Carcinogenesis vol.27 no.10 pp.2116–2123, 2006
doi:10.1093/carcin/bgl072
Advance Access publication May 16, 2006
Indole-3-carbinol in the maternal diet provides chemoprotection for the fetus
against transplacental carcinogenesis by the polycyclic aromatic hydrocarbon
dibenzo[a,l]pyrene
Zhen Yu1,2, Brinda Mahadevan1, Christiane V.Löhr3,4,
Kay A.Fischer3,4, Mandy A.Louderback1,
Sharon K.Krueger1,2, Clifford B.Pereira4,5,
Daniel J.Albershardt1, William M.Baird1,4,
George S.Bailey1,2,4 and David E.Williams1,2,4,
1
Department of Environmental and Molecular Toxicology,
The Linus Pauling Institute, 3College of Veterinary Medicine,
4
Environmental Health Sciences Center and
5
Department of Statistics, Oregon State University, Corvallis, OR, USA
To whom correspondence should be addressed at: Department of
Environmental and Molecular Toxicology, Oregon State University,
ALS1007, Corvallis, OR 97331-7301, USA. Tel: +1 541 737 3277;
Fax: +1 541 737 7966;
Email: [email protected]
2
The fetus and neonate are sensitive targets for chemically
induced carcinogenesis. Few studies have examined the
risk/benefit of chemoprotective phytochemicals, given in
the maternal diet, against transplacental carcinogenesis.
In this study, B6129 SF1/J (AHRb-1/d) and 129Sv/ImJ
(AHRd/d) mice were cross-bred. The polycyclic aromatic
hydrocarbon, dibenzo[a,l]pyrene (DBP), was administered
to pregnant mice (15 mg/kg, gavage) on gestation day 17,
and 2000 p.p.m. indole-3-carbinol (I3C), a chemoprotective phytochemical from cruciferous vegetables, was fed to
half of the mice from gestation day 9 until weaning.
Offspring born to dams fed I3C exhibited markedly fewer
mortalities (P < 0.0001). Maternal dietary exposure to I3C
also significantly lowered lung tumor multiplicity (P ¼
0.035) in offspring surviving to 10 months of age. The I3C
chemoprotection was independent of either maternal or
fetal AHR genotype. The bioavailability of DBP to fetal
target tissue was demonstrated by assessing DNA covalent
adduction with a 33P-post-labeling assay. The bioavailability of I3C was determined by dosing a subset of pregnant
mice with [14C]-I3C. Addition of chemoprotective agents
to the maternal diet during pregnancy and nursing may
be an effective new approach in reducing the incidence of
cancers in children and young adults.
Introduction
Childhood cancers represent <1% of all cancers, yet these
12, 400 annual cases result in 2300 deaths. This represents
the greatest cause of disease-related deaths and is second
only to accidents, in US children, among all childhood
deaths. Lymphomas and leukemias are the most common
cancers in children born in the United States (1,2). The fetus
and neonate are sensitive targets for chemically induced
toxicity including carcinogenesis (reviewed in ref. 3). The
polycyclic aromatic hydrocarbons (PAHs) are environmental
Abbreviations: AHR, aryl hydrocarbon receptor; CYP, cytochrome P450;
DBP, dibenzo[a,l]pyrene; FET, Fisher’s exact test; I3C, indole-3-carbinol;
PAH, polycyclic aromatic hydrocarbon.
#
pollutants produced from the incomplete combustion of many
organic materials including cigarettes, coal, cooking oil,
wood and diesel [4,5 (http://www.atsdr.cdc.gov/toxprofiles/
tp69.html)]. Epidemiology demonstrates that maternal exposure to PAHs through smoking is a risk factor for increased
childhood cancers and for increased incidence of adult cancers
(6). Transplacental exposure to PAHs has been shown to cause
DNA damage in newborns (7,8) and is also associated with
increased cytogenic damage linked to childhood leukemia (9).
In the past 20 years, a great deal of research has
documented the potential for phytochemicals to provide
chemoprotection against cancer (10–12). However, to our
knowledge, none of the chemoprotective phytochemicals
under test have been evaluated in a transplacental model.
These phytochemicals are consumed in the diet or taken as
supplements by women of child-bearing age. The relative
risk/benefit for the fetus is unknown. Indole-3-carbinol (I3C),
a major component of cruciferous vegetables, is chemoprotective in a number of animal studies (reviewed in ref. 13), is
under evaluation for chemoprotection of women against
breast cancer (14,15) and is available to the public as a
dietary supplement. A study by Wattenberg and Loub
provided the first evidence of I3C protection against PAHinduced cancer in animal models by showing that I3C
inhibited 7,12-dimethylbenz[a]anthracene-induced mammary
tumor formation in female Sprague–Dawley rats and in
benzo[a]pyrene-induced neoplasia of the forestomach in
female ICR/Ha mice (16). Several mechanisms have been
postulated for chemoprevention by I3C (reviewed in ref. 17).
