Carcinogenesis vol.17 no.9 pp.2061-2067, 19%
Inhibitory effects of 6-phenylhexyl isothiocyanate on 4(methylnitrosamino)-l-(3-pyridyl)-l-butanone metabolic activation
and lung tumorigenesis in rats
Stephen S.Hecht1, Neil Trushin, Jeffrey Rigotty,
Steven G.Carmella, Anna Borukhova, Shobha Akerkar,
Dhimant Desai, Shantu Amin and Abraham Rivenson
American Health Foundation, 1 Dana Road, Valhalla, NY 10595, USA
'To whom correspondence should be addressed
This study examined the effects of 6-phenylhexyl isothiocyanate (PHl'l'C) on lung tumorigenesis in F344 rats
induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK). Two biomarkers
of NNK metabolism, 4-hydroxy-l-(3-pyridyl)-l-butanone
(HPB)-releasing hemoglobin adducts and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanol (NNAL) and its glucuronide
(NNAL-Gluc) in urine, were also quantified during the
course of the tumor induction experiment. Rats were
divided into groups as follows: (1) NNK, 2 p.p.m. in
drinking water, 60 rats; (2) NNK, 2 p.p.m. in drinking
water and PHITC, 1 \tmoVg NIH-07 diet, 60 rats; (3)
PHTTC, 1 umol/g NIH-07 diet, 20 rats; (4) control, 20 rats.
PHITC was added to the diet for 1 week prior to and
during 111 weeks of NNK treatment. There were no effects
of PHITC on body weight, mortality, blood chemistry or
hematology. Seventy percent of the rats treated with NNK
had adenoma or adenocarcinoma of the lung. In the rats
treated with NNK plus PHITC, the total percent incidence
of lung tumors was 26% (P < 0.01 compared with NNK).
PHITC had no effect on the total incidence of exocrine
pancreatic tumors induced by NNK. The rats treated with
PHITC and NNK had significantly lower levels of HPBreleasing hemoglobin adducts throughout the course of the
bioassay than did those treated with NNK alone and
significantly higher levels of NNAL plus NNAL-Gluc
excreted in urine at two time points during the bioassay.
These results demonstrate that near lifetime administration
of PHITC to rats strongly inhibits the metabolic activation
and lung tumorigenicity of NNK.
Introduction
The tobacco-specific lung carcinogen, 4-(methylnitrosamino)l-(3-pyridyl)-l-butanone (NNK*) occurs in significant quantities in tobacco products and is believed to be one of the
principal agents which causes lung cancer in smokers (1-3).
Chemoprevention is one approach to potentially prevent or
delay lung cancer in addicted smokers who have failed smoking
cessation (4). Isothiocyanates (R-N=C = S) are the most potent
known chemopreventive agents against mouse lung tumorigenesis induced by NNK (4,5). Inhibitory potency increases with
lipophilicity of the isothiocyanate and decreases with increasing
•Abbreviations: NNK, 4-{methy lnitrosamino)-1 -(3-pyridyl)-1 -butanone;
PHITC, 6-phenylhexyl isothiocyanate; PEITC, phenethyl isothiocyanate;
NNAL,
4-(methylnitrosamino)-l-(3-pyridyl)-1-butanol;
NNAL-Gluc,
[4-<methylnitrosamino)-1 -<3-pyridyl)but-1 -yl]-j)-0-D-glucosiduronic
acid;
HPB, 4-hydroxy-1 -(3-pyridyl)-1 -butanone.
© Oxford University Press
reactivity toward glutathione (6). For example, 6-phenylhexyl
isothiocyanate [C 6 H 5 (CH 2 ) 6 N=C=S; PHITC] is 25-50 times
more potent as an inhibitor of NNK-induced mouse lung
tumorigenesis than is phenethyl isothiocyanate (PEITC)
(6—8). Indeed, 50% inhibition of lung tumor multiplicity in
mice treated with a single dose of 10 nmol NNK was achieved
with a single dose of only 0.1 umol PHITC (6). Other
lipophilic isothiocyanates which are potent inhibitors of lung
tumorigenesis by NNK include 10-phenyldecyl isothiocyanate,
dodecyl isothiocyanate and 1,2-diphenylethyl isothiocyanate
(6). Although the chemopreventive activities of a variety of
isothiocyanates against lung tumorigenesis have been extensively studied in mice treated with NNK, limited data are
available in the rat. In the present study, we examined the
effects of chronic administration of dietary PHITC on lung
tumorigenesis in F344 rats treated with NNK in the drinking
water.
