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This is to certify that the

thesis entitled

Effect of Diet on Xenobiotic Metabolism
by Intestinal Tissues

presented by

Lorraine Platka-Bird

has been accepted towards fulfillment
of the requirements for

Ph.D. Human Nutrition

degree in

 

 

Major professor

June 12, 1980
Date

 

0-7639

 

 

 

 

 

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RETURNING UBRARY MATERIALS ;

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Place in book return to remove
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EFFECT OF DIET ON XENOBIOTIC METABOLISM

BY INTBSTINAL TISSUES

By

Lorraine Platka-Bird

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1980

ABSTRACT

EFFECT OF DIET ON XENOBIOTIC METABOLISM
BY INTESTINAL TISSUES

BY

Lorraine Platka-Bird

A.model system was developed to study xenobiotic metabolism by the
intestinal tissue-microflora ecosystem. Interaction between drug metab-
olizing pathways in colon cells and intestinal microflora may be important
in production of compounds which act as carcinogens or cocarcinogens in
colonic tissues. Benzo(a)pyrene (BP) was used as the model carcinogen
because (a) its metabolism is representative of metabolism in a group of
common environmental contaminants, polycyclic aromatic hydrocarbons,

(b) it can be both mutagenic and carcinogenic, and (c) there is a vast
literature on the metabolism of BP. Charcoal-broiled hamburger (CBH)

was chosen as the source of dietary xenobiotics based on the presence, in
CBH, of measurable levels of compounds which induce drug metabolizing
enzymes.

BP metabolism was measured in the colon, small intestine, and liver
of rats fed purified diets or diets containing CBH. Microsomal aryl
hydrocarbon hydroxylase (AHH) activity, as measured by fluorometric and
radioactive assays, was significantly greater in all three tissues from
rats fed the CBH diet than in tissues from rats fed the purified diet.
Feeding CBH resulted in greater enzyme induction than did feeding 0.1 or

0.5 mg BP/gram of diet. AHH induction by CBH-feeding was related to both

Lorraine Platka-Bird

the level of fat in the hamburger before charcoal-broiling, and the total
level of CBH in the diet. Enzyme activities were maximal after feeding
CBH for 4 days in the small intestine and liver, and by 10 days in the
colon. Addition of various fiber sources to the purified or the CBH
diets had minimal effects on enzyme activity.

The relevance of dietary-induced alterations in AHH activity was
assessed by measuring in_vit£g_mutagenicity of BP metabolites produced,
and the ability of the metabolites to bind to cellular macromolecules
in 1212' These determinations were based on concepts relating mutagenesis
and macromolecular binding to basic mechanisms of carcinogenesis. Feeding
CBH did not result in increased activation of BP to mutagenic derivatives
in the Ames test for any of the tissues tested. Binding of BP to cellular
macromolecules was increased in all three tissues from rats fed the CBH
diet, but BP was not bound to DNA. The results show that alterations in
intestinal metabolism of xenobiotics produced by diet may influence
mechanisms which are basic to carcinogenesis. These alterations may

represent an additional role for diet in the etiology of colon cancer.

ACKNOWLEDGMENTS

I would like to express sincere gratitude to Dr. M. R. Bennink fer
his guidance, his patience and the values he has instilled in me over the
past several years. I would also like to thank Dr. D. R. Romsos for his
helpful suggestions and my Ph.D. committee members Drs. S. Aust, C. welsch,

M. Yokoyama, and 1. Gray.

ii

TABLE OF CONTENTS

Page

LIST OF TABLES . . . . . . . . . . . . . . . . . . iv
LIST OF FIGURES. . . . . . . . . . . . . . . . . . v
INTRODUCTION. . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE . . . . . . . . . . . . . . . . 3
Mechanisms of Carcinogenesis. . . . . . . . . . . . . 3
Nutrition and Cancer . . . . . . . . . . . . . . . 10

Nutrition and Colon Cancer . . . . . . . . . . . . . 15

MATERIALS AND METHODS. . . . . . . . . . . . . . . . 23

Animals. . . . . . . . . . . . . . . . . . 23
Diets . . . . . . . . . . . . . . . . . . . . 23
Enzyme Induction. . . . . . . . . . . . . . . . . 23
Preparation of Tissue Microsomes . . . . . . . . . 25
Enzyme Assays. . . . . . . . . . . . . . . . . 25
Ames Test . . . . . . . . . . . . . . . . 26

Isolation of Colonic Epithelial Cells. . . . .
Cell Cultures. . . . . . . . . . . . . . . . . . 27
Binding of BP to Cellular Macromolecules. . . . . . . . . 28
Isolation of DNA. . . . . . . . . . . . . . . . . 28

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 29

Enzyme Activity . . . . . . . . . .
Cell Cultures. . . . . . . . . . .
Ames Test . . . . . . . . . . . .
Binding of BP to Cellular Macromolecules.

O O O O 57
CONCLUSIONS . . . . . . . . . . . . . . . . . . . 69

REFERENCES 0 O O O O O O O I O O O O O O O O O O 70

iii

Table

LIST OF TABLES

Composition of Experimental Diets . . . . . . . .

Hyperplasia of the Liver, Small Intestine and Colon of
Rats Fed Charcoal-broiled Hamburger. . . . . . .

Benzo(a)pyrene Metabolism by Isolated Colonic Epithelial
cells. 0 O O O O O I O O O O O O O O 0

Activation of Benzo(a)pyrene by Colon, Small Intestine
811d Liver 8-9 FraCtionS. o o o o o o o o o 0

iv

Page
24

37

S7

59

LIST OF FIGURES

Figure Page
1. Benzo(a)pyrene . . . . . . . . . . . . . . . . 7
2. Metabolism of Polycyclic Aromatic Hydrocarbons . . . . . 8

3. Aryl Hydrocarbon Hydroxylase Activity in Colon Microsomes
as Related to Incubation Time . . . . . . . . . . 31

4. Relationship of Aryl Hydrocarbon Hydroxylase Activity in
Colon Microsomes to Protein Concentration . . . . . . 33

5. Effect of Feeding Charcoal-broiled Hamburger on Aryl
Hydrocarbon Hydroxylase Activity . . . . . . . . . 35

6. Time Required fer Aryl Hydrocarbon Hydroxylase Induction
and Return to Control Levels. . . . . . . . . . . 40

7. Aryl Hydrocarbon Hydroxylase Activity as Related to Ham-
burger Content of the Diet . . . . . . . . . . . 42

8. Effect of Dietary Benzo(a)pyrene on Aryl Hydrocarbon
Hydroxylase Activity . . . . . . . . . . . . . 45

9. Effect of Fat Level in Hamburger on Aryl Hydrocarbon
Hydroxylase Activity . . . . . . . . . . . . . 49

10. Effect of Feeding Heated Fats on Aryl Hydrocarbon
Hydroxylase Activity . . . . . . . . . . . . . 51

11. Effect of Feeding Various Fiber Sources on Aryl Hydrocarbon
Hydroxylase Activity . . . . . . . . . . . . . S4

12. Binding of Benzo(a)pyrene to Cellular Macromolecules in
Tissues of Rats Fed Charcoal-broiled Hamburger. . . . . 62

13. Effect of Feeding Charcoal-broiled Hamburger and Pretreat-
ment with B-naphthoflavone on Binding of Benzo(a)pyrene
to Cellular Macromolecules I. . . . . . . . . . . 64

14. Effect of Feeding Charcoal-broiled Hamburger and Pretreat-
ment with B-naphthoflavone on Binding of Benzo(a)pyrene
to Cellular Macromolecules 11 . . . . . . . . . . 66

INTRODUCTION

Cancer of the colon is the second.most common malignancy and the
second most common cause of death from cancer in the United States (1).
Colon cancer rates in different geographical locations implicate environ-
mental factors as being important in development of large bowel cancer.
Among the environmental factors, diet has been pr0posed to be of signi-
ficant importance in the etiology of this disease.

The dietary components which have been associated with colon cancer
are fat, animal protein, fiber, and certain vegetables. There are several
mechanisms by which these dietary constitutents may exert their effects.
Fat and animal protein affect neutral sterol and bile acid metabolism
by the liver and intestinal microflora. Steroid derivatives may be
important as promoters of carcinogenesis. Fiber may influence carcino-
genesis by decreasing exposure of the intestinal epithelium to carcinogens
or promoters, and cruciferous vegetables can alter activity of enzymes
involved in metabolism of xenobiotics.

Many studies in experimental colon carcinogenesis have utilized long-
term feeding studies to determine how various factors in the diet influ-
ence chemically-induced colon carcinogenesis. A second area has focused
on the effects of diet on the taxonomic grouping and enzyme activity of
intestinal microorganisms involved in metabolism of carcinogens and other
compounds entering the colon. Little emphasis has been placed on the

role of intestinal tissue itself in metabolism of xenobiotics. Elucidation

of the role of diet in intestinal tissue xenobiotic metabolism could pro-
vide insight to additional mechanisms by which nutrition can influence
colon carcinogenesis.

The objectives of this study were: (1) to develop a model system to
study dietary-induced alterations in xenobiotic metabolism; (2) to investi-
gate whether enzymes which metabolize xenobiotics can be induced in
intestinal tissues; and (3) to determine how dietary variables influence

intestinal tissue xenobiotic metabolism.

REVIEW OF LITERATURE

It has become increasingly apparent over the past several years that
environmental factors play an important role in the development of a
significant proportion of human cancers. Current estimates of cancer
incidence related to the environment range from 60 to 90 percent. These
estimates are based on differences in mortality rates from specific cancers
in different geographical locations (2).

Based on observations linking cancer to the environment, a major
focus in cancer research has been directed toward (a) identifying chemical
agents in the environment which can initiate carcinogenesis, and (b) deter-
mining other variables in the environment which can modify the carcinogenic
process. To further clarify the significance of environmental factors in
the etiology of cancer, it has become especially important to elucidate

the mechanisms by which these factors exert their effects.