One hypothesized mechanism of action of I3C and its
acid condensation products is that they can act as
blocking agents via aryl hydrocarbon receptor (AHR)
modulation of phase I [cytochrome P450s (CYPs)] and
phase II enzymes [glutathione-S-transferases (GSTs)
and UDP-glucuronosyltransferases (UGTs)]. These phase I
and phase II enzymes play important roles in the carcinogenesis of PAHs. The action of CYPs often results in
bioactivation of PAHs to reactive intermediates that covalently bind to DNA, leading to gene mutations and cancer;
induction of CYPs that lead to detoxication metabolites by
I3C could provide protection. Likewise, AHR-dependent
induction of GSTs and UGTs usually represents detoxication
(18). In mice, the AHR gene exists as four alleles (AHRb-1,
AHRb-2, AHRb-3 and AHRd). Various strains of mice have
differential responsiveness to PAHs and other AHR ligands.
AHRb-1, AHRb-2 and AHRb-3 encode high-affinity receptors
in ‘responsive’ strains (e.g. C57BL/6J, BALB/c, A/J). The
AHRd allele encodes a low-affinity receptor in ‘nonresponsive’ strains (e.g. DBA/2J, 129/svJ). Crosses between
AHRb and AHRd strains have shown that ‘responsiveness’ is
dominant (19).
PAHs are known mouse transplacental carcinogens in
C57BL6·DBA/2 (B6D2) crosses, and tumorigenesis is
related to both maternal and fetal AHR genotype (20–22).
The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 2116
Maternal dietary I3C reduces lymphoma mortality
To evaluate the risk/benefit of maternal dietary I3C against
PAH transplacental carcinogenesis, we utilized the heterozygous B6129 SF1/J (AHRb-1/d) mouse crossed with the
129Sv/ImJ (AHRd/d) mouse. When reciprocal crosses are
made, an advantage of this model is that it provides an equal
number of AHRb-1/d and AHRd/d offspring within two separate
maternal AHR environments. Thus, we can directly examine
the effect of both maternal and fetal AHR genotypes on I3C
chemoprotection.
Dibenzo[a,l]pyrene (DBP) is the most potent PAH
carcinogen in rodent models and is a multiorgan carcinogen
in the mouse, producing cancers of the ovary, skin, uterus
and liver in addition to production of lymphomas (23–29).
The carcinogenesis of DBP has also been linked to CYP
enzymes regulated in part through the AHR (28,30–34). We
recently demonstrated, for the first time, that DBP is a
transplacental carcinogen producing mortality as early as 3
months of age owing to an aggressive T-cell lymphoblastic
lymphoma with lung and liver tumors also seen in
survivors at 10 months (35). Maternal, as well as fetal,
AHR genotype influenced the risk for DBP-dependent
mortality (35). Offspring born to mothers with the AHRd/d
(non-responsive) genotype exhibited higher DBP-dependent
lymphoma, regardless of offspring genotype. If the mother
was AHRb-1/d, offspring that were also AHRb-1/d exhibited
higher mortality than their AHRd/d siblings (35).
In this study, we demonstrate that maternal dietary I3C
significantly reduces lymphoma mortality (and also reduces
the number of lung tumors in survivors) in offspring caused
by transplacental DBP and that the mechanism(s) of I3C
chemoprotection in this model is not associated with
AHR-dependent signaling.
Materials and methods
Chemicals and diets
Indole-3-carbinol (I3C) was purchased from Sigma (St Louis, MO). [14C]I3C (specific activity of 10 mCi/mmole, labeled at the carbinol carbon
(14CH2OH)) was custom-synthesized by American Radiolabeled Chemicals,
(St Louis, MO). The radiochemical and chemical purity was 99% based on
HPLC and TLC analysis. DBP was obtained from the NCI chemical
carcinogen repository, Midwest Research Institute (Kansas City, MO) at a
purity determined to be >98% by HPLC analysis. AIN93G and AIN93M diet
was obtained from Dyets (Bethlehem, PA).
