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Cunningham, Mary Jane

THE INDUCTION AND INHIBITION OF BENZO(A)PYRENE METABOLISM IN HUMAN EPIDERMAL KERATINOCYTES AND DERMAL FIBROBLASTS

The Ohio State University Ph.D. 1985

University Microfilms

International 300 N. Zeeb Road, Ann Arbor, Ml 48106

Cunningham, Mary Jane

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University M icrofilm s International THE INDUCTION AND INHIBITION OF BENZO(A)PYRENE METABOLISM IN HUMAN EPIDERMAL KERATINOCYTES AND DERMAL FIBROBLASTS

DISSERTATION

Presented 1n Partial F ulfillm ent of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Mary Jane Cunningham, B.A.

*****

The Ohio State University

1985

Reading Committee: Approved By

Dr. George E. Milo Dr. Ronald W. Trewyn Dr. Dorothy E. Schumm Dr. Donald T. Witiak Adviser Department of Physiological Chemistry Copyright by Mary Jane Cunningham 1935 Dedicated To My Family

i i ACKNOWLEDGEMENTS

I would like to thank Dr. Milo for his guidance and support and

Lalitha, Raman, Ponamma, Irene, and Marty for th e ir advice and input.

I am grateful to Inge for the use of her slides of the immunofluorescent

staining of human keratinocytes and for teaching me the fine art of

tissue culture and also to Prashant and Gerold who aided in starting

primaries. I am appreciative to Jeff for sharing his mechanical and

technical knowledge of the HPLC (especially in times of malfunctions),

to Jenny for showing me how to culture human lung c e lls, and to Aline for typing this dissertation. Finally, I am indebted to my family for th e ir constant support and encouragement, especially Mom, Dad, Caryl,

B ill, and Blatts, and in memorium, Grandma Mary and Grandpa, Grandma

Jane and Aunt Hilda. VITA

October 17, 1958 Born - East Cleveland, Ohio

1980 B.A. (Biology, Chemistry), Western Reserve College, Case Western Reserve University, Cleveland, Ohio

1980-1981 Teaching Associate, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio

1981-1984 Research Associate, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio

PUBLICATIONS

Cunningham, M.J., Kurian, P., Tejwani, R., and Milo, G.E., Metabolism of Benzo(a)pyrene in Human Skin Fibroblasts under Transforming and Non-Transforming Conditions. Mol. Pharmacol. Submitted.

Cunningham, M.J., and Milo, G.E., The Effect of Cytochrome P-450 Inducers on the Metabolism of Benzo(a)pyrene in Human Epithelial Keratinocytes in Vitro. In preparation.

Cunningham, M.O., Kurian, P., and Milo, G.E., Benzo(a)pyrene Metabolite Distribution and Delivery of a Carcinogenic Insult in Human Fibroblasts. Fed. Proc. 43: 2203 (1984).

FIELDS OF STUDY

Major Field: Biochemistry

Studies in Metabolism and Transformation of Human Cells in V itro by Chemical Carcinogens. Professor George E. Milo TABLE OF CONTENTS

Page

DEDICATION...... i i

ACKNOWLEDGMENTS ...... i i i

VITA...... iv

LIST OF TABLES...... vi

LIST OF FIGURES...... v iii

LIST OF ABBREVIATIONS...... xi

CHAPTER:

I. BENZO(A)PYRENE METABOLISM IN RANDOMLY-PROLIFERATING, SYNCHRONIZED, AND CONFLUENT HUMAN NEONATAL FORESKIN FIBROBLASTS ...... 1

A. Introduction ...... 1 B. Materials and Methods ...... 16 C. Results and Discussion ...... 23

I I . GROWTH AND MORPHOLOGICAL CHARACTERISTICS OF HUMAN EPITHELIAL KERATINOCYTES ...... 40

A. Introduction ...... 40 B. Materials and Methods ...... 48 C. Results and Discussion ...... 52

I I I . EFFECTS OF CYTOCHROME P-450 INDUCERS ON BENZO(A)PYRENE METABOLISM IN HUMAN EPITHELIAL KERATINOCYTES ...... 68

A. Introduction ...... 68 B. Materials and Methods ...... 73 C. Results and Discussion ...... 78

CONCLUSIONS...... 128

BIBLIOGRAPHY...... 131

v LIST OF TABLES

Table Page

1. Indices of Transformation in the Different Populations of Human Neonatal Foreskin Fibroblasts ...... 14

2. Retention Times of Benzo(a)pyrene Metabolite Standards on a DuPont Zorbax ODS HPLC Column (6.2 mm ID x 25 c m ) ...... 24

3. Organic-Extractable Metabolites of Benzo(a)pyrene in the Extracellular Fractions of the Different Fibroblast Populations 30

4. Organic-Extractable Metabolites of Benzo(a)pyrene in the Cytoplasmic Fractions of the D ifferent Fibroblast Populations . 34

5. Organic-Extractable Metabolites of Benzo(a)pyrene in the Nuclear Fractions of the D ifferent Fibroblast Populations . . . 37

6. Equimolar Aliquots of Stock Solutions of Pretreatment Chemicals Added to Epidermal Keratinocyte Medium ...... 75

7. Groups of Pretreated and Nonpretreated Epithelial Cultures. . . 76

8. Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular Fractions from Human Epithelial and Fibroblast Populations ...... 82

9. Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular Fractions from Human Epithelial and Fibroblast Populations ...... 84

10. Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular Fractions of Epithelial Cells Pretreated with 3-Methylcholanthrene ...... 88

11. Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular Fractions of Epithelial Cells Pretreated with 3-Methylcholanthrene ...... 93

12. Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular Fractions of Epithelial Cells Pretreated with Phenobarbital ...... 96

vi Table Page

13. Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular Fractions of Epithelial Cells Pretreated with Phenobarbital ...... * ...... 100

14. Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular Fractions of Epithelial Cells Pretreated with Isosafrole ...... 103

15. Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular Fractions of Epithelial Cells Pretreated with Isosafrole ...... 107

16. Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular Fractions of Epithelial Cells Pretreated with Arochlor 1254 ...... I l l

17. Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular Fractions of Epithelial Cells Pretreated with Arochlor 1254 ...... 114

18. Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular Fractions of Epithelial Cells Pretreated with Butylated Hydroxyanisole ...... 119

19. Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular Fractions of Epithelial Cells Pretreated with Butylated Hydroxyanisole ...... 122 LIST OF FIGURES

Figure Page

1. Chemical Structures of Pyrene, Benzo(a)pyrene, and Benzo(e)pyrene ...... 4

2. The Most Active Metabolic Pathway for Benzo(a)pyrene According to the Bay-Region Theory ...... 6

3. Known Metabolic Pathways for Benzo(a)pyrene ...... 9

4. Formation of Tetrols from BPDE Stereoisomers by Epoxide Hydrolase ...... 10

5. Chemical Structures of DMBA and TH-DMBA ...... 13

6. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Extracellular Fractions of the Different Fibroblast Populations ...... 28

7. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Cytoplasmic Fractions of the D ifferent Fibroblast Populations ...... 33

8. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Nuclear Fractions of the D ifferent Fibroblast Populations ...... 36

9. The D ifferent Layers of the Epidermis of the Skin ...... 41

10. Changes in the Morphology of Epidermal Keratinocytes as They Migrate Upward Through the Layers of the Epidermis ...... 43

11. Phase-Contrast Photomicrograph of Preconfluent Epithelial Colonies Growing in Vitro (X 4 0 ) ...... • ...... 56

12. Phase-Contrast Photomicrograph of Preconfluent Epithelial Colonies Growing in Vitro (X 100) ...... 56

13. Phase-Contrast Photomicrograph of a Preconfluent Fibroblast Culture (X 4 0 ) ...... 57 Figure Page

14. Phase-Contrast Photomicrograph of a Preconfluent Fibroblast Culture (X 100) ...... 57

15. Phase-Contrast Photomicrograph of a Confluent Epithelial Culture (X 200) ...... 59

16. Phase-Contrast Photomicrograph of a Confluent Fibroblast Culture (X 4 0 ) ...... 59

17. Epifluorescent Photomicrograph of an Epithelial Culture Stained with Rhodanile Blue (X 100) ...... 61

16. Phase-Contrast Photomicrograph of an Epithelial Colony Stained with the Mallory Stain (X 1 0 0 ) ...... 61

19. Phase-Contrast Photomicrograph of an Epithelial Culture Stained with the Mallory Stain (X 2 0 0 ) ...... 63

20. Phase-Contrast Photomicrograph of an E pithelial Culture Stained with the Mallory Stain (X 40) ...... 63

21. Phase-Contrast Photomicrograph of a Confluent Fibroblast Culture Stained with the Mallory Stain (X 1 0 0 ) ...... 64

22. Immunofluorescent Staining of Epidermal Keratinocytes with Anti serum against Human Keratin (X 400) ...... 65

23. Immunofluorescent Staining of a Cross Section of Human Neonatal Foreskin with Anti serum against Human Keratin (X 160) ...... 65

24. The Mechanism of Action of Cytochrome P-450 ...... 69

25. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Extracellular Fractions of the Epidermal Keratinocyte Cultures and Control Samples ...... 80

26. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Extracellular Fractions of the Epidermal Keratinocyte Cultures Pretreated with 3-Methylcholanthrene . . 87

27. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the In tra ce llu la r Fractions of the Epidermal Keratinocyte Cultures Pretreated with 3-Methylcholanthrene . . 91

28. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Extracellular Fractions of the Epidermal Keratinocyte Cultures Pre treated with Phenobarbi ta l ...... 95

i x Figure Page

29. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Intracellular Fractions of the Epidermal Keratinocyte Cultures Pretreated with Phenobarbital ...... 99

30. Histograms of Organic-Extractable Radiolabelled Benzo(aJpyrene Metabolites in the Extracellular Fractions of the Epidermal Keratinocyte Cultures Pretreated with Isosafrole ...... 102

31. Histograms of Organic-Extractable Radiolabelled Benzo(aJpyrene Metabolites in the Intracellular Fractions of the Epidermal Keratinocyte Cultures Pretreated with Isosafrole ...... 106

32. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Extracellular Fractions of the Epidermal Keratinocyte Cultures Pretreated with Arochlor 1254 ...... 110

33. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Intracellular Fractions of the Epidermal Keratinocyte Cultures Pretreated with Arochlor 1254 ...... 113

34. Histograms of Organic-Extractable Radiolabelled Benzo(aJpyrene Metabolites in the Extracellular Fractions of the Epidermal Keratinocyte Cultures Pretreated with Butylated Hydroxyanisole 118

35. Histograms of Organic-Extractable Radiolabelled Benzo(a)pyrene Metabolites in the Intracellular Fractions of the Epidermal Keratinocyte Cultures Pretreated with Butylated Hydroxyanisole 121

36. Histograms of Organic-Extractable Radiolabelled Benzo(aJpyrene Metabolites in the Extracellular Fractions of Three Epidermal Keratinocyte Cultures Pretreated with 10 ug/ml Phenobarbital . 125

x LIST OF ABBREVIATIONS

AHH Aryl Hydrocarbon Hydroxylase

BHA Butylated Hydroxyanisole

BHT Butylated Hydroxy toluene

BP Benzo(a)pyrene

BPDE Benzo(a)pyrene Diol Epoxide

DMBA 7,12-Dimethylbenz(a)anthracene

DTT Dithiothreitol

EDTA (Ethylenedinitrilo)-tetraacetic Acid Dicalcium Salt

EGTA [Ethylenebis(oxyethylenenitrilo)]- tetraacetic Acid

EKM Epithelial Keratinocyte Medium

FBS Fetal Bovine Serum

GSH Glutathione

HBSS Hanks' Balanced Salt Solution

HPLC High-Pressure Liquid Chromatography

ISF Isosafrole

3-MCA 3-Methylcholanthrene

MCG Membrane-Coating Granules

MEM Minimum Essential Medium

MeOH Methanol

ODS Octadecylsilane xi NaHC03 Sodium Bicarbonate

Na2SO^ Sodium Sulfate b - nf B-Naphthoflavone

PB Phenobarbi tal

PBS Phosphate Buffered Saline

PCBs Polychlorinated Biphenyls

PCN Pregnenolone-16a-carboni t r i

PDL Population Doubling Level

PMSF Phenylmethylsulfonyl Fluori

RLI Radiolabelling Index

TH-DMBA 1,2,3,4-Tetrahydro-7,12- dimethyl benz( a Janthracene

UT Untreated CHAPTER 1

INTRODUCTION

Benzo(a)pyrene or BP is the most studied compound belonging to the class of polycyclic aromatic hydrocarbons (PAH). I t is formed during pyrolysis of fuels and other organic materials and is a pollutant ubiquitous in the environment. Its association with cancer firs t began in 1775 when Sir Percivall Pott noticed an increased incidence of scrotal cancer in chimney sweeps. Previously, this disease, also known as "soot-wart" was thought to be veneral in origin and was treated using mercurials. However, Pott reported the cause as the "lodgment of soot in the rugae of the scrotum" (86,239). Later, his son-in-law, Sir James

Earle, republished his findings and noted that the cancer occurred also at other sites of the body such as the face, neck, and arms (86,78).

Then, in 1875, Volkmann reported three cases of scrotal cancer in workers of the coal tar and paraffin industries which were s im ilia r to the cancer seen in the chimney sweeps (314,47). Other studies by Bell

(29) and Butlin (47) also reported similiar results. In addition,

Butlin published a series of lectures in which he observed a lowered incidence of cancer in chimney sweeps in some European countries and advocated that this was due to protective clothing and daily baths (46).

The firs t investigators to take material from these trades and successfully demonstrate that i t did lead to cancer were Yamagiwa and

1 Ichikawa in 1915. They painted the ears of rabbits with coal tar every

two to three days for several months. Later, tumors were observed at

the sites of application (106,337). By detailing observations from

these types of experiments, Yamagiwa and Ichikawa classified tumor

development into four stages: atypical growth of the epithelium,

appearance of folliculoepitheliom a, production of carcinoma, followed by

metastasis (336).

Next, there were attempts to identify the carcinogenic component of

coal tar. Kennaway, in 1924, concluded that the substance was

predominantly aromatic (161) and probably a polycyclic aromatic

hydrocarbon of unknown structure (160). In 1933, Cook et a l. purified

two compounds from coal tar with a molecular formula of C20H12' a major

component (melting point 176°C) and a minor component (melting point

187°C). They then synthesized two new PAH compounds (Figure 1),

benzo(a)pyrene and benzo(e)pyrene, and found th e ir fluorescence spectra

to be identical with the major and minor substances, respectively.

Also, both the isolated and synthesized benzo(a)pyrene were discovered to be highly tumorigenic (63,238).

The only in vivo human skin painting experiment recorded to date was performed in 1939. After a previous report by Haddow (116) who advocated that benzo(a)pyrene treatment regressed transplantable tumors,

Cottini and Mazzone treated 26 volunteers with a 1.02 BP solution. Even though no effects of BP were observed in skin already involved in basal cell carcinoma or psoriatic lesions, areas of normal skin showed

"definite manifestations". These areas were concluded to be 3

Figure 1. Chemical Structures of Pyrene, Benzo(a)pyrene, and

Benzo(e)pyrene. The nomenclature for benzo(a)pyrene denotes

that the additional benzyl group is attached to the "a" side

of the pyrene molecule. In benzo(e)pyrene, the benzyl group

is attached to the "e" side of pyrene. P y r e n e

B e n z o ( a ) p y r e n e

Be n z o ( e ) p y r e n e preneoplastic and it was suggested that if treatment had beer prolonged,

they eventually would have become neoplastic (67).

Various theories of the action of BP were developed next and have

been reviewed extensively (238,271,147,65,98). The firs t theory was put

forth by the French scientists, the Pullmans, who suggested that the

K-region (Figure 2) of the aromatic molecule was responsible for its

activity because of a localization of a high degree of electron density

(245,238). Then, four years la te r, Boyland (39) proposed that,

theoretically, the formation of epoxides from the parent compound, BP,

could account for a ll the known metabolites. In comparison to BP, these

epoxides would be highly reactive molecules and could account for

biological a c tiv ity that cannot be explained by the parent compound.

However, there was no direct evidence a t this time to support this

theory.

Shortly afterward, M ille r (202) demonstrated that BP applied to

mouse skin became bound to ce llu la r protein and Heidelberger and

Davenport (123) showed that another PAH could bind to c e llu la r DNA.

However, Brookes and Lawley (43) determined that the amount of PAH bound

to DNA, not RNA or protein, was responsible for the carcinogenic

a c tiv ity . This report led the M illers (203,204) to propose that BP and

other PAH must f ir s t be activated through metabolism to e le ctro p h ilic molecules which then bind covalently to ce llu la r macromolecules to exert

their biological effects.

In addition, direct evidence was accumulating that epoxides were

intermediates in the metabolism of PAH, including BP (146,22,315,108). 6

BAY REGION 10 ^ o o 9 Oo UEQ> oo o K REGION o o o 7 6 ~5

Ben zo(a ) pyrene 7 ,8 -E po xide

o o

7, 8-Dio l- 9 , 10-Epo xid e 7 ,8 -D io l

Figure 2. The Most Active Metabolic Pathway for Benzo(a)pyrene

According to the Bay-Region Theory. In 1974, Sims et a l. (272) synthesized 7,8-dihydro-7,8-dihydroxybenzo-

(a)pyrene 9,10-oxide (7,8-dio1-9,10-epoxide) and incubated i t with DNA in the presence of ra t liv e r microsomes. The chromatographic p rofile of the DNA hydrolysates was identical to the profile of DNA hydrolysates from BP-treated cells. This experiment provided substantial support for the bay-region theory proposed by Jerina et a l. (145) in which a high degree of chemical re a ctivity is located where the fused aromatic rings of a PAH form an angle or are nonlinear (Figure 2).

Today, the bay-region theory is the most widely accepted explanation of the pathway in the metabolism of BP which leads to its toxic, mutagenic, and tumorigenic activity (270,30,7,153,102,214,136,228,125).

