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This is to certify that the

dissertation entitled

EFFECT OF DIETARY FAT ON THE GROWTH OF
HUMAN BREAST CARCINOMA TRANSPLANTED TO THE ATHYMIC NUDE MICE:
ROLE OF LIPID PEROXIDATION IN THIS GROWTH PROCESS

presented by
Michael J. Gonzalez

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in Human Nutrition

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,a/Céfifi x/M

Rachel A. Schemmel

Major professor

 

 

Date 10/7/93

 

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EFFECT OF DIETARY FAT ON THE GROHTH OF
HUMAN BREAST CARCINOMA TRANSPLANTED TO THE ATHYMIC NUDE MICE:
ROLE OF LIPID PEROXIDATION IN THIS GROHTH PROCESS

By

Michael J. Gonzalez

Advisors:
Dr. Rachel A. Schemmel
Dr. Clifford II. Helsch

AN ABSTRACT OF 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

1993

ABSTRACT
EFFECT OF DIETARY FAT ON THE GROWTH OF

HUMAN BREAST CARCINOMA TRANSPLANTED TO THE ATHYMIC NUDE MICE:
ROLE OF LIPID PEROXIDATION IN THIS GROHTH PROCESS

BY

Michael J. Gonzalez

In athymic nude mice, altering the Ievels and/or types of dietary
fat, markedly influences the deveIopment and growth of transplanted
human breast carcinomas (MCF-7 and MDA-M823l cell lines). This
phenomenon has also been demonstrated in an array of spontaneous,
carcinogen-induced, transplantable benign and carcinomatous
experimental mammary tumor systems. In general, diets high in
unsaturated fatty acids (i.e., corn oil) were more effective enhancers
of mammary tumorigenesis than diets high in saturated fatty acids
(i.e., butter). In contrast high-fat diets with elevated amounts of
long-chain polyunsaturated fatty acids, such as eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA), which are found in certain fish
oils (i.e. menhaden oil), did not enhance mammary tumorigenesis, and
had a suppressive effect on tumor growth.

Various mechanisms have been proposed for this fish oil-induced
inhibitory action; we have concentrated our efforts on lipid
peroxidation. Our results demonstrate that this neglected mechanism,
at Ieast in part, is responsible for the suppressive action exerted by
fish oils (menhaden) on the growth of human breast carcinomas (MDA-
M8231) maintained in athymic nude mice. In addition, evidence is
provided that in the preparation of fish oil diets, the addition of

antioxidants at the amounts recommended by the American Institute of

Nutrition, or even substantially higher levels, did not completely
suppress oxidative deterioration of experimental diets. In terms of
tumor growth kinetics, the question arises as to what aspect of tumor
growth dynamics can be affected by dietary fish oils: tumor cell
proliferation or tumor cell loss. Our preliminary results suggest that
tumor cell loss is more significantly affected than tumor cell
proliferation in human breast carcinoma transplanted to athymic nude
mice fed a diet high in fish oil compared to a diet high in corn oil.
This increase in cell loss may partly explain why tumor growth is

suppressed in animals fed diets high in fish oil compared to diets high

in corn oil.

Dedication
To the memory of my grandparents
Juan and Luz Maria Guzman
whom in earth and now in heaven
will always guide my steps.

(Both died victims of cancer)

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ACKNOWLEDGMENTS

I would like to express my deepest gratitude to Dr. Rachel A.
Schemmel, chairperson of my graduate committee, for her encouragement
and needed support. In reality, there are not enough words to thank
her kindness. She has a very special place in my heart.

I would also like to thank Dr. Clifford W. Welsch, who gave me an
excellent opportunity to do first-class research. I would also like to
thank him for letting me know my strengths and weaknesses in research
and for permitting me to try my ideas, some that worked and some that
did not. In many instances I looked up to him as the father figure
that was absent in my life.

Another sincere thank you goes to Dr. J. Ian Gray, who provided me
with a magnificent laboratory setting in which my ideas took reality
form. Also I would like to thank him for the lessons on lipid
peroxidation and for his positive encouragement.

My thanks to Dr. LeRoy Dugan, Jr. for his time, encouragement and
informal but excellent teachings of the lipid peroxidation process. I
consider him my definite academic grandfather.

Thanks to Dr. Maurice R. Bennink, without whom I would have not
attended Michigan State and obtained this excellent education. I would
like to thank him for being an important part of my guidance committee.

Appreciation also goes to Dr. Nikolay Dimitrov for being part of

this excellent graduate committee.

 

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I would like to give a special thank you for my grandparents, Juan
and Luz M. Guzman, who are no longer here but who encouraged me to get
a quality education and guided my steps toward this goal. This
dissertation is dedicated to their memory.

I would also like to thank my dear mother, Daisy Guzman, who always
provided continuous support for me in every way possible, for always
believing in me. Without her help, love and support this goal would
have been unattainable.

I would also like to thank Enid J. Gonzalez, my dear wife, without
whom it would have been nearly impossible to obtain this goal; her
help, encouragement and companionship were the basis of my support
system.

I would also like to thank my uncle Adolfo Guzman, the memories of
my greatgrandmother, Agustina Diaz, my uncle John Guzman, my friends
Mario Garcia, Roberto Zayas and my adoptive grandmother, Monserrate
Feliciano, for serving as inspiration for me.

A special appreciation goes to Daisy Gonzalez, my sister, Dely
Santana, my niece, and to my new motivator, my son Michael John, Jr.

AIso thanks to my formal mentors: Dr. Alan M. Preston,

Dr. Elizabeth Sanchez, Dr. Gabriel Infante, Prof. Juan L. R. Ricart,
Dr. Alejandro Ruiz, Prof. Nivea Benitez, Dr. Dave Copson, Prof. William
Whiting, Prof. Fernando Cofresi, Dr. Richard Squire, Dr. Robert Bard,
Dr. Cruz Maria Nazario, Dr. Flavio Padovani, Prof. Virginia Baez, and
to MSU faculty members Dr. Dale Romsos, Dr. Dana Ott and Prof. Rhonda
Crackel for pulling out the best of me.

I would also like to thank my extended family: Dr. Jose R.
Rodriguez, Prof. CarIos and Jackie Ricart, Dr. Carlos Benitez,

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Dr. Larry G. Martin, Krystina Kras, Dr. Carol Friesen, Fernando and
Mari Herrera, Dr. Carlos M. CeboIlero, Dr. Ramon D. Martinez, Prof.
Carlos Ramos, Dr. Dave McConnell, Graciela CeboIlero, Rafael A.
Rodriguez, Dr. Lilian Ramos, Pedro and Gladys Bauza, Agnes, Pepe, Quico
and Javier Bauza, Violeta and Hernan Marin, Nando, Oscar and Charlie
Rodriguez, Angel Melendez, Sister Nildred Rodriguez, Dr. Tirso Pena,
Hector Rodriguez, Guille Feliciano, Chit Leong, Cheng Hsuing Chen,
Fumin Chu, Liang-Ting Chang, Su-Fen Weng, my adoptive aunt Leo Colon
and Alba Rivera.

Appreciation goes to the people and agencies that gave me financial
support: Administration of Economic Development of the Government of
P.R., The University of P.R., Dept. of Food Science and Human Nutrition
at MSU, the Office of Equal Opportunity of Urban Affairs at MSU,

Dr. Clifford W. Welsch, Senators Miguel Hernandez Agosto, Victoria
Munoz, Velda Gonzalez, former Governor Rafael Hernandez Colon, Mayor of
Ponce Rafael Cordero Santiago, President of the Municipal Assembly Luis
wito Morales, Francisco Negron, Jaime Fuster, Jose Tito Colorado and
Peyo Tata.

Last but not least a real special thanks to my East Lansing family:
Marlene Green, Mary Schneider, Dr. Lois Lund, Murry Edwards, Vonnie
Vanderploeg, Maggie Welsch, Cori and Jessica Oakley, Sandra, Rei and
Ricky Torres, Alba and Lee Echeverry, Gilmartin and Yon Pares.

God bless them all!
Above all, thanks to God the Almighty, to whom I hope one day I can
return this favor using this bountiful knowledge for the good of
mankind.

vii

 

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TABLE OF CONTENTS

Page
List of Tables .......................... ix
List of Figures ......................... xi
Introduction ........................... 1
Review of Literature ....................... 9
Chapter One ........................... 48
Chapter Two ........................... 72
Chapter Three .......................... 93
Chapter Four ........................... 118
Summary ............................. 147
Conclusions ........................... 149
Recommendations ......................... 151
Appendix ............................. 157

viii

Che

Chapter
1.
2.
3.

Chapter
1.
2.

Chapter
1.
2.
3.

LIST OF TABLES

1 Page
Diet composition ..................... 67
Predominant fatty acids in oils and fats (percentage) . . 68

Effect of dietary fat on growth of MCF-7 and MDA-M8231
human breast carcinomas transplanted to athymic nude
mice ........................... 69

Effect of dietary fat on MDA-MBZ31 human breast carcinoma
growth and carcinoma lipid peroxidation product levels. . 70

2
Diet composition ..................... 86

Effect of supplemented dietary antioxidants on peroxide
values of fish (menhaden) oil diets ........... 87

Effect of supplemented dietary antioxidants on TBARS
levels of fish (menhaden) oil diets ........... 89

Effect of supplemented dietary antioxidants on TBARS
levels of organs in mice fed fish (menhaden) oil diets

for 4 weeks ....................... 91
3

Diet composition ..................... 111
Predominant fatty acids (1) ............... 112

Effect of different ratios of dietary corn oil and fish

oil on the growth and lipid peroxidation product

concentrations of MDA-MBZSI human breast carcinomas in

athymic nude mice .................... 113

Effect of different ratios of dietary corn oil and fish

oil and the influence of antioxidants on the growth and

lipid peroxidation product concentrations of MDA-MBZBI

human breast carcinomas in athymic nude mice ....... 114

ix

Table

Chapter
1.
2.
3.

Effect of amount of time the fish oil diet remained in
the food Jar under usuaI feeding conditions on the growth
and lipid peroxidation product concentrations of
MDA-M8231 human breast carcinomas in athymic nude mice. .

4
Diet composition .....................
Predominant fatty acids in oils and fats (percentage) . .

Effect of dietary fat (corn oil and fish oil) on DNA
synthesis (H ~thymidine autoradiography and Brdu) of

human breast carcinoma MDA-MBZ31 maintained in athymic
nude mice ........................

Effect of dietary fat (corn oiI and fish oil) on DNA
synthesis (Brdu and PCNA) of human breast carcinoma
MDA-MBZ31 maintained in athymic nude mice ........

Effect of different ratios of dietary fats (corn oil and
fish oil) on DNA synthesis (Brdu) of human breast
carcinoma MDA-MBZ31 maintained in athymic nude mice . . .

Effect of dietary fat (corn oil and fish oil) on loss of
cells in human breast carcinoma MDA-M8231 maintained in
athymic nude mice ....................

Page

115

139
140

141

142

143

144

LIST OF FIGURES

Chapter 3 Page

1.

Chapter
1.

Relationship between mean carcinoma volume (cm3) and

mean carcinoma TBARS concentration per individual
MDA-MBZ31 human breast carcinomas in athymic nude
mice fed a 1% corn oil/19% fish oil diet (N-24) ..... 116

4

Rate of 1125Urd Toss from human breast carcinomas in

athymic nude mice fed corn oil (CO), fish oil (F0) and
fish oil supplemented with antioxidants (FO+A) for a
period of 6 weeks .................... 146

xi

 

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INTRODUCTION

Numerous authors have reported that there is a strong association
between dietary fat intake and cancer prevalence (see 1 for review).
Various epidemiological studies indicated that the consumption of high
levels of dietary fat by humans is associated with increased incidence
of cancer, particularly of the breast (2), colon (3) and prostate (4).
In addition, in experimental animal studies, mammary tumorigenesis
enhancement by high levels of dietary fat has been achieved in
virtually every rodent model that has been examined (5-8). In these
studies, the type and amount of fat ingested influences the occurrence
and development of mammary tumors. High dietary levels of
poTyunsaturated fats have been more effective in enhancing mammary
tumorigenesis than high Ievels of saturated fats (9-11). It has been
suggested that linoleic acid may be the fatty acid primarily
responsibIe for the tumorigenesis enhancing effect of the
polyunsaturated fatty acids (12-14). It seems that in order to achieve
maximal tumor growth a requirement for essential fatty acids (EFA) must
be met (12). This requirement appears to be in the range of'3% to 8%
of the total kilocalorie intake for rodent mammary tumor production
(12). It is believed that once the EPA requirement is met, further
enhancement of tumorigenesis depends on the amount and not the type of
fat, although evidence exists that may challenge these criteria (15).

Recent evidence has pointed to linoleic acid and arachidonic acid as

 

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the most necessary fatty acids for mammary tumor growth in rats (14).
It is also important to mention that linoleic acid is often included in
synthetic tissue culture media of normal and neoplastic cell lines in
order to obtain rapid cell proliferation and optimal cell growth in
vitro (16).

Moreover, diets high in fat have been reported to have an
immunosuppressive action, especially when high in polyunsaturated fats
(l7). Diets high in polyunsaturated fatty acids (PUFA) enhance
immunosuppressive therapy following renal transplantation (17).
Additionally, there is evidence that feeding linoleic acid prolongs
skin allograft survival in rodents (18). This immunosuppressive
activity of PUFAs, primarily those high in linoleic acid, has been
suggested as a possible mechanism by which polyunsaturated fats enhance
tumorigenesis (19,20).

Another type of polyunsaturated fat that has been studied in
relation to mammary carcinogenesis is fish oil (11). Mammary tumors in
rats fed diets rich in polyunsaturated long-chain fatty acids [such as
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)], which are
abundantly found in fish oils, resulted in a marked inhibition of
growth (21,22). The mechanism(s) for this tumor suppressive action has
not been clearly elucidated. In most of the earlier studies, menhaden
oil was used as the only source of dietary fat. Menhaden oil is a very
poor source of linoleic acid and the possibility of a linoleic acid
deficiency in the menhaden oil-fed animals cannot be ruled out. The
effect of fish oils in the generation of different series of hormone-
like substances (prostaglandins) as compared to corn oil has been a

popular proposed mechanism for the suppressive action exerted by fish

 

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oils (19-22). Although seemingly important, this extensively studied
prostaglandin mechanism may not be the sole one operating in this
complex process of tumor growth enhancement. It has been reported that
prevention of prostaglandin synthesis has not suppressed the inhibitory
effects of PUFAs (23). When inhibitors of cyclooxygenase
(indomethacin) or lipoxygenase (nordihydroguaiaretic acid) were added
to neoplastic cells along with the fish oil, there was no decrease in
the inhibitory effects of the oil. This suggests that there is no
major involvement of prostaglandins or leukotrienes in mediating fish
oil inhibition of tumor growth.

Another attractive mechanism that has not been adequately examined
in relation to the fish oil exerted growth suppressive action on tumors
is lipid peroxidation. Considering that the mammary tumor cell growth-
promoting effects of fat have been closely associated with PUFAs, it is
conceivable that certain PUFAs and/or their oxidative metabolites may
have an important effect on mammary tumor cell proliferation (15,24).

In relation to this, it is known that antioxidants are effective
suppressors of lipid peroxidation radicals and/or oxygen radicals.
Thus, various laboratories have added different antioxidants to diets
high in PUFAs; for example, vitamin E plus selenium (25), vitamin A
derivatives (26), and butylated hydroxytoluene (BHT) (27) to decrease
free radical production. Interestingly, the addition of antioxidants
to diets high in PUFAs has been reported to inhibit
dimethylbenzanthracene (DMBA) induced mammary gland tumorigenesis in
rats (25-27). This antioxidant-induced inhibitory action on mammary
tumorigenesis is contrasted with reports providing evidence of a

cytostatic/cytolytic action of certain PUFAs and/or their oxidative

 

 

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products in an in vitro (16) and in an in vivo (15,28) setting. In
these experiments, PUFAs underwent lipid peroxidation, generating an
increased amount of secondary products of oxidation restricting cell
proliferation and/or increasing cell loss resulting in a diminished
tumor growth. Given the nature of the fatty acid composition of fish
oils, this mechanism seems a very attractive one to explain the fish
oil exerted tumor growth suppressive action compared to rodents fed
diets high in corn oil.

Another important kinetic feature of tumorigenesis, in addition to
tumor cell proliferation, which has not been adequately examined, is
tumor cell loss. There is evidence that the increase in growth (size)
of rodent mammary tumors as a result of feeding diets rich in PUFAs
could not be solely accounted for by an increase in tumor cell
proliferation, which suggests an important role of cell loss on tumor
growth (29,30). The study of dietary fat effects on tumor growth
dynamics (cell loss vs cell proliferation) has been neglected. An
understanding of these kinetic phenomena might result in more effective
cancer therapies.

Most of the experiments designed to examine the effect of dietary
fat on mammary tumorigenesis have been carried out using rodent models,
in order to evaluate the effects of dietary fats on human
tumorigenesis, studies utilizing human tissue are essential.

Our experimental model consists of human breast tumor cell lines
(MDA-M8231), an estrogen receptor negative cell line derived from
pleural effusions of a patient with medullary carcinoma of the breast
and (MCF-7), an estrogen and progesterone receptor positive cell line

derived from a pleural effusion of a patient with an infiltrating

5

ductal breast carcinoma transplanted subcutaneously to female athymic
nude mice. The nude mouse is a mutant deficient in T-lymphocytes and
therefore suitable for foreign tissue grafts without rejection. This
model facilitates the study of human breast cancer in an in vivo
setting. It is also a means of providing important information on the
influence of dietary manipulations on the in vivo growth of human
breast cancer.

According to the two-stage theory of carcinogenesis (31), tumors
are initiated by a mutational change in the cellular genome. This
stage can occur rapidly and is essentially irreversible. Subsequent
proliferation of tumor cells is a slower process (promotion), and it is
thought to depend considerably on local environmental conditions. This
concept offers the chance of preventing or suppressing carcinogenesis
by modifying the cellular environment during the promotional stage of
carcinogenesis. This can occur by creating an unfavorable or even
hostile environment for proliferation and growth of tumor cells. The
understanding of the role of dietary fat in human tumor cell metabolism
during tumorigenesis may be a very relevant step to accomplish the
suppression of malignant growth. Experiments and studies conduscent to
a better understanding of nutritional influences on kinetic parameters
in tumor biology are of great importance. Research involving
nutrition-mediated cellular changes may provide new and different
avenues of effective treatment for cancer, and may serve as a pivotal

stage for new approaches against other degenerative diseases.

10.

11.

12.

13.

14.

15.

6

References

. Committee on Diet, Nutrition and Cancer. Assembly of Life

Sciences, National Research Council: Diet, Nutrition, and Cancer.
1982, National Academy Press, Washington, D.C.

. Carroll KK. Influence of diet on mammary cancer. Nutr Cancer

1981:2:232-236.

. Enstrom JE. Colorectal cancer and consumption of beef and fat.

Brit J Cancer 1975;32:432-439.

. Blair A and Fraumeni JF, Jr. Geographical patterns of prostate

cancer in the United States. J Natl Cancer Inst 1978:61zl379-1384.

. Carroll KK and Khor HT. Effects of level and type of dietary fat

on incidence of mammary tumors induced in female Sprague-Dawley
rats by 7,12 dimethylbenz(a)anthracene. Lipids 1971:6:415-420.

. Chan PC, Ferguson KA and Dao TL. Effect of different dietary fats

on mammary carcinogenesis. Cancer Res 1983:43:1079-1083.

. Rogers AE and Wetzel WC. Mammary carcinogenesis in rats fed

different amounts and types of fat. Cancer Res 1987:41:3735-3737.

. Welsch CW and Aylsworth CF. Enhancement of murine mammary

tumorigenesis by feeding high levels of dietary fat: A hormonal
mechanism? J Natl Cancer Inst 1983:70:215-221.

. Carroll KK and Hopkins GJ. Dietary polyunsaturated fat versus

saturated fat in relation to mammary carcinogenesis. Lipids
1977:14:155-158.

Carroll KK. Lipid and carcinogenesis. J Environ Pathol Toxicol
1980:3:253-271.

Selenskas SL, Ip MM and Ip C. Similarity between trans fat and
saturated fat in the modification of rat mammary carcinogenesis.
Cancer Res 1984:44zl321-1326.

1p C. Fat and essential fatty acid in mammary carcinogenesis. Am
J Clin Nutr. 1987:45:218-224.

Carrol] KK, Hopkins GJ, Kennedy TG and Davidson MB. Essential
fatty acids in relation to mammary carcinogenesis. Prog Lipid Res.
1981:20:685-690.

Sauer L and Dauchy R. Identification of linoleic and arachidonic
acids as rate limiting nutrients for tumor growth in rats
(abstracts). FASEB J 1988:2:(4) abstract # 3269. A858.

Gonzalez MJ, Schemmel RA, Dugan L Jr, Gray J1 and Welsch CW.
Dietary fish oil inhibition of human breast carcinoma growth: a
function of increased lipid peroxidation. Lipids 1993:28z827-832.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

7

Begin ME. Effects of polyunsaturated fatty acids and of their
oxidation products on cell survival. Chem Phys Lipids 1987:45:269-
313.

Vitale JJ and Broitman SA. Lipids and immune function. Cancer
Res. 1981;41:3706-3710.

Mertin J and Hunt R. Influence of polyunsaturated fatty acids in
the survival of skin allografts and tumor incidence in
mice. Proc Natl Acad Sci USA 1976:73:928-931.

Welsch CW. Enhancement of mammary tumorigenesis by dietary
fat:review of potential mechanisms. Am J Clin Nutr 1987;45:192-
202.

Hopkins GJ and West CE. Effect of dietary polyunsaturated fat on
the growth of a transplatable adenocarcinoma in C3H AFB mice. J
Natl Cancer Inst 1977;58:753-756.

Karmali RA, Marsh J and Fuchs C. Effect of omega-3-fatty acids on
growth of a rat mammary tumor. J Natl Cancer Inst 1984:73:457-461.

Jurkowski JJ and Cave WT. Dietary effects of menhaden oil on the
growth and membrane lipid composition of rat mammary tumors. J
Natl Cancer Inst 1985;74:1145—1150.

Rice-Evans C and Burdon R. Free radical-lipid interactions and
their pathological consequences. Prog. Lipid Res. 1993:32:71-110.

Bull AW, Nigro ND, Goremoleski WA, Crissman JD and Marnett LJ. In
vivo stimulation of DNA synthesis and induction of ornithine
decarboxylase in rat on colon by fatty acid hydroperoxides,
autoxidation products of unsaturated fatty acids. Cancer Res
1984;44:4924-4928.

Horvath PM and 1p C. Synergistic effect of vitamin E and selenium
in the chemoprevention of mammary carcinogenesis in rats. Cancer
Res 1983:43:5335-5341.

Aylsworth CF, Cullum ME, Zile MH and Welsch CW. Influence of
dietary retinyl acetate on enhancement of 7,12
dimethylbenz(a)anthracene-induced rat mammary tumorigenesis by high
levels of dietary fat. J Natl Cancer Inst 1986:75:339-345.

King MM, Bailey DM, Gibson DD, Pitha JV and McCay PB. Incidence
and growth of mammary tumors induced by 7,12
dimethylbenz(a)anthracene as related to the dietary content of fat
and antioxidant. J Natl Cancer Inst 1979:53z657-663.

Gonzalez MJ, Schemmel RA, Gray IJ, Dugan L, Jr., Sheffield LG and
Welsch CW. Effect of dietary fat on growth of MCF-7 and MDA-MBZ31
human breast carcinomas in athymic nude mice: relationship between
carcinoma growth and lipid peroxidation product levels.
Carcinogenesis 1991:12:1231-1235.

29.

30.

31.

Hillyard LA and Abraham S. Effect of dietary polyunsaturated fatty
acids on growth of mammary adenocarcinomas in mice and rats.
Cancer Res 1979:39:4430-4437.

Gabor H, Hillyard LA and Abraham S. Effect of dietary fat on
growth kinetics of transplantable mammary adenocarcinoma in Balb/C
mice. J Natl Cancer Inst 1985:74:1299-1305.

Iversen OH. Initiation, Promotion: critical remarks on the two
stage theory Part 3, Chapter 10:119-126. In Theories of
Carcinogenesis. Iversen OH. (ed) 1988: Hemisphere Publishing Co.,
Washington.

REVIEW OF LITERATURE

It is very well established that the level and type of dietary fat
can significantly influence the development and/or growth of mammary
gland tumors in experimental animals (see 1 for review). In addition
the amount and/or type of dietary fat can markedly affect the growth of
human breast carcinoma (cell lines) maintained in immune deficient mice
(2-7). Increasing the level of fat in the diet results in an increase
in mammary gland tumorigenesis (1). Dietary fats containing large
quantities of omega 6-polyunsaturated fatty acids [i.e. corn oil z 50%
linoleic acid (18:2n-6)] are the most effective enhancers of the
tumorigenic process while fats containing high amounts of omega 3-
polyunsaturated long-chain fatty acids [i.e. menhaden oil, which
contain elevated concentrations of eicosapentaenoic acid (EPA, 20:5n-3)
and docosahexaenoic acid (DHA, 22:6n-3)] are less stimulatory and often
suppressive to mammary gland tumorigenesis.

