin1 Liver1 Control N.D. 0.57 ± 0.08" (n-3) Aroclor 1221 N.D. 0.91 ± 0.08b" (n-3) Aroclor 1254 N.D. 2.57. ± 0.21c" (n-3)

1 = nmoles P-450/mg protein ± SEM * = Xmax at 450 nm b = A max at 449 nm c = Xmax at 448 nm = Statistically different from control (p < 0.10) N.D. - Not Detected (n) values are from 3 independent experiments 3-4 animals/group 105 3.3 NADPH Cytochrome c Reductase Determination

As with the species comparison studies, the effects of protein concentration on the activity of the reductase were investigated. This investigation was prompted from the increase in protein content of liver microsomes from the Arocior treated rats. Linearity with respect to time and protein were generated; however, only the protein data are presented here. 20 -r •— • Control rat a — A A1221 rot • • A1254 rat 15 -- TD03 3O "a

o w ^ 5 -f o Ecz 0 0:00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 Protein (mg/ml)

300 t • • Control rat A • A1221 rat • • A1254 rat - 250--

B X3QJ O 200 -- 13 tu 150--

O 100 -- v(n O E 50-- c

0.00 0.25 0,50 0.75 1.00 1.25 1.50 1.75 2.00 Protein (mg/ml)

Figure 18: The effects of Aroclor pretreatment on the protein dependence of NADPH cytochrome c reductase activity in rat skin (A) and liver (B) microsomes. 107 Linearity was established for both skin and liver with respect to protein content. Aroclor pretreatment did not modulate the activity of the skin microsomal reductase activity. There was no statistical difference found between the control and

Aroclor treated skin microsomes. A1221 did not effect any significant difference on in the reductase activity in liver microsomes. Likewise there was no statistical difference between the control and A1254 liver microsomes. The skin microsomal reductase activity was found to be 5% in the control and A1221 treated rats. In the

A1254 treated rats, the skin was found to posses 4% of the corresponding liver activity. These data are summarized in table 17.

Table 17: The Effects of Aroclor Pretreatment on Skin and Liver NADPH Cytochrome c Reductase Activity

Species Skin1 Liver1 Skin/Liver Ratio Control 6.54 ± 0.72 128.0 ± 12.6 0.05 Aroclor 6.64 ± 0.72 129.8 ± 13.6 0.05 1221 Aroclor 6.80 ± 1.66 159.6 ± 10.4 0.04 1254

1 = nmoles Cytochrome c reduced/min/mg protein ± SEM. 108 3.4 Alkoxycoumarin Metabolism

The metabolism of the alkoxycoumarins, 7-MC and 7-EC, was quantified fluorimetrically as described in the methods section. Linearity with respect to time and protein was determined for skin and liver microsomes from the various treatment groups. Time dependency was studied up to 60 min for all preparations; however, time ranges which were linear are presented here. Routine protein concentrations of

1.0 and 0.5 mg/ml microsomal protein for skin and liver, respectively. Time courses for the metabolism of 7-MC and 7-EC are presented on the following pages for rat

(figure 19). The values presented for skin microsomal metabolism are in pico-moles

(pmoles) 7-HC generated/mg microsomal protein and the liver activities expressed in nano-moles (nmoles) 7-HC generated/mg microsomal protein from 3 independent experiments (3-4 animals per group). C 1000 -r Control rat A1221 rat o. en A1 254 rat 750 --

500 --

250 --

CL 01

Time (min.)

c ' 125 - - E B x>a> 100 --

Figure 19: The effects of Aroclor on the metabolism of 7-EC (100 /iM) by skin (A) and liver (B) microsomes from F-344 rats. 110 3.4.1 7-EC Metabolism

As may be seen from the data presented, there was a differential induction

of O-deethylation of 7-EC by the administration of A1221 and A1254. A1221

pretreatment did not significantly alter the rate of O-deethylation by skin microsomes

upon comparison to controls. A1254 effected a 7 fold increase (significant at p <

0.10) in the O-deethylation of 7-EC in skin microsomes over control levels. A lack

of induction of O-deethylation was obtained with the liver microsomal preparations

from A1221 pretreated rats. Liver O-deethylase activity was increased 4.5 (significant

at p< 0.10) fold over control levels in the A1254 treated rats. A comparison of the

skin and liver O-deethylase activities yielded that the skin had 0.2%, 0.1% and 0.4%

of the liver activity in the control, A1221, and A1254 treatment groups. The

significant increase in the percentage of skin activity indicates a greater percentage

increase in the skin activity compared to liver was effected by A1254 pretreatment

(see results in table 18).

3.42 7-MC Metabolism

The rat skin microsomal O demethylase activity was not demonstrable in any

of the treatment groups. The fluorescent signal was not quantifiable over the

background in incubations up to 60 min. However, a differential induction pattern was observed in liver microsomal preparations from these rats. A1221 effected a 2.0 fold increase in the liver O-demethylase activity over control levels (significant at p< Ill 0.10). A1254 effected a 1.5 fold increase in the liver O-demethylase activity over control levels (significant at p < 0.10). A comparison of the liver and skin O- demethylase activities was not possible since no skin microsomal activity was detected. However, a comparison of the liver O-deethylase and O-demethylase demonstrated that the O-demethylase activity was lower in all the groups tested.

Hepatic O-demethylase activity in the control and A1254 rats was significantly lower than hepatic O-deethylation in the control and A1254 rats (p < 0.10). Hepatic O- demethylation was found to be significantly different than hepatic O-deethylation in

A1221 treated rats.