Shown in Figure 2, BP is firs t metabolized by a mixed-function oxygenase

(MFO) to an 7,8-epoxide. (The enzyme system, MFO, w ill be discussed in greater detail in Chapter 3). Then, an epoxide hydratase (EH) reduces the epoxide to a 7,8-diol which is further acted on by MFO to form a

7,8-diol-9,10-epoxide or BPDE. BPDE has been shown by numerous investigators to form the major DNA adducts in mouse lung and liv e r

(173), ra t lung and liv e r (114,12), mouse submandibular gland (328), mouse skin (20,12,109,13,11), rat skin (20,14), hamster embryo cells

(21,263), and various human tissue explants, such as bladder (70,283), bronchus (70,283,109,296), liver (36), colon (16), esophagus (119), lymphocytes (36), skin fibroblasts (23,295), and epidermal keratinocytes

(299,230). Two stereoisomers (the a n ti- and syn-isomers) of BPDE have been detected and the predominant DNA adduct results from the reaction of the anti-isomer with the 2-amino group of deoxyguanosine. In addition to BPDE, several other classes of metabolites can be formed: phenols, quinones, te tro ls , trio ls , other diols, as well as sulfates, glucuronides, and glutathione conjugates. These pathways are shown in Figure 3 (98) and involve not only MFO and EH but also other enzymes including sulfotransferase, glutathione S-epoxide transferase, and UDP-glucuronic acid transferase. MFO, EH, and UDP-glucuronic acid transferase have been found in the endoplasmic reticulum (microsomal fraction) and in the nucleus (85,312,157,162,76,300,218,25,148,237).

Another form of EH is located in the cytosol as well as is sulfotransferase and glutathione S-epoxide transferase (65,218,115).

The reactions catalyzed by these enzymes have been divided into two classes: 1.) activation reactions which form metabolites which are more harmful or toxic to the cell than the parent compound and

2.) detoxification reactions in which metabolites are conjugated or converted to more polar compounds which can be easily excreted. This interplay of reactions can be seen in the above example of BPDE. The

7,8-diol is activated by MFO to give BPDE which can further bind to DNA,

RNA, and protein, or the two stereoisomers of BPDE can be detoxified by

EH to form the four te tro ls shown in Figure 4.

Besides the bay-region theory, other theories of BP metabolism leading to its biological effects have been proposed. In 1976, King et a l. (163,301) reported that DNA adducts can be formed from a

9-hydroxy-4,5-epoxide. However, compared to BPDE-DNA adducts, these adducts are detected in smaller amounts in various systems

(114,12,88,36,299,11,13,14,254,311) with the exception of one in vivo study in rat lung and liver (37). Nevertheless, the greater amounts of 9

Phenols Giucu'onides B in d in g UDPGA 1 1 0 G 3 0 G 7 0-G 9 0 G 6 0 G 7.8 D>oi G 9 10 Droi G 4.5 Oioi G

S u lla lrs

9.10 S

Tenoi 117.10 8.91 T th o l 217/8.9,101 Tnol

MFO - Muad Function Osidaut IH - Eponde Hydraiaae D id Epo««1<-1 GSH 7 ■ GMathiontS Erxmdf Translriaw 12 3 01 |r7 .r 8 dioi r 9.10 o«y UDPGA T UDPGkjcotouc Acid Tian»r*raa* 14.6 01 Mr-4.5 SI SuHouantlrate DNA NE • Non Enrymanc 17.8 01 H r 7.8 \ RNA SS • Stncotpecitic 19.10 01 M r 9.10 Piote*n I ■ Trans X Mulaprn DiOl Epoudc II C»»c*oopen I GSH T |f 7.f 8 dtoi c 9.10 o*v

GSH Conrupatcs NADPH NAOH 4.5 SG 7.8 SG 9.10 SG Tanol 1(7.9.10 81 Tnol 17.9 81 Tauol 217,9/8,10)

Figure 3. Known Metabolic Pathways for Benzo(a)pyrene (98). 10 DIOKJQlp- OH o o (7/8,9,10)- Te o o o o o o o o 7 , 8 -D iol-9 ,1 0 -Epo xide ( I )

(a n ti isomer) (7,10/8,9)- Tetrol ( I ) o o o o o

o o (7,9,10/8)- Tetrol ( I I ) o OH o o o o

7 ,8 -D iol-9 , 10-Epoxide (II) o o o (SYN ISOMER)

(7,9/8,10)- Tetrol (II)

Figure 4. Formation of Tetrols from BPDE Stereoisomers by Epoxide

Hydrolase. 11

BPDE-DNA adducts found and th e ir lower rate of excision gives further

support to the bay-region theory (114,88,311).

Three quinones, BP-1, 6-dione, BP-3, 6-dione, BP- 6,12-dione, have been detected as metabolites of BP (65,266,267,132). They are believed to be

formed by oxidation of BP to 6-hydroxybenzo(a)pyrene ( 6-OH-BP) which is

then converted to a 6-oxo-BP radical intermediate (192,183). This intermediate further reacts with water to form the three quinones. In addition, BP has been activated in microsomal preparations to

6-hydroxymethylbenzo(a)pyrene (BP- 6-CH2OH) (278,91) which has been shown to bind to DNA resulting in adducts (257,306). The presence of these metabolites suggested that the C -6 position of BP is a very reactive site in the molecule and led to the proposal of one-electron oxidation

(49,256,51,55,50,255). By this theory, only one electron was needed to oxidize BP to a radical cation intermediate which can then form quinones or covalently bind to DNA. These reactions were not catalyzed by MFO because they occurred even in the presence of MFO in h ib ito rs (278). An enzyme referred to as aryl hydroxymethyl synthetase has been isolated both as a microsomal-bound form and a soluble form (278,280) and requires NADPH (279). Recently, in another study, the MFO-associated reductase (which requires NADPH) catalyzed one-electron oxidation of quinones when the MFO pathway was inhibited (55). Therefore, the aryl hydroxymethyl synthetase and the MFO-associated reductase may be the same enzyme.

Another theory which has been proposed to explain PAH metabolism was derived from studies involving another molecule in this class of compounds, 7,12-dimethylbenz(a)anthracene (DMBA). Huberman et a l. (137) 12 reported that DMBA (Figure 5) was a potent mutagen but that the trans-3,4-diol was even more potent. In addition, Slaga et a l. (276) found this diol to be more tumorigenic in mouse skin than the parent compound and proposed that DMBA exerted these mutagenic and carcinogenic effects through the bay-region intermediate, 3,4-diol-l,2-epoxide (275).

However, in 1980, Inbasekaran e t a l. (140) synthesized two A-ring reduced analogues of DMBA, one of which was 1,2,3,4-tetrahydro-

7,12-dimethylbenz(a)anthracene (TH-DMBA). From studies using the Ames test, TH-DMBA (Figure 5) was found to be as mutagenic as DMBA but did not require metabolic activation as DMBA did. Furthermore, this compound induced neoplastic transformation in human neonatal foreskin fibroblasts in v itro (294,52). Since TH-DMBA cannot form a bay-region epoxide intermediate because it does not contain an aromatic A-ring, another metabolic pathway seems to be active and has been suggested to involve the D-ring (209). Therefore, these last two theories do have supportive evidence that alternate routes of metabolism besides the bay-region pathway may exist.

In previous studies, human neonatal foreskin fibroblasts were neoplastically transformed with BP when they were in logarithmic growth

(randomly-proliferating) and were treated in the S phase of the cell cycle (synchronized) (294,207,52,205). In contrast, confluent or contact-inhibited cultures were not transformed by BP (Milo, unpublished data). Several parameters indicative of transformation for these three populations of cells are shown in Table 1. The radiolabelling index

(RLI) is the percentage of radiolabelled nuclei observed for cells treated with 30 min pulses of [ H]-thymidine for a period of 24 hours. 13

CH 3

7 , 12-Dimethylbenz(a)anthracene (DMBA)

CH3

1 ,2 ,3 ,4-T etrahydro-7 , 12-di methylbenz (a) anthracene (TH-DMBA)

Figure 5. Chemical Structures of DMBA and TH-DMBA. 14

TABLE 1

Indices of Transformation in the Different Populations

of Human Neonatal Foreskin Fibroblasts

Population RLIa AIGb NAC IBd

Randomly-Proliferati ng 20-23* 0-2 4-8 3.0

Synchronized 80-93% 86 4.2+4.7 3.0

Confluent <1% NDe ND ND

aRLI-Radiolabelling Index bAIG-Anchorage-Independent Growth in soft agar, number of colonies per 5 10 cells at 16-20 population doubling levels after treatment

CNA-Nuclear Adducts present per 10® bases of DNA dIB-Induced Breaks by BP in 10®daltons of DNA after 10® ergs of 313 nm

lig h t eND-Not Detectable Randomly-proliferating cells had a RLI of 20-30% while less than 1% of the nuclei in confluent cells were labelled. The synchronized population had a RLI of less than 1% after release from block but

10-12 hours la te r, exhibited a peak with a maximum RLI of 80-93% indicating the cells were going through S phase (208). The next column indicates one of the assays used to show carcinogenic transformation, anchorage-independent growth in soft agar. In this assay, cells treated with a carcinogen w ill continue to divide and form colonies while suspended in soft agar whereas untreated cells w ill not continue to proliferate. The highest number of colonies was obtained for the synchronized population, 86 colonies per 10 cells (294,52,295). A small number of colonies or none were observed for the randomly-proliferating and confluent populations, respectively. The th ird column shows that the nuclear BP-DNA adducts detected were quantitatively (and also qualitatively) similiar for the randomly-proliferating and synchronized cells whereas adducts were not detected in the confluent cells (295,174). Finally, after treatment with BP followed by exposure to 313 nm lig h t, 3.0 breaks per 10® dal tons of DNA were reported for cells in the synchronized and randomly-proliferating cultures (205). The number of breaks observed in the confluent cultures, i f any, were below the lim its of detection.

In addition to the above data, previous results (296,83,293) demonstrated that prolifera tin g cultures take up BP into the cytoplasm and transport it via a carrier lipoprotein complex to the nucleus within

24 hours. This transport was not observed to occur in contact-inhibited cultures (205). Therefore, cells which were randomly-proliferating or synchronized transported BP to the nucleus where its interactions resulted in a transformed phenotype. Cells which were contact-inhibited did not transport BP and thus, seemed to be refractory to its effects.

These differences led to the following experiments in which the metabolism of BP was investigated in each population of fibroblasts.

MATERIALS AND METHODS

Preparation of Primary Fibroblast Cultures:

Immediately after circumcision, human neonatal foreskins were stored at 4°C in sterile vials containing 10 ml of 5% Fetal Bovine Serum (FBS,

Reheis Chemical Co., Phoenix, Arizona) in complete MEM (CM). CM refers to Eagle's Minimum Essential Medium (MEM) with Hanks' Salts and 25 mM

HEPES buffer (formula #78-5221, Grand Island Biological Co., Grand

Island, New York) supplemented with IX non-essential amino acids (M.A.

Bioproducts, Walkersvi1le, Maryland), 1 mM sodium pyruvate (M.A.

Bioproducts), 2 mM ^-glutamine (M.A. Bioproducts), 50 mg/1 Gentocin

(gentamicin sulfate, Schering Veterinary Corp., Kenilworth, New Jersey) and 0.132X sodium bicarbonate (NaHCOg, MCB Reagents, Cincinnati, Ohio).

The final pH was 7.2. If stored in this manner, the skin tissue was s till viable for culturing of fibroblasts up to four to five days later.

A ll subsequent tissue processing and cell culture maintenance were done under a s te rile atmosphere in a vertical laminar a ir flow hood

(Model NCB4, The Baker Co., Sanford, Maine). F irs t, 5 ml CM was added to each of four wells in a Linbro tissue multi-well plate (Flow

Laboratories, Inc., McLean, Virginia) with one plate used per skin tissue. Using sterile forceps, the skin was transferred from the vial to one of the wells, swirled in the medium to rinse off excess blood c e lls, and transferred to the next w ell. This was repeated u n til the

fourth w ell, where the medium was pipetted o ff and 2.5 ml of a 1%

collagenase (w/v, from Clostridium histolyticum , type I, Worthington

Diagnostic Systems, Inc., Freehold, New Jersey) solution was added. The 2 skin was minced into 2 mm pieces with disposable scape!s and the

collagenase-skin mixture transferred to a sterile 15 ml centrifuge tube

(S cie n tific Products Division, Corning Glass Works, Corning, New York)

containing 7.5 ml of 20% FBS in CM to make a final concentration of

0.25% collagenase. The tube was incubated at 37°C for four to six

hours.

Afterwards, the solution was vortexed u n til homogeneous and the

cells pelleted at 650-700 rpm for seven minutes at room temperature in

an IEC centrifuge (Model CRU-5000, Damon/IEC Division, Needham,

Massachusetts), (ca. 120 x g). The supernatant was pipetted off and the

cell p e lle t was rinsed with 10 ml CM, recentrifuged, and resuspended in

10 ml 20% FBS-CM. Then, 5 ml of the cell solution was added to 10 ml 2 20% FBS-CM already in a 75 cm tissue culture flask (Falcon Labware,

Becton Dickinson and Co., Oxnard, C alifornia). The cells were allowed

to attach for 48-72 hours (2-3 days) in a water-jacketed 4% COg-enriched

atmosphere incubator (Model 3172, Forma Scientific, Marietta, Ohio) at

37°c. Most of the cells which remained flo a tin g in the seeding medium

and unattached to the plastic substratum were blood cells and nonviable

skin cells.

Refeeding and Subpassaging of Cultures;

After two to three days when the cells had attached, the cultures were refed by pipetting o ff the medium, rinsing with 10 ml CM and adding 18 10 ml 20% FBS-CM. Subpassaging of the cultures took place when the cells were 80-90% confluent, as observed by phase contrast microscopy,

(X 100). A stock solution of 1% trypsin (w/v, from bovine pancreas,

Worthington Diagnostic Systems) was made up in incomplete MEM (MEM without supplements), doled out into 1 ml aliquots in sterile test tubes and froze. To subpassage the c e lls, the medium was pipetted o ff and each flask was rinsed with 10 ml CM. One ml of a 0.1% trypsin (a thawed

1 ml 1% aliquot was diluted by 9 ml CM) solution was added to each flask. In two to three minutes, the fibroblasts were observed to have rounded up and detached from the plastic substratum leaving behind a small number of epithelial colonies. The trypsin activity was then neutralized with 9 ml 20% FBS-CM and the cells were s p lit 1:2 by adding

5 ml of the cell solution to 10 ml 20% FBS-CM in a 75 cm 2 flask. When the cell culture in the latter flask was confluent, the cells were designated as having gone through two population doubling levels or were at PDL 2. The fib ro b la st cultures were routinely refed every two to three days and subpassaged u n til PDL 4 or 5 at which time they were used for the following experiments. 20% FBS was used in the medium up to and including the f ir s t subpassage a fte r which 10% FBS was used. In addition, just prior to treatment, the cultures were split out into 150 p mm diameter (177 cm ) dishes for easier manipulation.

Treatment with Benzo(a)pyrene:

For the succeeding experiments, three d iffe re n t types of fibroblast cultures were used: randomly-proliferating, synchronized, and confluent populations. Randomly-proliferating cultures consisted of cells active in the logarithmic phase of the growth cycle and were 50-60% confluent at the time of treatment. Synchronized cells were cultures blocked in

the phase of the cell cycle by treating with In stitu te of Basic

Research (IBR) modified Dulbecco's MEM without L^-glutamine, L-arginine

HC1, NaHC0 3 (synchrony medium, formula #83-5019-contains further

modifications, GIBCO) supplemented with 1 mM sodium pyruvate, 50 mg/1

Gentocin, 0.132% NaHCO^, and 10% dialyzed FBS for 24 hours. I t should

be noted here that dialyzed FBS was obtained by loading 500 ml of serum

into cellulose dialysis tubing with a cutoff of 12,000-14,000 MW (VWR

Scientific, Inc., San Francisco, California) and dialyzed against a

total of 16 lite rs of synchrony medium for 48 hours with changes of 4

lite rs at 2, 4, and 16 hours. The cells were later released from the

24-hour block by rinsing with 10 ml CM and then adding 15 ml of 10%

FBS-CM with 0.5 U/ml insulin (from bovine pancreas, Sigma Chemical Co.,

St. Louis, Missouri). After 10-12 hours or at the start of S phase, the

cultures were treated with benzo(a)pyrene added to 10% FBS-CM plus

insulin and the cells harvested at 24 hours after release. Last of all,

the confluent population was made up of cells which were confluent

(contact-inhibited), were refed every two days, and were treated on the

f if t h day after they had reached confluency.

A ll subsequent procedures were performed under gold lig h t (40W,

General E lectric, Cleveland, Ohio). F irs t, [G- H]benzo(a)pyrene (BP,

21-36.7 Ci/mmole, 5 mCi/ml, Amersham Corporation, Arlington Heights,

Illinois) was blown to dryness with argon (g, prepurified, 0SU Stores,

Columbus, Ohio) and taken up in acetone (SpectrAR grade, Mallinckrodt,

Inc., Paris, Kentucky). Then this radiolabelled BP along with unlabelled BP (NCI Chemical Carcinogen Repository, Chicago, Illin o is ) at 20 >95% purity were added to 10% FBS-CM for a final concentration of 9.4,jCi per 0.25 ug and all cultures were treated with this BP preparation for

24 hours with the exception of the synchronized population as described above. The fin a l concentration of acetone in the treatment medium was less than 1.0%. For control samples, 10 ml of the BP medium was used and extracted as described below.

Harvesting of Cells and Preparation of Fractions;

Three d iffe re n t fractions were analyzed for each cell population: the extracellular, the cytoplasmic, and the nuclear fractions. For each extracellular sample, one 150 mm dia. dish was used. The treatment medium (10 ml) was pipetted o ff, extracted by the method described in the next section, and a count of the cells in that dish was taken using a hemocytometer.