Mechanisms of Carcinogenesis

 

The mechanisms of chemically-induced cancer have been studied exten-
sively in experimental animals. One of the more accepted theories of
carcinogenesis, based primarily on experiments with.mouse skin, is that
carcinogenesis is a multistage process requiring at least two critical
steps, initiation and promotion (3,4,5). In initiation, the administra-
tion of a chemical compound to the skin of experimental animals results

in rapid alterations in some of the cells exposed to the initiating agent.

These changes, which are thought to be irreversible, are not alone suffi-
cient to result in tumor growth. However, if the altered cells are
repeatedly exposed to promoting agents, uninhibited cell replication
occurs resulting in neoplastic growth. For tunors to develop it is
essential that application of the promoter follows application of the
initiator, and that there is repeated application of the promoter (1).
Some chemical carcinogens, such as 7,12-dimethy1benz(a)anthracene can
produce cancer at high doses without promoting agents. The mechanisms
involved in this type of carcinogenesis have not been elucidated.

A large number and great variety of both organic and inorganic com-
pounds are capable of initiating the carcinogenic process. These chemical
agents can be broadly classified as (a) direct-acting carcinogens, which
do not require modification to exert their deleterious effects (such as
alkylating or acetylating agents), and (b) procarcinogens, which require
metabolic activation to produce more reactive metabolites. The majority
of environmentally important carcinogens fall into the second classifi-
cation (6).

Activation of procarcinogens to their proximate or ultimate carcino-
genic form often involves the same metabolic pathways which are used for
conversion of xenobiotics to forms which can be excreted from the body.

In this detoxification process, lipid-soluble xenobiotics are metabolized
to more polar water-soluble compounds which can be conjugated for excre-
tion. The metabolites produced in this process can be electrophilic
reactants which can be acted upon by conjugating enzymes, or they can
attach to electron-rich or nucleophilic sites upon cellular macromolecules.
Because nucleophilic sites are relatively abundant in proteins and nucleic

acids, production of electron-deficient species can result in protein- or

nucleic acid-adducts which make the cell susceptible to initiation of
neoplasia. Administration of a procarcinogen to experimental animals
produces protein-, RNA-, and DNA-bound derivatives which can be isolated
from target tissues (7).

The role of promoters has been less clearly defined. The most com-
monly used promoter in animal studies has been croton oil from the seeds
of Croton tiglium (S). The active ingredients in croton oil are the
12,13-diesters of diterprene alcohol phorbols, the most active of which
is tetradecanoly-phorbol acetate (8). These compounds produce several
phenotypic changes in promoted cells including (a) increased synthesis of
RNA, DNA, protein, and phospholipids, (b) increased mitotic rates, (c)
increased protease activity, and (d) a 200- to 400-fold induction of
ornithine decarboxylase activity (9). One current theory proposes that
promoters act through increased ornithine decarboxylase levels. According
to this theory, repeated administration of promoters to initiated cells
results in an inability of the cells to return ornithine decarboxylase
activity to control levels, producing elevated levels of polyamines in
transformed cells (9).

Two major theories concerning mechanisms of carcinogenesis have
emerged from numerous studies on chemically-induced cancer in experimental
animals. The more widely accepted theory is the genetic mechanism where
tumorigenesis results from alterations in the genes themselves. The
genomic abberations can be produced by reaction of electrophilic species
directly with DNA, through modification of RNA which is to be transcribed
and integrated into DNA, or through alteration of cellular proteins which

are involved in DNA repair (1). The role of nucleic acids in storage and

translation of genetic information makes these cellular organelles the
more likely targets to produce alterations in the genome of the cell.

Interaction of carcinogens with cellular macromolecules can occur
through covalent binding or through noncovalent binding. Covalent binding
occurs with electrophilic reactants capable of sharing electrons with
target sites in the cellular macromolecules. Noncovalent binding is more
likely to occur with compounds which have a planar aromatic ring approxi-
mately the size of a base pair, allowing for intercalation or stacking of
the carcinogen between base pairs in the DNA (6). Several studies have
shown a strong correlation between binding of carcinogens to DNA and
carcinogenicity (10,11,12). The less prominent theory of carcinogenesis
is the epigenetic mechanism in which the fundamental premise is that the
abnormality exists in gene expression and no alteration in genomic infor-
mation of the cell is required for production of tumors (13,14).

One of the more prevalent groups of environmental contaminants which
fall into the classification of chemical carcinogens is the polycyclic
aromatic hydrocarbons (PAH). These compounds are produced from incomplete
combustion of organic fuels and are released into the environment at sig-
nificant levels in highly industrialized areas. They are carcinogenic in
several tissues in many species including man (15). The most widely
studied PAH include benzo(a)pyrene (BP), 3-methycholanthrene dibenz(§h)-
anthracene, and 7,12-dimethylbenz(a)anthracene.

PAH require metabolic activation by a group of nonspecific mixed
function oxidases (MFO's) of the cytochrome P450 (cyt P450) family to be
converted to the ultimate carcinogenic form (1,16). The enzyme system
involved in PAH metabolism is located on the end0plasmic reticulum or the

microsomal fraction of the cell. The MFO system has also been associated

with the nuclear membrane, but the qualitative and quantitative importance
of activity in the nucleus has not been elucidated (17). In both parts of
the cell the enzyme system consists of two protein components, cyt P450

and NADPH cyt P reductase, and a lipid component, phosphatidyl choline.

450
Enzyme activity requires NADPH as a cofactor and molecular oxygen as a
substrate. The rate-limiting step of the reaction is the reduction of

cyt P The enzyme system is inducible and shows large variation in

450°
inducibility between different tissues and between different species.
Inducibility of the enzyme system is regulated genetically (18).

Much of the information about the cyt P MFO's has been gained

450
from studies on BP (see Fig. 1). BP is considered to be one of the most
carcinogenic PAH's, and the level of this xenobiotic.irlenvironmental

pollutants is considered to reflect overall PAH contamination. Metabolism

of BP has been studied extensively.

Figure l. Benzo(a)pyrene

The cyt P450 MFO involved in BP metabolism has been termed aryl
hydrocarbon hydroxylase (AHH). The form of cyt P450 which metabolizes
native BP is the same form which is induced by 3-methy1cholathrene and

B—naphthoflavone, and is known as cytochrome P448 (cyt P448;15).

AHH activity on BP results in the production of arene oxides which
can have several metabolic fates including: (a) nonenzymatic conversion
to phenols; (b) enzymatic conversion to dihydrodiols by the action of
epoxide hydratase; (c) enzymatic or nonenzymatic conjugation with
glutathione, glucuronic acid, or sulfate; and (d) NADPH-dependent reduction
to the parent compound (15). The dihydrodiols produced by the action of
epoxide hydratase can again be acted upon by AHH resulting in the pro-
duction of highly-reactive dial-epoxides. These derivatives can also be
conjugated to glutathione, glucuronic acid, or sulfate by the conjugating

enzymes to ferm soluble, detoxified metabolites (see Fig. 2).

further

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Figure 2. Metabolism of Polycyclic Aromatic Hydrocarbons (after
DePierre and Ernster, 1978).
BP metabolites produced by these pathways have been examined fer
mutagenicity and carcinogenicity. Based on calculations by the Pullmans

(19), K-reion epoxides (4,5 position of BP) were first implicated to be

the most carcinogenic. This area was calculated to have the most double
bond-like character. The 4,5-epoxide derivative is highly mutagenic for
bacteria and for mammalian cells in culture, but it does not show high
carcinogenic activity when painted on mouse skin (20,21). It has since
been recognized that the most critical site fer BP metabolism leading to
potentially carcinogenic derivatives is at the 7,8 position (22,23).

A.major ultimate electrophilic, mutagenic, and carcinogenic metabolite
of BP is the 7,8-diol-9,10-epoxide. This epoxide is very unstable and
is usually isolated from biological systems as the tetrol formed by the
addition of water (24). The major product produced by reaction of this
metabolite with DNA is a BP-DNA adduct between the 2-amino group of guanine
and the C-10 position of BP (25). Secondary hydrocarbon-nucleic acid
adducts are fermed by the reaction of the epoxide with cytosine and adenine
residues of DNA (26).

The relevance of the various metabolic pathways to carcinogenesis lies
in the balance between activation and detoxification of procarcinogens.

The water-soluble derivatives produced through conjugation probably
represent the major in 3132 detoxification mechanism, whereas DNA-bound
derivatives represent the carcinogenic mechanism. As the ultimate car-
cinogen is probably a transient intermediate, the relative activity of
these competing mechanisms determines the amount of reactive intermediates
which a given tissue is exposed to.

Steady state levels of the ultimate carcinogen will vary in different
tissues and in different species according to genetically determined dif-
ferences in enzyme activity, and according to diet, xenobiotic exposure,
and the physiological state of the organism (15). The nongenetic variables

may act through their effects on enzyme induction by altering the relative

10

rates of synthesis and degradation of the carcinogenic species. The most
susceptible cells to carcinogenesis may be those with either an increased
rate of activation to the ultimate carcinogen or those with a decreased
ability to detoxify xenobiotics to water-scluble derivatives. This wouId
result in prolonged survival of the species, increasing the chances for
reaction with cellular macromolecules.

A final concept of fundamental importance to carcinogenesis is DNA
repair. Reaction of cellular macromolecules, including DNA, with chemical
compounds capable of producing deleterious alterations occurs continually.
Mammalian cells have several systems capable of repairing altered DNA (27).
The rate of DNA repair varies in different tissues and may also vary
within tissues depending on the carcinogen involved and the site of adduct
fermation. Whether neoplastic growth occurs depends on the relationship
between the rate of DNA repair and the time of expression of the altered

genome.

Nutrition and Cancer

 

One environmental factor which has been strongly implicated in modifying
carcinogenesis is nutrition. It has been estimated that diet is related to
60 percent of cancers in females and to greater than 40 percent of cancers
in males (28).