Since the mid-1930’s, fish oils have been reported to have a
suppressive effect on tumor growth (8,9). Four decades later, in the
early seventies, further evidence was reported on the reduction of the

incidence of spontaneously-developing mammary carcinomas in female C3H

mice fed diets high in fish oil (menhaden oil) when compared to mice
fed diets high in corn oil or safflower oil (10). Since then,
inhibition or suppression of mammary gland tumorigenesis has been

achieved by feeding fish oil in carcinogen-induced rat mammary

9

10

carcinoma model (II-l7) and in a carcinogen-induced mouse mammary
carcinoma model (18). In these studies corn oil or safflower oil
containing diets were used as controls. Moreover, diets high in fish
oil (menhaden oil) have been reported to suppress growth of BN472
transplantable rat mammary carcinoma when compared to tumor-bearing
rats fed a sunflower seed/cacao butter/olive oil diet (19). Also
supplementation of laboratory stock diet with fish oil (MaxEPA) has
been shown to suppress growth of R323OAC transplantable rat mammary
carcinoma (20). Furthermore, the growth of the IX transplantable mouse
mammary carcinoma has been reported to be reduced in mice fed high
dietary levels of fish oil (menhaden oil), compared to mice fed a diet
high in corn oil (21). In three of these studies (ll-l3), mammary
gland tumorigenesis in rodents consuming high dietary levels of fish
oil (menhaden oil, 20% by weight) was substantiaIly less than that in
animals consuming a diet very low in total fat (corn oil, 0.5-3.0% by
weight). The fish oil diets used in these studies were rich in omega-3
long chain polyunsaturated fatty acids 20:5n-3 (EPA) and 22:6n-3 (DHA).
Feeding rats a diet in which the fat content is virtually only EPA
(4.7%.by weight, 20:5n-3/0.3%, 18:2n-6) substantially suppressed
carcinogen-induced mammary gland tumorigenesis when compared to a diet
in which the sole source of fat was linoleic acid (LA, 18:2n-6, 5% by
weight) (22).
’o o - “-1.4. H 1' ‘0 I I‘ .0 - .0, ‘4 (“p o. - o.

The modulatory activities of omega-3 PUFAs on eicosanoid metabolism
have been the most extensively studied mechanism to explain the
inhibitory or suppressive activities of omega-3 PUFAs on mammary gland

tumorigenesis. PGEz, TXB2 and 6-keto-PGF1. are metabolic products of

11
the action of cycloxygenase on arachidonic acid (20:4n-6). The

PGEz/PGE1 ratios in mammary tumors from fish oil (menhaden oil or Max

EPA) fed rats have been reported to be lower than such ratios in
mammary tumors obtained from control rats fed corn oil or sunflower
seed oil diets (15,21,23). Concentrations of metabolites such as
PGEz/TXBz and/or 6-keto-PGF1, have also been reported to be lower in

mammary tumors of fish oil-fed rats than in tumors from control rats
(15,16,21,23). These eicosanoid metabolites have also been shown to be
lower in mammary tumors of rats fed a diet containing primarily EPA as
its fat source compared to animals fed a diet consisting solely of

linoleic acid as the source of fat (24). In addition plasma PGEz and
TXBz levels have also been shown to be reduced in mammary tumor-bearing

rats fed a fish oil diet compared to plasma levels in rats fed a
sunflower seed oil diet (20). Therefore, these metabolites are lower
in mammary tumors of rodents fed diets high in omega-3 long chain PUFAs
(i.e. EPA and DHA). Despite these results, Rice-Evans and Burdon (25)
reported that prevention of prostaglandin synthesis failed to suppress
tumor growth inhibitory actions of fish oils. When inhibitors of
cycloxygenase (indomethacin) or lipoxygenase (nordihydroguaiatetic
acid) were provided along with the fish oil, no decrease in tumor
growth inhibitory actions of the oil was observed.

Other mechanisms have also been examined in relation to the
inhibitory effects of fish oil on tumor growth. For example, rats fed
a diet high in fish oil (type not specified), compared to those fed
sunflower seed, had reduced liver microsomal fatty acid desaturase

activities (As and A6 desaturase). No significant decrease in the

12
activity of these enzymes was observed when rats were fed a diet rich

in linseed oil (26). Both A5 and .5 desaturase activities are
important in the metabolism of linoleate to arachidonte, ‘5 desaturase

is believed to be rate limiting for these sequential reactions.
Dietary fish oil (menhaden oil) has been reported to reduce the
activity of rat liver fatty acid synthase compared to a diet rich in
safflower seed oil (27). Fatty acid synthase is an important lipogenic
enzyme system involved in the synthesis of palmitic acid from acetyl
CoA and malonyl CoA. Evidence has also been provided indicating that
fish oil diets (menhaden oil), compared to corn oil diets, may decrease
plasma triglycerides and free fatty acids in rats possibly by
increasing the activity of skeletal muscle lipoprotein lipase (28).
Lipoprotein lipase stimulates the hydrolysis of triglycerides to free
fatty acids thus possibly increasing the availability of free fatty
acid substrates for skeletal muscle oxidative processes. Diets high in
fish oil (menhaden) have also been reported to influence hepatic phase
I and phase II detoxifying enzyme systems (e.g. mixed function
oxidases, glutathione-s-transferase, etc.) (29). In mammary gland
chemical carcinogenesis, this modification of hepatic enzymes may be
particularly relevant when fish oil diets are fed during the time
period of carcinogen treatment. Modulation of hepatic enzymes can
markedly influence carcinogen metabolism, thus, influencing the
carcinogenesis process itself.

Another mechanism that has been largely ignored is the effect of
dietary fish oil on energy accumulation. In animals fed high levels of
fish oil (menhaden) a reduced carcass energy and reduced adipose tissue

was observed when compared to animals fed equal amounts of corn oil or

13
lard (30,31). In addition, Mermier and Baker (32) reported that fatty
acids, utilized for tumor energy and growth, are derived from host
tissue, and it is known that fatty acid composition of tissues is
influenced by dietary fat (1). In relation to this aspect of energy
efficiency, Pariza and Boutwell (33) introduced the terms metabolizable
energy and recovered energy. These terms differ in that energy is lost
during the conversion of metabolizable energy into recovered energy.
These authors also mentioned that the extent of energy loss depends on
the energy source. These terms were described studying different
energy sources, for instance more energy from carbohydrate is lost
during this conversion than is lost when fat is used as an energy
source. Nevertheless, it seems likely that certain fats such as corn
oil may have a more efficient use of energy than others (i.e. menhaden
oil) hence contributing more to nourishment and growth of neoplasms
than do fish oils. In accordance with this hypothetical concept, it
has been reported that there is a reduced lipogenic activity plus an
increased mitochondrial efficiency in rats fed menhaden oil compared to
rats fed corn oil (34-36). These studies provide evidence of a
menhaden oil effect on intermediary metabolism. Tisdale (37) reported
that EPA can induce a blockage on the production of a metabolic factor
at the level of the adipocyte involved in cancer cachexia.

Fatty acids are important in the maintenance of cell membrane
structure and are key determinants of membrane bound enzyme activity
and receptor expression. It is recognized that an important role in
the regulation of enzymatic activity and receptor expression by
membranes is played by the constitutive lipids (38). In addition, the

oxidation of membrane phospholipids in tissues can decrease their

14
fluidity, thus affecting membrane permeability, osmosis and activity of
certain bound enzymes, receptors and transport systems (39-41). It has
been reported that omega-3 fatty acids increase spontaneous release of
cytosolic components from tumor cells (42). This probably occurs
because of the unique shape of the omega-3 PUFAs (EPA, DHA), which when
incorporated into cellular constituents of tumor cells, are able to
change properties associated with the plasma membrane. The authors
suggested that dietary manipulation may be used to enhance tumor cell
permeability and contribute to tumor cell destruction. Tumors obtain a
substantial amount of fatty acids from the host (43), which are
primarily supplied from circulating free fatty acids (44), the mixture
of fatty acids provided by the host (after only minor structural
modifications) is incorporated into the complex lipids formed by the
neoplastic cells (45). If the composition of circulating fatty acids
can be sufficiently changed, the phospholipids formed will have a
different fatty acid composition and possibly altered physical and
functional properties. It is conceivable that the change in membrane
fatty acids and its consequences can be sufficient to alter the tumor
growth pattern or increase its sensitivity to certain forms of therapy.
For exampIe, fatty acid modification might make the neoplastic cell
more sensitive to adriamycin chemotherapy. Adriamycin has been
reported to accumulate in neoplastic cells enriched with
polyunsaturates (46). In addition, fatty acids (LA, EPA) have been
reported to modulate gene expression in human breast cancer cells (47).
Fatty acids can exert these functions directly and, therefore, may
themselves be important regulators of another possible mechanism: the

immune response (48). The concentration and the degree of unsaturation

15

of dietary fats have been reported to influence several indices of
immune status in a number of animal models (49-56). Diets high in
certain PUFAs have demonstrated immunosuppressive potential (51).
Furthermore, diets high in PUFAs enhance immunosuppressive therapy
following renal transplantation (57). In addition there is evidence
that feeding PUFAs high in linoleic acid prolongs skin allograft
survival in rodents (58). In contrast, conflicting results regarding
the effects of dietary omega-3 PUFAs in animal models were reported and
only limited human studies on this topic have been conducted (49-51).
Nevertheless the omega-3 enhancement of certain immunological
parameters has been suggested as a possible mechanism for the
inhibitory action of dietary fish oil (1).
W1

Yet another possible mechanism for the suppressive action exhibited
by omega-3 fatty acids on tumor growth is lipid peroxidation (59).
Lipid peroxidation, or more specifically, the oxidation of
polyunsaturated fatty acids refers to the oxidative deterioration of
unsaturated fatty acids. It is a catalytic chain reaction probably
initiated and propagated by free radicals (60), aided by molecular
oxygen and bivalent cations. Susceptible substrates for this radical
attack in biomembranes are PUFAs containing two or more double bonds
(50,61). The groups between these double bonds are particularly
susceptible to hydrogen abstraction (62). The radicals formed react
rapidly with oxygen to form oxy-free radicals which convert to
hydroperoxides (primary products of oxidation) that can break down to a
wide variety of carbonyl compounds and to other radicals capable of

further perpetuating the process (60). Most primary products of

16

peroxidation are unstable in vivo (63). The high concentrations of
PUFA’s in cell membranes make cells very susceptible to peroxidative
damage (64,65) and ultimately the damage can be of such an extent that
it can result in the death of cells, this accomplished by the
interaction of peroxidation products with biomolecules of important
and/or regulatory functions (39,66-69). Ingestion of a diet containing
high levels of fish oil may increase the tendency for a tissue to
undergo lipid peroxidation (70). Several laboratories have reported
increased peroxidation in tissues of animals fed high levels of fish
oil (71-77).
Won

Of special interest are various aldehydes which are secondary
products of omega-3 fatty acid oxidation that are highly cytotoxic,
capable of producing a wide variety of inhibitory effects on cell
proliferation (78-88). These aldehydes are mainly formed by B-cleavage
reactions of alkoxy radicals arising from hydroperoxides of PUFAs based
in phospholipids, glycolipids, triglycerides and cholesteryl esters
(84). Also these aldehydes have been detected in many foodstuffs (i.e.
microwave food containing fat) (87). No method has been developed that
completely separates and identifies all the components of the complex
mixture of substances that can possibly arise of the interaction of
lipid-aldehyde species. These secondary products of lipid peroxidation
consist of high molecular weight polymers and decomposed products.
These decomposed products are mainly low molecular weight aldehydes
(83) which can be easily absorbed from the diet. Examples of these
aldehydes are alkanals, alkadienals, 2-alkenals and 4-hydroxyalkanal
(89,90). Other examples of cytotoxic aldehydes that can be formed in

 

me
th

V3

17
relatively large quantities in vivo are 4-hydroxynonenal, hexanal and
propanal (89). Of special interest is 4-hydroxynonenal, the major
cytotoxic product of lipid peroxidation (84), which has been reported
to suppress the expression of specific genes involved in the metabolic
cascade that leads to cell proliferation (88). Although there are a
variety of enzymes (i.e. aldehyde dehydrogenases) that evolved to
metabolize aldehydes to less reactive forms (i.e. carboxylic acids),
these species are still formed in vivo in enough quantities to modulate
various physiological and/or biochemical functions (87). In addition
to some of these mentioned, other aldehydes capable of producing a
chromagen in reaction with 2-thiobarbituric acid (thiobarbituric
reactive substances, TBARS) which can be measured by the 2-
thiobarbituric acid assay for detection of lipid peroxidation (TBA
assay) are 2-hexanal, 2,4-hexadienal and malondialdehyde (MDA). These
products, especially MDA, have been reported to be cytotoxic to cell
lines at physiological concentrations (90).
MW;

MDA is a secondary product of PUFA peroxidation and is most
frequently measured by the TBA assay (91,92). The TBA assay is used
for evaluating the degree of lipid peroxidation and is one of the most
commonly used methods for detection of lipid peroxidation products
(91,93). The TBA assay is a colorimetric test in which a pink
chromagen is formed by the condensation of 2 molecules of TBA and 1
molecule of MDA (94). The MDA measurement is an indicator of the
secondary products of peroxidation in the sample in question: the
greater the number of double bonds in a PUFAs the greater the chances

of forming more MDA molecules plus other secondary reactive substances.

 

m

al
al
as
is

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18
MDA can react with amino and sulfhydryl groups (SH) of RNA, DNA and
proteins (39,95) causing inter and/or intramolecular linkages that can
lead to inactivation or damage of these biomolecules. Also MDA has
been reported to inactivate DNA polymerases directly by blocking SH
groups (69). Furthermore, it has been documented that MDA can
accumulate in sufficient quantity in cells to exert various biological
negative effects as the ones mentioned above (95).

During lipid peroxidation other compounds besides MDA (other
aldehydes) are formed which may have a similar absorbance and which may
also be capable of inhibiting cell proliferation by the same mechanisms
as MDA (63,96). These products may be detected by the TBA assay. This
is the main reason why secondary products of peroxidation are now
referred to as TBARS instead of MDA as it was referred to in the past.
In addition it should be mentioned that TBARS can also be formed from
monoxidized PUFAs, carbohydrates and amino acids (97).
L1n1d_Eeroxination_and_1umor_§routh

Various factors may affect the oxidation of unsaturated lipids in
complex biological tissues. Among these are pH, light, temperature,
quantity of antioxidants present in the tissue, quantity and nature of
metal ions present in the tissue, efficiency of oxidation inhibitory
enzymes (glutathione peroxidase, catalase, superoxidase dismutase),
presence of reducing agents in tissue membranes and fatty acid
composition of the tissue (59).

Despite the variety of protective mechanisms [such as inhibitory
enzymes and antioxidants (98)] against peroxidation, it still occurs in
complex biological systems. If lipid peroxidation overwhelms the

normally efficient cellular protective mechanisms, it can then cause

 

gen
pru
fac

eff

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impo
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deco:
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PUFAs,
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19
genotoxicity or cytotoxicity and subsequently cell death (99). It is
prudent to state that the interplay between enhancing and inhibiting
factors of peroxidation is what probably determines its extent and its
effect on biological systems.

It has been documented that various PUFAs are effective in killing
cancer cells in vitro (69,81,100-118) but the specific mechanism of
this action is yet to be determined (106). Peroxidation may be a very
important factor in PUFA cytotoxicity and also in determining the rate
of tumor cell proliferation (100). Peroxidation of lipids followed by
decomposition of the peroxides into aldehydes or other secondary
decomposition products may be a crucial step on the path leading to a
reduction of the rate of cell proliferation and/or increasing the rate
of cell loss of tumors. Such products have been shown to restrict cell
proliferation and to cause cell death (119).

Various investigators (81,104,105,116-118) reported that the
addition of iron salts to cell cultures increases the rate of cell
death and TBARS number in cultured tumor cells fed high levels of
PUFAs, suggesting the involvement of lipid peroxidation products in the
mode of action. It is well known that transition metals can exert a
major role in oxidative damage occurring in biological systems
(41,120,121). In the case of lipid peroxidation, the metal involvement
is dual: it can first react with molecular oxygen generating oxygen
active species able to initiate lipid peroxidation by abstracting a
hydrogen atom from the PUFAs; second, it can react directly with the
hydroperoxide generating organic radicals that can initiate and
propagate the peroxidation reaction (41). Also it is well established
that iron and copper salts are prooxidant catalysts (120,121). At 37'C

 

and

dec

as

bre.

pH i
ferr
cont
and
inve:
and a
to ad
additi
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et al.
than in
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Contrary
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20
and a pH near 7.4, iron may become critical in promoting hydroperoxide
decomposition to MDA. In this environment, iron serves the dual role
as both initiator of lipid peroxidation and promoter of lipid peroxide
breakdown (122). It is conceivable that in an environment with a lower
pH (<6), as can possibly be found in tumors, iron may dissociate from
ferritin and/or hemoglobin and increase aldehyde formation (123). In
contrast, the addition of alpha-tocopherol reduces both cell killing
and levels of TBARS in cell culture (101,105,125). Also, several
investigators (126-128) reported that there is less lipid peroxidation
and a higher level of antioxidants in the cancerous tissue as compared
to adjacent normal tissue in breast cancer patients (113,128-130). In
addition, a rather interesting observation was reported by L’Abbe’
et al. (131), in which feeding a high fish oil diet induced higher
lipid peroxidation in female rats compared to male rats. Meydani
et al. (132) reported higher concentrations of plasma TBARS in older
than in younger women supplemented with omega-3 fatty acids. These
reports suggest a sex-age associated difference in incorporation or
utilization of omega-3 fatty acids. In relation to antioxidants,
earlier studies found antioxidants inhibited chemical carcinogenesis in
a variety of tissues (see 133 for review). This antitumor activity
exerted by antioxidants was probably due to an interference with
initiating or promoting events in chemical carcinogenesis (134,135).
Contrary to this mechanism, the addition of antioxidants to already
malignant cells in the presence of PUFAs seems to protect the cells and
enhance proliferation (100,105,124). Furthermore, there is some

evidence of antioxidants enhancing cell proliferation (136,137).

 

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21

Evidence from cell culture studies also indicates that certain
PUFAs such as eicosapentaenoic, docosahexaenoic and arachidonic acid
have been effective in killing cancer cells, and this effect is
correlated with intracellular TBARS levels (81,100,101,105,106); this
killing of malignant cells is accelerated by iron and copper while
inhibited by antioxidants (105,122). The generation of oxygen radicals
and the stimulation of lipid peroxidation, seem to be necessary steps
leading to cell death, in these experiments. Also, Burns and Wagner
(138) reported that there is a heightened susceptibility of fish oil
PUFA-enriched neoplastic cells to generate ethane during lipid
peroxidation (138).

Does cell death cause lipid peroxidation or does lipid peroxidation
cause cell death? This question has been addressed by Takeda et al.
(118) and Benchekroun et al. (139). They reported that there was no
evidence of any substantial rise in lipid peroxidation in response to
cell killing by other mechanisms such as temperature, pH changes and
non-peroxidation drug induced cell death. The addition of an
antioxidant suppressed the formation of lipid peroxidation in
neoplastic cells and the killing effect was inhibited (118). This
report has been substantiated by results of our laboratory (158).

Other clues of the importance of lipid peroxidation in relation to
cell proliferation can be derived from data about regenerating liver in
which the content of lipid peroxidation products are low (140-142).
This can suggest a possible involvement of lipid peroxidation products
in the control of cell division (126,143). It is documented that
embryonic tissue has a very low peroxidation content and it is also

known that certain tumor cells resemble embryonic cells; also highly

 

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144
mar

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22

undifferentiated tumor cells have a very low degree of peroxidation
which seems to be inversely related to the growth rate of tumors (113,
144-147). Moreover, rapidly growing normal tissue such as testis, bone
marrow and intestinal epithelium are resistant to peroxidation and
their lipid peroxidation product content is very low (120).

Conceivably, lipid peroxidation can inhibit tumor growth by several
mechanisms; these include (1) interference with various phases of the
cell cycle by damaging important cell compounds that would enable the
cell to proceed normally through its cycle. For example, the
inhibition of tubulin production by secondary products of peroxidation.
Tubulin is the main functional protein involved in the formation of the
mitotic spindle (148). (2) Inhibition of polyamine synthesis (148)
which can directly affect the cell division process. (3) Increasing
DNA synthesis time and decreasing the growth fraction of the tumor
(149), thereby increasing overall cell cycle time. Secondary products
of peroxidation have been shown to be diffusible (150,151), possessing
the capability of traveling along the cells producing damage to
susceptible sites through their pathway. Nevertheless some of these
aldehydes are fairly stable (151) which permit them to escape the cell
and attack susceptible targets far away from its originating initial
site. In this manner these aldehydes can be considered second toxic
messengers of the initial free radical event (89). However, tissue or
cell injury caused by lipid peroxidation cannot be attributed to a
single factor, since various radical species are formed with different
cytotoxic, mutagenic, genotoxic and carcinogenic potential (87).
Although lipid aldehyde levels in neoplastic cells or tissue may equate

increased levels of absorption or production of aldehydes, it may also

lean an a
There
conpositi
as compai
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initiate
levels 0
conparat
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(18:1,
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1%

23

mean an alteration in removal pathways (84).
WW

There is evidence that tumor cells have an abnormal lipid
composition (i.e. lower phospholipid content and low levels of PUFAs)
as compared to their normal counterparts (60). Tumor cells have also a
low level of enzymes of the cytochrome P450 system. These enzymes can
initiate and propagate lipid peroxidation (60). In addition, higher
levels of antioxidants have been reported in tumors as compared to
comparable normal tissue (60). The low rate of lipid peroxidation and
the low cytochrome P450 mediated activity can be associated with an
increased rate of cell division (144). Apparently, secondary products
of lipid peroxidation are inversely related to tumor growth. In
related studies (152-154), it was reported that LA and oleic acid
(18:1, n-9), in vitro, partially inhibited lipid peroxidation by
chelating available free iron. The importance of this report to an in
vivo setting remains to be determined. The content of nonesterified
fatty acids in vivo may be insufficient to obtain this interesting
effect. Two different investigators (80,155) have shown that lipid
peroxidation products were too low to be detected or decreased in vivo
when feeding diets high in linoleic acid content.
WWW

Cancer is a multistep, polygenic process involving various events.
The induction phase is composed of two steps, initiation and promotion.
At the initiation step, a change occurs in the cell genes resulting in
a deregulation of the normal pathways. The cell is then endowed with
the capacity to proliferate without control. In other words, the cell

is transformed or 'initiated'. This occurs after a mutation, an

nplificati
repressor g
step and fi
against Illi
Generally l
the pronot'
apronoter
inducing p.
cancer pro
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promotion
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hypothesiz
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the Proces
addition,
Conceivab]
SDGcific p
enhancers ;
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dependent ‘
A Very
in "hich tl

24

amplification, a translocation of a cellular oncogene or a loss of a
repressor gene. Chemical or physical mutagens can be involved in this
step and free radicals are a common link in this process. Protection
against the initiation phase can be provided by antioxidants.
Generally the transformation is completed by successive mitosis during
the promotion step. Any mechanism inducing the cell cycle will act as
a promoter. Certain reactive free radicals are known to play an
inducing part in cell activation (127) and are expected to act as
cancer promoters. However, to further complicate the picture, various
experiments in order to avoid the toxic effect of the free radicals
onto the cell, the use of antioxidants was necessary to ensure the
promotion phase (156). In tumor progression (the phase during which
tumor cells become organized as a growing mass), it might be
hypothesized that a senescent environment characterized by a high lipid
peroxidation and a low antioxidant level may negatively influence tumor
growth (127). Thus, it seems that free radicals can differently affect
the process of carcinogenesis depending on the phase considered. In
addition, to even further increase the complexity of this issue, it is
conceivable that different species of free radicals may influence a
specific phase of carcinogenesis in different ways. Some may be
enhancers of cell proliferation while others can be highly toxic. If
this concept is correct, then the importance is based on which species
is formed in larger amounts by which dietary fat (which might be
dependent on the specific composition of fatty acids).
ummnmmmm

A very interesting finding was reported by Gabor and Abraham (157)
in which they state that the cancer-promoting effect exhibited by corn

pH is m
the high
fish oil
years ti
but alsc
dietary
and 159
growth I
(6,7,59
and inc:
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and doc
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(7.160,
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the C81
may be
c“Donn

25
oil is mainly related to a decrease in tumor cell loss as compared with
the higher cell loss resulting in tumor of rodents fed a diet high in
fish oil. It has become increasingly evident over the past several
years that tumors are not just a reflection of the rate of cell growth
but also the rate of cell loss. Many reports utilizing high levels of
dietary fish oils have demonstrated a decrease in tumor growth (see 1
and 159 for reviews). Dietary fish oils may decrease the rate of tumor
growth by increasing lipid peroxidation products in tumor cells
(6,7,59,158,159), which can decrease the growth fraction of the tumor
and increase the percent of cell loss. Animals fed high levels of
dietary fish oil will have a higher intake of eicosapentaenoic (EPA)
and docosahexaenoic (DHA) acid that results in a higher content of
these highly unsaturated fatty acids in the phospholipid fraction of
their cell membrane (160). These fatty acids have 5 and 6 double bonds
respectively. It has been documented that the autoxidation of fish oil
in purified diets is rapid, within 24 hours (7,162-164). Furthermore,
it has been suggested that the increase in tissue TBARS after feeding
diets high in PUFAs are not formed in vivo but absorbed from the diet
(7,160,161).

Recently it has been reported that lipid peroxidation plays an
important role in tumor cell eradication by hematoporphyrin derivative
sensitized phototherapy (165-166).

Secondary products of peroxidation when constantly in contact with
a cancerous cell may induce profound damage, making it impossible for
the cell to survive. The inhibitory or suppressive effect of fish oil
may be due, at least in part, to lipid peroxidation formation of toxic
compounds.

Cell
Ti
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26
WWII

The growth rate of tumors is influenced by diverse cell cycle
kinetic factors (I for review), namely: 1) cell cycle time (time
necessary for the tumor cells to reproduce); 2) growth fraction (part
of the tumor in which tumor cells are actively proliferating); 3) cell
loss (tumor cells that die or leave the tumor). The understanding of
these kinetic factors and their interplay is of ultimate importance if
a successful treatment approach against cancer is to be accomplished.

To date, cancer therapy has focused mainly on targeting cell
proliferation with limited results in most tumors, while the phenomenon
of cell loss has been largely ignored. However, cell loss is an
important factor in determining the dynamic state of cell
proliferation. Cell loss is a biological phenomenon which takes place
in normal and neoplastic tissues (167). Usually an equilibrium exists
in normal tissue of adults between cell proliferation and cell loss
which results in a steady state growth pattern without any increase in
tissue size. However, when cell proliferation exceeds cell loss, the
result is an increase in size (hyperplasia). The innate ability to
replace cells in a tissue is a highly successful evolutionary
development (168), but perturbations in these processes can cause
excessive loss or over-production of cells.