Previous studies have shown that isothiocyanates inhibit
NNK-induced tumorigenicity by inhibiting its metabolic activation (9-14). An overview of NNK metabolism is presented in
Figure 1. In laboratory animals and humans, NNK is extensively
converted by carbonyl reduction to 4-(methylnitrosamino)-1-(3pyridyl)-1-butanol (NNAL) (15-18). NNAL, like NNK, is a
potent pulmonary carcinogen in rats and mice (16,19). NNAL
undergoes glucuronidation to produce [4-(methylnitrosamino)l-(3-pyridyl)but-l-yl]-p-0-D-glucosiduronic acid (NNALGluc), which is one of the urinary metabolites of NNK
(17,18,20). Levels of NNAL and NNAL-Gluc can be quantified
in human urine as biomarkers of NNK uptake (17,18). The
metabolic activation of NNK proceeds by a-hydroxylation via
intermediates 1 and 2, which ultimately give rise to methanediazohydroxide (4) and 4-(3-pyridyl)-4-oxobutanediazohydroxide
(5). These electrophiles methylate and pyridyloxobutylate cellular macromolecules, including DNA and hemoglobin (3,21,22).
The DNA adducts formed by methylation and pyridyloxobutylation are critical in the initiation of lung tumorigenesis by NNK
(23-26). A biomarker for the pyridyloxobutylation pathway is
formation of hemoglobin adducts, which release 4-hydroxy-1(3-pyridyl)-1 -butanone (HPB) (6) upon base hydrolysis (27).
PHITC inhibits the cytochrome P450-mediated metabolic
activation of NNK in mouse and rat tissue microsomes (12).
When mice or rats were pretreated with PHITC, substantial
inhibition of the metabolic activation pathways illustrated in
Figure 1 was observed. In mouse lung, PHITC is a competitive
inhibitor of a-hydroxylation of NNK, with K{ values in the
range 10-20 nM. Similar activity has been observed in rat
tissue microsomes. In mice treated with PHITC and NNK,
levels of pulmonary C^-methylguanine in DNA were decreased
compared with those in mice treated with NNK alone (7). In
the present study, two biomarkers of NNK metabolism, HPBreleasing hemoglobin adducts as well as NNAL and NNALGluc in urine, were assessed during the bioassay of PHITC
and NNK in order to provide further insight into the mechanisms which might be involved in modification of NNK-induced
tumorigenesis by PHTTC.
2061
S.S.Hecht et al
Materials and methods
Chemicals
PHITC was synthesized (7). Its purity was >99%, as established by HPLC,
GC-MS, IR and 'H-NMR. NNK was synthesized and its purity was assessed
by HPLC as >99%.
Bioassay
One hundred and seventy two male F-344 rats, aged 6 weeks, were purchased
from Charles River Laboratories (Kingston, NY). They were maintained on
NIH-07 diet on arrival. They were randomly divided into five groups as
follows: (1) NNK, 60 rats; (2) NNK plus PHTTC, 60 rats; (3) PHITC, 20 rats;
(4) control, 20 rats; (5) sentinels, 12 rats. Groups 1 and 4 were also the
controls for a study of the effects of PEITC on NNK biomarkers and
tumorigenesis. The results of that study have been submitted in a separate
paper (28), but the relevant biomarker and tumor data from groups 1 and 4
are also included in the present report to allow comparison with the PHITCtreated groups.
Initially and at 6 month time points during the bioassay, sentinels were
examined for ectoparasites, endoparasites, dermatophytes, blood parasites,
respiratory bacteria and enteric bacteria. Routine serology tests were also
performed. There were no remarkable findings. The rats were housed in
groups of three in solid-bottomed polycarbonate cages with hardwood bedding.
Other conditions were standard as previously described (9). One week after
arrival, groups 2 and 3 were placed on NIH-07 diet to which PHITC [1 umol
(219 Hg)/g diet] had been added, while groups 1 and 4 were maintained on
NIH-07 diet for the entire experiment. The diet containing PHTTC was
prepared weekly and stored at 4°C. Analysis of the diet by HPLC demonstrated
that PHTTC was stable in the diet for at least 8 days under these conditions.