Table 18: Aroclor Effects on Alkoxycoumarin Metabolism by Skin and Liver Microsomes from F-344 rats.

O-Demethylase1 O-Deethylase1 Skin Liver Skin Liver Control N.D. 376 ± 27 2.0 ± 0.8 990 ± 150 A1221 N.D. 761 ± 115' 1.7 ± 0.4 1197 ± 147 A1254 N.D. 556 ± 76* 15.7 ± 2.4* 3740 ± 121*

1 = pmol 7-HC Generated/min/mg protein ± SEM N.D. = Not Detectable over background n =3 experiments (34 animals/pool) = Statistically different from control values (p< 0.10) 112 4.0 Culture of Rat Skin Explants

Cellular xenobiotic metabolism by F-344 rat skin explants in a short term

culture incubation system was investigated (see methods). This method represents,

in the author's opinion, a simple system with which to study the metabolism of

xenobiotics in the skin. The media used was a simple buffered essential salt solution

(Krebs-Henseleit supplemented with HEPES) which contained glucose for energy

requirements. Krebs-Henseleit does not contain amino acids in its composition, nor

does it contain hormones or other growth related factors. Implementation of the

current incubation system involved substantial development and considerable

frustration. For the sake of brevity, preliminary study data will not be presented here,

but a brief overview of the methods will be given.

Initially studies were performed with an adaptation of the Dynamic Organ

Culture System (DOCS) which was developed for use with liver slices. This

incubation technique consists of an incubation vial (liquid scintillation vial, 20 ml

volume), a stainless steel mesh insert and a temperature controlled incubator

equipped with a roller mechanism. Rat skin sections (2x2 cm), prepared as

described in the methods, were placed on the steel mesh and inserted into incubation

vials. Incubation buffer containing 100 nM substrate was added to the vial, the vial

capped and placed in the incubator (37 °C). The rotation of the rollers (approximate­ ly 1.5 rpm) enabled the tissue to be in contact with the buffer and the atmosphere 113 of the vial. The tissue, dermal or epidermal side, was found not the adhere to the inserts and fell into the media. In addition to this, the skin would absorb, i.e. hydration of the tissue, the media from the vial thus leaving no media for analysis of metabolites. Alterations in the size of the vial and tissue to buffer ratios all proved unsuccessful for the generation of detectable levels of metabolites.

Investigations were also performed with 2 x 2 cm squares or strips incubated as described in the methods section (see methods section 6.0). These investigations provided positive results, i.e. detectable metabolites in the media. Alterations in the tissue to volume ratios were investigated and optimized to the systems for which results are presented. The data are presented as product formed/mg dry weight of the tissue used. Expression of results using dry weights was found to provide more consistency to the data than normalization using skin wet weights. Tissue wet weights were dependent upon the hydration status of the tissue which was found to be extremely variable (results not shown). 114 4.1 Xenobiotic Metabolism by Rat Skin Explants in Swirly Culture

A study of the effects of the phase I and phase II metabolism of the model

substrates, 7-EC and 7-HC was undertaken. Incubations were performed as indicated

in the methods section (Swirly Culture). Substrate concentrations employed were 100

nM in all incubations. Control and blank incubations were generated to account for

any fluorescent interferences. The results presented are from two animals from which four replicate incubations were used.

4.1.1 Phase I 7-EC Metabolism: A Comparison of Squares and Strips of Rat Sldn

The time dependent metabolism of 7-EC (100 juM) was quantified in both the

tissue squares and strips (4 cm2/10 ml incubation). The results presented here are from the phase I metabolite generation only; however, tight integration of the phase

I and phase II systems was observed in these experiments as indicated by the low levels of free 7-HC present in the media (results shown in figure 22). Phase I production of 7-HC was found to be time dependent in both types of incubations, but the skin strips produced more 7-HC than the squares (see figure 20). This result may have been anticipated from the fact the square type incubations had surface area of exposure ten fold less than the strip type incubations. Initial rates of phase I 7-EC metabolism were considerably lower than those produced at later time points.

Differential rates of metabolite production may be due time required for the 115 movement of metabolites from the tissue into the media.

sz CTi 20 t 'a>S 1 H Total Phase I (Strips) -a •• Total Phase I (Squares) u> 15-- E •o0>

10 -- (L) cOT o 1 £ a. 8 10 14 TIME (Hours)

Figure 20: The effects surface area of exposure on the metabolism of 7-EC (100 pM) by untreated rat skin explants from F-344 rats. Values represented are the means from two animals (four replicates/animal).

4.1.2 Phase I and Phase II Integrated Metabolism by Rat Skin.

The phase I metabolism of 7-EC by thin strips of rat skin was found to be time dependent with conjugates of 7-HC detectable as early as one hour (figure 21).

Glucuronide conjugates were 0.0S and 0.13 pmoles 7-HC generated/mg dry tissue for 116 the 1 and 2 hour time points respectively. The sulfates conjugates at these same time points were found to be 0.06 and 0.12 pmoles 7-HC generated/mg dry tissue. No free

7-HC was detectable until four hours. A somewhat linear increase was observed over a 10 hour incubation period. However, between the 10 and 12 hour time points there was a doubling of the amount of metabolite present in the media, i.e. from 5.6 to

13.8 pmoles 7-HC generated/incubation/mg dry weight.

-C 20 s Total Phase I Activity

T> 01 c o0) c_> X 5 • • r-.