Each cytoplasmic fraction was prepared using the cells from five 150 mm dia. dishes of confluent cultures or 10-12 dishes of synchronized or randomly-proliferating cultures. The following procedures were performed at 4°C. To s ta rt, the cells of each dish were lig h tly trypsinized using 1.5 ml of a 0.067% trypsin solution and neutralized with 8.5 ml 10% FBS-CM. They were pooled into 50 ml centrifuge tubes

(Corning Glass Works), the dishes rinsed two times with CM, and the tubes centrifuged at 650-700 rpm for 7 min at 4°C, (ca. 120 x g). The pellet was then resuspended in 30 ml of CM, centrifuged twice, and the final p e lle t was suspended in a solution of 0.02 M Na phosphate (a mixture of Na 2HP04 and NaH 2P04), 0.03 M Tris HC1, 2.5 mM Na 2EDTA, and

0.5 mM dithiothreitol (DTT) at pH 7.5 to obtain 10^ cells/ml. After a cell count was taken, the fibroblasts were homogenized 1 ml at a time 21 using a 5 ml prechilled stainless steel homogenizer with a teflon pestle

(5(10) 4 to 1.7(10) ^ clearance) for 10-12 strokes and the nuclei examined by phase contrast microscopy, (X 100). The homogenate was centrifuged at 40,000 rpm for 1 hr in a Beckman ultracentrifuge (Model

L8-55, Beckman Instruments) using a Ti 75 rotor, (ca. 105,000 x g).

Afterwards, the supernatant was poured off and extracted as described below.

Finally, the nuclear fraction was prepared from 10-12 150 mm dia. dishes of confluent cultures or 20-21 dishes of randomly-proliferating or synchronized cultures. The method employed was the same as the cytoplasmic procedure described above with the following exceptions.

Before homogenization, the cell p e lle t was suspended in a solution of

10 mM Tris maleate, 1 mM DTT, 3 mM calcium acetate (Ca(AC)2), 2 mM magnesium acetate (Mg(Ac)2), and 50 yM phenylmethylsulfonyl fluoride

(PMSF) at pH 7.5 (hereafter, referred to as "Buffer A") plus 0.25 M sucrose (ultrapure, ribonuclease free, Schwarz/Mann Inc., Spring Valley,

New York) to obtain 10^ cells/m l. To 3 ml of homogenate, 15 ml of 1.8 M sucrose made up in Buffer A was added and the two solutions mixed thoroughly. Six ml of this solution was gently layered on top of 6.5 ml of 1.8 M sucrose in 9/16" x 3 1/2" ultra-clear ultracentrifuge tubes

(Beckman Instruments) and centrifuged at 15,000 rpm for 1 hr in a SW 41 rotor, (ca. 38,000 x g). At th is speed, the nuclei pelleted while the cytoplasmic material and whole cells remained at the interface of the homogenate and 1.8 M sucrose layers.

Afterwards, the supernatants from a ll tubes were pipetted o ff and the pellets resuspended in 10 ml of 1.0 M sucrose in Buffer A and 22 centrifuged at 10,000 rpm for 10-20 min in an IEC refrigerated

centrifuge (Model B-20, Damon/IEC D ivision), (ca. 13,000 x g). Two ml

of Buffer A was added to the nuclear pellet and a nuclear count was

taken. F inally, the suspension was extracted as described below.

Extraction of Benzo(a)pyrene and Metabolites:

All extracellular, cytoplasmic, and nuclear fractions were extracted

in the same manner. Three volumes of ethyl acetate (HPLC grade, Fisher

Scientific Co., Fair Lawn, New Jersey) plus 0.8 mg/ml butylated

hydroxytoluene (BHT, Sigma Chemical Co.) was added to each fraction,

vortexed for about 1 min, and the aqueous and organic layers separated.

The organic layer was dried over approximately 20 g of anhrydrous sodium

sulfate (Na 2S04, Fisher S cie n tific Co.), taken to dryness with argon,

resolubilized in 400 yl methanol (MeOH, HPLC grade, Fisher S cien tific

Co) and stored in a micro-centrifuge tube (Labconic, San Rafael,

California) under argon at -20°C. In order to determine yields of each

step throughout the procedures, aliquots were taken for liquid

s c in tilla tio n counting using Insta-Gel cocktail (Packard Instrument Co.,

Downers Grove, Illin o is ) and a LS-9000 liq u id s c in tilla tio n counter

(Beckman Instruments) with an efficiency of 36*.

Analysis by High-Pressure Liquid Chromatography:

Due to a small amount of precipitate in the MeOH samples of a ll

fractions, the tubes were centrifuged at 3000 rpm for 3 min in a microfuge centrifuge (Model 11, Beckman Instruments), (ca. 680 x g).

After the solid material had pelleted, 20 yl aliquots were injected onto a Zorbax Octadecylsilane (0DS) column (6.2 mm ID x 25 cm, DuPont Co.,

Wilmington, Delaware) and analysis of benzo(a)pyrene and its metabolites 23

was obtained with a 100 min gradient of 60% to 100% MeOH vs H20 linear

over 85 min with a high-pressure liquid chromatograph (HPLC, Model 334,

Beckman Instruments). The flow rate was 0.8 ml/min and the chart speed was 0.5 cm/min. Radioactivity was monitored and quantitated using a

Flo-One on-line s c in tilla tio n counter (Model HP, Radiomatic Instruments and Chemical Co., Tampa, Florida) with an overall efficiency of 24% for

RESULTS AND DISCUSSION

Previous studies have reported the use of high-pressure liquid

chromatography (HPLC) for the separation of organic-soluble

benzo(a)pyrene metabolites (266,267,132,264,298,339). However, the best

separation of a ll metabolites with one gradient was achieved with a

DuPont Zorbax 0DS column using a 60-100% methanol vs. water gradient at a flow rate of 0.8 ml/min (338). In the following studies, the gradient was linear for 85 min followed by a plateau at 100% methanol for 15 min.

Retention times of the metabolites varied even between Zorbax 0DS columns, the more recently-purchased columns giving better separation.

For this reason, the same column (serial number H709) was used for a ll analyses in this chapter and the last chapter. Identification of the radiolabelled metabolites from the ce llu la r samples was made by a comparison of retention times to those of unlabelled standards (NCI

Chemical Carcinogen Repository) whose UV absorbance was monitored at

254 nm.

A l i s t of the metabolites and th e ir retention times are shown in

Table 2. With th is gradient, the more polar metabolites eluted f ir s t beginning with the highly polar sulfates, the 3-sulfate and the 24

TABLE 2

Retention Times of Benzo(a)pyrene Metabolite Standards on a

DuPont Zorbax ODS HPLC Column (6.2 mm ID x 25 cm)

Retention Retention Time Time Metabolite (min) Metabolite (min)

Benzo(a)pyrene-3-sulfate 6.8 trans-7,8-Dihydrobenzo(a)pyrene- 52 7,8-diol Benzo(a)pyrene- 6-sulfate 6.8 Benzo(a)pyrene-l, 6-dione 74 Benzo(a)pyrene-9-sulfate 8.1 9-Hydroxybenzo(a)pyrene 75 Benzo(a)pyrene Tetrol I- (7,10/8,9) 21 1-Hydroxybenzo(a)pyrene 77

Benzo(a)pyrene Tetrol I- 3-Hydroxybenzo(a)pyrene 77 (7/8,9,10) 26 7-Hydroxybenzo(a)pyrene 77 9,10-Dihydrobenzo(a)pyrene- 9,10-Diol 28 Benzo(a)pyrene-3, 6-di one 77

Benzo(a)pyrene Tetrol I I - Benzo(a)pyrene-6,12-dione 81 (7,9/10,8) 31 6-Hydroxybenzo(a)pyrene 83 Benzo(a)pyrene Tetrol II- (7,9,10/8) 35 Benzo(a)pyrene 98 cis-4,5-Dihydrobenzo(a)- pyrene-4,5-diol 48 trans-4,5-Dihydrobenzo(a)- pyrene-4,5-diol 48 ci s-7,8-Dihydrobenzo(a)- pyrene-7,8-diol 50 25 6-sulfate at 6.8 min and the 9-sulfate at 8.1 min. The te tro ls eluted

next and the standards were prepared from hydrolysis of the two isomers of BPDE, a n ti-BPDE or BPDE-I, and syn-BPDE or BPDE-II, according to the method of Yang, et a l. (341). The (7,10/8,9)-tetrol (21 min) and the

(7/8,9,10)-tetrol (26 min) formed from BPDE-I were detected f ir s t ,

followed by the 9,10-diol and then the te tro ls from BPDE-II, the

(7,9/10,8)-tetrol (31 min) and the (7,9;10/8)-tetrol (35 min). The ci s- and trans-isomers of the 4,5-diol eluted together while the cis- and

trans-isomers of the 7,8-diol could be separated. The phenols were resolved into three groups: 9-OH at 75 min, 1-OH, 3-OH, and 7-OH at 77 min and 6-OH at 83 min. Last of a ll, BP was detected at 98 min.

Baseline separation was not observed in the radiolabelled samples for

the sulfates or the two isomers of 7,8-diol using the on-line scintillation counter.

The quinone standards, 1,6-quinone, 3,6-quinone, and 6, 12-quinone, had retention times very close to those of the phenols. The 1,6-quinone eluted before 9-OH, the 3,6-quinone eluted with 3-OH, and the

6, 12-quinone was detected ju s t before 6-OH. Using a sim ilia r HPLC system, quinones were only detected in very small amounts in a previous study (293) and th e ir formation by autooxidation was minimized by preparing a ll samples under yellow lig h t and storing them under argon at

-20°C. In addition, 0.8 mg/ml BHT was present in all ethyl acetate layers. Peaks corresponding to these substances were found as impurities in the radiolabelled BP used (>95 purity). Therefore, control samples ([ H]-BP added to the treatment medium) were extracted and analyzed for each experiment and the value of impurities detected in 26 the region of the quinones and phenols was subtracted from the amount

detected for the phenols in the ce llu la r samples.

For each population of human neonatal foreskin fibroblasts, the extracellular, the cytoplasmic, and the nuclear fractions were analyzed.

In Figure 6, representative histograms of organic-extractable radiolabelled BP metabolites in the extracellular fractions for the randomly-proliferating, synchronized, and confluent cultures are shown.

The major metabolites were the 9,10-diol, the 7,8-diol and the phenols,

9-OH and 3-OH. Minor amounts of 1-OH and 7-OH may be present but cannot be detected with this system. 6-OH was not detected and the peaks appearing between 3-OH and BP were found in the control samples. This data is in agreement with Baird and Diamond (23) except that they did not detect any phenols in WI-38 cultures. BP metabolism has been previously studied in human skin by several investigators who were concerned with the amount of total metabolism of BP and not with the specific identity of the metabolites (213,135,331,332,258). In addition, most studies involved mixed fibroblast and epithelial cultures and most cultures were treated at confluency with only extracellular metabolites examined. Selkirk et a l. (265) and Fox et a l. (92) studied the BP metabolism in human skin organ cultures and detected in addition to the phenols, the two diols, and a small amount of the 4,5 -d io l, a large amount of quinones. However, not as many precautions to prevent autooxidation were taken as in the following experiments.

In examining Figure 6 closely, the metabolic profiles seem qualitatively similiar in all three populations, though a noticable increase is observed for the confluent cultures. A quantitative 27

Figure 6. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the Extracellular Fractions of

the Different Fibroblast Populations. A representative

profile is illustrated for the randomly-proliferating, the

synchronized, and the confluent cultures. 9,10-DIOL BENZO(a)PYRENE 10 CONFLUENT

8 7 ,8 -D IO L LU

UJ 6 UJ ro

ro 4 o LJ iZ

2 CO Q_ O 0

2 RANDOMLY- PROLIFERATING

0 / T ______n_____

2 SYNCHRONIZED

0 10 20 30 40 50 60 70 80 90 100

I i'juro 6. TIME (MINUTES) r\o CO 29 evaluation of these metabolites is presented in Table 3 with the

9,10-diol, the 7,8-diol, the phenols, and a large amount of unmetabolized BP present in a ll cultures. In addition, small amounts of

sulfates were detected. Previously, Cohen et aK (56) demonstrated that a sulfate was extracted into the ethyl acetate layer and in human lung cultures, the 3-sulfate was found to be a major metabolite. This compound may be responsible for the sulfate peak observed in these fibroblast cultures although the presence of other sulfates, such as the

6-sulfate and the 9-sulfate, cannot be discounted. In a ll cultures, the metabolite detected in the greatest amount was the 9,10-diol, followed by the 7,8-diol and then the phenols. Radioactivity eluting at the position of the 4,5-diol was not significantly above background to state that this compound was present.

In addition, only in the confluent cultures were all four tetrols present. However, they accounted for only 7% of the total metabolites formed. For the confluent cells, the total metabolites excluding BP were 1.5 times more than those detected for the synchronized cells and

2.7 times more than those of the random ly-proliferating cells. This type of correlation had been reported earlier in studies by Wohler et a l. (332,331) and Rudiger et a l. (258) in which contact-inhibited skin fibroblast cultures had metabolized BP 10 times more than cultures which were exponentially growing. However, Baird and Diamond (23) did not find any difference in metabolism between confluent and proliferating cultures of WI-38 cells. They did notice that the ability of these cells to metabolize BP dropped with subpassaging. In addition, they were working with middle- and late-passage cells which may have been 30

TABLE 3

Organic-Extractable Metabolites of Benzo(a)pyrene in the

Extracellular Fractions of the Different Fibroblast Populations 9

Metabolites RPb Sc Cd

Sulfates 31.8 83.6 48.6

Tetrol I (7,10/8,9) NDe ND 46.7

Tetrol I (7/8,9,10) ND ND 31.1

9,10-Diol 373.5 631.0 943.3

Tetrol II (7,9/8,10) ND ND 17.2

Tetrol II (7,9,10/8) ND ND 62.3

7,8-Diol 279.6 485.7 539.3

Phenols 140.0 301.7 512.6

Benzo(a)pyrene 10,639.7 16,650.0 6,029.6

Total Metabolites 824.9f 1,502.09 2,207.3h aMean values are in picomoles of metabolites per 10^ cells bRP-Randomly-Proliterating Population cS-Synchronized Population dC-Confluent Population eND-Not Detectable f Range (708.6-958.1), n=5 gRange (287.6-2,496.7), n=5 hRange (1,819.0-2,575.0), n =8 31 approaching senescence. Indeed, i t was also demonstrated previously that high-passage fibroblasts were unable to take up and metabolize BP to as great an extent as low passage fibroblasts (297). In the experiments presented in this chapter, only fibroblast cultures less than PDL 6 (or low-passage cells) were used. Therefore, the data in

Table 3 correlates with the studies by Wohler et a l. and Rudiger et a l.

Representative histograms of radiolabelled BP metabolites extracted from the cytoplasmic fractions for each population are illustrated in

Figure 7. Again, these profiles were q u a lita tive ly sim ilia r wfth a large amount of unmetabolized and unbound BP present which was taken up into the cytoplasm. The major metabolites detected in a ll cultures were the 7,8-diol and the phenols with two unknown peaks eluting between the phenols and BP in the confluent ce lls. The data is presented quantitatively in Table 4. Small amounts of sulfates, two tetrols and

9,10-diol were present in some cultures. However, the confluent cells did metabolize BP to a greater extent than the other two types of cells.

In fact, the total metabolites formed minus BP were 4.3 times more than those formed in the synchronized population and 4.5 times more than those in the randomly-proliferating population.

Figure 8 presents representative metabolite profiles for the nuclear fractions of each type of fib ro b la st culture. The predominant metabolites are, again, the 7,8-diol and the phenols with two unknown peaks in the confluent c e lls. The amounts detected for each group of metabolites are shown in Table 5. The values for the randomly-proliferating and synchronized populations are similiar and are

1.3 and 1.8 times less, respectively, then those for the confluent 32

Figure 7. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the Cytoplasmic Fractions of

the Different Fibroblast Populations. A representative

profile is illustrated for the randomly-proliferating, the

synchronized, and the confluent cultures. Two unknown peaks

eluted between the phenols and BP. 10 CONFLUENT 7,8-DIOL 9_0H \ BENZ0(a)PYRENE 3-OH X o 8 a t o on o cr 6 ki

00 4 o s."

2 U y ! ( 1 Fj 0 a . . ^ A k i VI' > fU_ 2 RANDOMLY- PROLIFERATING 'A 0 -JiA . Jun__ J I U' jlIL.

2 SYNCHRONIZED ni i! !l

0 j l . i L U L A_.„. _L _L _i____ 10 20 30 40 50 60 70 80 90 100

TIME (MINUTES) 34

TABLE 4

Organic-Extractable Metabolites of Benzo(a)pyrene in the

Cytoplasmic Fractions of the D ifferent Fibroblast Populations 3

Metabolites RP^ Sc Cd

Sulfates 0.36 NDe 0.44

Tetrol I (7,10/8,9) ND ND 0.99

9 ,10-Diol 0.60 ND 3.10

Tetrol II (7,9/8,10) ND ND 0.46

7,8-Diol 5.90 6.27 31.53

Phenols 12.48 14.17 51.41

Benzo(a)pyrene 152.70 196.79 152.08

Total Metabolites -19.34f 20.449 87.93h aMean values are in picomoles of metabolites per 10 7 cells (n=2)

^RP-Randomly-Proliterating Population

cS-Synchronized Population

dC-Confluent Population eND-Not Detectable

fRange (18.74-19.33)

9Range (20.36-20.51)

hRange (64.06-110.36) 35

Figure 8 . Histograms of Organic-Extractable Radiolabelled

Benzo{a)pyrene Metabolites in the Nuclear Fractions of the

Different Fibroblast Populations. A representative profile

is illustrated for the randomly-proliferating, the

synchronized, and the confluent cultures. Two unknown peaks

eluted between the phenols and BP. 3-OH 10 CONFLUENT 7,8-DIOL 9- OH / 1 BENZOfa'PYRENE— >

l 6

i ’li > 'J i! i* V|, { '.3. jf T 1 0 ’■-TTv „ - IT (jrjtu.u . l iu L iT jk 2 AZLi i RANDOMLY- 2 1 PROLIFERATING ( i v. fl: i5 i ■ 1 m , nH L 0 — L.JL _Aa_ luJ T^jW jLilbTTi.Aj > l i SYNCHRONIZED 2 it i ' i ^iilift. Li i V M J 0

10 20 30 40 50 60 70 80 90 100

8 . TIME (MINUTES) CO O'* 37

TABLE 5

Organic-Extractable Metabolites of Benzo(a)pyrene in the

Nuclear Fractions of the D ifferent Fibroblast Populations9

Metabol ites RPb Sc Cd

7,8-Diol 311 318 670

Phenols 982 612 963

Benzo(a)pyrene 15,033 30,087 2,483

Total Metabolites 1,293e 930f 1.6339 aMean values are in pi comoles of metabolites per 107 cells bRP-Randomly-Proliferating Population

cS-Synchronized Population dC-Confluent Population en= l f Range (841-1,147), n=3 gRange (912-2,355), n=2 38

population. Unmetabolized BP could represent the BP which was

transported to the nucleus. The presence of the other metabolites could

be due to cytoplasmic contamination or the activity of the nuclear MFO

and EH on the translocated BP.