Treatment of mice
Eight-week-old B6129SF1/J and 129S1/SvImJ mice were purchased from
The Jackson Laboratory (Bar Harbor, Maine) and housed in the Laboratory
Animal Resources Center at Oregon State University under controlled
conditions of 20 ± 1 C and 50 ± 10% humidity and a light/dark cycle of 12 h
in micro-isolator cages (Super Mouse 750 Micro-Isolator, Life Products,
Seaford DE) with CareFRESH bedding. After 1 week, reciprocal crosses
between B6129SF1/J and 129S1/SvImJ mice were carried out to produce
fetuses gestating in both environments. From the 9th day of gestation (the
day the vaginal plug appeared was marked as gestation day 0), pregnant mice
were fed with 2000 p.p.m. I3C diet or control diet (AIN93G) and gavaged
with either vehicle (corn oil, 5 ml/kg body wt) or DBP (15 mg/kg body wt in
corn oil) on gestation day 17. The preparation, storage and analysis of I3Ccontaining AIN synthetic powdered diet (Dytes, AIN 93G recommended for
growth, pregnancy and lactational phases for rodents and AIN 93M for
maintenance of adult rodents) was described previously (36). Pregnant mice
were continued on I3C diet or control diet till the end of nursing (21 days
post-parturition). The offspring were weaned onto pelleted AIN93G diet for
the first 3 months, and then continued with AIN93M diet ad lib until
euthanized. The mice were housed in micro-isolator cages by sex and litter
(up to five per cage). Sentinels were housed in the same colony and used to
test for viral or bacterial pathogens and parasites; these tests (MU Research
Animal Diagnostic Laboratory, Columbia, MO) were negative throughout the
study. Upon signs of morbidity, pain or distress the mice were euthanized
with an overdose of CO2 and necropsied. Surviving mice were euthanized at
10 months of age and necropsied. For the study on bioavailability of [14C]I3C to the fetus, on day 17 of gestation pregnant mice were gavaged with a
dose of 1 mCi (3 mmol)/kg body wt (2.5% DMSO in corn oil) (36). The
mice were housed individually in metabolism cages. At the end of 8 h, fetal
tissues were collected and digested with 1 ml of BTS-450 tissue solubilizer.
All samples were digested overnight at room temperature and then heated at
55 C for one additional hour to complete the digestion. A few drops of 30%
H2O2 were added for decolorization. Liquid scintillation cocktail (3a70B,
Research Products, Intl., Mt Prospect, IL) was added and the samples stored
in the dark for 24 h before quantifying with a liquid scintillation counter. All
procedures for treatment, housing and euthanasia of the mice were approved
by the Oregon State University Institutional Animal Care and Use
Committee.
Histopathology
The following tissues were collected at necropsy: heart, thymus, lung, spleen,
liver, kidney, abnormal lymph node, testes or ovaries, colon and skin.
Tissues were fixed in 10% formalin, stained with H&E and analyzed by light
microscopy. The lymphomas were diagnosed as a T-cell lymphoma, lung
tumors as hyperplasia, adenomas, adenomas with progression and carcinomas (35). In the liver, lesions identified as tumors by gross examination were
histologically determined to be foci, areas of hyperplasia and hypertrophy
and occasionally hepatomas (35).
Genotyping for AHRb-1 and AHRd alleles
An ear-punch was collected at necropsy for genotyping, as already described
(35). Briefly, tissue was lysed in DirectPCR Lysis Reagent (Viagen Biotech,
Los Angeles, CA) containing proteinase K, and the resulting lysate was used
directly in a PCR with allele-specific primers to permit one-tube genotyping
of the AHR alleles. The common forward primer was 50 GAAGCATGCAGAACGAGGAG. Allele-specific reverse primers were
AHRb-1, 50 -caagcttataTGCTGGCAAGCCGAGTTCAG; and AHRd, 50 TGCTGGCAAGCGGAGTTCAT. Lowercase nucleotides are non-mouse
sequences added to allow allele-specific size discrimination of products,
whereas underlined nucleotides distinguish the AHR alleles, on the basis of
fixed strain differences at AA589 and AA591. PCR products were visualized
after separation on Novex 8% TBE gels (Invitrogen Life Technologies,
Carlsbad, CA). AHRb-1/d heterozygotes yielded two PCR products of 158 and
148 bp, respectively, whereas AHRd/d homozygotes yielded a single product
of 148 bp.
Quantification of DBP–DNA adducts in maternal and fetal lung by 33P-postlabeling
Lung tissue from maternal and fetal mice exposed to DBP was homogenized
in a glass homogenizer with 2 ml EDTA–SDS buffer [10 mM Tris, 1 mM
Na2EDTA, 1% SDS (w/v), pH 8.0]. Homogenates were treated with DNasefree RNase (1 U at 50 U/ml, Boehringer–Mannheim, Indianapolis, IN) and
RNase T1 (20 U at 1000 U/ml, Boehringer–Mannheim) at 37 C for 1 h,
followed by treatment with proteinase K (2 mg at 20 mg/ml, Sigma, St
Louis, MO) at 37 C for 2 h. The DNA was extracted with an equal volume
of Tris-equilibrated phenol (Boehringer–Mannheim) followed by extraction
with 1 : 1 volume of Tris-equilibrated phenol and chloroform : isoamyl
alcohol (24 : 1) and finally with an equal volume of chloroform : isoamyl
alcohol (24 : 1). The aqueous layer was treated with 500 mM NaCl and twice
the volume of cold 100% ethanol to precipitate the DNA, which was stored
overnight at 20 C. The DNA was washed in cold 70% ethanol before being
dissolved in double-distilled water. The concentration of the DNA was
determined by UV absorbance at 260 nm (37).
