Prostate Carcinogenesis in N-methyl-N-nitrosourea
(NMU)–Testosterone-Treated Rats Fed Tomato Powder,
Lycopene, or Energy-Restricted Diets
Thomas W.-M. Boileau, Zhiming Liao, Sunny Kim, Stanley Lemeshow,
John W. Erdman, Jr., Steven K. Clinton
Background: Consumption of tomato products or lycopene
and energy restriction have been hypothesized to reduce the
risk of human prostate cancer. We investigated the effects of
these dietary variables in a rat model of prostate carcinogenesis.
Methods: Male rats (n 194) treated with N-methyl-
N-nitrosourea and testosterone to induce prostate cancer
were fed diets containing whole tomato powder (13 mg lycopene/
kg diet), lycopene beadlets (161 mg lycopene/kg diet),
or control beadlets. Rats in each group were randomly assigned
to either ad libitum feeding or 20% diet restriction.
Differences between Kaplan–Meier survival curves for diet
composition or restriction were tested with the log-rank test.
Cox proportional hazards models were developed to examine
the combined effect of diet composition and restriction
on survival. Statistical tests were two-sided. Results: Risk of
death with prostate cancer was lower for rats fed the tomato
powder diet than for rats fed control beadlets (hazard ratio
[HR] 0.74, 95% confidence interval [CI] 0.59 to 0.93;
P .009). In contrast, prostate cancer–specific mortality of
the control and lycopene-fed rats was similar (P .63). The
proportions of rats dying with prostate cancer in the control,
lycopene, and tomato powder groups were 80% (95% CI
68% to 89%), 72% (95% CI 60% to 83%), and 62% (95%
CI 48% to 75%), respectively. Rats in the diet-restricted
group experienced longer prostate cancer–free survival than
rats in the ad libitum–fed group (HR 0.68, 95% CI 0.49
to 0.96; P .029). The proportion of rats that developed
prostate cancer was 79% (95% CI 69% to 86%) for ad
libitum–fed rats and 65% (95% CI 54% to 74%) for rats
fed restricted diets. No interactions were observed between
diet composition and dietary restriction. Conclusions: Consumption
of tomato powder but not lycopene inhibited prostate
carcinogenesis, suggesting that tomato products contain
compounds in addition to lycopene that modify prostate
carcinogenesis. Diet restriction also reduced the risk of prostate
cancer. Tomato phytochemicals and diet restriction may
act by independent mechanisms. [J Natl Cancer Inst 2003;
Both prospective epidemiologic and case– control studies
(1–5) have associated increased consumption of tomato products
and greater blood concentrations of lycopene with a reduced risk
of prostate cancer. These observations have led many to hypothesize
that lycopene, the principal carotenoid in tomatoes, may be
the active component in tomato products (6). Lycopene is found
in human prostate tissue, further suggesting the plausibility of a
direct effect on prostate biology (7–11). In addition, results of a
recent case– control study (12) revealed that pre-diagnosis blood
lycopene concentrations were lower in men who developed
prostate cancer than in men who remained disease-free. Results
of in vitro studies suggest that lycopene inhibits the growth of
human prostate cancer cell lines (13), is a potent antioxidant
(14,15), influences expression of gap junction proteins (16), and
inhibits growth factor signaling (17,18). Two recent studies of
men with prostate cancer who were given a lycopene-enriched
supplement (8) or fed tomato products (9) for several weeks
prior to prostatectomy demonstrated that lycopene concentrations
in the prostate can change rapidly in response to dietary
intake and that biomarkers of oxidative stress and tumor biology
can be altered.
Although none of these studies alone establishes a causal
relationship between tomato products or lycopene consumption
and prostate cancer risk (19), they constitute a growing body of
evidence supporting a continued research effort to further dissect
these relationships (1–18). It is of particular interest to determine
whether lycopene itself is associated with reduced risk or
whether it is simply a biomarker that is indicative of exposure to
tomato products that may contain other phytochemicals with
anti–prostate cancer properties (20). A laboratory animal model
of prostate carcinogenesis is an ideal system in which to address
To assess the role of lycopene in an experimental animal
model, it is important to consider the ability of the species used
to achieve biologically relevant tissue concentrations of lycopene.
Our laboratory (21,22) and others (23–25) have shown that
rats accumulate dietary lycopene in prostate, blood, and other
tissues at concentrations that overlap those reported for humans
if the dietary concentrations are sufficient to compensate for the
lower bioavailability of carotenoids by rodents compared with
humans. We have also observed that the pattern of lycopene
Affiliations of authors: Division of Nutritional Sciences, University of Illinois,
Urbana-Champaign, IL (TWMB, JWE); Division of Hematology and Oncology,
Department of Internal Medicine, James Cancer Hospital and Solove Research
Institute (ZL, SKC), School of Public Health (SL), Center for Biostatistics (SK),
and Department of Human Nutrition (SKC), The Ohio State University, Columbus,
Correspondence to: Steven K. Clinton, MD, PhD, A434 Starling Loving Hall,
320 West 10th Ave., The Ohio State University, Columbus, OH 43210 (e-mail:
See “Notes” following “References.”
