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IMMUNOHISTOCHEMICAL ANALYSIS OF CYTOCEROME P4SO ISOZYMES (CYP2B1& CYPlA1) AND NADPH-CYTOCHROME P450 REDUCTASE
DURING LUNG TUMOUR DEVELOPMENT IN S W J MICE
JASON ALLAN LORD
A thesis submitted to the Department of Anatomy and Cell Biology in conformity with the requirements
for the degree of Master of Science
Queen's University Kingston, Ontario, Canada
copyrighto Jason Allan Lord, April, 1997.
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ABSTRACT
The cytochrome P450 enzymes represent a family of hemoproteins, which
together with the flavoprotein NADPH-cytocbrome P450 reductase, are responsible for
the oxidative metabolism of multiple xenobiotics and endogenous compounds. These
enzymes are classified according to proposed evolutionary relationships. As such, an
italicized root symbol CYP denotes the cytochrome P450 gene (C' for mouse), an
Arabic number identifies the family, a letter distinguishes the subfamily and an Arabic
numeral denotes the individual gene. Gene products include only capital letters, without
italics. Previous studies have postulated that murine lung tunourigenesis may be
correlated with decreased cytochrome P450 expression. The present study utilized
polyclonal antibodies and avidin-biotin horseradish peroxidase, to
immunohistochemicaIly detect CYPZB 1, CYP 1 A1 and NADPH-cytochrome P450
reductase in developing tumours and non-neoplastic tismes within SWR/J murine lung.
Mice were administered the procarcinogen urethane (1 mg/g) and sacrificed at designated
stages of neoplastic development. Developing tumours were categorized as hyperplastic
foci (6-10 weeks), solid or papillary adenomas (16-22 weeks) and solid or papillary
carcinomas (52 weeks). To induce CYPlAl, mice were administered &naphthoflavone
(P-NF), in corn oil (80 rngkg) prior to sacrifice. Control mice received only the vehicle.
In control mice, CYP2Bl was localized in Clam and type II cells, while CYPl A1 was not
irnrnunohistochemically detected. Following treatment with P-NF, CYP2B 1 localization
was not altered; however, CYPlAl was detected in endothelid and type II cells.
NADPH-cytochrome P450 reductase was detected in Clara and type II cells of all mice.
CYP2B1 was weakly detected in al l developing tunours. In p-NF-treated mice,
CYPl A l and NADPH-cytochrorne P450 reductase were detected in hyperplastic foci,
solid adenomas and to a lesser extent in papilIary adenomas, but were absent in
carcinomas. Furthermore, localization of both CYPlAl and NADPH-cytochrome P450
reductase was most pronounced in hyperplastic foci within carcinoma-bearing lungs.
These results suggest that turnour formation is associated with diminished expression of
CYPZB 1, CYP IA1, and NADPH-cytochnnne P450 reductase.
ACKNOWLEDGMENTS
I would like to thank Dr. P.G. Fodcert for her efforts and persistence in this
endeavor. I would also like to extend my appreciation to Henry Verstappen and Anita
Graham for their technical assistance and advice.
My appreciation goes out to my colleagues and fiends within the department of
Anatomy and Cell Biology. I am also gratefbl to J. Maarhuis for her graphical advice and
personal support, in helping me complete this project. I would especially like to thank
Dr. W.J. Forrest, Dr. C.W. Reifel, Dr. RJ). Andrew and Dr. S.C. Pang for helping me see
the forest fkom the trees, while providing me with invaluable advice throughout my
education at Queen's University.
Finally, I would like to dedicate this body of work to my parents. Without your
wisdom, patience and encouragement, this thesis would not have been possible. Your
belief in me was always there. Thank you.
TABLE OF CONTENTS
Abstract ................... ...... ............ ..CC ........................................................................... i . . * Acknowledgments .............................................................................................................. m List of Tables ...................................................................................................................... .v List of Figures .................................................................................................................... vi . . List of Abbreviations ................... .. ....... .................... . w
........................................................................................................ . I INTRODUCTION 1 ................................................................ 1 . 1 PULMONARY CARCINOGENESIS 1
1.2 TUMOUR DEWLOP= ........................................................................... ....2 1.3 MOUSE LUNG CARCINOGENESIS .......................................,.......................... 4
......... 1.4 INTRODUCTION TO TKE CYTOCHROME P450 ENZYME SYSTEM 6 1.5 NOMENCLATURE OF THE CYTOCHROME P450 ENZYME SYSTEM ...... 7
...... 1.6 MECKANISMS OF CYTOCHROME P450 DRUG METABOLISM ...... .. 9 ........................................ ................... 1.7 CYTOCHROME P450 INDUCTION ,.., LO
1.8 CYP1A1/CYP1A2(CYTOCHROMEP450 1AlllA2) .................... ... .......... 11 ...................................... 1.9 CYP2B 1 /CYP2B2 (CYTOCHROME P450 2B 112B2) 14
................................. .......... 1 . 10 NADPH CYTOCHROME P450 REDUCTASE .., 1 6 1-1 1 RATIONALE AND OBJECTIVES OF PRESENT STUDY ................ ........... .. 17
2 . MATERIALS AND METHODS ................... ... ...................... .. . 18 ..................................................................... 2.1 CHEMICALS AND REAGENTS 1 8
.............................................................. 2.2 ANIhrLALS ........................... ....... 19 2.3 TREATMENTOFANIMALS ......................................................................... 19
.............................................................. 2.4 PREPARATION OF MICROSOMES -20 2.5 PROTEIN GEL ELECTROPHORESIS ............................................................ -21
..................................................................... 2.6 PROTEIN IMMUNOBLOTTING -22 ......... ..... 2.7 TISSUE PREPARATION FOR IMMINOHISTOCHEMISTRY ,,, -24
.................................... 2.8 AVIDIN-BIOTIN IMMUNOHISTOCHEMIS TRY..... -25
3 . RESULTS .................... ...... ................ .....-.. 28 3.1 PROTEIN IMMUNOBLOTTING ...................... ... .......... 28
.*... ...... .. 3.2 AVIDIN-BIOTIN IMMUNOFXISTOCHEMISTRY ...., .,.- 29 .. ................ . .............. 3.2.1 PULMONARY DISTRlBUTfON OF CYPZB 1 .,. .. .. 29
3 -2.2 PULMONARY DISTRIBUTION OF CYPl A1 ....................................... 30 3.2.3 PULMONARY DISTRIBUTION OF NADPH-CYTOCHROME
........................ ....... ....... P45O REDUCTASE ., , I
............................................................................ 4 . DISCUSSlON ....................... l, .. ...... 64 4.1 SUMMARY AND CONCLUSION ................................................................... 71
................................................................................. 5 . REFERENCES ............. .......,. 73
6 . CURRICULUM VITAE ...................................................................... ............ ........ 85
iv
LIST OF TABLES
Table 1. Relative content of CYP2B 1 and NADPH-cytochrome P450 reductase in normal lung tissues fiom vehicle-treated SWWJ mice ................................ 33
Table 2. Relative content of CYP2B 1 in neoplastic lung tissues h m untreated S W J mice ............ ..................................... ..................................................... 34
Table 3. Relative content of CYPl A1 and NADPH-cytochrome P450 reductase ..... in normal lung tissues b m vehicle-treated and P-NF-treated S WWJ mice -35
Table 4. Relative content of CYPl A1 in neoplastic lung tissues fhm vehicle-treated and P-NF-treated SWIUI mice ............................................................................ 36
Table 5. Relative content of NADPH-cytochrome P450 reductase in neoplastic ..................... lung tissues h m vehicle-treated and P-NF-treated SWWJ mice 37
LIST OF FIGURES
I. A,
B.
2. A.
B.
3.
4.
5.
6.
7.
8.
9.
LO.
11.
12.
13.
Protein immumblat demonstrating CYP2B 1 in lung microsomes from vehicle-treated SWR/J mice .................................................................................. 38 Protein immunoblot demonstrating NADPH-cytochrome P450 reductase in lung microsomes h m vehicle-treated S W J mice ...................................... .3 8
Protein immunoblot demonstrating CYPlAl in lung microsomes fkom P-NF-treated SWWJ mice ..................................................................................... 40 Protein immunoblot demonstrating NADPH-cytochrome P450 reductase in lung mictosomes from p-NF-treated SWWI mice .......................................... -40
Immunohistochemical control and detection of CYP2B 1 in normal lung tissues h r n vehicle-treated S W J mice .................................... ./
Immunohistochemical detection of CW2B I in n o d lung tissues h m vehicle-treated SWRU mice ................ ............................................*........-...... 44
Immunohistochemical detection of CYP2B 1 in neoplastic lung tissues £tom vehicle-treated S WR/J mice .................................. .,. ......................... 46
Immunohistochemical detection of CYPl A1 in normal lung tissues &om P-NF-treated S WRIJ mice .................. ... ...... .......................................... 48
Immunohistochemical detection o t CYP LA1 in hyperplastic foci b m P-NF-treated S W J mice ................................................................................... 50
hunohistochemical detection of GYP 1 A1 in papillary-type tumours fhm P-NF-treated S W J mice ........................................................................... S2
Tmmunohistochemicai detection of CYPl A1 in hyperplastic foci fiom carcinoma-bearing, P-NF-treated SWRD mice ..................................
Immunohistochemical detection of NADPH-cytochrome P450 reductase in normal lung tissues h m vehicle-treated SWR/J mice ................. ... ........... 56
Immunohistochemical detection of NADPH-cytochrome P450 reductase in neoplastic lung tissues b m vehicle-treated S W J mice ............... .. ........... 58
Immu110histochemical detection of NADPH-cytochrome P450 reductase in neoplastic lung tissues h m P-NF-treated SWWJ mice ................. .. ............. 60
Immunohistochemical detection of NADPH-cytochrome P450 reductase ............. in hyperplastic foci h m carcinoma-bearing, P-NF-treated SWWJ mice 62
LIST OF ABBREVIATIONS
A
Ah
Arnt
P-NF
C
CO
CYP
DAB
EDTA
F A D
FMN
g
h
hsc'
L
M
mA
mg
P8
irm
ampere
aryl hydrocarbon
aryl hydrocarbon nuclear translocator
fhaphthoflavone
celsius
carbon monoxide
cytochrome P450
diaminobenzidiae tetrachloride
disodium ethylenediamine tetraacetate
flavin adenine dinucleotide
flavin mononucleo tide
gram
how
heat shock protein
litre
molar
milliampere
milligram
microgram
micrometre
minute
millilitre
millimetre
messenger n~nucleic acid
nicotinamide adenme dinucleotide
nicotinamide adenine dinucleotide phosphate
p-nitrobluetetrazolium chloridel5-bromo-eChIom-3-
indolylphosphate p-toluidine salt
polyacrylamide gel electrophoresis
polycyclic aromatic hydrocarbon
phenobarbital
phosphate-buffered saline
sodium deodecyl sulphate
seconds
tris buffered saline
2,3,7,8-tetracMofOdibenzo-p-dioXin
3 -methylcholanthrene
tris buffered saline + Tween-20 *
uridyl diphosphate
1. INTRODUCTION
1.1 PULMONARY CARCINOGENESIS
The term "carcinogenesist' is commonly used to define tumour development,
characterized by abnormal cell division coupled with a loss of specialized tissue
characteristics (Fox, 1990). For more than 200 years, chemicals have been implicated in \
carcinogenesis and tumour formation, especially within skin, liver and lung tissues.
