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MSU I. An Affirmative Action/Equal Opportunity Institution
_ amp-nag:

THE EFFECT OF THE HOPS CONSTITUENT COLUPULONE
ON HEPATIC CYTOCHROME P450
IN THE MALE RAT

BY
Elizabeth Brownell Shipp

A-Thesis

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

Master of Science

Department of Food Science and Human Nutrition

1992

A

/77O

4

702-

ABSTRACT

The Effect of the Hops Constituent Colupulone
on Hepatic Cytochrome P450
in the Male Rat

BY

Elizabeth Brownell Shipp

The cytochrome P450 enzyme superfamily metabolizes a
number of xenobiotics. Some of these are carcinogenic,
either in their native state or after activation by
cytochrome P450 isozymes. However, a number of these
xenobiotics are detoxified by other cytochrome P450
isozymes. The possibility exists that cytochrome P450
metabolism could be manipulated to increasethe

detoxification of food-borne carcinogens and procarcinogens.

To determine the ability of a hops constituent to alter
cytochrome P450 metabolism, colupulone, one of the
components of hops, was added to purified diet and fed to
rats for a period of five days. Microsomal cytochrome P450
concentrations, metabolic activities, and steady-state RNA
abundance for three cytochrome P450 isozymes were determined
in control and treated animals. Colupulone treatment
produced an increase in RHA abundance for two of the
isozymes of interest. Total cytochrome P450 content and
microsomal cytochrome P450 metabolism did not differ between

treated and control animals.

This thesis is dedicated to Dr. Kathryn Grove Shipp

and to Mrs. Emily McPherson, for all of their inspiration.

iii

ACKNOWLEDGEMENTS

I would most of all like to thank my thesis advisor,
Dr. Bill Helferich, for his help and understanding during my
Master's degree program. I also want to thank the other
members of my committee, Dr. Bursian and Dr. Gray, for their
guidance and support. I owe a large debt to Dr. Christine
S. Mehigh for her generous help with RNA isolation
techniques and with the Northern blot hybridizations. My
thanks also go to Dr. Denison for his willingness to share
one of his figures for use in this manuscript. I would like
to thank Kalsec Inc. for their generosity and support for
this research. I appreciate the help and friendship I have
received from the other students in Dr. Helferich's lab, and
from the rest of the students in the department. I would
also like to thank the Department of Food Science and Human

Nutrition for their assistance during my program.

iv

TABLE OF CONTENTS

List Of Tables 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O Vii
List Of Figures O O O O O O O O O O O O O O O O O O O O O 0 O O O O O O O O O O O O O O O O O O O O O Viii
List of Abbreviations..... ..... ...........................ix

Introauction.OOOOOOOOOOOOOOOOOOOOO0000......00.0.0000000001
Environmental Factors Affecting Human Health.........1

Diet.0..OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOZ

Diet and cancerOOOO0.0.0....0.0.00.00.00.0000000000003

Literature Review.........................................6
Cytochrome P450......................................6
Discovery and Isolation of Cytochrome P450........6
Induction and Repression of Cytochrome P450.......15

Role of Cytochrome P450 in Xenobiotic
Detoxification and Activation...................22

Mutagenesis and Carcinogenesis.......................25
Chemically Mediated Mutation......................25
Mutation in Carcinogenesis.... ........ ............26

Hops componentSOOOOOOO0.0.0.0... 0000000000000 0.0.0.000000029

Methods and Materials... ........ . ............ .............32
Chemicals............................................32
Animals..............................................32

Diets...........................................33
Treatments......................................33
Tissue Collection...............................34
Microsomal Assays....................................34
Microsome Preparation...........................34
Protein and Cytochrome P450 Determination.......34
Substrate Metabolism Assays.....................36
RNA Isolation and Analysis...........................37
RNA Isolation...................................37
RNA Gel Electrophoresis.........................38
Northern Blotting...............................39
Northern Hybridization..........................40
Statistical Analysis.............. ..... ..............4l

Results and Discussion....................................43
Feed Intake and Daily Body Weight....................43
Final Body Weight and Liver Weight...................43
Microsomal Protein and Cytochrome P450 Isolation.....44
Aminopyrine and Erythromycin

N-Demethylase Activities........................44
Northern Blot Hybridization..........................45

summary and conCIUSionSOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.057

List of References ...... . ................... . ...... . ..... .59

vi

Table

Table

Table

Table

Table

1.

2.

3.

LIST OF TABLES

The occurrence of cytochrome P450 isozymes
in a few different tissues and species..........11

Nomenclature and organization of cytochrome
P450 isozymes in higher organisms (Nebert,
at al., 1991)0.0.00.00...OOOOOOOOOOOOO...O 000000 16

Initial body weights, final body weights,

liver weights, and relative liver weights in

rats treated for five days with colupulone or

for four days with PCN..........................51

The effect in the rat of administration of
colupulone for five days or PCN for four days

on hepatic microsomal protein and cytochrome

P450 concentrations.............................52

The effect in the rat of administration of
colupulone for five days or PCN for four days

on hepatic microsomal aminopyrine

N-demethylase and erythromycin N-demethylase
activities......................................53

vii

Fi

Fig

 

 

 

 

 

Jim},

 

Figure

Figure

Figure

Figure

Figure

Figure

Figure

LIST OF FIGURES

The induction of cytochrome P450 proteins by
polycyclic aromatic hydrocarbons as mediated

by the Aromatic Hydrocarbon Receptor

(Denison, 1992)................................19

The structures of three hops B-acids:
a) colupulone; b) lupulone; c) adlupulone
(Mannering et a1., 1992).......................30

Average grams of feed eaten per animal per

day prior to and during colupulone and PCN
administration (error bars represent standard
error of the mean) with treatment beginning

at 10:30 AM on Day 0...........................49

Average body weight in grams of each animal

each day prior to and during colupulone and

PCN administration (error bars represent

standard error of the mean) with treatment
beginning at 10:30 AM on Day 0.................50

Northern blot hybridization of cytochrome
P4SOIA2 mouse cDNA to RNA from livers of
untreated, colupulone- and PCN-treated rats....54

Northern blot hybridization of cytochrome
P4SOIIB1 rat cDNA to RNA from livers of
untreated, colupulone-and PCN-treated rats ..... 55

Northern blot hybridization of cytochrome

P4SOIIIA4 human cDNA to RNA from livers of
untreated, colupulone- and PCN-treated rats....56

viii

LIST OF ABBREVIATIONS

AFB1'-- aflatoxin B,

AFM1 —-- aflatoxin M1

AFQ1 --- aflatoxin Q1

AIA --- aminoimidoazaarene

AIN 76 --- American Institute of Nutrition 1976 diet

ATCC --- American Type Culture Collection

B[a]P --- benzo[a]pyrene

BPDE --- benzo[a]pyrene diol epoxide

EDTA --- ethylenediaminetetraacetic acid

HEPES --- N[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic
acid

3-MAB --- 3-methyl-4-monomethylaminobenzene

MAE --- MOPS, sodium acetate, EDTA

3-MC --- 3-methylcholanthrene

MOPS --- 3-[N-morpholino]propanesulfonic acid

NADP -—- nicotinamide adenine dinucleotide phosphate

PB --- phenobarbital

PCB --- polychlorinated biphenyl

PCN --- pregnenolone-léa-carbonitrile

a-32P-dCTP --- a-32phosphorus-deoxycytidine triphosphate

PhIP --- 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

SDS --- sodium dodecyl sulfate

SSC --- sodium chloride, sodium citrate

TCDD --- 2,3,7,8-tetrachlorodibenzo-p-dioxin

ix

INTRODUCTION

W

A number of factors in the environment are capable of
impacting human health, either beneficially or
detrimentally. Contaminants in the environment can have
either acute or chronic effects. One class of these
contaminants is the polychlorinated biphenyls (PCBs). These
were once used in electrical transformers, paints, and in
non-carbon copy paper. PCBs can enter the environment
through improper disposal of contaminated oil or paint, from
factory waste-water, and aerosolization of the PCBs
themselves (Tatsukawa, 1976). PCBs cause chronic liver
damage, skin pigmentation and irritation, and may be
carcinogenic. Another example is methylmercury, which is
used in a number of manufacturing industries, including
paint and pharmaceutical formulation, as well as in
dentistry. Methylmercury is released in wastes and in
industrial accidents, as well as from mining of mercury
itself. This neurotoxin can have both acute and long-
lasting effects and can also produce birth defects after

prenatal exposure.