Post-labeling was carried out as described previously (37). Briefly, 10 mg
DNA isolated from lung tissue was digested with nuclease P1 and prostatic
acid phosphatase, post-labeled with [g-33P]ATP (3000 Ci/mmol), reduced to
adducted mononucleotides with snake venom phosphodiesterase I, and prepurified with a Sep-Pak C18 cartridge (Waters, Milford, MA). Subsequent
separation by analytical HPLC (Varian Systems, Walnut Creek, CA) was
carried out using a C18 reverse-phase column (5 mm Ultrasphere ODS, 4.6 ·
250 mm). DBP–DNA adducts were resolved by elution at 1 ml/min with
0.1 M ammonium phosphate, pH 5.5 (solvent A), and 90 : 10 methanol :
acetonitrile (solvent B). The elution gradient was 20–44% solvent B over 20
min, 44–60% solvent B over 40 min, 60–80% solvent B over 15 min and 80–
20% solvent B over 1 min. The radiolabeled nucleotides were detected by an
online radioisotope flow-detector (Packard Instruments, Downers Grove, IL),
and the level of DNA binding was calculated on the basis of the labeling
efficiency of a [3H]B[a]P-7,8-dihydrodiol 9,10-epoxide standard (38). At
least two independent sets of the post-labeling reaction were carried out for
every sample treated, to determine the total PAH–DNA adduct levels.
2117
Z.Yu et al.
Results
there was no significant DBP-dependent reduction in litter
size or birth weight (data not shown). The B6129F1 mothers
had significantly larger litters (35). Beginning at 3 months
of age, offspring exposed to DBP in utero had difficulty
breathing and exhibited anemia and hypoxia that resulted in
morbidity requiring euthanasia. Most mortality occurred
between 3 and 6 months of age. Gross necropsy revealed
large thoracic masses and enlarged spleens, livers and lymph
nodes. There was no sex difference with respect to DBPdependent mortality. The cause of death was an aggressive
T-cell lymphoma that involved numerous organs (35).
Maternal dietary exposure to I3C did not change the litter
size or birth weight in control or DBP-treated groups, with
the exception that DBP-treated AHRb-1/d dams fed I3C diet
had slightly larger litters compared with the control diet
group (8.7 ± 0.3 and 7.1 ± 0.6, respectively; Table I). There
was no significant difference in the gender ratio between
groups (P > 0.5). The genotype ratio of AHRb-1/d/AHRd/d
varied from 0.56 to 1.50 (Table I), but did not appear to be
related to either DBP or I3C treatment (Table I). I3C in the
maternal diet enhanced the survival of all offspring (Table I
and Figure 1). A single death, out of 98 mice, was observed
in offspring born to dams dosed with vehicle and fed I3C,
compared with 9 deaths, out of 100 mice, in offspring born to
dams dosed with vehicle and fed control diet.
Maternal dietary exposure to I3C increased the survival of
offspring exposed to DBP in utero. In offspring born to
AHRb-1/d dams, I3C enhanced survival from 31 to 64% (P <
0.0001, Cox regression with litters as clusters). Similarly, in
offspring born to AHRd/d dams, I3C enhanced survival from
11 to 41% (P < 0.0001, Cox regression with litters as
clusters). As we reported previously (35), maternal as well as
fetal AHR genotype influenced DBP-dependent mortality.
Offspring born to AHRd/d mothers had greater susceptibility
to lymphoma, irrespective of offspring genotype. If the
mother was AHRb-1/d, an AHRb-1/d genotype increased
mortality 2-fold. However, the chemoprotective effect of
maternal dietary I3C was not related to maternal or fetal
genotype (note similarity of arrow lengths in Figure 2 and
P > 0.5 for all interactions with the I3C factor).