Journal of the National Cancer Institute, Vol. 95, No. 21, © Oxford University
Press 2003, all rights reserved.
1578 ARTICLES Journal of the National Cancer Institute, Vol. 95, No. 21, November 5, 2003
isomers in the rat prostate is indistinguishable from that in the
human prostate (7,21,22), with the majority of lycopene present
as multiple cis-isomers (7,21). Thus, the rat appears to be a
reasonable in vivo model in which to evaluate the biologic
actions of lycopene during prostate carcinogenesis.
Energy balance is another dietary variable that is linked to
prostate cancer risk. For example, recent reports suggest that
frequent exercise (26) and lower body mass index (27) are
associated with reduced prostate cancer risk. However, human
studies alone have not allowed investigators to precisely quantitate
the role of energy balance during prostate carcinogenesis
due to the relative difficulty in measuring the key variables
throughout the life cycle as well as the complex relationships
among energy intake, sources of energy, basal energy expenditures,
activity-related energy expenditures, body weight, and
body composition. Experimental models have proven useful in
this regard, because many interacting variables can be controlled.
For example, a role for energy restriction in prostate
tumor growth has been clearly demonstrated in transplantable
rodent models (28,29). Both modest total diet restriction and
selective limitation of energy intake from carbohydrate or lipid
sources statistically significantly reduced the growth of the welldifferentiated
hormone-sensitive Dunning R3327H prostate adenocarcinoma
Several new animal models of prostate cancer are being
developed that have unique characteristics that may be relevant
to specific aspects of the carcinogenic process or to
subtypes of prostate cancer exhibiting specific molecular defects
and/or biologic properties (30 –32). One such model is
the N-methyl-N-nitrosourea (NMU)–androgen-induced rat
model of prostate cancer developed by Bosland (31,32) and
used in several recent chemoprevention studies (33–35).
Whereas many rodent prostate cancer models result in cancers
that predominantly affect the seminal vesicle and ventral
prostate (36,37), the NMU–androgen-induced model causes
tumors of the dorsolateral and anterior prostate (33). These
lobes of the rat prostate are generally considered homologous
to the areas of the human prostate that are most susceptible to
cancer (30 –33). In this model, the incidence of prostate
carcinomas approaches 75% by approximately 52 weeks, with
many showing evidence of androgen dependence and histopathologic
features similar to human prostate cancer (30 –
37). In addition, the host experiences limited toxicity and
does not exhibit a high frequency of malignancies at nontarget
sites (33). Thus, the NMU–androgen-induced rat model is
an anatomically and physiologically relevant system for the
preclinical evaluation of substances that are hypothesized to
inhibit or enhance human prostate carcinogenesis (33–35).
In this study, we assessed the individual and interactive
effects of precisely controlled dietary interventions on the survival
of rats treated with NMU androgens to stimulate prostate
carcinogenesis. One goal was to determine whether freeze-dried
whole tomatoes (tomato powder) or pure lycopene could enhance
survival in this model. A second goal was to assess the
ability of diet restriction to enhance survival in a prostate carcinogenesis
model as we have observed in transplantable systems
(28,29). Finally, we aimed to determine whether interactions
between energy intake and tomato powder or lycopene
intake could be observed.
MATERIALS AND METHODS
Animals and Diet Formulations
Male Wistar-Unilever rats (HsdCpb:Wu) (n 194) (Harlan,
Indianapolis, IN) were obtained at 5 weeks of age and fed a
standard AIN-93G–based diet for 1 week of adaptation. At 6
weeks of age, rats were randomly assigned to one of three
semi-purified AIN-93G–based experimental diets (38) prepared
according to our formulations (Dyets, Bethlehem, PA). One diet
contained control beadlets (Hoffmann-La Roche, Basel, Switzerland)
(n 64 rats), the second contained lycopene beadlets
(Hoffmann-La Roche) (n 65 rats), and the third contained
tomato powder (Armour Foods, Springfield, KY) (n 65 rats)
(Table 1). The control beadlet diet was prepared by incorporating
water-dispersible beadlets into the experimental diets at a
concentration of 2.5 g of beadlets per kilogram of diet. The
lycopene beadlet diet was prepared similarly. The tomato powder
(Armour Foods) is a spray-dried product made from heatprocessed
tomato paste (prepared from whole tomatoes including
seeds and skins) (Del Monte Foods, San Francisco, CA). The
tomato powder contains 12.9 g of protein, 74.6 g of carbohydrate,
and 0.44 g of fat per 100 g of powder. Diets were stored
at 4 °C in the dark. Rats were weighed weekly. The University
of Illinois Laboratory Animal Care Advisory Committee approved
all animal procedures.