This review will focus on the importance of cell loss to solid
(carcinomas, sarcomas, adenomas) tumor growth. Obviously, if cell loss
(by means of cell death) can be enhanced in a solid tumor in such a way
that it will overcome cell proliferation; solid tumor regression can be

accomplished.

Tumor
exfoliati
The relat
site of t
growing i
vessel, e
chance it
organ suc
oetastasi
circulati
is the 01
circulati
(171). I
transplai
Primary 1
doubling
by Ietasi
growth.
Viable pa
SUCh as 1
sloughim
1169).

1 ea'
Cell
of the m
death (a'

spatial .

27

Tumor cell loss can occur through various mechanisms such as
exfoliation, metastasis, differentiation and necrosis (cell death).
The relative contribution of the various modes of loss depends on the
site of the tumor and its histological type. Tumors that have surfaces
growing into a lumen such as in the peritoneum, pleurage, major blood
vessel, etc. or growing at the surface of the body have a greater
chance for cell loss by exfoliation than a tumor enclosed within an
organ such as liver or kidney (169). Another mechanism of cell loss,
metastasis, effectively promotes overall cancer spread. The
circulating cancer cells have several effects on the host, one of which
is the overcoming of immune resistance (170). However, most
circulating cancer cells die in their journey throughout the body
(171). But, if the metastatic cells are established, they behave like
transplants that have the capacity to grow faster than the original
primary tumor because of a shorter or absent lag phase or a shorter
doubling time (172). Nevertheless, there is no evidence that cell loss
by metastasis is a predominant mechanism affecting primary tumor
growth. Differentiation of cells within tumors can provide another
viable pathway for cell loss. For example, well differentiated tumors
such as those of the skin and colon lose cells by keratinization or
sloughing them off to the lumen of crypts (exfoliation), respectively
(169).
W

Cell death is encountered in early embryogenesis since it is part
of the morphogenesis of several organs (168). The programmed cell
death (apoptosis) in the embryo is a vital process providing a strict

spacial and temporal control. Its failure may result in congenital

abnormal

In ti
and/or 3
However.
cell 105
mechaniSI
tumor ma
in cell .
Counmn p
associatu
lysosoma‘
Various 1
the ChI‘Ol
El 0 :

Disc1
doubling
focus On
growth r;
n“Ch $101
of the u
been stag
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the follo
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of "mm

I'll, this

28

abnormalities of varying severity (168).

In tumor growth, there appears to be a reduction of the death index
and/or a prolongation of the average life span of the cells (172).
However, cell death in tumors appears to be the most relevant form of
cell loss. Cell death in solid tumors can be promoted by numerous
mechanisms, for example: insufficient blood supply with increasing
tumor mass (169), immune reactions against the tumor (173), anomalies
in cell division (174), and/or local effects of toxic products (59).
Common pathways for cell injury that may reach a lethal outcome are
associated with respiratory failure, inhibition of ATP synthesis,
lysosomal release and alteration of membrane permeability (175).
Various of these events may have a genetic component which may affect
the chromosomes and ultimately DNA.
Management

Discrepancies between tumor cell doubling time and the volumetric
doubling time of the tumor containing those cells led investigators to
focus on the possibility of a negative effect of cell loss on tumor
growth rate. Specifically, tumors are able to double in volume at a
much slower rate than one would expect by monitoring cell cycle times
of the tumor cells. The importance of cell loss in tumor growth has
been stagnated by its measurement difficulty. An accurate assessment
of cell loss in tumors has proven to be a very difficult task due to
the following: it can only be measured in situations in which a tumor
grows as a well defined mass which can be measured (176). In general,
when carcinomas reach a large size this is often associated with a core
of necrosis (dead center) and tumor growth is restricted to the outer

rim, this would underestimate cell death substantially; especially if

cell loss ‘
inrdathv
different
limhatior
tumors.
loss fact
cells as
hmnr vol
of growtl
A ce
neither
lost, tl
producec
regress¢
affectii
time.
increas
IHIlUEn
be 9m
factor
dUE to
tUmor ‘
undere:

necrota

29
cell loss is equated to cell death. Additional sources of variability
in relation to tumor growth are that tumors proliferate at very
different rates in different areas of the same tumor. In spite of this
limitation, there have been various attempts to measure cell loss from
tumors. In order to estimate cell loss, Steel (176) developed a cell
loss factor method. The cell loss factor measures the rate of loss of
cells as a fraction of the rate at which cells are being added to the
tumor volume by mitosis. Steel's cell loss factor expresses the loss
of growth potential by the tumor on the basis of production.

A cell loss factor of 100% describes a tumor in which there is
neither growth nor regression. If more cells are being generated than
lost, the cell loss factor is less 100% and if more cells are lost than
produced the cell loss factor is more than 100% and the tumor
regresses. Tumor growth is affected by small fluctuations in elements
affecting growth as well as by overall changes in growth patterns with
time. Estimates of cell loss factor usually differ over time as tumors
increase in size. There are a number of elements capable of
influencing the cell loss factor calculations which can only, at best,
be guides to the degree of cell loss. The estimations of the cell loss
factor is an approximation and must be interpreted with reservation
due to the impreciseness of the method. The simple measurement of
tumor volume doubling time in the calculation of cell loss factor will
underestimate cell loss due to the inclusion of blood, tissue fluid and
necrotic material in the measurement of tumor volume. Impressions of
the magnitude of cell loss has been obtained by comparing potential
doubling times of tumors (expected doubling time without cell loss)
determined from H3-thymidine labelling indices (176-178). In other

studies
the exp
factors
These r
methodo
reutili
prolife
tumors.

Cel

loss of
deoxyur
princi;
associa
wide di
means I

these 1

l"f0rmi

that f¢
Provide
Contra:
thymjd.
the 11:
Elimin;
p°DUla1

are Bf1

30

studies using human carcinomas, there was a marked disparity between
the expected and observed doubling times, which suggested cell loss
factors of around 60% (179) and in the region of 90% or higher (180).
These results again point out the imprecision in the cell loss
methodology (utilization of cell loss factor and isotopes of high
reutilization potential). Also affected by local variations in tumor
proliferative activity and varying distribution of cell cycle times in
tumors.

Cell loss has also been studied with some success by following the

loss of radioactivity from cells labelled with radioactive 1125-

deoxyuridine (178-188). However, this procedure follows the same

principle of the H3-thymidine and also had similar problems as those
associated with H3-thymidine labelling technique. For example, still a

wide dispersion of individual tumor doubling times among their group
means providing an excessive variability. It has been argued that

these two methods (H3-thymidine and Ile-deoxyuridine) can provide

information about different aspects of cell loss. Begg (183) reported
that for tumors with a high cell loss rate, the H3-thymidine method

provides more meaningful data than the Ilzs-deoxyuridine technique. In
contrast, Dethlefsen et al. (184) considered that the use of H3-

thymidine was well suited to the study of intratumor events, whereas

the Ile-deoxyuridine was considered better suited to investigate the

elimination of intact cells and necrotic products from a tumor

population regardless of rate. Both Ilzs-deoxyuridine and H3-thymidine

are efficiently incorporated into DNA of tumor cells, but reutilization

 

of I125_
After in

cells ur
deterior
limiting
equate 1

death tl

deoxyur'

cells);
they ar.
labelle«

excrete.

and 5)

method
Althoug
'noise'
cItocid
Suitab]
Vlvo.
Showing
Hell Wi
dUe to
ridiQSQ
Cells b,
Cell 10

“"1919“,

31
of Ilzs-deoxyuridine is much lower than that of H3-thymidine (185-188).

After incorporation into DNA, Ilzs-deoxyuridine remains bound within

cells until they die (178,186). When a labelled cell dies, rapid
deterioration and excretion of the DNA breakdown products occur
limiting the reutilization by other cells to a minimum. In order to

equate the loss of 1125 activity to naturally occurring tumor cell
death the following assumptions must be accepted as true: 1) the 1125-

deoxyuridine is associated exclusively with the DNA (mainly of tumor

cells); 2) Ilzs-deoxyuridine is not released from labelled cells while
they are alive; 3) Ilzs-deoxyuridine does not damage or kill the
labelled tumor cells; 4) Ilzs-deoxyuridine is rapidly broken down and

excreted (mostly in the urine) following the death of labelled cells;

and 5) chemical effects of IlZS-deoxyuridine are minimal. Hence, this

method has the advantage of not perturbing the tumor in question.
Although this method, when used in vivo, possesses a high degree of
'noise' (interfering variables) which makes it unfit to measure any
cytocidal effect quantitatively. Nevertheless, it seems to be the most
suitable way of determining cell loss occurring in solid tumors in
vivo. It has been reported (189) that the volumes of carcinomas
showing shrinkage 24 hours after being irradiated, did not correlate
well with their cell loss factor numbers. The discrepancy is probably
due to the fact that tumor regression depends not only on the
radiosensitivity of the tissue but also on the rate of removal of dead
cells by autolysis and phagocytosis. In order to attain a significant
cell loss to incur into tumor regression, cell death needs to be

complemented by an effective removal of dead cells. Tumor regression

 

also de
brings
are not
mass of
tumor v
tumor v
loss or
tumor 9
provide
it is ;
measure
Sol
death.
loss It
cell an
Chain I
unfavov
death 1
Previol
oil su;
deCTeas
It

kinetic

32
also depends on blood supply which reabsorbs products of autolysis and
brings phagocytes into contact with dead cells. When cells in a tumor
are not effectively removed, they remain to form a necrotic mass. The
mass of necrotic tissue is included with the live tissue when measuring
tumor volume; thereby overestimating tumor volume. This suggests that
tumor volume is not a useful measurement in the determination of cell
loss or cell loss factor. However, tumor volume as used in studies of
tumor growth kinetics is a simple straightforward procedure which
provides an insight into tumor growth despite its limitation. Because
it is possible to measure tumor volume in vivo, it can be used to
measure tumor growth over time which cannot be done by other means.

Solid tumors have high rates of cell loss (14) especially cell
death. It remains to be determined how we can take advantage of cell
loss to be used in solid tumor eradication. The manipulation of tumor
cell membrane with nutritional substances (for example, omega-3 long
chain polyunsaturated fatty acids) which may provide a hostile or
unfavorable environment for tumor cell growth thereby promoting cell
death is an attractive approach to this problem. As mentioned
previously (151,190), Abraham et al. have proposed that dietary fish
oil suppression of mammary tumor growth in mice is not a function of
decreased cell proliferation but a function of increased cell death.

It is conceivable that a better understanding of tumor growth
kinetic factors may enable us to shift the intricate balance from tumor
growth stimulating factors to inhibiting ones. This action may prove
to be a pivotal step in controlling the neoplastic expression and
improving the response to therapy. This principle might be relevant in

improving current therapeutic approaches and in developing or designing

33

newer and complementary therapeutic strategies against cancer. The

biological modification of cell loss instead of cell proliferation

might prove to be a valuable turn in the strategy used against cancer.

In conclusion, a better understanding of tumor growth dynamics

(cell loss and cell proliferation) and its dietary manipulation studied

in an vivo model of human tumors may provide important clues for

effective cancer therapy.

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Effect

Chapter 1

Effect of dietary fat on growth of MCF-7 and MOA-M8231 human breast
carcinomas in athymic nude mice: relationship between

carcinoma growth and lipid peroxidation product levels.
(Carcinogenesis 1991, 12:1231-1235)

 

 

NflWl

Lm

Dapa
Depa

Hict

East

EFFECT OF DIETARY FAT ON GROWTH OF NCF-7 AND MDA-N823]
HUMAN BREAST CARCINONAS IN ATHYMIC NUDE NICE: RELATIONSHIP
BETWEEN CARCINOMA GROWTH AND LIPID PEROXIDATION PRODUCT LEVELS

Michael J. Gonzalezl, Rachel A. Schemmell, J. Ian Grayl,

Leroy Dugan, Jr.1, Lewis G. Sheffield2 and Clifford W. Welsch2

1Department of Food Science and Human Nutrition
2Department of Pharmacology and Toxicology

Michigan State University
East Lansing, MI 48824 U.S.A.

49

 

 

and .
prepi
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Board

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Reprin

De]
Mic
Ea:

50
WE:

We acknowledge the technical assistance of Chris Heil, Deb O’Connor
and Steve Wysong in the experimental studies and Marlene Green for the
preparation of this manuscript. These studies were supported by
research grants NIH-CA 42876, AICR-9OBW05, Wisconsin Milk Marketing
Board 111-17, and Ladies Auxiliary to the Veterans of Foreign Wars -
Michigan Division to C.W. Welsch.

mm:

Dietary Fat and Human Breast Carcinoma Growth

musing:
C.W. Welsch
Department of Pharmacology and Toxicology
Michigan State University
East Lansing, MI 48824

 

 

Huma
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51
mm

Human breast carcinoma cell lines MCF-7 and MDA-M8231 were
transplanted s.c. to female athymic nude mice at 3-4 weeks of age.
Seven to 10 days after transplantation, the mice were divided into
groups and fed for 6-8 weeks one of the following semi-purified diets
containing different amounts and types of fat, i.e., 5% corn oil, 20%
corn oil, 20% butter, 19% beef tallow/1% corn oil and 19% fish
(Menhaden) oil/1% corn oil. In additional experiments, the fish oil
diets were supplemented with antioxidants (vitamin E, 89 or 2000 IU/kg
diet plus tertiary butyl hydroquinone-TBHQ, 4g/kg diet) or ferric
citrate (3g/kg diet). Tumor peroxidation product levels were assessed
by measuring 2-thiobarbituric acid reactants (TBA assay). At the
termination of the studies (6 to 8 weeks of diet feeding) mean human
breast carcinoma volume (MCF-7 and MDA-M8231) was the largest in mice
fed the 20% corn oil diet, intermediate in mice fed the butter or beef
tallow diets and the least in mice fed the fish oil diet. The
difference in mean tumor volumes among mice fed the 20% corn oil diet
and those fed the fish oil diet was significant (P < 0.01). When
comparing low (5% corn oil) and high (20% corn oil) fat diets,
numerical increases in human breast carcinoma volume (MCF-7 and MDA-
M8231) were consistently observed in the high fat diet groups but these
differences were not always significant. Tumor lipid peroxidation
product levels were determined on the MDA-M8231 tumors; tumor lipid
peroxidation levels were significantly (P < 0.01) increased only in
mice fed the fish oil diets. Supplementation of the fish oil diets
with antioxidants (vitamin E + TBHQ) significantly reduced the level of

tumor peroxidation products and significantly increased tumor volume

 

 

(P < 0.(
oil plus
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52

(P < 0.05). When tumor lipid peroxidation product levels in the fish
oil plus antioxidant fed mice were reduced to the level of that
observed in the tumors of the corn oil fed mice, no significant
differences in tumor volumes were observed in these two groups. In
contrast, supplementation of the fish oil diets with ferric citrate,
significantly (P < 0.05) increased tumor lipid peroxidation product
levels and decreased tumor volume. Thus, the type of dietary fat can
clearly affect the growth of human breast carcinomas (MCF-7 and MDA-
MBZ31) maintained in athymic nude mice. The consistent and significant
inhibition of dietary fish oil (Menhaden) on growth of these carcinomas
appears to be due to an increased accumulation of tumor lipid
peroxidation products.

MIMI]!!!

In experimental animals, altering the level and/or type of dietary
fat markedly influences the development and growth of mammary tumors.
This has been demonstrated in an impressive array of spontaneous,
carcinogen-induced, transplantable, benign and carcinomatous
experimental rodent mammary tumor systems (1). In human populations,
several epidemiological studies have suggested that diets high in fat
may increase the risk of breast cancer development (2). To date, there
is mainly epidemiological data to link dietary fat and human breast
cancerigenesis. In an effort to resolve this relationship, we have
embarked upon a series of studies to assess the influence of dietary
fat on growth of human breast carcinomas maintained in athymic nude
mice. In this communication, we report the influence of the amount (5%
corn oil/20% corn oil) and type (corn oil/butter/beef tallow/fish oil)

of dietary fat on growth of hormone responsive (MCF-7) (3) and hormone

 

 

non-res
these ‘
of the
relatil
deterii
oxidat
replic.
concen'
fats, .
oxidat
the in
of fat
implic

diets

Tw
Spragu
studie
owerhe
temper
Surgic
aSEpti
Chow (
Until
Thetea
a Cal:

”Iner;

53
non-responsive (MDA-M8231) (4) human breast carcinoma cell lines in
these immune deficient animals. In addition, we assessed the influence
of the level of lipid peroxidation products in these carcinomas in
relation to carcinoma growth. Lipid peroxidation is an oxidative
deterioration of unsaturated fatty acids that give rise to a variety of
oxidation products with the potential of interfering with cell
replication and/or cell survival. The presence of higher
concentrations of highly unsaturated fatty acids in certain dietary
fats, e.g., fish oil brings into the context the possibility that
oxidation products of these fatty acids may play an important role in
the inhibitory or growth rate limiting action exhibited by these types
of fat. In studies reported herein, we are providing evidence
implicating lipid peroxidation products in the inhibitory action of
diets high in fish oil on human breast carcinoma growth.
HAIEBIAL§_AND_NEIHQD§
Two hundred and fifty-eight female athymic nude mice (Harlan
Sprague Dawley Inc., Madison, WI) 3-4 weeks old were used in these
studies. The mice were housed under aseptic conditions (enclosed
overhead laminar flow hood, sterilized cages, bedding and water) in a

temperature controlled (24°C) and light controlled (14 hr/day) room.

Surgical procedures for transplantation of tumors were performed under
aseptic conditions in a laminar flow hood. Autoclaved laboratory mouse
chow (Purina Mills Inc., St. Louis, MO) was fed ad_11b11um before and
until 7 to 10 days after human breast carcinoma transplantation.
Thereafter, mice were fed ad libitum semi-purified diets (Table 1). On
a caloric basis, the diets contained the same amount of protein,

minerals, vitamins and fiber. Energy calculations were based on 4

 

 

Kcal/g 0
ingredie
except 5
Mesa, Cl
Corp. (1
Univers
predomi
of the

prepare

packed
consumv
steril-
19/1it
§gggr§

Pa
Cultur
athymi
to 4 5
Nice ‘
transF
condit
IntegL
dlStar
breast
Has ta

Spec1"

54
Kcal/g of protein and carbohydrate and 9 Kcal/g of fat. All dietary
ingredients were obtained from U.S. Biochemicals, Inc. (Cleveland, OH)
except sucrose which was obtained from ICN Biochemicals, Inc. (Costa
Mesa, CA), fish oil (Menhaden) which was obtained from Zapata Haynie
Corp. (Reedville, VA) and butter which was obtained from Michigan State
University Food Stores (East Lansing, MI). The percentage of the
predominant fatty acids (1% or greater, manufacturers specifications)
of the dietary oils and fats are shown in Table 2. The diets are

prepared in our laboratory weekly and stored at -20°C, individually

packed with nitrogen prior to feeding. Mice were fed daily and non-
consumed food discarded daily. Since the semi-synthetic diets were not
sterilized, antibiotics (bacitracin, streptomycin and neomycin,
lg/liter) were added to the drinking water.
IieneraLEmedure

Palpable MCF-7 and MDA-M8231 human breast carcinomas (American Type
Culture Collection, Rockville, MD) were surgically excised from female
athymic nude mice and transplanted to 3-4 week old athymic nude mice, 3
to 4 slices (2x4 mm, 0.1 to 0.3 mm thick) of tumor tissue per mouse.
Mice were anesthetized with sodium pentobarbital prior to
transplantation; all surgical procedures were done under aseptic
conditions in a laminar flow hood. An incision was made in the
integument, the tumor slices were placed s.c. in the dorsum at
distances from each other of at least 2 cm. One to 3 palpable human
breast carcinomas provided the tissue slices for each experiment; care
was taken to randomize the carcinoma slices from pooled tumor

specimens.

 

Sin!
for groi
implantl
cholest
Ins/Ll-
describ
indepen
growth
diets 7
the car
the ons
bearing
for a p

tumors

vernier
Stu
3. The
amount
fat (co
growth
mice.
itu
Study w
fit aff

55

Since the MCF-7 human breast carcinoma is dependent upon estrogen
for growth in athymic nude mice, mice bearing these tumors were
implanted with a single estrogen pellet (2 mg 17 B-estradiol/38 mg
cholesterol) s.c. and were given estrone in their drinking water (0.5
mg/L). The preparation of the hormone pellets is as previously
described (5). The MDA-M8231 human breast carcinoma is hormone
independent; such tumors do not require supplemented hormones for
growth in athymic nude mice. Mice were placed on the experimental
diets 7 to 10 days after carcinoma transplantation. This period allows
the carcinoma grafts to become established in the host animals before
the onset of experimental dietary treatments. Athymic nude mice
bearing human breast carcinoma grafts were fed the experimental diets
for a period of 6 to 8 weeks. At the termination of the studies, all

tumors were excised and their volume determined (V - 4/3 TT r3) with a

vernier caliper. Three separate studies were performed (Studies 1-3).

Study_1 The experimental design of this study is provided in Table
3. The purpose of this study was to determine whether or not the
amount of dietary fat (5%.corn oil/20% corn oil) or the type of dietary
fat (corn oil/butter/beef tallow/fish oil) could differentially affect
growth of MCF-7 and MDA-M8231 human breast carcinomas in athymic nude
mice.

Stugy_z (Experimental design - see Table 4). The purpose of this
study was to determine whether or not the amount and/or type of dietary
fat affects the level-of human breast carcinoma lipid peroxidation
products. Excised tumors were assayed for lipid peroxidation products
by measuring Z-thiobarbituric acid reactants using a modified TBA assay

as described below.

 

51
study
(vitat
oil d‘
lipid
peroxi
Yitami
89 or
Chemic
oil di
added
carcin
assay.

51
EXperii
lice).
feeding
Peroxic
Weight
Val'l'anc
'35 set

%

SUbl
excised,
at .200(
tumor sa

Samples

56

Study_3 (Experimental design - see Table 5). The purpose of this
study was to determine whether or not the addition of antioxidants
(vitamin E and TBHQ) or an oxidizing agent (ferric citrate) to the fish
oil diets would decrease and increase, respectively, the level of tumor
lipid peroxidation products and whether such changes in lipid
peroxidation products could influence human breast carcinoma growth.
Vitamin E (DL-tocopheryl acetate, U.S. Biochemical Co., Cleveland, OH,
Bg or 2000 IU/kg diet) and tertiary butyl hydroquinone (TBHQ, Eastman
Chemical Products, Kingsport, TN, 4g/kg diet) were added to the fish
oil diets. Ferric citrate (Sigma Chemical Co., St. Louis, MO) was
added to the fish oil diet at a level of 3g/kg diet. Excised breast
carcinomas were assayed for lipid peroxidation products by the TBA
assay. This study was performed twice, experiments #1 and 2.

Studie§_1;3 Athymic nude mice that became ill and/or died prior to
experiment termination were excluded from all studies (< 3% of the
mice). All mice were weighed at the beginning and termination of diet

feeding. Mean human breast carcinoma volume (cm3), mean lipid

peroxidation product levels (ug TBA reactants/g tissue) and mean body
weight gains were statistically analyzed by one way analysis of
variance and the Newman-Keuls multiple comparison test. Significance
was set at P < 0.05.
188.853!

Subcutaneously growing human breast carcinomas were surgically
excised, placed in a 0.9% NaCl solution, immediately frozen and stored
at -20°C. Immediately before the TBA assay, the tumors were thawed,

tumor samples were pooled yielding 109 of tumor tissue; the 109 tumor

samples were used in the assay. An antioxidant was added (TBHO, 0.2%

 

of tota

homogen
homogeh
contair
suffici
The dis
of fres
solutic
triplit
pigment
2000, l
were mi

reports

ffect
H05;ggg
MEI

at term
corn oi
and the
differ,
oil dis
butter
l'nterme
decreaS
c°mDare

Statlst

57
of total fat) and samples were homogenized (4°C) in a polytron

homogenizer (Ultra Turrax SOT 1810, Tekmar Co., Cincinnati, OH). The
homogenates were transferred into prepared distillation flasks
containing 2.5 ml of 4N HCl plus 0.5 ml silicone antifoam with
sufficient distilled water to yield a total volume of 100 ml/flask.
The distillate was collected (50 ml); 5 ml of this distillate plus 5 ml
of freshly prepared 2-thiobarbituric acid solution (TBA, 0.2 N aqueous
solution) were used for the assay. Samples were prepared in
triplicate, heated in boiling water for 35 minutes to develop the
pigment, cooled and absorbance read on a spectrophotometer (Spectronic
2000, Bausch 8 Lomb, Rochester, NY). The absorbance values obtained
were multiplied by an adjustment factor of 6.2 (6). Results were
reported as ug of TBA reactive substances per g of sample.
RESULIS
. ,- .- 1 . .Ug_, . . - . , .I . . f, . I - . .

u'A'w; 1-11s] o -. . In“. I ‘11-. - . IIII Im‘ 11

Mean human breast carcinoma volume of MCF-7 or MDA-M8231 carcinoma
at termination of these studies was the largest in mice fed the 20%
corn oil diet, intermediate in mice fed the butter or beef tallow diets
and the least in mice fed the fish oil diet (Tables 3 & 4). The
differences in final tumor volumes of mice fed the corn oil and fish
oil diets were consistent and significant (P < 0.01). In mice fed the
butter or beef tallow diets, final tumor volume was consistently
intermediate to those fed the corn oil or fish oil diets. This
decrease in tumor volume in mice fed the butter or beef tallow diets
compared to corn oil, however, did not reach the 5% level of

statistical probability.