The diet is also able to affect the status of human

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health. Long-term overnutrition can lead to several chronic
diseases, including heart disease, high blood pressure, and
obesity. A chronic intake of excess calories may also be
linked to the development of cancer. Many components of the
food supply have been demonstrated to be rodent carcinogens.
These include natural components of food, such as caffeine
and many other compounds in coffee (Ames and Gold, 1990);
cooking by-products such as benzo[a]pyrene (B[a]P) and the
aminoimidoazaarenes (AIAs); and food contaminants such as
the mycotoxin aflatoxin 31 (AFBI). Recent research has
focused on the potential for dietary components to aid in
the prevention of cancer (0mmen, et a1., 1988; Santamaria,
et a1., 1988; von Hofe, et a1., 1991; DeWys, et al., 1986)).
These may either alter the metabolic activation of
carcinogens or interfere with the ability of carcinogens to

damage cells.

Dis;
A high fat intake has been conclusively linked to a

number of chronic health conditions. The development of
arterial plaques during arteriosclerosis can lead to high
blood pressure, weakening of the heart muscle, and lack of
sufficient blood supply to major organs, including the heart
itself. High calorie and high fat diets additionally
provide more nutrition than is needed for energy
maintenance. This extra energy is stored in the adipose

cells, leading to significant weight gains over time. The

3
added body mass requires extra work by the heart and may be
another cause of heart disease. However, nutrition in
general has been linked to other chronic diseases as well.
Laboratory animals fed high-calorie diets developed more
tumors in response to one dose of diethylnitrosamine than
did their counterparts whose caloric intake was restricted
(Lagopoulos and Stalder, 1987). This would imply that
general overnutrition can lead to the development of cancer.
In similar experiments, animals maintained on an ad libitum
(ad lib), high-fat diet developed more spontaneous tumors

than animals fed an ad lib low-fat diet (Reddy, 1986).

Wee:

A number of specific or potential carcinogens have been
identified in the human diet. Heterocyclic amines,
especially common in cooked meats, have been shown to cause
a wide variety of cancers in laboratory animals (Sugimura,
1985). Another food-borne carcinogen is AFBH,.a mycotoxin
found in many grain crops and products (thter and Krfihm,

1988; Dvorackova, 1990).

Some dietary carcinogens are capable of damaging DNA in
their native state. These direct-acting carcinogens need no
metabolic activation after they are ingested. Vinyl
chloride is one compound that falls into this category.
Indirect-acting carcinogens, or procarcinogens, must be

chemically altered before they are mutagenic. Two examples

4
of these chemicals are AFB1 and Bta]P. Metabolic activation
or detoxification of these and other compounds is often
carried out by the hepatic cytochrome P450 enzyme system.
A great deal has been discovered about this group of enzymes
since its activity was first observed in 1954. The
information developed since then has demonstrated the‘
abundance and variety of isozymes in the cytochrome P450
superfamily. In humans, for example, twenty-four genes and
three pseudogenes have been classified into ten enzyme
families based on gene and protein sequence data (Nebert et
a1., 1991). Both endogenous and exogenous xenobiotic
substrates have been identified for a number of the isozymes
characterized. Most xenobiotics metabolized by the
cytochrome P450 system are detoxified by this metabolism.
0n the other hand, some substrates are activated to more
reactive and mutagenic forms by cytochrome P450-mediated
reactions. Most cytochrome P450 isozymes can be induced or
inhibited by chemicals in the diet or in the environment,
thereby altering the abundance and metabolic activity of
specific isozymes, as well as the activation and
detoxification of xenobiotics. This alteration of
cytochrome P450 metabolism may be of benefit to human
health, as modifying the human diet to include more of the
compounds able to increase detoxification of xenobiotics may
help to decrease the risk posed by food-borne or
environmental procarcinogens. One group of compounds that

may be useful in altering xenobiotic metabolism is the B-

5
acids found in hops. These have been shown to induce
certain cytochrome P450 isozymes in the mouse (Mannering et

a1., 1992).

The objectives of this study were to evaluate the
inductive effect of dietary colupulone, one of the hops B-

acids, on hepatic cytochrome P450 metabolism in the rat.

LITERATURE REVIEW

Q!!QQEBQE£.2£§Q

W

The hepatic xenobiotic-metabolizing enzyme system first
received attention in the mid-19503. Brown et a1. (1954)
indicated that the type of diet fed to experimental animals
could affect the enzyme activity observed in liver
homogenates. These studies were an outgrowth of a series of
experiments on the metabolism of carcinogenic dyes. The N-
demethylation of 3-methyl-4-monomethylaminoazobenzene (3-
MAB), a carcinogenic aminoazo dye, was used as an indicator
of xenobiotic metabolism in rats and mice. Altering the
diet from a purified chemical diet to a commercially
available chow produced striking increases in N-demethylase
activity. The factor suggested as the cause of this
increase was sterols formed in the commercial diet during
storage. Since that time, a number of components naturally
present in the diet (rather than processing by-products)
have been shown to alter xenobiotic metabolism, including
dietary protein (Butler and Dauterman, 1988), corn oil (Yoo
et a1., 1990), carbohydrate, vitamins A and C, and some

minerals (Yang et a1., 1992).

In another series of experiments, liver homogenates
from rats administered 3-methylcholanthrene (3-MC, a
polycyclic aromatic hydrocarbon) by intraperitoneal
injection possessed increased aminoazo dye N-demethylase
activity (Conney et a1., 1956). The specific enzyme
responsible for this N-demethylation had not yet been
identified. It was also not clear how 3-MC could produce
the increase in demethylase activity. Combining heat-
treated homogenates possessing no enzymatic activity with
preparations from 3-MC treated rats did not change the
demethylase activity seen in the 3-MC homogenates. This
result ruled out the possibility that the liver homogenate
itself contained a soluble effector of enzyme activity

(Conney et a1., 1956, 1957).

It was then hypothesized that the inducer, such as 3-
MC, might be increasing (inducing) N-demethylase activity by
activating previously existing enzyme molecules. If this
were the case, then both in vitro and in vivo addition of
the inducer should produce the same effect. To test this,
known N-demethylase inducers such as phenobarbital (PB),
benzo[a]pyrene (B[a]P), and 3-MC were added to the
homogenate. No inductive response was seen in these in
vitro experiments (Conney and Burns, 1959; Conney et a1.,
1956; Conney et a1., 1957; Conney and Burns, 1963; Conney,

1967), demonstrating that induction was an in vivo process.

8

Another avenue of research investigated the effect of
endogenous hormones on induction of cytochrome P450 by
xenobiotics. Adrenalectomy, hypophysectomy (Conney et a1.,
1956), or ovariectomy (Conney, 1967) did not affect
induction by 3-MC. Similarly, the response to PB treatment
was not altered by adrenalectomy and castration,
hypophysectomy, or thyroidectomy (Conney, 1967). Thus,
enzyme induction by these xenobiotics did not depend on the

hormonal status of the animals.

These experiments demonstrated that enzyme induction
was not due to allosteric effects by soluble compounds, or
to activation of pre-existing enzyme molecules. Hormonal
status also did not have a role in enzyme induction by
xenobiotics. It was therefore hypothesized that induction
was the result of increased protein synthesis. This was
first tested by using ethionine, an amino acid antagonist
and protein synthesis inhibitor. Administration of
ethionine in vivo before treatment with an inducer prevented
any increase in enzyme activity (Conney and Burns, 1959;
Conney et a1., 1957; Conney et a1., 1956, Conney and Burns,
1963). Adding ethionine to liver homogenates from 3-MC
treated animals had no effect on enzyme activity (Conney et
a1., 1956). This confirmed that the inducer acted before
the homogenate was prepared. Animals treated with ethionine
a short time after 3-MC administration demonstrated a higher

N-demethylase activity than animals given ethionine and 3-MC

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9
simultaneously. However, they possessed enzyme activities
lower than did animals treated with 3-MC alone (Conney et
a1., 1956). These results supported the hypothesis that
inducers such as 3-MC and PB increased enzyme activity by
increasing protein synthesis and thus enzyme abundance.
Radiolabelling studies using 1"'C-leucine given in vivo after
treatment with PB confirmed this (Kato et a1., 1965).
Incorporation of 1"C-leucine doubled in liver microsomes
after PB treatment, but did not change in other subcellular
fractions. In vivo treatment with PB also increased
endoplasmic reticulum (ER) volume and total protein in liver

cells (Remmer and Merker, 1965).