All rats initially consumed one of the three diets, with unlimited
access to food (defined as ad libitum). When they
reached 10 weeks of age (3 days after carcinogen administration;
see below), the rats in each dietary group were further subdivided
and randomized to ad libitum or 20% total dietary restriction
for the remainder of the study. Diet-restricted rats were fed
daily a quantity of food equal to 80% of the average daily intake
Table 1. The formulation and composition of AIN-93G-based diets
containing control beadlets, lycopene beadlets, or tomato powder (g/kg)
Casein 200 200 200
L-cysteine 3 3 3
Corn starch 381.24 381.24 297.49
Sucrose 99.99 99.99 99.99
Dextrinized cornstarch 115.75 115.75 101.98
Cellulose fiber (solka floc) 50 50 50
Soybean oil 100 100 100
Mineral mix (AIN-93G-MX) 35 35 35
Tertbutylhydroquinone 0.02 0.02 0.02
Vitamin mix (AIN-93G-VX) 10 10 10
Choline bitartrate 2.5 2.5 2.5
Placebo beadlets 2.5 0 0
Lycopene beadlets 0 2.5 0
Tomato powder 0 0 100
Supplemental (total) vitamin E‡ None
Total 1000 1000 1000
Lycopene beadlets are 10% wt/wt lycopene.
†The freeze-dried tomato powder contains approximately 0.01% lycopene.
‡Placebo and lycopene beadlets both contain 1% wt/wt vitamin E. The tomato
powder diet was supplemented with vitamin E ( -tocopherol acetate) to provide
equivalent final concentrations of vitamin E in each diet. The final vitamin E
concentration in the diet includes the amount in the vitamin mix (AIN-93G-VX)
and the vitamin E provided in beadlets or supplement form.
Journal of the National Cancer Institute, Vol. 95, No. 21, November 5, 2003 ARTICLES 1579
of ad libitum–fed rats, which was recalculated weekly until rats
were 17 weeks of age, when food intake had stabilized. From
that point on, food intake in the ad libitum–fed group was
precisely measured every 4 weeks, and the amount of food
provided to the diet-restricted groups was adjusted accordingly.
Hormone and Carcinogen Treatment
Starting at 6 weeks of age and continuing for the next 3
weeks, all rats received daily intraperitoneal injections of the
luteinizing hormone–releasing hormone antagonist cyproterone
acetate (CA) (50 mg/kg body weight) (Sigma Chemical, St.
Louis, MO). CA inhibits androgen secretion from the testis,
thereby causing atrophy of prostatic epithelial cells. Starting on
the day after the last injection of CA, the rats, which were then
9 weeks old, received daily subcutaneous injections of 100 mg
of testosterone propionate (TP) (Sigma Chemical) per kilogram
of body weight in 0.5 mL of soybean oil to maximally stimulate
proliferation of prostatic epithelial cells. On the day after the last
TP injection, the rats were anesthetized with metofane and
injected intravenously (via the tail vein) with the carcinogen
NMU (Ashe Stevens, Detroit, MI) at a dose of 50 mg per
kilogram of body weight. The NMU was initially wetted with
3% acetic acid and stored at –20 °C until use. Immediately prior
to injection, the NMU was dissolved in saline at 10 mg/mL,
yielding a final pH of 5.5. One week after NMU administration,
rats received testosterone as two subcutaneous implants (1.0-mm
inner diameter 2.2-mm outer diameter 2.54-cm-long silastic
laboratory tubing; Dow Corning, Midland, MI), each containing
crystalline testosterone (Sigma Chemical) that had been
drawn into the implants under vacuum pressure before the ends
of the tube were sealed with silicone adhesive (Dow Corning).
Implants were inserted subcutaneously in the dorsolumbar region
of the back using sterile technique, and wounds were sealed
with surgical glue.
Survival and Necropsy
All rats were monitored daily, and rats showing any signs or
symptoms of morbidity, including reduced food intake or weight
loss, were killed. The remaining rats were killed at 73 weeks of
age, when the study was terminated. Death in healthy-appearing
rats was rare, and such rats were necropsied immediately on
discovery. Rats that were going to be killed were first anesthetized
by exposure to CO2; blood was then collected by cardiac
puncture into heparinized tubes. Blood was centrifuged in the
dark at 250g for 10 minutes to separate plasma, which was
stored at –20 °C in the dark for lycopene analysis (see below).
Rats were then killed by further CO2 inhalation, and the prostate
(dorsolateral, ventral, and anterior lobes [coagulating gland])
and seminal vesicle were removed en bloc immediately and
placed in 10% neutral-buffered formalin. The liver was removed,
cooled rapidly on ice (with protection from light), and
stored at –20 °C in the dark for lycopene analysis.
The fixed prostate and seminal vesicles were examined, and
those found to be normal at a gross level were dissected into
seven components for microscopic evaluation: 1) bladder, 2)
right ventral lobe, 3) left ventral lobe, 4) right dorsolateral lobe,
5) left dorsolateral lobe, 6) right anterior prostate and seminal
vesicle, and 7) left anterior prostate and seminal vesicle. The
right and left dorsolateral and ventral lobes were dissected
longitudinally and embedded in paraffin. Each anterior prostate
and seminal vesicle complex was dissected into four or five
sequential pieces and embedded in paraffin. Those tissue specimens
with tumors large enough to disrupt normal anatomic
structure were dissected into three to five pieces, which were
embedded in paraffin. The carcass of each rat was examined, and
any tissues showing abnormalities were also fixed in 10% neutral
buffered formalin and embedded in paraffin. Step sections
(3.5 m thick) were prepared from all of the blocks and stained
with hematoxylin–eosin. Sections were blindly and independently
evaluated by two investigators to classify the lesions, and
any discrepancies in the interpretation were discussed and resolved.