Two weeks after arrival of the rats, NNK was added to the drinking water of
groups 1 and 2 at 2 p.p.m. (9.66 \xM). The drinking water solutions were
prepared weekly and were stored at 4°C, conditions under which NNK is
known to be stable. Drinking water was added to amber bottles twice weekly
and dispensed to the rats. Food and water consumption were measured weekly.
At various intervals during the experiment (see Figure 2), three rats from
groups 1 and 2 were selected at random and 0.5—1 ml blood was withdrawn
from the orbital sinus of each rat under halothane anesthesia. The blood was
collected in tubes containing EDTA. The rats were returned to their cages. At
intervals as summarized in Table IV, three rats from groups 1 and 2 were
selected and each rat was placed in a metabolism cage. They were maintained
on their appropriate diets and dnnkjng water. Urine was collected for 24 h,
then the rats were returned to their cages.
One to two months before termination of the experiment, hematology and
blood chemistry were carried out on some rats from groups 3 and 4.
The analyses were performed in the American Health Foundation Clinical
Biochemistry Facility and by Ani Lytics (Gaithersburg, MD).
Rats were killed by CO2 inhalation when moribund or at termination of
the bioassay after 111 weeks of treatment with NNK. Complete autopsies
were carried out. Histology slides were prepared for all gross lesions and for
lung, liver, pancreas, spleen, kidney, testes, stomach and nasal cavity.
Biomarker analyses
HPB-releasing hemoglobin adducts were quantified by GC-MS (27). NNAL
and NNAL-Gluc in urine were assayed by GC with nitrosamine selective
detection (17,18).
Statistical analyses
Food and water consumption, hematology, blood chemistry and NNAL
biomarker data were evaluated using Student's r-test. Tumor incidence data
were compared with the x 2 test- Body weights, survival data and hemoglobin
adduct levels were analyzed using analysis of variance with repeated
measures (29).
Results
Food and water consumption are summarized in Table I. There
were no significant differences in food consumption among
the groups. The rats in the groups treated with PHTTC consumed
significantly more water than the corresponding control groups.
Body weights and survival did not differ significantly among
the groups. Hematology and blood chemistry data for the rats
treated chronically with PHITC and the corresponding controls
are summarized in Tables II and III. There were no significant
differences between the PHITC-treated and control rats.
HPB-releasing hemoglobin adducts were measured at inter2062
vals during the study in the rats treated with NNK or NNK
plus PHITC. The results are illustrated in Figure 2. Adduct
levels in the rats treated with NNK plus PHITC were 2.1 ±
0.65 times lower than in the rats treated with NNK alone. This
difference was significant (P < 0.0001).
Levels of NNAL and NNAL-Gluc in the urine of the rats
treated with NNK or NNK plus PHITC were assessed after
68 and 79 weeks of treatment with PHITC (Table IV). A
significant 6.0-fold increase in levels of NNAL plus NNALGluc in the urine of the rats treated with NNK plus PHTTC
was observed compared with the NNK-treated rats at week
68. At week 79, an 8.0-fold increase was observed. In the rats
treated only with NNK, the levels of NNAL plus NNAL-Gluc
represented ~6.1 and 5.3% of the dose at 68 and 79 weeks
respectively, while the corresponding figures for the rats treated
with NNK plus PHITC were 34.7 and 36.5%.
The results of the tumor study are summarized in Table V.
The lung was the major target organ in the rats treated with
NNK in the drinking water, as seen previously (19,30). The
incidences of adenoma (P = 0.04), adenocarcinoma (P =
0.001) and total lung tumors (P < 0.00001) were significant
compared with controls. Tumor incidence in the NNK-treated
group at the other sites summarized in Table V was not
significant compared with controls. PHITC strongly inhibited
lung tumor induction. The rats treated with NNK plus PHITC
had significantly lower incidence of adenoma (P = 0.015),
adenocarcinoma (P = 0.003) and total lung tumors (P <
0.00001) than the animals treated with NNK. Although some
lung tumors were observed in the rats treated with NNK plus
PHITC, their incidence was not significantly greater than that
in control animals.
The incidence of malignant pancreatic tumors was lower in
the rats treated with NNK plus PHITC than in the rats treated
with NNK only, but the difference was not significant. The
incidence of pancreatic acinar adenoma was significantly higher
in the NNK plus PHITC-treated rats than in the rats treated
with NNK (P = 0.04). There was no difference between these
two groups in the incidence of total exocrine or islet tumors
of the pancreas.