In summary, both MFO and EH have been detected in human skin

(217,4,54,3,186,31) and fibroblast cultures (219) and in other species,

have been detected both in microsomal preparations and in the nucleus.

Recently, several investigators have attempted to study the balance

between MFO and EH in their a c tiv itie s towards BP and its binding to DNA

(113,184,103,220,313,221). In these studies, MFO seems to play a

greater role in activation of BP and enhances its binding to DNA while

EH takes on a more inactivating and detoxifying role. Large variations

were observed in the a c tiv itie s of these enzymes between species,

tissues, and even individuals. In the experiments detailed in this

chapter, the a c tiv itie s of these enzymes were in d ire ctly observed. All

three populations of fibroblasts were shown to metabolize BP to the

9,10-diol, the 7,8-diol, and the phenols, of which the la tte r two

metabolites also were present in the cytoplasmic and nuclear fractions.

In addition, the confluent cultures metabolized BP to much a greater extent than either the randomly-proliferating or the synchronized

cultures, both of which formed similiar amounts of metabolites. The

confluent population even formed tetrols which were detected in the extracellular fraction. Preliminary studies indicated that these cells were not transformed by BP nor did they transport i t to the nucleus.

Therefore, although all three populations metabolized BP, the contact-inhibited cultures, which are the model for the in vivo state of fibroblasts may be more concerned with the detoxification of BP rather than its activation. In contrast, the synchronized and logarithmically-growing cells may be more concerned with converting BP to activated intermediates which lead to transformation. CHAPTER 2

INTRODUCTION

In Chapter 1, a comparison of the benzo(a)pyrene metabolism in

d iffe re n t human skin fib rob la st populations was made. Yet the tumors which occur most frequently in adults are carcinomas (922), whereas

sarcomas comprise only 22 (40,48). Carcinomas are malignant tumors which are epithelial in origin; sarcomas originate in the supporting

tissues of the body (48). Until recently, attempts to establish, maintain, and subpassage epithelial cells in vitro free of fibroblasts were unsuccessful. However, before describing these early attempts, an overview of the histology of skin e p ith e lia ls w ill be presented.

The skin (integument) is composed of two major layers, the epidermis and the dermis (corium). The majority of the cells in the dermis are fibroblasts whereas the cells which compose most of the epidermis are epidermal keratinocytes, the e p ith e lia l cells of the skin. The epidermis, in turn, is further subdivided into five layers as shown in

Figure 9 (281). The two layers closest to the dermis are the stratum spinosum and the stratum germinativum, which together are sometimes referred to as the stratum m alpighii. The stratum germinativum (the basal layer) is separated from the dermis by the basement membrane

(basal lamina) and consists of only one row of cells which are cuboidal or columnar in shape. In the next layer, the stratum spinosum, the epithelial cells are polyhedral with small processes or spines extending 41

-Strarum tomrum

■ Stratum lucidum -Stratum granulotum

Epidermis

—Stratum ipinosum \

$ — Strarum germinativum

hPapillary p*P‘ layer Dermis

Melanocyte Biial lamina Langerhans cell

Figure 9. The D ifferent Layers of the Epidermis of the Skin (281). 42

from their edges. These spines lie in between adjacent cells and are

responsible for another name given to this layer, the prickle-cell

layer. Above the stratum spinosum, lie s the stratum granulosum (the

granular layer). The epithelial cells here are more flattened and

contain dense-staining cytoplasmic granules or keratohyalin granules.

These cells are more closely packed and appear to be transparent in the

next layer, the stratum lucidum (the clear layer). Finally, the topmost

layer is the stratum corneum (the horny layer). Its cells are

completely filled with keratin, devoid of all organelles and nuclei, and

eventually are lost from the skin surface (34).

In order to replace these lo s t cells, the cells of the basal layer

undergo mitosis and one of each pair of newly-formed daughter cells

migrates upward through the various epidermal layers simultaneously

changing its c e llu la r morphology (168,107). This process is referred to

as keratinization and is shown schematically in Figure 10 (281). From a

columnar shape in the basal layer, the e p ith e lia ls become more flattened

and polyhedral. Next, they begin to synthesize the precursors for

keratin which are neatly packaged into keratohyalin granules. These

granules then merge with cytoplasmic tonofilaments to form keratinized masses within the cells and keratin envelopes ju st underneath the plasma

membranes (19). As the keratin accumulates, these e pithelials lose

their organelles and nuclei and eventually become completely cornified.

At this point, they are referred to as squames and w ill ultimately

detach from the epidermis (19,180).

There are several differences in morphology between the epidermal keratinocytes and the dermal fibroblasts. These differences include: 43

Ruptured vesicle Desquamation of cells of desmosome

Keratinized Stratum corneum dead cells

Desmosomal body

Thick plasma membrane Strarum granulosum Nuclear remains

Keratohyalin granules

Bundles of tonohbrils Stratum spinosum

Tonofibrils

Desmosome % ¥ .

Melanin granules Stratum I germinativum %

Tonofilaments Basal lamina Hemidesmosome

Figure 10. Changes in the Morphology of Epidermal Keratinocytes as They

Migrate Upward Through the Layers of the Epidermis (281). cell shape, and the existence of tonofilaments, desmosomes, and granules. The polyhedral shape of the epithelials is in sharp contrast to the spindle form of the fibroblasts which is apparent not only in vivo but also in vitro as well. In addition, in vivo, the keratinocytes have ce llu la r features not observed in the fibroblasts. Tonofilaments which are 70 to 80 ft filaments exist in all epithelial cells from the stratum germinativum to the stratum corneum. These structures are infrequent and randomly-oriented in the basal cells, become more organized into bundles in the p rickle -ce ll layer, and along with the keratohyalin granules, eventually form keratin in the horny layer. Also some tonofilaments have been observed to connect at the plasma membrane forming dense thickenings or desmosomes. The epithelial cells are attached to each other by these desmosomes and to the basal lamina by hemidesmosomes. An important part of the keratinization process involves the disappearance of the desmosomes in the cells of the stratum corneum leading to their desquamation. Last of a ll, the keratinocytes contain three types of granules: keratohyalin granules, melanosomes, and lamellar granules. The keratohyalin granules have already been discussed and play a major role in keratinization. Melanosomes are granules of melanin produced by melanocytes (another cell found in the epidermis) and transferred to the keratinocytes. Melanin is the primary pigment of the skin which serves as protection against the u ltra v io le t rays of the sun. Finally, lamellar granules are found only in the epidermal cells of the stratum spinosum and the stratum granulosum and have been known by d iffe re n t names such as Odland bodies, keratinosomes, cementsomes, and membrane-coating granules (MC6). Less is known about 45 these granules compared to the melanosomes and keratohyalin granules above but what is known is that they are composed of alternating layers of lipid and protein and stain positive for acid phosphatase activity.

I t has been suggested that these lamellar granules serve a dual function: they secrete the lip id substance over the plasma membrane of the epithelial cell for protection against proteolytic enzymes until the final stages of keratinization and at this point, they release the protein portion which hydrolyzes this protective layer so desquamation can occur (34,180,216).

In addition to keratinocytes, the stratum germinativum of the epidermis also contains other cell types such as melanocytes, Langerhans cells and Merkel cells (41). The function of the melanocytes has been well established and th e ir origins traced to the neural crest (149).

The Langerhans cells are thought to be derived from the bone marrow

(41,159,158). These cells have lysosomal a c tiv ity and seem to function like macrophages by playing an important part in immunological reactions

(41,269,268,334). On the other hand, the origin of the Merkel cells is unknown but i t has been suggested that they function as mechanoreceptors detecting sensations in the skin and transmitting the messages to the nerve complexes (41,139). To date, there have been no published attempts to cultivate these ce lls. However, both the Langerhans cells

(243) and the melanocytes (243,165,155,117,118,133,329,307,170,81) have been maintained in culture.

In contrast, there are many publications dealing with the in vitro growth of human epidermal keratinocytes (for reviews, see 19,243,169,

249,94,154,130,198). The fir s t experiment ever recorded in which human skin was maintained in v itro was in 1898. Ljungren floated a piece of skin in ascites flu id for several days and demonstrated its v ia b ility by reimplantation into its donor (154,191). In 1914, Kreibich was the f ir s t investigator to be successful in explanting a piece of human skin on blood agar and describing the outgrowth of the e p ith e lia l cells

(172,198). Later, a trypsin digestion was used to separate the epidermis from the dermis by Szabo in 1955 (289). Skin grafts were treated with 0.5% trypsin and 0.05% NaHCOg overnight at 4°C and the dermis of each graft separated with fine forceps. The epidermis was then established in an explant culture. By this procedure, attempts to obtain a pure epithelial cell culture were unsuccessful because the cultures were eventually overrun by fibroblasts, presumably from a small amount of dermis s till attached to the explant (187,26,134). It was not until 1967 that Briggaman was able to establish a human skin epithelial culture without dermal contamination (42).

However, in 1975, Rheinwald and Green demonstrated that epidermal keratinocytes not only could be established in culture but also could be se ria lly subpassaged (248). Biopsy samples of human skin were digested with trypsin and the epithelial cells seeded along with 3(10)^ lethally irradiated 3T3 cells. Observations on the growth of these keatinocytes led to the following conclusions. The epithelials proliferate and keratinize (terminally differentiate) in vitro similiar to the way they do in vivo. They also have a finite life span in culture of 20-50 generations. Hydrocortisone at 0.4 pg/ml had no effect on primary cultures but enhanced the growth rate of subsequent cultures and therefore, was routinuely added to the growth medium. By culturing the 47 epithelial cells on a feeder layer of 3T3 cells, their growth seemed to be enhanced while the growth of fibroblasts was inhibited. This deduction served to further the idea that epidermal cells are dependent on dermal components for continued growth (199,211,201,75,323,324).

Following the publication of this paper, several variations and modifications of the procedure have appeared. Equally viable epidermal cultures have been obtained free of fibroblasts with dispase (167) and collagenase (127) digestions. Feeder layers of irradiated 3T3 cells

have been replaced in some instances by collagen gels (189,190,122,241) fibronectin-coated dishes (99,197,100), or just the plastic surfaces of

tissue culture flasks (80,206). Specific growth media

(240,231,232,305,197,100) have been developed including attempts to

identify the exact requirements of the epithelial cells by growing them in a "defined" medium. This medium is supplemented with a variety of hormones and extracts instead of serum removing the need of the epithelial cells for dermal components. In addition, several substances have been added to the mediums to enhance e p ith e lia l growth including cholera toxin (222,105) and epidermal growth factor (305,100,233,250).

Hydrocortisone enhances growth in dispersed cultures (248,233,189,305) but has been found to induce terminal d iffe re n tia tio n in human skin organ cultures (291,292). Finally, the keratinocytes were shown to grow better at a more acidic pH (pH 5.2 to 6 ) (80,81,82,87) although there

have been disagreements (122,240).

For some investigators, contaminating fibroblasts are s till a formidable problem. Several solutions have been proposed. Treating the epidermal keratinocytes with geneticin (117), spermine (142), or cholera 48 toxin (81), or growing them at a lower temperature of 32-33°C (143) instead of 37°C was found to inhibit the growth of fibroblasts.

In 1980, Milo <2t aK (206,215) reported the a b ility to grow human epidermal keratinocytes on a plastic substratum without the addition of extrinsic factors such as the hormones and substances described above.

The cells were grown at 37°C and at pH 7.2 and could be serially subpassaged up to PDL 20. Also, fibroblasts were not detectable at any time in these cultures. However, this procedure has been further modified as presented below.

MATERIALS AND METHODS

Establishing Primary Cultures of Human Epidermal Keratinocytes:

As described in Chapter 1, human neonatal foreskins were collected in 20 ml glass vials containing 5 ml of 5% FBS-CM and refrigerated.

Only foreskins which had been obtained by circumcision one to two days before were used to establish epidermal keratinocyte cultures and again, a ll cell culture procedures were done under a s te rile atmosphere.

First, with sterile forceps, the foreskins were transferred from their collecting vials to 60 mm dia. dishes (28 cm , Falcon Labware, Becton

Dickinson and Co.) each containing 3 ml HBSS(EGTA) plus p e n ic illin and streptomycin, one skin per dish. HBSS(EGTA) refers to prechilled Hanks'

Balanced Salt Solution without calcium or magnesium (HBSS-Ca,-Mg,GIBC0) with 0.02% [Ethylenebis(oxyethylenenitrilo)]tetraacetic Acid (EGTA,

Eastman Kodak Co., Rochester, New York) to which was added 200 U/ml penicillin-G (benzylpenicillin, sodium sa lt, Sigma Chemical Co.) and

0.2 mg/ml streptomycin sulfate (Sigma Chemical Co.). Each skin was swirled to remove excess blood cells and then transferred to another 49

60 mm dia. dish also containing 3 ml HBSS(EGTA) plus penicillin and 2 streptomycin. The skin was cut into four to six 5 mm pieces and

subsequently transferred to a s te rile glass vial with 3 ml of a 0.125%

trypsin solution made up in HBSS(EGTA). The vial was refrigerated for

48 hours at 4°C.

After refrigeration, the solution of skin pieces in the vial was

transferred to a 60 mm dia. dish. The top layer (epidermis) of each

piece was separated from the bottom layer (dermis) with the aid of

ophthalmic forceps. The dermal pieces were then transferred to another

60 mm dia. dish containing 3 ml prechilled HBSS(EGTA), swirled to rinse

off any remaining epithelial cells, and discarded. (It should be noted

here that these dermal pieces could be redigested in 0.25% collagenase as described in Chapter 1 for approximately one to two hours and used to establish primary fibroblast cultures.) The cell solutions from both

60 mm dia. dishes were then pipetted into a sterile 15 ml centrifuge

tube and the dishes rinsed with 5 ml prechilled HBSS(EGTA) which was also added to the tube (fin a l volume-11 ml). The solution was

centrifuged at 1000 rpm for 10 min (ca. 260 x g), the supernatant

pipetted off, and the cell pellet rinsed with 10 ml HBSS(EGTA) and

recentrifuged. The p e lle t was resuspended in 5 ml EKM and seeded into a o 100 mm dia. dish (79 cm , Corning Glass Works). EKM refers to Epidermal

Keratinocyte Medium which is a 11:9 mixture (v/v) of CM and Eagle's MEM with Modified Earle's Salts for Suspension Culture without ^-glutamine

(supplemented in the same manner as CM except no NaHCO^ was added,

GIBC0) with IX vitamins (M.A. Bioproducts), 2 mM ^-glutamine, 10% FBS,

100 U/ml p e n ic illin , and 0.1 mg/ml streptomycin. The cultures were 50 grown at 37°C and under 3.5% CO 2 . After two to three days, most of the keratinocytes which had previously been the basal layer of the epidermis had attached to the plastic substratum and the cultures were refed as described below.

Refeeding and Subpassaging Keratinocyte Cultures:

The keratinocyte cultures were refed by pipetting off the medium, rinsing each dish with 10 ml of CM, and then adding 10 ml EKM. These cells were routinely refed every two days. When the cultures were

70-80% confluent, they were subpassaged. Again, the medium was pipetted o ff, the dishes rinsed twice with prechilled versene (Ethylenedinitrilo) tetraacetic Acid Dicalcium Salt (EDTA, Eastman Kodak Co.), and 1 ml of a

0.25% trypsin solution made up in versene was added. After incubating for five to ten minutes, the cells were observed by phase contrast microscopy (X 100) to have detached from the plastic surface and the trypsin activity was neutralized with 10 ml EKM. The cell solution was transferred to a sterile 15 ml centrifuge tube and following centrifugation at 1000 rpm for 10 mins (ca. 260 x g), the supernatant was pipetted o ff, the cell p e lle t resuspended in 10 ml EKM, and 5 ml added to each of two 100 mm dia. dishes. Only 1:2 s p lits were used to subpassage these epith e lia l cells.

Characterization of Epidermal Keratinocytes:

Three d iffe re n t procedures were used to characterize the epidermal keratinocytes: phase contrast photomicrography, a modification of the

Mallory connective tissue stain by Ayoub and Shklar (18), and epifluorescence photomicrography with Rhodanile blue (166). The f ir s t procedure, phase contrast photomicrography, was done using an inverted 51

microscope (Nikon Diaphot-TMD, Nippon Kagaku K.K., Tokyo, Japan) with a

35 mm camera (Nikon FE, Nippon Kagaku K.K.) attached. The following

settings were used: a film speed of 80 or 100 ASA and an exposure value

of +1 on the camera and a voltage setting of 9 for the lamp of the

microscope. Panatomic-X film (FX 135-20, Eastman Kodak Co.) was

developed by direct positive processing.

Next, a modified Mallory connective tissue stain was applied to the

epithelial cultures. Cells which contained keratin were distinguished

from those which did not by staining red whereas the la tte r cells

stained gray. The cultures were firs t treated with the fixative, 3%

formalin in Phosphate Buffered Saline (PBS), for 20-25 min and then

rinsed several times with double-distilled water (dd H 20 ) and air dried.

To each dish, 5 ml of a 5% acid fuchsin solution (MCB Manufacturing

Chemists, Norwood, Ohio) was added and three minutes later, poured o ff.

Next, 5 ml of a second stain solution containing 0.5% aniline blue

(water-soluble), 2.0% orange G (both from MCB Manufacturing Chemists),

and 1.0% phosphotungstic acid (J.T. Baker Chemical Co., Phillipsburg,

New Jersey) was added for 45 min. The dishes were then rinsed twice with dd H 20, several times with 95% ethanol and twice with absolute

ethanol. Photographs of the cultures were obtained similiar to the method described above except that Ektachrome film (160 Tungsten, ET

135-20, Eastman Kodak Co.) was used along with a ND2 f ilt e r .