Lesions were classified using previously described histopathologic
criteria (32,33). We categorized lesions in the prostate
and seminal vesicle complex as carcinoma in situ, as
microscopic adenocarcinomas ( 0.4 cm), or as macroscopic
advanced adenocarcinomas ( 0.4 cm). The intraprostatic site of
origin was defined for the smaller lesions, whereas the precise
origin of larger invasive carcinomas often could not be determined.
In agreement with previous reports (32,33) using this rat
model of prostate carcinogenesis, we saw no evidence that the
ventral lobe was involved in proliferative lesions or a primary
source of carcinomas. A total of nine rats developed malignancies
in tissues other than the prostate (Zymbal’s gland tumors,
leukemia/lymphoma, and sarcoma).
Extraction and High-Performance Liquid
Chromatography Analysis of Lycopene
Lycopene was extracted from the diet and the plasma and
quantitated as previously described (21,22). Briefly, an ethanol–
hexane solution was used to extract lycopene from the dietary
and biologic samples, and the extracts were then subjected to
high-performance liquid chromatography (HPLC) analysis with
separations performed on a C30 column (YMC, Wilmington,
NC) and detected at 470 nm on a UV/VIS detector (model
UV-DII; Rainin Dynamax, Walnut Creek, CA). Standard curves
were prepared using crystalline lycopene extracted from a tomato
oleoresin (LycoRed Natural Products Industries, Beer-
Sheva, Israel) and purified on a C30 column. Lycopene was
quantified using an external standard curve. Our laboratory
participates quarterly in the National Institute of Standards in
Technology micronutrient measurement proficiency testing program.
The coefficient of variance for lycopene analysis in our
laboratory is less than 12%.
The experiment was designed as a survival study, in which
rats were monitored carefully and killed at the first sign of
morbidity. Thus, the most appropriate outcome events to address
the efficacy of the dietary treatments were prostate cancer–
specific survival and death from any cause. For survival analysis,
time to death was defined as age (in weeks) at the time the
rat died with prostate cancer. Kaplan–Meier survival estimates
were calculated for each diet composition group (tomato powder,
lycopene, and control) and for each food intake group (ad
libitum and restricted). The log-rank test was used to test the
equality of the survival curves for the treatment groups. This test
weights each time point equally in the comparison of survival
curves. The Wald test (39) was used to assess statistical signif-
1580 ARTICLES Journal of the National Cancer Institute, Vol. 95, No. 21, November 5, 2003
icance of coefficients in the Cox proportional hazards model.
Cox proportional hazards regression was then used to investigate
the effects of diet composition, controlling for diet restriction.
The proportional hazards assumption was tested for each model.
When the proportional hazards assumption was violated, a series
of analyses was used, each analysis examining two diet composition
variables at a time. A Bonferroni adjustment was used to
assess statistical significance (i.e., instead of declaring statistically
significant a result whose P value is less than .05, we use
the .017 level as an indicator of statistical significance). In this
study, survival time was defined as the age (in weeks) at death
or, for those rats that were still alive at the end of the study, 73
weeks of age (i.e., 64 weeks on study). Rats surviving until the
end of the study were entered into the statistical model as
censored observations (39). All survival analyses were conducted
using STATA Statistical Software (release 8.0; Stata
Corporation, College Station, TX).
Differences in food intakes, body weights, and plasma lycopene
concentration between the rats on the different diets were
tested by two-way analysis of variance (ANOVA). Data were
log-transformed for analysis if they were found not to be normally
distributed but are expressed as original values in the text
and tables for ease of interpretation. Differences in mean plasma
lycopene concentrations at the time of death were tested by
two-way ANOVA, with assessment of main effects of diet
composition and dietary intake as well as interactions among
diet composition and dietary intake. Statistically significant (i.e.,
P .05) main effects were further tested by two-way post hoc
Fisher’s protected least-square difference test to identify differences
in mean plasma lycopene between any two groups. All
statistical tests were two-sided.
Food Intake and Growth
Food intake of rats fed the control beadlet (mean standard
deviation of 16.6 2.0 g/day), lycopene beadlet (16.1 2.1
g/day), or tomato powder (16.7 2.1 g/day) diets did not differ
(P .757) during the 4 weeks between initial assignment to
diets and the randomization to continued ad libitum feeding or
diet restriction. Three days after carcinogen administration, rats
in each dietary group were divided into diet restriction and ad
libitum subgroups. Diet-restricted rats received 80% of the dietary
intake of ad libitum–fed rats for the duration of the study.
Food intake of rats having ad libitum access to food gradually
increased until they reached 17 weeks of age (19.6 1.7 g/day)
and remained stable ( 2 g/day) until individual rats began to
show symptoms of prostate cancer. Body weights (Fig. 1) did
not differ statistically significantly among rats fed the control
beadlet, lycopene beadlet, or tomato powder–containing diets ad
libitum or among the diet-restricted subgroups. Within a week
following initiation of dietary restriction, rats in all restricted
groups weighed less than the corresponding ad libitum–fed rats
and remained smaller (approximately 18%–20% less in body
weight) for the duration of the experiment. There was greater
variation in rat weights after 60 weeks of age due to the smaller
number of surviving animals and the high prevalence of morbidity
associated with prostate cancer.