* * 2 4 6 6 10 12 14 16 TIME (Hours)

Figure 21: The phase I metabolism of 7-EC (100 mM) by thin strips of raf skin in Short Term Culture. 117 Integrated phase I and phase II metabolism of 7-EC is presented in figure 22, as evidenced by low levels of free 7-HC (less than 30% of the total conjugates from glucuronidation and sulfation of 7-HC). Additionally, rat skin was found to preferentially for the glucuronide conjugate of 7-HC. The total metabolism of 7-EC at 8 hours was determined to be 0.09%.

cr> 10-r 'ID •• Free >,3 •H Glucuronides "O 8 - - E3 Sulfates cn E \ <1> 6 - - o c 4 -- O

Figure 22: The integrated phase I and phase II metabolism of 7-EC (100 /tM) by thin strips of rat skin in Short Term Culture. 118 4.1.3 Phase II conjugation of 7-HC by Rat Skin.

The results from figure 23, demonstrate that the increase in the detection of formed conjugates in the media of the skin explant incubations was time dependent.

In all cases, with the exception of 0.5 hour time point, the glucuronide conjugate was found to be greater than the sulfate. The glucuronide conjugates accounted for greater than 75% of the total metabolism of 7-HC over the incubation period examined. This preference for the formation of the glucuronide conjugate was consis­ tent with the conjugation profile observed from the integrated metabolism presented earlier. The use of 7-HC as a substrate resulted in a 150 fold increase for the formation of glucuronides and 93 fold higher for the sulfates. 119 Q) 3 cn 100 -r to Glucuronides EZJ Sulfates ~u ao- OI E T3 a) 60-- a> c OCD 40 -- o X 20 • in _a; o E Q. 0.5 1.0 2.0 8.0 TIME (Hours)

Figure 23: The phase II metabolism of 7-HC (100 mM) by thin strips of rat skin in Short Term Culture. 120 4.1.4 Bacterial Contribution to the Skin Metabolism of 7-EC

Contamination of the incubation media with bacteria of cutaneous origin became a concern due to the extended time of incubation necessary to quantify metabolism. To answer this question, thin strips were incubated as described in the methods section for 12 hours and then the tissue was removed. Bacterial presence was obvious from the stench and turbidity of the media. No attempts were made to characterize the bacterial flora present in the media. The media was then supplemented with glucose ( 2.0 g/liter of media) and 100 /nM 7-EC. These supplemented incubations were incubated for an additional 12 hours at 37°C. No metabolites were detectable in these incubations over the background levels.

In a subsequent study, Gentamicin (0.1,0.2 and 0.5 mg/ml),-a broad spectrum antibiotic, was added to the incubation medium. The results from this study demonstrated that the presence of this antibiotic in the media did not alter the metabolism profile significantly from control levels (no antibiotic). These experiments indicated that the contribution of the bacteria (of cutaneous origin) to the phase I and phase II metabolism of 7-EC was negligible or nonexistent.

4.1.5 The Deconjugation of Para-Nitrophenol (PNP) Derivatives by Rat Skin.

The deconjugation of glucuronide and sulfate conjugates of PNP were determined as described in the methods section. A comparison of the square and strip type incubations for the assessment of the deconjugation of PNP conjugates was 121 examined. As previously seen with the alkoxycoumarin metabolism, increased surface area (with the strip type incubations) effected an increase in the generation of PNP

(results not shown). However, the rat skin strips were found to preferentially deconjugate the sulfate moiety. A time dependency was noted with the appearance of PNP in the media from the sulfate conjugates. Up to 2 hours, a relatively linear profile of PNP appearance was noted; however, by the 4 hour time point a tailing off was observed. From these experiments, it was found that an endogenous aryl- sulfatase activity was present in rat skin. Additionally, no detectable endogenous (3- glucuronidase activity was detected (see figure 24). No attempts were made to determine if the deconjugation activity observed was bacterial or cellular in nature. 122

JZgi 600 -r *d> 3 Giucuronides 500 - - T> Sulfates CT> 400 - • "O1> 300 - - V cQ> O 200-

w 100-- a; O ECL

TIME (Hours)

Figure 24: The deconjugation of PNP derivatives by thin strips of rat skin in Short Term Culture.

4.2 Xenobiotic Metabolism by Rat Skin Explants in Long Term Culture

These studies developed as an extension of the previous work with swirly culture. Inspiration from the work of Trowell (1958) gave rise to the idea of incubations using support matrix which would allow for the maintenance of a more physiological separation, i.e. dermal contact with media and epidermal contact with the atmosphere. In order to more systematically examine this incubation technique, a modification of the swirly culture was compared to this new approach. To this end, the metabolism of the alkoxycoumarins (phase I and phase II) was compared 123 between the use of an agar support block and the placement of the tissue square submerged in the media. Further details of the incubations may be found in the

Methods section of this work.

4.2.1 Phase I and Phase II Metabolism of Alkoxycoumarins: A Comparison of Supported and Submerged Incubations

Whole skin explants were incubated with 7-EC and the production of 7-HC and its glucuronide and sulfate conjugates was monitored. Overall metabolism was

0.4% and 0.7% of total substrate metabolism (100 juM) for the agar supported and submerged skin explants, respectively. The glucuronide conjugate was the major metabolite in both incubation situations (see figure 25). The submerged tissue, after

72 hours of incubation, had a "gel-like" appearance in both the epidermis and the dermis. These submerged tissues were also found to be 10% heavier in wet weight than corresponding tissues on the agar support. The agar support tissues maintained a more "normal" skin appearance with a definite difference between the epidermis and dermis. The hair on the agar supported incubations had a noticeable yellow tinge and could be removed by gentle abrasion; whereas removal of hair on the submerged tissues required more effort. 124

15 T •Free 7-HC 1Glucuronides of 7- •HC X)

Agar Support Submerged

Figure 25: The phase I and phase II integrated metabolism of 7-EC (100 #iM) by rat skin in Long Term Culture

The addition of 7-MC as a substrate (100 ^M) did not produce detectable metabolites in any of the incubations. These results were found to be consistent with those from the skin microsomal metabolism of alkoxycoumarins, i.e. no detectable

O-demethylase activity.