It was observed that bubbling carbon monoxide through a
reduced microsomal suspension resulted in the formation of a
spectral absorption peak at 450 nm (Klingenberg, 1958).

This peak was not due to the cytochrome bs already
identified in microsomal suspensions. Dissociation
experiments with the carbon monoxide-complexed hemeprotein
showed that it was not myoglobin or cytochrome b5. This new
carbon monoxide-binding pigment was a hemeprotein with a
spectral absorption maximum at 450 nm (Omura and Sato,
1964). Like other cytochromes, the main function of the new
hemeprotein was electron transport to oxygen from an NADPH-
linked reductase, activating the oxygen to allow its
addition to a substrate molecule. The protein was therefore

labelled cytochrome P450.

10

Cytochrome P450 was originally found in the greatest
concentration in liver microsomes, and has since been
located in many tissues throughout the body in lower
concentrations. Many of the xenobiotics that are
metabolized by cytochrome P450 enter the body in the diet,
either from plant foods or dietary contaminants.
Localization of the primary cytochrome P450 activity in the
liver would allow these compounds to be metabolized close to
the site of first exposure. However, the presence of
specific cytochrome P450 isozymes in the epithelial tissue
of the lung and in the skin can also serve an important
protective metabolic function by facilitating detoxification
of toxicants inhaled or absorbed through the skin. The
distribution of cytochrome P450 within a few different
tissues and species is listed in Table 1. The
identification of cytochrome P450 in adrenal cortex
microsomes led to«one of the earliest suggestions of a role
for cytochrome P450, in the metabolism of endogenous
compounds such as steroids. The in vitro steroid C-21
hydroxylase activity in these microsomes was inhibited by
carbon monoxide. This inhibition could then be reversed by
light at 450 nm (Omura and Sato, 1964). These results
confirmed the involvement of cytochrome P450 in steroid

metabolism.

In further experiments on the function and inducibility

of cytochrome P450, Remmer and Merker (1965) observed that

11

Table 1. The occurrence of cytochrome P450 isozymes in
a few different tissues and species.

 

Organism Tissue Reference
Cow Adrenocortical Tsubaki et a1.,
Mitochondria 1987

Herring Gull Liver Peakall et a1.,
1986

Human Granulocytes Mungikar and

Gothoskar, 1986
Placental Pasanes and
Mitochondria Pelkonen, 1986

Pig Kidney Bergman and
Postlind, 1990

Rabbit Lung, Liver Parandoosh et a1.,
1987

Rat Brain Walther et a1.,
1986

Avocado Mesocarp Bozak et a1., 1990

Soybean Entire bean Kochs and
Griseback, 1989

nzgsgpnila Entire animal Sundseth et a1.,
1990

Streptomyggg Entire culture O'Keefe et a1.,
1988

xgntngbagtg; Entire culture Warburton et a1.,

1990

 

12
in vivo administration of PB increased the magnitude of the
450 nm absorption peak in reduced and carbon monoxide-
treated microsomes. This increase in the 450 nm peak was
accompanied by an increase in both in vitro and in vivo
barbiturate metabolism. Thus the elevated cytochrome P450
content corresponded to the induction of enzymes involved in
drug metabolism. This was the first observation linking
microsomal cytochrome P450 to drug oxidation and metabolism.
The use of difference spectra and photodissociation of
carbon monoxide complexes in microsomal preparations
demonstrated that cytochrome P450 was the final oxidase in
the drug/xenobiotic-metabolizing enzyme systems (Cooper et

a1., 1965).

Because administration of an inducer produced an
increased 450 nm peak and simultaneous increases in various
enzyme activities, the cytochrome P450 system was originally
thought to be one enzyme capable of several different
reactions. However, the existence of more than one form of
the N-demethylase enzyme was eventually hypothesized.
Intraperitoneal administration of B[a]P increased some
enzyme activities while having little or no effect on others
(Conney et a1., 1959). N-Demethylation of 3-MAB was
inhibited by SKF 525A, a very specific, very potent
demethylase inhibitor, in microsomes from untreated and PB-
treated rats. In microsomes from 3-MC treated rats, this

same inhibitor had no effect on N-demethylase activity.

13
This suggested that the enzyme induced by 3-MC was different
from that induced by PB (Sladek and Mannering, 1966).
Similar inhibition was observed using 7,8-benzoflavone to
inhibit metabolic activity in microsomes from untreated, 3-
MC-treated, or PB-treated animals (Wiebel et a1., 1971),
further suggesting the existence of at least two cytochrome
P450 isozymes. Parke (1975) further verified the existence
of multiple forms of cytochrome P450 when a spectral
absorption profile for 3-MC microsomes had an absorption
peak at 448 nm, rather than 450 nm. This 448 nm peak was
not due to formation of a complex between 3-MC and the
normal cytochrome P450 molecule (Fujita et a1., 1973). The
second hemeprotein was labelled cytochrome P448. When the
two proteins were purified and characterized, they were
found to have different substrate specificities (Parke,
1975). This finding confirmed that cytochrome P448 and

cytochrome P450 were two different proteins.

The question still remained of exactly how the two were
different from each other. To investigate this, inducers
could be used to selectively induce cytochromes P450 with
differing substrate specificities and enzymatic activities.
These purified forms could then be analyzed to determine
their relationships to each other. Botelho et a1. (1979)
showed that at least three forms of cytochrome P450 were
induced by Aroclor 1254 (a polycyclic aromatic hydrocarbon).

This inducer is actually a mixture of many different

14
polychlorinated biphenyl congeners, each with a different
pattern of chlorination on the parent molecule, but
averaging 54% chlorine. The congeners have differing
inductive abilities, explaining the induction by Aroclor
1254 of more than one form of cytochrome P450. These
studies showed that the induced isoforms were different from
each other on the basis of total amino acid composition and
partial amino acid sequence data. The differences in amino
acid sequence among the three proteins demonstrated that the
proteins were the products of different genes rather than
alternate splicing of thestNA transcript or post-
translational modification of the protein itself.

With the evidence from these and similar studies, Coon
and Persson (1980) suggested that there were only five or
six forms of cytochrome P450. They further hypothesized
that inducers altered substrate specificity and
regioselectivity of the enzymes toward the substrate. For
example, one inducer would stimulate production of one group
of metabolites from a given substrate, while another set of
metabolites would be seen after treatment with another
inducer. It was suggested that the inducer somehow changed
the site of metabolism on the substrate, without altering
the type of cytochrome P450 enzyme present. It has since
been demonstrated that there are many different forms of
cytochrome P450, and that inducers alter the type of
metabolites produced from a given substrate by altering the

15
type of number of cytochrome P450 isozymes present.
Research has focused on characterizing new forms of
cytochrome P450 by means of their substrate specificity,
their relationship to previously identified forms in amino
acid sequence and composition, and their constitutive and
induced levels of expression. The genes coding for a number
of the cytochrome P450 isozymes have also been identified
and mapped to specific locations of the chromosome. Using
these new approaches, more than one hundred and fifty
distinct forms of cytochrome P450 have been characterized in
a number of species and tissues (Coon et a1., 1992). The
isozymes identified to date have been organized within the
cytochrome P450 superfamily on the basis of protein and gene
sequence data. The organization developed for cytochrome
P450 isozymes is listed in Table 2 (Nebert et a1., 1991).
The nomenclature suggested in that review will be used for

the remainder of this discussion.

16

 

Table 2. Nomenclature and organization of cytochrome P450
isozymes in higher animals. (Nebert et a1., 1991)
Organism Family Subfamily Genes
Rat I A 1,2
II A 1,2,3
B 1,2,3,8
c 6,6p*,7,11,12,
13,22,23
D 1,2,3,4,5
E 1
G 1
III A 1,2,9
IV A 1,2,3,8
B 1
VII
XI A l
B 1,3
XVII
XIX
XXVII
Human I A 1,2
11 A 6,7
B an?
C 8,9,10,17,18,19
D 6,7 ,8P
E 1
F 1
III A 3,4,5,7
IV A 9
B 1
VII
XI A 1
B 1,2
XVII
XIX
xxx A 113*,2
XXVII
Rabbit I A 1,2
II B 4,4p‘,5
c 1,2,3,4,5,14,
5,16
E 1,2
G 1
III A 6

 

'P indicates a pseudogene present in the designated family
and subfamily.