Lycopene Content of the Diet
HPLC analysis showed that the control beadlet diet (Table 1)
did not have detectable lycopene or other carotenoids (Table 2).
Fig. 1. Body weight of all rats surviving at each time point after N-methyl-Nnitrosourea
(NMU) treatment and fed a placebo beadlet (control), lycopene
beadlet, or tomato powder–containing AIN-93G diet with food provided ad
libitum (Ad lib) or with a 20% diet restriction. Rats fed a 20% restricted diet
weighed statistically significantly (P .01) less than control rats with free access
to food, regardless of diet composition by 1 week after NMU administration.
Body weights were not statistically significantly altered by diet composition
(control, lycopene, or tomato powder) between 0 and 60 weeks of age. The
variation in body weights after week 50 following NMU treatment was due to the
lower number of surviving rats in each group because of death and/or the
presence of tumors.
Table 2. The mean concentrations (and 95% confidence intervals) of lycopene isomers in the lycopene beadlet and tomato powder diets (mg/kg)
Diet Total lycopene All-trans lycopene 5-cis lycopene Other-cis lycopene
Lycopene beadlet, fresh† 161 (143 to 179)a 68 (64 to 72)a 71 (69 to 73)a 21 (17 to 25)a
Lycopene beadlet, exposed‡ 106 (104 to 108)b 51 (49 to 53)b 46 (44 to 48)b 13 (7 to 18)b
Tomato powder, fresh†§ 13 (11 to 15)c 5 (5 to 5)c 4 (4 to 4)c 4 (4 to 4)c
Tomato powder, exposed‡ 7 (7 to 7)c 3 (3 to 3)c 2 (2 to 2)c 2 (2 to 2)c
Values were determined by high-performance liquid chromatography. Lycopene was not detectable in the control beadlet diet. Lycopene concentrations in the
lycopene and tomato powder diets were compared using one-way analysis of variance followed by post hoc Fisher’s protected least-squares difference test. Values
in the same column with different superscripts are statistically significantly (P .05) different. Data are means and 95% confidence intervals for four diet samples
per group. Means and 95% confidence intervals are rounded to the nearest milligram.
†The diet was analyzed immediately after removal from storage at 4° C.
‡The diet was exposed to the atmosphere, temperature, and light of a rat cage for 2 days and then analyzed.
§The tomato powder diet also contains all-trans -carotene (approximately 0.001 g/kg diet) as well as 9-cis -carotene and other polar carotenoids eluting before
-carotene (see Fig. 2).
Journal of the National Cancer Institute, Vol. 95, No. 21, November 5, 2003 ARTICLES 1581
The lycopene concentration in the lycopene beadlet diet was 161
mg lycopene/kg diet, and the tomato powder diet contained 13
mg lycopene/kg diet) (Table 1). The tomato powder and lycopene
beadlet diets had similar patterns of lycopene isomers and
percentages of total lycopene in the cis configuration (Table 2
and Fig. 2). Both lycopene-containing diets showed a decline in
lycopene ( 40% reduction) after exposure to the atmosphere,
temperature, and lighting of the rat housing for 2 days. The
tomato powder diet also contained other carotenoids typically
found in tomatoes, such as all-trans -carotene (approximately 1
mg/kg) as well as 9-cis -carotene and other unidentified polar
carotenoids (Fig. 2).
Plasma Lycopene Isomer Concentrations
Lycopene was not detected in plasma of rats fed the control
beadlet diet. Rats with unrestricted access to food and fed the
lycopene beadlet diet had greater plasma concentrations of total
lycopene (37% higher, P .017) and all-trans lycopene (61%
higher, P .003) than rats fed the tomato powder diet (Table 3).
Rats fed under restricted conditions and consuming lycopene
beadlet or tomato powder diets accumulated approximately 15%
less lycopene in plasma than rats with ad libitum access to the
same diet, although the difference was not statistically significant
for any of the diets. Although -carotene was present in the
tomato powder diet, it was not detectable in the plasma of rats
consuming this diet, which suggests that it was converted completely
to retinol (vitamin A).
Observations From Gross Dissection and Histopathology
Of the 194 rats that were subjected to the tumor induction
protocol, seven died within the first week after NMU administration
due to complications of anesthesia or acute toxicity. Of
the remaining 187 rats, 165 (88%) died or were killed before
reaching 73 weeks of age, when the study was terminated. A
total of 134 of the 187 rats (72%) were killed because they
displayed symptoms of prostate cancer before 73 weeks of age.
A total of 151 (81%) of the 187 rats developed some form of
cancer (adenocarcinoma, sarcoma, or carcinoma in situ) in the
prostate and seminal vesicle complex. Of the 22 rats that were
killed at the end of the study but showed no morbidity, 17 had
histologically detected prostate cancer. In addition to using
histopathologic criteria, we classified the tumors based on size.