Maternal dietary I3C protects against mortality in offspring
from transplacental DBP-induced lymphoma
The experimental design of the tumor study resulted in 8
groups of offspring and a total of 654 mice (Table I). As we
presented in a previous paper (35), treatment of pregnant
mice with a single 15 mg/kg dose of DBP by gavage on day
17 of gestation did not elicit acute maternal or fetal toxicities;
Maternal dietary I3C protects against DBP-dependent
transplacental lung cancer in mice surviving to 10 months
of age
Lung (Table II) and liver tumors in mice surviving to 10
months of age were examined by histopathology. The
spontaneous lung tumor incidence in control groups was 4–
10%, whereas all the offspring born to DBP-treated dams had
Statistical analysis
For comparing diet treatments, the experimental unit is the pregnant female
and the litters represent clusters for analyzing measurements made on the
offspring. Cox (proportional hazard) regression was used for survival
modeling with litters as clusters (marginal model) to get robust standard
errors and z-tests for comparing groups [coxph function in S-plus version 6.2
as described by Therneau and Grambsch (39)]. To examine the consistency
of the I3C effect on survival across genotypes, the significance of
interactions between I3C and genotypes (two- and three-way interactions)
was assessed in a three-factor proportional hazard model [(i) I3C, (ii) dam
genotypes and (iii) offspring genotype]. For analysis of gender and genotype
ratios of offspring, the proportion in one of the two categories (males and
AHRb-1/d, respectively) was analyzed. Each treatment group was analyzed for
evidence of litter effects using Fisher’s exact test (FET) for r · c tables. For
gender ratio, no evidence of litter effects was found (P > 0.09 for all groups)
and data were subsequently pooled across litters for comparison of groups at
the offspring level by FET. For genotype ratio, there was evidence of litter
effect within the last two groups (AHRd/d dams treated with DBP with or
without I3C; P ¼ 0.04 and 0.024, respectively), but not in any other groups
(P > 0.16 in all cases, FET). Owing to the evidence of litter effects, litter
level comparisons were conducted using FET to compare groups with the
litter (rather than the offspring) as the basic unit. The response analyzed was
the proportion of litters with more AHRd/d offspring surviving to weaning.
Lung tumor multiplicity in offspring was modeled on the square root scale
to obtain reasonably symmetrical and homogeneous residuals from the mixed
procedure in SAS (40). When there was evidence of litter effects
(comparison between with and without I3C for AHRb-1/d dams receiving
DBP), a linear mixed model was used to compare treatment groups with
random litter effects allowed to differ between treatment groups (because
only one group exhibited strong litter effects). When there was no evidence
of litter effects (comparison between with and without I3C for AHRd/d dams
receiving DBP), the linear model reduced to a simple t-test.
Lung DBP–DNA adduct formation at 24, 48, 96 and 144 h post-dosing for
three litters per time point was analyzed using linear mixed models (mixed
procedure in SAS). For comparing fetal genotypes there were 11 litters with
both genotypes present. The 22 genotype means over 2–4 replicate reactions
were log-transformed and analyzed using a model with litters (within time
point) as a random factor and with fixed factors for time, genotype and their
interaction. For comparing mothers and offspring all 12 litters could be used
and the 24 means over 2–4 replicate reactions were log-transformed and
analyzed with the same model except that the genotype factor was replaced
by a mother-versus-offspring factor. (In all models main effects and
interactions involving time were not significant and the general conclusions
regarding all factors would not change if the data were analyzed without log
transformation.)
Table I. Effect of treatment and genotype of dams on litter size, genotype, gender ratio and survival of offspring
Genotype and treatment
of dam/no. of offspringa
Litter size
Genotype ratio
(AHRb-1/d : AHRd/d)
Gender ratio
(male : female)
Percent survival
at 10 months (n)
AHRb-1/d, DBP-I3C/n ¼ 55
AHRb-1/d, DBP+I3C/n ¼ 51
AHRb-1/d, +DBP-I3C/n ¼ 121
AHRb-1/d, +DBP+I3C/n ¼ 130
AHRd/d, DBP-I3C/n ¼ 53
AHRd/d, DBP+I3C/n ¼ 47
AHRd/d, +DBP-I3C/n ¼ 102
AHRd/d, +DBP+I3C/n ¼ 95
7.8
7.3
7.1
8.7
4.8
4.7
4.1
5.0
1.50
1.12
1.35
1.01
1.08
1.35
0.71
0.56
1.20
0.82
1.09
1.06
1.30
1.14
0.82
0.86
94.5
100
31.4
63.8
88.7
97.9
10.8
41.0
±
±
±
±
±
±
±
±
0.5
0.5
0.6
0.3
0.6
0.7
0.4
0.3
(32 : 21)
(27 : 24)
(65 : 48)
(64 : 63)
(27 : 25)
(27 : 20)
(39 : 55)
(31 : 56)
(30 : 25)
(23 : 28)
(63 : 58)
(67 : 63)
(30 : 23)
(53 : 46)
(46 : 56)
(44 : 51)
(52/55)
(51/51)
(38/121)
(83/130)
(47/53)
(46/47)
(11/102)
(39/95)
a
AHRb-1/d, DBP-I3C represented offspring born to dams of AHRb-1/d, without DBP initiation on gestation day 17, without I3C in maternal diet; similar:
AHRd/d, +DBP+I3C represented offspring born to dams of AHRd/d, with DBP initiation on gestation day 17, with I3C in maternal diet.