Table 2 (cont.)

17

 

Organism Family Subfamily Genes
Rabbit (cont.) IV A 4,5,6,7
B 1
VII
XVII
Trout I A 1
Dog I A 1,2
II B 11
C 21
Monkey I A 1
II C 20
E 1
III A 8
Mouse I A 1,2
II A 4,5
B 9,10
D 9,10,11,12,13
E 1
XVII
XXI A 1,2
Hamster I A 2
II A 8,9
III A 10
Chicken II H 1,2
XVII
XIX
Cow VII
XI A 1
B 1
XVII
XXI A 1
Pig XI A 1
XVII
XXI A 1

 

18

WW

Enzyme induction is defined (Gelboin, 1967) as a
process that increases the rate of synthesis of new enzyme.
Similarly, enzyme repression decreases the rate of synthesis
below the usual rate. Several mechanisms exist that explain
how inducers may affect cytochrome P450 synthesis (Conney,
1967). The inducer may bind to a receptor that then
interacts with the DNA to increase transcription from the
cytochrome P450 gene. It may interact antagonistically with
repressors made by regulator genes, or may alter histone
structure around the DNA. The former would prevent down-
regulation of the gene, allowing increased transcription to
occur, while the latter would make the DNA more accessible
to the transcription complex. The inducer may act at the
endoplasmic reticulum to increase protein translation from
the mRNA on the ribosomes. One example of the processes
involved in induction of a cytochrome P450 isozyme is the
well-studied induction of cytochrome P4SOIA1 by the potent
inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The
mechanisms and pathways of induction by TCDD are shown in
Figure 1 (Denison, 1992). The polycyclic aromatic
hydrocarbon ligand, such as TCDD, binds to the cytoplasmic
receptor, causing receptor transformation and translocation
into the nucleus. Here, the ligand-receptor complex binds
to an enhancer sequence upstream of the cytochrome P4SOIA1
gene and increases mRNA transcription from the gene. The

mRNA is exported to the cytoplasm, where the increased mRNA

19

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Detoxification
and Activatio

i
[New PoiypeptidgHIt-anslation 1

 

 

 

 

7 Increased Cytochrome
P-‘isolAl
l
J

 

Figure 1. The induction of cytochrome P450 proteins by
polycyclic aromatic hydrocarbons as mediated by
the aromatic hydrocarbon receptor (Denison,
1992).

20
abundance produces increased translation of the message into
cytochrome P4501A1 protein at the ribosomes on the
endoplasmic reticulum. The mechanism of induction most
frequently seen is an increase in transcription of the gene
in question (Okey, 1989; Bock et a1., 1990). Cytochrome
P4SOIIB induction in response to PB (Adesnik et a1., 1981)
and methylenedioxybenzenes (Marcus et a1., 1990) is the
result of both increased gene transcription rates and
increased protein synthesis rates. 3-MC induces cytochrome
P4SOIA by increasing the processing rate of the mRNA (Silver
and Krauter, 1990). Pyridine induces the cytochrome P4501A
subfamily by increasing transcription rates (Kim et a1.,
1991). All these mechanisms of induction lead to an

increase in at least one cytochrome P450 isozyme.

Induction of one cytochrome P450 isozyme may also be
accompanied by a decrease or stabilization in the levels of
other isozymes (Harada and Omura, 1981). This can be seen in
the large increase of one isozyme, such as the induction of,
cytochrome P450118 by PB, with a smaller increase in total
cytochrome P450. This is also seen in treatment with
aminoazotoluene, where cytochrome P450IA increases ten-fold
and cytochrome P4501IB increases five-fold by twenty-four
hours after treatment, while total cytochrome P450
concentration is only slightly increased (Tagashira et a1.,

1985).

21

Several compounds that are inducers of cytochrome P450
are also substrates for the isozyme they induce. Some of
the compounds in this group are toluene (Okey, 1989), B[a]P
(Conney et a1., 1957), and naphthoflavones (Juchau, 1990).
Increasing the metabolism of a toxic compound to less toxic
products would reduce the toxicity of the chemical.
Selective induction of the cytochrome P450 isozyme
responsible for detoxifying a specific xenobiotic is an
ideal method for increasing the excretion of the foreign
compound. The oxidative reactions catalyzed by cytochrome
P450 tend to detoxify foreign compounds and increase their
excretion. However, they also have the potential to
activate xenobiotics and make them more reactive towards DNA
and proteins. The activation and detoxification of
xenobiotics such as B[a]P by cytochrome P450 metabolism can
often be simultaneous reactions whose outcomes depend on the
induction state of the animal. Anything that changes the
abundance of cytochrome P450 isozymes may alter the
detoxification or activation of their substrates and thus
have dramatic effects on their toxicity. In addition,
inducing one cytochrome P450 isozyme to increase the
detoxification of a specific xenobiotic may actually
increase the activation of another foreign compound by the
same isozyme. The decrease or stabilization in other
cytochrome P450 isozymes that can accompany specific
induction could also result in a lack of detoxification of

some xenobiotics by the repressed or non-induced isozymes.

Three primary outcomes of cytochrome P450-mediated
reactions have been suggested (Ryan and Levin, 1989). The
first is the metabolism of endogenous substrates, such as
the steroid hormones by cytochrome P4SOIIIA and the fatty
acids by cytochrome P450IVA (Juchau, 1990). Another is the
detoxification of lipophilic xenobiotics by hydroxylating
them to more polar and less toxic metabolites. A closely
related third result is the activation of some xenobiotics,
such as B[a]P or AFB" to more toxic products by these same

hydroxylation reactions.

Cytochrome P450 induction can be accompanied by the
induction of several conjugating (Phase II) enzymes (Bock et
a1., 1990).~ These enzymes add endogenous, highly polar
compounds such as glutathione to reactive sites on
xenobiotic substrates. This addition makes the xenobiotics
more polar and more easily excreted. The ability of these
Phase II enzymes to be induced by a foreign compound would
be advantageous in reducing the toxicity of xenobiotics.
Both cytochrome P450 (Phase I) and Phase II reactions are
important in the metabolism of foreign compounds. However,
further discussion of Phase II enzymes is beyond the scope

of this discussion.

One well-studied xenobiotic metabolized by cytochrome

23
P450 is AFB,. This mycotoxin is produced primarily by
Aspergillus flavus on grain crops, either in the field or in
storage (Palmgren and Ciegler, 1983). To exert its toxic or
mutagenic effects, AFB, must undergo cytochrome P450
metabolism (Shimada et a1., 1987). The ultimate mutagenic
potential of the metabolites is determined by the site of
metabolism on the aflatoxin molecule. This in turn is
determined by the relative abundance of specific cytochrome
P450 isozymes. The most mutagenic product is AFB,-2,3-
oxide. Studies using human liver microsomes have shown that
this metabolite is primarily produced by cytochrome
P4SOIIIA4 (Shimada and Guengerich, 1989; Aoyama et a1.,
1990; Guengerich et a1., 1991). However, antibodies to this
isozyme only inhibited the activation of AFB, by 65% in
human liver microsomes (Aoyama et a1., 1990). This
indicates that other cytochrome P450 isozymes are involved
in activating AFB,. One of these forms has been identified

as cytochrome P450IIB1 (Doehmer et a1., 1988).

Cytochrome P450 is also responsible for the
detoxification of AFB,. The 4-hydroxylation of AFB, to form
AFM, has been assigned to cytochrome P450IA2 on the basis of
cDNA expression and activation studies (Koser et a1., 1987;
Faletto et a1., 1988; Koser et a1. , 1988) . AFM, is markedly
less mutagenic in the Ames assay, possessing only about 3%
of the mutagenicity of its parent compound, AFB, (Wong and
Hsieh, 1976). In the rat, induction of the cytochrome

24
P450IIIA2 isozyme has been shown to increase the 9-
hydroxylation of AFB, to AFQ, (Halvorson et a1. , 1988) ,
which has only 1% of the mutagenicity of the parent (Wong
and Hsieh, 1976). Induction of either cytochrome P4501A2 or
of cytochrome P4SOIIIA2 would thus shift the metabolism of
AFB, in the direction of the less mutagenic hydroxylated
products that can also be easily excreted. If the presence
of AFB, in food were confirmed, then supplementation of the
diet with some inducer of either of these two isozymes could
potentially increase the detoxification of AFB,, thereby

reducing the risk of mutagenesis posed by the mycotoxin.