Among the 187 rats, microscopic adenocarcinomas ( 0.4 cm)
were observed in 57 rats (31%), whereas 88 rats (47%) developed
locally advanced adenocarcinomas ( 0.4 cm). Two of the
187 rats (1.1%) developed prostate sarcomas, and only four
(2.1%) were found to have pathology limited to prostatic carcinoma
in situ at the time of killing. Nine of the 187 rats (4.8%)
had extensive metastatic disease from their prostate carcinoma
to lymph nodes, liver, or peritoneum. Another nine rats (4.8%)
developed cancers at sites other than the prostate (including
Zymbal’s gland), leukemia, and lymphoma. The frequency and
types of cancer observed are consistent with those previously
reported in this model system (30–33).
Diet Composition and Prostate Cancer–Specific Survival
The primary outcome evaluated was prostate cancer–specific
survival. This outcome was chosen over other outcomes, such as
Fig. 2. High-performance liquid chromatography analysis with C30 column
separations of lycopene isomers and other carotenoids in the diet and plasma of
rats fed lycopene beadlets and tomato powder. The control beadlet diet contained
no detectable lycopene or other carotenoids and is not shown. A) The lycopene
beadlet diet contained only lycopene as all-trans (at lycopene), 5-cis (5c lycopene),
and several other isomers eluting before all-trans (labeled as other-cis
lycopene [oc lycopene]). B) The tomato powder diet contained the same lycopene
isomers as the lycopene beadlet diet and also contained all-trans -carotene
(at -carotene), 9-cis -carotene (9c -carotene), and other polar carotenoids
eluting before all-trans -carotene. C) Lycopene isomers were the only carotenoids
detected in the plasma of lycopene beadlet–fed rats. D) Tomato powder–
fed rats also accumulated a similar array of lycopene isomers in plasma.
Table 3. Concentrations of lycopene isomers in the plasma of ad libitum–fed and diet-restricted rats consuming the lycopene beadlet and tomato powder
Diet Total lycopene All-trans lycopene 5-cis lycopene Other-cis lycopene
Lycopene, ad libitum 118 (75 to 161)a 52 (32 to 72)a 38 (24 to 52) 28 (19 to 37)a
Lycopene, restricted 99 (80 to 118)a,b 43 (32 to 54)a 30 (24 to 36) 25 (22 to 28)a,b
Tomato, ad libitum 85 (74 to 95)b 31 (27 to 35)b 30 (26 to 35) 23 (20 to 25)a,b
Tomato, restricted 74 (64 to 83)b 28 (23 to 32)b 26 (22 to 30) 20 (15 to 25)b
Main effect of lycopene source P .017 P .003 NS P .027
Main effect of dietary intake NS NS NS NS
Interaction NS NS NS NS
No lycopene was detectable in plasma from rats fed the control beadlet, and such rats were omitted from the analysis. Data represent means of 11–19 rats per
group with 95% confidence intervals. Two-way analysis of variance was used to test for main effects of lycopene source (lycopene and tomato powder) and dietary
intake (ad libitum and restricted) as well as their interaction. When statistically significant main effects were found, each group was further tested by one-way analysis
of variance and post hoc Fisher’s protected least-square difference test to determine the statistically significant differences among the individual treatment groups.
Values in the same column with different superscripts are statistically significantly (P .05) different. NS not significant (P .05).
1582 ARTICLES Journal of the National Cancer Institute, Vol. 95, No. 21, November 5, 2003
tumor incidence, because rats were killed as they became moribund,
and thus a time-related variable was critical to the analysis.
Therefore, Kaplan–Meier survival curves were initially
used to evaluate the influence of dietary treatments on prostate
carcinogenesis. Survival functions for the three different diet
composition groups (tomato powder, lycopene, or control) were
not equal (log-rank test, P .042). Kaplan–Meier survival
curves indicated that rats fed tomato powder experienced longer
prostate cancer–free survival than rats in the other two dietary
groups (Fig. 3, A). A Cox proportional hazards model, restricted
to the control and lycopene beadlet diets (Table 4), showed that,
after controlling for diet restriction, rats fed the control and
lycopene beadlet diets experienced similar survival (P .63).
However, the proportional hazards assumption was violated and
P values obtained from this statistical model should therefore be
interpreted cautiously. By contrast, for the model restricted to
rats fed the control and tomato powder diets, rats fed the tomato
powder diet experienced a statistically significantly longer survival
than the rats fed the control diet (hazard ratio [HR] 0.74,
95% confidence interval [CI] 0.59 to 0.93; P .009) after
controlling for diet restriction. This result remained statistically
significant even after applying the Bonferroni adjustment. Finally,
the Cox model comparing survival of tomato powder– and
lycopene-fed rats suggested that rats fed tomato powder had
prolonged survival compared with lycopene-fed rats after controlling
for diet restriction, but the difference did not reach
statistical significance (P .07). The percentages of rats dying
with some form of prostate cancer (adenocarcinoma, carcinoma
in situ, or sarcoma) were 80% (95% CI 68% to 89%), 72%
(95% CI 60% to 83%), and 62% (95% CI 48% to 75%) for
the control, lycopene, and tomato powder groups, respectively.