These organ systems are susceptible to the effects of xenobiotics and p r o c ~ o g e n s
because they represent important routes of exposure, especially in the occupational
setting (Miller, 1978; Tomatis, 1988).
During the 1930s, lung cancer ranked 8th in cancer mortalities in the United
States, killing 3 out of 100,000 people annually (Prescott and Flexer, 1986). By the
1970s, annual deaths resuiting from lung cancer had reached 47 per 100,000. In 1980,
this figure reached 70 per 100,000 (Prescott and Flexer, 1986). Today, lung cancer
remains a global medical concern. It is now the leading cause of cancer deaths (Pitot,
1990b). Its world-wide incidence of mortality continues to rise faster than any other
cause of death, while mortality rates of newly diagnosed patients remain high, with
overall 5-year survival estimates between 5 and 10 percent (Rescott and Flexer, 1986;
Carney et al., 1994). This dramatic increase in mortality has been repeatedly associated
with the effects of tobacco smoking (Boyle and Maissonewe, 1995). In fact, it has been
speculated that over 90% of all lung cancer deaths could be prevented with cessation of
cigarette smoking (Boy le and Maiso~euve, 1 995).
1.2 TUMOUR DEVELOPMENT
Theories of chemical carcinogenesis had their earliest beginnings in the latter part
of the 1930s and early 1940s (Haddow, 1938). By the late 1960s, clonal models of
carcinogenesis indicated tumour development to be a multi-step process involving stages
of initiation, promotion and progression (Boutwell, 1964; Foulds, 1969; Pitot et ul., 1978;
S laga et al., 1982). These models suggest that tumour initiation leads to the irrevemble
formation of neoplastic cells that are able to expand clonally under the influence of a
promoting agent (Farber, 1982).
The first interactions between chemical carcinogens and target tissues were
identified in the late 1940s and early 1950s (MXIer and Miller, 1947, 1952, 1966). It is
still unktlown precisely how chemicals and other etiological agents induce genetic change
(Farber, 1984; Farber and Sarma, 1987; Pitot, 1990a). The complexity of a multi-stage
model of carcinogenesis implies that interaction with multiple genetic sites may be
necessary for tumour initiation and maintenance of malignant cells (Weinstein et al.,
1 984). Molecular interaction between reactive metabolic intermediates and crucial
genetic sequences involved in cell signalling and replication could potentially disrupt
regulated cellular growth. In fact, disruption of several murine proto-oncogenes, such as
myc and K-ras, as well as tumor suppressor genes, such as Rb-1 and p53, have been
implicated in the process of tumourigenesis (Nuam, et al., 1990; Ohmori et nl., 1992;
Clarke, 1995; Li et al., 1995; Massey et ul., 1995). These genetic interactions must be
followed by a complete round of cellular proliferation for the initiation process to be
rendered permanent and hrmour development to proceed (Cayama et 02.. 1978; Yig et a[,
1979; Farber, 1982; Pitot, 1990b).
Hepatic models of carcinogenesis indicate that cellular initiation leads to tumour
promotion, involving the selective growth of f d prolifitive nodules, some of which
may act as precursors for cancer development Farber, 1984; Farba and Satma, 1987).
The expansion of initiated cells to form focal nodules is the most obvious feature during
this promotion stage of tumourigenesis (Farber, 1984; Farber and Sarma, 1987). In
carcinogenic models involving the liver and skin, these hyperplastic nodules follow two
lines of development (Farber, 1984; Clark et al., 1984). The majority (up to 98%) revert
back to a phenotype resembling the sunomding parenchyma However, some
proliferations persist and may eventually undergo fbrther cellular changes believed to be
necessary for the development of cancer.
As early as 1938, investigators suggested that certain carcinogens were capable of
inhibiting cellular proliferation in normal tissue (Haddow, 1938). From this, it was
postulated that initiated cells developed a resistance to the cytotoxic effects of the
carcinogenic agent responsible for their formation (Haddow, 1 93 8). This induced
resistance enabled the hyperplastic nodule to grow in an enviro~lment that was hostile to
the surrounding tissues (Farber, 1973, 1984; Judah et al., 1977). In this way, "cancer
cells arise and commence their career of proliferation under conditions which impair the
life of nomal cells" (Haddow, 1938).
In hepatic models of tumourigenesis, hyperplastic nodules are consistently seen
prior to metastasis. These cytotoxic-resistant nodules show consistent patterns of
physiological behaviour, cellular organization, and molecular composition (Roomi et al.,
198 5). Physiologically, these hypexplasia demonstrate resistance to the cytotoxic effects
of multiple compounds including those respomile for their generation. Furthermore,
hyperplastic foci show an ability to proliferate in an environment which is typically
inhibitory to non-neoplastic tissues. In addition, these nodules exhibit decreased
localization of some phase I drug-metabolizing enzymes, such as cytochmme P450-
containing monooxygenase systems, generally coupled with increased activation of phase
II enzymes systems, including epoxide hydratases, DT-diaphorases, and glutathione-S-
transferases (Faher, 1990). This altered metabolic profile decreases the foci's ability to
activate encountered xenobiotics and generate reactive metabolites which may in turn
damage the nodule itself (Farber, 1984; Roomi et al., 1985). This inability to activate and
metabolize foreign compounds may explain how tumour tissues acquire resistance to
various cytotoxic xenobiotics, including those responsible for their formation.
The h a 1 step in tumour development involves the progression of a small minority
of initiated cells to metastatic cancer (Farber, 1984). Progression involves both invasion
of the growing adenoma as well as cellular metastasis (Farber et al., 1989). It is during
this ha1 stage of tumourigenesis that persistent hyperplastic nodules acquire the ability
to proliferate without the evident need for an external influence. This process of
unregulated cellular proliferation ultimately leads to malignancy (Farber et al., 1 989).
1.3 MOUSE LUNG CARCINOGENESIS
Studies with mouse lung models of tumourigenesis have described the
development of hyperplastic foci to adenomas and ultimately to carcinomas (Shimkin and
Stoner, 1975; Foley et al., 1991). Furthermore, individual mouse strains have been 4
shown to possess differing potentids to deve1op spontaneaus tumows (Andervont, 1938;
Shimlcin and Stoner, 1975). Relative to other more resistant strains, such as C3H and
DBA, SWR strains demonstrate a high incidence of spontaneous and chemically induced
pulmonary tumours. Furthermore, carcinogens often elicit differing dosedependent
responses in tumour initiation, patterns of development and multiplicity (average number
of tunours per lung) betwem strains (Shimldn and Stoner, 1975). While solid turnours
tend to predominate in An strains of mice, SWR strains tend to demonstrate a
proportionately higher rate of papillary tumour formaton (Beer and Mallcinson, 1985)
Mouse lung tumour development leads to the formation of two major histological
types of turnours: those demonstrating a solid pattern, and those manifesting a papillary
pattern. Solid tumours arise in alveolar septae and subsequently proliferate to produce a
spherical, compact mass of W o r m cuboidal cells with no distinct border (Mostofi and
Larsen, 1951; Ka*an et id., 1979; Witschi, 1984; Thaete et al., 1991). Due to their
morphologic and biochemical profiles, it is generally agreed that these turnours arise fiom
alveolar type II cells ('l'haete and Malkinson, 1991). Papillary tumours, on the other
hand, arise as finger-like projections in or adjacent to bronchioles, exhibit a tubular or
papillary pattern and are Lined by columnar epithelial cells similar to Clara cells
(Kauflban et al., 1979; Witschi, 1984; Thaete and Malkinson, 1991). A pseudocapsule
of compressed tissue is characteristically seen along their border. The cellular origin of
these papillary tumours is controversial. It has been proposed that they represent a more
advanced stage of solid tumour progression (Mostofi and Larsen, 1951; Kimura, 1971).
An alternate proposal is that they arise from a non-ciliated Clara cell (Thaete and
Malkinson, 1991). This proposal is supported by similarities between the phenotypical
5
arrangement of papillary tumour cells and bronchiolar Clara cells, including plaomorphic
nuclei, abundant and large mitochondria and extensive smooth endoplasmic reticulum
(Thaete and Malkinson, 1991). It has not yet been determined which developmental
pathway ultimately leads to malignancy. As such, the sequential events involved in the
formation of these turnours, as outlined by their biochemical profiles, may be examined
in an attempt to elucidate their cellular origins and contn%utions to the development of
cancer.
1.4 INTRODUCTION TO THE CYTOCHROME P450 ENZYME SYSTEM
In the late 1950's, the cytochrome P450 enzyme was identified as a hemoprotein
that exhibited a spectral peak at 450nm when it was reduced and bound to carbon
monoxide (CO) (Klingenberg, 1958; Gamnkel, 1958; Omura and Sato, 1964 a & b).
Furthermore, it was recognized that these P450 enzymes were the primary enzymes of
drug metabolism, participating in many biochemical reactions, including steroid and drug
hydroxylations (Estabmok et al., 1963).
The cytochrome P450 enzymes are a superfamiIy of hemoproteins, ranging in
molecular weight between 45 to 60 kDa (Eisen et al., 1983). Together with NADPH-
cytochrome P450 reductase or cytochrome b5, and a suitable phospholipid matrix, they
comprise the microsomal cytochrome P450-containing monooxygenase system (Okey et
al., 1986). In mammalian species, this enzyme system is principally located within the
smooth endoplasmic reticulum, mitochondria1 and nuclear envelopes (Eisen et al., 1983;
Guengerich, 1 987).
The cytochmme P450 enzyme system is responsible for the oxidative metabolism
of a variety of xenobiotics, such as drugs, chemical carcinogens, environmental
pollutants, plant products and alcohols (Nebert et al., 1991). It is also involved in the
activation and metabolism of multiple endogenous compounds, such as steroids, bile
acids, fatty acids, prostaglandins, leukotrienes, and biogenic amines (Nebert et al., 199 1 ;
Nebert and Gonzalez, 1987). The cytochrome P4SO enzymes are involved in activating
and degrading such a wide variety of substrates that they comprise the largest class of
phase I enzymes in the phase Vphase iI drug-metabolizing system (Eisen et al., 1983).