Epidemiological studies provide some insights concerning environment
and cancer. Japanese immigrants to the United States develop a shift in
cancer incidence patterns toward those prevalent in the United States
within two to three generations. This shift is represented by an increase
in the incidence of breast and colon cancer and a decrease in the incidence

of stomach cancer (29,30). The dietary modifications which correlate with

11

this shift include an increase in meat and fat consumption, a decrease in
fiber and vegetable intake, an increase in the intake of refined carbohyd-
rates, and an increase in overall caloric consumption.

Nutrient imbalances can influence several areas related to the
development of’neoplasia including tumor nutrition, cell-mediated immu-
nological destruction of neoplastic cells, hormonal control of tumor
growth, and carcinogen availability (31). Carcinogen availability can be
affected by both the actual level of carcinogens or potential carcinogens
in the foods consumed and through modification of the enzyme systems
involved in the conversion of xenobiotics to carcinogenic forms.

Dietary-induced alterations in drug metabolism have been studied in
many tissues in several species. The MFO's are greatly influenced by diet
and it appears that most nutrients are capable of altering MFO activity
when consumed at either deficient or excessive levels.

Animal studies concerning protein intake and tumor incidences have
yielded inconsistent results; chemically-induced tumor incidences have
been decreased by both low protein and high protein diets (32,33). Con-
flicting results on the effects of protein on tumorigenesis may be due to
protein effects on different mechanisms of tumor development. Dietary
protein could influence tumor initiation through its effects on MFO
activity, tumor promotion through its effects on cell replication, tumor
growth rate through its effects on cell-mediated immunity, or tumor
nutrition through the supply of amino acids required by the tumor (34).

Protein deficiency, due to either low levels of dietary protein or
poor protein quality, inhibits MFO activity (35). Clinton et a1. (32)
showed dramatically decreased AHH activities when the protein level of the

diet was decreased from 45 percent to 7.5 percent. Protein deficiency can

12

also result in increased glucuronyltransferase activity in livers of
experimental animals (36,37). The combined effects of decreased MFO
activity and increased transferase activity may produce more conjugated
derivatives of carcinogens and less reactive electrophiles, and thereby
decrease the risk fer carcinogenesis.

In several studies chemically-induced tumor production in experimental
animals has been increased by high fat diets (38-43). Although dietary
fat may influence MFO activity, it has been proposed that the major role
for fat is related to its effects on promotion (40,44,45).

Cruciferous vegetables such as Brussels sprouts, cabbage, and cauli-
flower contain indoles which induce AHH activity and decrease risk from
PAH-induced cancers in experimental animals (46-48). The most active
indole compound in inducing AHH is indole-3-carbinol. The derivatives
3,3'-diindoy1methane and indole-3-acetonitrile produce lower levels of
induction (49). In DMBA-induced mammary tumorigenesis and BP-induced
forestomach neoplasia Wattenberg and Loub (47) feund a decrease in the
number of animals developing tumors and in the number of tumors per animal
when isolated indoles were added to the diet or injected as a single dose
prior to carcinogen administration. Graham and Mettlin (50) feund a
decreased risk for colon cancer associated with vegetable consumption.
However, other investigators (51,52) found no relationship between cancer
incidence and total vegetable consumption.

Other dietary variables which have been shown to inhibit chemically-
induced carcinogenesis include selenium, vitamin E, butylated hydroxy
anisole, butylated hydroxytoluene, and vitamin A. The antioxidant pro-
peties of some of these compounds are believed to inhibit carcinogenesis

by interfering with metabolic transfermation of procarcinogens to ultimate

13

carcinogens. Some of these agents may also act by combining with free
radicals to prevent tumorigenic cellular reactions (53). The mechanism
fer vitamin A probably involves its role in cell turnover and in main-
tenance of integrity of epithelial cells.

Several agents capable of causing cancer have been identified in the
food chain. Mycotoxins, such as aflatoxin Bl, are extremely toxic com-
pounds which are produced by molds in certain foods such as nuts, oilseeds,
and grains (54). These agents can enter the food supply directly from
mold growth on food or indirectly through the use of contaminated ingre-
dients in processed feeds or by feeding contaminated feed to animals which
produce consumable products. Aflatoxin B1 is the most potent liver
carcinogen known and its effects can be seen at very low levels of con-
sumption.

Sodium nitrite and sodium nitrate are common fbod additives. The
hazard of these agents to human health derives from conversion of nitrates
to nitrites and combination of nitrites with secondary or tertiary amines
at the acid pH of the stomach. This results in the formation of carcino-
genic N-nitrosamines (55).

Compounds which have been shown to be mutagenic in inugitrg'mutagene-
sis assays have been isolated from cooked meat and fish (56-59). The pro—
duction of’mutagens is related to the length of time and the temperature
at which the meat is cooked. In the experiments of Matsumoto et al. (56),
heating of peptides and proteins resulted in production of mutagenic
metabolites at temperatures above 400°. Mutagenic potential was maximum
at 500° for dipeptides containing tryptophan and at 600° fer proteins in
general. Nagao et al. (60) and Commoner et a1. (61) showed that cooking

proteinaceous foods at relatively high temperatures produced mutagenic

14

activity distinguishable from BP or the pyrolysis products of amino acids
(62). These mutagenic agents have yet to be shown to be carcinogenic in
man (57) .

PAH contamination of consumable products can occur through pollution
of the atmosphere, soil, or water, during fbod processing, or with use of
petroleum based food additives. Measurable levels of PAH have been
detected in many commonly consuned foods such as charcoal-broiled meats
and fishes, smoked foods, and vegetables and fruits grown in polluted
environments (63-65).

The level of PAH in meat and fish depends largely on the method of
processing or cooking. Smoking and charcoal-broiling appear to produce
higher levels of PAH than other processing and cooking methods (64,66).
It has been proposed that the occurrence of these compounds in charcoal-
broiled foods results from fats dripping down onto the hot coals and pro-
ducing pyrolysis products which rise in the smoke to condense on the sur-
face of the grilled meat (67). The level of PAH produced depends on the
time and temperature of cooking, the closeness of the food to the coals,
and the level of fat in the meat or fish (66). Studies by Pariza et a1.
(62) indicate that the cooking temperature is more important than the
length of time of cooking in producing PAH. Rappaport et a1. (68) found
that cooking ground meat at high temperatures in closed systems (systems
in which volatilized agents are recirculated over the food) resulted in
significantly greater PAH levels than when the meats were cooked in open
systems. These authors concluded that over 90 percent of the mutagens
formed in open cooking procedures are volatilized. Furthermore, cooking
procedures which allow for redisposition of airborn substances increase

the level of mutagens in cooked foods.

15

The level of BP in fbods is considered to be an adequate reflection
of overall PAH contamination (69). Levels as high as 121 ppb BP have
been detected in cooked beef containing approximately 40 percent fat.

It is likely that consumption of food products containing high levels of
BP results in exposure to a large number of PAH and possibly other
xenobiotics. These compounds may differ in ferm and level of induction
of drug metabolizing enzymes. A synergistic effect on enzyme induction

may result from ingestion of mixed food products.

Nutrition and Colon Cancer

 

One of the most prevalent forms of cancer which is related to diet
is colon cancer. Cancer of the large bowel is second only to skin cancer
as the most common malignancy and is second only to lung cancer as the
most common cause of death from cancer in the United States (1).

Diet could potentially modify colon carcinogenesis in several ways
including: (a) provision of carcinogens directly in the diet; (b) modifi-
cation of the taxonomic grouping or enzyme activity of intestinal bacteria
resulting in altered metabolism of potential carcinogens; (c) modification
of the enzyme activity or histological characteristics of the intestinal
mucosa resulting in increased susceptibility to carcinogenesis; (d) modi-
fication of enzyme activity in other tissues producing an altered profile
of metabolites reaching the colon; and (e) modification of promoting agents
which reach the colon.

Epidemiological studies show important trends in the relationship
between food consumption and colon cancer. The incidence of this disease
is greater in North America and Western Europe (where diets high in animal

protein and fat and low in fiber are consuned) than it is in Japan, Asia,

16

Africa, South America and rural India (where diets are low in animal
products and higher in fiber) (51,70-72). The incidence of colon cancer
in Japanese consuming Western diets is higher than in Japanese consuming
Oriental diets (73), and in Japan colon cancer incidence rates are in-
creasing in line with gradual westernization of the Japanese diet (74).
Japanese immigrants to Hawaii have a significantly greater chance of
developing colon cancer than do Japanese in Japan (51).

That diet is more important than environmental pollution is shown by
the fact that the pollution level in Japan is higher than in Hawaii, but
the incidence of colon cancer is lower in Japan (53). Also, colon cancer
incidence does not correlate with smoking. This indicates that the environ-
mental pollutants which are most important in the development of lung cancer
do not play a major role in the etiology of colon cancer. K

Interpretation of results from epidemiological studies must be care-
fully considered due to problems associated with isolating specific feed
components in the diet and variation in the presence of additives, preser-
vatives, fertilizers, pesticides, and other contaminants in the diet. How-
ever, the evidence to date strongly suggests important interaction between
nutritional habits and large bowel cancer.

The majority of animal studies support epidemiological studies impli-
cating diet as a major etiologic factor in colon cancer. Animal models
have been described which produce a reasonably accurate representation of
human colon cancer (75-78). These models allow investigators to manipulate
specific dietary variables to study their effects on the mechanisms
involved in colon carcinogenesis.

Four classes of chemical agents capable of producing large bowel cancer

have been identified. These include some PAH, 3-amino-4-biphenyl derivaties,

l7

alkylnitrosoureido derivatives, and 1,2-dimethy1hydrazine (DMH) deriva-
tives (79). DMH or metabolic derivatives have been the most widely used
due to their specificity for the colon. It is not clear whether the
majority of DMH derivatives reach the colon via the bile or whether they
are transported in the blood and taken up by the colon (79,80).