The incidence of leukemia/lymphoma was significantly less
in the group treated with PHITC and NNK than in the NNKtreated rats (P = 0.002). Other tumors are summarized in
Table VI. There were no significant incidences of tumors in
any of the groups compared with controls.
Discussion
Our results clearly show that PHITC is an effective inhibitor
of NNK-induced pulmonary carcinogenesis in the F344 rat.
Addition of PHITC to NIH-07 diet caused a significant
reduction in lung tumor incidence in rats treated with NNK,
compared with rats treated with NNK and maintained on NIH07 diet. Lung tumor incidence in the rats treated with PHTTC
and NNK was low and was not significantly different from
that in untreated controls. The results of the biomarker studies
are fully consistent with the hypothesis, developed in previous
studies (7,12), that PHITC inhibits NNK carcinogenesis by
inhibiting its metabolic activation. The consistent reduction
in levels of hemoglobin adducts throughout the experiment
indicates that the metabolic activation of NNK by
a-hydroxylation of its methyl group is being inhibited. Moreover, the increased excretion of NNAL plus NNAL-Gluc in
urine is also consistent with inhibition of other metabolic
PHITC inhibition of NNK cardnogenesis
COOH
N.0
N.O
iNNK-
NNK
N-oxide
N.0
DNA methylation
DNA pyridyloxobutylab'on
[QM
-CH,
+
NNAL-Gluc
NNAL
[CH,-N.NOH|
CHt-o
Hb, .DNA
DNA
G — T transversions
G — A transiUoas
PyridyloxoDBtyl addacts
Mathvl adducts
7-mathylguanine
O(-methylguanlne
04-methylttiyinlne
G — A transitions
0 H
/»'
-\
ftHPB
Fig. 1. Overview of NNK metabolism.
160
140 JD
2o
120 -
O)
E
100 -
-L
s
CD
D.
O
40 20
12
14
16
18
Months
Fig. 2. Levels of HPB-releasing hemoglobin adducts in rats treated with NNK or NNK plus PHITC. Values are mean ± SD for three rats per group at each
time point.
2063
S.S.Hecht et al
Table L Food and water consumption in the bioassay
Group
No. of rats
Food consumption
per day (g)
(mean ± SD)"
Mean daily PHITC dose
[mg (nmol)]
1 NNK
2 NNK+PHITC
3 PHITC
4 Control
60
60
20
20
16.81
16.90
17.02
16.65
3.70 (16.9)
3.73 (17.0)
±:
±:
±:
±:
1.37
1.46
1.65
1.25
Water consumption
per day (ml)
(mean ± SD)1
Mean daily NNK dose
[mg (jimol)]
22.49
23.91
24.50
21.94
0.045 (0.217)
0.048 (0.231)
±
±
±
±
4.43
5.73b
6.69C
4.00
•Food consumption was measured weekly during the bioassay; values are mean ± SD of 111 weekly determinations.
b
Significantly greater than NNK group, P = 0.04.
c
Significantly greater than control, P = 0.007.
Table IL Hematology of rats treated with PHITC and control rats'
Parameter
PHITC
(mean ± SD)b
Control
(mean ± SD)b
White blood cells (lOfyl)
Red blood cells (10*^1)
Hemoglobin (g/dl)
Hematocrit (%)
Mean corpuscular volume (fl)
Mean corpuscular hemoglobin (pg)
Mean corpuscular hemoglobin
concentration (%)
Platelets (lO 3 /^)
Segmented neutrophils (lO 3 /^)
Lymphocytes (l6*/\l\)
Monocytes (lO 3 /^)
Eosinophils (lCP/jll)
13.7
6.71
13.9
38.2
57.3
21.0
36.6
5.6 ± 1.36
7.22 ± 2.18
14.9 ± 2.64
42.1 ± 8.46
61.6 ± 12.43
22.0 ± 5.05
35.5 ± 0.95
± 13.2
± 0.97
± 0 92
± 3.45
± 3.73
± 1.89
± 1.18
906 ± 92.1
5.18 ± 4.55
8.1 ± 13.2
0.24 ± 0 . 1 3
0.17 ± 0.27
716 ± 193
2.83 ± 1.41
2.57 ± 0.42
0.09 ± 0.08
0.1 ± 0.07
"n = 6 rats each from groups 3 and 4, maintained on NIH-07 diet
containing PHITC (1 nmol/g diet) or NIH-07 diet with no additions
respectively.
b
There were no significant differences between the groups.