 

AFB,-2,3-oxide, produced by metabolic activity of
cytochrome P450, binds covalently to macromolecules such as
DNA or proteins. The DNA adduct formed most often by AFBf-
2,3-oxide is AFBrdVFguanine. A number of other chemicals
can also form DNA adducts after activation by cytochrome
P450. One of these is B[a]P, which is activated to B[a]P-

diol epoxide (BPDE).

DNA-xenobiotic adduct formation can destabilize the
structure of the affected nucleosides (Moran and Ebisuzaki,
1991), and lead to their rearrangement or depurination.
Neither the adduct formation nor the alteration in
nucleoside structure is in itself a mutation. Mutation, a
permanent change in DNA sequence, will only occur if the
lesion is not repaired correctly before DNA replication
occurs. If repair does not occur, the damaged area will be
unable to serve as a template for replication. An incorrect
base may be substituted at the lesion during replication,

producing a permanent mutation.

25

26
H ! Ii . g i .

If a lesion is left unrepaired in non-replicating DNA,
the persistent damage will not produce a mutation. It has
been demonstrated that unrepaired DNA damage cannot initiate
carcinogenesis without cell proliferation, which is
necessarily accompanied by DNA replication and mutation at

the damaged sites (Farber, 1982).

A mutation may have no effect on the cell's physiology
or function, or it may prove lethal. Between these two
extremes, however, the mutation may give the cell a
selection advantage when responding to a growth stimulus.
This increased growth ability, initiation, is seen as the
first step in chemical carcinogenesis (Farber, 1982).
Environmental factors termed promoters allow clonal
expansion of the initiated cell. This then increases the
number of cells that can be initiated a second time (Farber,
1982). The cycle of initiation and promotion may be
repeated more than once, each time allowing the cell to
acquire new properties (Scherer, 1989). The number of
mutational events required for the development of cancer
from a progenitor cell is not currently known, although it
has been suggested that more than one is necessary (Scherer,
1989). As the initiated cells respond to promotion and then
continue to proliferate independently, the genome may
undergo more drastic changes such as chromosomal

rearrangement or deletion. This can lead to growth

27
deregulation and transformation to a neoplastic state. This

stage of carcinogenesis is termed progression.

All of these steps are regulated to some extent by
environmental factors. Initiation can be affected by the
dose and type of carcinogen. If the xenobiotic is a
promutagen requiring cytochrome P450-mediated activation,
the environment can affect mutation rates by altering the
abundance of specific cytochrome P450 isozymes responsible
for this activation. This would then change the activation
of the chemical to its DNA-binding form. Initiation by
mutation may occur repeatedly due to continued exposure to
the same carcinogen, or to others. After initiation, either
exogenous or endogenous factors may act as promoters and
select for clonal expansion of the initiated cells (Farber,
1982). This expansion can lead to further initiation by
environmental compounds (Farber, 1982; Scherer, 1989). This
series of changes can finally lead to the removal of the
cell from normal growth controls, allowing its progression

into neoplasia.

Because the environment can have such a large impact on
chemical carcinogenesis, a major area of research is focused
on modifying the environment to decrease cancer risks. One
avenue of risk reduction is through dietary modification.
The goals of altering the diet would be to both reduce the

intake of carcinogens and to increase the consumption of

28
compounds able to favorably alter xenobiotic metabolism.
These alterations could include either increasing
detoxification of activated carcinogens or decreasing

activation of procarcinogens.

HOPS CONSTITUENTS

One group of compounds that may have potential for
altering cytochrome P450-mediated activation and
detoxification of xenobiotics are the B-acids found in hops.
The structures of some of these compounds are shown in

Figure 2.

Hops constituents have been studied previously
(Mannering and Deloria, 1988; Mannering et a1., 1992).
Interest in hops compounds was initiated by the finding that
hops B-acids adsorbed onto brewer's yeast could induce
cytochrome P450 metabolism in the mouse (Mannering and
Deloria, 1988). .Further research showed that colupulone was
the primary B-acid responsible for this induction (Mannering

et a1., 1992). This inductive ability was demonstrated by
measuring total cytochrome P450 concentration, by using
antibodies to several cytochrome P450 isozymes, and by
measuring cytochrome P450 metabolic activity using B[a]P,
aniline, and ethylmorphine as substrates for cytochromes

P4SOIA, P4501IE1, and P4SOIIIA4 respectively.

B-acids are present in both alcoholic and non-alcoholic

29

3O

Cflflflflfifiz

a) Colupulone

cnzcmcus),

b) Lupulone

(”NOTEQCTQGH,

c) Adlupulone

 

Figure 2. The structures of three hops B-acids:
a) colupulone; b) lupulone; c) adlupulone
(Mannering et a1., 1992).

31
hopped beverages. Because these beverages can be ingested
in high quantities (several liters per day), it is essential
to define the effect of hops B-acids on cytochrome P450

abundance and metabolic activity.

The isozymes examined in the current experiment were
selected because of their involvement in the activation and
detoxification of a number of important procarcinogens. For
example, cytochromes P4501A1 and P4501A2 activate the
heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine (PhIP) (Wallin et a1., 1990), and the
carcinogenic dye 3-methoxy-4-aminoazobenzene (Yamazaki et
a1., 1991). Cytochrome P4SOIA2 is known to metabolize AFB,
to the less mutagenic AFM,, and also deactivates the
carcinogen 1,3-dinitropyrene (Shimada and Guengerich, 1990).
Cytochrome P450IIBl is involved to some extent in the
activation of AFB, to the mutagenic, reactive AFB,-2,3-
oxide. This reaction is also carried out by cytochrome
P4501IIA4, as is some of the activation of 3-methoxy-4-
aminoazobenzene (Yamazaki et a1., 1991). This isozyme also
deactivates 1,6-dinitropyrene to a non-carcinogenic
metabolite (Shimada and Guengerich, 1990). Examining the
ability of colupulone to induce these forms of cytochrome
P450 may produce a clearer understanding of the potential

chemopreventive effects of dietary colupulone.

METHODS AND MATERIALS

e 'c s

Colupulone (Kalsec Inc., Kalamazoo, MI) was provided as
95% pure by high-pressure liquid chromatography.
Pregnenolone-16a-carbonitrile was a gift of UpJohn Inc.
(Kalamazoo, MI). Grade I glucose-6-phosphate dehydrogenase
was obtained from Boehringer Mannheim (Indianapolis, IN) as
a 5 mg/ml solution in ammonium phosphate and stored at 4%:
in a desiccator between uses. Glucose-6-phosphate,
monosodium salt (98% purity) was also from Boehringer
Mannheim Biochemicals. Grade II, 97% pure NADP from Sigma
Chemical Co. (St. Louis, MO) was stored at -20°C. Carbon
monoxide was obtained from AGA Gas Co. (Lansing, MI) at 99%
purity. All other chemicals were of the highest grade

obtainable.

Animals
Male Sprague Dawley rats (Harlan Sprague Dawley Co.,

Indianapolis, IN) weighing 200-225 g were used for this
experiment. Animals were housed in polycarbonate boxes on
hardwood chip bedding. The room was temperature- and
humidity-controlled with a 12-hour light/dark cycle.

Animals were allowed free access to food and water

32

33

throughout the study.

Diets. Animals were acclimatized to a modified American
Institute of Nutrition (AIN) 76 diet for six days prior to
treatment. The original composition of the AIN 76 diet was:
cellulose, 5%; cornstarch, 15%; casein, 20%; sucrose, 50%;
DL-methionine, 0.3%; AIN vitamin mixture, 1%; AIN mineral
mix, 3.5%; choline, 0.2%; corn oil, 5% (AIN, 1977). In the
modified diet, cornstarch was increased to 49.2%, while
sucrose was decreased to 10%. This new formulation provided
the same nutrient density as the original composition.
Purified diet was mixed and stored in 1-kg lots at 4°C. All

feedings and treatments took place at 10:30 each morning.

Treatments. Colupulone and pregnenolone-16a-carbonitrile
(PCN) were each given to a separate group of four animals.
PCN, a fairly specific inducer for cytochrome P4SOIIIA4, was
suspended in corn oil to give a concentration of 93.75 mg/ml
(Wrighton et a1., 1985) This suspension was administered
daily by oral gavage to provide 300 mg PCN/kg body weight
per day for four days. Colupulone was mixed into modified
AIN 76 diet at a level of 0.36% (w/w) (Mannering et a1.,
1992). Colupulone-supplemented diet that was not used
immediately was stored at -20°C. This diet was fed ad lib
for five days. A third group of four animals were
untreated and allowed ad lib access to modified AIN 76 diet

and water.