4.2.2 Phase II conjugation of 7-HC by rat skin

As may be observed from the results presented in figure 26, the extent of metabolism was 10 fold higher when 7-HC was employed as a substrate (100 /iM). 125 The total metabolism of 7-HC was determined to be 10.9% and 11.6% for the agar support and submerged incubations respectively. The glucuronide conjugate of 7-HC

accounted for greater than 80 % of the total phase II metabolites, which was

consistent with previous observations of the glucuronide > > sulfate. The generation

of phase II metabolites was not affected by the mode of incubation, i.e. agar support

or submerged.

120 t Glucuronide conjugates E3 Sulfate conjugates "O o o 60 • z:

to 40 -- JD o E c 20--

Agar Support Submerged

Figure 26: The phase II metabolism of 7-HC (100 pM) by rat skin in Long Term Culture. Discussion 127 The major objective of this work was the development of procedures for the quantification of the cutaneous microsomal Mixed Function Oxidase (MFO; i.e.,

Cytochrome P-450 and related flavoproteins) system. A study of the MFO must be established in a model species and characterized with respect to basal activities. The

F-344 rat was the species of choice for the early work in this study, primarily due to its extensive use in toxicological studies. Secondly, there were no methods which were readily applicable for the study of cutaneous P-450 mediated metabolism in normal adult rat tissue. Reasons contributing to the latter situation include difficulties in the preparation of subcellular fractions and low levels of metabolism resulting from these fractions.

The skin, particularly the normal ("hairy") rat, was found to be extremely resistant to conventional homogenation techniques, i.e., mincing with scissors and then using a teflon pestle glass homogenizer. Initial attempts using this method resulted in a viscous glue like material which did not sediment a pellet after centrifugation. After numerous failures, it was found that pulverization of the tissue under liquid nitrogen resulted in a homogenate more amenable to sedimenting a microsomal pellet. A further refinement of the homogenation method was the use of a Polytron homogenizer. Inclusion of a Waring Blendor for the initial pulverization of the skin greatly reduced the time required for homogenation and subcellular fractionation. Using the Blendor, up to 50 g of rat skin may be powdered in less than

30 seconds. An extension of the microsomal work in the rat entailed the use of agents known to induce cytochrome P-450. The effects of these inducing agents, Aroclor compounds (i.e. different preparations of PCB's), have been demonstrated and characterized in the liver with respect to microsomal marker enzymes and substrate metabolism (Bickers, et.aL, 1972; Alvares, etal, 1974; Alvares and Kappas, et.al.,

1977 and Guengerich, 1977). These inducing agents also have demonstrated effects on the extrahepatic P-450 mediated metabolism of certain compounds. The work detailed in this thesis demonstrated the differential induction abilities of the Aroclor isomers, with respect to model substrate metabolism. It was found that the higher chlorinated mixture, Aroclor 1254 (A1254) was found to induce both O-de- methylation and O-deethylation in hepatic microsomes, but effected an induction of only O-deethylation in skin microsomes. The lower chlorinated mixture, Aroclor 1221

(A1221) was found only to induce O-demethylation in hepatic microsomes.

In addition to these microsomal studies with skin and liver, investigations into coupled phase I and phase II metabolism of the model substrates were undertaken in full thickness adult rat skin. The swirly culture technique provided an incubation system which did not provide for a separation of epidermal and dermal exposure of a compound. This system was found to support metabolism over an extended incubation period (up to 12 hours). The long term culture (LTC) incubation system provided a more versatile exposure situation (up to 72 hours). These situations included the dermal exposure of a compound (as investigated) and the possibility of 129 the topical application of a compound (not investigated in this work).

Protein Determinations

As alluded to in the results section, there was a concern with respect to the

method of protein determination and the use of these values in the expression of

results. It was therefore decided, for the ease of comparison to the literature, that the

two most common protein assays be evaluated (Lowry and Bio-Rad assays). Both

methods of protein determination rely on a dye based color development and subsequent comparison to a standard protein.

Both the Lowry and Bio-Rad assays were found to be linear and predictive

between 10 and 80 Mg of protein, which is in agreement with the literature (Lowry,

1958 ; Bradford, 1976). However, the choice of standard was found to be critical for

the Bio-Rad. The use of bovine serum albumin (BSA, standard for the Lowry) as a

standard in the Bio-Rad resulted in statistically significant differences in the

prediction of the protein content of samples when compared to ovalbumin (OA; standard for the Lowiy assay). No significant differences between OA and BSA values were noted when a similar comparisons were made using the Lowry assay.

Presumably, the observed differences were due to the amino acid contents of the proteins and the interaction of the dye with certain of those amino acids. The Bio-

Rad assay, using OA as a standard reference, was chosen for use in this work based upon its relative insensitivity to interferences (for review see Peterson, 1979). A 130 conversion factor of 1.3 was used to allow for the comparison of Bio-Rad and Lowry protein values. This was calculated from the assay comparison data using standard and sample preparations. [It should be clearly stated that the term "Bio-Rad equivalent" refers to the conversion of published or obtained Lowry protein values by the factor referred to earlier].