Table III. Blood chemistry of rats treated with PHITC iand control rats*
Parameter
PHITC
(mean ± SD)b (n)
Control
(mean ± SD)b (n)
Ca 2+ (mg/dl)
K + (mmol/1)
Na + (mmol/1)
C r (mmol/1)
Alanine aminotransferase (U/l)
Aspartate aminotransferase (U/l)
Cholesterol (mg/dl)
Blood urea nitrogen (mg/dl)
PO?f (mg/dl)
Triglycerides (mg/dl)
Alkaline phosphatase (U/l)
Y-Glutamyl transpeptidase (U/l)
Bilirubin (mg/dl)
Glucose (mg/dl)
High density lipoproteins (mg/dl)
Total protein (g/dl)
Creatinine (mg/dl)
Albumin (g/dl)
11.1 ± 0.48 (7)
4.76 ± 0.23 (7)
145 ± 2.41 (7)
107 ± 3.87 (7)
42.9 ± 4.81 (7)
84.3 ± 40.4 (7)
188 ± 51.8 (7)
45.7 ± 34.9 (7)
6.62 ± 1.76(6)
118 ± 89.1 (7)
83.8 ± 14.2 (6)
7.57 ± 1.4(7)
0.39 ± 0.25 (7)
98.4 ± 5.83 (7)
100 ± 25.2 (7)
7.24 ± 0.32 (7)
1.21 ± 0.9(7)
3.4 ± 0.27 (7)
10.9 ± 0.56 (8)
4.6 ± 0.44 (10)
155 ± 17.7(10)
98.7 ± 22.8 (10)
52.6 ± 27.0 (8)
82.3 ± 23.2 (7)
168 ± 102 (9)
23.3 ± 15.9 (10)
6.66 ± 3.95 (10)
119 ± 91.4 (10)
108 ± 76.9 (8)
12.1 ± 9.74 (10)
049 ± 0.49(10)
88.8 ± 18.3 (10)
75.2 ± 40.8 (10)
6.4 ± 1.34 (10)
0.73 ± 0.5 (10)
3.21 ± 0.49 (10)
•Rats were maintained on NIH-07 diet containing PHTTC (1 (imol/g diet) or
NIH-07 diet with no additions (control group).
There were no significant differences between the groups.
b
pathways, such as a-hydroxylation and/or pyridine N-oxidation. Collectively, the presently available results demonstrate
that PHITC is an effective inhibitor of the metabolic activation
and tumorigenicity of NNK in both mice and rats (6-8,12).
2064
In a recently completed study, PHITC was administered to
F344 rats (2 or 4 umol/g ATN-76A diet) 1 week before, during
20 weeks of NNK administration by s.c. injection and 1 week
after NNK treatment (31). The experiment was terminated
after 98 weeks. As in the present study, PHTTC significantly
inhibited NNK-induced lung tumorigenesis in these rats. There
were no apparent toxic effects of PHITC. In another previous
study, PHTTC was administered to F-344 rats (1.5 or 2.9 (imol/
g ATN-76A diet) for 55 weeks in order to determine its effects
on colon carcinogenesis (31). This study is discussed further
below. However, no toxic effects of PHITC were observed
other than some bleeding in the cecum and intestine in ~30%
of the animals at the higher dose. The present study is the first
in which PHITC (1 |imol/g NTH-07 diet) has been administered
to rats virtually for their entire lives. As judged by body
weight, survival, hematology and blood chemistry data, there
were no overt toxic effects of PHITC in these rats. There were
also no carcinogenic effects of PHITC, as summarized in
Tables V and VI. Thus, the strong inhibitory effects of PHITC
on lung tumorigenesis in this study were obtained without
apparent toxicity. Overall, the presently available results firmly
establish the chemopreventive activity of apparently non-toxic
doses of PHITC against lung cancer induced in rats by NNK.
The drinking water protocol for NNK administration
employed in this study allowed us to examine the effects of
PHITC on pancreatic carcinogenesis (19,30). The incidence of
malignant exocrine pancreatic tumors in the NNK-treated
group was low and this was diminished by PHITC treatment,
although the difference was not significant. Benign exocrine
pancreatic tumor incidence was significantly enhanced by
PHITC, while the total of benign and malignant tumors in the
two groups was not different. These results suggest that PHITC
may inhibit the progression of benign to malignant pancreatic
tumors in NNK-treated rats.