34
Tissue Collection. Animals were euthanized by C02
asphyxiation and cervical dislocation. Livers were quickly
removed, rinsed in ice-cold 150 mM KCl, and quickly frozen
in liquid nitrogen. The tissues were then stored in liquid

nitrogen until use.

M os a ss s

Microsome Preparation. Two to three grams frozen liver
tissue were allowed to thaw on ice in 6 ml of 19.8 mM
Tris/1.5% KCl and homogenized on speed setting 5 of a
Polytron homogenizer (Brinkmann Instruments, Westbury, NY)
for 20 to 30 seconds. Homogenates were centrifuged for 20
minutes at 10,000x g in a Sorvall RC-SB centrifuge. The
supernatant was filtered through two layers of cheesecloth
and centrifuged at 105,000x g for 60 minutes in a Beckman
L7-65 ultracentrifuge. The resulting pellet was resuspended
in 4 ml of 0.39 M sucrose/0.77 mM pyrophosphate buffer, pH
7.6, and homogenized with a Polytron on speed setting 3 for
10 seconds. The suspension was then centrifuged at 105,000x
g for 60 minutes in the Beckman L7-65. The second pellet
was resuspended in 1.5 ml of 150 mM KCl and homogenized on
speed 3 of the Polytron for ten seconds. All steps in the

preparation were carried out on ice.

Protein and Cytochrome P450 Determination. Microsomal

protein concentrations were determined by the Biuret protein

35
concentration method (Gornall et a1., 1949). Microsomal
suspension (100 pl) was diluted in 1.9 ml of distilled water
and 2 ml of 6% sodium hydroxide. Biuret color reagent (200
pl) (Biuret reagent: 1.19 M sodium bicarbonate, 69 mM copper
sulfate) were added. The reaction mixture was allowed to
incubate at room temperature for 10 minutes. The tubes were
centrifuged in a Beckman clinical centrifuge for five
minutes to sediment any precipitate. The absorbance of the
supernatant was then measured against a blank of distilled
water. Absorbance values were then compared to a standard
curve of bovine serum albumin in 150 mM KCl, prepared along
with each protein concentration determination. Microsomal

protein concentrations were calculated by linear regression.

Microsomes were diluted to 1 mg protein/ml in 100 mM
Tris, pH 7.4, and analyzed for cytochrome P450 concentration
by the method of Omura and Sato (1964). Approximately 5 mg
sodium dithionite were added to the suspension and mixed
well. The absorption of this reduced suspension was then
recorded from 500 to 400 nm, against a blank of the same
reduced suspension. Carbon monoxide was bubbled through the
sample solution for 20 seconds, and absorbance was again
measured between 500 and 400 nm, with the same reduced blank
as before. Peak height at 450 nm was used to calculate
cytochrome P450 concentration, using an extinction

coefficient of 91 mM"cm" (Omura and Sato, 1964) .

36
Substrate Metabolism Assays. Two specific substrates were
used. Each was incubated with the microsomal fraction and
an NADPH-generating system. Aminopyrine (cytochrome
P4501IB1) and erythromycin (cytochrome P4SOIIIA4) N-
demethylase activities were determined by the formation of
formaldehyde, which was quantified spectrophotometrically
(Nash, 1953). The incubation mixture consisted of 250 ul
183 mM HEPES buffer, pH 7.6; 10 pl 197 mM MgC12; 20 pl 354
mM glucose-6-phosphate, pH 7.0; and 1 unit glucose-6-
phosphate dehydrogenase. NADP (0.5 mg) was added to the
sample incubations but omitted from the blanks. Microsomal
protein (0.8 mg), diluted in 150 mM KCl, was added to the
incubation mixture on ice. The mixtures were pre-incubated
for three minutes at 37°C in a shaking water bath. The
reaction was initiated by the addition of the substrate
(aminopyrine, 20 umole; erythromycin, 0.5 umole), followed
by vortexing. The reaction mixtures were then allowed to
incubate for 20 minutes at 37°C in a shaking water bath.
The reactions were stopped by adding 1.0 ml of 20% zinc
sulfate and 1.0 ml saturated barium hydroxide, vortexing,
and replacing the tubes on ice. The tubes were centrifuged
in a Beckman clinical centrifuge for five minutes at 1000x
g. 1.0 ml of the supernatant was combined with 1.0 ml of
Nash reagent (1.95 M ammonium acetate, 37.2 mM
acetylacetone, 50 mM acetic acid; Nash, 1953) and incubated
for 10 minutes at 60°C. The tubes were centrifuged as

before and allowed to cool. Color development was measured

37
at 412 nm on a Varian Cary-3E dual beam spectrophotometer.
A new formaldehyde standard curve was prepared each time the
assays were performed. Absorbance values for blank and
sample incubations were compared to the standard curve, and
nanomoles of formaldehyde produced were calculated by linear

regression.

s 'o a 8
RNA Isolation. All glassware used was baked overnight at
300°C. All plasticware was handled with latex gloves before
autoclaving and was autoclaved for 25 ‘minutes at 250°C, or
was used from pre-wrapped, sterile individual packages.
Solutions were filter-sterilized through a 0.2 pm filter
(Nalgene Inc., Rochester, NY) into sterile glass bottles.
The homogenizer probe was autoclaved before use, and was
rinsed with approximately 5 ml chloroform between tissues to
remove debris without introducing RNAse contamination. RNA
was isolated using a modification of the Chirgwin procedure
(Chirgwin et a1., 1978), as previously described (Helferich
et a1., 1990). Approximately 0.25 g liver tissue was
completely homogenized in 4 M guanidinium isothiocyanate
buffer, pH 7.0 (4 M guanidinium isothiocyanate,1 M sodium
citrate, 0.1 M B-mercaptoethanol, 0.5% N-laurylsarcosine).
The homogenate was centrifuged at 10,000 rpm for 10 minutes
in a Sorvall SS-34 rotor. Four ml of the supernatant were
layered over 1 ml of 5.7 M cesium chloride (5.7 M cesium

chloride, 0.1 M EDTA, pH 8.0) and centrifuged for 12 hours

38
at 150,000x g at 20°C in a Beckman L7-65 ultracentrifuge and
SW 50.1 rotor. After centrifugation, the cesium chloride
and guanidinium isothiocyanate were decanted and the tube
walls were wiped with a Kimwipe“. The pellet was
resuspended in 1 ml of 7 M guanidine hydrochloride buffer,
pH 7.0 (7 M guanidine.hydrochloride, 19.8 mM sodium acetate,
0.97 M dithiothrietol, 1 mM EDTA) and transferred to
microcentrifuge tubes. RNA was precipitated by the addition
of 0.5 volume absolute ethanol and 0.05 volume 0.3 M sodium
acetate at -20°C for 60 minutes. RNA pellets were then
resuspended in 3 M sodium acetate and precipitated as
before. The resulting pellet was washed with 66% ethanol
and 33 mM sodium acetate at 4°C, and washed again with
absolute ethanol at -20°C. The final pellet was resuspended
in 1 ml 10 mM Tris, 1 mM EDTA, pH 8.0. The RNA suspensions
were scanned with a Varian Cary-3E dual beam
spectrophotometer from 220 to 320 nm, and the concentrations
were determined from the absorbance at 260 nm using an
extinction coefficient of 40 ug/ml, as described in Maniatis

et a1. (1986). The solutions were stored at -80W:.

RNA Gel Electrophoresis. RNA solutions were allowed to thaw
on ice and diluted with 10 mM Tris, 1 mM EDTA, pH 8.0, to 1
ug/ul. The RNA dilutions (20 ul of each) were mixed with 60
ul of RNA denaturing buffer (10x MOPS (N-morpholino-propane-
sulfonic acid) buffer: 0.4 M MOPS, pH 7.0, 100 mM sodium

acetate, 10 mM EDTA; RNA denaturing buffer: 13% 10x MOPS,

39
23% formaldehyde, 64% formamide) and heat-denatured at 50%:
for 15 minutes. Twenty pl of RNA loading buffer (50%
glycerol, 1 mM EDTA, 0.4% xylene cyanol, 0.4% bromphenol
blue) were added and the solutions were mixed by pipeting
repeatedly. Five pg of each RNA sample were loaded onto one
of three 1.2% agarose-10% formaldehyde denaturing gels made
up with 1x MAE buffer (10x MOPS, 100 mM sodium acetate, 10
mM EDTA) in running buffer of 1x MAE buffer. The gels were
electrophoresed at a constant voltage of 20 volts for 16
hours. The gels were then stained by the addition of 0.02
pg ethidium bromide per ml of running buffer and placing the
gels on a rotating shaker for 60 minutes. Destaining was
accomplished by replacing the staining buffer with 1x MAE
buffer for three hours on a rotating shaker. The gels were
prepared for Northern blotting by denaturation in 50 mM
sodium hydroxide and 10 mM sodium chloride for 45 minutes
followed by neutralization in 0.1 M Tris, pH 7.5, for 45

minutes.