Cytochmme P450 enzymes act to oxygenate hydrophobic substances into more
water soluble intermediate compounds through the introduction of polar groups, such as
alcohols (White and Coon, 1980). These oxygenated intermediates are more readily
accessible to other drug-metabolizing enzymes, known as phase II enzymes. The phase II
drug-metabolizing enzymes, such as epoxide hydrases, UDP glucuconosyl-transferases,
glutathione transferases and sulfotransferases, are able to conjugate these intermediate
metabolites to other water-soluble carrier compounds. In this way, initial substrates are
rendered hydrophilic and may be more easily excreted by the organism (Okey et ni.,
1986; Nebert and Gonzalez, 1987).
1.5 NOMENCLATURE OF THE CYTOCHROME P450 ENZYME SYSTEM
During the first three decades of research involving the cytochrome P450
enzymes, nomenclature was based on various factors, including species and inducibility
differences, substrate specificities, spectral properties and molecular weights (Gonzalez,
1989). In 1987, the Committee for Human Gene Mapping together with the Committee
7
on Standardized Nomenclature of Mouse Genes aligned and classified the many P450
isoymes on a basis of proposed evolutionary relationships (Nebert et al., 1987, 1989,
1 99 1, Nelson a al., 1996).
The cytochrome P450 genes are designated according to the following: an
italicized root symbol CYP is used to denote the cytochrome P450 gene in humans (C'
for mouse), followed by an Arabic number identifying the cytochrome P450 fiuniy. A
letter is used to distinguish the cytocbrome P450 subfamily (when two or more exist
witbin the family), and an Arabic numeral to denote the individual gene within the
subfamily. Gene products in all species, including mouse, should include all capital
letters without italics. For example, CElPlAl designates the h u m gene (Cjplal in
mouse), while CYPlAl refers to the cDNA, mRNA and enzyme in both species (Nelson
et al., 1996). Protein sequences between families possess 5 40% similarity, whereas,
protein sequences within a single family possess > 40% identity. In mammalian
sequences, proteins that demonstrate > 55% homology are designated into the same
subfamily (Nebert et al., 199 1 ; Nelson et al., 1996).
As of 1996, 481 cytochrome P450 genes had been identified in 85 eukaryotes
(including 14 mammalian species) and in 20 prokaryotes. Fourteen of the 74 identified
gene families existed in all mammals. Furthennore, these 14 mammalian families
comprised 26 separate subfamilies (Nelson et al., 1996).
1.6 MECHANISMS OF CYTOCaROME P4SO DRUG METABOLISM
Once a target substrate is bound to cytochrome P450, the enzyme accepts an
electron fbm either NADPH (nicotinamide adenine dinucleotide phosphate) or
cytochrome bS (White and Coon, 1980; Guengerich, 1983; Pompon, 1987). This electron
exchange is usually mediated by the flavoprotein NADPH-cytochrome P450 reductase.
This protein contains two flavin molecules as prosthetic groups, flavin adenine
dinucleotide (FAD) and flavin mononucleotide 0, invoIved in the transfer of
reducing equivalents h r n the electron-carrier cofactor to the bound substrate (Smith et
al., 1994). Consequently, the cytochrome P450 enzyme is reduced and molecular oxygen
is able to bind to this newly formed me-subs t ra te complex. Following the addition
of a second electron, donated by either NADfH-cytochrome P450 reductase or
cytochrome b5, the oxygen atom dissociates into two oxide anions. This helps create an
unstable trimolecular intermediate. The first oxygen atom is incorporated into the
substrate, while the second atom is used to generate water. It is the insertion of this hrst
atmospheric oxygen atom into the substrate that is common to all oxidative reactions
mediated by the various cytochrome P450 isozymes. The oxidized substrate is then
released, allowing the P450 enzyme to repeat the cycle (see Guengerich, 1993 for
review).
Cytochrome P450 enzymes exist in multiple forms and constitute a group of
isozymes with differing, but ovedapping specificities for many exogenous and
endogenous compounds (Nebert, 1979). Hence, organisms possessing these enzymes are
able to metabolize nearly all small, hydrophobic molecules they encounter (Okey et aL,
1986). However, depending on the rate of formation of reactive intermediates, the
9
presence of nearby phase 11 enzymes, and the inherent chemical instability of the
oxygenated intermediate, two different metabolizing pathways exist. In the first pathway,
the parent molecule is tramfomed into a chemically-inert, water-soluble substance,
which is easily excreted, thereby, benefiting the organism. in the second paihway,
covalent binding of the unstable oxygenated intermediate to cellular structures may occur,
leading to necrosis, mutagenesis, drug toxicity, teratogenesis and carcinogenesis (Wright,
1980; Miller and Miller, 1985; Pekonen and Nebert, 1982; Minchin and Boyd, 1983;
Okey et al, 1986).
1.7 CYTOCHROME P450 INDUCTION
Inducibility is an important property of several cytochrome P450 enzymes
(Adesnik and Atchison, 1985). W e not all cytochrome P450 isozymes are inducible in
all tissues, this adaptive response plays a significant role in increasing the rate at which
some target substrates are metabolized. Following initial studies of cytochrome P450
induction, inducing agents of drug metabolism were divided into two classes: those
resembling phenobarbital (PB) and those resembling 3-methylcholanthrene (3-MC)
(Comey, 1 967). However, some polychlorinated biphenyls exhibited both classes of
induction, elevating the levels of many differing P450 species (Stonard, 1975; Parkinson
et ai., 1980a, 1980b). It is now evident that many structurally diverse compounds can
induce the same form of cytochrome P450 (Adesnik and Atchison, 1985). Furthemore,
many inducers are substrates for the P450 species that they induce, thereby stimulating
their own metabolism. Since most inducible species of P450 demonstrate substrate
versatility, induction often stimulates the metabolism of multiple compounds, in addition
to the inducing agent. This is present to some extent in nearly all tissues (Okey, 1990).
Induction of drug-metabolizing enzymes enables the cell to deal with the many
toxic compounds it encounters. By elevating levels of specific enzymes in response to an
introduced xembiotic, the cell is better eqyipped to detoxify and r&ove the cytotoxic
compound before it causes cellular damage. However, this process is not universally
beneficial. While detoxiiication requires the presence of both phase I and phase I1 drug-
metabolizing enzymes, the degree of inducibility of the phase I enzymes is often higher
than that of the phase II enzymes (Okey, 1990). If the balance between these enymes is
altered, the cell's ability to eliminate formed reactive intermediates may be compromised
(Okey, 1990). The cell may not be able to dispose of these toxic metabolites via its
conjugating enzymes, leading to genomic alterations and various toxicities through
covalent binding of the intermediates to various cellular proteins, nucleic acids, or other
biomolecules (Eisen et al., 1983; Okey, 1990).
1.8 CYPIAl/CYPlA2 (CYTOCHROME P450 lAlflA2)
The CYPl family is comprised of two subfamilies, designated A and B. The
CYP 1 A subfamily contains two corresponding gene sequences, 1 and 2. These sequences
are approximately 70% homologous indicating a high degree of conservation between
their genetic sequences ( A d d and Atchison, 1985; Gonzalez, 1990). The two genes
lie in tandem on mouse chromosome 9 and human chromosome 15 (Gonzalez, 1990).
Furthennore, both genes have been isolated and sequenced fkom rat, mouse and human
( G o d e z , l99O). Various terms have been used for the CYP l A1 isozyme. In rat tissue,
11
this enzyme has previously been termed P450c, or type BNF-B. It has also been referred
to as P,-450, and P450c in human tissue, and P450 form 6 in both human and rabbit
tissue; it has been designated P450 IA1 in trout, Dahl in dog and MKahl in monkey
tissue ( A d d and Atchison, 1985; Nebert and Gonzalez, 1987; Gonzalez, 1990; Nebert
et aL, 1991).
Both CYPlAl and CYPlA2 are inducible by agents such as polycyciic aromatic
compounds and aromatic hydrocarbons (Gonzalez, 1990). CYP LA2 is fuaher involved in
the activation of several heterocyclic amine promutagens, derived from pyrolysates of
proteins (Johnson et ul., 1980; Goldstein et al., 1982; Kamataki et al., 1983; McManus et
al., 1988).
The first identified inducer of CYPlAl was the polycyclic aromatic hydmcarbon,
3-methylcholanthrene (3-MC) (Richardson et al., 1952). Many other inducers of this
gene have since been identified. These select agents include several other polycyclic
aromatic hydrocarbons such as benzo[a]pyrene and benz[a]anthracene, several
p heno thiazines, P-naphtho flavone, various plant indoles, polychlorinated and
polybrominated, halogenated dibenzo-p-dioxins, and halogenated dibenzohuaas, as well
as several compounds found in charcoal-broiled beef, cigarette smoke, crude petroleum
production, cosmetics and pedbmes (Eisen et al., 1983). This cytochrome P450 isoform
has received much attention, primarily because its metabolism of aromatic hydrocarbons
is often a key step in the activation of these compounds to mutagenic and carcinogenic
byproducts (Eisen et al., 1983).
The first step in the induction of CYPl A1 involves diffusion of a lipid soluble
xenobiotic through the cell plasma membrane to the cytosolic compartment of the cell.
This substrate initially binds to the Ah (Aromatic hydrocarbon) receptor, which is present
as a heterodimer coupled with heat shock protein (hsp90). This heterodimer complexes
with the xenobiotic substrate, and releases hsp90 into the cytoplasm (Okey et al., 1986;
Guengerich, 1993). The next step has been postulated to be the interaction of the ligand-
receptor complex with the Ah receptor nuclear translocator (Arnt) protein, to form the
Ah-receptorxuyl hydrocarbon nuclear translocator protein heterodimeric complex. This
complex migrates into the nuclear compartment, acting as a Ligand-activated transcription
factor, and binds to a xenobiotic regulatory genetic sequence (Safe and Krishnan, 1995;
Sogawa et al., 1995). This regulatory gene sequence has been identifled as the xenobiotic
responsive element and is located in the 5'-regulatory region, 1200 to 1500 base pairs
upstream fhm the initiation start site. Transactivation and gene transcription are
initiated, leading to the synthesis of messenger ribonucleic acid (mRNA). This mRNA is
translated into CYPlAl apoprotein, which binds to a heme prosthetic group within the
cytosol (Okey et al., 1986; Guengerich, 1993; Safe and Krishnan, 1995). The newly
formed hemoprotein is incorporated into the membranes of various organelles, most
notably the smooth endoplasmic reticulum, where it becomes catalytically active,
increasing the rate of activation and metabolism of the initial PAH that initially
stimulated its production.