Finally, the la st procedure involved staining the epidermal

keratinocyte cultures with Rhodanile blue and observing them with epifluorescence photomicrography. First, the cultures were fixed with

10% formalin in PBS for 20 min, rinsed twice with PBS, and 10 ml of a 1% 52 Rhodanile blue (Pfaltz and Bauer, Inc., Stamford, Connecticut) solution was added for 45 min. The cells were then rinsed several times under cold water and viewed using an epifluorescence attachment with a 100W Hg lamp on the inverted microscope. Ektachrome color film (400 Daylight,

Eastman Kodak Co.) was used along with a f ilt e r whose excitation wavelength and barrier wavelength was 546 nm and 580 nm, respectively.

RESULTS AND DISCUSSION

From the procedure by Milo e t a l. (206,215), several steps were modified. F irs t, the human neonatal foreskins obtained from circumcisions were frequently contaminated with bacteria. This contamination was controlled by rinsing the skin pieces twice in

HBSS(EGTA) with 200 U/ml p e n ic illin and 0.2 mg/ml streptomycin before treatment with trypsin. In addition, 100 U/ml p e n ic illin and 0.1 mg/ml streptomycin were added to the growth medium routinuely except during chemical treatment of the cells as in Chapter 3. 50 yg/ml gentamicin was also present in the growth medium and in the vials used to collect the foreskins. These antibiotics were not observed to effect the seeding or growth of the epidermal keratinocytes. Bacterial contamination of human skin samples had previously been reported (156) and was minimized by soaking the skin tissue in HBSS with 400 U/ml penicillin and 0.2 mg/ml streptomycin for one hour a t 12°c (189,190).

The use of p e n ic illin and streptomycin in the growth medium of keratinocyte cultures is widespread (81,127,80,100,291,142,82,143,77,

310). However, i t has been suggested that the use of streptomycin is outdated since many bacteria are resistant to it (10). In this case, it was recommended that streptomycin be replaced with 20 yg/ml o xa cillin 53 and used in addition to 100 U/ml p e n ic illin and 50 pg/ml genamicin.

This recommendation s t i l l remains to be investigated.

Secondly, the digestion with 0.25% collagenase was replaced with a digestion in 0.125% trypsin in HBSS(EGTA) for 48 hours at 4°C. This trypsin solution was not buffered and the pH was 3.7. Nevertheless, the epidermis was easily separated from the dermis with cleavage at the basement membrane. Recently, more e p ith e lia l cells were observed to attach to the plastic substratum if the trypsin treatment was only for

24 hours. This method is si mi lia r to the procedure used by Eisinger et a l. (80,82) and Kitano and Hata (165) where a 0.25% trypsin solution was used for 12-15 hours at 4°c. In addition, the dermis could be redigested in 0.25% collagenase for one to two hours at 37°C to obtain viable fibroblast cultures which had also been noted by Eisinger et a l.

(80).

Third, an increase in the attachment of the epidermal keratinocytes was noted when only 5 ml EKM was used to seed the cells into the 100 mm dia. dishes rather than the 10 ml volume used with the fibroblasts. The lesser volume lowered the air-liquid interface so that it was closer to the cells. Prunieras (244) demonstrated that e p ith e lia l cultures more closely resembled the in vivo structure of the epidermis i f they were more directly exposed to a ir oxygen.

In addition, when the keratinocyte cultures were grown at 3.5% C02, the cells exhibited enhanced growth than when they were cultured at 4%

C02. The culture temperature was 37°C which was found to be the optimum temperature for growth by previous investigators (122). Also, the growth medium was buffered to pH 7.2 and was supplemented with 10% FBS. 54 Both conditions were demonstrated by Hawley-Nelson et a l. (122) and

Karasek (156) to be responsible for maximum growth of human

keratinocytes in culture.

Next, the combination of media used to culture the e p ith e lia l cells

improved th e ir growth compared to the media used by Milo et a l.

(206,215). Based on the amount of anhydrous CaCl 2 used in the

formulations and excluding the free calcium present in the serum, the

calcium concentration of this combination of media was calculated to be

approximately 0.7 mM. In contrast, the calcium concentration of the

medium used for the fib rob la st cultures (Chapter 1) was on the order of

1.3 mM. This level is in agreement with Whitefield et a l. (77,325) who

demonstrated that the optimum calcium level for fib rob la st p ro life ra tio n

was 1.0 mM. However, the lowered calcium concentration of the

e p ith e lia l medium has been found by several investigators to enhance

keratinocyte growth (122,232,305,77,223,321). Calcium levels on the

order of 0.03-0.4 mM were responsible for increasing cell proliferation

while higher calcium levels (~1.2mM) induced these cells to undergo

terminal d iffe re n tia tio n , although there is some disagreement (241).

Finally, in subpassaging the epidermal keratinocytes, trypsin in

versene was more e ffic ie n t in detaching these cells from the plastic

substratum than trypsin in HBSS(EGTA). The cells were removed by the

former solution in 5-10 min whereas with the la tte r solution, detachment was as long as 30 min.

By using the method of trypsin digestion, a pure culture of epidermal cells could be obtained. No fibroblasts were detected by

phase contrast microscopy or by electron microscopy (206). An 55 approximate population of 86-90% epidermal keratinocytes, 5-7% melanocytes, and 5-7% Langerhans cells was observed by Eisinger and

Marko (81). However, the predominantly keratinocyte cultures differed markedly from cultures of fibroblasts and resembled closely the in vivo structure of the epidermis.

When epith e lia l cells were disaggregated from the dermis and seeded into culture, groups of cells attached to the plastic substratum and within 24-48 hours, spread out and formed colonies. The cells were polyhedral in shape and somewhat flattened as Seen in Figures 11 and 12.

In contrast, fibroblasts had a spindle shape in culture and tended to attach as single cells as in Figures 13 and 14. Fibroblasts also lacked desmosomes which were a prevalent feature observed in cells in an e p ith e lia l culture. Using time-lapse cinematography, Sun and Green

(285) noted that these la tte r cells moved in groups not as individual cells as fibroblasts did. These groups of keratinocytes were observed to move around the periphery of a colony or between the center and the periphery. The cells were attached to one another presumably by desmosomes. Also, previously i t was demonstrated that single epithelials which attached to the culture surface isolated from the colonies did not survive (240).

In addition, keratinocytes in culture underwent two different processes: cell p rolifera tio n and keratinization or terminal differentiation. The cells which first attached to the plastic surface closely resembled the basal cells of the epidermis. Most of the time, they appeared polyhedral and flattened and only appeared in some very confluent cultures as cuboidal (166). These cells had numerous 56

Figure 11. Phase-Contrast Photomicrograph of Preconfluent Epithelial

Colonies Growing in Vitro (X 40).

Figure 12. Phase-Contrast Photomicrograph of Preconfluent Epithelial

Colonies Growing in Vitro (X 100). Figure 13. Phase-Contrast Photomicrograph of a Preconfluent Fibroblast

Culture (X 40).

Figure 14. Phase-Contrast Photomicrograph of a Preconfluent Fibroblast

Culture (X 100). desmosomes and were richin organelles but there was an absence of

hemidesmosomes and basal laminae (206,166). The e p ith e lia ls cells in

this layer proliferated causing the colonies to grow in size and to

eventually merge asshown in Figure 15. In contrast, confluent

fibroblast cultures were seen as a swirling or whorling pattern as in

Figure 16. It had been noted by Milo et a l. (206) that a confluent 2 population of keratinocytes was approximately 50,000 cells/cm while 2 confluent cultures of fibroblasts were about 20,000 cells/cm . The

larger density recorded for the e p ith e lia l cells may be explained by the

process below.

Simultaneous with p ro life ra tio n , keratinocytes also became detached

from the culture surface and migrated upward forming multilayered

colonies. These colonies had been observed to be from 5 to 11 cell

layers in thickness (206,82,285). The cells in these layers underwent

terminal d iffe re n tia tio n s im ilia r to the process which occurs in vivo.

Layers resembling the stratum spinosum and the stratum granulosum had

been observed with cells which contained an increased number of

tonofilaments, keratohyalin granules, lamellar granules and a decreased

number of desmosomes (80,206,166,144). Also, in the most superfical

layers, fully keratinized cells resembling cells of the stratum corneum were seen. These cells were found to be approximately 35 ym in diameter

compared to the 14 ym diameters of the basal cells (285). They also

possessed a cornified envelope ju st beneath the plasma membrane and

underwent desquamation as in vivo. However, the squames which were shed

from the cultures into the medium were discovered by Green (104) to

contain nuclei, although they were pyknotic. In vivo, squames digest Figure 15. Phase-Contrast Photomicrograph of a Confluent Epithelial

Culture (X 200).

Figure 16. Phase-Contrast Photomicrograph of a Confluent Fibroblast

Culture (X 40). 60

their nuclei with proteolytic enzymes before they become detached from

the skin surface. Later, digestion of the nuclei in vitro was found to

be dependent on the presence of serum. Green showed a direct

correlation which was concentration-dependent between serum plasminogen

and the destruction of the nuclei. The ce llu la r membranes became

permeable as a result of d iffe re n tia tio n allowing plasminogen to enter

and followed shortly afterward by nuclear digestion induced by plasmin

(154).

The process of keratinization which the epidermal cells undergo in

vitro has attracted much interest in recent years. Two stains have been modified and widely used to distinguish these cells in culture.

Rhodanile Blue (195,196) and the Mallory connective tissue stain (18).

Rhodanile Blue was f ir s t used on human keratinocytes in v itro by

Rheinwald and Green (248) who also used i t to characterize keratin

formation in a mouse teratoma cell line (247). It consists of a

combination of two stains, Rhodamine B and Nile Blue (196) and on fixed cells, a red color denotes keratin while everything else is blue.

Another way in which this stain can be used is in conjunction with epifluorescence microscopy (166). Cultures of epidermal keratinocytes were fixed and stained with Rhodanile Blue and the results are shown in

Figure 17. A predominantly monolayer culture fluoresces orange and the beginnings of a keratin sheet can be seen. This sheet contains cells which are undergoing terminal d iffe re n tia tio n and also fluoresces more

intensely than the cells beneath.

The second stain, the Mallory connective tissue stain, was firs t used on human keratinocytes by Steinberg and Defendi (282). Not only Figure 17. Epifluorescent Photomicrograph of an Epithelial Culture

Stained with Rhodanile Blue (X 100).

I w m M s *

'ia&wt ‘1 s>. „ % m - f < . « . . i ' l

1 8 ^ *

Figure 18. Phase-Contrast Photomicrograph of an Epithelial Colony

Stained with the Mallory Stain (X 100). 62 does it stain keratin a bright red, but it also stains a prekeratin

substance orange and fibroblasts and cells not containing keratin blue.

Preconfluent epithelial colonies in Figure 18 stained red showing the

presence of keratin. Both orange and red colors are seen in Figure 19

in a more confluent epithelial culture denoting prekeratin and keratin,

respectively. Next, in Figure 20, a very bright or deep red color

corresponds to a discontinuous keratin sheet over a confluent e p ith e lia l

culture. As expected, a fibroblast culture illustrated in Figure 21

stained blue.

In addition, immunofluorescent staining of keratin in human

epidermal keratinocytes grown on 3T3 feeder layers was done by Sun and

Green (286,287). Noyes and Milo (unpublished data) developed a rabbit

anti serum made against human keratin extracted from excised callus and

attached to a fluorescent tag. This anti serum was used to treat fixed e p ith e lia l ce lls cultured on a plastic substratum and in Figure 22,

these cells were shown to fluoresce indicating the presence of keratin.

This fluorescence in some cells was observed to be greater than in

others and was shown to be greater around the nuclear membrane compared

to the periphery of the cells. In Figure 23, a cross section of a human

neonatal foreskin is shown. Only the epidermis fluoresced indicating

that keratin was present only in this portion of the skin.

Recent studies on terminal differentiation of human keratinocytes in vitro have shown that keratin peptides between the molecular weights of

44,000 to 58,000 were synthesized (144,287,101). However, the largest

class of keratin molecules (MW=63,000) was not synthesized in culture as

i t was in vivo. Cultured cells have been shown by Fuchs and Green (96) 63

Figure 19. Pbase-Contrast Photomicrograph of an E pithelial Culture

Stained with the Mallory Stain (X 200).

Figure 20. Phase-Contrast Photomicrograph of an E pithelial Culture

Stained with the Mallory Stain (X 40). 64

■ s ^ 1 ’ -ft*;8 V % *’V :" -s’*'K-'- .!?■"«*****&»*£■' •“

i •• v n N > ' i > < > ' -., ’ 4 A \ \ W \S \A -: i.

‘ V ' ’ \ t V '9 * \ ->VX , w* ' . -.. *. • ' t * a A V -V -' ?’. :•■'•'■•' XVN'- > > -,v‘ <.> i\ • ’ , * v '• *»A v ’ v ' 1 *■ ^ 5l . 1 * - 1 ^ A\\\^A*Vl~, ' :■'''- •••

Figure 21. Phase-Contrast Photomicrograph of a Confluent Fibroblast

Culture Stained with the Mallory Stain (X 100). Figure 22. Immunofluorescent Staining of Epidermal Keratinocytes with

Anti serum against Human Keratin (X 400).

Figure 23. Immunofluorescent Staining of a Cross Section of Human

Neonatal Foreskin with Anti serum against Human Keratin

(X 160). 66 to possess mRNAs only for the smaller molecular weight keratins. As the

cells differentiated, the total amount of keratin increased while the relative proportions of each type of peptide remained the same (290).

These molecules in the fin a l stages of d iffe re n tia tio n were cross-linked by disulfide bonds and completely fille d the cytoplasms of the epithelials (104). So far, nine classes of keratins have been identifed

(79).

In addition, epithelial cells formed a cornified envelope directly underneath the plasma membrane. The precursor to this envelope was a protein, involucrin, firs t described by Rice and Green (251,252). It was present in a ll cells of epidermal cultures except the basal layer and also in vivo in a ll layers except the basal layer and the lower portion of the pickle-cell layer (24). When the intracellular calcium ion concentration to the individual cells was increased by the use of high salt solutions, nonionic detergents, or ionophores, the activity of tranglutamlnase was increased. Cells in the basal layer began to synthesize involucrin and were immediately expelled from this layer to more superficial layers (321). As the cells grew in size, more involucrin was synthesized and cross-linked with e-(Y-glutamyl)lysine bonds by transglutaminase forming the cornified envelope (252,320).

In summary, methods were shown to be available now by which human epidermal keratinocytes can be cultured in v itro without contamination by fibroblasts from the dermis. These cells could be se ria lly subpassaged and formed multilayered cultures which closely resembled the in vivo structure of the epidermis. They could be readily distinguished from fibroblasts by the presence of desmosomes and tonofilaments, the a b ility to synthesize keratin, the formation of cornified envelopes, and

the manner in which they proliferated and differentiated in culture.

Much information has been obtained and w ill continue to be collected about the inner ce llu la r processes of the human epidermal keratinocytes. CHAPTER 3

INTRODUCTION

The mixed-function oxygenases (MFOs) or monooxygenases are a group

of enzymes which metabolize a variety of compounds ranging from steroids

and bile acids to drugs and carcinogens (330). The principal enzyme

complex was f i r s t demonstrated to be substrate-inducible in 1957 by

Conney et a l. (59,58). Pretreatment of rats with benzo(a)pyrene (BP)

caused an increase in the synthesis of the BP-metabolizing enzyme found

in liv e r microsomes. I t required NADPH and 02 for maximal a c tiv ity and was referred to as "benzpyrene hydroxylase" because the major metabolites identified were the monohydroxy derivatives. Later, as the

range of substrates for this enzyme broadened, i t was referred to as aryl hydrocarbon hydroxylase. It was also shown to be inhibited by carbon monoxide (171,97) and was named "cytochrome P-450" by Omura and

Sato (224,225) because its CO-difference spectrum obtained from reduced

liv e r microsomes showed a Soret peak at 450 nm.

Cytochrome P-450 is closely associated with NADPH-cytochrome P-450 reductase and a phospholipid, phosphatidylcholine (284,152). In 1965,

Cooper et a l. (66) f ir s t deduced that this cytochrome was the terminal oxidase of MFO systems and the mechanism of action of this enzyme complex is shown in Figure 24 (330). The substrate (R) is bound to the iron (III) protoporphyrin IX moiety and an electron is transferred by the reductase from NADPH to the cytochrome P-450-substrate complex

68 69

P450(Fe’ *) P450(FeJ+) • R

ROH e- (NADPH)

XOOH H,0.

H,0 e- (NADPH)

Figure 24. The Mechanism of Action of Cytochrome P-450 (330). 70 reducing the iron atom. Next, a molecule of oxygen is bound forming

oxycytochrome P-450 and another electron is transferred. One oxygen

atom is then inserted into the substrate molecule to give R-OH and the

other atom is reduced to water. Phosphatidylcholine is necessary

because i t binds the reductase and the substrate to cytochrome P-450

(64).

Further studies by Orrenius and Ernster (226) and Remmer and Merkel

(246) reported that this particular cytochrome, cytochrome P-450, was

increased in ra t liv e r by phenobarbital (PB) causing an increase in the

smooth endoplasmic reticulum within the hepatocytes. However, studies with 3-methylcholanthrene (3-MCA) showed that i t induced a hemoprotein whose difference spectrum showed a s h ift in the Soret peak to 448 nm

(1,274). This new enzyme was synthesized de novo (6) and was called cytochrome P-448 or £ytochrome P^-450. Even though multiple forms of

P-450 had previously been suggested (60,62,17,322), these reports were the f ir s t to demonstrate conclusively that more than one form or isozyme did exist.

As more compounds were investigated for th e ir effects on cytochrome

P-450, they were classified into two groups: those which acted like PB in stimulating a wide variety of actions and those which were similiar to 3-MCA in inducing only a few specific reactions (61,176). The firs t group included not only PB but also trans-stilbene oxide (200) and xylene (304,303) and these compounds were found to induce several oxidation, reduction, and rearrangement reactions catalyzed by P-450, such as hexobarbital oxidation, ethylmorphine N-demethylation, and aminopyrine N-demethylation. In the 3-MCA group, other compounds such 71 as 2,3,7,8-tetrachlorodibenzo-£-dioxin (188), benz(a)anthracene (129),

BP and B-naphthoflavone or 5,6-benzoflavone (229) stimulated specific activities like BP hydroxylation, 7-ethoxyresorufin o-deethylation, and acetanilide hydroxylation. However, some compounds behaved neither like

3-MCA or PB and were classified as atypical inducers (58). These chemicals included rifampicin (128), pregnenolone-16a-carbonitrile

(33,194), isosafrole (89,90,74), and Arochlor 1254 (259,5).