58

When comparing a high fat diet (20% corn oil) to a low fat diet (5%
corn oil), numerical increases in MCF-7 and MDA-M8231 carcinoma volumes
at termination of the experiments were consistently seen in mice fed
the higher level of fat (Tables 3 & 4). Such volume increases were
significant (P < 0.01) in 1 of the 2 studies utilizing MDA-M8231 cells
(Table 3). Thus, a trend toward increased human breast carcinoma
volume is observed, albeit marginally significant, when these animals

are fed the higher level of fat.
I l' 0' :1. 1H' 1 a a ' . . oI o 0 .I0 a.o

a‘ 0‘ a. oI o oo_ ‘ ‘ a u'i'w: I “.I l ‘. . Io“. I

As in the preceding study, human breast carcinoma volume at
termination of this study was largest in mice fed the 20% corn oil
diet, intermediate in mice fed the butter diet, and the least in mice
fed the fish oil diet (Table 4). The difference in tumor volumes of
mice fed the 20% corn oil and fish oil diets was significant
(P < 0.01). Numerical increases in tumor volumes were observed in mice
fed the 20% corn oil diet compared to mice fed the 5% corn oil diet,
however, these differences did not reach the 5% level of statistical
probability.

Mean lipid peroxidation product levels (ug TBA reactants/g tissue)
in the human breast carcinomas derived from the fish oil fed mice were
significantly higher when compared to tumor lipid peroxidation products
in each of the other diet groups (P < 0.01). No significant difference
in the level of mean lipid peroxidation products in carcinomas derived
from animals fed the 5% corn oil, 20% corn oil or butter diets was

observed (Table 4).

 

 

59

As in the preceding studies, human breast carcinoma volume at the
termination of study was significantly (P < 0.05) reduced in mice fed
the fish oil diet compared to mice fed the corn oil. The addition of
antioxidants (vitamin E and TBHQ) to the fish oil diet caused a
striking increase (6 fold, P < 0.05) in final tumor volume, a final
tumor volume, however, that was still quantitatively less than that
observed in the corn oil fed mice (P < 0.05). This increase in tumor
volume in mice fed the fish oil diet supplemented with antioxidants was
accompanied by a significant (P < 0.05) reduction in tumor lipid
peroxidation products (ug TBA reactants/g tissue). The level of tumor
lipid peroxidation products in the antioxidant supplemented fish oil
diet fed mice was still significantly (P < 0.05) more than the level of
lipid peroxidation products in the tumors of the corn oil fed mice.

The addition of ferric citrate to the fish oil diet, in contrast,
resulted in a further reduced final tumor volume (P < 0.05). Such a
reduction in tumor volume was accompanied by a significant (P < 0.05)
increase in the level of tumor lipid peroxidation products.
Willem

The study described above was repeated, the results are identical
to those observed in experiment #1 with but one important exception.

The addition of the antioxidants (vitamin E and TBHO) to the fish oil

 

diet C
not si
oil fe
produ<
supplt
level

diet.
EffeC‘

among
to th
citra
throu
less

nice.

0f hu
trans
the t
leVel
fats

(Henh
lines
°bSer
Certa

SQed

60
diet caused an increase in final human breast carcinoma volume that was
not significantly different than the tumor volumes observed in the corn
oil fed mice. Furthermore, the level of tumor lipid peroxidation
products in the tumors derived from the mice fed the antioxidant
supplemented fish oil diet was not significantly different than the
level of tumor lipid peroxidation products in mice fed the corn oil
diet.
Effest_oIED1ets_on_flodx_fle19ht_§ains.

There were no significant differences in mean body weight gains
among any of the dietary groups (data not shown). The only exception
to this are the mice of study 3 (Table 5) who were fed the ferric
citrate supplemented fish oil diet. While these mice gained weight
throughout the study, their rate of gain was significantly (P < 0.05)
less than the corn oil, fish oil or the fish oil plus antioxidant fed
mice.

DISCUSSION

The results of our study demonstrate very clearly that the growth
of human breast carcinoma cell lines MCF-7 and MDA-M8231, when
transplanted to female athymic nude mice, can be markedly influenced by
the type of dietary fat. Tumor growth is greatest in mice fed high
levels of a vegetable oil (corn oil), intermediate in mice fed animal
fats (butter, beef tallow) and the least in mice fed fish oils
(Menhaden oil). Thus, the growth of these human breast carcinoma cell
lines, as a function of the type of dietary fat, mimics what has been
observed in numerous rodent mammary tumor models, i.e., diets rich in
certain vegetable oils (e.g., corn oil, sunflower seed oil, safflower-

seed oil) are the most stimulating to mammary tumor growth, diets high

 

 

in fish
in satu
stimula
and the
to alts
import;
human s
Ous
growth
interes
while s
reports
lines I
levels
process
xenogr;
laborat
(breast
focus s
pr0005s
oil is
liberal
eXplm.E
fish 0i
aCCUmuI
l” the

ObserVe

61
in fish oils (e.g., Menhaden oil) the least stimulatory and diets high
in saturated animal fats are of intermediate potency in the growth
stimulation of these tumors (1,7). That the rodent mammary tumor cells
and the human breast carcinoma cells have similar growth responsiveness
to alterations in dietary fat provides additional support for the
important utility of rodent mammary tumor systems as models for the
human disease.

Our observation that high levels of dietary fish oil suppress the
growth of human breast carcinoma cell lines in athymic nude mice is an
interesting and potentially important observation. Very recently,
while our studies were in progress, other laboratories (8-12) have
reported similar observations utilizing human breast carcinoma cell
lines (MDA-330, MX-I and MCF-7) in athymic nude mice. Clearly, high
levels of dietary fish oil suppress mammary tumorigenic growth
processes, not only in rodent mammary tumor models (13-15) but in
xenografts of human breast carcinomas maintained in immune deficient
laboratory mice as well. How dietary fish oil suppresses mammary
(breast) tumorigenic growth processes is not known and is currently the
focus of attention of a number of laboratories. Many laboratories have
proposed that the inhibition of mammary tumorigenesis by dietary fish
oil is via alterations in eicosanoid metabolism (13-15). Our
laboratory, in contrast and as reported in this communication, has
explored another potential mechanism, i.e., that high levels of dietary
fish oil suppress mammary tumorigenic processes by causing an increased
accumulation of cytotoxic and/or cytostatic lipid peroxidation products
in the tumor tissue. In athymic nude mice fed the fish oil diets, we

observed a substantial increase in the level of lipid peroxidation

 

ps
in
ad
ans
prs
grc
the
lev
dif‘
Furi
i.e.
prod
Thus.
supp]
part,

Peroa

fed a
level
butts
PTOdL
large
(DHA)
diet
llpic

dIrEC

62
products (TBA reactants) in the breast carcinomas; a level of
peroxidation products approximately 4 times greater than that observed
in the breast carcinomas of mice of the other dietary groups. The
addition of high levels of antioxidants to the fish oil diet (vitamin E
and TBHQ) substantially reduced the level of tumor lipid peroxidation
products and simultaneously caused a substantial increase in tumor
growth. Indeed, when tumor lipid peroxidation products were reduced in
the fish oil fed mice by dietary antioxidant supplementation, to the
level observed in the tumors of the corn oil fed mice, no significant
difference in tumor growth in these two groups of mice was observed.
Furthermore, the addition to the fish oil diet of a pro-oxidant cation,
i.e., ferric citrate, caused an increase in tumor lipid peroxidation
product levels and a simultaneous further decrease in tumor growth.
Thus, these data provide compelling evidence that the tumor growth
suppressing activities of a diet high in fish oil is due, at least in
part, to the generation of fish oil derived products of lipid
peroxidation.

Of the dietary groups examined in these studies, only the fish oil
fed mice had substantial increases in tumor peroxidation product
levels. Animals fed low or high levels of corn oil or high levels of
butter had approximately 1/4 the level of tumor lipid peroxidation
products than that observed in the tumors of mice fed fish oil. The
large amounts of eicosapentaenoic (EPA) (20:5) and docosahexaenoic acid
(DHA) (22:6) in the fish oil diets, compared to the corn oil or butter
diet groups, is most likely the major reason for this increase in tumor
lipid peroxidation products. It is well established that there is a
direct relationship between the number of double bonds in a fatty acid

 

and the
oxidat‘
Hos
influe
second
report
substa
(17).
amino
inacti
polyam
DEFOXII
cell p‘
Indeed
S“Ppre
decrea
(15).
(20.21
certas
linoli
the ge
cancer
stimu]
Sisuit

63
and the potential formation of lipid peroxidation products; such
oxidation can occur via a chemical or an enzymatic reaction (16).

How the accumulation of lipid peroxidation products in tumor tissue
influences tumor growth processes is not entirely certain. The
secondary products of lipid peroxidation measured by the TBA assay are
reportedly capable of affecting cell proliferation. For example, these
substances can inactivate polymerase reactions by blocking SH group
(17). They can cause inter- and/or intramolecular linkages between
amino acid sulfhydryl groups of RNA, DNA and proteins leading to the
inactivation or damage of these molecules (18). They can inhibit
polyamine synthesis (19). Such biochemical interactions of lipid
peroxidation products with cellular macromolecules can not only reduce
cell proliferation processes but can directly lead to cell death.
Indeed, Abraham and colleagues have proposed that dietary fish oil
suppression of mammary tumor growth in mice is not a function of
decreased cell proliferation but a function of increased cell death
(15). In a series of interesting studies, Horrobin and colleagues
(20,21) have reported that the addition of relatively large amounts of
certain polyunsaturated fatty acids, e.g., EPA, arachidonate,
linolineate, to cultures of malignant cells causes a sharp increase in
the generation of TBA reactive products and a parallel increase in
cancer cell death. The addition to the culture media of inhibitors or
stimulators of lipid peroxidation, e.g., vitamin E or iron, caused a
significant inhibition or enhancement, respectively, of cell killing.
Interestingly and of potential pragmatic importance is their
observation that the enhancement of cell death by supplementation of

polyunsaturated fatty acids is repeatedly seen in an array of cancer

cell ‘
currei
repor‘
mammal
level
of can
cells
is an

II
can is
nude i
dietai
0f the

to an

64
cell lines but is notably absent in normal cells (21). We are
currently in the process of determining whether or not our previously
reported observation of dietary fish oil induced inhibition of normal
mammary gland growth processes (22) is a function of increasing the
level of mammary gland lipid peroxidation products. The vulnerability
of cancer cells to certain polyunsaturated fatty acids, while normal
cells appear resistant as reported by Horrobin and colleagues (20,21),
is an observation that merits further examination.

In summary, we report in this communication that the type of fat
can influence growth of human breast carcinomas maintained in athymic
nude mice. Most impressive was the consistent inhibitory effect of
dietary fish oil on growth of these carcinomas. The inhibitory effect
of the fish oil diets on growth of these carcinomas appears to be due

to an accumulation of lipid peroxidation products in the tumor tissues.

10.

11.

12.

I ‘ 4“ ‘

3111/1

10.

ll.

12.

65

References

. Welsch, C.W. (1987) Enhancement of mammary tumorigenesis by

dietary fat: Review of potential mechanisms. Am. J. Clin. Nutr.,
45, 192-202.

. Armstrong 8. and Doll, R. (1975) Environmental factors and cancer

incidence and mortality in different countries, with special
reference to dietary practices. Int. J. Cancer, 15, 617-631.

. Sheffield, L.G. and Welsch, C.W. (1985) Cholera-toxin-enhanced

growth of human breast cancer cell lines in vitro and in vivo:
Interaction with estrogen. Int. J. Cancer, 36, 479-483.

. Arteaga, C.L., Tandon, A.K., Van Hoff, 0.0. and Osborne, C.K.

(1988) Transforming growth factor Beta: Potential autocrine growth
inhibitor of estrogen receptor-negative human breast cancer cells.
Cancer Res., 48, 3898-3904.

. Soule, H.D. and McGrawth, C.M. (1980) Estrogen responsible

proliferation of clonal human breast carcinoma cells in athymic
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. Crackel, R.L., Gray, J.I., and Pearson, A.M. (1988) Some further

observations on the TBA test as an index of lipid oxidation in
meats. Food Chem., 28, 187-196.

. Carroll, K.K. and Hopkins, G.J. (1979) Dietary polyunsaturated

fat versus saturated fat in relation to mammary carcinogenesis.
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. Fernandes, G., Frieduchs, W., and Shultz, J. (1989) Modulation of

MCF-7 tumor cell growth in nude mice by omega-3 fatty acid diet.
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. Pritchard, G.A., Jones, O.L., and Mansel, R.E. (1989) Lipids in

breast cancer. Brit. J. Surg., 76, 1069-1073.

BIink, E.W. and Ceriani, R.L. (1989) Fish oil enhancement of
I-conjugated anti-human milk fat globule monoclonal antibody

experimental radio-immunotherapy of breast cancer. J. Steroid
Biochem., 34, 149-153.

Borgeson, C.E., Pardini, L., Pardini, R.S., and Reitz, C. (1989)
Effects of dietary fish oil on human mammary carcinoma and on
lipid-metabolizing enzymes. Lipids, 24, 290-295.

Gabor, H., Blank, E.W., and Ceriani, R.L. (1990) Effect of
dietary fat and monoclonal antibody therapy on the growth of human
mammary adeno-carcinoma MX-I grafted in athymic mice. Cancer
Lett., 52, 173-178.

13.

14.

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66

Karmali, R.A., Marsh, J., and Fuchs, C. (1984) Effect of omega3
fatty acids on growth of a rat mammary tumor. J. Natl. Cancer
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Jurkowski, J.J. and Cave, W.T. (1985) Dietary effects of menhaden
oil on the growth and membrane lipid composition of rat mammary
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Gabor, H. and Abraham, S. (1986) Effect of dietary menhaden oil
on tumor cell loss and the accumulation of mass of a transplantable

mammary adenocarcinoma in BALB/c mice. J. Natl. Cancer Inst., 76,
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Cheeseman, K.H., Burton, G.W., and Ingold, K.U. (1984) Lipid
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Roubal, W.T. and Tappel, A.L. (1966) Damage to proteins, enzymes
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Reiss, U., and Tappel, A.L. (1973) Fluorescent product formation
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Dianzani, M.U. (1982) Biochemical effects of saturated and
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Begin, M.E., Ells, G., and Horrobin, D.F. (1988) Polyunsaturated
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Horrobin, D.F. (1990) Essential fatty acids, lipid peroxidation,
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351-377.

Welsch, C.W., and O’Connor, D.H. (1989) Influence of the type of
dietary fat on developmental growth of the mammary gland in
immature and mature female BALB/c mice. Cancer Res., 49, 5999-
6007.

 

 

Table 1

———-——-

Composi
Fat
Casein
Dt-meth
Dextrin
Sucrose
AIN Sal
AlN Vit
Cellulo

*The ca'
diet 1:

“Al pha
conten‘

***The l
tal l 0W,
fit dis

 

67
Table 1. Diet composition

 

 

Composition* Low fat (5%) High fat (20%)
diet*** diet***
Fat 5.00% 20.00%
Casein 17.10 20.17
OL-methionine 0.30 0.35
Dextrin 45.50 32.18
Sucrose 22.70 16.09
AIN Salt Mixture** 3.50 4.13
AIN Vitamins** 1.00 1.18
Cellulose __§,QQ __§,29
100.00 100.00

 

*The calculated energy value (metabolizable energy) for the low fat
diet is 3.87 kcal/g, for the high fat diets, 4.55 kcal/g.

**Alpha tocopherol level content of the diet is 0.2 g/100 g diet. Iron
content of the diet is 0.004 g/100 g diet.

***The high fat diets consisted of 20% corn oil, 20% butter, 19% beef
tallow/1% corn oil or 19% fish (Menhaden) oil/1%.corn oil; the low
fat diet consisted of only 5% corn oil.

68

Table 2. Predominant fatty acids in oils and fats (percentage)

 

 

Fatty acids* Corn Butter Beef Menhaden
oil tallow oil
Caprylic (8:0) -- -- -- --
Capric (10:0) -- -- -- --
Lauric (12:0) -- 2.5 -- --
Myristic (14:0) -- 10.5 3.4 8.0
Palmitic (16:0) 10.1 31.0 25.5 28.9
Palmitoleic (16:1) -- 2.5 2.8 7.9
Stearic (18:0) 1.6 13.6 24.9 4.0
Oleic (18:1) 31.4 29.9 35.7 13.4
Linoleic (18:2) 56.3 1.8 1.6 1.1
Linolenic (18:3) -- -- -- 1.0
Eicosapentaenoic (20:5) -- -- -- 10.2
Docosahexaenoic (22:6) -- -- -- 12.8

 

*Fatty acid concentrations less than 1%

are not included.

 

Ti

20%

20%

+11

19%
+1%

Treat

5% Cs
20% Cs
20% BL

19% Be
+11 Co

19% Me
+11 Co

*Female
'eeks
tI'anSp

i/b, p
d/b, p

 

69

Table 3. Effect of dietary fat on growth of MCF-7 and MDA-M8231 human
breast carcinomas transplanted to athymic nude mice.

INZF-7 human breast carcinoma

 

 

Treatment* No. of No. of Mean
mice carcinomas carcigoma volume
(cm ,1 S.E.)
5% Corn oil 9 36 3.40 i 0.28“
20% Corn oil 8 28 4.02 i 0.58“
20% Butter 9 35 2.36 i 0.32“
19% Beef tallow 9 35 2.36 i 0.36“

+1% Corn oil

19% Menhaden oil 9 36 0.20 1 0.16”
+1% Corn oil

KIA-118231 human breast carcinoma

 

 

Treatment* No. of No. of Mean
mice carcinomas carcipoma volume
(cm 1 S.E.)
5% Corn oil 8 24 0.52 1 0.12”
20% Corn oil 8 22 1.54 1 0.37“
20% Butter 8 24 1.16 1 0.19cl
19% Beef tallow 6 19 0.84 1 0.23

+1% Corn oil

19% Menhaden oil 7 24 0.56 z 0.50”
+1% Corn oil

 

*Female athymic nude mice were fed diets for 8 weeks, from 5 to 13
weeks of age, commencing 7-10 days after human breast carcinoma
transplantation.

a/b, P < 0.01
d/b, P < 0.05

 

Tab

Tre

5%
20%

20%

+1%

*Fen
wee
tra

a/b,

70

Table 4. Effect of dietary fat on MDA-M8231 human breast carcinoma
growth and carcinoma lipid peroxidation product levels.

 

Mean
Mean carcinoma lipid
carcinoma peroxidation
Treatment* No. of No. of Iolume products (ug/

,1 S.E.) TBA reactants/g
tissue i S.E.)

mice carcinomas (cm

 

5% Corn oil 5 19 0.20 i 0.06 0.16 i 0.04“
20% Corn 011 8 20 0.37 i 0.11a 0.22 i 0.04a
20% Butter 6 19 0.10 i 0.03 0.20 1 0.07a
19% Menhaden Oil 7 21 0.03 10.01b 0.82 i 0.01b

+1% Corn oil

 

*Female athymic nude mice were fed diets for 6 weeks, from 5 to 11
weeks of age, commencing 7 to 10 days after human breast carcinoma
transplantation.

a/b, P < 0.01

 

Tabl

Tree

Lane
20%

19%
+1%

19%

+1% 1
anti
(Vit

19% |

+1% I
+ Fe

£102
202:

19% l
+12 1

19% I
+11 1
antis
(Vit

191 I
+1: I
+ Fe

*Fems
l"Eel
tray

*‘Uei

71

Table 5. Effect of dietary fish oil plus antioxidants or iron
supplementation on MDA-M8231 human breast carcinoma growth
and carcinoma lipid peroxidation product levels.

 

 

Mean
Mean carcinoma lipid

carcinoma peroxidation

Treatment* No. of No. of v lume products
mice carcinomas (cm 1S.E)** (ug/TBA reactants/g
tissuetS.E.)**

Emenimentil
20%.Corn oil 15 40 2.8210.41“ 0.4410.01“
19% Menhaden oil 16 50 0.24:0.04b 1.411007b
+1% Corn oil
19% Menhaden oil 15 39 1.45:0.14c 0.701003c
+1% Corn oil and
antioxidants
(Vit E & TBHO)
19% Menhaden oil 16 40 0.181003“ 2.13:0.09d
+1% Corn oil
+ Fe citrate
20% Corn oil 22' 55 3.2610.82“ 0.39:0.03“
19% Menhaden oil 24 60 0.34_+_0.04” 13010.03”
+1% Corn oil
19% Menhaden oil 25 70 2.3910.35“ 0.4510.02“
+1% Corn oil and
antioxidants
(Vit E G TBHO)
19% Menhaden oil 18 57 0.15:0.04c 1.52:0.02b

+1% Corn oil
+ Fe citrate

 

*Female athymic nude mice were fed diets for 6 weeks, from 5 to 11
weeks of age, commencing 7 to 10 days after human breast carcinoma

transplantation.

**Means with different letters are significantly different at P < 0.05.

Chapter 2

Lipid peroxidation products are elevated in fish oil diets
even in the presence of added antioxidants
(J. Nutr. 1992, 122:2190-2195)

LIPID PEROXIDATION PRODUCTS ARE ELEVATED IN FISH OIL DIETS
EVEN IN THE PRESENCE OF ADDED ANTIOXIDANTS1

Michael J. Gonzalez, J. Ian Gray, Rachel A. Schemmel, LeRoy Dugan, Jr.
and Clifford W. Welsch*

Departments of Food Science and Human Nutrition and
*Pharmacology and Toxicology

Michigan State University, East Lansing, MI 48824

Request for reprints: C.W. Welsch
Dept. Pharmacology and Toxicology
Michigan State University
East Lansing, MI 48824
Telephone 4 (517) 353-4549
Fax 4 (517) 353-8195

73

 

C0

11
p1

di

74
ABSTRACT
Purified corn and fish oil diets with different types and
concentrations of antioxidants were evaluated for oxidation products.
In addition, a determination of different organ and carcass oxidation

product levels was performed. PV2 and TBA2 assays were done on the

diets immediately after mixing (0 h) and 24, 48 and 72 h after being
fed to mice. The AIN recommended level of antioxidant addition (BHTZ,

0.02%.w/w of oil) and even the addition of 100 times this level (BHT,
2.0% w/w of oil), although decreasing the level of oxidation products,
failed to totally prevent oxidative deterioration in diets high in fish
oil. Furthermore, other antioxidants added in excess to the fish oil
diets also failed to completely suppress oxidative deterioration of the
diet and, in addition, when fed daily to mice for a period of 4 weeks,
caused an increased accumulation of lipid peroxidation products in
certain organs (e.g., heart, skeletal muscle, mammary glands) and in
the carcass. Thus, these results provide evidence that in the
preparation of fish oil diets, the addition of the AIN recommended
level of antioxidants, or levels even substantially higher than that
recommended does not completely suppress oxidative deterioration of

experimental diets.

Indexing keywords: fish oil, lipid peroxidation, antioxidants, Balb/c

mice.

 

Pol:
unambh

problem

 

 

packagi
recogni
synthei
experis
Howeve
than d
becaus

acids,

Johns‘
added
(1988
also
0T or

auto:

diet
DTOC
r900
of a
by a
has
auts

I‘eS‘

75
INTRODUCTION
Polyunsaturated fats can quickly autoxidize at ambient or
subambient temperatures (Chan 1987). In the food industry, this
problem has been reduced by the addition of antioxidants and vacuum
packaging of foods containing these fats (Buford-Coulter 1988). In
recognition of this problem, the AIN has recommended the addition of a
synthetic antioxidant at a level of 0.02%, (by weight of oil) to
experimental animal diets containing polyunsaturated fats (AIN 1980).
However, diets containing fish oil are more susceptible to autoxidation
than diets containing other polyunsaturated fats (e.g., corn oil),
because of their high concentration of long chain polyunsaturated fatty

acids, e.g., EPA“ (20:5, n-3) and DHA“ (22:6, n-3). Fritsche and

Johnston (1988) reported an extremely rapid autoxidation of diets with
added fish oil as measured by PV. This report by Fritsche and Johnston
(1988) plus an earlier publication by Rasheed et al., (1963), which
also reported a high autoxidation in diets containing fish oil, are two
of only a few publications dealing with the important issue of
autoxidation in fish 011 containing diets.

It is our opinion that in most studies dealing with the role of
dietary fish oil in physiological or pathological processes, the
process of dietary autoxidation has been largely ignored. In a recent
report, our laboratory has provided evidence that the inhibitory effect
of a fish oil diet on tumorigenic processes may be mediated primarily
by a lipid peroxidation process (Gonzalez et al. 1991). Such evidence
has provided the basis for our belief that the importance of
autoxidation or lipid peroxidation has been underestimated by those

researchers interested in the health impact of fish oil diets. With

this in
autoxid
oxidati

under I

Diets
Pus
modifh
of the
used f
arriva
with n
after
(~20°c
of pre
Henhad
was ad
essent
corn 0
"‘51 a
in EXp
V/w (
T
or 3H
the f
TBHQ

76
this in mind, we felt it necessary to further evaluate the issue of
autoxidation of fish oil containing diets and the accumulation of
oxidation products in organs of mice given a diet rich in fish oil
under normal feeding circumstances.
MATERIALS AND METHODS

01:15

Purified diets (Table 1) were formulated according to a slight
modification of the guidelines developed by AIN (1977). Dry components
of the diet (Table 1) were mixed for 15 min and stored at -20°C until

used for diet preparation. The fish (Menhaden) oil was used upon
arrival with the remaining oil stored in small dark containers sealed
with nitrogen gas. Mean PV of the Menhaden oil when received was 7.25,
after 7 days it was 7.76 (-20°C) and after 14 days it was 10.45

(-20°C). The Menhaden oil diet was prepared the following way: 800 g

of previously mixed dry ingredients were combined with 190 g of
Menhaden oil and 10 g of corn oil (19% fish oil/1%»corn oil). Corn oil
was added to fish oil to increase the amount of linoleic acid, an
essential fatty acid for normal growth and development. The use of
corn oil, a polyunsaturated vegetable fat (:60% linoleic acid, 18:2,
n-6) as a control comparison for fish oil was based on its frequent use
in experimental animal dietary studies. These diets, utilizing 20% fat
w/w (200 g/kg diet), are considered high fat diets.