The inhibition of leukemia/lymphoma by PHITC is noteworthy. In the recent study by Chung et al. (31), PHITC also
inhibited leukemia/lymphoma incidence in NNK-treated rats
as well as decreasing the incidence of leukemia/lymphoma in
control rats. Thus, PHITC should be evaluated further as a
chemopreventive agent against leukemia/lymphoma.
In a parallel study which has been reported separately, we
evaluated the effects of PEITC (3 \imo\Jg NIH-07 diet) on
biomarkers and tumorigenicity in rats treated with 2 p.p.m.
NNK in the drinking water (28). Although the doses of PEITC
(3 (imol/g diet) and PHITC (1 u.mol/g diet) were different, their
effects were remarkably similar. PEITC inhibited formation of
HPB-releasing hemoglobin adducts to a similar extent to
PHTTC, enhanced excretion of NNAL plus NNAL-Gluc in
urine, completely inhibited lung tumorigenesis and had a
PHITC inhibition of NNK carclnogenesis
Table IV. Levels of urinary NNAL and NNAL-Gluc in rats treated with NNK or NNK plus PHITC1
Group
Metabolites (nmol/24 h)
NNK
68 weeks
79 weeks
NNK plus PHITC
68 weeks
79 weeks
Fold increase in total
NNAL
NNAL-Gluc
Total
4.1 ± 1.0
3.1 (3.5, 2.7)
9.4 ± 3.8
8.8 (12, 5.3)
13.5 ± 4.8
12(16, 8.1)
21.3 ± 13.3
25.1 ± 18.1
58.5 ± 28.9b
70.1 ± 49.0
80.4 ± 41.l b
95.1 ± 66.9
NNAL-Gluc/NNAL
2.21 ± 0.51
2.7 (3.5, 2.0)
6.0"
8.0
2.88 ± 0.55
2.77 ± 0.39
"After 68 or 79 weeks of treatment, rats from groups 1 and 2 of the bioassay were placed in metabolism cages and urine was collected for 24 h. Three rats
per group, except NNK, 79 weeks, two rats.
Significantly greater than NNK group, P < 0.05.
Table V. Tumorf of the lung, pancreas, liver, nasal cavity ind leukemia/lymphoma in rau treated with NNK, NNK plus PHITC, PHITC or nothing*
Group
(initial no of rau)
Number of rmu with tumors/number of rats examined (*)
Lung
Adenoma
NNK (60)
NNK+PHITC(60)
PHTTC (20)
Control (20)
Leukemia/
lymphoma
Nasal cavity
16756(29)
6/57 (11/
1/16(6)
1/18 (6)
Adenocarcinoma
6
Total
1
23/56 (41T
9/57 (16)"
0/16 (0)
0/18 (0)
39/56 (70)
15/57 (26)b
1/16 (6)
1/18 (6)
b
Malignant
Bcnjgn
Total
4/52
1/50
0/17
0/16
1/52 (2)
6/50(12)"
3/17 (18)
3/16(19)
6/52 (12)
7/50 (14)
3/17(18)
3/16(19)
(8/
(2)
(0)
(0)
•Mile F-344 nils WOT treated witb NNK (2 p.p.m in the drinking witer for 111
di« for 112 week*) or N1H-07 diet only as described in Materials and methods.
b
Adnar adenomas
•Benign ulet tumors
''includes one bronchial squamous carcinoma.
*ln addition to these tumon, one rat bad • potsiWe ductular carcinoma.
r
Less than NNK, P = 0.015.
•Less than NNK, P = 0.003
b
Las than NNK, P < 0.00001.