Diet Restriction and Prostate Cancer–Specific Survival
The percentages of rats that died with prostate cancer were
79% (95% CI 69% to 86%) and 65% (95% CI 54% to 74%)
for the ad libitum and diet-restricted groups, respectively.
Kaplan–Meier survival functions for the two groups were statistically
significantly different (log-rank test, P .03), indicating
an increase in prostate cancer–free survival in the rats
assigned to diet restriction (Fig. 3, B). The two factors (diet
composition and level of intake) and their interaction term were
entered into the Cox model. The interaction term for the type of
diet and the amount of dietary intake was not statistically significant
(Wald test, P .38) and was therefore removed from
the model. The model results suggest that diet-restricted rats had
a statistically significantly lower risk of dying with prostate
cancer over their lifespan than ad libitum–fed rats, after controlling
for diet type (HR 0.68, 95% CI 0.49 to 0.96; P .029)
(Table 4). The precision of this P value is somewhat questionable,
however, because a statistically significant violation of the
proportional hazards assumption had occurred (P .02). We
believe that this P value is conservative because the crossing of
the Kaplan–Meier survival curves may well be the reason for the
rejection of the proportional hazards assumption.
Diet and Risk of Death From Any Cause
A second Cox model was fitted to the survival data, using
death from all causes as the outcome. The final model coefficients,
along with hazard ratio estimates and 95% confidence
intervals, are shown in Table 4. The interaction term was not
statistically significant (Wald test, P .55) and was removed
from the final model. The test for the proportional hazards
assumption indicated no statistically significant violations (P
.13). No statistically significant effect of diet composition or
intake on all-cause mortality was noted.
This study focused on survival, and thus this design would
bias an interpretation of tumor grade and stage because these are
time-dependent outcomes of carcinogenesis that are best evaluated
in a study with a fixed termination point. Nevertheless, we
provide these data for descriptive purposes. Among rats dying
Fig. 3. Kaplan–Meier prostate cancer–specific survival curves. A) Curves for
rats fed tomato powder, lycopene beadlet, and control beadlet diets. The duration
of prostate cancer–free survival was greater for the rats fed tomato powder than
for the rats fed either purified lycopene or control diets. At 50 weeks, the
surviving fractions were 37% (95% confidence interval [CI] 24% to 50%) for
controls, 39% (95% CI 27% to 51%) for lycopene fed, and 54% (95% CI
39% to 67%) for the tomato powder–fed rats. B) Curves for rats fed ad libitum
or under conditions of 20% diet restriction. The rats consuming a restricted diet
experienced statistically significantly longer prostate cancer–free survival than
rats with unlimited access to food. At 50 weeks, the surviving fractions were
35% (95% CI 25% to 45%) for controls and 52% (95% CI 40% to 62%)
for the diet-restricted rats.
Journal of the National Cancer Institute, Vol. 95, No. 21, November 5, 2003 ARTICLES 1583
with prostate cancer, 70%, 71%, and 45% of the total cancers in
rats fed the control beadlet diet, lycopene beadlet diet, and
tomato powder diet, respectively, were macroscopic, poorly
differentiated lesions. We observed that 55% of the rats in the
diet-restricted group had macroscopic prostate adenocarcinomas
as compared with 69% of the rats with ad libitum access to food.
The majority of lycopene intake (82%) by American men is
from a single food source, tomato products (1,6,20). Thus, it is
not possible for epidemiologic studies to differentiate whether
intake of lycopene alone, as opposed to intake of one or more of
the vast array of phytochemicals found in tomatoes, is related to
risk of disease outcomes. In contrast, a laboratory animal model
allows investigators to address this critical question. Our study is
the first, to our knowledge, that compares the abilities of purified
lycopene and tomato powder to alter the risk of prostate cancer
in a highly controlled model of prostate carcinogenesis. In
addition, we examined the interaction between intake of lycopene
or tomato products and modest (i.e., 20%) dietary restriction
on prostate cancer–specific survival because a previous
study (28) suggested that the response to dietary variables may
change with different levels of energy intake.