The antioxidants TBHQ“ (Eastman Chemical Products, Kingsport, TN)

or BHT (Sigma Chemical Co., St. Louis, M0) were immediately added to
the fish oil at a level of 0.02%.(BHT) (w/w oil) or 2.0% BHT or 2.0%
TBHO,1 8 g/kg dl-alpha tocopherol acetate (U.S. Biochemical, Cleveland,

0H).
plasti
nitrog
signil
storas
Agaly
D
exper
(Balb
mice/
light
the I
Trip'
afte

for

Peru

(197

385a
the
291
to l
tre.

eQLi

506:

77
OH). Sufficient diet for one day’s feeding was packed into small
plastic zipper sealed bags. Each bag containing diet was flushed with
nitrogen gas and stored at -20°C for no longer than 7 days (no

significant increase in diet peroxidation is observed during the 7 day

storage period).

:1- 0 sons. I..I.'-.,.-.-I,.1-
Diets were fed (free access) to mice to expose diets to actual

experimental conditions. One hundred twenty mature female mice

(Balb/c), from our breeding colony, were housed in 24 cages (5

mice/cage). Mice were housed in a temperature controlled (24°C) and

light controlled (14 hr. light/day) room. Our protocol complied with
the Guide for the Care and Use of Laboratory Animals (NRC 1985).
Triplicate samples of each diet were obtained for analysis immediately
after mixing (0 h) and 6 diet samples of each diet group were analyzed
for each time period, i.e. 24, 48 and 72 h after being fed.

Lipid peroxidation was determined by two methods, the PV (mmol of
peroxides/kg oil) by the method of the American Oil Chemist's Society
(1973) and by measuring TBARS“ (ug TBARS/g sample) using a modified TBA

assay as previously described (Gonzalez et al. 1991). Modification of
the TBA assay is as follows: to 10 g of diet, TBHO (0.2% of total fat,
2 g/kg sample) was added to diet samples before homogenization at 4°C

to prevent further oxidation during the assay. The homogenates were
treated with 4N HCl. A distillate was collected and combined with
equal amounts of freshly prepared TBA solution. Samples were heated in
boiling water, cooled and absorbance read on a Spectronic 2000

spectrophotometer (Bausch 8 Lamb, Rochester, NY). Once diet samples

were ob
were d0
map;

Li;
glands
(10 an'
at al.
consum

oil/1%

and tr
SigniI

Al
01‘] (,4
E (Tat
hlghei
on d;
thrOug

0f 81'1“.

78

were obtained, they were analyzed immediately for PV and TBARS which
were done in duplicate and triplicate, respectively.
Wham

Lipid peroxidation was assessed in liver, heart, kidney, mammary
glands and skeletal muscle (15 animals/group) and in whole body carcass
(10 animals/group) of female mice by a modified TBA assay (Gonzalez
et al. 1991). Mice were fed one of the following diets daily (non-
consumed diet discarded daily) for 4 weeks: 20% corn oil, 19% fish
oil/1%.corn oil or 19% fish oil/1% corn oil with added antioxidants,
i.e., TBHO, 2.0%.w/w of oil and dl-alpha tocopherol acetate, 8 g/kg or
2000 IU/kg diet. At termination, organs were collected and stored in a
NaCl solution (9g/L) at -20°C. These were analyzed for TBARS the

following day. Animals used for whole body carcass TBARS were
terminated by carbon dioxide inhalation and stored intact in a plastic
zipper bag at -20°C and analyzed for TBARS the following day.

WW

Results were statistically analyzed by one-way analysis of variance
and the Newman-Keuls multiple comparison test (Wilkinson 1989).
Significance was set at p < 0.01.

RESULTS

At 0 h, the PV of the corn oil diet was lower than any of the fish
oil diets except for the fish oil diets containing 2.0% TBHQ 1 vitamin
E (Table 2). At 0 h, the PV of the non-supplemented fish oil diet was
higher than the PV of the corn oil diet or the PV of each of the fish
oil diets supplemented with antioxidants. This trend was observed
throughout the other time periods (24, 48, 72 h). Increasing the level
of BHT from 0.02% to 2.0% reduced the PV at all time points. TBHQ (2%)

was sli

of vita

 

values.
compare
in the
(0 h) a
rate 0
diet 9
48 ans
A1
highe
3).
decre
fish
TBAR:
BHT
redu
'TBAJI
that
‘lovn
Sum
finer
i11c
at,

anc

3k.

79
was slightly more effective than BHT (2%) in reducing PV. The addition
of vitamin E to the TBHO group further reduced, albeit slightly, PV
values. PV at 0 h (immediately after diet preparation) was the lowest
compared to all other time points (24, 48, 72 h) both in the corn and
in the fish oil diets. Oxidation occurring between diet preparation
(0 h) and after being fed for a day (24 h) was the period in which the
rate of PV increase was at its highest. PV levels increased in all
diet groups over time throughout the entire experimental period (0, 24,
48 and 72 h).

At 0 hour, the dietary TBARS were lowest in the corn oil diet and
highest in the fish oil diet not supplemented with antioxidants (Table
3). The addition of antioxidants (BHT or TBHO) to the fish oil diet
decreased the levels of TBARS when compared to the non-supplemented
fish oil diet. Increasing the level of BHT from 0.02% to 2.0% reduced
TBARS at all time points. TBHO (2%) was slightly more effective than
BHT (2%) in reducing TBARS. The addition of vitamin E to TBHO further
reduced, albeit slightly, TBARS. However, at 0 and 24 h, the levels of
TBARS were higher at all levels of antioxidant supplementation than
that observed in the corn oil diet. Dietary TBARS values at 72 h were
lower than those at 48 h except for the fish oil diet which was not
supplemented with antioxidants. In all of the foregoing, the TBARS
were measured at the standard wavelength of 532 nm. However, an
increase in dietary TBARS was observed at wavelengths 455 nm and 495 nm
at 72 h as compared to 455 nm and 495 nm wavelength readings at 0, 24
and 48 n time points (data not shown).

TBARS values of organs (liver, heart, kidney, mammary gland,

skeletal muscle) and whole bodies of mice fed a fish oil diet without

antioxl
whole I
levels
organs
skelet
fish 0

mice f

Th
air an
to 0cc
the oi
radica
higher
Perios
likely
(0.02:
Perox-
Concel
0.021
TEduc
the up
NEVEr'
“Yen s
Synth.
manif.

Prod”

80
antioxidants were consistently higher than that observed in organs and
whole bodies of corn oil fed mice (Table 4). The addition of high
levels of antioxidants to the fish oil diet reduced TBARS values in all
organs and in whole bodies (carcass). However, TBARS values in heart,
skeletal muscle, mammary glands and whole bodies (carcass) of mice fed
fish oil containing antioxidants were still higher than TBARS values of
mice fed a corn oil diet.
DISCUSSION

The PV of fish oil diets constantly increased with time exposure to
air and under normal feeding conditions. Peroxide formation is likely
to occur as susceptible polyunsaturated fatty acids are available in
the oil (Esterbauer et al. 1986). PY increases are catalyzed by free
radical attacks at multiple sites. This might explain the observed
higher rate of dietary peroxide formation attained during the 0 to 24 h
period in which more susceptible sites for free radical attack are
likely to be present. The addition of the synthetic antioxidant BHT
(0.02%.w/w oil) to the fish oil diet significantly reduced lipid
peroxidation when compared to the control fish oil diet. The higher
concentration of BHT (2.0%.w/w of oil) was more effective than the
0.02%ilevel. TBHO, at the 2.0% level was slightly more effective in
reducing peroxide formation. TBHO has been reported previously to be
the most effective antioxidant for fish oil (Ke et al. 1977).
Nevertheless, peroxide formation still occurred in the fish oil diet
even when 100 times the AIN recommended level (0.02%.w/w of oil) for a
synthetic antioxidant (2.0% TBHO or BHT) was added. Since peroxides
manifest a transitory nature, over time conversion to secondary

products of oxidation is favored.

81

Secondary products of lipid peroxidation (TBARS) measured by the
TBA assay also displayed an increasing trend over the first 48 h.
However, this trend was not apparent at 72 h where a decline in TBARS
values was observed. In contrast to PV, TBARS formation depends on
specific site oxidation accompanied by serial decomposition reactions
(Dahle et al. 1962). Further examination of TBARS at shorter
wavelengths (455 nm and 495 nm) suggest the possibility of a subsequent
conversion of these secondary products of lipid peroxidation to other
related compounds, most probably aldehydes such as alkanals and
alk-2-enals (Kosugi et al. 1987) in the 72 h sample. Such TBARS
determined at shorter wavelengths, should be expected to increase as
secondary reactions proliferate with continuing oxidation. It is also
possible that the observed reductions in TBARS values at 72 h may be
due, at least in part, to possible reactions between aldehydes (i.e.,
malondialdehyde and/or alkanals) and proteins (Cuppett et al. 1989).
The addition of synthetic antioxidants to the fish oil diets resulted
in a substantial decrease in dietary TBARS, as was observed for PV,
i.e., 2.0% TBHO was most effective, followed by 2.0% BHT, then 0.02%
BHT. Once again, and importantly, the addition of synthetic
antioxidants at a level 100 times greater than the AIN recommended
level (0.02%.w/w of oil) did not totally prevent the formation of
oxidation products in the fish oil diet. TBARS values in the
antioxidant supplemented fish oil diets were still higher than that
observed for corn oil. In regard to the oxidative susceptibility of
the dietary oils used in this study, it is clear that corn oil (2 60%
linoleic acid, 18:2,n-6) is considerably less prone to autoxidation

than is fish oil. This difference is no doubt due to the high content

82
of long chain polyunsaturated fatty acids, i.e., EPA and DHA, in fish
oil; such fatty acids are absent in corn oil (Table 2).

Significant differences in lipid peroxidation were found among fish
oil diets prepared from oils originating from separate batches from the
same supplier (data not shown). The mean PV in freshly obtained fish
oil in our studies was 7.25 (for comparison purposes, fresh stripped
corn oil had a mean PV value of 0.97). This particular problem may
prove to be very difficult to solve and may be due to an array of
variables such as storage, processing, air and light exposure,
temperature and seasonal variations (e.g. type of phytoplankton
available for fish consumption). The rapid addition of a synthetic
antioxidant to the oil as soon as the oil container is opened should
deter or decrease further deterioration of the oil.

Organs and whole body carcasses of mice fed the corn oil and fish
oil diets (1 antioxidants) were also evaluated for TBARS. TBARS values
obtained from certain organs and carcasses were directly related to the
oxidation content of the ingested diet, although it is conceivable that
this process can be further enhanced 1n_§11u. Even large doses of
antioxidants (100x the AIN recommended level of a synthetic antioxidant
and approximately 30x the amount of tocopherol acetate of that supplied
by the vitamin mix) added to the fish oil diet failed to prevent the
accumulation of secondary products of oxidation in certain organs
(heart, skeletal muscle, mammary gland) and in whole bodies (carcass)
of mice fed the supplemented fish oil diet. Lebovitz et al. (1990)
reported similar results in tissue slices obtained from rats consuming
a fish oil diet. They reported a decrease in secondary oxidation

products in tissue slices of organs of rats receiving high levels of

83

dietary vitamin E, but also were unable to fully suppress the formation
of these oxidation products. In contrast to what was observed in our
study with heart, skeletal muscle and mammary glands, the livers and
kidneys of mice fed fish oils containing antioxidants had significantly
lower TBARS values than did the livers and kidneys of mice fed corn
oil. Although the exact explanation is not certain, it is conceivable
that at least for the liver, the capacity for storage of antioxidant
lipid soluble components and/or the function of this organ in the
production of lipoproteins may explain this phenomenon.

In summary, based on the results presented in this communication,
the AIN recommended level of addition of a synthetic antioxidant (0.02%
w/w of oil) to diets containing high levels of polyunsaturated fatty
acids appears to be insufficient to completely inhibit oxidative
deterioration when such diets have fish oil as their fat base. Thus, a
distinct recommendation for fish oil diets compared to other
polyunsaturated fats seems appropriate. Meanwhile, in order to
minimize oxidative deterioration in experimental diets containing high
levels of fish oil, a synthetic antioxidant should be added in
quantities substantially above the AIN recommendations. PV and TBARS
values should be monitored throughout all feeding experiments involving
dietary fish oil. Animals must be provided a fresh diet at least every
24 h in clean food jars; the remaining non-consumed diet should be
discarded. In addition, the mixing of diets in an inert atmosphere

(e.g. N2 or C02) should be mandatory. Freshly prepared diets should be

stored in individual zipper bags in an amount sufficient for a single

day’s feeding and kept at -20°C until fed. These recommendations will

substantially reduce, albeit not completely eliminate, excessive

84
oxidative deterioration of experimental diets incorporating high levels
of fish oil. The multitude of research laboratories studying the
effects of dietary fish oil on physiological and/or pathological
processes should be cognizant of these concerns.
LITERATURE CITED

American Institute of Nutrition (1977). Ad hoc committee on standards
for nutritional studies. J. Nutr. 107: 1340-1348.

American Institute of Nutrition (1980). Ad hoc committee on standards
for nutritional studies. J. Nutr. 110: 1726.

American Oil Chemist’s Society (1973). Official and recommended
practices of the American Oil Chemist’s Society. Vol. 1, Methods: cd
8-53 and Ba 3-38, AOCS, Champaign, IL.

Buford-Coulter, R. (1988). Extending shelf life by using traditional
phenolic antioxidants. Cereal Foods World 33: 207-210.

Chan, H.W.S. (1987). The mechanism of autoxidation: In Autoxidation of
Unsaturated Lipids. (Chan, H.W.S., ed.) pp. 1-16, Academic Press,
Lon on.

Committee on Care and Use of Laboratory Animals, Institute of
Laboratory Animal Resources Commission on Life Sciences, National
Research Council (1985) Guide for the Care and Use of Laboratory
Animals. National Institutes of Health publication No. 85-23 (rev.).
Washington, D.C.

Cuppett, S.L., Gray, J.I., Booren, A.M., Price, J.F. and Stachiw, M.A.
(1989). Effect of processing variables on lipid stability in smoked
Great Lakes Whitefish. J. Food Sci. 54: 52-54.

Dahle, L.K., Hill, E.G. and Holman, R.T. (1962). The thiobarbituric
acid reaction and the autoxidation of polyunsaturated fatty acid methyl
esters. Arch. Biochem. a Biophys. 98: 253-255.

Esterbauer, H., Benedetti, A., Lang, J., Fulceri, R., Fauler, G. and
Comporti, M. (1986). Studies on the mechanism of formation of 4-
hydroxy-nonenal during microsomal lipid peroxidation. Biochem.
Biophys. Acta 876: 154-166.

Fritsche, K.L. and Johnston, P.V. (1988). Rapid autoxidation of fish
oil in diets without added antioxidants. J. Nutr. 118: 425-426.

Gonzalez, M.J., Schemmel, R.A., Gray, J.I., Dugan, L., Jr., Sheffield,
L.G. and Welsch, C.W. (1991). Effect of dietary fat on growth of MCF-
7 and MDA-MB 231 human breast carcinomas in athymic nude mice:
Relationship between carcinoma growth and lipid peroxidation levels.
Carcinogenesis 12: 1231-1235.

85

Ke, P.J., Nash, D.M. and Ackman, R.G. (1977). Mackerel skin lipids as
an unsaturated fat model system for the determination of antioxidative

potency of TBHO and other antioxidant compounds. J. Am. Oil Chem. Soc.
54: 417-420.

Kosugi, H., Kato, T. and Kikugawa, K. (1987). Formation of yellow,
orange and red pigments in the reaction of Alk-2-enals with 2-
thiobarbituric acid. Analytical Biochem. 165: 456-464.

Leibovitz, B.E., Hu, M.L. and Tappel, A.L. (1990). Lipid peroxidation
in rat tissue slices: effect of dietary vitamin E, corn oil - lard and
menhaden oil. Lipids 24: 125-129.

Rasheed, A.A., Oldfield, J.E., Kaufmes, J. and Sinhuber, R.O. (1963).
Nutritive value of marine oils: menhaden oil at varying oxidation

levels with and without antioxidants in rat diets. J. Nutr. 79: 323-
332.

Wilkinson, L. (1989). Systat: the system for statistics. Evanston,
IL. Systat, Inc.

86
Table 1. Diet Composition

 

 

Ingredients1 g/100 g diet“
Fat“ 20.00
Casein“ 20.17
dl-Methionine 0.35
Dextrose 32.18
Sucrose 16.09
AIN Mineral nix5 4.13
AIN Vitamin nixs5 1.18
Cellulose6 5.90

 

1All ingredients (dry components) were obtained from U.S. Biochemical

Cos, Cleveland, OH except sucrose (ICN Biochemicals, Inc., Costa Mesa,
CA .

“High fat diets (20%iw/w) consisted of 20% corn oil or 19% fish oil

(Menhaden)/1% corn oil. Menhaden oil contains 50 IU/kg of dl-alpha
tocopherol.

3Corn oil (tocopherol stripped) was obtained from U.S. Biochemical Co.,
Cleveland OH. Fish oil (Menhaden) was obtained from Zapata Haynie
Corp., Reedville, VA.

“Vitamin free, high nitrogen (14.5%).
”J. Nutr. 107: 1340-1348, 1977.

6Celufil, non nutritive bulk.

87

 

 

Table 2. Effect of supplemented dietary antioxidants on peroxide
values of fish (Menhaden) oil diets.
Peroxide Yalu
(mmol/kg oil)
Time (hours)
0.1.9.1. 03 2.4 so 12
20%.corn 1.3310.17“ 8.67:0.34“ 11.67:0.88“ 19.67i1.46“
oil
19% fish 13.57¢1.2s 45.331220“l 712012.89“ 262.671;7.27ff
oil/1%
corn oil
19% fish 6.67:0.88c 25.671088c 39.6711.46“ 78.20¢2.09°
oil/1%
corn oil 3
0.02%.BHT
19% fish 4.01_+_0.se” 17.03¢0.se” 306712.48“ 48.66i4.42°
oil/1%
corn oil
+2xesn3
19% fish 2.07:0.29a 14.0811.“b 22.011058c 38.67¢1.20c
oil/1%
corn oil 4
+ 2% TBHO
19%.fish 1.50:0.29“ 11.671088a 17.051116b 30.151050b
oil/1%
corn oil
+2%TBHO‘
+ 2000 IU

Vitamin E5

 

88
Footnotes for Table 2

1Statistical comparisons made at each time point between diets (values

are means 1_SEM, n-6) determined by ANOVA and Neuman-Keuls multiple
comparison test.

“Value with different superscript letters in the same column are
significantly different at p < 0.01.

3Butylated hydroxytoluene
“Tertiary butyl-hydroxyquinone

Sdl-alpha-tocopherol acetate

89

 

 

Table 3. Effect of supplemented dietary antioxidants on TBARS levels
of fish (Menhaden) oil diets.
Thiobarbituric acid reactive substances
(ug TBARS/ g diet)
Time (hours)
miss 93 21 is 12
20% corn 0.3510.01“ 0.49:0.03“ 0.74:0.04“ 0.50:0.03b
oil
sex fish 2.931033f 6.53:0.15“ 13.95¢0.45° 19.06:0.67“
oil/1%
corn oil
19%.fish 1.18:0.03“ 1.6810.14° 1.951009d 0.97:0.05d
oil/1%
corn oil 3
0.02% BHT
19% fish 0.70:0.03d 0.951004d 1.10:0.04c 0.80:0.05c
oil/1%
corn oil3
+ 2% BHT
19% fish 0.63:0.03“ 0.84:0.05c o.91;0.03” 0.47:0.04”
oil/1%
corn oil ‘
+ 2% TBHO
19% fish 0.49:0.03b 0.671004b 0.7910.05“ 0.3710.03“
oil/1%
corn oil
+ 2% TBHQ‘
+ 2000 IU

Vitamin :5

 

90
Footnotes for Table 3
1Statistical comparisons made at each time point between diets (values
are means 2 SEM, n-6) determine by ANOVA and Neuman-Keuls multiple
comparison test.

2Value with different superscript letters in the same column are
significantly different at p < 0.01.

3Butylated hydroxytoluene
‘Tertiary butyl-hydroxyquinone

Sdl-alpha-tocopherol acetate

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Footnotes

1Supported by research grants AICR-9OBH05 and NIH CA-42876 to C.W.

Welsch and by a Predoctoral mini grant of FSHN Department of MSU, an
Equal Opportunity Fellowship and government of Puerto Rico, and
University of Puerto Rico Fellowships to H.J. Gonzalez. Thanks to
Marlene Green for the preparation of this manuscript.

2Abbreviations used: BHT, butylated hydroxytoluene; DHA,

Docosahexaenoic acid; EPA, eicosapentaenoic acid; PV, Peroxide value;
TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive
substances; TBHQ, tertiary butylhydroxyquinone.

Chapter 3

Dietary fish oil inhibits human breast carcinoma growth:
A function of increased lipid peroxidation
(Lipids 1993: 28,827-832)

Dietary Fish Oil Inhibits Human Breast Carcinoma Growth:
A Function of Increased Lipid Peroxidation

Michael J.'Gonzaleza, Rachel A. Schemmel“, LeRoy Dugan Jr.a,

J. Ian Gray‘, and Clifford H. Nelschb*

aDepartment of Food Science and Human Nutrition and
bDepartment of Pharmacology and Toxicology, Michigan State University,

East Lansing, Michigan 48824 USA
Running title: DIETARY FISH OIL AND HUMAN BREAST CARCINOMA GRONTH

Key words: Fish oil, breast carcinoma, lipid peroxidation, linoleic

acid, antioxidants

Mailing Address:Dr. Clifford H. Helsch
Department of Pharmacology and Toxicology
Michigan State University
East Lansing, MI 48824, USA
Phone: 517-353-4549
Fax: 517-353-8915

94

95
*To whom correspondence should be addressed at Dept. of Pharmacology
and Toxicology, 357 Giltner Hall, Michigan State University,
East Lansing, Michigan, 48824, USA

Abbreviations:
CO, corn oil; EPA, eicosapentaenoic acid; FO, fish oil; DHA,
docosahexaenoic acid; TBA, thiobarbituric acid assay; TBARS,
thiobarbituric acid reactive substances; TBHO, tertiary butyl

hydroquinone

96

ABSTRACT

Female athymic nude mice were implanted 5,9, with human breast
carcinoma MDA-M8231. Seven to 10 days later, the mice were divided
into groups and fed a purified diet containing the following types of
fat (% of diet): 1) 20% corn oil (CO); 2) 15% CO/5% fish (menhaden) oil
(F0); 3) 10% CO/10% F0; 4) 5% CO/15% F0; 5) 1%»CO/19% F0 and 6) 1%
CO/19% FO plus antioxidants [alpha tocopherol acetate, 2000 IU/kg diet
and tertiary butyl-hydroquinone (TBHO), 2% of total fat]. The linoleic
acid levels (% of diet) of the groups were 12.0%, 9.1%, 6.2%, 3.3%,
0.9% and 0.9%, respectively. After 6-8 weeks, the carcinomas were

assessed for tumor volume (cm3) and assayed for thiobarbituric acid

reactive substances (TBARS). Human breast carcinoma growth was
suppressed in mice consuming FO diets without antioxidants compared to
mice fed CO; the greater the amount of dietary FO fed, the greater the
carcinoma growth suppression (p<0.05). The addition of antioxidants to
the FO diet significantly (p<0.05) reversed the F0 induced carcinoma
growth suppression. Concentrations of TBARS in the human breast
carcinomas were increased in all the FO (without antioxidants) fed
mice, compared to mice fed CO; the level of increase in TBARS was
directly related to the increase in the level of F0 fed (p<0.05). The
addition of antioxidants to the FO diet significantly (p<0.05) reduced
the concentration of TBARS in the breast carcinomas. Thus, these
results provide evidence that dietary FO can significantly suppress
growth of human breast carcinoma MDA-MB231, even in the presence of
substantial amounts of linoleic acid (3.3%.to 9.1%). The inhibitory
effect of F0 on growth of these carcinomas was associated with an

increased concentration of TBARS in the tumor tissue. In conclusion,

97

dietary F0 induced suppression of human breast carcinoma growth is a
function, at least in part, of an accumulation of lipid peroxidation
products in the tumor tissues.
INTRODUCTION

Fish oil (F0), which contains elevated levels of long chain omega-3
polyunsaturated fatty acids, in particular, eicosapentaenoic acid (EPA,
20:5) and docosahexaenoic acid (DHA, 22:6), has been shown to suppress
mammary gland tumorigenesis in rodent models (1-4). In addition, F0
has been shown to inhibit the growth of human breast carcinomas
maintained in immune deficient animals (5-9). To explain how F0
inhibits mammary tumorigenic processes, many investigators (2-4) have
focused on EPA and/or BHA-induced alterations in eicosanoid metabolism.
Other investigators (IO-11) suggest that dietary FO inhibits mammary
tumorigenesis because it may not provide a sufficient amount of
essential fatty acids such as linoleic acid (18:2). Indeed, a direct
relationship between the amount of dietary linoleic acid and mammary
tumor growth has been clearly shown (12). In contrast, we have
explored yet another potential mechanism, i.e., that high levels of
dietary F0 inhibit mammary tumorigenesis by causing an increased
accumulation of cytostatic and/or cytotoxic lipid peroxidation products
in the tumor tissue (9). In this communication, we further examine the
relationship between dietary F0 and growth of human breast carcinomas
in athymic nude mice by pursuing the following objectives: 1) to
determine whether or not high levels of dietary F0, in the presence of
substantial amounts of linoleic acid, can inhibit growth of these
tumors and 2) whether or not there is a correlation between tumor

growth and the quantity of lipid peroxidation products in the tumor

98
tissues of mice fed FO.
MATERIALS AND METHODS
An1m31§_3ng_d1gt‘ One hundred sixty-four female athymic nude mice
(Harlan Sprague Dawley Inc. Madison, NI) 4 to 5 weeks old were used in
these studies. The mice were housed under aseptic conditions (enclosed
overhead laminar flow hood, sterilized cages, bedding and water) in a

temperature (24°C) and light (14 h/day) controlled room. All

experimental procedures pertaining to the athymic mice were performed
under aseptic conditions. Autoclaved laboratory mouse chow (Purina
Mills Inc., St. Louis, MO) was fed an 11bitum before and up to 7-10
days after human breast carcinoma transplantation. Thereafter, mice
were fed purified diets (Table 1) 3d,11hitum. The percentages of the
predominant fatty acids (1% or greater, manufacturer's specifications)
of the dietary oils are shown in Table 2. The diets were prepared
weekly in our laboratory, individually packed in small plastic zipper-
sealed bags of sufficient size for one day’s feed, flushed with
nitrogen, sealed and stored at -20°C. Mice were fed daily and non-

consumed food discarded daily. Since the purified diets were not
sterilized, antibiotics (bacitracin combined with streptomycin or
neomycin, 1 g/L) were added to the distilled drinking water.
flgman_brgast_carcingma_tran§plantatign, Palpable MDA-M8231 human
breast carcinomas (American Type Culture Collection, Rockville, M0)
were surgically excised from female athymic mice, cut into slices (2x4
mm, 0.1-0.3mm thick) and implanted into recipient mice. Mice were
anesthetized with sodium pentobarbital (60 ug/g, 1,9,) prior to
transplantation. An incision was made in the integument, the tumor

slices were placed 5‘s. in the dorsum at distances from each other of

99
at least 2 cm, 3 to 4 slices/mouse. Between 1 and 3 palpable human
breast carcinomas were pooled to provide the tissue slices. Mice were
placed on the experimental diets 7-10 days after carcinoma
transplantation. This period allows the carcinoma grafts to become
established in the host animals before the onset of experimental
dietary treatments. Athymic mice bearing human breast carcinoma grafts
were fed the experimental diets for a period of 6-8 weeks. At the
termination of the studies, all tumors were excised and their volume

determined (V - 4/3 x r3) with a vernier caliper. Four separate

studies (1-4) were carried out, each study was a complete individual
experiment.