Northern Blotting. The gels were soaked in 20x sodium
chloride-sodium citrate (20x SSC: 3 M sodium chloride, 0.3 M
sodium citrate) for 10 minutes, then the RNA was transferred
to Hybond N nylon membrane (Amersham Inc., Arlington
Heights, IL) for 24 hours with 10x SSC. The transfer
apparatus was assembled as described in Maniatis et a1.
(1986). After transfer, the blots were rinsed briefly in

20x SSC, then allowed to air dry for two hours. The RNA was

40
cross-linked to the membrane by two minutes of ultraviolet

radiation.

Northern Blot Hybridisation. The blots were hybridized with
cDNAs of mouse cytochrome P4501A2 from ATCC (Kimura et a1.,
1984), rat cytochrome P4SOIIB1 (Doehmer et a1., 1988), or
human cytochrome P4SOIIIA4 (Bork et a1., 1989). The probes
were labelled with a Dupont-NEN (Boston, MA) random-prime
labelling kit. Fifty microcuries of a4RP-dCTP from Dupont
NEN were used per probe, with a specific activity of 3097
curies/mmole. Labelled probe was purified from the
unincorporated nucleotides by centrifuging through Sephadex
G-50 in 1 M Tris, 1 mM EDTA, 100 mM sodium chloride, pH 8.0.
The radiolabelled probe was then stored at -20°C until use.
All prehybridizations, hybridizations, and washes were
carried out in a Robbins hybridization oven. Blots for
cytochromes P4501A2 and P4SOIIIA4 were prehybridized at
36.5°C for four hours in 15 ml 45% formamide
prehybridization solution (45% formamide, 3x SSC, 5x
Denhardt's solution, 50 mM phosphate buffer). 5 mg herring
sperm DNA was heat-denatured at 95°C for five minutes before
addition to the pre-hybridization solution. The
prehybridization solution on each blot was replaced with 7
ml of 45% formamide hybridization solution (45% formamide,
3x SSC, 5% dextran sulfate, 50 mM phosphate buffer, 5x
Denhardt's solution, 0.1% SDS), 2.5 mg heat-denatured

herring sperm DNA, and heat-denatured probe. Hybridization

41
hours. The blots were washed with 100 ml of 2x SSC, 0.1%
sodium dodecyl sulfate (SDS) at 45°C for 30 minutes,
followed by 100 ml of 2x SSC, 0.1% SDS at 45°C for 60
minutes. The cytochrome P4SOIIB1 blot was prehybridized in
15 ml of 50% formamide prehybridization solution (50%
formamide, 3x SSC, 5x Denhardt's solution, 50 mM phosphate
buffer) and 5 mg heat-denatured herring sperm DNA at 42%:
for four hours. The prehybridization solution was then
replaced with 7 ml 50% formamide hybridization solution (50%
formamide, 3x SSC, 5% dextran sulfate, 50 mM phosphate
buffer, 5x Denhardt's solution, and 0.1% SDS), 2.5 mg heat-
denatured herring sperm DNA, and heat-denatured probe.
Hybridization was carried out at 42°C for sixteen hours.
Washing was conducted in 100 ml of 2x SSC, 0.1% SDS at 65%:
for sixty minutes, followed by a twenty-five minute wash at
65°C in 100 ml 0.3x SSC, 0.1% SDS. All blots were allowed
to air-dry for two hours, and exposed to X-Omat
autoradiography film (Kodak Inc., Rochester, NY) at -80%:.
Autoradiographs were developed according to Kodak directions

for X-Omat film.

W

Means and standard errors were calculated for: feed
eaten per animal; body weight and liver weight; microsomal
protein and cytochrome P450 isolated; and aminopyrine and
erythromycin N-demethylase activities. Differences between

the means of control and treated groups were determined by

42
the one-tailed Student's t test (Bhattacharyya and Johnson,

1977). The criterion for significance was set at 0.05.

RESULTS AND DISCUSSION

F ed e a W '

Daily feed intake for control, colupulone-treated, and
PCN-treated animals is presented in Figure 2. Days -3 to -1
are the acclimatization period, with treatment starting at
10:30 AM on day 0. Before treatment, the amount of feed
consumed by animals in all three groups was similar. After
treatment began, feed intake by the colupulone treatment
group dropped from an average of 25.8 g per animal before
treatment to 20.1 g per animal during treatment. This
decrease was significant at P < 0.05. This level of feed
consumption was also significantly less than the 26.8 g per
animal eaten by the control animals on days 1-5. Feed
consumption by the PCN treatment group showed no real change

during treatment.

Average daily body weight in grams for each treatment
group is shown in Figure 3. Neither treatment group was
significantly different from the controls either before or

during the course of the experiment.

43

44
We 'v We'

Neither the initial nor the final body weights were
significantly different between the control and the treated
animals, at a significance level of P < 0.05. Liver weights
also were not different between groups. However, liver
weight expressed as a percentage of body weight was
significantly increased at P < 0.05 in the PCN-treated group
(6.4% for PCN compared to 5.6% for the control group).

These data are presented in Table 3.

so 0 e

For the control group, 18.3 mg microsomal protein was
isolated per gram of liver used. As presented in Table 4,
this was slightly but not significantly higher than the
yield in the colupulone-treated group (17.8 mg/g) and in the
PCN-treated group of animals (17.9 mg/g). Microsomes from
control animal livers also had a higher cytochrome P450
concentration (0.5 ng/g protein) than microsomes from either
colupulone-treated or PCN-treated animals (0.3 ng/g protein
for both). Using a significance level of P < 0.05, however,

this difference was not significant.

in 909 1‘ :12 $4.! -u '9 -l‘u‘ ! a;- ; °-=
Measurements of the metabolic activities of two

cytochrome P450 isozymes, presented in Table 5, did not show

significant differences between untreated and treated

animals. For aminopyrine N-demethylation, there was a

45
nonsignificant trend towards increased activity in both
colupulone-treated (5.60 nanomoles formaldehyde/mg
protein/minute) and in PCN-treated animals (5.63
nanomoles/mg protein/minute) when compared to control

animals (4.57 nanomoles/mg protein/minute).

The same trend was apparent for erythromycin N-
demethylase activity, although the increase was not as
large. Erythromycin N-demethylase activity in control
microsomes was 1.68 nanomoles formaldehyde/mg
protein/minute. Enzyme activity in microsomes from
colupulone-treated animals was 2.06 nanomoles/mg
protein/minute, slightly higher than the activity in
microsomes from PCN-treated animals, 2.02 nanomoles/mg

protein/minute.

o t B
Figure 4 is a photograph of an autoradiogram of

cytochrome P4501A2 hybridization to hepatic RNA isolated
from untreated, colupulone-treated, and PCN-treated animals.
The signal produced by hybridization in the colupulone-
treated lanes is somewhat more intense than that seen in the
control lanes. This indicates that colupulone may have
effected a slight increase in the level of hepatic
cytochrome P4SOIA2 RNA. On the other hand, some reduction
in signal strength can be seen in the PCN-treated lanes,

suggesting that PCN somehow decreased message abundance for

46

cytochrome P4501A2.

A similar figure for the hybridization of cytochrome
P450IIB1 cDNA is presented in Figure 5. Here, however, the
difference between signal strength in untreated and
colupulone-treated lanes is more intense. This increased
hybridization by the labelled cDNA probe to RNA from
colupulone-treated animals indicates that the RNA level for
this isozyme is increased by colupulone administration. The
hybridization signal is also more intense in the PCN-treated
lanes than in the control lanes, indicating increased

message levels in response to PCN treatment.