The mechanism of action of DMH in producing colon carcinomas has not
been elucidated. DMH binds to several other tissues such as the liver,
small intestine, and kidneys, but carcinogenesis in these tissues is signi-
ficantly lower than in the colon (81). It has been postulated that the
higher incidence of DMH-induced tumors in the colon may be due to a delayed
or incomplete repair system. Kanagalingam and Balis (82) found slower
repair of DNA in the colon than in the small intestine when rats were
given a single dose of DMH.

Based on the results from epidemoiological studies and from experi-
mental studies in animals and humans the major dietary factors which have
been implicated in colon cancer are fat, cholesterol, animal protein, fiber
and certain vegetables. The fead component which has been most strongly
implicated is dietary fat. Populations consuming high-fat western diets
are at greater risk for colon cancer (83). Animal studies demonstrate
that colon carcinogens are more effective in animals on high fat diets
(79,84). High levels of dietary fat can increase both the percentage of
animals developing tumors and the number of tumors produced per animal
(42,43,85,86).

The importance of fat content in the diet appears to be related to
its effect on secretion of bile acids and neutral sterols. Reddy and
Nynder (87) demonstrated good correlation between fat consumption, the

level of bile acids and neutral sterols excreted, and risk for colon

18

cancer. Evidence indicates that bile acids and their metabolites may be
important as tunor promoters (88-97) . Because it is necessary for tumor
promoters to be consistently present in appreciable amounts to be effec-
tive, steroid derivatives are likely candidates for this role based on
their constant presence in the large bowel.

High levels of dietary fat increase bile flow and increase production
of bile acids required fer fat absorption from the intestine. The end
result is more bile acids and neutral sterols reaching the large bowel.
The effects of dietary fat on steroid metabolites reaching the colon may
be related to both composition and metabolic activity of the intestinal
microflora (87,92,93).

Studies to investigate the effects of diet on gut flora have yielded
extremely inconsistent results. Some reports suggest that high risk p0pu-
1ations excrete more anaerobes, while low risk populations excrete more
aerobic bacteria (92,93). However, other studies demonstrate no differ-
ence in the number or species of bacteria in feces from high and low risk
populations. Experiments designed to alter the intestinal flora through
dietary modification within individual subjects have been largely un-
successful (94-98).

More recent evidence indicates that alterations in the enzyme activity
of large bowel microorganisms are more important than taxonomic groupings
(95,99,100). Cholesterol dehydrogenase (conversion of cholesterol to
coprostanone), 7 o-dehydroxylase (conversion of primary to secondary bile
acids), and B-glucuronidase (hydrolysis of conjugated compounds) are
inducible enzymes and may be controlled by substrates reaching the colon
and other dietary factors. Populations consuming high fat diets excrete

more secondary bile acids and cholesterol metabolites than p0pulations

19

consuming low fat diets (93). This indicates increased activity of
7o-dehydroxy1ase and cholesterol dehydrogenase. Numerous studies report
an increased ability of intestinal microbes to hydrolyze glucuronides in
populations at high risk for colon cancer (92,101,102).

Colon cancer patients compared with control patients excrete more
secondary steroids and have increased cholesterol dehydrogenase and 7a-
dehydroxylase activity in their feces (99,103). However, it is not clear
whether these changes are etiologic or occur as a result of the carcino-
genic process.

Studies on protein consumption and large bowel cancer indicate that
there is a relationship between animal protein consumption and bile acid
metabolism. Subjects consuming high meat diets excrete higher levels of
acid and neutral steroids and have higher rates of microbial conversion of
these steroids to their secondary products (104). B-glucuronidase activity
is higher in rats fed diets enriched with ground beef than in rats fed
non-supplemented diets (84,93,101,102). However, Graham et al. (105)
report little effect of meat consumption on neutral and acid steroid
excretion or risk for large bowel cancer.

Interpretation of these studies has been complicated by the diffi-
culty in separating meat consumption from fat consumption, as most meat
products are relatively high in fat. The limited number of studies which
have isolated these variables indicate that the importance of high meat
intake may be more related to the level of fat intake than animal protein
consumption per se (87,92).

In addition to diet, other factors such as stress and age appear to
be important in determining the type and quantity of steroid metabolites

reaching the colon. Meore et al. (106) feund that fear or stress produced

20

significantly greater alterations in intestinal microflora than could be
produced by dietary modification. These authors suggested that bacterial
response probably involves epinephrine which affects both bile flow and
intestinal motility. Other studies have shown that the microbial enzymes
cholesterol dehydrogenase, 7a-dehydroxylase, and B-glucuronidase are
significantly affected by age (107).

The inconsistent results relating diet to bacterial species and to
enzyme activity of intestinal microflora makes it difficult to interpret
the relevance of these findings. Failure to standardize methods of col-
lection, storage, and analysis between investigators are responsible for
many of the inconsistencies noted above. Until these technological problems
can be resolved, such studies will provide minimal useful information
related to this aspect of colon carcinogenesis.

Early hypotheses relating dietary fiber intake to colon cancer were
developed by Burkitt (108). There are several possible effects of fiber
as a protective factor against colon carcinogenesis. These include:

(a) decreased intestinal transit time resulting in shorter contact of
potential carcinogens with the intestinal mucosa; (b) increased fecal bulk
resulting in decreased concentrations of potential carcinogens exposed to
the intestinal mucosa; (c) altered composition and metabolic activity of
the intestinal flora; (d) binding of bile acids resulting in increased
excretion; and (e) binding of carcinogens for excretion and consequent
decreased absorption by intestinal cells (108).

Epidemiological evidence suggests that the incidence of colon cancer
is lower in populations consuming high fiber diets (50,109-111). In a
Finnish population examined by Reddy et a1. (89) diet was characterized

by both high-fiber and high-fat consumption. In this population the

21

incidence of colon cancer was low. In the subjects daily neutral

steroid output was higher and daily bile acid output was similar to levels
found in American subjects. However, greater fecal bulk due to increased
fiber intake resulted in decreased steroid concentrations and consequent
dimunition of exposure of these compounds to the intestinal mucosa.

Because there were other dietary variables such as meat and dairy product
consumption which differed between the two population groups, the Specific
effects of fiber could not be isolated in this study. However, the results
do provide support for fiber as one of the dietary modifiers in colon
carcinogenesis.

Experimental studies on fiber intake and colon cancer have yielded
inconsistent results. The protective effect of dietary fiber toward colon
cancer appears to depend on the type of fiber and the particular carcinogen
examined (112-114). In DMH-induced colon carcinogenesis, addition of
wheat bran to the diet was effective in decreasing the total number of
tumors, but not the number of malignant tumors in experimental rats (114).
However, other studies report no effect of other fiber sources on tumor
incidence in the colon.

Investigation of the effects of fiber on colon cancer has been
hindered by difficulties in separating fiber effects from other dietary
variables and problems associated with the use of retrospective diet
histories in epidemiological investigations. Also, dietary fiber has not
been adequately defined, creating problems for both epidemoiological and
experimental studies. Clarification of these issues will be beneficial in
elucidating the effects of dietary fiber on colon carcinogenesis.

In addition to the effects of fat, fiber and other dietary consti-

tuents on substrates which pass through the large bowel and the metabolism

22

of these substrates by endogenous microflora, food intake may affect the
ability of the colonic mucosa to metabolize compounds which it is exposed
to. Several studies demonstrate that intestinal mucosa has a MFO system
which can metabolize drugs and carcinogens (46, 115-118). Autrup et al
(115) have cultured human colon explants which can convert native BP to a
metabolite (7,8-dihydroxy BP) which can bind to colonic DNA. There was
considerable interindividual variation in BP binding to DNA. In these
studies the total level of metabolites produced by human colon explants
was similar to the level produced by rat colon explants, but there was a
higher ratio of organic-soluble derivatives and binding was higher in
human colon explants. These results indicate that rats detoxify more of
the primary metabolites of BP. Human colon explants fermed only guanine
adducts (N-Z amino group) while rat colon explants formed adducts with
both guanine and adenine.

The data to date clearly indicate that nutrition is a determining
factor in the etiology of colon cancer. Based on the evidence which has
accumulated, the current focus in colon cancer research must be directed
toward identifying causative, protective, and modifying factors in the
diet and determining the mechanisms by which they exert their effects.

In this study I will describe a model system used to study dietary-
induced alterations in metabolism of xenobiotics by intestinal tissues.
Within this model system charcoal-broiled hamburger was used as a dietary
source of xenobiotics, and BP was used as a model substance to evaluate
xenobiotic metabolism. The relevance of dietary-induced alterations in
BP metabolism to colon carcinogenesis was evaluated by measuring the
mutagenicity of BP metabolities and by measuring binding of these metabo-

lites to cellular macromolecules.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats weighing 100-125 grams were obtained from
Spartan Research Corporation, Haslett, Mi. 48864. The rats were housed
individually in wire-bottom cages and maintained at 20: 2° with a 12/12

hour light/dark cycle. Food and water were provided ad libitum.

mats
Compositions of the basic experimental diets are shown in Table 1.
These diets were used for all studies except with the modifications as
noted in Results and Discussion. Unless otherwide indicated experimental
diets were fed for two weeks. Analytical compositions of the experimental
diets are described in Results and Discussion. Diets were dried at 80°
to a constant weight. Lipids were extracted from the dried diets with
diethyl ether. Protein was determined by Kjeldahl nitrogen (119) and is

expressed as crude dietary protein.

Enzyme Induction

 

Animals induced with B-napthoflavone (Aldrich Chemical Co., Inc.,
Milwaukee, Wi.) received one intraperitoneal injection of B-naphthoflavone
(80 mg/kg body weight) in corn oil for 3 successive days befOre sacrifice.
Animals induced with Aroclor (Monsanto Co., St. Louis, Mo.) received one
intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) in corn

oil for 3 successive days before sacrifice.

23

24

Table l.--Composition of Experimental Diets.