Our observations support the concept that tomato products
contain components in addition to lycopene that may inhibit
prostate carcinogenesis. Although we can conclude that lycopene
alone, in this model system and at this dose, did not
statistically significantly alter the risk of prostate cancer, it
remains possible that lycopene, when provided in combination
with the other phytochemicals found in whole tomato powder
may contribute to the benefits observed. Tomatoes contain an
array of phytochemicals, including other carotenoids in addition
to lycopene, that could potentially modulate prostate cancer risk
(40–46). These substances include all-trans -carotene and
9-cis -carotene, polyphenolic compounds such as quercetin
(47,48), other phenolic compounds (40), and vitamin C and
folate (20). Results of recent in vitro studies suggest that several
of the phytochemicals found in tomatoes, such as other carotenoids
(13,41), vitamin C (42)–44), and vitamin E (45), can
influence prostate tumor cell growth and quench reactive oxygen
species (46). Additional efforts to characterize bioactive phytochemicals
in tomatoes, their mechanisms of action, and, most
important, any additive or synergistic effects on prostate carcinogenesis,
Lycopene concentrations in the plasma provide some insights
into the outcomes of this study. Rats fed the lycopene beadlets
had statistically significantly higher plasma lycopene concentrations
(means of 99 and 118 nmol of lycopene per liter of plasma
for diet-restricted and ad libitum–fed rats, respectively) than rats
fed tomato powder (means of 74 and 85 nmol of lycopene per
liter of plasma for diet-restricted and ad libitum–fed rats, respectively)
(Table 3). It is interesting that the plasma lycopene
concentrations were so similar, given that the lycopene beadlet
diet contains more than 10 times more lycopene than the tomato
powder diet. These data have several possible explanations: that
the efficiency of lycopene absorption declines as the lycopene
concentration in the diet increases or that the bioavailability of
lycopene from tomato powder is much greater than that from
beadlets. In addition, because the lycopene beadlets achieved the
greatest plasma lycopene concentrations but did not protect
against prostate cancer, these data further support the hypothesis
that tomatoes must contain phytochemicals in addition to lycopene
that may modulate prostate carcinogenesis.
Rats that experienced a modest total diet restriction demonstrated
a statistically significant reduction in prostate cancer risk
in the NMU model of prostate carcinogenesis. This observation
complements, reinforces, and extends our results previously
reported using transplantable prostate cancer models (28). It is
important to recognize that a dietary restriction of this extent
(i.e., 20%) allows continued growth of the rats and does not
result in malnutrition but rather in the prevention of obesity (28).
Interestingly, no statistically significant interactions were observed
between energy intake and diet composition. Thus, tomato
products and diet restriction may have additive independent
In summary, our results suggest that tomato products may be
more effective for the inhibition of prostate carcinogenesis than
Table 4. Proportional hazards analysis of risk of dying with prostate cancer and risk of death from any cause
Variable SE ( ) P value† HR (95% CI)
Risk of death with prostate cancer
Restricted vs. ad libitum-fed‡
0.38 0.17 .029 0.68 (0.49 to 0.96)
Lycopene diet vs. control diet§
0.10 0.20 .630 0.91 (0.61 to 1.35)
Tomato powder diet vs. control diet
0.30 0.11 .009 0.74 (0.59 to 0.93)
Tomato powder diet vs. lycopene diet¶
0.40 0.22 .071 0.67 (0.44 to 1.04)
Risk of death from any cause
Restricted vs. ad libitum-fed group
0.19 0.16 .220 0.83 (0.61 to 1.12)
Lycopene diet vs. control diet§
0.15 0.19 .410 0.86 (0.59 to 1.24)
Tomato powder diet vs. control diet
0.24 0.10 .018 0.79 (0.65 to 0.96)
Tomato powder diet vs. lycopene diet¶
0.29 0.20 .138 0.75 (0.51 to 1.10)
SE standard error; HR hazard ratio; CI confidence interval. The interactions between dietary restriction and tomato phytochemicals were not statistically
significant (Wald test, P .38 for risk of dying with prostate cancer and P .55 for risk of death from any cause) and were thus removed from the final statistical
model for both outcomes.
†Based on Wald test. Because of multiple looks (in the case of analyses of diet by type), a Bonferroni correction was used such that the cutoff for statistical
significance is P .017. All P values are two-sided.
‡Estimates from model containing factors for diet type and level of intake. Proportional hazards assumption was violated in this model, and P value should be
interpreted with caution.
§Estimates from models restricted to lycopene and control groups.
Estimates from model restricted to tomato powder diet and control groups.
¶Estimates from model restricted to tomato powder diet and lycopene diet.
1584 ARTICLES Journal of the National Cancer Institute, Vol. 95, No. 21, November 5, 2003
supplementation with pure lycopene. This observation is consistent
with epidemiologic findings (1–3) and recent results from
small clinical trials (8,9). Our results do not rule out the possibility
that lycopene is one of several phytochemicals in the
tomato that contributes to an inhibition of prostate carcinogenesis.
At the present time, many men are consuming lycopenecontaining
supplements with the hope that they may prevent
prostate cancer or enhance the treatment of their prostate cancer.
We suggest that a focus on interventions with whole tomato
products and energy balance should be a priority while clinical
studies simultaneously investigate the risks and benefits of lycopene
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Supported by Public Health Service grants KO7-CA01680 (to S. K. Clinton),
RO1–72482 (to S. K. Clinton and J. W. Erdman, Jr.), and P30CA16058 (to The
Ohio State University Comprehensive Cancer Center) from the National
Cancer Institute, National Institutes of Health, Department of Health and
J. W. Erdman, Jr., and S. K. Clinton contributed equally to this publication.
We thank Dr. Maarten C. Bosland and Dr. David L. McCormick for guidance
regarding establishing this model in our laboratory. We thank Kimberly Carter,
Dahlys Hoot, and Valerie DeGroff for technical assistance.
Manuscript received May 15, 2002; revised August 21, 2003; accepted
September 2, 2003.
1586 ARTICLES Journal of the National Cancer Institute, Vol. 95, No. 21, November 5, 2003
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