The rank order for microsomal protein per gram of tissue was found to be maintained between the liver and skin comparisons of the various species, i.e, rat > rabbit > mouse > human. Literature comparisons of the obtained values were cumbersome, to say the least. Of the literature reviewed, many investigators did not cite the method of protein determination, which made the normalization to a Bio-

Rad equivalent not possible. Litterst, et.al, (1975) presented a rank ordering of mouse > rat > rabbit, although the strains of mouse and rat differed from those used in our work the order was consistent with our results. Microsomal yields from

B6C3F1 male mouse liver were found to be in agreement with those reported elsewhere (Caldwell, et.aL, 1985).

Value comparisons for skin were not available for rat, rabbit and human skin microsomal yields. A comparison of skin microsomal protein yields in Swiss Webster mice obtained from Pannatier, et.aL, (1981) was found to be similar to our work when corrected to Bio-Rad equivalents.

Microsomal pellets from all species were found to contain a whitish core which was distinct from the remaining opaque microsomal pellet. The rat and human 131 skin were the most difficult to homogenize. Rat and human skin microsomes were found to contain more of the whitish core substance in the microsomal pellets than rabbit or mouse skin microsomes. Difficulties encountered with resuspension of microsomal pellets from rats and humans containing the white core of material was reminiscent of difficulties associated with the structural matrix in the early stages of the development of homogenation procedures. The skin from rabbits and mice was less resilient to homogenation than rats and humans, presumably due to a reduced amount of support matrix. Based upon these observations and personal judgements, the core was probably support matrix in origin* i.e. keratin or collagen.

Aroclor induction was shown to increase the protein contents of liver microsomes; although upon initial examination, these increases in liver microsomal protein were not found to be statistically significant. Since Aroclor induction also increases liver size and weight, an alternative method of the expression of protein results was used (total microsomal protein/liver). This means of expression did demonstrate statistically significant increases in hepatic microsomal protein contents in the Aroclor pretreated animals. The results from our studies, for liver, were consistent with earlier reports examining these parameters (Alvares, et.aL, 1974;

Alvares and Kappas, 1977). There were no measurable differences between the treatment groups as far as skin microsomal protein content was concerned with either expression method (i.e. per mg protein and per g tissue). To reiterate a point from difficulties associated with the preparation of homogenates of rat skin, the skin 132 microsomes may have been contaminated with support protein which may mask any

alterations in protein content. No relevant literature was available to allow for the

comparison of skin microsomal protein contents of the control or Aroclor pretreated

animals.

Cytochrome P-450

The P-450 content of skin microsomal preparations from rat, rabbit, mouse and human was not demonstrable spectrophotometrically in our work. The inability

to spectrophotometrically determine P-450 was found to be consistent with previous investigations (Norred and Akin, 1976; Pannatier, et.aL, 1978; Rettie, et.aL, 1986;

Mukhtar and Hasan, 1989). In the work of Pannatier, et.aL, it was stated that the peak at 450 nm in skin microsomal preparations was not to be considered definitive for the presence or absence of P-450. Spectrophotometric masking of the P-450 spectra from cytochrome oxidase was prevalent in the skin microsomal preparations.

Pohl, et.aL,(1976) reported cutaneous P-450 contents of 10 pmol/mg protein from

NMRI mice and 22 pmol/mg protein in Swiss mice. These measurements were determined to be one order of magnitude below the detection limits of the instrumentation available for our work.

Contamination of the microsomal pellets with support proteins may have diluted the specific microsomal protein content, thus contributing to the observed reduced enzyme activity. Various protein dilutions from 0.125 to 2 mg/ml were 133 assayed spectrally to determine if the protein dilution was responsible for the reduced activity; however these efforts met with no success. As may be readily appreciated,

the confirmation of P-450 spectrophotometrically in skin microsomal fractions was complicated by the presence of many systematic variables beyond investigator control.

A literature review of hepatic P-450 contents, expressed as nmol P-450/mg protein, from untreated male F-344 rats were found to range between 0.57 and 1.22

(Gold and Windell, 1975; Kao and Hudson, 1979). The values reported in our work were consistent with the lower end of the range for age and weight matched rats (i.e.

0.57 nmol P-450/mg protein). Similarly, literature citings of male New Zealand

Rabbit hepatic P-450 contents were found to have a three fold range of values, i.e.,

0.52 to 1.50 (El Amri, et.aL, 1986, Kaul and Novak, 1987; Chhabra, et.aL, 1974;

Miranda and Chhabra, 1980; Litterst et.aL, 1975). The values obtained in our studies were consistent with those of El Amri, et.aL (0.52 nmol P-450/mg protein). The P-

450 content obtained for male B6C3F1 mice in our work (0.60 nmol P-450/mg protein) was in agreement with those cited in the literature. The literature values showed an almost two fold difference in the range of values for this strain, i.e, 0.38 to 0.61 (Degawa, et.aL, 1984; Caldwell, et.aL, 1985; Mitchell, et.aL, 1987). Surprisingly, the literature review revealed that human P-450 contents did not span as great a range of values. Values were found to range between 0.16 and 0.24 nmoles P-450/mg protein (Alvares, 1969; El Amri, et.aL, 1986). The results of our P-450 content quantified for human liver (0.21 nmol P-450/mg protein) were found to be within the 134 range of published values.

Increases in hepatic P-450 content and the shift of the absorbance maxima to

448 nm were consistent with the results previously reported using A1254 (Alvares and

Kappas, 1977; Parkinson, et.aL. 1983). A1221 pretreatment produced a moderate increase in the total hepatic P-450 content and an absorbance maxima at 449 nm.