 

% Composition by Weight

 

 

Control CBH
Cornstarch 68 --
Casein 20 --
Charcoal-broiled Hamburgera -- 88
Sucrose, Minerals, Corn Oil, Vitaminsb 12 12

 

aTen pound logs of ground hamburger containing 25 percent fat
before charcoal-broiling were thawed and sliced into 1 cm thick patties.
The meat patties were grilled approximately 5 cm from the coals for
approximately 10 min per side until very well done.

bSucrose, 5 percent; minerals, 4 percent (salt mix, 4164, Teklad,
Inc., Madison, Wi.; in g/100 g: calcium acetate. H20, 6.293; calcium
diphosphate ° 2 H 0, 28.525; dipotassium phosphate, 28.443; ferric
citrate - 5 H20, 2.44; magnesium sulfate - 7 H20, 10.053; potassium iodine,
0.65; sodium diphosphate - 12 H20, 14.630; sodium chloride, 9.546); corn
oil, 2 percent (Archer Daniels Midland Co., Decatur, 11.); vitamins,
1 percent (thiamine, HCl, llg; pyridoxine, 11g; riboflavin, 11g; calcium
pantothenate -'33g; p-aminobenzoic acid; 55 g; menadione, 25g; inositol,
50g; ascorbic acid 100g; niacin, 50g; vitamin B12, 15mg; biotin, 0.3g;
folic acid, 2g; retinol acetate, 1x107 IU; a-tocopheIOI, 50,000 IU;
vitamin 03, 110,000 IU; and cerelose to 5kg).

25

Preparation of Tissue Microsomes

Rats were sacrificed by decapitation and microsomes were obtained
by a modification of the method of Fang and Strobel (116). Colons were
excised at the ileal-cecal junction and the rectum, and small intestines
were excised at the pyloric sphincter. The tissues were rinsed with tap
water and then with a 154 mM NaCl, 1 mM dithiothreitol solution, weighed,
and placed in ice-cold Tris buffer (10 mM, pH 7.4) containing 140 mM KCl,
10 mM EDTA, 1 mM dithiothreitol, and 0.25 mM phenyLmethylsulfonylfluoride.
Livers were removed, blotted, weighed, and placed in ice-cold Tris buffer.
All tissues were homogenized in a Polytron tissue homogenizer (Brinknan,
Westbury, NY) and centrifuged at 8,500g for 6 min. The supernatant was
centrifuged at 105,000g for 60 min to obtain the microsomal pellet. The
pellet was resuspended in 0.25 M sucrose, 10 mM EDTA solution and stored

at -40°.

Enzyme Assays

 

The fluorometric assay for aryl hydrocarbon hydroxylase (AHH) was
perfbrmed as described by Nebert and Gelboin (120). The reaction mixture,
in a total volume of 1.0 m1, contained 50 umoles Tris buffer, pH 7.5,

0.36 umole NADPH, 3 umoles MgClZ, 0.8 mg bovine serum albumin, 75 nmoles
benzo(a)pyrene (BP) and 200-600 ug microsomal protein. The mixture was
incubated with shaking at 37° for 30 min (unless otherwise specified).
The reaction was stopped by addition of 1.0 ml cold acetone. BP metabo-
lites were extracted in 3.25 ml hexane by shaking fer 10 min at 37°. A
1.0 ml aliquot of the organic phase was extracted with 3.0 m1 of l N NaOH
which had been previously extracted with benzene to remove fluorescent

contaminants. After centrifugation, BP metabolites in the alkali phase

26

were measured spectrofluorometrically with excitation at 396 nm and
emission at 522 nm. Blank values, obtained for each tissue by addition
of acetone to the reaction mixture before incubation, were subtracted
from reported values.

The radioactive assay for AHH was performed by a modification of the
method of VanCanfort et al. (121). The reaction mixture, in a total volume
of 0.5 ml, contained the same reagents described for the fluorometric assay

3H BP (0.116 uCi/ug) substituted for the

at 50% the level described, with
unlabeled BP. After incubation at 37° for 30 min with shaking the reaction
was stopped with 1.0 ml KOH (0.15 M in 85% DMSO). The unmetabolized BP

was extracted in 5.0 m1 hexane by mechanical shaking for 5 min. After
centrifugation the upper and interphases were removed and the sample was
re-extracted with hexane. A 0.5 ml aliquot of the aqueous phase was
neutralized with HCl, brought up in 20 ml Triton-toluene scintillation
cocktail (1:2; 4 g 2.5-diphenyloxazole, 0.6 g 1,2-bis [2(4-methyl-5-
phenylazoxyl)] and counted by liquid scintillation spectrometry. Blank
values were obtained by addition of KOH to the reaction mixture before

incubation. Protein concentrations were determined by the method of Lowry

et al. (122).

Ames Test

The Ames test was perfOrmed according to the standard procedure
described by Ames et al. (123) using the S-9 fraction from the colon, small
intestine, and liver from rats fed the various diets. To obtain the S-9
fractions, tissues were homogenized in 3 volumes of 0.15 M KCl, centrifuged
at 9,000g for 10 min, and the supernatant was collected. The fractions

were frozen at -40° until analysis. The procedure was carried out as

27

previously described using Salmonella typhimurium strain TA 100. S-9
fractions were tested at several protein concentrations, and the level

resulting in maximum number of revertants was used fer subsequent assays.

Isolation of Colonic Epithelial Cells

 

Epithelial cells were isolated from colons of experimental rats as
described by Skrypec and Bennink (124). The colon was removed, flushed
with a 154 mM NaCl, 1 mM dithiothreitol solution and tied at the ends with
thread to hold the solutions required fer dissociation of the epithelial
cells. Residual intestinal contents were removed by solution A (1.5 mM
KCl, 96 mM NaCl, 27 mM Na3C6HSO7, 8 mM KH2P04, 5.6 mM NaZHPO4, pH 7.3) by
incubation at 37° for 15 min. The mitotically active epithelial cells
were dissociated by solution B (phosphate buffered saline, Ca and Mg free,
1.5 mM EDTA, 0.5 mM dithiothreitol) by incubation at 37° for 52 min. The
colon was rinsed twice with phosphate buffered saline to obtain the maximum
number of cells. The solutions were combined and cells were collected by
centrifugation. The cells were then immersed in culture medium [CMRL-
1066 with glutamine (GIBCO)] supplemented with 5 mg/ml glucose, 0.05 mg/ml
fetal bovine serum, 125 U/ml penicillin, 125 meg/m1 streptomycin, 2 meg/ml
amphotericin, 0.67 mcg/ml hydrocortisone, and 10 mM minimal essential

median nonessential amino acids.

Cell Cultures

 

Isolated epithelial cells were incubated with BP for 24 hours by
addition of l ug/ml 3H BP (0.116 uCi/ug) to the media. BP metabolism was
determined by analyzing a 5.0 ml aliquot of the media according to the

method of VanCanfort et al. (121) described above.

28

Binding of BP to Cellular Macromolecules

3n BP (0.2 uCi/ng, dissolved in so ul methanol)

Rats were given 50 ng
in 2 ml saline by gavage 12 to 48 hours before sacrifice. The tissues
were removed, rinsed thoroughly with water, and homogenized in 3 volumes
of water. Cellular macromolecules were isolated from an aliquot of the
homogenized tissues using a series of extractions. Cellular macromolecules
were precipitated twice with ice cold 10% trichloroacetic acid. The pre-
cipitate was washed twice with ice cold 1% potassium acetate in ethanol
to remove lecithins, twice with ethanol:chloroform (3:1,v:v) and once each
with ethanol:diethy1 ether (3:1,v:v) and diethyl ether to remove remaining
lipids (125). The resulting precipitate was solubilized by addition of
NCS tissue solubilizer (Amersham, Arlington Heights, 11.) and heating at
50° for 12 hr with shaking. The solubilized sample was neutralized with
HCl, added to 20 ml Triton-toluene scintillation cocktail described above
and counted by liquid scintillation spectrometry. In another study
epithelial cells were isolated (as described above) from experimental

rats which had been administered 3H BP by gavage. The cells were solubi-

lized and counted as described above.

Isolation of DNA

 

DNA was isolated from the tissues of rats intubated with 3

H BP by
phenol extraction as described by Jernstrom et al. (126). DNA was quanti-
tated on a Gilfbrd Spectrophotometer (Model 240; Gilford Instrument
Laboratories, Oberlin, Ohio) with absorbance at 260 nm. DNA.was resuspended

in water and binding of BP to DNA was determined by liquid scintillation

spectrometry as described above.

RESULTS AND DISCUSSION

The purpose of these studies was to develop a model system to study
dietary-induced alterations in intestinal metabolism of potentially toxic
agents entering the body. In this model system charcoal-broiled hamburger
(CBH) was chosen as the source of dietary xenobiotics and benzo(a)pyrene
(BP) metabolism was chosen as the model for microsomal xenobiotic
metabolism. AHH activities in the colon, small intestine, and liver
of rats fed diets containing CBH were compared to AHH activities in

tissues from rats fed purified diets.

Enzyme Activity

 

Results from rats pretreated with B-naphthoflavone, an inducer of
AHH, showed that B? metabolism (according to the fluorometric assay) was
linear both with time (up to 40 min; Fig. 3) and with protein concentra-
tion (up to 600 ug of microsomal protein/reaction; Fig. 4). Freezing the
microsomes did not affect enzyme activity.

The fluorometric and radioactive assays produced similar results
(Fig. 5) and were consistent between studies. The difference in liver
AHH activity between control and CBH-fed rats was greater when measured
by fluorometry than by radioactivity, whereas in the colon and small
intestine the difference in dietary-induced AHH activity was greater
when measured by radioactivity. These results may reflect differences in
the metabolites produced, as the fluorometric assay measures primarily

3-hydroxy BP, while the radioative assay measures all metabolites of BP.

29

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Because the direction and degree of response showed a high correlation
(r2=0.92) between the two assays, only the results of the radioactive assay
will be presented for the remaining studies (with the exception of one
study for which only the results of the fluorometric assay are available).