'Greater than NNK, P - 0.04
*Less than NNK, P - 0 002
Endocrine
Adenoma
Carcinoma
Total
PapUloma
Carcinoma
Total
5/52 (10)
10/50 (20)
1/17 (6)
1/16 (6)
2/55 (4)
4/50(8)
0/20(0)
1/15(7)
3/55 (5)
1/50 (2)
0/20(0)
0/15 (0)
5/55 (9)
5/50(10)
0/20(0)
1/15(7)
1/48
1/45
1/19
0/18
0/45
1/45
0/19
1/18
1/48 (2)
2/45 (4)
1/19(5)
1/18 (6)
(2)
(2)
(5)
(0)
(0)
(2)
(0)
(6)
26/56(46)
11/57 (19)1
4/20(20)
4/15 (27)
) or NNK (2 pp m, in the drinking water for 111 weeki) and PHITC (I urool/g diet for 112 weeks) or PHITC (1
Tkb4e VL Vtnaa tumoa In rau treated with NNK, NNK plus PHITC, PHITC or nothing1
Group (initial no of rats) Number of rats with tamoo/number of rats examined (%)
NNK (60)
NNK + PHITC (60)
PHITC (20)
Control (20)
Leydig
testkutar
Mammary tumon
Thyroid tumon
lumon
Fibroadenoma
Fibroma
Ccell
Papillary
adenocarciDomj
Fobcular
•denocarcinoma
49
51
16
18
0
3
0
0
6
6
2
0
1
1
1
0
1
0
0
0
0
1
0
0
Osteosarcoma
Kidney
adenoma
Skin
tumors
AdreoaJ
tumors
Peritoneal
mesothelioroa
Other
3
0
0
0
2
0
0
1
3
0
0
1
2
2
1
0
1
2
0
0
3"
y
0
•Male F-344 ratt were orated with NNK (2 p.p m in the drinking water for 111 weeks) or NNK (2 p.pjn. in the drinking water for 111 weeks) and PHTTC (1 fimol/g diet for 112 wcekl) or PHITC (I pjnol/g
diet for 112 weeks) or N1H-07 diet only as described in Materials and methods
b
One stomach squamoos cell papillontt, one prostate carcinoma hi situ, one brain meninpoma.
c
One jejunum adeoocarcinoma, ooe colon adenocarcinoma, ooe rbabdomyourcoma.
d
Ooe colon leiooivosarcoma, one Zymbal gland tumor.
similar effect to PHITC on pancreatic carcinogenesis. PHITC
is a stronger inhibitor of lung tumorigenesis induced by NNK
in the mouse than is PEITC, but their relative efficacies in
rats are difficult to compare at present because comparative
dose-response studies have not been carried out (28,31).
The results reported here are encouraging with respect to
the effect of PHITC on NNK carcinogenesis. However, the
effects of PHITC on tumorigenesis in some other systems are
not so favorable. In the azoxymethane-induced rat colon
carcinogenesis model, relatively high doses of PHTTC (1.5
and 2.9 n.mol/g diet) enhanced colon carcinogenicity when
given during and after carcinogen administration (32). PHITC
also enhanced certain pathways of arachidonic acid metabolism
in the colonic mucosa of these rats. The mechanism of the
promoting effect of PHITC on colon tumorigenesis in that
experiment is not known, but could be related to the intestinal
toxicity of the high dose of PHITC employed or to the effect
of PHITC on arachidonic acid metabolism. The relevance of
these observations is unclear at present, since azoxymethane
is a synthetic carcinogen to which humans are not known to
2065
S.S.Hecht et al
be exposed and the doses of PHTTC employed were higher
than required for inhibition of carcinogenesis by NNK. In
another study, PHITC (0.4-2.5 n.mol/g diet) enhanced esophageal tumorigenesis in rats treated with N-nitrosomethylbenzylamine (33). These results contrasted with the strong
inhibitory effects of PEITC and 3-phenylpropyl isothiocyanate
in the same model system (34). PHITC also enhanced skin
tumor multiplicity in mice treated with benzo[a]pyrene (35).
The results of these studies indicate that, under certain conditions, PHITC can enhance carcinogenesis. Nevertheless, in the
study described here, as well as in several previous studies,
PHITC has shown strong chemopreventive activity against
NNK. Further research is required to delineate the precise
circumstances in which PHITC might be employed in chemoprevention trials in humans.
Acknowledgements
This study was supported by National Cancer Institute Grant CA-46535. The
bioassay was performed in the American Health Foundation Research Animal
Facility, supported in part by National Cancer Institute Cancer Center Support
Grant CA-17613. This grant also partially supports the Clinical Biochemistry
Facility, which performed some of the analyses in this study, the Biostatistics
and Computer Facility, which carried out the statistical evaluations, and the
Organic Synthesis Facility, which provided the PHITC and NNK. We thank
Rachid Hamid for preparing the diet, Chang-In Choi for supervising the
bioassay and Brian Pittman and Edith Zang for statistical consultation.
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Received on March 6, 1996; revised on May 9, 1996; accepted on May 23, 1996
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