Activated transcription, ultimately leading to increased protein synthesis of
CYP 1 A I, is the primary mechanism involved in 3-MC-type induction. However, it must
be emphasized that no single mechanism can account entirely for P450 induction. Other
13
post-transcriptional events involved in the regalation of P450 cytochromes art also
important, especially in response to inducing agents other than PAHs. Other sites of
regulation include mRNA processing, mRNA transport, mRNA stability and altered rates
of degradation of both the parent cornpound and the P450 enzyme itself (Guengerich,
1993).
1.9 CYPZBI/CYPZB2 (CYTOCEROME P450 2Bll2B2)
The C(P2B subfamily is comprised of 16 separate gene members across multiple
species, which are inducible by phenobarbital (PB) and other structmdly dissimiIar
compounds, including 5-ethyl-5-phenyfiydantoin, tetrachlomethylene and methoxychlor
(Nebert and Gonzalez, 1987; Nims et al., 1994; Hanioka et al., 1995; Li et al., 1995).
Within this subfamily, two members have been extensively examined - CYP2B1 and
CYP2B2. CYP2Bl has previously been referred to as rabbit form 2, rat b, PB-4, PB-B
(Adesnik and Atchison, 1985; Nebert and Gonzala, 1987; Gonzalez, 1990). CYP2B2
has been termed rat e, PB-5 and PB-D (Adesnik and Atchison, 1985; Nebert and
Goozalez, 1987; Gonzalez, 1990). These two genes are very closely related. On the basis
of amino acid sequence data for tryptic peptide hgrnents, representing 75% of their
enzymatic sequences, they differ by only 13 amino acids, alI of which are clustered in the
carboxy terminal halves of the polypeptide chains (Gonzalez, 1990). These two genes,
inducible in rat liver, are thought to have diverged fhm a common ancestral gene
approximately 12 million years ago (Sogawa et al., 1985). Interestingly, the rat and
mouse species are believed to have separated fiom one another approximately 17 million
years ago (Miyate et al., 1982). Therefore, species other than rat, such as mouse, may not
14
express these two P450 genes within the CYP2B subfamily (Nelson et al., 1996). In the
mouse, the CYP2B subfsmily has been localized to chromosome 7 and in humans to
chromosome 9 (Gonzalez, 1990).
Mechanisms by which these genes are regulated are poorly understood. Although
it is known that increased activity of CYP2B1 and CYP2Bl primarily results h m
transcriptional activation and not protein stabilization, little is known of the mechanism
by which the cell recognizes the inducer or how the inducer affects transcription
(Bresnick, 1993; Hodgson, 1994). In fact, a PB receptor has yet to be identified and
investigators have hypothesized that induction of the CYP2B subfamily may involve a
direct interaction between the inducing agent and the cytochrome P450 molecule
(Waxman and AzamtT, 1992). This interaction may release an endogenous ''suppressor",
ultimately resulting in the increased expression of CYP28l and CYP2B2 (Waxman and
Azaroff, 1992). Although these two isozymes are genetically similar, they are differently
expressed across several organs. In rat Liver, both CYP2B 1 and CYP2B2 are inducible by
PB. However, while CYP2B1 is a major constitutively expressed cytochrome P450
isozyme in rat lung, CYP2B2 is not expressed in pulmonary tissues h m either untreated
or PB-treated animals (Guliaeva et al., 1994). Furthermare, CYPZBl is constitutively
demonstrated in rat testis and is inducible in rat kidney (Paolini, et al., 1995). On the
other hand, CYP2B2 is constitutively present in the small intestine, but not in the kidney
or testis of either untreated or PB-treated rats (Omiecinski, 1986; Traber et al., 1988;
Guliaeva et al., 1994).
1-10 NADPH-rVTOCHROME P450 REDUCTME
NADPH-cytochrome P450 nductase is a flavoprotein that shuttles electrons,
either directly or via cytocbrome b5, from NADPH or NADH to the microsoma1
membrane-bound cytochrome P450 unit (White and Coon, 1980). As such, it is involved
in mediating the NADPH-dependent reduction of multiple cytochrome P450 isozymes
(Wilsocki et al., 1980; Guengerich, 1983). As a co-enzyme of the P450 drug-
metabolizing system, this protein is co-ordinately regulated together with cytochrome
P450 to maximize the drug-metabolizing action of this enzyme system (Emster and
Orrenius, 1965; Masters and Okita, 1980 for review; Dees et al., 1980; Comey, 1982).
1.1 1 RATIONALE AND OBJECMVES OF PRESENT STUDY
Tumourigenesis occurs as a result of xenobiotic-induced cellular modifications
within target tissues (Miller and Miller, 1977; Miller, 1970; Farber, 1980). These
modifications are believed to alter the expressions of drug-metaboiizing enzymes within
the developing tumour (Farba, 1984, 1990). Of relevance to this study is the temporal
association between neoplastic development and perturbations of the CYP2B 1, CYP 1 A 1
and NADPH-cytochrome P450 reductase. It has been postulated that altered metabolic
profiles within developing tumours provide these neoplastic tissues with an increased
resistance to numerous cytotoxic xenobiotics (Farbet et al., 1976). Consequently, they
are abie to proliferate in an endnment which is toxic to surrounding non-neoplastic
tissues (Farber, 1984; Judah et aL, 1976). While the physiologic, metabolic and
biochemical profiles of these carcinogen-induced "resistant" tissues have been
extensively studied in other tissues such as the liver, immmohistochernical identification
of the cytochrome P450 enzymes and NADPH-cytocbrome P450 reductase within the
lung has received little attention.
The primary objective of the present study is to test the following hypothesis:
solid- and papillary-type lung turnour development in SWWJ mice is associated with
decreased localization of CYP2B 1, CYPl A1 and NADPH-cytochrome P450 reductase.
2. MATERIALS AND METHODS
2.1 CaEMICALS AND REAGENTS
Polyclonal a n t i i e s raised in rabbit against rat liver CYP2B1/2B2, CYPl A l
and NADPH cytochrome P450 reductase, were generously donated by Dr. A. Parkinson
(Department of Pharmacology, Toxicology and Therapeutics, University of Kansas
Medical Centre, Kansas City). Chemicals and reagents were obtained as follows: Vector
Laboratories, Inc. (Burlingame, California) - Biotinylated goat anti-rabbit IgG (H+L),
normal goat serum, avidin-biotin 'Elite' conjugating kits and avididbiotin blocking kits;
Sigma Chemical Co. (St. Louis, Missouri) - P-naphthoflavone, urethane,
paraformaldehyde, pepsin, and 3,3'-diaminobenzidine tetrahydrocMoride; Bio-Rad Labs
(Richmond, California) - sodium deodecyl sulphate polyacrylamide gel electrophoresis
standards (SDS-Page) (low and high molecular range); Ametsham Life Science (Oakville
Ontario) - rainbow-coloured high molecular weight SDS-Page standards; ICN
Biomedicals (Cleveland, Ohio) - Tris-HCl; BDH Chemicals (Toronto, Ontario) -
acrylamide and sodium deodecyl sulphate; Schleicher & Schuell Inc. (Keene, New
Hampshire) - nitrocellulose transfer and immobilization membrane; MTC
Pharmaceuticals (Mississauga, Ont) - Somnotol (sodium pentobarbital); Fischer
Scientific (New Jersey) - hematoxylin stain (Gill's formulation #I), Tissue Prep;!
par& embedding medium, Histo-prep mold releasing agent, and 30% hydrogen
peroxide; National Diagnostics Ltd. (Manville, New Jersey) - Histoclear histological
clearing agent. All other chemicals of reagent grade were obtained &om standard
commercial suppliers.
2.2 ANIMAL,S
In order to facilitate comparisons between solid and papillary tumours over the
course of development, SWR/J mice were chosen for the study. Not only is this strain
relatively susceptible to turnour induction, but the proportion of solid and papillary
tumours is more evenly distriiuted than other murine strains (Shimkin and Stoner, 1975;
Malkinson, 1989; Chen et al., 1994). Mice were bred h m three breeding pairs, which
were established using six-weeksld SWWJ mice weighing 20-22 g (Jackson
Laboratories, Bar Harbour, Maine). These breeding pairs provided all mice used in the
present study. Two females and one male were housed together in small plastic cages
over hardwood bedding (Eletachip). These breeding pairs were maintained in a controlled
environment, at a temperature of 25 f 1°C, under a 12 h light/dark cycle. Mice were
given £ice access to food (Purha rodent chow) and drinking water. Pups were allowed to
remain with their mother for 7-10 days. Subsequently, these pups were weaned and
separated by sex and age.
2.3 TREATMENT OF ANIMALS
Mouse pulmonary tumours were induced with urethane as previously described
(Forkert et al., 1992). AU mice were administered I mg/g body weight urethane in saline
(i.p.) at six weeks of age. To induce CYPlA1, mice were administered P-naphthoflavone
(P-NF) in corn oil (80 mgkg body weight) at 72 h and 48 h prior to sacrifice. Control
mice received only the vehicle. All protocols involving the use of animals were approved
by the Animal Care Committee at Queen's University.
2.4 PREPARATION OF MICROSOMES
Twenty mice were sadiced for protein immunoblotting studies. Of these mice,
10 were administered prior to sacrifice, while 10 received only the vehicle. Mice
were sacrificed by cervical dislocation, and the lungs were removed. Microsomes were
prepared by differential cenagation by using procedures described previously
(Matsubara et al., 1976; Forkert et al.. 1987). AU procedures were conducted on ice,
using cold solutions. Lungs were hrst rinsed in cold phosphate buffered KC1 (1.15%
KCI, 100 m M K2HP04, pH 7.4). The tissue was weighed and minced with a razor blade
in 4 volumes of buffer. The tissue was homogenized, using 6 strokes of a Teflon pestle
over ice. The homogenate was transferred to a cold, screw-topped, Beckman
ultracentrifuge tube and centrifuged at 40,000 g, at O°C, for 20 min. The supernatant was
dispensed into another ultracentrifbge tube, which was centrifuged at 105,000 g, at O°C,
for 60 min. Centrifigation was carried out in a L860M Beckman Ultracentrifbge using a
Ti-50 rotor. The supernatant was discarded, while the pellet was resuspended in 4 mL
cold 100 mM K2HP04 buffer, pH 7.4, and re-homogenized with six strokes of the pestle.
This homogenate was then centrifuged again at 105,000 g, at O°C, for 60 min. Following
this second centrifbgation, the supernatant was once more discarded. The pellet was re-
suspended in 4 mL of lOOmM &wo4 buffer, pH 7.4, containing 1.5 mM EDTA, and
homogenized with a glass rod. The hornogenate was aliquoted into Eppendorf tubes (200
PI), frozen in Liquid nitrogen, and stored at -70°C.
Protein concentrations of the dcrosomal suspensions were determined using the
method of Lowry et al. (1951). AU experiments were conducted at mom temperature.