Feeding the CBH diet resulted in significantly increased AHH activity
in the colon, small intestine, and liver (Fig. 5). These diets also
resulted in small intestinal and liver hyperplasia (Table 2). Small
intestine weight was approximately 1.6 times the control and liver weight
was approximately 1.5 times the control when expressed per gram of body
weight. Colon weight was increased in rats fed the CBH diet, but these
differences were not significant. Due to this hyperplasia, the differ-
ences 32.!itrg_BP metabolism which are expressed per mg protein in these
studies are even greater when expressed on an organ weight basis.

Enzyme activity in colon and small intestinal microsomes from rats
fed the control diet was minimal. Liver microsomes from rats fed the
control diet metabolized BP in_!itrg_indicating the presence of constitu-
tive enzyme in this tissue. This is supported by previous studies on
cyt P448 activity in livers from non-induced experimental animals (127).

When the CBH diet was fed, the colon and small intestine had marked
increases in in vitrg metabolism of BP over control levels. Livers from
rats fed the CBH diet showed a 3 to S-fold increase in enzyme activity.

It is probable that the CBH contained high levels of compounds which are

normally metabolized by microsomal cyt P enzyme pathways resulting in

448
significant induction of AHH. These results indicate that the liver has
a moderate basal level of AHH activity which can be increased by exposure

to environmental xenobiotics, while the colon and small intestine have

37

Table 2.--Hyperp1asia of the Liver, Small Intestine and Colon of Rats
Fed Charcoal-broiled Hamburger.

 

Organ Weight(g)/100 g Body Wgt

 

 

Small
Colon Intestine Liver

Study A (175-200 g rats)

Control 0.68:0.03 3.42:0.16 5.04:0.13

can 0.73:0.06 5.24:0.20a 7.11:0.17a
Study B (225-250 g rats)

Control 0.47:0.02 2.64:0.11 4.36:0.24

can 0.55:0.02 4.29:0.17a 6.84:0.19a

 

8Significantly different from control (P<0.05).

38

very low basal levels of AHH, but respond to xenobiotic exposure by dra-
matically increasing microsomal metabolism,

The time required to induce AHH with CBH feeding and the time
necessary for enzyme activity to return to control levels when rats were
refed the control diet are shown in Figure 6. Enzyme activity was in-
creased in all tissues by feeding the CBH diet for two to four days, or
after consumption of 20 to 50 g of diet. In the liver maximal AHH activity
was reached by consuming the CBH diet for four days. When rats were refed
the control diet, liver enzyme activity returned to control levels within
three days. Enzyme induction was also maximal by four days in the small
intestine, but return of enzyme activity to control levels required five
days of refeeding the control diet. The colon was the slowest tissue to
adapt to dietary-induced changes in AHH activity. IE zitgg BP metabolism
was maximal by the tenth day of CBH consumption and return to control
levels required five days of refeeding the control diet. Since enzyme
activity in the colon adapted to refeeding the control diet similarly to
the small intestine, the slower rate of adaption in the colon was not due
solely to the time required for the dietary constituents to reach this
tissue.

Figure 7 shows a dose-response curve for the level of CBH in the diet
and in XEEEE BP metabolism. AHH activity was directly proportional to and
showed high linear correlation with the level of CBH in the diet for the
colon (r2=0.99), small intestine (r2=0.97), and liver (r2=0.85). The
lower coefficient of determination in the liver resulted from maximal
stimulation of enzyme activity in this tissue at a lower level of CBH in
the diet. Based on the high correlation between tissue microsomal enzyme

activity and the level of CBH in the diet it can be concluded that it was

39

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.e.me .meeaoue> one mfioeoeea .ooeaoacooeoo “H.e~ .ewouoed me.o~ .eedefi

mm.mH .onSHmwoa umwv :owpwmomeou .uofip :mu ca :mu mo ucmwoz Hence you peanufiumnom goaepmehoo wane

.n.mo .mceeeufi> pee maeuocfis .ouenvznonneo mo.~H .cwouonm me.m .ewmwa
"H.0H .onzumeos "flay :ofiuwmoQEOU .uoflv mmu a“ mac mo ucwfloz Hence Hem popspwumnom :oneumcaoo anon

.uofin on» we 9:09:00 Homnnoeem ou popeaom mm Suw>wuo< ommfixxonvx: :ooneoonex: Hxh< .h ousufim

RADIOACTIVE ASSAY

42

 

 

     
   
 

   
 

 

 

3W

 

 

 

 

 

 

.—F~

 

I
a
D liver

"F" Al

I I L l J l J
§ § § § § § §
1- 1-

Illlll oz [ulelold 6m
[puzuoqeloul 49 Du

50°/ob 75%° P-F Had

level at hamburger In dlel

25%"11

O

43

the CBH which caused AHH induction in these three tissues. However, it
is not clear what factors within the CBH are responsible for the increase
in enzyme activity.

AHH induction in the tissues of rats fed diets containing 88% pan-
fried hamburger (P-F HB; Fig. 7) varied when compared to induction in rats
fed diets containing 88% CBH (Fig. 5). In the P-F HB-fed rats enzyme in-
duction in the liver and small intestine was similar to that in CBH-fed
rats. In the colon, feeding the P-F HB diet produced a lower level of
induction than did feeding the CBH diet. This difference in enzyme in-
duction may represent differences in the level and type of pyrolysis
products produced by pan-frying the hamburger.

To determine whether it was the meat itself or agents produced when
the meat was cooked which induced AHH, rats were fed a diet containing
uncooked hamburger which had been extracted with hexane and acetone to
produce a moisture and lipid content comparable to the CBH. Results from
the fluorometric assay fer the colon and small intestine are presented in
Figure 8. Feeding the raw meat diet did not increase enzyme activity over
control levels fer any of the tissues examined. This indicates that it
was cooking the meat which resulted in production of compounds which
induce microsomal metabolism. It also shows that differences in dietary
protein per se are not responsible fer the induced AHH activity observed
with feeding CBH.

Analysis of foods which have been cooked by various techniques have
shown readily detectable levels of BP and other PAH in charcoal-broiled
meats (57,64,67,68). To determine the effects of feeding isolated PAH on
enzyme activity, the addition of two levels of BP to the experimental diets

was examined. Addition of BP (0.1 mg BP/g of diet) to the control diet

44

.onoHH one onnNNm .mr
.eene “Home one anode .w\ne man. + new ”meme one meson .nnu tonne one eeenm .M\nn one. + u tonne
one nnennm .m\nn one. + u «anew one meeom .u "one: new ennee enmeoz one “we nonennnenoo ooon

.m.mH .mnfiaopo> one mHononHe .ouonoxnenneo ”H.0o .noopenm mo.m~ .owmflfi mo.m~ .onnumoea "meg
neopomenneo .uooo :mu no :mu new m: npxo me nonunpoumonm one on one .zmu onu ep oHnenomneo
unopneo ofimoa one onnpmnen o oonoenm ea onouooe one onoxon noes ooueenuxo Howannnon some

.xuo>opo< omefixxeno»: neoneoenoxm H>H< no ononznmeveenom Someone me ueomwm .w onnwfim

FLUOROMETRIC ASSAY

Intestine

fl small

1

45

l

unu oz [amend 6m
[mun 00000801011”

10-

 
  

a

BP/g

c +.1 mg 0+.5 mg CBH CBH +.1 mg extr. HB
BP/g

BP/g

C

46

significantly increased ipuyitgg' BP metabolism in the colon and small
intestine (Fig. 8). Enzyme induction was not as great as that seen with
feeding CBH even though the level of BP added was probably greater than
the level of BP in the CBH diet. Additional BP (0.5 mg BP/g of diet) did
not cause a greater increase in 32 yitrg_metabolism by colon microsomes.
Enzyme activities were not increased by addition of BP to the CBH diet.
In the study of Clinton et al. (128), addition of BP (1.0 mg BP/g of diet)
to a purified diet also increased AHH activity in the small intestine, but
enzyme levels were higher than levels reported here. It is not clear
whether this difference in results is due to differences in experimental
techniques or whether enzyme activity would have been greater if a higher
level of BP had been fed. Based on the results from my study alone, it
appears that there is a maximum level to which AHH can be induced by
feeding BP alone, but the presence of other inducing factors in the diet,
as would occur in the CBH, can produce greater levels of AHH activity.
This is supported by the study of Gielen and Nebert (129). These investi-
gators obtained an additive effect for in XEEEE AHH induction in cultured
liver cells when either phenobarbital or 2,2-bis(p-chloropheny1)-1,1,l-
trichloroethane (p,p'-DDT) was present with a PAH, but not when two PAH
nor when phenobarbital and p,p'-DDT were combined. In my study, the extent
of enzyme induction after feeding a mixture of inducing agents may be
maximal, and therefore, enzyme activity was not increased by feeding
additional amounts of one of the inducing components (BP).

It has been proposed that the occurrence of PAH in charcoal-broiled
meats results from fats dripping down onto the hot coals and producing
pyrolysis products which rise in the smoke to condense on the surface of

the grilled meat (67). To assess the importance of fat content in

47

hamburger on the production of inducing compounds, hamburger with varying
levels of fat was charcoal-broiled and fed to rats (Fig. 9). There was
high linear correlation between enzyme activity and the level of fat in

the hamburger for the colon (r2=0.97) and small intestine (r2=0.96).

Liver enzyme activity also increased with higher fat levels in the ham-
burger, but the coefficient of determination was lower (r2=0.6l) due to
maximal stimulation of enzyme activity in this tissue at a lower percentage
of fat in the hamburger. These results support the concept that fat in
hamburger is one of the major substrates fer pyrolysis leading to ferma-
tion of xenobiotics in CBH.

Consumption of diets containing heated fats was not sufficient to
induce microsomal metabolism in the manner seen with feeding CBH (Fig. 10).
When the drippings from the pan-fried hamburger of the earlier study
replaced cornstarch in the control diet to comprise 20% of the total diet,
in XEEIQ.BP metabolism increased in the colon, but not in the small
intestine or liver. Diets containing beef tallow which had been pan-fried
at conditions similar to the pan-frying of the hamburger, or beef tallow
which was held at 180° for 150 hrs did not increase enzyme activity above
control levels for any of the tissues tested.