Stydy_1, The experimental design of this study is provided in
Table 3. The purpose of this study was to determine if varying levels
of F0 mixed with varying levels of linoleic acid-rich corn oil (C0) are
capable of modifying the human breast carcinoma growth suppressive
effect of a F0 diet. In addition, we sought to determine if a
relationship exists between human breast carcinoma growth and the level
of lipid peroxidation products in the tumor tissue. The level of lipid
peroxidation products of excised 10 g tumor composites in each group
were determined by measuring 2-thiobarbituric acid reactive substances
(TBARS) using a modified macro-thiobarbituric acid (TBA) assay (9).

Magrg;IBA_a§§ay,__Subcutaneously growing human breast carcinomas
were surgically excised, placed individually in a 0.9% NaCl solution,
immediately frozen (-20°C) and stored for one day. Immediately before

the TBA assay, the tumors were thawed and tumor samples pooled to
obtain 10 9 composites. Once sufficient tumor homogenate was obtained

from pooling (10 g composite), this constituted one sample. To obtain

100
other samples, other tumors within the same group were also pooled to
form 10 9 samples. Three to 5 samples (10 9 composites) were done in
triplicate for each group. An antioxidant [tertiary butyl-hydroquinone
(TBHO), Eastman Chemical Products, Kingsport, TN], at 0.02% of total
fat, as determined by a dry column extraction method (13) was added to
the samples; such samples were homogenized (4°C) in a polytron

homogenizer (Ultra Turrax SDT 1810, Tekmar Co., Cincinnati, OH). The
homogenates were transferred into distillation flasks containing 2.5 mL
4N HCl plus 0.5 mL silicone antifoam (Thomas Scientific Co.,
Swedesboro, NJ) with sufficient distilled water to yield a total volume
of 100 mL/flask and distilled to collect 50 mL. Five mL of this
distillate plus 5 mL of freshly prepared TBA (0.2N aqueous solution,
Sigma Chemical Co., St. Louis, M0) were used for the assay. Samples
were prepared in triplicate, heated in a boiling water bath for 35 min
to develop the pigment, cooled and absorbance read on a
spectrophotometer (Spectronic 2000, Bausch G Lomb, Rochester, NY) at
532 nm. The absorbance values obtained were multiplied by an
adjustment factor of 6.2 (14). Results were reported as ug of TBARS
per g of sample.

§tugy_z, The experimental design of this study is provided in
Table 4. The purpose and experimental design of this study was similar
to Study 1 but two additional dietary groups (1% CO/I9%.FO and 1%
CO/19% F0 plus antioxidants) were added. A modified micro-TBA assay
(15) was used in this study in order to individually assay each excised
tumor for TBARS.

519:9;Ifia_gssay, Subcutaneously growing human breast carcinomas

were surgically excised, frozen (-20°C) for one day, thawed and

101
individually homogenized (4°C) in the presence of an antioxidant (TBHO,

0.02% of total fat) in a polytron homogenizer. One mL of homogenate
was combined with 2.0 mL of TBA (0.375% wt/vol), trichloroacetic acid
(15.0% wt/vol), and HCl (0.25N) and mixed thoroughly. The solution was
heated for 15 min in a boiling water bath. After cooling, the
precipitate was removed by centrifugation (1,000 xg for 10 min) and the

supernatant kept for analysis. The absorbance of the sample was

determined at 535 nm against a blank that contains all reagents minus
the homogenate. This TBA assay is a slightly modified version of the
Buege and Aust assay (15). TBARS concentration of the sample was

calculated using an extinction coefficient of 1.56 x 105 M’1 cm'1 (16)

 

and reported as nmoles TBARS per mg of protein. The protein
concentration of tumors was determined according to the method of Lowry
et a1. (17).

§1ndy_3. The experimental design of this study is shown in the
legend of Figure 1. The purpose of this study was to determine if the
concentration of TBARS in individual human breast carcinomas correlated
with tumor size from a single F0 dietary group (1%1CO/19% F0). Excised
tumors were individually assayed for TBARS by the modified micro TBA
assay.

§_1;_ugly__4__L The experimental design of this study is provided in
Table 5. The purpose of this study was to determine if the length of
time the F0 diet is allowed to remain in the food jar, under feeding
conditions, 1ggg, 24, 48 and 72 h after feeding, influences human
breast carcinoma growth and carcinoma concentration of TBARS. The jars
with diet were changed every 24, 48 or 72 h. Excised tumors were

individually assayed for TBARS by the modified micro-TBA assay.

102
Statist1cal_analy§g§, Data in Studies 1,2, and 4 were analyzed by
one-way analysis of variance and the Newman-Keuls multiple comparison
test (18). Significance was set at p<0.05. Data for Study 3 were
examined by Pearson’s correlation coefficient (18).
RESULTS
flgman_brga§t_ga[§1ngma_grgwth, In Study 1, mean human breast carcinoma

volumes (cm3) obtained from the 4 dietary groups at experiment

termination ranked (from largest to smallest) as follows: 20% CO > 15%
CO/5% FO > 10% CO/10% F0 > 5% CO/15% F0 (Table 3). Mean carcinoma
volumes in mice fed each of the 3 F0 diets were significantly (p<0.05)
smaller than the mean carcinoma volume of mice fed CO as their sole
source of fat (20% CO). Body weight gains were not significantly
different among the mice in the different dietary groups.

In Study 2, mean human breast carcinoma volumes (cm3) obtained from

the 6 dietary groups at termination of the experiment ranked (largest
to smallest) as follows: 20% CO > 15% CO/5%.FO > 1% CO/I9% F0 plus
antioxidants >10% CO/10% F0 > 5% CO/15% FO > 1%.CO/19% F0 (Table 4).
Mean carcinoma volume in mice fed 20% CO was numerically higher than
those of the 15% CO/5% F0 and 1% CO/19% F0 plus antioxidants fed mice,
but this difference did not reach a level of significance. However,
each of these 3 groups had mean carcinoma volumes which were
significantly (p<0.05) larger than those produced by the 10%.C0/10% F0,
5% CO/15% F0, 1%.C0/19% F0 dietary groups. Body weight gains were not
significantly different among the mice in the different dietary groups.

In Study 4, mean human breast carcinoma volumes (cm3) obtained from

the 3 dietary groups at termination of the experiment ranked (largest

 

103
to smallest) as follows: 24 h - 1% CO/19%.FO > 48 h - 1% C0/19% FO >
72 h - 1%.CO/19% F0 (Table 5). Mean carcinoma volumes in mice in which
the diet (1%.CO/19% F0) was changed every 24 h was significantly larger
(p<0.05) than in the 48 h or 72 h diet groups. The mean carcinoma
volume of the 48 h diet group was significantly larger (p<0.05) than
that of the 72 h diet group. Body weight gains were significantly
lower (p<0.05) for the 72 h diet group compared to the 24 hour diet
group.
1 Mil - '- a non. . - .- n. n: '1 . '9. ' ' =:: The
ranking of mean human breast carcinoma TBARS concentrations in the 4
dietary groups of Study 1 at termination was: 5% CO/15% F0 > 10% CO/10%
FO > 15‘% CO/5% F0 > 20% CO (Table 3). Mean human breast carcinoma
TBARS concentrations in mice fed each of the 3 F0 diets were
significantly (p<0.05) greater than the mean carcinoma TBARS value in
mice fed CO as their sole source of fat (20% CO). In Study 2, the
ranking of the mean human breast carcinoma TBARS concentrations in the
6 dietary groups at termination was: 1% CO/19% F0 > 5% CO/15% FO > 10%
CO/10%FO > 15% CO/5% F0 > 20% CO > 1% CO/19% F0 plus antioxidants
(Table 4). Mean tumor TBARS concentrations were virtually identical in
'the 1%.CO/19% F0 plus antioxidants and 20% C0 fed animals. Mean tumor
“TBARS concentration in mice fed each of the four non-antioxidant
{supplemented F0 diets were significantly (p<0.05) greater than the mean
‘tumor TBARS concentrations in mice fed the antioxidant supplemented F0
{and in the group fed only CD as their sources of fat. In Study 3, an
inverse relationship is shown between volumes of individual human
breast carcinomas and carcinoma TBARS concentrations in mice fed the 1%

(IO/19% FD diet (p<0.05) (Figure 1), Le... the smaller the tumor

104

volumes, the greater the concentration of TBARS. In Study 4, mean
carcinoma TBARS concentration in mice in which the diet (1%.CO/19% F0)
was changed every 24 h was significantly lower (p<0.05) than in the 48
or 72 h diet groups (Table 5). The mean carcinoma TBARS of the 48 h
diet group was significantly less (p<0.05) than that of the 72 h diet
group.
DISCUSSION

This study provides evidence that supplementation of a high fat
(CO) diet with even a modest amount of F0 (gygg, 15% CO/5% FO) can
result in a suppression of growth of a human breast carcinoma (MDA-
M8231) maintained in athymic nude mice. Other laboratories have
provided evidence that FO containing diets inhibit mammary gland
tumorigenesis in rodents (1-4) and suppress growth of human breast
carcinomas in athymic nude mice (5-9). Such studies often utilize
diets containing FO as their only source of fat. F0 is extremely low
in the essential fatty acid linoleic acid (18:2). It is conceivable,
therefore, that the suppression of mammary tumorigenesis exerted by
diets high in F0 may be via essential fatty acid (linoleic acid)
insufficiency. Indeed, supplementation of F0 diets with modest amounts
of linoleic acid, in certain mammary tumor animal models, has been
reported to block (4) or partially block (19) the tumorigenic
inhibitory activities of a diet high in F0. In this study, we were
still able to attain growth inhibition of MDA-M8231 human breast
carcinomas in athymic nude mice by feeding FO diets containing
substantial amounts of linoleic acid (3.3% -9.1%). Hhile the estimated
nutrient requirement for adequate growth and reproduction of mice for

linoleic acid is 0.3%.wt/wt of diet (20), the level required for

 

105

optimal tumorigenic processes may be substantially higher. For
example, Ip gt al. (12) have reported that approximately 4.4% of
linoleic acid in the diet is required for optimal (maximal) mammary
tumor development in a rodent model. In our study, diets containing F0
and substantial amounts of linoleic acid (up to 9.1% of diet) still
suppressed growth of this human breast carcinoma. Thus, it is likely
that dietary FO-induced suppression of growth of human breast carcinoma
MDA-M8231 is not due to insufficient dietary linoleic acid.

Inhibition of growth of human breast carcinoma MDA-MB231 by dietary
FO may be yin the accumulation within the tumor of cytostatic and/or
cytolytic lipid peroxidation products. Evidence presented in our study
which supports this conclusion are as follows. 1) As the FO content of
the diet is increased, a concurrent significant increase in carcinoma
TBARS concentrations is observed. 2) Increasing the FO content of the
diet decreases carcinoma growth. 3) Those carcinomas that have the
highest concentrations of TBARS have the least tumor growth.
4) Allowing the FO diet to remain in the food jar for as long as 72 h,
a feeding protocol that increases substantially the level of TBARS and
the peroxide values in the diet (21), results in an increased
accumulation of TBARS in the carcinomas and a concordant suppression of
carcinoma growth. 5) The addition of efficacious antioxidants to the
F0 diet substantially decreases carcinoma TBARS concentrations and
concurrently increases carcinoma growth. Collectively, these results,
clearly support the concept that lipid peroxidation products are
relevant to the process of dietary FO-induced inhibition of breast
carcinoma growth. Recent evidence from our laboratory (21) indicates

that dietary F0 derived lipid peroxidation products are formed

 

106
extensively in the food jar under normal feeding conditions. After
feeding FO, such products are not only found in tumor tissue but are
found in normal tissues as well.

The mechanism by which lipid peroxidation products retard or
inhibit tumor growth processes is not certain (22). Secondary products
of lipid peroxidation (measured by the TBA assay) are capable of
decreasing cell proliferation through damaging cell membranes, by
changing cellular composition and/or cytoskeleton assembly. These
modifications in the molecular architecture of the membrane can lead to
the inactivation of membrane transport systems and/or membrane bound

enzymes (23-25). This phenomenon may adversely affect the entering of

 

cells into the cell cycle or it may accelerate their exit from it (1‘g,
cell death). Furthermore, secondary products of lipid peroxidation can
decrease tumor cell survival by inactivating polymerase reactions (26),
forming inter- and/or intramolecular linkages between amino acid
sulfhydryl groups and biomolecules (DNA, RNA and proteins) (27) and
inhibiting polyamine synthesis (28). Such processes may not only
result in inhibition of cell proliferation but may also lead to an
increase in cell death. Indeed, Gabor and Abraham (4) reported that
dietary F0 inhibits mammary tumor growth processes by increasing
mammary tumor cell death rather than suppressing mammary tumor cell
proliferation.

Secondary products of lipid peroxidation, while under certain
conditions may decrease tumor growth via a cytostatic/cytolytic
mechanism, under other circumstances such products could stimulate
tumor growth. For example, a peroxidation product of linoleic acid,

1yg‘, 13-hydroxy (hydroperoxy) octadecadienoate is more mitogenic to

107
rat colonic mucosa cells (29) or to hamster embryo fibroblasts (30)
than is the parent compound. In addition, a peroxidation product of
arachidonic acid, 1‘g; hydroxy (hydroperoxy) eicosatetraenoic acid
stimulates rat colonic mucosa cell proliferation while the parent
compound itself lacks stimulatory activities (29). Thus, peroxidation
products derived from fatty acids in dietary F0 (particularly EPA, DHA
and possibly linoleic acid) could be stimulatory to tumor growth
processes. In the tumor model used in this study (MDA-M8231 human
breast carcinoma maintained in athymic nude mice), however, we find no
evidence of dietary FO induced stimulation of tumor growth.

The results of our study clearly show that the addition to the FO
diet of large amounts of antioxidants (Vit E and TBHO) significantly
counteracted the inhibitory effects of dietary F0 on human breast
carcinoma growth. In essence, high levels of antioxidants, in the
presence of dietary FO, stimulated tumor growth. Other laboratories
have provided evidence that supplementation of the diet with
antioxidants inhibits tumorigenesis. Clearly dietary antioxidants can
suppress carcinogen activation in experimental tumor models (31).
Occasionally it is reported that antioxidant supplementation can
suppress tumor growth (32,33), although there is considerable evidence
to the contrary (34-36). Thus, it is likely that various antioxidants
can suppress genotoxic induced initiation and/or progression events in
tumorigenesis; evidence that antioxidants can effectively suppress
tumor growth processes, however, has not been consistent or compelling.

In conclusion, the results of this study provide evidence that
dietary FO can significantly suppress the growth of MDA-MB231 human

breast carcinomas maintained in athymic nude mice, even in the presence

 

108

of substantial amounts of dietary linoleic acid. The suppressive
effect appears to be directly related to the accumulation of TBARS in
the tumor tissues.
ACKNONLEDGEMENTS

This work was supported by research grants AICR-9OH05 and
NIH-Ca42876 to C.M. Nelsch and an Equal Opportunity Fellowship from
Michigan State University, a scholarship from the Administration of
Economic Development of the Government of Puerto Rico and a Fellowship
from the University of Puerto Rico (Medical Sciences) to M.J. Gonzalez.
He also thank Mrs. Marlene Green for her excellent work in typing this

manuscript.

10.
11.

12.

13.
14.

15.
16.
17.

18.

19.

109
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Hilkinson, L. (1989) Systat: The system for statistics. SYSTAT,
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Helsch, C.H. (1992) J. Nutr. 122, 2190-2195.

Gonzalez, M.J. (1992) Med. Hypotheses 38, 106-110.

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1035.

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Dianzani, M.U. (1982) Free Radicals, Lipid Peroxidation and Cancer
(McBrien, D.C., and Slater, T.F., eds.) pp. 129-158, Academic
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1771-1776.

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Cancer 56, 445-449.

111
Table 1. Diet Composition

 

 

Ingredientsa Percentb
fatc 20.00
caseind 20.17
dl-methionine 0.35
dextrose 32.18
sucrose 16.09
AIN mineral mix° 4.13
AIN vitamin nixf 1.13
celluloseg 5.90

 

aAll ingredients (dry components) were obtained from U.S. Biochemical

Co. (Cleveland, OH) except sucrose (ICN Biochemicals, Inc., Costa
Mesa, CA).

bHigh fat diets (20% wt/wt) consisted of 20% corn oil or a fish oil
(menhaden)/corn oil combination totaling 20%.

cCorn oil (tocopherol stripped) was obtained from U.S. Biochemical Co.

(Cleveland OH). Fish oil (menhaden) was obtained from Zapata Haynie
Corp. (Reedville, VA).

dVitamin free, high nitrogen (14.5%).

°The mineral mixture supplied, per kg diet: CaHPO‘, 20.659; NaCl, 3.06
kg; potassium citrate, 9.09 g; KZSO‘, 2.159; MgO, 991.20 mg; manganous

carbonate, 144.55 mg; ferric citrate, 247.8 mg; zinc carbonate, 66.08
mg; cupric carbonate, 12.39 mg; K103, 0.41 mg; sodium selenite, 0.41

mg; chromium potassium sulfate, 22.72 mg; sucrose, 4.87 g.

1!The vitamin mixture provided, per kg diet: thiamine HCl, 7.08 mg;
riboflavin, 708 mg; pyridoxine HCl, 8.26 mg; nicotinic acid, 35.40 mg;
D-calcium pantothenate, 18.88 mg; folic acid, 2.36 mg; DL-a-tocopheryl

acetate, 236.00 mg; cholecalciferol, 0.03 mg; menaquionine, 0.06 mg;
sucrose, 11.48 g.

“Celufil, non nutritive bulk.

112
Table 2. Predominant Fatty Acids (%)

 

 

 

Fatty acidsa Corn oil Fish (menhaden) oil
Myristic (14:0) --- 6.7
Palmitic (16:0) 10.8 15.7
Palmitoleic (16:1) --- 8.7
Stearic (18:0) 2.1 2.7
Oleic (18:1) 26.5 14.3
Linoleic (18:2) 60.0 1.8
Linolenic (18:3) --- 1.5
Arachidonic (20:4) --- 2.2
Eicosapentaenoic (20:5) --- 15.5
Docosahexaenoic (22:6) --- 12.1

 

aFatty acids concentrations <1% are not included.

and lipid permcidatim

growth
athymic rude mice.

oilmthe

minutes

'I‘able3. Effectofdifferattratinsofdietaxymoilardfish

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Mean tum
lipid permddatim

product. levels 1:
(ug TBARS/g sanple):S.E.

2.55:9.31“

 

20% mail

28.1:O.5

(12.0% linoleic acid)

15%camoil/
5%fishoil

0.31:9.02d

(9.1% linoleic acid)

10% com 011/
10% fish oil

113

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o.sq:o.oa°

O.8810.lld

27.x:o.3

5% com 011/
15% fish oil
(3.3% linoleic acid)

 

bValues with different superscript letters in the same column are significantly

different at p<0.05.

M to 25 animals/gram.

114

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Figure l.

TBARS (nmol TBARS/mg protein)

lie
Relationship between mean carcinoma volume (cm?) and mean
carcinoma TBARS concentrations per individual MDA-M8231
human breast carcinoma in athymic nude mice fed a 1% corn
oil/19% fish oil diet (N-24). Eight animals in this dietary

group. Curved lines represent a 95% confidence interval.
Pearson correlation coefficient - 0.725 (p<0.05).

 

 

l L

O 5 10 15

Tumor Volume (cm3)

Chapter 4

Dietary fish oil induced inhibition of human breast carcinoma growth:
a phenomenon of reduced cellular DNA synthesis or increased cell loss

ABSTRACT
Diets high in unsaturated fatty acids, especially those containing
high levels of linoleic acid, e.g., corn oil, enhance mammary gland
tumorigenesis in experimental animals. In contrast, diets high in
long-chain polyunsaturated fatty acids such as eicosapentaenoic (EPA)
and docosahexaenoic (DHA), e.g. menhaden oil, appear to have a
suppressive effect on this tumorigenic process. Many mechanisms have

been proposed to explain the tumor inhibitory action exerted by

menhaden oil and other fish oils, e.g., differences in prostaglandin
metabolism, energy efficiency, alterations of the immune system,

changes in lipid peroxidation, etc. Fundamental to a mechanistic

 

‘F- T

understanding of this phenomenon, however, is an understanding as to
whether or not the tumor inhibitory activities of dietary fish oil is
mediated via an inhibition of tumor cell proliferation or mediated via
an enhancement of tumor cell loss. Hhether the amount of dietary fat
or the type of fat effects mammary tumorigenic processes, via an effect
on tumor cell proliferation or tumor cell loss, has not been clearly
established. In the studies described in this communication, three
methods were utilized to study tumor cell proliferation, i.e.,

H3-thymidine autoradiographic analysis, 5-bromo 2'-deoxyuridine (Brdu)

flow cytometric analysis, and proliferative cell nuclear antigen (PCNA)
flow cytometric analysis. Two methods were used to study tumor cell

loss, i.e., a determination of the 1125Urd tumor emission rate and a

determination of a cell loss factor from the formulas of Steel and
Begg. The tumor examined was the human breast carcinoma cell line MDA-
M3231 maintained in athymic nude mouse. No significant difference in

cell proliferation between carcinomas of mice fed a high corn oil diet

118

119
(20%.w/w) and a diet high in fish oil (19% menhaden oil, 1% corn oil).

In contrast, a significant (p<0.05) increase in the rate of 1125Urd

emission rate and cell loss factor from the carcinomas in the fish oil
fed mice compared to the corn oil fed mice was observed. In summary,
the decreased tumor volume in the human breast carcinomas maintained in
athymic nude mice fed a fish oil diet as compared to those fed a corn
oil diet, appears to be due, at least in part, to an increased rate of
carcinoma cell loss rather than a decreased rate of carcinoma cell
proliferation.