2118
Maternal dietary I3C reduces lymphoma mortality
100
+I3C
- I3C
Corn Oil
% survival
80
60
+I3C
40
+I3C
DBP
-I3C
20
Table II. Lung tumors in offspring surviving to 10 months of age
Group
Incidence
Multiplicitya
AHRb-1/d dam DBP I3C
AHRb-1/d dam DBP +I3C
AHRb-1/d dam +DBP I3C
AHRb-1/d dam +DBP +I3C
AHRd/d dam DBP I3C
AHRd/d dam DBP +I3C
AHRd/d dam +DBP I3C
AHRd/d dam +DBP +I3C
5/50
6/51
38/38
82/82
3/46
2/46
11/11
39/39
1.2
1.3
14.0
8.9
1.3
1.0
13.5
11.9
(10%)
(12%)
(100%)
(100%)
(7%)
(4%)
(100%)
(100%)
±
±
±
±
±
±
±
±
0.2
0.2
1.5b
0.6b
0.3
0.0
1.6
1.0
a
Data are present as mean ± SE for multiplicity (number of tumors per
mouse).
I3C significantly reduced multiplicity (P ¼ 0.035).
-I3C
b
0
10
20
30
Age (weeks)
40
Fig. 1. Effect of maternal dietary treatment, carcinogen treatment and AHR
genotype on survival of offspring. % Survival ¼ live offspring/total offspring
of this group · 100%. Solid symbols represent offspring born to AHRb-1/d
dams; open symbols represent offspring born to AHRd/d dams.
A
100
% survival
80
d/d
+ I3C, AHR
60
b-1/d
+ I3C, AHR
d/d
- I3C, AHR
40
b-1/d
- I3C, AHR
20
0
10
20
30
Age (weeks)
40
B
100
% survival
80
60
d/d
+ I3C, AHR
b-1/d
+ I3C, AHR
40
lung tumors at 10 months of age. The tumor multiplicity
(tumors per tumor-bearing animal) in control groups was
1.0–1.3, whereas it ranged from 8.9 to 14.0 in DBP-treated
groups (Table II and ref. 35). I3C in the maternal diet
significantly reduced the lung tumor multiplicity in offspring
from AHRb-1/d dams from 14.0 to 8.9 (P ¼ 0.035 mixed
model described in Materials and methods). There was a
reduction also in lung tumor multiplicity in offspring from
AHRd/d dams (13.5 ± 1.6 to 11.9 ± 1.0), but this did not reach
statistical significance (P ¼ 0.34, t-test). Lung tumor
multiplicity was not statistically related with AHR genotype
(data not shown).
Maternal exposure to I3C had no significant effects on
liver tumor incidence or multiplicity in mice surviving to
10 months of age (data not shown). No liver tumors were
observed at 10 months of age in offspring born to dams
treated with vehicle. Exposure to DBP resulted in high
incidences of liver tumors in male offspring born to mothers
of both genotypes. The incidence was 70% (14 out of 20 and
28 out of 40) in offspring from AHRb-1/d dams fed control or
I3C diet, respectively. In offspring born to dams of the
AHRd/d genotype, treated with DBP, the incidence was 67%
(4 out of 6) and 39% (7 out of 19) from dams receiving
control and I3C diet, respectively. However, as noted earlier,
there were few survivors in these groups at 10 months
compared with the other groups and some evidence of litter
effects not observed in the other groups (see Materials and
methods). As previously seen in this (35) and most strains of
mice, liver tumors were much more prevalent in males than
females.
33
20
0
d/d
- I3C, AHR
b-1/d
- I3C, AHR
10
20
30
Age (weeks)
40
Fig. 2. Effect of maternal and offspring AHR genotype on survival of
offspring born to DBP-treated dams. % Survival ¼ live offspring at 10
months of age/total offspring of the same genotype of this group · 100%.
(A) Offspring born to AHRb-1/d dams and (B) offspring born to AHRd/d dams.
Open triangle, AHRd/d offspring born to dams fed I3C diet. Closed triangle,
AHRb-1/d offspring born to dams fed I3C diet. Open circle, AHRd/d offspring
born to dams fed control diet. Closed circle, AHRb-1/d offspring born to dams
fed control diet. The arrows indicate the degree of I3C survival enhancement
for each offspring genotype; note that the degree of I3C chemoprotection is
similar, thus supporting our conclusion that I3C chemoprotection against
DBP-induced lymphoma-dependent mortality is not dependent upon the AHR
genotype.