These results are in support of the findings of Rappaport et a1. (68).
In their studies, when ground meat was cooked at high temperatures in open
or closed systems, greater than 90% of the mutagens fermed were volatilized
into the air. The level of PAH in meats cooked in closed systems was
significantly higher than in meats cooked in open systems. In my study,

a high percentage of the compounds which result in AHH induction were
probably volatilized under the conditions of both pan-frying and charcoal-

broiling. In charcoal-broiling the volatilized components which rose in

48

.meen one onenme .eme tonne one eenem
.emm tonne one memmm .em “None one oeomm .u "one: new ennem oneees one new noeonnnenoo ooon

.o.nH .mnonepo> one maenonon .oueeoxnenneo

“n.em .nneeoen an.o~ .oenen m~.ee .oenoeeon ”noeoneonnou .eoeo one no one nee: nonenoeoenne

one on one .mnofifienouaoeenone onomon pom wmo nomeneo op zeHHou woos ooooe no“: Homnnnaomn

.m.oH .mnoeouo> one maeaonon .oponoxneonno.
mo.mm .noouenn an.o .ofinwfi mo.mm .onnpmoen "neopflmemneu .uooo :mo no :mu nun: nomenpopm
room one on one .mnfifioennuaoeoneno onemoo pom em nfiopneo op onexon new: ooueonuxo nowannnemo

.xufi>opo< omofixxonoxm nonnooenox: H>n< no nomnnnnem no Ho>og you me poommm .m enamom

49

39.552. 5 =3 .e .03.

Horne

can“ erm

 

 

 

  

 

 

 

 

 

 

 

 

 

 

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0532:.
=25. a

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ulw oz [umold Bun
[pozuoqeleul :18 Du

50

.meme one eneone .nnu ”meme one eenmm .en own .oen nee .oenm one eeeee .oeo n-n
mononn one meson .m: n-n none oeo meemoH one eeone .u "one: new eneee oneeoz one new nonennnenoo ooon

. .o>ene e oom "newpomemneu
.uooo u no noneumnneo we unmooz Hence new oounpwumonm an omH .oowH we oHon zefifieu moon aowo

.o>eoe e oom "newuomenneu

.uowo u no nonoumnnee me unmwoz Hence Hem oounuoumonm an: N .ooomu reflfieu moon oooHMunom acme

.o.~m
.mnoneuo> one mHenonoa .ouenoxnennee no.5H .nwouenn mo.- .ownofi mo.m Honeymoon "may neopwmenneu
.uowo o no noeeumnnee me unwooz Hence new oounuoumnnm nonhuman: ooonmnnon nenm manomnono new acme

.xuo>ouo< omofixxenoxm nonnooenoxz th< no menu ooueo: mnoooom we ueomwm .ofi enamom

51

:3

Our—OM? mm: n—afl

.8. .2 an. a... 52. .e.

 

 

 

 

 

 

 

 

 

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52

the smoke could combine with some non-volatile component (such as protein)
on the surface of the meat on the grill. This is further supported by the
studies of Hecht et al. (130) where formation of BP in charcoal-broiled
meat was prevented by placing foil between the meat and the coals. In my
study, when the fats alone were subjected to high temperatures, the
majority of inducing compounds were probably volatilized with minimal
levels retained in the fat itself. These findings may partially explain
the higher incidence of certain forms of cancer in kitchen workers (131).
The results shown in Figure 10 also demonstrated that fat content of the diet
was not responsible for increased enzyme activity observed with higher
dietary fat in the basic CBH diet compared to the control diet.

To study the effects of other dietary components on AHH activity,
various fiber sources were added to either the control or the CBH diet.
Fiber has been proposed to be involved in colon cancer through several
mechanisms. Fibrous compounds which act to dilute colon contents and to
decrease transit time are believed to be beneficial in decreasing exposure
of the intestinal epithlium to potentially carcinogen agents. Fiber
sources which contain indole compounds or chlorOphyll increase mixed
function oxidase activity and consequently modify the metabolism of xeno-
biotics.

Figure 11 shows the effects of addition of alfalfa (a fiber contain-
ing compounds which have been shown to induce AHH), wheat bran (a fiber
which decreases transit time and dilutes colon contents), or cellulose to
the various diets. Replacement of 10% of the cornstarch in the control
diet with alfalfa meal increased.in“!itrg'BP metabolism in the small
intestine, but not in the liver or colon. Substitution with 10% cellulose

had not effect on enzyme activity in any of the tissues studied.

S3

.onnw one women .m: + :nu tonne one neemn .nno “meme one mnmoe .noo + u
meenOn one meemm .one + 0 «women one neene .e "one: new ennee onenen one new nonennnenoo ooon

.m.- .mnonopo> one mfienonon .ouenoxnenneo “H.5m .noopenn mm.oH noonwa
mm.m~ .onnumoen "neoufimemneo .uooo :mu no Inc we unmfloz Amoco new oounuopmonm none neon: wage

..o>ene e oou "nefiuwmenneu .pooo :mu no man we usage: Hence new oounufiumonm omeHnHHoo wean

.o.wo .mnfinouo> one mHenonon .ouenoxnenneo no.5H .nwouenm mo.N .ofinfifi
mm.- .onnumoen “neouomenneu .uowo :mu no :mu me unwooz Hence Hem ooanpopmnnm anon awaomHn *oHe

.xuo>wpo< omefixxeeoxz neonoeenokz HxH< no meannem Henna mnewno> unwooom me ueommm .HH onnmom

RADIOACTIVE ASSAY

, small
" Intestine

12M-

54

g
g
0

51’1’1’7///////////////////

 

 

CBH

  

 

+-

C-t-celb

 

-F

c +aIF°

 

 

S
0 liver
0

l

L
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150

1050 l-
900 h-
750

liltfl oz [Ulflimd bur
linoleum“: ea 6"

55

When wheat bran replaced 10% of the CBH in the CBH diet the expected
decrease in microsomal metabolism did not occur. Instead enzyme activity
increased in the small intestine and remained the same in the colon and
liver even though less meat was fed. These results are in contrast to
the findings of Clinton et al. (128). When they added 10% wheat bran to
a purified diet containing 1 mg BP/g diet, AHH activity in the small
intestine was significantly lower than when wheat bran was not present
in the BP diet. In their study colon and liver enzyme activities were
not reported. It is probable that feeding an isolated xenobiotic in a
purified diet results in more specific enzyme induction than does feeding
a diet containing a mixture of xenobiotics. When rats consumed the CBH
diet numerous enzyme pathways were probably induced to metabolize the
various compounds to which the tissues were exposed. Thus, it is plausible
that wheat bran added to the CBH diet did not lower AHH activity, while
addition of wheat bran to a purified diet containing BP did lower AHH
activity.

The mechanism by which wheat bran decreased AHH activity in the study
of Clinton et al. (128) probably involved a decrease in the absorption of
BP which would be beneficial to the host. However it is not clear whether
wheat bran had a specific effect in decreasing AHH activity independent of
carcinogen absorption. If so, addition of wheat bran to a diet containing
agents which are metabolized by AHH may not benefit the host. Decreased
AHH activity could decrease conversion of xenobiotics to forms which can
be excreted from the body. The proposed benefits of fiber in relation to
colon cancer probably come from its action in diluting the concentration

of potential carcinogens, decreasing intestinal transit time, and

56

decreasing carcinogen absorption rather than any possible direct action
on AHH activity.

The combined results of the time-induction study, the dose response
study, and the study with varying levels of fat in the hamburger provide
insight to differences in enzyme response of the colon, small intestine,
and liver to dietary xenobiotics. Upon exposure to compounds capable of
inducing AHH, liver enzymes respond rapidly with lower levels of inducing
compounds in the diet. On the other hand, enzyme response in the colon
and small intestine is slower and requires higher levels of inducing agents
to reach maximum. However, the relative change in enzyme activity between
the control and the induced colon and small intestine is many times greater
than the relative change in the liver.

A principal function of the liver is to metabolize xenobiotics, so it
is able to carry out this function with moderate changes in enzyme activity.
However, in other tissues, such as the colon and small intestine, drug
metabolism is not a principal function. In these tissues enzyme systems
must adapt dramatically to handle increased exposure to compounds which

require metabolism to be excreted from the body.

Cell Cultures

 

To determine whether microsomal metabolism was representative of
whole cell metabolism under the conditions of these studies, an in XEEEQ
system was devised to measure BP metabolism in isolated colon epithelial
cells. Using the technique developed in our laboratory (124), epithelial
cells were isolated from the colon of rats fed the control or the CBH
diet fer two weeks. Cells were cultured and incubated with 3H BP for

24 hours. Results are shown in Table 3. Colon epithelial cells from

57

Table 3.--Benzo(a)pyrene Metabolism by Isolated Colonic Epithelial Cells.

 

ug BP metabolized/24 hrs

 

Control 0.76:0.05

CBH 1. 1720.04ail

 

aSignificantly different from control (P<0.05).

rats fed the CBH diet produced a greater level of BP metabolites than did
cells from rats fed the control diet. The higher level of BP metabolites
may have been due to an increase in the number or viability of cells or
due to increased metabolism by the same number of viable cells. Regard-
less of the reason for increased metabolism, the results of this study
support the results of the previous studies, although the magnitude of
response was not as great as that seen in the microsomal incubations.

The importance of the alterations in enzyme activity which has been
described in these studies is not well-defined. AHH induction can serve
two opposing roles. As a normal mechanism of drug metabolism, increased
AHH activity allows for conversion of fereign compounds to metabolites
which can be excreted from the body. This results in decreased toxicity.
However, many of the metabolites produced through AHH activity are more
toxic or more carcinogenic than the parent compound. Thus, increased

AHH activity may also increase toxicity.