Lower chlorinated mixtures of Aroclor (A1221) were found to be less potent than the

A1254 mixture in their total hepatic P-450 inducing abilities. This reduced induction capacity may be related to the fact that lower chlorinated mixtures are subject to metabolism to a greater extent than the higher chlorinated mixtures (such as A1254) and thus may not be able to attain effective concentrations for induction to occur

(Bickers, et.aL, 1972). Furthermore, the absorbance maxima shift to 449 nm in the

A1221 pretreated rats would indicate that the isozyme profile in the livers from these animals would be distinct from those of control and A1254 pretreated rats.

Cytochrome c Reductase

The activity of Cytochrome P-450 reductase (reductase) was indirectly measured by the reduction of cytochrome c in subcellular fractions of skin and liver.

The reductase has been shown to be an integral part of the functional P-450 system

(Williams and Kamin, 1962) and is therefore used as an indicator for the presence of the MFO system. Homogenate, S-9, cytosolic and microsomal fractions, were all analyzed for reductase activity. Reductase activity was found to be the greatest in the 135 microsomal fractions isolated from both skin and liver in all species examined

(results not shown). The reductase was therefore used as a convenient marker enzyme for the isolation of the microsomal fraction from liver and skin.

The stoichiometiy of the reductase to P450, from liver, has been reported to be between 1:10 and 1:30 (Sipes and Gandolfi, 1986). This stoichiometry would favor conditions for the detection of the P-450 spectrophotometrically. In the skin microsomal fractions, the reductase was preferentially detected (Finnen, et.aL, 1985).

This would suggest that there may be an altered stoichiometry of reductase and P-450 in the skin. Differences in reductase-P450 stoichiometry have previously been correlated with differences in the MFO related metabolism of certain compounds

(Strobel, et.aL, 1980).

The rat and human skin reductase activities were observed to be the lowest of the species examined, possibly due to systematic reasons associated with the preparation of skin microsomes. Stoichiometric differences between species may also be responsible for the differences observed. The lack of literature on this subject did not allow for comparisons of the reductase values attained in our work.

The rat liver values from untreated F-344 rats reported here were found to be consistent with those reported by Gold and Windell (1975). However, values were

30% lower than those reported by Kao and Hudson (1980). The range of values from the literature was between 103 and 178 nmoles cytochrome c reduced/min/mg protein. Rabbit liver reductase values were found to range between 88 and 142 136 nmoles cytochrome c reduced/min/mg protein (Bio-Rad equivalent) (El Amri, et.al,

1986; Litterst, et.aL, 1975; Chhabra, et.al, 1974). Our values were approximately 36% lower than those cited in the literature. Human liver reductase values (87 nmol cyt c reduced/ min/mg protein) were found to be consistent with those reported by El

Amri, et.aL, (1986) ,i.e. 76 ± 11 (± S.D.). No such value comparisons were available for the mouse liver reductase activities. The rank order for reductase activities in the liver were found to be different from those of the P-450 contents. As previously mentioned, this may suggest that these stoichiometric differences may contribute to differences in the metabolism of certain compounds by different species.

The pretreatment of F-344 rats with Aroclor mixtures (A12S4 and A1221) produced interesting effects in the skin and liver reductase activities. Skin reductase activities showed no statistically significant difference between the control and treatment groups. A1254 treatment did not result in a statistically significant increase in hepatic reductase activity; however, other investigators have found statistically significant increases in the reductase activity following A12S4 pretreatment. These increases were found to be characteristic of phenobarbital (PB) type P-450 inducing agents (Alvares, et.aL, 1974: Alvares and Kappas, 1977). A1221 was not observed to effect any alterations in activity. It may be therefore suggested by these findings that

A1221 does not express PB type induction qualities that A1254 does. The inability of A1221 pretreatment to effect any measurable changes in the parameters examined may also be related to the reduced chlorine content, as discussed earlier. In the 137 absence of confirmatory spectrophotometric results, reductase activity was instrumental in confirming the presence of the MFO system from skin and liver subcellular preparations.

Alkoxycoumarin Metabolism

Alkoxycoumarins have been widely used as substrates for the assessment of hepatic P-450 metabolism in both control and induced animals (Matsubara, et.aL,

1982, 1983; Guengerich, et.aL, 1985; Rettie, et.aL, 1986). The utility of various the alkoxy derivatives for cursory evaluation of the P-450 isozyme profile was described by Matsubara, et.aL. (1982). From these studies it was found that the liver O- dealkylase activity was increased towards the O-ethyl, O-propyl, and O-butyl derivatives of 7-HC in rats pretreated with 3-MC or j9NF. These inducing agents are known to induce a series of P-450 isozymes classified as "P-448" or "Aryl Hydrocar­ bon Hydroxylase" (AHH). Basal activities for the cutaneous metabolism of alkoxycoumarin derivatives have not been clearly elucidated (Goerz, et.aL, 1979).

Hepatic O-deethylation was demonstrated to proceed at a significantly higher rate than O-demethylation in control rat, rabbit and mouse microsomes. This observation was found to be consistent with the work of Matsubara, ef.ai, (1982,

1983). Curiously, in humans, the hepatic O-demethylase activity was found to be significantly higher than the O-deethylase activity. However, results from our studies with human hepatic microsomal metabolism of alkoxycoumarins were found to be 138 consistent with the work of Matsubara, et.aL (1986).

Liver O-deethylase activities were found to be the highest in the rat, which was also found to have the highest P-450 content. The rabbit and mouse contained essentially similar amounts of P-450.O-deethylation, in mouse liver microsomes, was found to proceed at a much higher rate than in the rabbit liver microsomes.