In addition, the reactions stimulated by these various inducers are inhibited by several compounds. SKF 525-A was one of the f ir s t inhibitors studied (274) and was found to specifically block

BP hydroxylation (176). Butylated hydroxyanisole (BHA) was demonstrated by Hennig et a l. (126) to lower the content of cytochrome P-450 not only in microsomes but also in the nuclear fraction in mice. Other compounds which inhibit MFO activity include a-naphthoflavone or 7,8-benzoflavone

(319), metyrapone and norharmane (44).

To date, rat liver and rabbit liver have been the most studied tissues in investigating the presence of cytochrome P-450 isozymes. In rat liver, eight forms of P-450 have been isolated and purified to homogeniety (111,110). Five isozymes (PB-B, PB-C, PB-D, UT-A, and

PB/PCN-E) are inducible by PB and three forms (UT-F, BNF-B, and

BNF/ISF-G) are stimulated by B-naphthoflavone (b_NF). In addition, several of these isozymes are also induced by Arochlor 1254, 3-MCA, pregnenolone-16a-carbonitrile (PCN), isosafrole (ISF), or appear in large enough amounts to be observed in live rs of untreated (UT) rats.

These differences in inducers, molecular weight, immunochemistry, and 72

spectral data led to the detection of these eight different cytochrome

P-450s. However, in rabbit liv e r, at least 10 forms of P-450 have been identified (9,84) but only seven have been isolated and purified and are

referred to as LMj y, LM2, LM3a* LM3b» LM3C’ L^4’ LM5’ and LM6

(72,253,210,35,121). As in the rat liver, different inducers stimulated

the synthesis of these various P-450s. Also, more than one cytochrome

P-450 has been found in several other species and tissues including mouse liver (212), mouse lung (309), rat lung (261,318), and rabbit lung

(112,277,333).

In contrast to the wealth of information in mammalian species, little is known about P-450 in human tissue. The firs t isolation of human liv e r microsomes was done by Creaven and Williams who observed the presence of an aromatic hydroxylase (69,68). Then, in 1966, Kuntzman et a l. . (175) studied the hydroxylation of BP by human liv e r enzymes and found them to be sim ilia r to those in the ra t. Three years later,

Alvares et a l. (2) and Darby et a l. (71) isolated microsomes from adult liv e r and detected the presence of P-450. Cytochrome P-450 was also detected by Yaffe et a l. (335) in fe ta l liv e r. Since then, several investigators have isolated and attempted to characterize this cytochrome in both adult and fetal liver (316,38,28,164,151), but only recently was it reported that there are at least six forms of P-450 in adult liver (317). Different isozymes have also been strongly implicated in human placenta (227,236,53) with as many as four forms possible (236) and monooxygenase a c tiv ity has been demonstrated in other human tissues such as lung (242,193), leukocytes (326,45,27), kidney, adrenal gland, intestine, spleen, and pancreas (235,234,150). 73 Therefore, cytochrome P-450 has been found not only in mammalian systems

but also in human tissues.

Aryl hydrocarbon hydroxylase a c tiv ity which is associated with P-450

has been reported in human skin (31,3,186) and was induced by various

PAH. As described e a rlie r, AHH is responsible for the major pathways in

BP metabolism and therefore has been in d ire ctly implicated in several

reports on BP metabolism of human epithelial cells in culture

(213,299,230,179,178,124). In the following experiments, BP metabolism was again investigated in human epidermal keratinocytes comparing

extracellular metabolites to intracellular metabolites. In addition,

the effects of five compounds (BHA, 3-MCA, PB, isosafrole and Arochlor

1254) which were previously found to in h ib it or induce P-450 in mammalian species, were observed on the formation of BP metabolites in

these cells.

MATERIALS AND METHODS

Pretreatment of Epidermal Keratinocytes:

Five chemicals were evaluated for th e ir effects on the

benzo(a)pyrene metabolism p ro file of human epidermal keratinocytes:

3-methylcholanthrene (3-MCA, practical grade, Sigma Chemical Co.),

sodium phenobarbital (PB, Elkins-Sinn, Inc., Cherry H ill, New Jersey),

isosafrole (ICN Pharmaceuticals, Inc., Plainview, New York), Arochlor

1254 (NCI Chemical Carcinogen Repository), and butylated hydroxyanisole

(BHA, Sigma Chemical Co.). For each compound, a stock solution of

1 mg/ml was made up in SpectrAR grade acetone with the exception of PB, which was dissolved in s te rile double-distilled water. The stock

solution of 3-MCA was stored under argon(g) in a foil-wrapped vial at 74

-20°C, whereas the other stock solutions were stored also under argon but refrigerated. The keratinocytes were established and grown in culture as described in Chapter 2. Only primary cultures which were

20-60% confluent were used in the following experiments.

The treatment protocol was the same for the administration of all five compounds. To begin with, the medium was pipetted o ff the cultures growing in 100 mm dia. dishes and the cells rinsed twice with 10 ml CM.

Next, 10 ml EKM (without p e n ic illin or streptomycin but which contained the chemical to be studied) was added to each dish and the cultures incubated for 24 hours and 37°C. Afterwards, the dishes were rinsed twice with 10 ml CM and then treated with benzo(a)pyrene as described in the next section.

The f ir s t compound which was studied, 3-MCA, previously had been shown to be nontoxic in human skin fib rob la st cultures at 1 yg/ml

(3.73 y M) and 10 yg/ml (37.3 yM) (205). Therefore, the epithelial cultures were divided into two groups with each group treated at one of the concentrations. For example, in the case of 3-MCA, 100 ul (100 ug) of the stock solution was added to 100 ml EKM to give a final concentration of 3.73 yM. For 37.3 yM, 1000 yl (1000 yg) of the stock solution was used per 100 ml EKM. Equimolar amounts of the remaining four chemicals were calculated using the molecular weight for each compound and the aliquots which were added to 100 ml EKM are shown in

Table 6. The molecular weight of Arochlor 1254 was estimated using the molecular formula ci2H10-xC1x where x=0*54 or 54* chlorine. In addition, all manipulations with 3-MCA in the treatment procedure detailed above were completed under gold light (40W, General Electric) TABLE 6

Equimolar Aliquots of Stock Solutions of Pretreatment Chemicals

Added to Epidermal Keratinocyte Medium

______yil Stock Solutions Added to 100 ml Molecular Final Final Weight Concentration Concentration Chemi cal (g/mole) 3.73 tjM 37.3 yM

3-Methylcholanthrene 268.34 100.0 1000

Phenobarbi tal 254.22 94.7 947

Isosafrole 162.18 60.4 604

Arochlor 1254 340.22 126.8 1268

Butyl a ted Hydroxyani sole 180.00 67.1 671

because of the s e n sitivity of this compound to photodecomposition in the presence of white light (95,177). This precaution was not necessary for the other chemicals studied.

Treatment with Benzo(a)pyrene:

In these experiments, there were six groups of cultures treated with benzo(a)pyrene as shown in Table 7. The profiles of the f ir s t group were used to compare not only with the profiles of the other five groups but also with profiles previously published (179,178,299). For each group, the procedure used was s im ilia r to the method described in

Chapter 1. B rie fly , [G-3H]BP (21-36.7 Ci/mmole, 5 mCi/ml) was blown to dryness with argon and redissolved in SpectrAR grade acetone. Both radiolabelled and unlabelled BP were added to 100 ml EKM (without penicillin or streptomycin) to make a final concentration of 6.9 uCi per

0.25 Mg. Ten ml of this medium was then added to each dish of cells and 76 TABLE 7

Groups of Pretreated and Nonpretreated Epithelial Cultures

Group Pretreatment Treatment

1 None BP

2 3-MCA BP

3 PB BP

4 Isosafrole BP

5 Arochlor 1254 BP

6 BHA BP

the cultures incubated fo r another 24 hours and subsequently harvested.

Again, 10 ml aliquots of the treatment medium were extracted and designated as control samples.

Preparation of Extracellular and Intracellular Fractions:

For each group above, only extracellular and cytoplasmic fractions were studied.1 The preparation of the extracellular portion was sim ilia r to the method described in Chapter 1 for the fibroblasts.

After 24 hours' treatment with BP, the medium from the culture dish

(total volume-10 ml) was pipetted o ff and extracted. The cells were trypsinized and a cell count taken using a hemocytometer.

For the intracellular fraction, three to four 100 mm dia. dishes of keratinocytes were rinsed twice with 10 ml versene and 1 ml of a 0.25* trypsin solution made up in versene was added to each dish as detailed

1I t was not possible at the present time to obtain cytoplasmic and nuclear fractions of the keratinocytes. The keratin formation in these cells make it difficu lt to obtain nuclei without any attached cytoplasmic material. in Chapter 2. When the e p ith e lia l cells had detached, 9 ml EKM (without penicillin or streptomycin) was added. The cell solutions from all dishes were pooled in s te rile centrifuge tubes, the dishes rinsed twice with 10 ml versene, and the tubes centrifuged at 1200 rpm for 30 min

(ca. 350 x g). A ll pellets were pooled into a single tube, recentrifuged at 1000 rpm for 10 min (ca. 260 x g), rinsed once with

10 ml versene, and the fin a l p e lle t resuspended in 10 ml HBSS(EGTA) for a cell count. After centrifugation, the cells were transferred to a 1.5 ml polypropylene micro test tube (Brinkmann Instruments, Inc., Westbury,

New York) and pelleted at 5000 rpm for 5 min (ca. 1660 x g). The supernatant was pipetted off and to extract BP within the cells, ethyl acetate with 0.8 mg/ml BHT was added. The tube was vortexed at maximum speed for 3 min and recentrifuged. The supernatant was pipetted off and saved and a 10 yl aliquot was used for liquid scintillation counting.

This extraction procedure was repeated u n til the counts in the ethyl acetate supernatants matched the counts from an ethyl acetate blank or approximately nine more times. Finally, the ten 1 ml aliquots were pooled for one in tra c e llu la r fraction.

The ethyl acetate layers from both the extracellular and in tra c e llu la r fractions were prepared for HPLC analysis in the same manner as in Chapter 1. B rie fly , the layers were dried with sodium sulfate, blown to dryness with argon and resolubilized in 400 ul MeOH.

Analysis by HPLC was done as before with an 0DS column and a linear gradient of 60* to 100* MeOH vs H20 with the radioactivity quantitated using an on-line s c in tilla tio n counter. 78

RESULTS AND DISCUSSION

A representative histogram of the organic-extractable radiolabelled

benzo(a)pyrene metabolites present in the extracellular fractions of

human epidermal keratinocyte cultures is presented in Figure 25.

Keratinocytes metabolized BP to the phenols, the 9,10-diol and the

7,8-diol with smaller amounts of the sulfates, the (7,10/8,9)-tetrol,

and the (7/8,9,10)-tetrol detected. The (7,9,10/8)-tetrol was present

in other preparations but was not present in the one shown in Figure 25 and the (7,9/8,10)-tetrol was not observed in any of the fractions evaluated including those samples from cells which had been pretreated.

However, the retention time of th is la tte r tetrol (31 min) is very close

to the retention time of the 9,10-diol (28 min) and baseline separation of these two peaks can be obtained using unlabelled standards detected

by UV absorbance a t 254 nm but baseline separation may not be achieved with detection by the on-line scintillation counter. In some of the

pretreated cell fractions, shoulders on the 9,10-diol peak were observed which may be due to the presence of a small amount of the

(7,9,10/8)-tetrol.

In addition, a small amount of the 4,5-diol which eluted ju st before

the 7,8-diol was observed in the extracellular fraction of the

BP-treated epithelial cells (Figure 25) along with three unknown peaks.

The f ir s t unknown peak, unknown A, eluted between the sulfates and the

(7,10/8,9)-tetrol and because of its very early retention time (12 min),

i t may be another type of conjugate. Two other kinds of conjugates, glucuronides and glutathione (GSH) conjugates, are known to occur during

BP metabolism (98). However, enzymatic digestion with 6-glucuronidase 79

Figure 25. Histograms of Organic-Extractable Radiolabelled

Benzo(aJpyrene Metabolites in the Extracellular Fractions of

the Epidermal Keratinocyte Cultures and Control Samples. m m m

oo 9 -O H (7,10/8,9)-TETROL (I) (7/8,9,10)-TETROL (1)01 SULFATES UNKNOWN UNKNOWN A UNKNOWN B UNKNOWN 4 , 5 - DIOL UNKNOWN C UNKNOWN (7,9,10/8)-TETROL (H) ■ ■ (7,9/8,10)TETROL — (I) DPM DPM x |0 : no no O no o O no

m —I —I m d co

Figure 25. 08 81 and arylsulfatase of the methanol layers blown to dryness, resolubilized in aqueous buffer according to Autrup's method (15) for digestion, reextracted, and rechromatographed, yielded a decrease in the sulfate peak to near background levels but had no e ffe ct on unknown A.

Therefore, unknown A could possibly be a glutathione conjugate.

The next two unknown peaks, unknown B and unknown C, eluted between the 7,8-diol and the phenols with retention times of 55 min and 64 min, respectively. Unknown B could be another type of d io l, such as the

11,12-diol, while unknown C could be a diol or an early eluting phenol.

These two peaks had been observed e a rlie r in the cytoplasmic and nuclear fractions of the confluent fibroblast populations.

The 9-OH and the 3-OH were the major phenols detected but small amounts of the 1-OH and 7-OH may also be present. The la tte r two phenols coeluted with the 3-OH and could not be quantitated separately.

There was a small amount (less than 5%) of im purities present in the control samples which eluted at the region of the phenols and was substracted from the value for the phenols in each fraction analyzed.

Again, quinone production due to autooxidation and photodecomposition was minimized using the precautions described in Chapter 1.

Pi comole amounts of these BP metabolites per 10^ e p ith e lia l cells present in the extracellular fractions are shown in Table 8. The phenols made up the largest portion of the metabolites found (32?) followed by the 9,10-diol (17%) and the 7,8-diol (14%). These results are in agreement with the findings of previous investigators. Early studies showed that indeed the isolated epidermis either in organ culture (92) or in monolayer culture using a feeder layer (230) could 82

TABLE 8

Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular

Fractions from Human Epithelial and Fibroblast Populations3 Epithelial Fibroblast Extracellular Extracellular Metabolites Fraction Fraction Sulfates 467.9 31.8

Unknown A 522.8 ND

Tetrol I (7,10/8,9) 507.6 ND

Tetrol I (7/8,9,10) 495.3 ND

9,10-Diol 1,298.5 373.5

Tetrol II (7,9/8,10) ND ND

Tetrol II (7,9,10/8) 270.0 ND

4,5-Diol 141.9 ND

7,8-Diol 1,043.2 279.6

Unknown B 236.0 ND

Unknown C 140.1 ND

Phenols 2,405.1 140.0

Benzo(a)pyrene 17,629.4 10,639.7

Total Metabolites 7,528.4e 824.9 aValues are expressed in picomoles per 10^ ce lls, lower lim it of detection was set at 0.2* of the total dpm per HPLC run bMean values, n=5 cRandomly-proliterating fib rob la st population, values are from Table 3, Chapter 1 dND-Not Detectable eRange (2,713.9-12,860.0) 83 metabolize BP. However, using confluent e p ith e lia l cultures without a feeder layer, Kuroki et a l. (179) were the firs t investigators to report the specific organic-extractable BP metabolites. The most abundant metabolites were the phenols followed by the 9,10-diol and the 7,8-diol.

They (179) were able to detect a small amount of quinones by HPLC and a peak corresponding to the tetrols but were not able to identify the individual tetrols or any of the conjugated metabolites. Later, their results were confirmed by Theall et a l. (299). In addition, Hukkelhoven et al. (138) isolated keratinocytes from human hair follicles and found the same metabolic profile.

Also, in Table 8, the BP metabolites from the randomly-proliferating fibroblast population described in Chapter 1 is presented for comparison. In these cultures, only the sulfates, the 9,10-diol, the

7.8-diol, and the phenols were detected and the total metabolites excluding the parent BP were only 11% of the total metabolites found for the preconfluent epithelial cultures. This difference in BP metabolism of these two cell types had previously been reported by Huberman and

Sachs (135), Nemoto et al. (213), and Karoki et a l. (178). In addition, in mouse skin, AHH activity in the epidermis was found to be four to five times higher than in the dermis (302) and this higher epidermal a c tiv ity could explain the higher rate of BP metabolism of the keratinocytes in human skin.

BP metabolites were also found in the intracellular fractions of the e p ith e lia l cells and the values are shown in Table 9. Only the

7.8-diol, unknown B, unknown C, and the phenols were detected along with a large portion of unmetabolized BP. A representative HPLC histogram is 84

TABLE 9

Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular

Fractions from Human Epithelial and Fibroblast Populations9 Epithelial Fibroblast In tra ce llu la r Cytoplasmic Metabolites Fraction Fraction

Sulfates NDd 0.4

9,10-Diol ND 0.6

7,8-Diol 41.6 5.9

Unknown B 43.9 ND

Unknown C 45.4 ND

Phenols 464.0 12.5

Benzo(a)pyrene 718.2 152.7

Total Metabolites 594.9e 19.3 aValues are expressed in picomoles per 10^ cells, lower lim it of detection was set a t 0.2% of the total dpm per HPLC run

^Mean values, n=2 cRandomly-proliterating fib ro b la st population, values are from Table 4, Chapter 1. dND-Not Detectable eRange (402.4-787.4) 85

illu s tra te d in the top portion of Figure 27. Again, the phenols

constitute the majority of the metabolites (78%). For comparison, the

BP metabolites detected in the randomly-proliferating fibroblast

population are shown in the table and are 3% of the total metabolites

found in the epithelial cells. In addition, neither unknown B nor

unknown C were detected in this fibroblast population but were present

in the epithelial cells.