Ames Test
To assess the importance of the enzyme alterations produced, in_vitro

mutagenicity of the BP metabolites produced by microsomes from tissues of

58

rats fed the control or the CBH diet and rats which were pretreated with
B-naphthoflavone was determined (Table 4). Native BP is not mutagenic in
the Ames test, but microsomes from induced mammalian tissues can convert
BP to derivatives which are mutagenic. Pretreatment of rats with Aroclor
1254 induces liver enzyme activity and results in production of mutagenic
metabolites when microsomes from this tissue are incubated with BP in the
Ames test. In these studies BP activation by liver S-9 fractions from rats
pretreated with Aroclor 1254 was used as a positive control. As can be
seen from Table 4, liver microsomes from Aroclor-pretreated rats metabolized
BP to highly mutagenic products. When the S-9 fractions from the colon,
small intestine, and liver of rats fed the CBH diet'were incubated with

BP, there was no increase in activation of BP to mutagenic derivatives
compared to rats fed the control diet. The S-9 fraction from the liver

of rats pretreated with B-naphthoflavone produced more revertants than

the S-9 fraction from control rats. S-9 fractions from colon and small
intestinal tissues produced a similar number of revertants for control
rats and rats pretreated with B-naphthoflavone.

These results are in contrast to the findings of Fang and Strobel
(116,117). In their studies the S-9 fraction from the colon of rats
pretreated with B-naphthoflavone was incubated with BP and strain TA 100
in the Ames test, and there was a four-fold increase in the number of
revertants produced over revertants produced by colon microsomes from
control rats. The difference in results between these two studies was
not due to differences in S-9 protein concentrations, as several protein
levels were assayed in my studies, and the level producing the maximum
number of revertants per plate was used. The reason for this discrepancy

in results is not clear.

59

Table 4.--Activation of Benzo(a)pyrene by Colon, Small Intestine and
Liver S-9 Fractions.

 

Revertants/mg S-9 Protein

 

 

Colon Small Intestine Liver
Control 44 1 9a 41 t 7 37 t 6
can 53 t 13 47 t 10 51 t s
B-naphthoflavone 37 t 12 40 t 6 190 t 18b
Aroclor 956 2 104b

 

aMean t SE for at least 5 separate determinations. Values cor-
rected for spontaneous revertants.

bSignificantly different from control (P<0.01).

60

The results from this study indicate that the metabolites which are
produced specifically by tissue microsomes from rats fed the CBH diet are
not mutagenic in 11332, However, in colon cancer the colon microflora
may play a prominent role in metabolism of compounds which enter the
colon. This makes it especially important to also investigate in X312

test systems related to potential mutagenicity or carcinogenicity.

Binding of BP to Cellular Macromolecules

 

One of the important concepts for initiation of carcinogenesis is the
binding of carcinogens to cellular marcomolecules. There is a high corre-
lation between carcinogenesis and binding of carcinogens to DNA (10-12).

To measure this parameter rats were given 3

H BP by gavage 12 to 48 hours
before sacrifice to assay binding of BP to cellular macromolecules.
Binding varied with time (Fig. 12), and was maximal at 24 hours. Figures
13 and 14 show the amount of BP bound per organ and the amount of BP bound
per gram of tissue at 24 hours. Tissues from rats fed the CBH diet bound
significantly more BP than tissues from rats fed the control diet. Colons
from all rats bound more BP than small intestines or livers.

When DNA was isolated from colons and livers which bound BP no radio-
activity could be detected. This indicates that binding was non-specific
and may explain why BP does not act as a carcinogen for these tissues.
Formation of adducts with non-critical target sites may represent a
mechanism of removal of BP in these cells. However, if metabolism of
compounds which do act as colon carcinogens is affected by diet in the
same manner as BP metabolism, the alterations in enzyme activity may
result in binding to DNA in addition to binding to RNA and/or protein.

The extent of BP binding to RNA and/or protein was not consistent

with AHH activity for either the CBH-fed rats or rats which had been

61

Figure 12. Binding of Benzo(a)pyrene to Cellular Macromolecules in
Tissues of Rats Fed Charcoal-broiled Hamburger.

aNanograms of BP bound to cellular macromolecules per organ.

62

 

 

12' 24 48

 

7//////A
7////

   

iNTESTINE

_ SMALL
fl LIVER

IICOLON

 

 

 

4
m

12 24 48

HOURS

63

Figure 13. Effect of Feeding Charcoal-broiled Hamburger and Pretreatment
with B-naphthoflavone on Binding of Benzo(a)pyrene to Cellular
Macromolecules I.

aNanograms of BP bound to cellular macromolecules per organ at 24 hours.

noBP‘

1.8

1.5

 

I colon

email
Intestine

liver

 

control

 

 

 

 

 

CBH

 

65

Figure 14. Effect of Feeding Charcoal-broiled Hamburger and Pretreatment
with B-naphthoflavone on Binding of Benzo(a)pyrene to Cellular
Macromolecules 11.

aNanograms of BP bound to cellular macromolecules per tissue at 24 hours.

no BP/o oi tissue a

d
b

.5
.N

d
o
O

 

66

I colon

. small
intestine

U liver

 

 

control CBH

 

 

67

pretreated with B-napthoflavone. Pretreatment with B-napthoflavone
results in maximum enzyme induction in colons approximately 1.5 times the
level of induction with feeding CBH. The colon from rats pretreated with
B--napthoflavone bound more BP than did the colon from rats fed the CBH
diet. In the small intestine and liver pretreatment with B-naphthoflavone
results in enzyme activity comparable to that feund with feeding CBH.
These tissues from rats pretreated with B-napthoflavone bound signifi-
cantly less BP than tissues from CBH-fed rats. With differences in repair
rates between colons, small intestines, and livers, and the probable
presence of more than one type of inducer in CBH, it was not unexpected

to see differences between AHH activity and binding of BP to cellular
macromolecules at 24 hours. By 48 hours after intubation the level of
binding decreased for all tissues (Fig. 12), probably due to further
repair of RNA and/or protein.

The higher level of binding of BP to RNA or protein in the colon
indicates that more reactive intermediates may have been exposed to the
colon than to other tissues. It is possible that these intermediates
were produced from interaction between tissue AHH and activity of colon
microflora. Conjugated metabolites produced in the liver and excreted
via the bile enter the colon where they are exposed to colon microflora.
Activity by the microorganisms in the colon can result in deconjugation
of the compounds, exposing epithelial cells of the colon to the uncon-
jugated metabolite.

The role of diet in this scheme could be two-fold. As shown in these
studies, diet can alter AHH activity in all tissues examined. The altera-
tions in enzyme activity produced by long-term exposure to dietary xeno-

biotics may be such that the metabolites produced are more reactive before

68

conjugation. This could be due to long-term modifications in activating
and detoxifying pathways. Arnott et a1. (18) feund that livers from male
rats induced with hydrocarbon produced different profile of BP metabolites
than livers from non-induced animals. Prior induction resulted in higher
levels of proximate carcinogens, whereas in non-induced animals primarily
detoxified derivatives of BP were produced.

Secondly, it has been shown (92,101,102) that high fat, high meat
diets can increase B-glucuronidase activity of the colon microbial popula-
tion. The conjugated metabolites entering the colon from the liver in
rats on this type of diet could be more susceptible to deconjugation.

This would result in increased exposure of colon epithelial cells to
reactive intermediates. The importance of this scheme could be deter-
mined by use of model carcinogens which are capable of producing cancer
in the colon.

In summary, these studies have shown that intestinal metabOlism of
BP, and presumably other xenobiotics, can be influenced by dietary modifi-
cations. AHH activity can be rapidly increased in the colon, small
intestine, and liver by feeding CBH, and the level of enzyme induction is
related to both the level of fat in the hamburger before charcoal-broiling,
and the total level of CBH in the diet. AHH induction is probably due to
retention in the CBH of volatilized pyrolysis products produced at the
cooking surface. Enzyme activity appears to reach maximal levels upon
exposure to a.mixture of inducing agents and is minimally affected by
dietary fiber at the dietary levels used in this study. The alterations
in AHH activity produced by feeding CBH do not result in increased acti-

vation of BP to mutagenic derivatives in the Ames test, but do modify

69

binding of BP by RNA and/or protein but not by DNA in the colon, small

intestine and liver.

Conclusions

 

These studies have contributed to three areas of colon cancer research:
(1) It has been shown that diet can alter at least one drug-metabolizing
system, and it is probable that others are affected also.
(2) A role for dietary xenobiotics (in this case coming from CBH) in colon
cancer has been suggested. It is hypothesized that repeated exposure to
dietary compounds which induce microsomal metabolism of xenobiotics may,
ever a period of time, alter metabolic pathways in such a manner that
there is increased susceptibility to colon cancer upon exposure to a given
colon carcinogen.
(3) A.mode1 fer use in colon cancer research which can be used to study
the effects of nutrition on intestinal metabolism of xenobiotics has been
described. Within this model system, other sources of xenobiotics could
be investigated. By adapting the techniques applied in these studies,
this model could be used to study the role of environmental contaminants
which enter the body through routes other than diet. Additionally, other
pathways of metabolism could be studied by use of different model carcino-
gens. Within this context it could be determined whether the principal
role for diet in colon cancer is related to the effects of diet on metabo-

lism, rather than diet as a source of carcinogenic compounds.

REFERENCES

l. Schottenfeld, D., and Hass, J. F. "Epidemiology of Colorectal Can-
cer." In Lipkin, M. and Good, R. A. (eds.), Gastromtestmal
Tract Cancer. New York: Plenum Publ., 1978.

2. Miller, E. C. "Some Current Perspectives on Chemical Carcinogenesis
in Humans and Experimental Animals." Cancer Res. 38, 1479-1496,
1978.

3. Berenblum, I. "The Cocarcinogenic Action of Croton Resin." Cancer
Res., 1, 44-48, 1941.

4. Friedewald, W. F., and Rous, P. "The Initiating and Promoting Ele-
ments in Tumor Production: An Analysis of the Effects of Tar,
Benzpyrene, and Methylcholanthrene on Rabbit Skin." J. Exptl.
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