P-Post-labeling analysis of DBP–DNA adducts in maternal
and fetal lung
We investigated the potential of the carcinogenic PAH, DBP,
to cross the placenta in mice to form genotoxic DNA adducts
in fetal lung tissue. Representative HPLC elution profiles of
both the maternal and fetal lung DBP–DNA adducts 2 and
6 days after exposure are shown in Figure 3. The fetal lung
exhibited lower adduct levels than maternal lung, but the
profile was similar (Figure 3). The peaks labeled 1, 2a, 2b
and 4 are derived from ()-anti-DBP-(11R,12S)-dihydrodiol
(13S,14R)-epoxide and peak 6 is from (+)-syn-DBP(11S,12R)-dihydrodiol (13S,14R)-epoxide (38). This pattern
is almost identical to that seen upon incubation of DBP with
expressed mouse Cyp1b1 (31). A time-course study showed
that lung DBP–DNA adducts peaked at 2 days in both
maternal and fetal lung after maternal dosing. An increase in
2119
Z.Yu et al.
4
2d Maternal
6d Maternal
2b
2a
Radioactivity
1
6
2d Fetal
6d Fetal
Retention Time
Fig. 3. Representative HPLC elution profiles of 33P post-labeled DBP–DNA adducts in maternal and fetal lung tissues 2 and 6 days after exposure to DBP.
On the basis of comparison of elution times to those reported by Ralston et al. (38) and the use of the same diol epoxide adduct markers, the DBPDE peak
labeled 1 was formed by the reaction of the (+)-syn-DBPDE with dA. Peaks that eluted with retention times of 60–80 min are ()-anti-DB[a,l]PDE adducts
(peaks 1, 2a, 2b and 4) and the single product that eluted around 90 min (peak 6) is a (+)-syn-DB[a,l]PDE adduct (31). HPLC conditions are described in
Materials and methods.
total DBP–DNA adducts was observed in maternal lung
tissue from 6 to 48 h, with an average of 24.5 pmol/mg of
DNA at 48 h. DBP–DNA adduct levels decreased in maternal
tissues after 48 h. Over the period of 24–144 h, there was
strong evidence of higher adduction (on average 10 pmol/mg
or greater) in maternal lung compared with fetal lung (P ¼
0.008 with n ¼ 3 litters per each of four time points). There
was some evidence of a difference between the fetal AHR
genotypes in total DBP–DNA adduction in the fetal/pup lung
(P ¼ 0.045, with adduct levels higher in the AHRb-1/d
offspring for 9 of the 11 litters having both types of
offspring). Over 24–144 h, lung DBP–DNA adduct formation
was, on average, 1.6 times higher for AHRb-1/d fetal/pup
than with the AHRd/d fetal/pups (data not shown). Preliminary results with fetal thymus indicate the presence of DBP–
DNA adducts derived from the ()-anti-DBPDE, but the
levels were too low to accurately quantify (adduct profile
data not shown here).
Bioavailability of [14C]-I3C to fetal tissues
Previous studies from our laboratory provided evidence that
I3C was bioavailable transplacentally in the rat (41). In the
present study, 8 h following administration of [14C]-I3C to
pregnant mice on day 17 of gestation, radiolabeled I3C was
detected in fetal liver, stomach, kidney, intestine and lung
(Figure 4). The 8 h time point was chosen on the basis of
previous studies in rat (36) and we have, as yet, not
performed time-course studies in the mouse. Although the
mass balance indicates that a small percent of the total dose
reaches the fetus 8 h following maternal dosing, on a
2120
dosimetry level, this represents 100–300 nmol/g tissue or
100–300 mM concentrations.
Discussion
I3C is chemoprotective against cancer in multiple animal
models (reviewed in ref. 13). This study demonstrates that
I3C also protects against DBP-dependent transplacental
carcinogenesis. One proposed mechanism for the chemoprotective action of I3C is as a blocking agent through activation
of AHR-dependent pathways. I3C was fed to pregnant mice
beginning at gestation day 9 to provide sufficient time for
AHR-dependent enzyme induction before DBP administration, but also to avoid any possible teratogenic effect of I3C
in the first trimester. Interestingly, I3C reduced the noncancer-related deaths in offspring born to mothers not treated
with DBP. A study by Auborn et al. (42) showed that diet
supplemented with 200 p.p.m. I3C, beginning at 1 month of
age, can prolong the lifespan of autoimmune-prone (NZB/
NZW) F1 mice. Our study also showed that prenatal and
lactational exposure to I3C may have some benefits to the
healthy growth of offspring, and may even prolong lifespan.