Hybridization of the cytochrome P4SOIIIA4 cDNA is shown
in Figure 6. While the hybridization of the probe in the
control lanes is more evident than in Figures 4 and 5, there
is also a noticeable increase in signal intensity in the
colupulone-treated lanes. This indicates an increase in
message level for cytochrome P4SOIIIA4 in response to
colupulone treatment. An even more intense is evident in
the PCN-treated lanes, demonstrating that PCN is able to

increase the message level of cytochrome P4SOIIIA4.

The results of the current study do not correspond to
the results described by Mannering et a1. (1992). In that
study, treatment of Swiss-Webster mice with 0.36% colupulone

in purified diet resulted in an 11-fold induction of

47
cytochrome P4SOIIIA4 metabolic activity, as well as doubling
cytochrome P4SOIIE1 metabolic activity and inducing that of
cytochrome P4501A1 by three-fold. An increase in the
content of hepatic cytochrome P450 and of the cytochrome
P45OIIIA4 isozyme itself, as indicated by Western blot

analysis, were also observed.

There are several possible explanations for the
differences between these studies. Cytochrome P4SOIIIA4
may be less sensitive to a given chemical, such as
colupulone, in the rat than it is in the mouse. This would
therefore require more of the chemical to produce the same
amount of induction in the rat. Alternatively, the time
required for translation of the cytochrome P4SOIIIA4 message
into protein may be longer in the rat than in the mouse.
Colupulone did produce an increase in cytochrome P4SOIIIA4
message in the rat. RNA levels for the isozymes in question
were not measured in the previous study (Mannering et a1.,
1992). It is thus possible that the increase in message
abundance would indeed have been translated into increased
cytochrome P4SOIIIA4 if treatment time in the rat had been

extended.

Erythromycin is the diagnostic substrate used to
measure cytochrome P4SOIIIA4 activity in the current
experiment, whereas Mannering et a1. (1992) used

ethylmorphine as the cytochrome P4SOIIIA4 substrate. It is

48
possible that after N-demethylation of the erythromycin
molecule, the reaction product complexes with the active
site of the cytochrome P450 protein and inhibits further
reactions (Pershing and Franklin, 1982). If this is the
case, formation of this complex could obscure any increase
in the metabolic activity of the cytochrome P4SOIIIA4.
isozyme. It is therefore possible that using ethylmorphine
would produce results demonstrating an induction of
cytochrome P4SOIIIA4 metabolic activity in response to
dietary colupulone supplementation. This would agree with
the cytochrome P450IIIA4 RNA data obtained in the current

experiment.

49

as! PCN

D.
u
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9%

   

 

O O
1

        

        

-2

-3

Average grams of feed eaten per animal per day

prior
adm

inning at

lupulone and PCN
(error bars represent standard

1ng co

tration
error of the mean) with treatment beg

10

to and dur

inis

30 AM on Day 0.

Figure 3.

50

PCN

-Control Colup.

 

      
    

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VI/I/ll/lllllr/l/l/J
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IEII_IenEIB_1III 225.23554

  
  

 

Average body weight in grams of each animal each
day prior to and during colupulone and PCN

administration

(error bars represent standard

30 AM on Day 0.

error of the mean) with treatment starting at

10

Figure 4.

51

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.ONHm

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52

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53

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35.36 H 34 V 3.3.6 H 5.4 Hopscoo

ouscwa\cwououm vs\oc>cocaoshou a: ouscHa\cHououm oa\opmcocaoshou a: ucosuooua
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Untreated Colupulone
34 1234

1 2

 

Figure 5.

54

 

Northern blot hybridization of cytochrome P450IA2
mouse cDNA to RNA from livers of untreated,
colupulone- and pregnenolone—l6a-carbonitrile-
treated rats.

Approximately 5 pg RNA was loaded into each lane.
After transfer and hybridization as described

in Methods and Materials, the blot was washed
with a low-stringency wash of 100 ml 2x SSC, 0.1%
SDS SSC, 0.1% SDS at 45°C for 30 minutes,
followed by a low-stringency wash in 100 ml 2x
SSC, 0.1% SDS at 45°C for 60 minutes. The washed
blot was exposed to X-Omat autoradiography film
for 11 days at -80°C. The increase in signal in
the colupulone-treated lanes compared to the
signal compared to the signal in the control
lanes indicates an increase in cytochrome

P4501A2 mRNA abundance in response to

colupulone treatment.

55

Untreated Colupulone PCN
1 2

Figure 6.

 

3 4 1 2_ 3 4

 

Northern blot hybridization of cytochrome
P450IIB1 rat cDNA to RNA from livers of
untreated, colupulone- and pregnenolone-16a-
carbonitrile-treated rats.

Approximately 5 pg RNA was loaded into each lane.
After blotting and hybridization as described in
Methods and Materials, the blot was washed in a
high stringency wash of 2x SSC, 0.1% SDS at 65°C
for 60 minutes, followed by a 25 minute wash in
0.3x SSC, 0.1% SDS at 45°C. The washed blot was
exposed to X-Omat autoradiography film at -80°C
for 11 days. There is an increase in signal
intensity visible in the colupulone-treated
lanes, indicating that colupulone induces an
increase in cytochrome P450IIB1 message
abundance. There is also an increase in signal
intensity in the PCN-treated lanes, demonstrating
that PCN is able to increase mRNA abundance for
cytochrome P4501IB1.

56

Untreated Colupulone PCN

1 2

' "‘ ml
, m
t

Figure 7.

3 4 1 2 3 4 1 2 3 4

 

,6

Northern blot hybridization of cytochrome
P4SOIIIA4 human cDNA to RNA from livers of
untreated, colupulone- and pregnenolone-16a-
carbonitrile-treated rats.

Approximately 5 mg RNA was loaded into each lane.
The blotting and hybridization techniques used
are described in Methods and Materials. After
hybridization, the blot was washed by low-
stringency washing in 2x SSC, 0.1% SDS at 45°C
for 30 minutes, followed by a 60 minute wash in
2x SSC, 0.1% SDS at 45°C. The blot was then
allowed to air dry for two hours and exposed to
X-Omat autoradiography film at -80°C for 11 days.
Colupulone produced an increase in cytochrome
P450IIIA4 mRNA level, as demonstrated by the
greater signal in the colupulone-treated lanes
when compared to the control lanes. The very
intense signal in the PCN-treated lanes
indicates that PCN increases the abundance of
cytochrome P450IIIA4 message.

SUMMARY AND CONCLUSIONS

In this experiment, administration of colupulone in
modified AIN 76 diet to rats had no significant effect (P <
0.05) on body weight, liver weight, or cytochrome P450-
mediated metabolism of aminopyrine and erythromcyin. These
data do not agree with earlier results demonstrating an
inductive effect of colupulone on cytochrome P450 in the
mouse (Mannering et a1., 1992). This difference in the
response to colupulone may be due to a number of factors,
such as species differences or the difference in cytochrome
P450 substrates used (erythromycin instead of
ethylmorphine). However, Northern hybridization in the
current study did reveal an increase in the abundance of
cytochrome P450IIIA4 mRNA after treatment with colupulone,
as well as a slight increase in the message level of
cytochromes P4501A2 and P4SOIIB1. This message level-
induction, of cytochrome P4SOIIIA4 especially, was not
observed at the protein level, as indicated by the lack of
increase in total cytochrome P450 concentration in response

to colupulone treatment.

Neither study examined the effect of colupulone

57

58
administration on metabolism of substrates which may be
activated or detoxified by cytochrome P450IIIA4. Further
research is required to determine whether inductive effects
of colupulone, as seen at the mRNA level, may have an impact

on human health.

The use of any agent to alter cytochrome P450-mediated
promutagen or procarcinogen metabolism needs to be evaluated
with reference to other xenobiotics entering the body, as
well as to endogenous cytochrome P450 substrates. Because a
compound able to induce one cytochrome P450 isozyme may
either induce or repress other isozymes at the same time,
directed induction to increase detoxification of one
xenobiotic may actually increase activation or decrease
detoxification of another xenobiotic. The possibility also
exists that induction of one isozyme to detoxify a given
promutagen may lead to the increased activation of other
xenobiotics which are substrates for the same isozyme. In
addition, altering xenobiotic metabolism may alter
cytochrome P450 metabolism of medications, anesthetics, and
endogenous compounds such as steroid hormones and
cholesterol. For these reasons, the effect of any potential
chemopreventive agent on overall cytochrome P450 metabolism
needs to be thoroughly defined before that agent is added to

the diet in pharmaceutical quantities.

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