INTRODUCTION

The mechanism by which diets high in certain fats such as corn oil

 

are capable of enhancing mammary tumorigenesis in rodents (I) and
increase human breast carcinoma size in athymic nude mice (2) is
unclear. Moreover, the mechanism by which diets high in long-chain
polyunsaturated fatty acids (PUFA) such as fish oils, can effectively
suppress mammary tumorigenesis in rodents (l) and inhibit human breast
carcinoma growth in athymic nude mice (2,3) also remains to be
determined. This raises a fundamental question in tumor biology that
has not been rigorously examined. How do dietary fats exert their
enhancing or suppressive activity at a tumor growth kinetic (tumor cell
loss vs tumor cell proliferation) level? Only a few research groups
(4-6) have examined cell proliferation in tumors of animals fed high
fat diets. One group (7) reports no difference in cell proliferation
rates in transplantable mouse mammary tumors from animals fed diets
composed of unsaturated vs saturated fats. In another study, Abraham
et al. (8) hypothesized that the increase in tumOr size induced by a

corn oil diet compared to hydrogenated cottonseed oil or fish

120
(menhaden) oil diet was due to a decrease in cell loss (cell death) as
a result of the high corn oil diet impairing immune system activity.
In other studies (5,6,9-10), an increase in cell proliferation was

observed by an increase in H3-thymidine incorporation into DNA of

carcinogen-induced rat mammary tumors from rats fed a high corn oil
diet compared to those fed lower levels of corn oil. They concluded
that this increase in mammary tumor growth was due to an increase in
carcinoma cell proliferation by providing diets high in corn oil. Our
study was designed to determine if the growth of a human breast
carcinoma cell line (MDA-M8231) in vivo (athymic nude mice), as a
function of feeding high levels of either corn oil or fish oil
(menhaden), is due to changes in carcinoma cell proliferation and/or
changes in carcinoma cell loss. The knowledge of how dietary fats can
affect mammary carcinoma growth dynamics is critical to a mechanistic
understanding of nutritional tumorigenesis.
MATERIALS AND METHODS

Animalmndlim

Female athymic nude mice (Harlan Sprague-Dawley Inc., Madison, NI)
4-5 weeks old were used in these experiments. The mice were maintained
under aseptic conditions which included an enclosed overhead laminar
flow hood and were housed in sterilized cages, with sterilized bedding
and provided sterilized drinking water in a temperature-controlled
(24'C) and light-controlled (I4 h/day) room. Autoclaved laboratory
mouse chow (Purina Mills Inc., St. Louis, MO) was fed ad libitum before
and until 7-10 days after human breast carcinoma transplantation.
Thereafter mice were fed ad libitum purified diets for 4 to 6 weeks
(unless indicated otherwise) (Table 1). All dietary ingredients were

 

121

obtained from U.S. Biochemicals Inc. (Cleveland, OH) except sucrose,
which was obtained from ICN Biochemicals Inc. (Costa Mesa, CA), and
fish oil (menhaden), which was obtained from Zapata Haynie Corp.
(Reedville, VA). The percentages of predominant fatty acids (1% or
greater, manufacturer’s specifications) of the dietary oils are shown
in Table 2. The diets were prepared weekly and stored at -20’C,
individually packed in small plastic sealed bags of sufficient size for
one day’s feed. Mice were fed daily and non-consumed food discarded
daily. Since purified diets were not sterilized, antibiotics
(Bacitracin combined with Streptomycin or Neomycin, 1 g/L) were added
to the distilled drinking water.
Human_BLeast_9arsinnma_lran§nlantatinn

Palpable MDA-M8231 human breast carcinomas (American Type Culture
Collection, Rockville, MD) were surgically excised from female athymic
mice, cut into slices (2x4 mm, 0.1-0.3 mm thick) and implanted into
recipient female athymic mice under aseptic conditions. Mice were
anesthetized with sodium pentobarbital (60 ug/g, i.p.) prior to .
transplantation. An incision was made in the integument, the tumor
slices were placed s.c. in the dorsum at distances from each other of
at least 2 cm, 3 to 4 slices/mouse (autoradiograph and flow cytometer
experiments). One slice per mouse was placed in the middle of the
upper back between the shoulder blades in the animals used for the cell
loss experiments. The carcinoma grafts were established in the host
animals before the onset of experimental dietary treatments.

... . .. . “u ~ - . . . .. ,,. ;_ . .. .. ,.,
After being fed the diet for 5 to 6 weeks mice were sacrificed.

The tumors were excised and cut into slices (1-2 mm). Tumor slices

122
were incubated in 10x30 mm Falcon disposable Petri dishes
(2 slices/dish) containing 2.5 ml of medium (10X Haysmouth MB 752/1
medium, GIBCO Labs, Grand Island, NY). Per 100 ml of media, the
following constituents were added: 35 mg glutamine, 3.5 mg penicillin
and 125 mg of sodium bicarbonate. Sterile M3-thymidine (45 Ci/nmol,

New England Nuclear, Boston, MA) was added at a concentration of
l uCi/ml of medium. The Petri dishes were placed in a small gassing
chamber, housed in an incubator at 37'C. The chamber was continuously

infused with gas 95% 02: 5% C02 for a 4 hr incubation period. The

slices were then fixed in Bouins Fluid, embedded in a paraffin

preparation (Tissue-prep, Fisher Scientific Co., Fairlawn, NJ),

 

sectioned at 5-7 pm and mounted on glass slides. Two series of tissue
sections were prepared; one series was stained with hematoxylin and
eosin (M a E) and the other series was used for the autoradiographs.
The slides for autoradiography were dipped in NTBZ nuclear tract
emulsion (Eastman Kodak Co., Rochester, NY), dried and stored away from
light in tight black boxes with a desiccant for 14 days at 4°C. After
two weeks, the slides were developed and stained by M a E using a
standard method (11). The slides were then coded (identity of
treatment unknown) and the number of M3-thymidine labelled breast

carcinoma cells per area was computed for each carcinoma of both
dietary groups. Group mean differences between labelled cells were

evaluated statistically by the students t-test.

123
0‘ in; ;|. 0 111.13“ . 1.“. i ll; "1'
WWW
Preparation and Dissociation of Tumor Tissue
One hour prior to sacrifice, mice were injected i.p. with 5-bromo
2’-deoxyuridine (Brdu) at a concentration of 50 mg/kg body weight
(Sigma Chemical Co., St. Louis, MO). After 1 hr, animals were

terminated by an overdose of CO2 and the tumors excised. Necrotic

tissue was trimmed from the tumor and a 7 mm biopsy punch was used to
obtain a tumor sample. The sample was minced with a single-edged razor
blade and placed in a 12x75 mm glass tube containing 2 ml of ice cold
70% ETOM. The tubes were then sealed with parafilm and stored at -20'C
until the dissociation step. Tissue samples were removed from -20'C
storage and approximately 40 mg of tissue was finely minced with a
scalpel or single-edged razor blade. The tissue was then
simultaneously dissociated and denatured by placing in a 25 ml
Erlenmeyer flask containing 2 ml of 0.4 mg/ml pepsin in 2 N HCl. The
flasks were placed in a shaking water bath at room temperature for
1.5 to 2 hours or until the cells could be easily dispersed by gentle
up and down pipetting with a pasteur pipette. The cell suspensions
were then filtered through a 50 ug mesh and washed twice with 2 ml of
PBS (pH 7.4) containing 0.1%.BSA and 0.05% Tween 20 (PBT buffer).
WWW

Approximately 5x105 to 1x106 cells were resuspended in 100 pl of
PBT buffer containing 2 ug/ml of anti-Brdu antibody (Boehringer-
Mannheim Co., Indianapolis, IN). The tubes were incubated for 30 min
at room temperature, washed with 2 ml of PBT buffer and resuspended in

100 pl of PBS containing 10 ug/ml goat antimouse IgG-FITC. The tubes

124
were incubated for 30 min at room temperature, washed twice with PBS
and resuspended in 1 ml of PBS containing 10 ug/ml propidium iodide
(Sigma Chemical Co., St. Louis, MD). A control for non-specific
binding was run for each sample by preparing a duplicate tube with no
anti-Brdu. The tubes were incubated overnight at 4'C and analyzed
using a flow cytometer (Ortho 50H, Ortho Diagnostics, westward, MA).

The amount of Brdu uptake was reported as the percent of cells with

F

$.13.”

green fluorescence intensity above that of the non-specific binding
control. Mice that were not injected with Brdu were used as a control
to eliminate background fluorescence.
-.. . ., . -- . - . - - (_ -, .1 .-. - x;
55531 _
The cold 70% ethanol-fixed tumors prepared for the Brdu assay were

 

 

also used for the PCNA assay. The tissue samples were removed from the
70% ethanol, rinsed and placed in an Erlenmeyer flask containing 2 ml
of pepsin (0.4 mg/ml in 0.1N HCl). Dissociation was carried out on a
shaking water bath at room temperature for 30-60 min or until the cells
were easily dispersed by gentle up and down pipetting with a Pasteur
pipette. Cells were washed twice in PBS (pH 7.4) containing 0.1%
Triton-X 100. Cells were then suspended in 100 pl PBS containing

25 ug/ml of PCNA (Boehringer-Hannheim Co., Indianapolis, IN) and

0.1% BSA. Cells were then incubated at room temperature for 30 min.
Cells were washed and resuspended in 100 pl of PBS containing 10 ug/ml
goat anti-mouse FITC. Tubes were incubated for 30 min at room
temperature, washed twice with PBS and resuspended in 1 ml of PBS
containing 10 ug/ml of propidium iodide. The cold 70% ethanol-fixed

tumors prepared for the Brdu assay were also used for the PCNA assay.

125
The tissue samples were removed from the 70% ethanol, minced and placed
in an Erlenmeyer flask containing 2 ml of pepsin (0.4 mg/ml in
0.1M HCl). Dissociation was carried out on a shaking water bath at
room temperature for 30-60 min or until the cells were easily dispersed

by gentle up and down pipetting with a pasteur pipette. An 1961

antibody of irrelevant specificity was used as control to monitor non-
specific binding. The cells were analyzed using a flow cytometer
(Ortho 50H, Ortho Diagnostics, Westward, HA). The amount of PCNA was
reported as percentage of green fluorescent cells.
AW

Cell loss is defined as the rate of loss of cells as a fraction of
the rate at which cells are being added to the tumor volume by cell
proliferation. Cell loss is an important factor in estimating the
growth potential of a tumor (12). In order to facilitate the study of
this phenomenon in our experimental model, we proceeded as follows.
The human breast carcinomas, maintained in athymic nude mice, were
measured weekly with a Vernier caliper. The weekly increase in volume

(cm3) was determined for each carcinoma. After the mice had been fed

diet for 6 weeks, the carcinoma-bearing athymic nude mice were injected

i.p. with 5 uCi of Ins-iodo 2’-deoxyuridine (1125Urd, 5 mCi/mg, Sigma

Chemical Co., St. Louis, H0). In order to prevent excess concentration

of 1125 in the thyroid, each mouse was given 0.1% K1 in the drinking
water commencing 3 days prior to IlaSUrd administration. Twenty-four
hours after 1125Urd injection, mice were lightly anesthetized with

ether and secured in a holding apparatus to allow for gamma emission

readings. Emissions were read using a Geiger counter with a

126
Mal crystal, 2 inch diameter and 0.04 inches thick, Model leg-1, low
energy gamma probe, 61% efficiency, Eberline Inc., Santa Fe, NM. Care
was taken to place the probe in an identical position on top of the
carcinoma in contact with the integument overlying the outer surface.
Duplicate 1 minute emission readings (cpm) were recorded for each
carcinoma for seven consecutive days, subtracting background emissions.

Mean rate of IleUrd loss from each tumor was calculated as follows:

y-mx+b y-natural log of the daily mean 1125Urd emissions (from
dupl;cate measurements) (cpm)-background emissions
cpm

m-slope (KL, rate constant)
x-time (days)
b-y intercept-activity at time zero
Using the above equation, a graph was generated for each carcinoma as
follows:
ln[cpm(1-7)-cpm(0)] vs time (days), ln-natural log
cpm(1-7)-counts per minute
(emissions) from day 1 to
day 7

cpm(0)-counts per minute
(emissions) at day 0

The resulting slope, or rate constant KL, was utilized to compute

carcinoma cell loss factor using the following formulas:
wig-Effi- Begg’s Formula (13)

i¢-cell loss factor-cell loss rate expressed as percent of the
cell birth rate

TD-tumor doubling time in days (calculated by determining the
number of days for tumor to double in size)

Tllz-time (days) for IleUrd emission from the tumor to reach
1/2 of initial (time 0) emission rate

127
«1%? Steel’ s Formula (12)
o

i¢-cell loss factor-cell loss rate expressed as percent of the cell
birth rate

TD-tumor doubling time in days (calculated by determining the
number of days for tumor to double in size)

“in:

PKL

Principles for assessing cell loss from growing tumors in situ using
these formulas have been validated by Kallman et al. (14).
RESULTS

In Table 3 after mice were fed corn oil (CO) and fish oil (F0)
diets for a period of only one week (Study 1), the difference in mean
tumor volumes did not reach a level of 5% significance. Also no
significant difference between mice fed C0 and F0 diets was obtained in
DNA synthesis parameters (H3-thymidine autoradiograph analysis and Brdu

flow cytometry analyses). This trend was also observed in tumors of
animals fed C0 and F0 diets for a period of two weeks (Study 2), in
which mean tumor volumes and mean tumor DNA synthesis parameters were
not significantly different. The animals fed the C0 diet for four
weeks (Study 3) had a significantly larger (p<0.05) tumor volume than
those fed a F0 diet; nevertheless no significant difference was

detected in mean H3-thymidine autoradiograph analysis. In another

study in which animals were fed diets for 4 weeks (Study 4) we also
observed a significantly larger tumor volume (p<0.05) in the C0 fed
animals compared to those fed F0. However, when F0 was supplemented
with excess antioxidants, mean tumor volume of animals fed the

supplemented F0 was comparable to the mean tumor volume of the C0 fed

128
animals. Again no significant difference was observed in tumor DNA
synthesis parameters. In addition, animals fed F0 supplemented with
iron, mean tumor volume were significantly less compared to the other
three experimental groups (C0, F0, f0+antioxidants) but no significant

difference in mean tumor H3-thymidine analysis was detected. After

feeding C0 and F0 diets for six weeks (Study 5), C0 fed animals had a
significantly larger (p<0.05) mean tumor volume than those fed F0.
However, no significant difference in tumor DNA synthesis parameters
was observed. After feeding diets for 10 weeks (Study 6), a
significant difference in mean tumor volume was not reached, neither
was a significant difference obtained in tumor DNA synthesis
parameters.

In Table 4 (Study 7), after mice were fed a C0 and F0 diet for six
weeks, animals fed a C0 diet had a significantly larger (p<0.05) tumor
volume compared to those fed F0 but not compared to those fed the
antioxidant supplemented F0 diet. No significant difference in tumor
DNA synthesis parameters (Brdu analysis and PCNA analysis) was detected
between these three dietary groups.

In Table 5 (Study 8), after feeding different ratios of C0 and F0
for a period of six weeks the animals fed 15% C0/5% F0 had a
significantly larger (p<0.05) tumor volume than the ones fed
10% 00/10% F0 and 5% C0/15%.F0. The animals fed 10% 00/10% F0 had a
higher tumor volume than those fed 5% C0/15% F0 but this difference did
not reach a level of 5% significance. Also no significant difference
was detected in tumor mean Brdu analysis between these three groups.

In Table 6 (Study 9), after feeding tumor bearing mice a CD and F0

diet for two weeks no significant difference was detected in mean tumor

 

129

volume, mean rate of 1125Urd loss from tumors nor mean tumor cell loss

factors between the two diet groups. In Study 10, after feeding a C0
and F0 diet for four weeks, no significant difference was detected in

mean tumor volume, mean rate of 1125Urd loss from tumors, nor mean

tumor cell loss factors. In Study 11, after feeding a C0 and F0 diet
for six weeks the C0 fed animals had a significantly larger (p<0.05)
mean tumor volume compared to the F0 fed animals; the tumor volume in
the C0 fed animals, however, was not significantly different from that
observed in the antioxidant supplemented F0 fed animals. Mean rate of

I125Urd loss from tumors was significantly (p<0.05) lower in the C0 fed

animals compared to the F0 fed animals and the antioxidant supplemented

 

F0 group. Mean tumor cell loss factor as determined by Steel (12) and
Begg (13) was also significantly lower in the C0 fed group compared to
the F0 fed group and to the antioxidant supplemented F0 group.

Figure 1 compares the mean slopes of the rate of IleUrd loss from

the tumors of the three dietary groups (C0, F0+A and F0). A
significant difference (p<0.05) in slopes between C0 and F0 and C0 and
F0+A was observed; the slopes of the F0 and F0+A dietary groups were
virtually identical.
DISCUSSION

Diets high in polyunsaturated fatty acids (e.g., corn oil), when
fed to rodents, causes an increase in size and number of mammary tumors
when compared to rodents fed low levels of the same fat or high levels
of other types of fat (e.g., beef tallow and certain fish oils) (15).
To examine the tumor growth kinetics of this phenomenon, only a few

laboratories (4-8) have investigated the differential effects of

130

dietary fat on tumor cell proliferation or on tumor cell loss
(cytolysis). Abraham et al. (8) reported a significantly smaller tumor
size of transplantable mouse mammary tumors in mice fed a high FO diet
compared to tumors of mice fed a high CO diet. They accounted for this
result by providing data of an increase cell loss in tumors of rodents
fed a high FO diet compared to the cell loss obtained from those tumors
of mice fed a high CO diet. Previously they reported (4,7) no
significant difference in tumor cell proliferation parameters when
feeding diets high in unsaturated and saturated fatty acids to mammary
tumor bearing rodents in spite of obtaining a significant difference in
tumor size. Hhen examining different levels of dietary fat, 0yaizu
et al. (5) and Noguchi and colleagues (6,9,10) reported a smaller
mammary tumor size in rats fed a low level of CO compared to rats fed a
high level of CD; a decrease in carcinoma cell proliferation parameters
in the tumors of rats fed the low level CO diet compared to those fed
the high level of CO was observed. Clearly, more studies are required
to have a more definitive understanding as to the effect of the type
and amount of dietary fat on tumor cell proliferation dynamics in
mammary tumors in order to have a better understanding of the
nutritional influence on tumor growth processes.

Thus, an important question remains unanswered. Is the decreased
size of tumors of animals fed high F0 diets due to a decrease in DNA
synthesis, or because of an increase in cell loss (cytolysis)? The

H3-thymidine autoradiographic methodology is a precise means to assess

DNA synthesis (16). Furthermore, this technique is an effective means
of providing a quantitative differentiation between carcinoma cell and

stromal cell proliferation processes (17). One drawback of the

 

l

r.

 

I31
H3-thymidine autoradiographic methodology is that it is extremely time

consuming. On the other hand, the flow cytometry method of measuring
DNA synthesis provides a fast (labeling and detection can be performed
the same day), sensitive and quantitative way to measure DNA synthesis
in suspended cells. Brdu is an analog of thymidine that is
concentrated only in cells in active DNA synthesis (18). Quantitation
of Brdu concentration in DNA is made possible by the development of a

monoclonal antibody against Brdu (19). The Ha-thymidine !

autoradiographic technique and the Brdu flow cytometric technique, as

methods of estimating cell proliferation have been reported to be in

 

close agreement with each other (20). The PCNA flow cytometric fj

V3:

technique has also been used to study cell proliferation processes.
PCNA possesses a temporal specificity which makes it a suitable marker

for cell proliferation. PCNA begins to accumulate during the 61 phase

of the cell cycle, is most abundant during the S phase and declines
during GZ/M phase (21). PCNA has been successfully used to selectively
identify proliferating cells in solid tumors (22).

 

The method for the assessment of cell loss from growing tumors was
originally described by Steel (12) and validated by Begg (13) and
Kallman et al. (14). Steel’s cell loss factor measures the rate of
loss of cells as a fraction of the rate at which cells are being added
to the tumor volume by cell proliferation (23). This factor,
therefore, expresses the growth potential of a tumor (ratio of cell
loss rate to the cell birth rate). The extent to which processes of
cell loss are competing with the process of cell proliferation can be

obtained utilizing the formula:

132

¢-1.]r:: , where ¢-the cell loss factor.

A modified version of Steel’s cell loss measurement concept was first
utilized in a dietary study of tumor growth by Abraham and colleagues
(18). The method of measuring cell loss from tumors in situ by using

the I125 deoxyuridine (1125Urd) technique was first described by Begg

(13). Begg’s derived tumor kinetic parameters originated from Steel’s

formula for cell loss factor (11). Begg equates the IleUrd emission
rate (KL, loss of radioactivity) to the rate of cell loss. The slope
of the 1125Urd emission rate in a semilogarithmic plot is defined as
T1/2 (time to halve the radioactivity). The modified adaptation of

Steel’s formula is:
The pragmatic difference between these two formulas is that Begg’s

derived formula takes into account only one point (point of 1/2

radioactivity) on the curve generated by the 1125Urd emission data,

whereas Steel’s formula takes into account the total curve generated by

the 1125 emission data. Therefore, by utilizing the whole curve,

Steel's formula provides a more precise assessment of cell loss factor.
In general, both formulas were in close agreement in these studies.
These techniques to measure cell loss are very attractive since they
provide a direct determination, and therefore, are superior to methods
which depend solely on calculated and measured doubling times of tumor
growth. Another advantage of these methods is that they require fewer
animals than methods requiring the excision of tumors, since each

animal contributes several time points. In addition, variation is

 

ll'

 

133
reduced since each tumor acts as its own control. Moreover, it allows
for cell loss rates of individual tumors to be determined. Currently,

the IIZSUrd technique offers the only direct non-invasive method of

assessing cell loss in individual tumors. Although errors of the in
situ technique are smaller and less frequent than those occurring with
other methods, the problems of reutilization of the isotope are still
present. The isotope could also be trapped in necrotic areas inside or
surrounding the tumor. For this reason, in our experiments, tumors
with overt necrotic areas were not used. Since our cell lines do not
elicit any substantial immune response, in athymic nude mice, we did
not have the problem of additional necrosis induced by immune cell
infiltration of the tumor tissue. Thus, our experimental model
utilizing athymic nude mice bearing human breast carcinoma cell lines

is suitable for determination of the cell loss factor by the IleUrd in

situ technique. In addition to reporting Steel’s and Begg’s cell loss

factor, we report the tumor 1125Urd emission rate which, indirectly can

be equated to cell loss rate.

No significant differences in DNA synthesis parameters between the
diet groups (C0 and F0) were observed (Studies 1-6, in Table 3, Study 7
in Table 4 and Study 8 in Table 5), despite the significant differences
in tumor size that were observed in a number of these studies (Studies
3-5,7,8). The small numerical decrease in DNA synthesis parameters in
tumors of F0 fed mice (3-5% decrease) may have relevant biological
significance. This very small decrease in DNA synthesis may prove of
importance, if this difference is real and can be extended throughout

the entire dietary feeding period.

134

In Study 4 (Table 3), two additional FO groups were added, an
antioxidant supplemented PD group (FO+A), and an iron-supplemented FO
group (FO+I). In this study, supplementation with antioxidants
significantly enhanced tumor volume of the F0 fed animals, while in
contrast, supplementation with iron significantly decreased tumor
volume of the F0 fed animals. This appears to be due to differences in
cytostatic/cytolytic lipid peroxidation product accumulation in the
tumors as we reported previously (2,3). Nevertheless, in spite of this
tumor volume difference, no significant difference in cell
proliferation parameters in the tumors was observed. Study 7 (Table 4)
followed a similar trend as Study 4 in which a significant difference
was observed in mean tumor volume between the CO and the F0 fed groups,
with the antioxidant supplemented F0 having a comparable tumor volume
to the C0 fed group; no significant difference in cell proliferation
parameters was once again observed. In Study 8 (Table 5), different
ratios of CO and F0 were fed which resulted in an inverse relationship
in which a decreasing tumor volume was evident as the FO content of the
diet increased; once again no significant difference in cell
proliferation parameters was observed. These results are in accordance
with those of Abraham et al. (4,7,8) in which the difference in mammary
tumor size between CO and F0 fed animals cannot be accounted for by the
fraction of tumor cells that were actively proliferating.

Our results suggest that parameters other than cell proliferation
may be the primary mechanism by which differences in tumor volume
between dietary CO and F0 fed animals is achieved. Studies 9,10,11
(Table 6) furnish a possible answer to the mechanistic question of how

dietary F0 affects tumor growth-related kinetic parameters by providing

 

135
yet another parameter that can substantially influence the size of
tumors; this parameter is cell loss. Cell loss is a biological
phenomenon that occurs in normal and neoplastic tissue and is an
important kinetic variable which can determine the dynamic state of
tissue growth (19). Importantly, when a significant difference in
tumor volume was not reached between the CO and F0 dietary groups, no
significant difference was observed in tumor cell loss parameters

(IleUrd emission rate and cell loss factors, Studies 9,10). In

contrast, when a significant difference was found in mean tumor volume,
a significant difference was also observed in tumor cell loss
parameters (Study 11). These studies are in agreement with those of
Abraham et al. (8) in which a significant difference in mammary tumor
size, in CO and F0 fed animals, was accompanied by a significant
difference in tumor cell loss; mammary tumor cell loss was greatest in
the F0 fed animals. The addition of antioxidants to a dietary F0 group
did not have a significant effect on tumor volume compared to either of
the two other dietary groups (C0 and F0) in Study 11. Nevertheless, in
Study 4 and 7, a significant increase in tumor volume was obtained when
feeding the FO+A diet compared to the group fed solely F0. Study 4 and
7 are in agreement with previous reports from our laboratory (2,3).
Also in Study 11, no difference in tumor cell loss parameters was
obtained between FO+A and the FO group. The reason for this is not
known. It is conceivable that the F0 used in Study 11 could have been
substantially oxidized prior to diet preparation. The already
excessively oxidized FO could prevent any substantial antioxidative
effect by the addition of antioxidants. This would result in similar

tumor volumes and cell loss parameters in animals fed F0 and FO+A

 

136

diets. These results, albeit preliminary, suggest that differences in
mammary tumor cell loss parameters in CO and F0 fed animals are very
important, perhaps more important than tumor cell proliferation, in
determining the extent of volume of these tumors. More studies are
needed to confirm these preliminary results in order to provide a
conclusive unifying concept to explain how dietary fat affects tumor
growth. Nevertheless, these experiments and those reported earlier
(2,3) support the concept that FO suppresses human breast carcinoma
growth in athymic nude mice by increasing the concentration of
secondary products of lipid peroxidation in the tumor; such products
(cytostatic/cytolytic) significantly increase tumor cell loss.

REFERENCES

1. Carroll KK and Braden LM. Dietary fat and mammary carcinogenesis.
Nutr. Cancer 1985, 6:254-259.

2. Gonzalez MJ, Schemmel RA, Gray J1, Dugan L, Jr., Sheffield LG and
Nelsch CH. Effect of dietary fat on growth of MCF-7 and MDA-M8231
human breast carcinomas in athymic nude mice: Relationship between
carcinoma growth and lipid peroxidation product levels.
Carcinogenesis 1991, 12:1231-1235.

.3. Gonzalez MJ, Schemmel RA, Dugan L, Jr., Gray J1 and Welsch CH.
Dietary fish oil inhibits human breast carcinoma growth: A
function of lipid peroxidation. Lipids 1993, 28:827-832.

4. Hillyard LA and Abraham S. Effect of dietary polyunsaturated
fatty acids on growth of mammary adenocarcinomas in mice and rats.
Cancer Res. 1983, 39:4430-4437.

5. 0yaizu N, Marii S, Saito K, Katsuda Y and Matsumoto J. Mechanism
of growth enhancement of 7,12 dimethylanthracene-induced mammary
tumors in rats given a high polyunsaturated fat diet. Japan J.
Cancer Res. 1985, 76:676-683.