Standards were prepared with bovine serum albumin, at concentrations ranging between
of 50 mg/mL and 400 mg/mL. Microsoma1 samples were diluted within this
concentration range at a h a 1 volume of 200 mL each. To each of the various dilutions,
200 mL of 0.5 N NaOH was added, vortexed and left to stand for 30 min. Each sample
received 2 ML of the copper reagent (1 mL 1.0% CuSO;5H20, 1 mL 2.0% sodium
potassium tartarate, 100 mL 2.0% sodium carbonate). The samples were vortexed and
left to stand for 10 min. Folin phenol reagent was added to each sample, vortexed and
incubated for 30 min. Finally, the absorbance of each sample was determined at 670 nm,
using a Novaspec I1 4040, single beam spectrophotorneter. Standards were then used to
plot a protein concentration c w e , and values for each samples were determined fkom this
curve.
2.5 PROTEIN GEL ELECTROPHORESIS
Separation of microsomal proteins was performed by SDS-PAGE using an 8.5%
separating gel together with a 4.5% stacking gel. The 8.5% separating gel was prepared
by adding 8.5 mL 30% acrylamide, 8.5 mL 1.5M Tris-HC1 buffer (pH 8.8), and 0.68 mL
10% SDS to 1 1.9 mL nanopure water. Immediately prior to use. 0.025 mL NyNyN',N'-
te tramethylethylenediamine (TEMED) and 0.32 mL 10% ammonium persulfate were
added to the saiution. This separating gel was poured between the glass plates of the gel
apparatus, covered with a layer of 0.5% SDS to ensure a smooth surface at the interface,
and allowed to polymerize for 60 min.
The 4.5% stacking gel monomer solution was then prepared, containing 2.66 rnL
30% acrylamide, 5.0 rnL 0.5M Tris-HC1 buffer (pH 6.8) and 0.20 mL 10% SDS in 12.2
mL nanopure water. Again, prior to pouring the gel, 0.02 mL TEMED and 0.120 mL,
10% ammonium p d a t e were added. The stacking gel was poured over the separating
gel and allowed to polymerize for 30 min. The combs were removed, and the wells
rinsed with nanopure water.
Equal parts of protein sample and sample buffer were combined and boiled for 2
min to solubilize the protein. Molecular weight standards and protein samples were then
loaded into the wells. Gel electrophoresis was performed for 1 h at a constant current of
80 mA, using a Hoefer Scientific 500 volt direct cunent power supply. Finally, the gel
was placed in the transfer buffer.
2.6 PROTEIN IMMUNOBLOTTING
Protein immunoblotting was carried out on both p-NF-treated and vehicle-treated
lung tissues as described in previous studies (Forkert et ai., 1994). The separated protein
samples were transferred electrophoretically onto a nitrocellulose transfer and
immobilization membrane. Prior to tnbsfa, the gel, nitrocellulose paper and all transfer
apparatus (i-e., sponges and transfer chamber), weze allowed to equilibrate in transfer
buffer for 5 min. Transfer buffer was prepared h m 6.06 g Tris, 28.8 g glycine and 400
mL MeOH, adjusted to a final volume of 2 L with aaeopure water. Following
equilibration, the gel and transfer membrane were placed within the Hoefer Trander
Chamber containing transfer buffer. Transfer was achieved at 1.0 A, for 1, h at 20°C.
Following transfer, the nitrocellulose paper was riwd for 10 min in Tris-buffered
saline (T5S). The membrane was then gently immersed into a solution of non-fat dried
milk (3.0%) in TBS for 2 h at mom temperature, to block non-specific antibody binding.
The membrane was transferred and rinsed 3 times for 10 min, with gentle agitation, in
TBS containing Tween 20 (T/TBS) (0.05%), pH 7.5. Agitation in all solutions was
performed on a Hoefer Red Rotor at room temperature.
Primary anh'body was suspended in T/TBS, containing gelatin (1.0%), at 1 : 1000,
and added to the membrane. The incubation was performed overnight at room
temperature. These primary polyclonal antibodies, which were raised in rabbit against rat
liver microsomal CYP2B 1/2B2, CYPl A1 and NADPH-cytochrome P450 reductase, were
generously donated by Dr. A. ~arlcinson. The next morning, the membrane was washed 3
times for 10 min in changes of TiTBS, to remove any residual antibody. The membrane
was then transferred to TlTJ3S containing alkaline phosphatase-conjugated goat anti-
rabbit secondary antiiody at 1:1000, and incubated for 2 h. Following incubation, the
membrane was again washed 2 times for 10 min in T/TBS, then an additional 10 min in
TBS, to remove any residual Tween-20 &om the membrane surface. Visualization of
protein bands reacting with the antibody was performed using p-nitrobluetetrazolium
c hloride/5-bromo-4-chlom-3 -indolylphosph p-toluidine salt (NBT/BCIP). This
colouring reaction is achieved by using the alkaline phosphatase enzyme conjugated to
the secondary antibody. This enzyme hydrolyzes BCIP, releasing two hydrogen atoms,
which reduce NBT to form a purple-coloured formazan dye (McGadey, 1970; Altman,
1976). The reaction was terminated by immersiug the blot in deionized water.
2.7 TISSUE PRF,PARATION FOR IMMUNOHISTOCHEMISTRY
In preparation for immu11ohistochemical studies, vehicle-treated and fbNF-treated
mice were sacrificed at various stages of tumour development. Fifteen mice received
only the control vehicle. Six of these mice were killed at 10 weeks post-urethane
treatment, while six were sacrificed between 16-22 weeks and three were killed at 52
weeks of age. On the other hand, 18 mice were admiaistered p-NF to induce CYPl Al
prior to sacrifice. Of these 18 mice, seven were sacrificed at 10 weeks post-urethane
treatment, five were killed between 16-22 weeks and 6 were sacrificed at 52 weeks.
Mice were anaesthetized with sodium pentobarbital (0.12 mdg) prior to sacrifice.
Lung tissues were fixed with 4.0% pdormaldehyde in Soreason's buffer (0.1 M) (12.0
mM NaH,P04, 69.0 mM Nqmo4, pH 7.4) by vascular perfusion for 5 min, followed by
tracheal instillation. The inflated lungs were removed en bloc with the heart and
immersed in hative for 4 h at room temperature. Individual pulmonary lobes were
separated in bufEer, immersed in fiesh fixative and refrigerated overnight at 4'C. The
tissue was dehydrated and processed in an Autotech Ultra Tissuematon, consisting of
seven hourly washes through an ethanol series (from 70% to loo%), 2 1-hour treatments
in Histoclear, and 2 1-hour treatments in parafk at 58OC. The tissue was embedded in
paraffin and sectioned at 4-6 p on a Reichert-Jug Biocut 2030 microtome using
disposable black. Step sectioning of both vehicle-treated and p-NF-treated lung tissues
was performed Every tenth tissue section was retained for immunohistochemical
analysis and mounted on a glass slide.
2.8 AVIDIN-BIOTIN IMMUNOHISTOCHEMISTRY
Tmmunohistochemistry was performed for the detection of CYP2B 1, CYP 1 A1,
and NADPH cytochrome P450 reductase in murine pulmonary tissues. This technigue
was performed using the avidin-biotin 'Elite' protocol supplied by the manufachuer
(Vector Laboratories, Inc.). Prior to staining, all slides were depadhked with heat at
60°C for 10 min. The sections were then hydrated through a series of graded ethanol
solutions according to the following:
Histoclear 1 x2min
Histoclear l x l m i n
100% EtOH 2 x I m i n
95% EtOH 2 x l m i n
70% EtOH 2xlmin
Following hydration, all sections were incubated in 0.01 N HCl, containing
0.004% pepsin at 37T for 30 min, to expose antigenic sites. Sections were briefly rinsed
in phosphate-buffered saline (PBS), pH 7.4. A blocking solution consisting of 5.0% skim
milk powder and 1.5% normal goat serum, was applied for 20 mi. to block non-specific
antibody binding. Sections were rinsed in PBS for 5 min, then incubated in an avidin-
blocking solution for 15 min. Following another PBS rinse for 2 min, the sections were
incubated in a solution containing biotin for 15 min. These two incubations were
necessary to inhibit endogenous biotin reactivity. Pzhaq antiibodies were administered
for 1 h at dilutions ranging h m 1 : 100 to 1 : 1,600 to determine concentrations for optimal
staining. Following two 10 min rinses in PBS to remove unbound primary antibody,
biotinylated goat anti-rabbit secondary antitbody (1 :200) was applied for 30 min. Sections
were rinsed twice in PBS for 5 min, then placed in hydrogen peroxide (1.0%) for 30 min,
to block endogenous peroxidase activity. During this incubation, the ABC 'Elite' avidin-
biotin conjugate was prepared according to the manufacws instructions and allowed to
stabilize at room temperature for 30 min. Following two 5 min rinses, the sections were
incubated in the ABC complex for 30 min, and again rinsed twice in PBS for 5 min each.
Visualization was achieved by incubating the sections for 4 min in PBS containing 0.05%
3 , 3 ' - b e e tetrahydrochloride (DAB), and 0.01% hydrogen peroxide. The
sections were briefly rinsed twice in tap water to stop the reaction. The tissues were
incubated in 0.5% copper sulfate in 0.15 M sodium chloride for 5 min, and rinsed in tap
water for 5 min. Selected sections were counterstained with Gill's #I hematoxylin for 20
sec, and rinsed in tap water. These counterstained sections were briefly destained,
dehydrated, cleared and mounted using Ro-Texx mounting medium.
Immunohistochemical controls included incubations in which the primary antibody was
omitted and incubations which contained HyHel-9, an antibody specific for egg white
lysozyme (Smith-Gill et dl., 1982). These controls were used to evaluate non-specific
background staining and to ensure that staining variability between immunohistochemical
experiments was minimalized.
Immunohistochemical localization of CYP2B 1, CYP 1Al and NADPH-
cytochrome P450 reductase, in neoplastic and non-neoplastic tissues, was performed by
light microscopy. Imm~11ohistochemical staining intecsities o f these enymes were
qualitatively assessed. Tissues failing to express these proteins were classified as
negatively stained (-), while tissues demonstrating the proteins were classified according
to their relative staining intensities. Minimally stained times were graded plus one (+),
while tissues with increasing imrnunohistochemical staining intensities were graded plus
two (++I or plus three (*). Tissues that expressed the proteins at high levels were
graded plus four (++++).
3. RESULTS
3.1 PROTEIN IMMUrYOBLOTTING
Protein immunoblotting was performed to ensure that polyclonal antibodies mised
in rabbit, against rat cytochrome P450 isozymes, detected orthologous enzymes in murine
pulmonary tissues. Rabbit serum IgG was purified to produce mti'bodies specific to rat
liver CYP2B 1/2B2, CYP 1 A1 and NADPH-cytochrome P450 reductase (Patkinson and
Gemzik, 1991). The first step in this purification technique involved precipitating out
non-IgG serum proteins with capryllic acid at pH 4.5, followed by centrifugation. The
next step involved fractionating the remaining proteins with ammonium sulfate, at pH
7-4, and fiutber centrifugation. The resulting IgG pellet was resuspended in PBS and
dialysed overnight. Following a final centrifugation, the concentration of purified
antibody was determined by its absorbance spectra at 280 nm.