Once the above experiments were completed and an extracellular and an intracellular BP metabolic profile obtained for the keratinocytes,

these cells were pretreated with five different chemicals: 1.) the inducers of P-450, 3-methylcholanthrene (3-MCA), phenobarbital (PB), isosafrole, Arochlor 1254, and 2.) the inhibitor, butylated hydroxyanisole (BHA). The f ir s t chemical studied was 3-MCA and in

Figure 26, representative histograms of the metabolites in the extracellular fractions of the epithelial cells pretreated with

1 yg/ml and 10 yg/ml 3-MCA are shown. The histogram from cells pretreated with 1 yg/ml 3-MCA is very s im ilia r to the histogram from cells treated with only BP. However, in cells pretreated with 10 yg/ml

3-MCA, the total metabolic p ro file is decreased and peaks corresponding to the sulfates, the te tro ls , the 4,5-diol, and the unknowns are almost completely gone.

In Table 10, 1 yg/ml 3-MCA pretreatment led to an increase in the diols, the phenols and unknown C whereas the values for the other metabolites are decreased. The total metabolites are increased by 22% over the total metabolites from cells treated with only BP. In cells 86

Figure 26. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the Extracellular Fractions of

the Epidermal Keratinocyte Cultures Pretreated with

3-Methylcholanthrene. n c -s rD ro DPM x I0; C T i

ro ro ro oo

> CD

SULFATES 5 a

f t ' 0 UNKNOWN A

Oro (7,10 /8,9)-TETROL (I)

(7/8,9,10)-TETR0L (I)

oj O (7,9/8,10)-TETROL (I)

(7,9,10/8)-TETROL (I)

o m 4. 5-D I0L

UNKNOWN B m co Ocr> UNKNOWN C

o-si _ 9 -OH

o(X»

o o

Z8 TABLE 10

Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular

Fractions of Epithelial Cells Pretreated with 3-Methyleholanthrenea BP 1 yg/ml 3-MCA 10 yg/ml 3-MCA Metabolites Alone + BP + BP

Sul fates 467.9 245.0 191.2

Unknown A 522.8 202.4 NDd Tetrol I (7,10/8,9) 507.6 399.0 29.8

Tetrol I (7/8,9,10) 495.3 438.2 37.1

9,10-Diol 1,298.5 1780.9 325.6

Tetrol II (7,9/8,10) NDb ND ND

Tetrol II (7,9,10/8) 270.0 ND ND

4,5-Diol 141.9 190.0 ND

7,8-Diol 1,043.2 1,536.3 336.3

Unknown B 236.0 231.3 ND

Unknown C 140.1 272.6 ND

Phenols 2,405.1 3,866.9 544.0

Benzo(a)pyrene 17,629.4 30,208.3 14,888.3

Total Metabolites 7,528.4 9,162.6C 1,464.0d a 7 Mean values are expressed in picomoles per 10 cells bND-Not Detectable cRange (6,015.1-10,812.1), n=4 dRange (665.2-2,166.2), n=3 89

pretreated with 10 yg/ml 3-MCA, the values for a ll the metabolites show

a decrease with the 4,5-diol and the three unknown peaks not even being

detected. Also, the total metabolites are only 19% of the total

metabolites for the BP-treated cells. Thus, 3-MCA at 1 yg/ml seems to

induce P-450 activity leading to production of more diols and/or

in h ib its EH a c tiv ity leading to te tro ls . In other words, at this

concentration, 3-MCA increases the activation pathways or decreases the

detoxification pathways, whereas at 10 yg/ml, 3-MCA in h ib its the

formation of BP metabolites.

3-MCA had previously been associated with cytochrome P-450 as an

inducer of its AHH a c tiv ity . Heimann and Rice (124) used keratinocytes

grown on feeder layers pretreated with 4 yM 3-MCA and observed an

overall enhanced rate of BP metabolism. However, specific metabolites

were not evaluated. Also, in a study by Holder et a l. (131), rats and

mice were pretreated with 3-MCA and BP metabolism was evaluated in vitro

using liv e r microsomes. An increase in the formation of the diols, the

phenols, and the quinones was observed. F inally, Wiersma and Roth (327)

pretreated rats in vivo with 3-MCA before administration of BP and

discovered that the pretreatment not only increased the distribution of

BP throughout the body and but also increased its excretion due to

enhanced extrahepatic metabolism. Toxic or carcinogenic effects were

not examined.

The intracellular fractions of cells pretreated with 3-MCA were also examined and the representative histograms are shown in Figure 27. The major metabolites seen were the 7,8-diol, unknown B, unknown C, and the

phenols along with unmetabolized BP. The specific amounts of these 90

Figure 27. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the In tra ce llu la r Fractions of

the Epidermal Keratinocyte Cultures Pretreated with

3-Methylcholanthrene. o INI m 03 m >0D 5 a CO DIOL UNKNOWN C 3-OH 9-O H UNKNOWN B 7,8- CD no D P M x I O ' no JD o rv> o •£» no O O oj O O r c> cn CJi o uo o CO o o o o

TIME (MINUTES) -5 ro c 16 peaks are shown in Table 11. These values have been corrected for yields and cell number and are the average values from two experiments

for each dosage level. Both 1 yg/ml and 10 yg/ml 3-MCA pretreatments

resulted in a lower amount of metabolites in tra c e llu la rly , with the

values for 1 yg/ml 3-MCA being s lig h tly lower than those for 10 yg/ml

3-MCA. Therefore, at 1 yg/ml 3-MCA, the majority of metabolites are

found extracellularly compared to cells treated only with BP, whereas at

10 yg/ml 3-MCA, decreased amounts of metabolites are found both extracellularly and intracellularly.

Next, the e p ith e lia l cells were pretreated with 1 yg/ml and 10 yg/ml

PB. Even though the specific concentrations used for all pretreatments

(equimolar amounts to 3-MCA) were those values in Table 6, the two dosage levels for the other four chemicals w ill be referred to as

1 yg/ml and and 10 yg/ml fo r sake of sim plicity throughout this discussion. For example, in pretreatment with 1 yg/ml PB, the specific concentration 0.947 yg/ml was used which is 3.73 yM or equimolar to

1 yg/ml used for 3-MCA pretreatment. The results from the pretreatment of the epithelial cells with PB are shown in Figure 28. At both dosage levels, the total metabolic profile is decreased with a greater reduction seen at 1 yg/ml PB. The quantities of the metabolites are

listed in Table 12. The total metabolites excluding parent BP for

1 yg/ml PB are 54% less than the value for the cultures treated with BP alone whereas the total metabolites for 10 yg/ml PB are only 30% less.

Therefore, PB seems to have an in h ib itin g e ffe c t on BP metabolism in these cells, more so at 1 yg/ml than at 10 yg/ml. 93 TABLE 11

Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular

Fractions of Epithelial Cells Pretreated with 3-Methylcholanthrenea

BP 1 ug/ml 3-MCA 10 Pg/ml 3-MCA Metabolites Alone + BP + BP

7,8-Diol 41.6 16.6 43.4

Unknown B 43.9 21.9 11.7

Unknown C 45.4 24.4 64.1

Phenols 464.0 287.1 300.7

Benzo(a)pyrene 718.2 647.5 2,268.2

Total Metabolites 594.9 350.0C 419.9d aValues are expressed in pi comoles per 107 cells

^Mean values, n=2 cRange (180.9-519.2) dRange (131.9-707.8) 94

Figure 28. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the Extracellular Fractions of

the Epidermal Keratinocyte Cultures Pretreated with

Phenobarbital. o 00 m m m > o co 9 - 0 H cn (7,10/8,9)- TETROL (I (7/8,9,10)-TETROL (I) UNKNOWN A UNKNOWN B UNKNOWN C (7,9/8,10)-TETROL (I) SULFATES (7,9,10/8)-TETROL (I) 4 , 5 - DI0L ro _> D P M X I 0 : ro ro O ro O X 00 sg CO-9 o o ro O CM O O O cn O CO O o o o

TIME (MINUTES) c Q PO 00 tO S6 96 TABLE 12

Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular

Fractions of Epithelial Cells Pretreated with Phenobarbitala BP 1 pg/ml.PB 10 pg/ml PB Metabolites Alone + BP + BP

Sulfates 467.9 201.3 520.5

Unknown A 522.8 122.0 296.6

Tetrol I (7,10/8,9) 507.6 85.5 508.7

Tetrol I (7/8,9,10) 495.3 90.9 389.2

9,10-Diol 1,298.5 512.1 772.6

Tetrol II (7,9/8,10) NDC ND ND

Tetrol II (7,9,10/8) 270.0 ND ND

4,5-Diol 141.9 118.3 ND

7,8-Diol 1,043.2 585.2 590.9

Unknown B 236.0 85.0 232.6

Unknown C 140.1 65.5 81.5

Phenols 2,405.1 1, 559.7 1,900.2

Benzo(a)pyrene 17,629.4 15, 644.4 5,698.2

Total Metabolites 7,528.4 3. 425.5d 5,292.8e aValues are expressed in pi comoles per 107 cells bMean values, n=3 cND-Not Detectable dRange (2,867.6-4,164.9) eRange (3,804.7-6,022.7) 97

Figure 29 illustrates the profiles from the intracellular fractions of cells treated with PB. Again, only the 7,8-diol, unknowns B and C, and the phenols are observed. However, a new metabolite can be seen which eluted at 40 min and w ill be referred to as unknown D. I t was only detected in the histograms of cells pretreated with 10 yg/ml PB as listed in Table 13. This peak may be due to another diol or possibly a trio l. At both concentrations of PB, an increase in all the metabolites detected in tra c e llu la rly is observed. The total amounts for 1 yg/ml and

10 yg/ml PB are 1.3 and 2.1 times more than the total metabolites in cells treated only with BP, respectively. Thus, the majority of metabolites formed due to PB pretreatment remain within the cell. The effects of PB are also observed in all metabolites formed and not in certain groups of metabolites as seen with 3-MCA which effected primarily the diols and the phenols. These results correlated with the data from the mammalian systems where PB induced a number of metabolic reactions associated with P-450 whereas 3-MCA was classified differently because i t induced a specific number of reactions.

The third compound studied for its effects on the BP metabolic pro file of the epidermal keratinocytes was isosafrole. The representative histograms from the extracellular fractions are shown in

Figure 30 and the data summarized in Table 14. After corrections were made for cell numbers and yields, the pi comoles of a ll metabolites were increased with the exception of unknown B which was greatly reduced at

10 yg/ml isosafrole. The greatest increases occurred at both concentrations in the diols, the phenols, and surprisingly, in unknowns

D and C of which the value for the la tte r unknown was increased over 98

Figure 29. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the In tra c e llu la r Fractions of

the Epidermal Keratinocyte Cultures Pretreated with

Phenobarbi t a l. 10 B(a)P ALONE 8

to o

CL o 0 B(a)P+l^q/ml PHENOBARBITAL

2 B(a)P+10yu.q/ml PHENOBARBITAL

o 10 20 30 40 50 60 70 80 90

TIME (MINUTES)

Figure 29. 100 TABLE 13

Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular

Fractions of Epithelial Cells Pretreated with Phenobarbitala BP 1 pg/ml,PB 10 yg/m l PB Metabolites Alone + BP + BP

Sulfates NDC ND 20.0

Unknown D ND ND 45.9

7,8-Diol 41.6 35.1 84.5

Unknown B 43.9 47.5 46.8

Unknown C 45.4 180.9 215.5

Phenols 464.0 473.2 865.4

Benzo(a)pyrene 718.2 507.1 1,174.3

Total Metabolites 594.9 751.ld l,278.1e aValues are expressed in picomoles per 107 cells bMean values, n=2 cND-Not Detectable dRange (360.4-1,132.2) eRange (633.4-1,922.9) 101

Figure 30. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the Extracellular Fractions of

the Epidermal Keratinocyte Cultures Pretreated with

Isosafrole. O 03 m m m OJ 00 9 -O H cn (7,10/8,9)"TETR0L (I) (7/8,9,10)-TETROL (I) UNKNOWN A UNKNOWN B UNKNOWN C (7,9/8,10)-TETROL (I) SULFATES 7,9,10/8)-TETROL (1) 4 , 5 - DI0L ro D P M x IO' ro ro O m — ro O o ro o o CD O O cn to o o o *"5 C n> o

g o TIME (MINUTES) UD 201 TABLE 14

Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular

Fractions of Epithelial Cells Pretreated with Isosafrole3

BP 1 yg/ml Isosafrole 10 yg/ml Isosafrole Metaboli tes Alone + BP° + BP

Sul fates 467.9 485.2 401.2

Unknown A 522.8 633.9 569.6

Tetrol I (7,10/8,9) 507.6 1,316.5 1,261.1

Tetrol I (7/8,9,10) 495.3 865.6 832.8

9,10-Diol 1,298.5 3,049.2 2,745.3

Tetrol II (7,9/8,10) NDd ND ND

Tetrol II (7,9,10/8) 270.0 ND ND

Unknown D ND 401.5 668.7

4,5-Diol 141.9 600.7 586.9

7,8-Diol 1,043.2 1,360.3 1,767.3

Unknown B 236.0 234.4 144.9

Unknown C 140.1 877.6 1,601.3

Phenols 2,405.1 4,371.5 4,626.5

Benzo(a)pyrene 17,629.4 9,668.4 22,800.9

Total Metabolites 7,528.4 14,196.4e 15,205.6f aValues are expressed in picomoles per 10^ cells

^Mean values, n=3 cMean values, n=3 dND-Not Detectable eRange (8,119.0-18,979.5) f Range (8,870.0-21,387.4) 104

10-fold. The total metabolites for 1 yg/ml and 10 yg/ml isosafrole were

1.9 and 2.0 times more, respectively, than the amount observed in cells treated with just BP.

The intracellular fractions obtained in experiments with isosafrole pretreatment are shown in Figure 31 and quantitated in Table 15. Again, increases are seen in all metabolites including the appearance of unknown D. Only unknown B is markedly decreased.

In mammalian systems, isosafrole has been demonstrated to be both an inhibitor and an inducer of P-450 activity. As an inducer, it behaved like both the 3-MCA and PB classes of inducers and as an inhibitor, it acted in a dual or "mixed" manner both competitively and non-competitively (89,90). The competive inhibition occurred during the formation of a complex of the isosafrole metabolite with the iron atom of P-450 while non-competitive inhibition occurred once the metabolite complex was formed (93). In ra t liv e r, isosafrole induced four forms of

P-450 isozymes including one form which is exclusively induced by this compound (74). However, in rabbit liv e r, i t induced the same isozyme as

3-MCA (73).

In human epithelial cells, isosafrole seemed to play more of an inducing role by increasing the formation of the BP metabolites. By specifically leading to significant increases in the diols and the phenols, i t tended to behave like 3-MCA. However, i t also led to the formation and increases in the unknowns C and D which was not observed to any great extent with PB or 3-MCA. Therefore, isosafrole may possibly induce P-450 activity in its own characteristic manner. 105

Figure 31. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the In tra ce llu la r Fractions of

the Epidermal Keratinocyte Cultures Pretreated with

Isosafrole. 1 0 B(a)P ALONE 8

ro 9 2

Cl O - B(a)P+lftq/ml ISOSAFROLE

o B(a)P+10/xq/ml ISOSAFROLE

o 10 20 30

TIME (MINUTES) F i rure 31. 107

TABLE 15

Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular

Fractions of Epithelial Cells Pretreated with Isosafrole9

BP 1 yg/ml Isosafrole 10 yg/ml Isosafrole Metaboli tes Alone + BPd + BPd

Sul fates NDC 21.0 34.6

Unknown D ND 57.9 55.5

7,8-Diol 41.6 100.1 103.7

Unknown B 43.9 43.9 35.1

Unknown C 45.4 204.3 171.2

Phenols 464.0 929.0 1,035.7

Benzo(a)pyrene 718.2 1,428.9 1,540.4

Total Metabolites 594.9 l,356.2d l,435.8e aValues are expressed in picomoles per 107 cells

^Mean values, n=2 cND-Not Detectable dRange (1,169.5-1,521.8) eRange (1,176.2-1,695.4) 108 Pretreatment with Arochlor 1254 resulted in the histograms shown in

Figure 32. Again, the whole range of BP metabolites is observed with a sig n ifica n t increase in unknown C. Compared to BP alone and 1 yg/ml

Arochlor 1254, there is a reduction in peaks with 10 yg/ml Arochlor

1254. The values for each metabolite are shown in Table 16. Slight decreases in a ll metabolites are seen at 1 yg/ml Arochlor 1254 with the exception of the (7,10/8,9)-tetrol, the 4,5-diol, and unknown C. Of these latter metabolites, unknown C showed the greatest increase. At

10 yg/ml Arochlor 1254, decreases were seen in a ll metabolites with the total disappearance of unknown B. The total metabolites for the two concentrations of Arochlor 1254 were 16% and 45% less than the total metabolites quantitated for treatment with BP alone.

In examining the histograms of the in tra c e llu la r fractions of cells pretreated with Arochlor 1254 in Figure 33, an increase was seen in all metabolites, especially in unknown C and the appearance of unknown D.