Differential rates of metabolism from livers with similar P-450 contents emphasizes the importance of the consideration of isozyme composition in explaining spe­ cies/tissue differences in the metabolism of a compound. These observations concerning isozyme content from our work were found to be supported by previous investigations of species differences in coumarin metabolism (Gangolli, et.aL, 1974;

Feuer, 1980).

The species rank order for cutaneous metabolism of alkoxycoumarins was found to parallel the relative ease of homogenation of skin tissue (i.e. mouse > rabbit > rat, with respect to ease of homogenation). Thre treatment of the tissue during preparation may be associated with the activity of the enzyme preparation.

That is to say, the harsh treatment that the rat required for homogenation may have inactivated or destroyed some of the enzyme activity in the skin samples. The mouse was found to posses the highest cutaneous O-deethylase activity of the species examined and it was the only species to express cutaneous O-demethylase activity.

Cutaneous O-deethylase activities for rabbit were significantly lower than those found in the mouse skin. Rat skin O-deethylase activity was significantly lower than that 139 found in rabbit skin. No relevant literature was available for comparison of these cutaneous metabolism results.

Mouse skin microsomal metabolism was observed to follow the trends of O- deethylation > O-demethylation observed in the liver microsomes. Differences observed in the hepatic metabolism of 7-EC and 7-MC may offer an explanation of the lack 7-MC metabolism in skin microsomes, i.e. skin O-demethylase activities are much lower than those of skin O-deethylase. In addition, tissue specific expression of certain isozymes or differential inactivation of them during preparation may offer an explanations for the lack of O-demethylase activity observed in skin microsomes

(Akin and Norred, 1976; Pannatier, et.aL, 1978; Adesnick and Atchison, 1981; Martin,

1987).

Reasons for the lack of activity in human skin microsomes towards 7-EC and

7-MC still remain elusive. We postulate that the lack of activity may be related to origin of the tissue, i.e. human cadaver skin removed at the time of post mortem.

Conclusions from these observations would have required investigations using skin biopsy samples from living donors. Unfortunately, this tissue source was not available during the course of this line of experimentation. Therefore, we put little weight in the data obtained using human skin preparations.

Pretreatment of rats with Aroclor mixtures resulted in a differential induction of the O-dealkylase activities. A12S4 is a well known broad spectrum inducing agent which has been shown to induce both 3-methylcholanthrene (3-MC) and PB type P- 140 450 isozymes. A1254 was found to induce cutaneous and hepatic O-deethylation;

furthermore, A1254 also induced hepatic O-demethylation. A1221 was found to

preferentially induce hepatic O-demethylation to a greater extent than A1254. The

hepatic alkoxycoumarin metabolism results obtained with A1254 pretreatment were consistent with the findings of Matsubara et.aL (1982,1983) who used 3-MC and PB

independently as inducing agents. From these studies, hepatic O-deethylation of 7-EC was found to be preferentially induced by 3-MC, with no induction of O-demethylase activity. PB pretreatment increased hepatic O-demethylase and O-deethylase activities. The effects observed from our studies would indicate that A1254 was acting as a mixed 3-MC and PB type P-450 inducing agent.

Intraperitoneal treatment with A1254 resulted in an induction of the cutaneous P-450 which was demonstrated by a 7 fold increase in cutaneous O- deethylase activity. This type of induction was found to be consistent with the work of Alvares, et.aL (1974). These investigators found increases in the both hepatic and cutaneous AHH activity (P-448 mediated metabolism) after either topical or intraperitoneal administration of inducing agents. In our work, skin O-demethylase activity was not detectable in any of the Aroclor pretreated groups. As discussed previously, tissue specific expression or differential inactivation of P-450 isozymes may account for the substrate specificity observed.

No clear identification of which isozyme(s) is(are) responsible for the metabolism of 7-EC is possible; however, increases in rat hepatic 0NF-B and PB-B 141 isoforms were found to correlate with an increase in hepatic O-deethylase activity

(Guen-gerich, etal 1982). These studies suggest that other isozymes were also found

to contribute to the O-deethylase activity. Information regarding specific isozymes

and O-demethylase activity, using 7-MC, as a substrate was not was not available.

Furthermore, P-450 isozyme profiles from A1221 pretreatment of rats were not

available from the literature. Therefore, any conclusions reached now would be based

upon the work presented here in light of previous work with higher chlorinated

Aroclors. The induction results presented herein and from kinetic parameter analysis

(data not given here) would suggest that in F-344 rat liver there are distinct isozymes

which catalyze the O-demethylation and O-deethylation of 7-MC and 7-EC,

respectively. The results from the species differences and induction metabolism studies also support the idea that adult rat skin does not possess the isozyme(s)

necessary for the O-demethylation of 7-MC.

Rat Skin Explant Metabolism: STC

As stated in the results section, the swirly culture incubation system was the

first successful attempt in the demonstration of both cutaneous O-dealkylase (phase

I) and phase II activity. The phase I metabolism of 7-EC was found to be time dependent and tightly integrated with phase II metabolism. The metabolically inert support matrix of the skin in both epidermal and dermal layers, may contribute to the low rates and linear divergence reported. For a compound to be metabolized, it 142 must first traverse the inert support matrix of the skin and then come into contact with the drug metabolizing enzymes. Once the compound has been metabolized (free, glucuronide or sulfate) it must diffuse or be transported out of the tissue into the media. A contributing factor to the diffusion/transport of metabolites would be the state of hydration. Changes in the support matrix due to hydration of the tissue may contribute to the non-linearity of metabolite appearance in the media. As may be readily appreciated, there are a number of variables to be considered in the process of cutaneous metabolism in a culture situation.