Polyclonal antibodies against CYP2BKYP2B2 were reacted against vehicle-
treated, S W J pulmonary microsomes (Fig. 1A). Optimal protein concentration was
observed at 3 pg. This antibody detected two proteins with molecular masses of
approximately 49 kDa. The amino acid sequence of rat CYP2Bl is 97% identical to that
of CYP2B2 (Ryan and Levin, 1990). In addition, the base promotor sequence of rat
CYP2B2 has been shown to be 83% similar to a corresponding murine sequence
involving Cp2b 10 (Honkakoski, et al., 1996) . Furthermore, the translational sequence
derived fiom a recently isolated hybrid enzyme incorporating both CYPZBlO and
NADPH-cytochrome P4SO reductase (currently termed p24) is approximately 97%
identical to that of mouse CYPZBlO (Damon et al., 1996). With such similarities in
molecular structure, across multiple cytochme P450 isofom, the polyclond antibody
against rat CYP2B1/2B2 may detect multiple proteins within the murine tissue.
Pulmonary microsomes fiom vebicle-treated mice were reacted with a polyclonal
antiiody against NADPH-cytochrome P450 reductase (Fig. 1B). At 3 pg, this antibody
detected a protein with a molecular mass of approximately 70 kDa Pulmonary
microsomes h m P-W-treated mice were subjected to electrophoresis, immunoblotted
and reacted with a polyclonal mtiiody against rat CYPlAl. At 5 pg, this antiiody
detected a protein with a molecular mass of approximately 55 kDa (Fig 2A). These
microsomes f?om P-NF-treated mice were reacted with a polyclonal antibody specific for
NADPKcytochrorne P450 reductase. At a concentration of 1 pg, this antibody
recognized a protein of approximately 70 kDa (Fig. 2B).
3.2 AVIDIN-BIOTIN IMMUNOHISTOCHEMISTRY
3.2.1 PULMONARY DISTRIBUTION OF CYP2B1
The dilutions of polyclonal primary antibodies against CYP2B1 which produced
optimal staining of the isozyme, while minimizing non-specific staining in tissues, ranged
between 1 :400 and 1 500. Within murine pulmonary tissues, CYP2B 1 was expressed in
non-ciliated Clara cells lining the pulmonary airways and alveolar type II cells within the
parenchyma (Table 1). Clara cells exhibited the most intense staining for the CYP2B1
protein, with preferential location in its apical aspect (Fig. 3). Type Il cells exhibited a
more homogenous, but less intense, staining pattern for the CYP2B 1 apoprotein (Fig. 4).
In murine lung tissues, urethane treatment initially led to the formation of
hyperpiastic foci (10 wks). With linther development, these hyperplasia proceeded as
either solid- or papillary-type adenomas (16-22 wks), and ultimately progressed to solid-
or papillary-type carcinomas (52 wks). Developing tumom were identified at each stage
of development. In fact, within the 15 vehicle-treated, hrmour-bearing S W J mice,
CYPZBl localization was assessed in 55 hyperplastic foci, 20 solid-type adenomas, 15
papillary-type adenomas, 25 solid-type carcinomas, 24 papillary-type carcinomas and 35
hyperplastic foci present in carcinoma-bearing lung tissues.
Immunohistochemical staining for CYP2B1 was weakly localized within all
hyperplasias and neoplastic tissues, at all stages of development. Furthermore,
localization of the CYP2Bl isoyme was maintained in non-oeoplastic lung tissues
surrounding these developing hlmours (Table 2) (Fig. 5).
3.2.2 PULMONARY DISTRIBUTION OF CYPlA1
Optimal staining for CYP LA1 was identified at a dilution of 1 :200. In pulmonary
tissues fiom vehicle-treated mice, CYP 1 A1 was not expressed in any cells. However, in
all lungs h m P-NF-treated mice, this isozyme was expressed in vascular endothelid
cells, and alveolar type I1 cells (Table 3) (Fig. 6 0 ) .
Identification of CYPlAl was examined in 15 vehicle-treated, turnour-bearing,
SWRIJ mice. From these mice, 10 hyperplastic foci, 7 solid-type adenomas, 8 papillary-
type adenomas, 6 solid-type carcinomas, 5 papillary-type carcinomas and 20 hyperplastic
foci within tumour-bearing lungs were assessed. CYPlAl was not expressed within any
of these tumous (Table 4). Of the 18 mice treated with f3-NF, 40 hyperpIastic foci 22
solid-type adenomas, 18 papillary-type adenomas, 14 solid-type carciaomas, 18 papillary-
type carcinomas and 34 hyperplastic faci within carcinoma-bearing lungs were examined
for CYP l A1 induction (Table 4). The CYPl A1 isoform was detected in hyperplastic foci
(Fig. 7) and solid-type adenomas, but localization of the apoprotein was decreased in
papillary-type adenomas (Fig. 8A). Furthermore, CYPlAl was not identified in either
solid- or papillary-type carcinomas (Fig. 8B). Interestingly, CYPlAl was prominently
expressed in hyperplastic foci developing within carcinoma-bearing lungs (Fig. 9).
3.2.3 PULMONARY D1STRIBUTI:ON OF NADPH-CYTOCHROME P450
REDUCTME
In all murine pulmonary tissues, optimal staining for NADPH-cytochrome P450
reductase was identified at an anti'body dilution of 1:200. In vehicle-treated mice,
NADPH-cytochrome P450 reductase was demonstrated in Clara cells and type II cells
(Table 1.) (Fig. 10). As with CYP2B1, immmohistochemical staining was localized
more prominently within the Clara cells; however, the reductase protein was evenly
distributed throughout the cytoplasm, and not restricted to the apical portion of the cell.
Treatment with fLNF failed to influence cell-specific staining for the reductase
enzyme. As with vehicle-treated tissues, this isozyme was restricted to Clara cells and
type II cells (Table 3). Furthermore, intra-cellular distribution of the isozyme was not
altered by treatment with P-NF.
In developing turnour tissues from mice receiving only the control vehicle, 23
hyperplastic f a 10 solid-type adenomas, 7 papillary-type adenomas, 4 solid-type
carcinomas, 6 papillary-type carcinomas and 19 hyperplastic foci in carcinoma-bearing
lungs were identified (Table 5). NADPH-cytochme P450 reductase was moderately
demonstrated in hyperplastic foci and solid-type adenomas, while papillary-type
adenomas showed diminished localization; on the other hand, solid- and papillary-type
carcinomas failed to express the protein (Table 5) (Fig 11). Nevertheless, hyperplastic
foci within carcinoma-bearing lung stained intensely for the reductase enzyme.
In developing hunour tissues h m mice treated with P-NF, 35 hyperplastic foci,
18 solid-type adenomas, 16 papillary-type adenomas, 14 solid-type carcinomas, 12
papillary-type carcinomas and 29 hyperplastic foci in carcinoma-bearing lung tissues
were identified (Table 5). NADPH-cytochrome P450 reductase was moderately
expressed in hyperplastic foci and solid-type adenomas while papillary-type adenomas
stained considerably weaker (Fig. 12). Furthermore, both solid- and papillary-type
carcinomas failed to demonstrate the reductase protein. As with control tissues,
hyperplastic foci developing within carcinoma-bearing lungs stained intensely for the
reductase enzyme (Fig. 13).
Table 1.
Relative content of CYP2Bl and NADPH-cytochrome P450 reductase in normal lung
tissues from vehicle-treated SWRD mice.
Tissue a CYPZB 1 NADPHqtochmme P450 reductaset
Airway
Parenchyma
Endo thelium
Normal lung tissues h m 15 vehicle-treated, tumour-bearing S W J mice.
' Content expressed as - (absent) or + (minimal immMohistochemical staining) to * (intense immunohistochemical staining) levels of CYP2B 1 and NADPH-cytochrome
P450 reductase proteins.
Table 2.
Relative content of CYP2B 1 in neoplastic lung tissues h m vehicle-treated SWWJ mice.
Tissue a CYP2.B 1'
Hyperplastic Foci (10 wks; n=55) +
Solid Adenoma (16-22 wks; n=20) +
Papillary Adenoma (16-22 wks; n=15) +
Solid Carcinoma (52 wks; n=25) +
Papillary Carcinoma (52 wks; n=24) +
Hyperplastic Foci (52 wks; n=35) +
' Neoplastic lung tissues fiom 15 vehicle-treated, turnour-bearing S W J mice.
' Content expressed as - (absent) or + (minimal immunohistochemical staining) to t ~ +
(intense immunohistochemical staining) levels of CYP2B1 and NADPH-cytochrome
P450 reductase proteins.
Table 3.
Relative content of CYPlAl and NADPH-cytocbrome P450 reductase in normal lung
tissues fkom vehicle-treated and p-NF-treated SWWJ mice.
Tissue CYPIAlt NADPH-cytochrome P450 reductaset
Vehicle-treated '
Airway
Parenchyma
Endothelium
P-Nap htho flavone-Treated
Airway -
Parenchyma tt
Endo thelium ++H-
" Normal lung tissues fkom 15 vehicle-treated, tumow-bearing S WRlJ mice.
Normal lung tissues fkom 18 P-NF-treated, tunour-bearing SWWJ mice.
+ Content expressed as - (absent) or + (minimal immunohistochemical staining) to * (intense immunohistochemical staining) levels of CYP 1 A l and NADPH-cytochrome
P450 reductase proteins.
Table 4.
Relative content of CYPl A1 in neoplastic lung tissues h m vehicle-treated and P-NF-
treated SWRlJ mice.
Tissue CYPIAlt
Vehicle-treated '
Hyperplastic Foci (10 wks; n=lO)
Solid Adenoma (16-22 wks; n=7)
Papillary Adenoma (16-22 wks; n=8)
Solid Carcinoma (52 wks; n=6)
Papillary Carcinoma (52 wks; n=5)
Hyperplastic Foci (52 wks; n=20)
p-Nap htho flavone-treated
Hyperplastic Foci (10 wks; n=40) tt
Solid Adenoma (16-22 wks; n=22) +t
Papillary Adenoma (16-22 wks; n=l8) + Solid Carcinoma (52 wks; n=14) - Papillary Carcinoma (52 wks; n=18) -
Hyperplastic Foci (52 wks; n=34) st++
a Neoplastic lung tissues h m 15 vehicle-treated, turnowbearing SWWJ mice.