In Table 17, the pi comoles calculated for unknown C at 1 yg/ml Arochlor

1254 increased 5 times over the pi comoles for cells treated with ju s t

BP. Also, the amounts observed for the 7,8-diol and the phenols were twice as much as for the values with BP alone and the total metabolites at 1 yg/ml Arochlor 1254 excluding the parent peak of BP were 2.3 times higher. However, at 10 yg/ml Arochlor 1254, even though the picomoles for the metabolites were increased by 32% over the values for BP alone except unknown B, the total picomoles per 107 cells were less by 38% than the picomoles calulated for each metabolite at 1 yg/ml Arochlor

1254. 109

Figure 32. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the Extracellular Fractions of

the Epidermal Keratinocyte Cultures Pretreated with Arochlor

1254. CQ -5 rt> D P M X I 0 :

CO no

SULFATES o UNKNOWN A

rv> (7,l0/8,9)-TETR0L (I) O

(7/8,9,10) - TETROL (I

oj (7,9/8,10)-TETROL (H) O X ) U J (7,9,10/8)-TETROL (I)

■fc> H O

m 4 , 5 - DIOL (ji o

UNKNOWN B —i m CT) co o

UNKNOWN C

9 - OH

CD o

<■0 o

o o Oil TABLE 16

Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular

Fractions of Epithelial Cells Pretreated with Arochlor 1254a

BP 1 vg/ml Arochlor 1254 10 vg/ml Arochlor 1254 Metabolites Alone + BP + BP

Sulfates 467.9 234.7 211.4

Unknown A 522.8 287.8 69.9

Tetrol I (7,10/8,9) 507.6 522.8 163.7

Tetrol I (7/8,9,10) 495.3 374.8 113.2

9,10-Diol 1,298.5 839.5 752.6

Tetrol II (7,9/8,10) NDC ND ND

Tetrol II (7,9,10/8) 270.0 ND ND

Unknown D ND 180.5 120.7

4,5-Diol 141.9 235.1 82.9

7,8-Diol 1,043.2 820.6 773.4

Unknown B 236.0 122.2 ND

Unknown C 140.1 436.5 296.1

Phenols 2,405.1 2, 290.6 1,534.9

Benzo(a)pyrene 17,629.4 8, 846.8 13 ,598.3

Total Metabolites 7,528.4 6, 345.l d 4 ,118.8e aValues are expressed in pi comoles per 10^ cells

^Mean values, n=3 cND-Not Detectable dRange (5,610.0-7,449.6) eRange (1,951.5-5,170.8) 112

Figure 33. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the In tra c e llu la r Fractions of

the Epidermal Keratinocyte Cultures Pretreated with Arochlor

1254. BENZO (a)PYRENE cr> '■o cr> u TIME TIME (MINUTES)

B(a)P+10/xq/ml AROCHLOR 1254 B(a)P+l^q/ml B(a)P ALONE - AROCHLOR 1254

OlxlAldQ ro Fi

Organic-Extractable Benzo(aJpyrene Metabolites in the Intracellular

Fractions of Epithelial Cells Pretreated with Arochlor 1254a

BP 1 ug/ml Arochlcr 1254 10 pg/ml Arochlor 1254 Metabolites Alone + BPd + BPd

Sul fates NDC 24.4 11.3

Tetrol I (7,10/8,9) ND 18.7 ND

Unknown D ND 34.0 22.0

7,8-Diol 41.6 99.4 63.1

Unknown B 43.9 49.7 28.6

Unknown C 45.4 236.1 141.9

Phenols 464.0 934.5 601.3

Benzo(a)pyrene 718.2 1,386.8 1,155.3

Total Metabolites 594.9 1,396.8d 868.2e aValues are expressed in picomoles per 107 cells

^Mean values, n=2

cND-Not Detectable

dRange (1,005.6-1,769.2) eRange (852.0-872.9) 115

Therefore, in the keratinocytes, 1 ^g/ml Arochlor 1254 pretreatment led to a decrease in metabolites extracellularly but an increase in tra c e llu la rly with the exception of unknowns C and D which were induced in both fractions. At this concentration, Arochlor 1254 may induce nuclear activity of P-450 firs t leading to an accumulation of metabolites within the cell while in h ib itin g somewhat microsomal P-450.

I f i t had induced microsomal a c tiv ity , an expected increase in extracellular metabolites would have been observed within the 24 hours of treatment time. However, if only nuclear P-450 was induced, an accumulation of metabolites would have been seen intracellularly with a lower amount in the extracellular fraction. Since there are low amounts seen for the more polar metabolites extracellularly, these metabolites are formed but not to as great an extent. Therefore, there may be some inhibition of microsomal P-450. In addition, Arochlor 1254 is a mixture of polychlorinated biphenyls (PCBs) and these compounds may be acting differently. One compound may inhibit microsomal activity while another may induce nuclear a c tiv ity . At 10 pg/ml Arochlor 1254, the same effects were seen as with 1 pg/ml but to a lesser extent. Unknowns C and D seemed to be specifically induced by Arochlor 1254 and isozymes leading to the formation of these metabolites may be specific for

Arochlor 1254 as in the case of isosafrole.

In mammalian systems, Arochlor 1254 was demonstrated to be an inducer as well as an in h ib ito r depending on the species and tissues examined. For example, in rabbit lung, this mixture of PCBs inhibited the synthesis of one P-450 isozyme but had no e ffe c t on the other (308).

In ra t lung, i t was shown to be a powerful inducer (5) and in rat liv e r, 116 i t induced three forms of P-450 (UT-F, PB-B, 8NF-B) which were also

induced either by 3-MCA or PB (259). In other words, Arochlor 1254 in

rat liv e r was classified as a mixed-type inducer behaving like both

3-MCA and PB (185). The effects of this chemical was also studied in

rat skin by Bickers et a l. (32) who reported a 7-fold increase in AHH

activity and a 3-fold increase in P-450 content when rats were

pretreated j_n vivo with PCBs.

Finally, the last chemical used for pretreatment in the epithelial

cells -was BHA. The representative histograms for the extracellular

fractions are shown in Figure 34 and the data liste d in Table 18.

Reductions in the BP metabolites were seen at both 1 yg/ml and 10 yg/ml.

The picomoles per 10^ cells were decreased for a ll metabolites except

unknown D and C which were induced at 1 yg/ml. At 10 yg/ml, the

formation of these unknowns was also inhibited with the complete

disappearance of unknown D. Overall, the total metabolites minus BP at

1 yg/ml and 10 yg/ml BHA were 60% and 44% of the total metabolites

observed in cells treated with BP alone.

In the in tra ce llu la r fractions, both doses of BHA resulted in

increases of the metabolites detected except unknown B which was

decreased. The histograms are illu s tra te d in Figure 35 and the

picomoles for the metabolites are quantitated in Table 19. Compared to

the values of metabolites in cells treated with ju st BP, the values for

total metabolites at 1 yg/ml and 10 yg/ml BHA were increased 1.5 and 1.4

times, respectively. Like Arochlor 1254, BHA causes an accumulation of metabolites intracellularly and seems to behave like Arochlor 1254 in

inducing both unknown D and C. 117

Figure 34. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the Extracellular Fractions of

the Epidermal Keratinocyte Cultures Pretreated with

Butylated Hydroxyanisole. L3 C -s DPM*IO' ro U> ro ro O ro CD CD O

> □□

SULFATES

UNKNOWN A

ro (7,IO/8,9)-TETROL (I) O

(7/8,9,10)-TETROL (I)

(7,9/8,10)-TETROL (I)

(7,9,10/8)-TETROL (I)

—I o s m 4 , 5 - DIOL CD o

c: UNKNOWN B m—i r~ co o

UNKNOWN C

9 -O H

CD CD o m

CO O m m

O o Cl I 119

TABLE 18

Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular

Fractions of Epithelial Cells Pretreated with Butylated Hydroxyanisole3

BP 1 yg/ml BHA 10 pg/ml Metabolites Alone + bpd + BPd

Sul fates 467.9 226.9 243.8

Unknown A 522.8 307.0 175.5

Tetrol I 507.6 343.0 207.5

Tetrol I 495.3 378.0 116.4

9,10-Diol 1,298.5 879.9 639.9

Tetrol II NDC ND ND

Tetrol II 270.0 ND ND

Unknown D ND 123.1 ND

4,5-Diol 141.9 113.7 ND

7,8-Diol 1,043.2 576.1 594.0

Unknown B 236.0 73.5 ND

Unknown C 140.1 251.2 203.6

Phenols 2,405.1 1,212.5 1,126.1

Benzo(a)pyrene 17,629.4 4,791.7 12,130.3

Total Metabolites 7,528.4 4,484.9d 3,306.8e aValues are expressed in picomoles per 10^ cells bMean values, n=3 cND-Not Detectable dRange (1,486.0-7,578.9) eRange (2,082.4-4,413.1) 120

Figure 35. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the In tra ce llu la r Fractions of

the Epidermal Keratinocyte Cultures Pretreated with

Butylated Hydroxyanisole. CD C D P M x IO' "5

CO cr> O no O no O no cn 00 O o - r - i T -< r - ~I I ^ ro ro 5 ro ro j> ro ^ 3: Q ? 5 Q 5 a S "D ro + m O - j r I ~p JO $JD

oj O }

I, - C, \ o ■f

m ? (J1 t o <— 7, 8 - DIOL

UNKNOWN B m CD I co O p a *— UNKNOWN C

C) C • 9 - OH - 3-OH

00 / > O CD 3 m s -r M o 1 }• u 5 ) T) b -< o i ‘I Zl r* m ’’TO 3______

O O

ICI 122 TABLE 19

Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular

Fractions of Epithelial Cells Pretreated with Butylated Hydroxyanisolea

BP 1 ug/ml BHA 10 yg/ml BHA Metabol i tes Alone + BPd + BP°

Sulfates NDe 30.5 ND

Unknown D ND 33.3 16.7

7,8-Diol 41.6 69.5 55.4

Unknown B 43.9 35.2 22.6

Unknown C 45.4 119.0 125.2

Phenols 464.0 582.2 602.4

Benzo(a)pyrene 718.2 623.9 1,115.1

Total Metabolites 594.9 869.7d 822.3e aValues are expressed in picomoles per 10^ cells

^Mean values, n=2 cND-Not Detectable dRange (844.6-894.5) eRange (757.9-886.7) 123

In mouse liv e r, forestomach, and lung, BHA has been found to lower

P-450 content and inhibit BP-DNA binding (126,8,141). Also, reductions in BP metabolism by BHA was observed in mouse lung and liv e r affecting the diols and phenols. Sydor e t a l. (288) observed decreases in the formation of the 9,10-diol while no effects were seen in the 4,5-diol or the 3-OH in mouse lung while Lam et a l. reported a decrease in the

4,5-diol, no effect on the 9,10-diol and increases in the 3-OH and 2-OH in mouse liv e r (181,182). The presence of this new metabolite, 2-OH, in the BP metabolism of mouse liv e r is interestingly and could possibly be unknown C in our system. Sawicki et a l. (262) cultured human skin explants and treated them with radiolabelled BP with and without BHA added simultaneously. They reported a 502 decrease in organic-extractable metabolites with BHA in the extracellular fraction and the amount of metabolites bound to DNA. However, the in tra c e llu la r metabolites were not analyzed. This data is in agreement with the data in Table 18 except Sawicki et a l . did not detect the metabolites unknowns C and D.

In the above experiments, interindividual differences were seen between keratinocyte cultures. Each extracellular fraction was obtained from the culture started from one foreskin. An example of these differences is shown in Figure 36 where the histograms from the three extracellular samples pretreated with 10 yg/ml phenobarbital are illustrated. Significant decreases in the 9,10-diol and the 7,8-diol are seen in Profile A while the (7,10/8,9)-tetrol and unknown A is greatly increased. In Profile B, only very small amounts of unknown A, the (7 ,10/8,9)-tetrol and the (7/8,9,10)-tetrol are observed. 124

Figure 36. Histograms of Organic-Extractable Radiolabelled

Benzo(a)pyrene Metabolites in the Extracellular Fractions of

Three Epidermal Keratinocyte Cultures Pretreated with

10 yg/ml Phenobarbital. COc -5 D P M * I C r a> ONCO o rv> O ro O ro CD oo O O

SULFATES

CD m m UNKNOWN A m o co m

ro CO ^— (7/8,9,101-TETR0L (I)

<— 9,10-DIOL (7,9/8,10) - TETROL (I) (7,9,10/8)-TETROL (I)

m m

7 ,8 -D IO L d —I 7> m UNKNOWN B co

UNKNOWN C

9-OH

INI

m m

O L 521 126 Therefore, after corrections for cell number and yields, it was

necessary to average the values for comparison with other data. These

large interindividual differences have been previously reported by

Harris et a l. (120) in the binding of BP to DNA in cultured human

bronchi and by Cohen et a l. (57) and Sabadie et a l. (260) in the BP

metabolism of d iffe re n t normal and tumorous lung tissue samples. A

range from 18-fold to 44-fold variations were reported.

In conclusion, human epithelial keratinocytes were demonstrated to

metabolize BP to a greater extent than human dermal fibroblasts which

included the whole range of organic-extractable metabolites from

sulfates to the phenols including three peaks whose identity is not

known, unknowns A, B, and C. Differences were also observed in the

effects of these metabolites when the cells were pretreated with five

chemicals shown to be inducers or in h ib ito rs in mammalian systems.

3-MCA induced the preferential extracellular formation of the diols and

phenols at 1 yg/ml but seemed to inhibit the formation of all BP metabolites at 10 yg/ml. In contrast, PB increased the intracellular

metabolites but decreased the metabolites detected in the extracellular

fractions. In addition, at 10 yg/ml, a new metabolite was formed,

unknown D. Isosafrole led to significant increases in both the

extracellular and intracellular BP metabolites especially in unknown C

and the appearance of unknown D. Both Arochlor 1254 and BHA behaved

similiarly and like PB in decreasing the formation of extracellular metabolites while leading to increases in the intracellular metabolites.

However, significant increases in unknowns C and D were observed at both

1 yg/ml Arochlor 1254 and BHA which were not observed with PB. 127

Therefore, four different schemes of induction seemed to be present.

3-MCA induction differed from induction by the other four chemicals and must be considered separately. PB, Arochlor 1254, and BHA behaved in a

similiar manner by inducing the intracellular formation of the BP metabolites which may suggest a preferential induction of nuclear P-450 activity and simultaneously inhibiting the extracellular metabolite

formation which may be a pa rtia l in h ib itio n of microsomal P-450. Next,

isosafrole pretreatment led to an overall increase in both extracellular and intracellular metabolites, not only in the formation of certain metabolites. Finally, isosafrole, Arochlor 1254, and BHA preferentially

led to the induction of unknowns C and D. Only in the in tra ce llu la r

samples did PB increase unknown C and a small amount of unknown D was only detected at 10 yg/ml. Thus, the above three chemicals may lead to the synthesis of an isozyme which preferentially metabolizes BP to unknowns D and C. In analysis of these findings, there is evidence of

P-450 a c tiv ity in human keratinocytes in v itro and suggestion for either differences in the a c tiv itie s of nuclear and microsomal cytochrome P-450 or for the existence of different P-450 isozymes. CONCLUSIONS

In Chapter 1, the effects of d iffe re n t growth conditions on the

metabolism of benzo(a)pyrene (BP) by human skin fibroblasts were

examined. A comparison of the extracellular, the cytoplasmic, and the

nuclear fractions of three different populations was made and the data

showed a higher amount of total metabolite formation in all fractions

from confluent cells as compared to the metabolites present in the

fractions from randomly-proliferating and synchronized cells. From

preliminary studies, these la tte r two cell populations were determined

to be transformable by BP as indicated by the positive results in

several transformation assays and by the demonstrated a b ility to take up

BP into the cytoplasm and transport i t to the nucleus. The confluent

cells gave negative transformation results and were unable to take up BP

into the cytoplasm. The higher metabolism of BP in these cells and

their inability to be transformed seemed to indicate that the state of

confluency induces more detoxification pathways (or a higher rate of

these pathways) leading primarily to metabolite excretion. In contrast,

the randomly-proliferating and synchronized cell populations seemed to be more concerned with inducing activation pathways leading to the formation of intermediates which subsequently result in a transformed phenotype.

Next, Chapter 2 dealt with several improvements in the available methods for maintaining human epidermal keratinocytes in v itro free of

128 129 contaminating fibroblasts. These epithelial cells derived from the epidermal layer of the skin possessed several unique tra its not found in the dermal fibroblasts: the presence of desmosomes and tonofilaments, the ability to synthesize keratin, the formation of cornified envelopes, their polyhedral shape, and the manner in which they proliferated and terminally differentiated in culture. Improvements in the culturing of these keratinocytes included the lack of a feeder layer, the use of a medium without added hormones or growth factors except a n tib io tics and which has a decreased calcium concentration, the digestion of the skin tissue with trypsin in HBSS(EGTA) at 4°C instead of collagenase at 37°C, a reduction in the volume of media used to in it ia lly seed the c e lls, a lowered *C02 to maintain the cultures and the use of versene instead of trypsin in HBSS(EGTA) to subpassage the cells. The above changes resulted in cultures of epidermal keratinocytes which more closely resembled their in vivo state and could be more easily established and routinuely maintained in vitro for a variety of studies.

F inally, in the la st chapter, the effects of known inducers and in h ib ito rs of cytochrome P-450 on the BP metabolism of the human keratinocytes was analyzed and compared. The pretreatment of these cells with the five chemicals, 3-methylcholanthrene, phenobarbital, isosafrole, Arochlor 1254 and butylated hydroxyanisole, resulted in changes in the BP metabolic profile which seemed to follow four distinct patterns. One pattern was demonstrated by 3-MCA which induced the specific extracellular formation of BP diols and phenols. Another pattern was indicated by pretreatment with PB, Arochlor 1254, and BHA resulting in the nonspecific formation of BP metabolites intracellularly 130 while their formation was decreased extracellularly. Next, isosafrole pretreatment induced increases in metabolites both extracellularly and intracellularly (nonspecific induction) and also resulted in significantly high increases in diols and phenols (specific induction).

In other words, isosafrole seemed to behave like both 3-MCA and PB leading to a mixed type of induction. Last of all, isosafrole-,

Arochlor 1254-, and BHA-pretreated cells showed specific increases in two unknown peaks, unknowns C and D, and this induction seemed to be characteristic of these three compounds only. Thus, these four patterns appeared to indicate the presence of several isozymes of P-450 and/or differences between the a c tiv itie s of microsomal and nuclear P-450s.

In summary, human skin fibroblasts and epithelials differed in their metabolism of BP. The fibroblasts formed large amounts of the 7,8-diol indicating metabolism proceeded primarily by the bay-region theory.

Transformable fib rob la st cultures induced the synthesis of P-450 over EH while nontransformable cultures induced EH activity over P-450 activity.

BP was more harmful to these la tte r cultures (resembling the normal in vivo state of the cells) than the proliferating cultures (resembling in vivo fibroblasts active in wound healing during injury). Compared to the fibroblasts, the epithelials induced more MFO activity leading to large formations of the phenols over the diols thereby indicating the primary pathway of BP metabolism does not proceed via the bay-region theory. Alternative pathways were also noted by significant increases in two unknown peaks during chemical pretreatments. Even though BP seemed to be harmful to skin cells (particularly when they are dividing), this toxic effect may be more apparent when this tissue is f ir s t exposed to other environmental chemicals. BIBLIOGRAPHY

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