The use of skin strips to increase in surface area of exposure showed that the access of the compound to the drug metabolizing enzymes was a limiting factor in the rate of metabolism. The metabolites detected in the media followed a linear pattern up through the 10 hour incubation. However, between the 10 and 12 hour incubation points there was a greater than 100 % increase in the amount of 7-HC detectable in the media. This would suggest that there may be changes in the tissue due to excessive hydration (i.e. tissue was submerged in buffer for up to 12 hours) which would permit a greater influx and outflow of metabolites.

Investigations into phase II metabolism by rat skin explants were possible with the use of 7-HC as a substrate. The levels of phase II metabolism were found to far exceed those of phase I which were found to be consistent with results obtained using other tissues (for review see Dutton, 1980; Mulder, 1980). In both cases, using 7-EC or 7-HC as the substrate, the glucuronide conjugate of 7-HC was found to predomi­ 143 nate in the media. This preference for cutaneous glucuronidation activity was also noted by Moloney, et.al, (1982). These investigators used Wistar rat skin which had been shaved and incubated in a rich tissue culture media (L15 medium containing fetal calf serum).

Since the skin has a bacterial flora, concerns were expressed as to whether the metabolism that was measured was cellular or bacterial in nature. No attempts were made to classify the bacterial flora since it was considered beyond the scope of this work. Investigations involving media and antibiotic additions supported the idea that the metabolism was cellular in origin and that the bacteria did not contribute to the metabolism of the alkoxycoumarins.

Previous investigators have reported the presence of ^-glucuronidase and aiylsulfatase activity in the media of skin incubations in vitro (Moloney, et.aL, 1982).

These investigators found /3-glucuronidase activity towards the glucuronide conjugate of 4-methyl umbelliferone, but no arylsulfatase activity towards the sulfate conjugate.

The results presented in our work supported the presence of an aryl sulfatase, but no detectable ^-glucuronidase activity. Deconjugation activity was seen to be increased by increases in surface area, i.e. square vs. strip studies. It must be remembered that in these incubation situations involving deconjugation, the quantification of metabolites is a measure of an equilibrium process. This deconjugation activity may have contributed to the higher levels of free 7-HC observed at later incubation time points. 144 Rat Skin Explant Metabolism: LTC

This system represented, in the author's opinion, a logical extension of the

swirly culture method. The information gained from the swirly culture was used to

assess rat skin explant metabolism of alkoxycoumarins up to 72 hours. Antibiotics,

antifungals were added to the media to suppress bacterial and fungal growth over the

extended incubation period.

The submerged tissue, "gel-like" in appearance, was found to be increased in

weight (approximately 10%) over the agar support tissue, which was primarily due

to excessive hydration. The hydration state of this tissue may have allowed for

increased substrate availability and metabolite exit. Thus demonstrating again that

the phase I metabolism of the model substrates was limited by substrate access to the

enzymes. The conjugation profiles for 7-HC from rat skin explants were found to be

in opposition to those observed from hepatic studies using these compounds. Barr, et.aL, (1990) found that liver slices incubated in dynamic organ culture showed a

definite preference for formation of the sulfate conjugate of 7-HC.

To better exemplify the magnitude of the support matrix effect, a comparison

of the rates microsomal and skin explant metabolism of alkoxycoumarins will be

presented. The skin microsomal metabolism of 7-EC yielded a rate of 120

pmol/hour/mg microsomal protein. The culture incubations yielded rates of 6.95 and

11.63 pmol/hour/mg microsomal protein (based upon average microsomal protein yield mg/g tissue) in agar and submerged incubations, respectively. The lack of O- 145 demethylase activity in rat skin microsomes was confirmed in the results from the

LTC rat skin explant studies.

The results of these investigations have shown the development of a microsomal isolation procedure for cutaneous tissue. The use of alkoxycoumarins, with the major fluorescent metabolite 7-HC, have allowed for a characterization of basal MFO activity in the microsomal fractions isolated from three species; rat, rabbit and mouse. Furthermore, increased microsomal O-deethylation of 7-EC after

A1254 pretreatment suggested this substrate may be a useful indicator of cutaneous

MFO induction. Additionally, developmental studies with rat skin explants in culture demonstrated that the rat skin was capable of integrated phase I and phase II metabolism. These explants were also found to possess an arylsulfatase that was released from the tissue and was able to deconjugate sulfate moieties at physiological pH. These studies also demonstrated the usefulness of the alkoxycoumarins, 7-EC and 7-HC, as model substrates for the assessment of phase I and phase II metabolism in skin explant cultures. 146 Appendix A

Human Liver Histories

Liver Age Sex Race Cause of Death/ Medical History I.D. D20 49 F Cauc Coronary artery aneurysm rup­ ture D21 23 M Cauc Motor vehicle accident/ recre­ ational drug use. D22 18 M Cauc Motor vehicle accident/ seizure disorder. D23 31 F Cauc Internal cerebral hemorrhage/ mutliple congential abnormailites D24 11 M Cauc V-P shunt obstruction malfunc­ tion/ hydrocephalus, seizures D26 29 M Cauc Self inflicted gunshot wound to head/ alcohol abuse, asthma D43 N.A. N.A. N.A. N.A. D44 NA N.A. N.A. N.A. D45 N.A. N.A. N.A. N.A. D46 N.A. N.A. N.A. N.A.

N.A. = Patient information was not available. 147 LITERATURE CITED

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