Neoplastic lung tissues fiom 18 P-NF-treated, tumour-bearing S W J mice.
' Content expressed as - (absent) or + (minimal immunohistochemical staining) to ++t-+
(intense immunohistochemical staining) levels of CYPlAl and NADPH-cytochrome
P450 reductase proteins.
Table 5.
Relative content of NADPH-cytochrorne P450 reductase in neoplastic lung tissues from
vehicle-treated and p-NF-treated SWWJ mice.
Tissue NADPH-cytochrome P450 reductaset - - -
Vehicle-treated '
Hyperplastic Foci (10 wks; n=23)
Solid Adenoma (16-22 wks; n=10)
Papillary Adenoma (1 6-22 wks; n=7)
So lid Carcinoma (52 wks; n=4)
Papillary Carcinoma (52 wks; n=6)
Hyperplastic Foci (52 wks; n=18)
P-Naphthoflavone-treated
Hyperplastic Foci (10 wks; n=35) ++ Solid Adenoma (16-22 wks; n=18) - -f+
Papillary Adenoma (16-22 wks; n=16) t
Solid Carcinoma (52 wks; n=14) - Papillary Carcinoma (52 wks; n=12) -
Hyperplastic Foci (52 wks; n=29) tttf
' Neoplastic lung tissues fiom 15 vehicle-treated, turnour-bearing S W J mice.
~eoplastic lung tissues fhm 18 P-NF-treated, twnour-bearing SWWJ mice.
Content expressed as - (absent) or + (minimal immunohistochemical staining) to +++
(intense immunohistochemical staining) levels of CYP 1 A1 and NADPH-cytochrome
P450 reductase proteins.
Figure 1 A, B.
(A) Protein irnmunoblot demonstrating CYP2B 1 expression in lung microsomes from
vehicle-treated S W J mice. Molecular weight standards and lung microsomal
proteins were loaded as follows: lanes 1 and 2, 10 pg; lanes 3 and 4 5 pg; lanes 5 and
6 , 3 pg; lane 7, 1 pg; lane 8, molecular weight standards.
(B) Protein immunobiot demonstrating NADPH-cytochrome P4SO reductase expression
in lung microsomes from vehicle-treated S W J mice. Molecular weight standards
and lung microsomal proteins were loaded as follows: lane 1, 10 pg; lanes 2-4, 5 pg;
lanes 5-7, 1 pg; lane 8, molecular weight standards.
Figure 2 A, B.
(A) Protein immunoblot demonstrating CYPl A1 expression in lung mimsomes h m P-
NF-treated SWWT mice. Molecular weight standards and lung microsoma1 proteins
were loaded as follows: lanes 1 and 2, 10 pg; lanes 3-5,s pg; lanes 6-8, 1 pg; lane 9,
molecular weight standards.
(B) Protein immunoblot demonstrating NADPH-cytochrome P450 reductase expression
in lung microsomes from p-NFbeated SWlUJ mice. Molecular weight standards and
lung mimsomal proteins were loaded as follows: lane 1, molecular weight standards;
lanes 2-4, 1 pg; lanes 5-7,s pg; lanes 8 and 9, 10 pg.
Figure 3 A, B.
Normal lung tissue f?om vehicle-treated S W J mice. A) Control incubations were
performed in the absence of the polyclonal primary antibody and demonstrated minimal
non-specific reactivity. B) CYP2Bl was prominently expressed in the apical portions of
the oon-ciliated Clara cells (arrows). Magnification: A) x 500; B) x 480 (Hernatoxylin
counterstain).
Figure 4 A, B.
Normal lung tissue from vehicle-treated S W M mice. CYP2B1 was detected in alveolar
type I1 cells (mows). Magnification: A) x 480; B) x 800 (Hematoxylin counterstain).
Figure 5 A, B.
Neoplastic lung tissues &om vehicle-treated SWRLT mice. A) CYP2B1 was weakly
detected in hyperplastic foci (10 weeks after urethane treatment). Staining was retained
in type I1 cells within the parenchyma (arrowheads). B) CYP2Bl was weakly localized
in papillary-type adenomas (16 weeks after urethane treatment). Magnification: A) x
500; B) x 200 (Hematoxylin counterstain).
Figure 6 A, B.
Normal Lung tissue from P-NF-treated SWR/J mice. A) CYPlAl was detected in
vascular endothelid cells (arrows). B) CYPlAl was expressed in alveolar type II cells
(arrows). Magnification: A) x 880; B) x 880 (Hematoxylin counterstain).
Figure 7 A, B.
hunohistochemical detection of CYPlAl in hyperplastic foci (10 weeks after urethane
treatment) h r n P-NF-treated S W J mice. Magnification: A) x 640; B) x 880
(Hematoxy lin counterstain).
Figure 8 A, B.
1rnmunohistochemica.I detection of CYP 1 A1 in papillary-type turnours fkom P-NF-treated
SWRU mice. A) CYPlAl was weakly detected in papillary-type adenomas (16 weeks
after urethane treatment). CYPlAl expression was retained in alveolar type II cells
(arrows). B) CYPlAl was absent in papillary-type carcinomas (52 weeks after urethane
treatment). Magnification: A) x 640; B) x 680 (Hematoxylin counterstain).
Figure 9 A, B & C.
Immunohistochemicd detection of CYPlAl in hyperplastic foci within carcinoma-
bearing, P-NF-treated, SWRU mice (52 weeks after urethane treatment). Magnification:
A) x 250; B),x 500; C) x 560 (Hematoxylin counterstain).
Figure 10.
Normal lung tissue &om vehicle-treated S W J mice. NADPH-cytochrome P450
reductase was detected in bronchiolar Clara cells (arrows), and alveolar type I1 cells
(arrowheads). Magmfication: x 500 (Hematoxylin counterstain).
Figure 11 A, B & C.
Neoplastic lung tissue h m vehicle-treated SWWJ mice. A) NADPH-cytochrome P450
reductase was expressed in hyperplastic foci (10 weeks after urethane treatment). B)
Papillary-type adenomas (16 weeks after urethane treatment) demonstrated weak specific
staining for NADPH-cytochrome P450 reductase. C) Solid-type adenomas (16 weeks
after urethane treatment) moderately exhibited the reductase protein. Magnification: A) x
740; B) x 180; C) x 250 (Hematoxylin counterstain: B & C only).
Figure 12 A, B.
Neoplastic lung tissue fiom P-NF-treated SWWJ mice. A) Papillary adenomas (16 weeks
after urethane treatment) expressed the NADPH-cytochrome P450 reductase protein at
low levels. B) Moderate staining of the reductase protein was found in solid-type
adenomas (16 weeks after urethane treatment). Magnification: A) x 180; B) x 200
(Hernatoxylin counterstain: B only).
Figure 13 A, B.
Immunohistochemical detection of NADPH-cytochrome P450 reductase in hyperplastic
foci fiom carcinoma-bearing, P-NF-treated, SWRD mice (52 weeks after urethane
treatment). Magnification: A) x 720; B) x 720 (Hematoxylin counterstain).
4. DISCUSSION
Previous studies of lung tumour promotion and progression indicate that most
tumours arise as hypcrplasias, progress to adenomas, and finally develop into carcinomas
(Foley et al., 199 1; Fotkert et of., 1992). Murine pulmonary hyperplastic nodules are first
detectable at approximately 3 weeks following urethane treatment (Foley et al., 1991).
At 16 to 22 weeks, a minority of these nodules develop into larger adenomas, which
compress mounding tissues. By 30 weeks, carcinomas have developed, demonstrating
nuclear atypia, metastatic invasion and dissemination (Witschi, 1984; Thaete et al.,
1987). In this study, pulmonary neoplasm in S W mice developed sequentially. At 6
- 10 weeks following urethane treatment, foci were grossly visible within the lung as
spherical hyperplasias. By 16 to 20 weeks, larger adenomas were m d e s t e d as either
papillary or solid histological arrangements. Carcinomas developed at 52 weeks, evident
by their enormous size and extensive invasion into surrounding lung tissues. This
sequential pattern of tumour development suggests the progressive growth of neoplastic
tissues, fiom hyperplasia to adenoma and ultimately carcinoma
As pulmonary neoplastic tissues progressed fiom hyperplastic nodules to
adenomas, they consistently developed in two histological patterns. The first pattern
involved a papillary arrangement in which tumours developed as fhger-like, tubular
extensions of non-ciliated columnar cells, surrounded by a pseudocapsule of compressed
tissue. The second pattern involved a solid arrangement, where the tumour depicted a
more focal and compact mass of cuboidal cells, which seemed to lack the extensive
organizational structure seen in papillary-type tumom. Two hypotheses have been
suggested in an attempt to r&olve whether these patterns of turnour development 64
represent growth h m diffe~ie~lt individual cell types, or growth h m a common cell
advancing through progressively inmasing stages of metastatic potential. The first
hypothesis suggests that solid and papillary turnours develop h m alveolar type II cells
and bronchiolar Clara cells, respectively- With fiuther neoplastic development, solid
tumours generally regress, while papiby turnouts are more apt to advance into
carcinomas (KaufEnan et al., 1979; Thaete et al., 1987; Rehm et al., 199 1). The second
hypothesis proposes that while both solid and papillary tumours arise h m alveolar type
II cells, papillary adenomas represent a more advanced stage of hnnour development,
ultimately leading to metastasis (Mostofi and Larsen, 1951; Kimura, 1971; Ward et al.,
1985). Cell-specific enzyme markers such as succinate dehydrogenase and
glyceraldehyde-3phosphate dehydrogenase have been unsuccessful in resolving this
controversy (Gunning et al., 1991; Thaete and Malkjnson, 1991). This study did not
identi@ significant differences in the localization of CYP2B1, CYPlAl or NADPH-
cytochrome P450 reductase between the two tumour types during their course of
development. As such, we conclrlde that CYPZB1, CYPlAl and NADPH-cytochrome
P450 reductase are not useful as markers in debeating the cellular origins of solid or
papillary tumours within murine lung.
A common feature to neoplastic tissues in many species, including mice and
humans, is an altered metabolic profile as compared to surrounding non-neoplastic
tissues. In comparison to non-neoplastic cells, investigators have demonstrated decreased
expression of cytochrome P450 monwxygenases, and their associated metabolic
activities, within neoplasms developing in liver as well as lung (Gerber and Thung, 1979;
Farber, 1984, 1990; Roomi et al., 1985; Forkert et al., 1992, 1996; Botto et al., 1994).