6. Kumaki T and Noguchi M. Effects of high dietary fat on the total
DNA and receptor contents in rats with 7,12-

dimethylbenz[a]anthracene-induced mammary carcinoma. Oncology
1990, 47:352-358.

7. Gabor H, Hillyard LA and Abraham S. Effect of dietary fat on
growth kinetics of transplantable mammary adenocarcinoma in Balb/C
mice. J. Natl. Cancer Inst. 1985, 74:1299-1305.

 

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. Gabor M and Abraham S. Effect of dietary menhaden oil on tumor

cell loss and the accumulation of mass of a transplantable mammary
adenocarcinoma in BALB/c mice. J. Natl. Cancer Inst. 1986,
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. Noguchi M, Taniya T, Koyasaki N, Kumaki T, Mizayaki I and Mizukami

Y. Effects of the prostaglandin synthetase inhibitor indomethacin
on tumorigenesis, tumor proliferation, cell kinetics and receptor
contents of 7,12-dimethylbenz(a)anthracene-induced mammary
carcinoma in Sprague-Dawley rats fed a high- or low-fat diet.
Cancer Res. 1991, 51:2683-2689.

Noguchi M, Ohta N, Kitagawa M, Earashi M, Thomas M, Miyazaki 1 and
Mizukami Y. Effects of switching from a high-fat diet to a low-
fat diet on tumor proliferation and cell kinetics of 7,12-
dimethylbenz(a)anthracene-induced mammary carcinoma in rats.
Oncology 1992, 49:246-252.

Nalker BE. Radioautographic observations on regeneration of
transitional epithelium. Tex. Rep. Biol. Med. 1959, 17:375-384.

Steel GG. Cell loss from experimental tumours. Cell Tissue
Kinet. 1968, 1:193-207.

Begg AC. Cell lggs from several types of solid murine tumour:
Comparison of I -deoxyuridine and tritiated thymidine methods.

Cell Tissue Kinet. 1977, 10:409-427.

Franko AJ and Kallman RF. Cell loss and influx ofllgbelled host
cells in these transplantable mouse tumors using I udR release.

Cell Tissue Kinet. 1980, 13:3881-393.

Nelsch CM. Relationship between dietary fat and experimental
mammary tumorigenesis: A review and critique. Cancer Res. 1992,
52:20405-20485.

Terz JJ, Curutchet MP and Lawrence NJr. Analysis of the cell
kinetics of human solid tumors. Cancer 1971, 26:1100-1110.

Silvestrini R. Feasibility and reproducibility of the [3H]-

thymidine labelling index in breast cancer. Cell Prolif. 1991,
24:437-445.

Giaretti M. Ploidy and proliferation evaluated by flow cytometry.
A; oggrxigw of techniques and impact in oncology. Tumori., 1991,

Hardonk MJ and Harms G. The use of 5’bromodeoxy uridine in the
iggdy of cell proliferation. Acta Histochem. 1990, Suppl.(39)99-

 

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23.

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Zatterstrom UK, Johansson M, Kallen A, Baldetorp B, Oredsgon S,
Nennerberg J and Killander D. Comparison of BrdUrd and [ H]TdR

incorporation to estimate cell proliferation cell loss and
potential doubling time in tumor xerografts. Cytometry 1991,
13:8 2-879.

Kurki P, Ogata K and Tan EM. Monoclonal antibodies to
proliferating cell nuclear antigen (PCNA)/cyclin as probes for
proliferating cells by immunofluorescence microscopy and flow
cytometry. J. Immun. Methods 1988, 109:49-59.

Smetana K, Gyorkey F, Chan PK, Tan E and Busch M. Proliferating
cell nuclear antigen (PCNA) and human malignant tumor nuclear

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Blut 1983, 46:133-141.

Steel GG. Cell loss as a factor in the growth rate of human
tumors. Eur. J. Cancer 1967, 3:381-387.

 

139
Table 1. Diet composition.

 

Ingredient1 Amount

 

g/100 g diet

 

Fat2 20.00
Casein3 20.17
DL-Methionine 0.35
Dextrose 32.18 E;_
Sucrose 16.09
AIN Mineral mix4 4.13
AIN Vitamin mix‘ 1.18 .3
CelluloseS 5.90

 

1All ingredients (dry components) were obtained from U.S. Biochemical
(Cleveland, OH) except sucrose (ICN Biochemicals, Costa Mesa, CA).

2High-fat diets (20% wt/wt) contained 20% corn oil or 19% fish oil

(menhaden) + 1%icorn oil. Menhaden oil contains 0.03 g/kg of all-rac
-a-tocopherol. Corn oil (tocopherol stripped) was obtained from U.S.
Biochemical. Fish oil (menhaden) was obtained from Zapata Haynie
(Reedville, VA).

3Vitamin free, high nitrogen (14.5%).
‘AIN (1977).
5Celufil, non-nutritive bulk.

 

 

140
Table 2. Predominant fatty acids in oils and fats (percentage)

 

Fatty acidsa Corn oil Menhaden oil

 

Caprylic (8:0) - -
Capric (10:0) - -
Lauric (12:0) - -

 

' Myristic (14 0) - 8.0
Palmitic (16:0) 10.1 28.9 E
Palmitoleic (16:1) - 7.9 'Mi
Stearic (18:0) 1.6 4.0
Oleic (18:1) 31.4 13.4 13
Linoleic (18:2) 56.3 1.1 j I
Linolenic (18:3) - 1.0
Eicosapentaenoic (20.5) - 10.2
Docosahexaenoic (22.6) - 12.8

 

aFatty acid concentrations less than 1% are not included.

 

 

141

Table 3. Effect of dietary fat (corn oil and fish oil) on DNA
synthesis (H -thymidine autoradiography and Brdu) of human

breast carcinoma MDA-M8231 maintained in athymic nude mice.

 

Number of Mean Mean H3-thymidine Brdu labeling-

Diet tumors tumor autoradiographs index (% tumor
v lume (# labeled tumors cells showing
(cm tS.E.) cells/mm2 of Brdu uptake
tissuetS.E.)° 1$.E.)e

 

1 week on diet (Study 1)

Corn oila 15 0.1210.03 n.d. 20.9611.69(15)

Fish oiib 16 0.09:0.03 n.d. 19.8511.93(16)
2 weeks on diet (Study 2)

Corn oila 15 0.12:0.03 138.01112.18(15) 7.26:1.30(14)

Fish oilb 9 0.0710.02 134.70115.07(9) 10.4211.92(9)
4 weeks on diet (Study 3) f

Corn oila 55 1.2410.11 43.54t2.40(55) n.d.

Fish oilb 40 O.60:tO.O7g 41.60i2.49(40) n.d.

4 weeks on diet plus diet supplementation (Study 4)
Corn oila 25 1.5010.15 45.3612.98(25) 16.65t2.19(25)

Fish Oilb 21 03110.10g 43.1912.47(21) 11.64il.l7(21)
Fish oil + 17 l.48:l:0.29f 45.4614.63(17) 13.8811.25(17)
antioxidantsc
Fish oil + 10 0.24:0.07h 43.8615.83(10) n.d.
irond.
6 weeks on diet (Study 5)
Corn oila 50 15510.16f 28.5212.04 14.17t0.85(42)
Fish oilb 55 03410.08g 25.8911.82 12.7610.80(33)
10 weeks on diet (Study 6)
Corn oila 8 1.2010.34 45.7916.29(8) 4.3111.11(6)
Fish oilb 9 1.19:0.23 40.5413.80(9) 4.3010.83(9)

 

a20% corn oil.
b19% menhaden oil/1% corn oil.
c19% menhaden oil/1%.corn oil + alpha tocopherol acetate (8 g/kg diet),

dtertiary butyl hydroquinone (4 g/kg diet).
19% menhaden oil/1% corn oil + ferric citrate (3 g/kg diet)

°number of tumors examined in parenthesis.
f/g/h/p<0.05.

 

142

Table 4. Effect of dietary fat (corn oil and fish oil) on DNA
synthesis (Brdu and PCNA) of human breast carcinoma MDA-MB231
maintained in athymic nude mice.

 

 

Diet Number of Mean Brdu labeling- PCNA labeling-
6 weeks tumors tumor index (% index (%
on diet vol me tumor cells tumor cells
(Study 7) (cm 1 showing Brdu PCNA positive
S.E.) uptake1S.E.)d 13.5.)d
Corn oila 24 1.4010.21° 4.0210.52 (24) 14.1710.85 (24)
Fish oiib 18 04310.11f 2.6310.38 (18) 12.7610.80 (18)
Fish oil +

antioxidantsc 7 1.0310.18° 2.1410.47 (7) 17.47t2.86 (7)

 

a20% corn oil .
b19% menhaden oil/1% corn oil.

c19% menhaden oil/1%.corn oil + alpha tocopherol acetate (8 g/kg diet)
and tertiary butyl hydroquinone (4 g/kg diet).

dnumber of tumors examined in parenthesis.

°/‘p<o.os.

143

Table 5. Effect of different ratios of dietary fats (corn oil and fish
oil) on DNA synthesis (Brdu) of human breast carcinoma
MDA-M8231 maintained in athymic nude mice.

 

 

Diet Number of Mean tumor Brdu labeling-

6 weeks tumors vol me index (% tumor
on diet (cm 1 cells showing
(Study 8) S.E.) Brdu uptaketS.E.)b
Corn oil 15%/ 20 l.241:0.23c 10.1111.55(l6)
fish oil3 5%

Corn oil 10%/ 18 0.6510.15d 7.5011.09(17)
fish oiia 10%

Corn oil sz/ 17 O.3410.08d 6.2111.30(9)

fish oila 15%

 

amenhaden oil.

bnumber of tumors examined in parenthesis.

c/dp<o.os.

 

 

144

 

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146

 

 

 

 

0.0
-0.5-
J
-1.0d
1.5-
q I (D
0 F0
I FO+A
'20 V I V l l ' fl
0 2 4 6 8

Time (days)

Rate of 1125Urd loss from human breast carcinomas in athymic
nude mice fed corn oil (CO), fish oil (F0) and fish oil
supplemented with antioxidants (FO+A) for a period of 6
weeks. Rates were 0.1741-0.0l (N-SZ), 0.22410.01 (N-45)

and 0.22310.02 (N-l4) for C0, F0 and FO+A, respectiveiy,
p<0.05.

 

 

SUMMARY

It is clear that diets high in omega-3 PUFAs suppress the
development and/or growth of mammary gland tumors in experimental
animals compared to omega 6-PUFAs. Long chain omega-3 PUFA as EPA and E?
DHA appear to be more effective suppressors of mammary tumorigenesis

than shorter chain omega-6 PUFAs such as linoleic acid, although more

 

E
work is needed to be done to totally verify this point. It is apparent :
in the athymic nude mice bearing human breast carcinoma MDA-M8231 the E”
omega-3 fatty acids present in menhaden oil have a suppressive effect
on tumor growth although various mechanisms may be operative in this
suppressive action exerted by the fish oils upon tumor growth. We
propose yet another mechanism that has been neglected and that, at
least in part, may explain this phenomenon, i.e., that long chain
omega-3 polyunsaturated fatty acids may inhibit mammary gland
tumorigenesis via a generation and/or accumulation of lipid
peroxidation products in the tumor tissue. Also evidence is provided
that in the preparation of fish oil diets, the addition of antioxidants
at recommended and substantially higher levels does not completely
suppress the oxidative damage occurring in experimental diets.

In terms of tumor growth kinetics we studied the effect of dietary
fat (corn oil and fish oil) on tumor cell proliferation and tumor cell
loss. Preliminary results suggest a higher tumor cell loss occurring

in the human breast carcinomas of athymic mice fed fish oil diets

147

 

148
compared to those fed corn oil diets. No significant difference was
detected in tumor cell proliferation between these two groups. This
increase in cell loss may partly explain why tumor growth is suppressed
in animals fed a high fish oil diet compared to those fed high corn oil
diets. It is conceivable that this increase in cell loss in the tumors
of mice fed fish oil is due to an increase in cytolytic secondary

products of lipid peroxidation in the tumor tissue.

 

 

CONCLUSIONS

Compared to athymic nude mice fed a corn oil diet, athymic nude
mice fed fish oil (menhaden) exhibited a significant decrease in
growth of human breast carcinomas ("CF-7, MDA-H8231).

Human breast carcinomas from athymic nude mice fed fish oil diets
contained at least 3 x the level of lipid peroxidation products as
that measured in carcinomas obtained from mice fed corn oil diets.
Addition of antioxidants (vitamin E and TBHQ) to the fish oil
diets significantly reduced the level of lipid peroxidation
products in human breast carcinomas and concurrently significantly
increased carcinoma growth.

Concentrations of lipid peroxidation products in diets high in
fish oil, under normal feeding conditions, increase substantially
as a function of time (0 hours to 72 hours).

The American Institute of Nutrition (AIN) recommended level of
addition of a synthetic antioxidant (0.02%.w/w of oil) to diets
containing high levels of fish oil are insufficient to completely
inhibit the accumulation of oxidative metabolites in such diets.
TBARS concentrations of organs (liver, heart, kidney, mammary
gland, skeletal muscle) and whole body carcass of mice fed fish
oil were consistently higher than those in organs and bodies of

mice fed corn oil.

149

 

10.

ll.

12.

150
Diets containing fish oil, with substantial amounts of linoleic
acid (up to 9.1% of diet), still suppressed growth of MDA—M8231
human breast carcinomas in athymic nude mice.
Increasing the fish oil content of the diet decreased human breast
carcinomas (MDA-M8231) growth in athymic nude mice.
As the fish oil content of the diet was increased, there was a
concurrent increase in human breast carcinoma TBARS concentration.
Human breast carcinomas that have the highest concentrations of
TBARS have the least tumor growth as measured in tumor volume.
Preliminary results suggest that cell loss from tumors has a more
important role in tumor growth than does tumor cell proliferation.
The decrease in mammary tumor development and/or growth reported
in animals fed high levels of fish oil may be due, at least in
part, to an accumulation of secondary peroxidation products in the

tumor tissue which can probably increase tumor cell loss.

 

 

RECOMMENDATIONS

The field of nutritional tumorigenesis has a potential for
producing an enormous impact in many areas of cancer research; in
relation to nutritionally relevant fatty acids such as linoleic acid
and eicosapentaenoic acid, it has been recently reported an apparent
regulatory function of these fatty acids on gene expression in vitro
(1).

The proposed mechanisms by which diets high in omega-3 PUFAs
inhibit mammary gland tumorigenesis are numerous and varied. The
omega-3 fatty acid-eicosanoid metabolism relationship has been the most
intensely examined mechanism. Although in a particular cell line used
in the studies reported herein (MDA-M8231), cell growth has been
reported to be more dependent on leukotrienes than on prostaglandins
(2). Nevertheless, in BHK-Zl transformed cells, when inhibitors of
cyclooxygenase (indomethacin) or lipoxygenase (nordihydroguaiacetic
acid) were added along with the fish oil, there was no diminution in
the inhibitor actions of the oil (3), suggesting that the involvement
of prostaglandins or leukotrienes may be negligible in this process.
At variance we have proposed the hypothesis that dietary fish oil
inhibits mammary gland tumorigenesis, at least in part, by the
generation and/or accumulation of secondary products of lipid
peroxidation in the tumor tissue, a concept we have first described in

recent publications (4-6).

151

 

 

152

These secondary products of lipid peroxidation (e.g., 4-
hydroxynonenal), besides affecting a whole array of cellular
biochemical and physiological functions (4), have been recently
reported to suppress the expression of specific genes involved in the
metabolic cascade leading to cell proliferation (7). Furthermore, fish
oils have also been reported to decrease oncogene expression when
compared to corn oil (8). This concept of modulating gene expression
by dietary fatty acids and/or their oxidative products, is without
doubt, the next logical step in nutritional tumorigenesis research.
This will clarify the specific molecular mechanism(s) of action
hindered in this phenomenon. This research might even help determine
what specific fatty acid products can affect what specific types of
cell, since the actions of fatty acid oxidation products appear to be
cell type dependent (9). Also of interest are studies addressing the
interaction of oxidative species with immune system components
(lymphocytes, neutrophils, natural killer cells).

Other areas may also require further research. The nature of the
different oxidative species formed by the oxidation of fish oil in vivo
or absorbed from the diet remains to be determined. Assays with the
capability to separate contributions of oxidative species generated
from nonenzymatic lipid metabolism from those generated by enzymatic
activity of the cellular lipoxygenase or cycloxygenase systems need to
be developed. Also, more studies are needed in the area of tumor
growth kinetics, specifically in relation to cell loss and cell
proliferation. Preliminary results pointing at cell loss as a more
important tumor growth kinetic parameter than cell proliferation need

to be verified. Also of interest is the effect of different fats on

153
fatty acid metabolism enzymes, such as the desaturases [6-desaturase
system has been reported (10) to be frequently modified in malignant
cells]. Further studies in these previously mentioned areas may
clarify important details of the physio-biochemical pathways that fats
undertake in order to affect tumor growth. It is conceivable that an
interplay of various mechanisms are operative in the suppression of
mammary gland tumorigenesis by omega-3 PUFAs.
W

At present, fats contribute close to 40% of the total kilocalories
in the average American diet. Fats are involved in numerous
biochemical processes. Thus, the amount and type of dietary fat can
influence normal physiological functions and also can contribute to the
pathogenesis of various diseases. General consensus supports the
recommendations that many Americans should consume less fat in their
diet. However, less settled is the issue of the appropriate type(s) of
fat. It is clear that different fats have distinct biochemical/
physiological functions in normal and/or pathological processes.

The substitution of foods with high levels of saturated fats with
marine products has been recommended for health promotion and even
treatment of certain diseases. Epidemiological and experimental data
indicate that fish oils, which contain high levels of omega-3 PUFAs may
help in the prevention and treatment of various clinical diseases (11-
13), which includes breast cancer. In relation to fish oil, it is
clear that the level of omega-3 PUFAs required to produce an antitumor
effect is much higher than would normally be achieved from the diet
alone. Nevertheless, the fish oil appears to have an anti-tumor effect

similar to many conventional cytotoxic drugs with the added benefit of

 

 

154
no toxic short term side effects. Furthermore, the possibility of
utilizing omega-3 PUFAs as an effective adjuvant therapy (thereby using
less chemotherapeutic agents, thus producing less side effects without
sacrificing treatment effectiveness) seems now within reach. This
renewed interest in fish oil as both a preventive and a therapeutic
substance for various conditions has overlooked potential detrimental
side effects. These secondary effects may include: oxidation of low
density lipoproteins (LOL) (14) which can be conduscent to coronary
artery disease, suppression of the immune response (15) via oxidation
of omega-3 fatty acids in the lymphocyte membrane and the oxidation of
infant lipid emulsions and formulas (16) which can contribute to
premature lung injuries and damage to the central nervous system and
retina. In contrast to these detrimental effects, supplementation with
fish oil has been found to be of benefit in reducing plasma
triglycerides, reducing platelet aggregability (l7) and possible
therapeutic roles in hypertension (18), inflammatory diseases (19) and
cancer (5). Although mechanisms for these actions remain uncertain, it
is clear that lipid peroxidation cannot be ignored in our assessment of
the role of long chain omega-3 PUFAs in normal and pathological
processes.
References

l. Tiwari RK, Mukhopadhyay B, Telang NT and Osborne MP. Modulation

of gene expression by selected fatty acids in human breast cancer

cells. Anticancer Res. 1991, 11:1363-1388.
2. Rose DP and Connolly JM. Effects of fatty acids and inhibitors of

eicosanoid synthesis on the growth of a human breast cancer cell

line in culture. Cancer Res. 1990, 50:7139-7144.

3. Rice-Evans C and Burdon R. Free radical-lipid interactions and
their pathological consequences. J. Lipid Res. 1993, 32:71-110.

 

 

10.

ll.

12.

l3.

14.

155

Gonzalez NJ. Lipid peroxidation and tumor growth: an inverse
relationship. Ned. Hypotheses 1992, 38:106-110.

Gonzalez NJ, Schemmel RA, Gray J1, Dugan L, Jr., Sheffield LG and
Helsch CH. Effect of dietary fat on growth of NCF-7 and NDA-NBZ31
human breast carcinomas in athymic nude mice: Relationship between
carcinoma growth and lipid peroxidation product levels.
Carcinogenesis 1991, 12:1231-1235.

Gonzalez NJ and Helsch CH. Inhibition of mammary gland
tumorigenesis by dietary fish oil: A phenomenon associated with
increased levels of tumor lipid peroxidation products. In: Drevon
CA, Baksaas K and Krokan HE (eds) Omega-3 Fatty Acids: Netabolism
and Biological Effects 1993, 315-326. Birkhauser Verlag Basel,
Switzerland.

Fazio VN, Barrera G, Nartinotti S, Farace NG, Giglioni B, Frati L,
Nanzari V and Dianzani NU. 4-hydroxynonenal, a product of
cellular lipid peroxidation, which modulates c-myc and globin gene
expression in K562 erytholeukemic cells. Cancer Res. 1992,
52:4866-4871.

Fernandes G and Venkatraman JT. Modulation of breast cancer
growth in nude mice by w-3 lipids. In: Simopoulos AP, Kifer RR,
Nartin RE and Barlow SN (eds.). Health Effects of w-3
Polyunsaturated Fatty Acids in Seafoods. 1991, 488-503. Horld
Rev. Nutr. Diet. Basel, Karger, Switzerland.

Zollner H, Schaur RJ and Esterbauer H. Biological activities of
4-hydroxy-alkenals. In: Hills ED (ed.) Oxidative Stress: Oxidants
and Antioxidants, 1991, 337-369, London, Academic Press.

Horrobin DF. Essential fatty acids, lipid peroxidation and
cancer. In: Horrobin DF (ed). Omega-G-Essential Fatty Acids:
Pathophysiology and Roles in Clinical Nedicine, 1990, 351-377,
Alan R. Liss, Inc., New York.

Glomset JA. Fish, fatty acids and human health. N. Engl. J. Ned.
1985, 312:1253-1255.

Knapp KR and Fitzgerald GA. The antihypertensive effects of fish
oil - a controlled study of polyunsaturated fatty acid supplements
in essential hypertension. N. Engl. J. Ned. 1989, 320:1037-1043.

Kaiser L, Boyd NF, Kriukov V and Tritchler 0. Fish consumption

andsbrggst cancer risk: An ecological study. Nutr. Cancer 1989,
2: - .

Levy Y, Amotz AB, Danker G, Brook JG and Aviran N. Enhanced lipid
peroxidation of low density lipoprotein from subjects supplemented
with fish oil. J. Optimal Nutr. 1993, 1:6-9.

 

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19.

156

Neydani SN, Endres S, Hoods NN, Goldin BR, 800 C, Norrill-Labrode
A, Dinarello CA and Gorbach SL. Oral (n-3) fatty acid
supplementation suppresses cytokine production and lymphocyte
proliferation: Comparison between young and older women. J. Nutr.
1991, 121:547-555.

Pitkanen 0N. Peroxidation of lipid emulsions: A hazard for the
premature infant receiving parenteral nutrition? Free Rad. Biol.
Ned. 1992, 13:239-245.

Leaf A and Heber PC. Cardiovascular effects of n-3 fatty acids.
N. Engl. J. Ned. 1988, 318:549-557.

Knapp HR and Fitzgerald GA. The antihypertensive effects of fish
oil: A controlled study of polyunsaturated fatty acid supplements
in essential hypertension. N. Engl. J. Ned. 1989, 320:1037-1043.

Kremer JN. Studies on the effect of omega-3 fatty acids in the
treatment of patients with rheumatoid arthritis. In: Drevon CA,
Baksaas I and Krokan HE (eds.) Omega-3 Fatty Acids: Netabolism
and Biological Effects 1993, 271-283. Birkhauser Verlag Basel,
Switzerland.

Appendix

 

Appendix
The concept of cell loss having on important role in tumor growth

was introduced by Steel (Cell Tissue Kinet. 1968, 1:193-207).

Fundamentally, there are two different ways of assessing cell loss:

1) indirectly - in which cell loss is estimated from the discrepancy
between potential doubling time calculated from the earlier quasi-
exponential growth of the tumor (in which cell loss should be
minimal ~0); and tumor doubling time calculated from the growth
curve graph of the tumor.

2) directly - (although can be also considered indirectly,
nevertheless we consider it the most direct way of assessment since
it can obtain the values directly from the tumor in question).
Entails monitoring the loss of radioactivity from the tumor after

injection of a radioactive labelled compound (preferably Ilzsurd)

which is incorporated into the DNA during the synthesis phase and
released when tumor cells die. Tumors are measured at subsequent
intervals as cells are lost, the total tumor radioactivity will
decrease and the rate of this decrease once corrected for natural
decay provides virtually a direct measure of cell loss rate. Cell
loss is obtained from the knowledge of the volume doubling time (by
periodic measures of tumor volume) and the slope of the
radioactivity loss curve. The corrected curve can be equated with
the rate of loss of cells from the tumors.
Many researchers have estimated cell loss by scanning techniques
which require invasive methods such as excision of the tumors and prior
chemical extractions of DNA of the tumor sample which is very time-

consuming. In addition to being time-consuming, the tumor sample can

158

159

only be measured once. This increases the variability plus increases

the number of animals and/or tumors needed for an experiment. The use

of external scanning techniques seems to be a more convenient way to

estimate the total radioactivity loss throughout the measuring period

in a localized superficial tumor. Some advantages of employing these

methods are:

1)

2)

3)

Is a simple virtually direct measurement, that perturbs
minimally the tumor in question. Also it does not rely on
calculated vs. measured parameters (such as doubling times).
In addition it does not require any time consuming methodology
as chemical extractions, autoradiography and/or microscopic
work.

Requires fewer animals than any invasive method that need to
excise tumors for the measurement of cell loss. Also provides
in vivo measurements.

Individual tumors are easily assessed. This individual
assessment of tumors decreases variation considerably. It
provides the opportunity to each tumor to serve as its own

control.

 

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