A significant portion of the total lifetime exposure to PAHs
and other AHR agonists, including PCBs and dioxins, occurs
transplacentally and through breast feeding (43). DBP, like
other PAHs, is highly lipophilic and probably excreted to
some degree through the breast milk to the newborn. As we
did not utilize a cross-fostering design, some of the DBP
exposure may have been lactational. For that reason, we
Maternal dietary I3C reduces lymphoma mortality
500
n mol I3C eq/g tissue
400
300
200
100
0
liver
stomach
kidney
intestine
lung
Fig. 4. Bioavailability of [14C]-I3C to fetal tissues following maternal
dosing. Data shown are the mean from a single pool of fetuses (liver,
stomach, intestine, n ¼ 8; kidney, n ¼ 7; lung, n ¼ 2).
continued maternal I3C dietary exposure until the offspring
were weaned.
Our experimental design tested the hypothesis that I3C
chemoprevention was mediated through AHR-dependent
blocking. The induction of AHR-regulated phase 1 and
phase 2 enzymes by I3C has been reported (44–46). Our
laboratory has shown that feeding pregnant rats I3C induces
CYP1A1 and CYP1B1 in livers of newborns (41). In that
previous study in the rat, we provided evidence for
transplacental bioavailability of at least one of the I3C acid
condensation products, 2,3-bis-[3-indolylmethyl]indole (LT1)
(41). In the present study in mice, we utilized [14C]-I3C to
determine bioavailability of I3C, and any acid condensation
product, to fetal tissues. We did not seek to identify the
individual I3C acid condensation products in fetal target
tissues. The 14C label was only available on the carbinol
(14CH2OH), which means that loss of at least one 14C during
most condensation reactions would make exact quantification
of each product difficult. There were no effects of maternal
or fetal genotype on the transplacental bioavailability of
[14C]-I3C.
The molecular dosimetry of DBP in maternal and fetal
target organs was determined by 33P-DNA post-labeling
assays. The major adducts in DNA, isolated from maternal
and fetal lung 48 h post-treatment with DBP, were derived
from ()-anti-DBPDE. DBP adduction was higher in lung
DNA from fetuses genotyped as AHRb-1/d than their AHRd/d
siblings. These results provide direct evidence of the
genotoxicity of DBP to the fetus following maternal dosing
and indirectly support bioactivation by CYPs in fetal target
tissues, possibly via Cyp1b1.
Our results suggest that the chemoprotection observed is
not dependent upon an AHR blocking mechanism. One
caution to be raised in this regard is that ‘responsiveness’ is a
relative term for the AHR alleles. AHR-dependent signaling
can be manifest under conditions of high receptor concentrations, high ligand concentrations or with ligands exhibiting
high affinity. The use of an AHR null model was initially
attempted, but these mice did not breed robustly and exhibit
an unusual placental phenotype that would have compromised this study (data not shown).
I3C and its acid condensation products exhibit other
potential chemoprotective mechanisms of action. For
example, I3C acts through several transcription factors such
as estrogen receptor, SP1, and NFkB (reviewed in refs
17,47). I3C and its acid condensation products induce a G1
cell cycle arrest and specifically downregulate expression
of CDK6 (48–50), increase apoptosis (51,52), inhibit
P-glycoprotein-dependent multidrug resistance (53) and
inhibit invasion and migration of tumor cells (54). Sarkar
and Li (55) recently found that I3C functions as an inhibitor
of Akt and NFkB, which play important roles in cell survival
and are potential targets in cancer therapy. By in utero and
lactational exposure to I3C, genes related to cell cycle or
apoptosis may change in early development and affect
responsiveness to carcinogenesis not just in juveniles but
also in later life.
In conclusion, in a mouse model of PAH-dependent
transplacental carcinogenesis, maternal diets containing I3C,
administered in the second and third trimester and through
lactation, significantly reduced lymphoma mortality in
offspring 3–6 months of age and lung tumor multiplicity in
mice surviving to 10 months of age. The degree of
chemoprotection of the fetus by maternal dietary I3C was
independent of the AHR genotype. The addition of chemoprotective agents to the maternal diet during pregnancy and
nursing may be an effective strategy in reducing the
incidence of cancers in children and perhaps in adults
as well.
Acknowledgements
The authors wish to thank Marilyn Henderson, Lisbeth Siddens and David
Castro for their technical help. In addition, we acknowledge the excellent
animal care provided by Ms Mandy Louderback. We also acknowledge
support by PHS NIH grants CA90890, ES03850 and ES00210.
Conflict of Interest Statement: None declared.
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Received November 28, 2005; revised April 25, 2006; accepted May 5, 2006
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