65
Characteristically, decreased levels ofcytochrome P450 monooxygenases in these tumour
tissues have been associated with variable localization of various phase II drug-
metabolizing enzymes, such as glutathione s-transfetases, DT-diaphorases and uridine-
5'-~phosphate-gul~rr)nlyltransfera~e I (Gerber and Thung, 1980; Becker and Stout, 198%
Farbet et al., 1984; Roomi et al., 1985; El Mouelhi et al., 1987). It has been suggested
that these biochemical alterations may confer a survival advantage to the growing tumour
(Farber, 1984). As the levels of drug-metabolizing enzymes in the neoplastic tissues fall,
their biochemical activities are diminished. Consequently, the tumoufs ability to
generate harmful reactive intermediates from environmental xenobiotics, which in turn
may damage or impair the genetic constitution of the tumour itseE, is reduced. In this
way, turnour tissues may survive and proliferate in an eavkoument that is deleterious or
even deadly to surrounding non-neoplastic tissues. This neoplastic resistance has
previously been demonstrated in murine lung tumour tissues (Forkert et al., 1992).
Pneumotoxicants such as 1, l dichloroethylene and naphthalene are known to selectively
damage Clara cells, while paraquat has been demonstrated as a cytotoxicant specifk to
alveolar type II cells in rats and Clara cells in mice (Mahvi et a[., 1977; Forkert and
Reynolds, 1982; Forkert et a[., 1992). Administration of these deleterious chemicals to
murine lungs containing solid and papillary neoplasms was shown to selectiveiy damage
surrounding lung tissues, leaving tumoui tissues relatively unaffected (Forkert et a[.,
1992). These studies demonstrated that cytochrome P450-dependent metabolic activation
of potential pneumotoxicants is diminished in pulmonary tumour tissues.
Whereas Forkert et d., (1992) demonstrated decreased localization of cytochrome
P450 in murine pulmonary neoplasms, the temporal relationship between this decreased 66
metabolic potential and tumour development has not beea previously identified In the
present study, we have examined, by immunohistochemical technique, the localkations
of CW2B I, CYP 1 A1 and NADPH-cytochrome P450 reductase in non-neoplastic lung
tissues and developing pulmonary neoplasms within SWRlJ mice. This
irnrnunohistochemical technique has allowed us to localize these drug-metabolizing
enzymes in correlation with tissue morphology and pathology. In addition, it has
provided a method for semiquantitatively assessing the relative enzymatic profiles, and
hence the relative degrees of xenobiotic resistance, within developing solid and papillary
pulmonary turnours. By identifying the cellular distniution of drug-metabolizing
enzymes in the lung we will better understand their roles in xenobiotic activation,
metabolism and carcinogenesis.
CYP2B1, CYPlAl and NADPH-cytochrome P450 reductase were detected in
non-neoplastic lung tissues of SRWJ mice. In these tissues, CYP2B1 was constitutively
expressed within Clara and alveolar type II cells. The apical l o c ~ t i o n of CYP2BI in
Clara cells is related to the abundant smooth endoplasmic reticulum present in this
region, within which this enzyme resides (Serabjit-Singh et al., 1987; O v d y et al.,
1992b). Localization of this isozyme was not altered following treatment with p-NF, an
agent commonly used to induce CYPlAl. These findings are in agreement with previous
immunohistochemical, electrophoretic and ultrastructural immunolabelling studies that
demonstrate CYP2B 1 to be constitutively expressed in pulmonary tissues across various
species including mouse, rat, rabbit and humans (Serabjit-Singh et al., 1980; 1987;
Deverew et al., I98 1; Baron and Kawabata, 1983; Domin et al., 1986; Plopper et al.,
1987; Forkert et al., 1986; 1989; Pairon et al., 1994; Lee and DinsdaIe, 1995). CYP 1A1,
on the other hand, was not constitutively expressed within untreated tissues. Yet,
following treatment with p-NF, this isoyme was detected in type 11 cells, and more
prominently, in endothelial cells within tissues from both tumouf-bearing and non-
tumour-bearing mice. The inducibility of CYPlAl in endothelial cells and type II cells
has been previously demonstrated in mouse, rat and rabbit (Dees et al., 1980; Forkert et
al., 1986, 1989; Keith et al., 1987; Plopper et ol., 1987; Overby et al., 1992% Pairon et
al., 1994; Lee and Dinsdale, 1995). NADPH-cytochrome P450 reductase was
constitutively expressed within murine non-neoplastic lung tissues. In these tissues, the
reductase was detected in Clara cells and type II cells, but was neither constitutively
expressed, nor induced by P-NF, in vascular endothelial cells. To activate and metabolize
foreign compounds, cytochrome P450 must receive at least the first of two electrons from
NADPH-cytochrome P450 reductase (Wiiams and Kamin, 1962; Phillips and Langdon,
1 962; White and Coon, 1980). Therefore, in order for a cell to be metabolically active, it
must be capable of producing a cytochrome P450 isozyme responsible for activating and
metabolizing a particular substrate, as well as NADPH-cytochrome P450 reductase.
Since murine pulmonary endothelial cells do not contain the reductase enzyme, these
tissues likely play a limited role, if any, in the activation, metabolism and excretion of
CYP 1 A1 -dependent substrates. Other investigators have demonstrated similar patterns of
CYP 1Al and reductase localization within vascular tissues fiom rat, rabbit and mouse
(Dees et al., 1982; Plopper et al., 1987; Overby et al., 1992a; Lee and Dinsdale, 1995).
In this study, innnunohistochemical detection of CYP;?BI, CYPlA1 a d
NADPH-cytochrome P450 reductase in hyperplastic and neoplastic tissues was
performed at all stages of tumour deveiopment. C ' 1 was minimally expressed within
all developing turnour tissues regardless of stage or histological arrangement*
Furthermore, treatment with p-NF failed to influence the localization of this enzyme.
Our findings confirm those of Forkext et al. (1992), which indicated that tumour
formation is associated with diminished CYP2B 1 localization. CYP 1 A1 was not
detectable in any tumourigenic tissues in vehicle-treated mice. Following treatment with
P-NF, CYPlAl was moderately expressed within hyperplastic nodules and solid
adenomas, although to a lesser degree than identified in surrounding tissues. CYPlAl
inducibility was further reduced in papillary adenomas, and absent in all carcinomas.
NADPH-cytochrome P450 reductase was constitutively identified within neoplastic
tissues of untreated mice and was not influenced by treatment with p-NF. In developing
turnours, this protein's staining pattern closely correlated with that of CYP1Al.
Although the reductase protein was moderately detected in hyperplastic nodules and solid
adenomas, localization was diminished in papillary adenomas, and nearly absent in all
carcinomas. This coordinate regulation of CYP 1 A1, together with NADPH-cytochrome
P4SO reductase, was maintained in hyperplastic nodules examined within carcinoma-
bearing lungs (52 wks). In these foci, levels of CYPlAl and NADPH-cytochrome P450
reductase were markedly elevated, and heterogenously distributed within the foci. These
nodules were consistently situated in the lung parenchyma and not associated with
pulmonary ainvays. Furthermore, they comprised of cuboidal cells, and were
morphoiogically similar to alveolar type LI cells. These findings, together with the
correlated IocaIization of CYPlAl and NADPH-cytochrome P450 reductase suggest that
these nodules represent adenomas undergoing regression and fedifferentiation back to a
more phenotypically-normal cell type, notably an alveolar type 11 cell. During this course
of regression, the outermost cells may be the first to undergo redifferentiation. As such,
neoplastic cells in the interior of the tumour may be more advanced and therefore take
longer to redifferentiate to the normal phenotype. This explains the heterogenous
staining patterns of both CYP l A1 and NADPH-cytochme P450 reductase demonstrated
in hyperplastic nodules present within carcinoma-bearing lungs. Evidence from previous
studies of murine pulmonary tumourigenesis support this conclusion. In a number of
genetic strains of mice, including SWWJ, solid tumom were found to be restricted in
growth aad even regressed while papillary tumom continued to grow (Thaete et al.,
1987). Further evidence is derived ftom hepatic models of carcinogenesis, which indicate
that the large majority of hyperplastic foci and neoplastic nodules revert back to a
phenotype that is morphologically similar to the surrounding parenchyma, while only a
few proceed to carcinoma and ultimately metastases (Solt and Farber, 1976; Farber and
Cameron, 1980; Farber, 1 984).
4.1 SUMMARY AND CONCLUSION
This study has demonstrated that SWRD murine pulmonary tumours progress
sequentially from hyperplastic foci to adeaomas, and ultimately carcinomas,
demonstrating a solid or papillary histological arrangement- Previous studies have
determined that tumour development is associated with decreased localization of phase I
drug metabolizing enzymes, such as cytochrome P450. As such, it is suggested that
tumour tissues may be less capable of effectively activating and metabolizing xenobiotics
and chemical compounds for which they may have been previously responsible.
Consequently, the tumour tissue becomes incapable of converting chemical substrates
into reactive intermediates that may in tum damage or destroy the tumour itself. This
neoplastic tissue, with its altered metabolic profile and acquired resistance to harrml
chemical compounds, may be capable of developing and proliferating in an environment
that is harmful or even deadly to the surrounding tissues; thereby, providing tumours with
a potential survival advantage over non-neoplastic tissues.
The immunohistochemical technique performed in this study has enabled us to
identify the cellular location of CYP2B1, CYPlAl and NADPH-cytochrne P450
reductase within non-neoplastic and neoplastic murine lung tissues. Ollr findings support
previous studies, which suggest that bronchiolar Clara cells and alveolar type II cells are
actively involved in drug metabolism within the mouse lung. Furthermore, by providing
a means to semi-quantitatively examine the levels of enzyme localization within
developing tumours, this technique has enabled us to more effectively assess the temporal
relationship between enzyme localization and tumour development In this study, we
have found tumour development to be consistently associated with decreased levels of
7 1
localization of CYP2B1, CYPlA1, and NADPH-cytochrome P450 reductase, when
compared to enzyme levels in surrounding tissues. Furthermore, while CYP2B1 was
absent in all developing tumour tissues, regulation of CYPlAl and NADPH-cytochrome
P450 reductase was highly coasaved throughout tumour development, iilustrating
coordinate localizatio11 during tumour initiation, growth and regression. Localization of
both enzymes decreased in all tumour tissues over the course of development h m
hyperplasias to carcinomas. Interestingly, CYP 1 A 1 and NADPH-cytocbmme P450
reductase were strongly, and hetmgenously, expressed in hyperplastic foci present in
carcinoma-bearing lungs. Due to the consistent location of these foci within the lung
parenchyma, together with their morphological and irnmunohistochemical resemblance to
alveolar type I1 cells, we have concluded that these hyperplastic nodules represent
previously formed adenomas regressing and redifferentiating back to alveolar type I1
cells.
The relative levels of cytochrome P450-dependant drug metabolizing enzymes in
developing tumour tissues may directly relate to the metabolic capabilities of these
neoplastic tissues. If so, the imrnunohistocbemical technique is invaluable in effectively
demonstrating the temporal relationship between tumour development and increased
xenobiotic resistance in murine pulmonary tissues.
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