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Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan
2To whom correspondence should be addressed. E-mail: fukuchan{at}med.osaka-cu.ac.jp.
ABSTRACT
The chemopreventive effects of five water-soluble organosulfur compounds, S-methylcysteine (SMC) and four analogs, were examined on the promotion stage of diethylnitrosamine hepatocarcinogenesis in male F344 rats, using the medium-term bioassay (Ito test), which is based on the two-step model of hepatocarcinogenesis. In addition, we investigated the modifying effects of SMC and cysteine on the initiation stage of rat hepatocarcinogenesis. Carcinogenic potential was scored by comparing the numbers and areas of a putative neoplastic lesion, glutathione S-transferase placental form (GST-P)–positive hepatocellular foci. SMC and cysteine significantly decreased the number and area of GST-P–positive foci when given in the promotion stage of the Ito test. When given during the initiation stage, these two organosulfur compounds also significantly inhibited focus formation. Liver ornithine decarboxylase activity after two thirds partial hepatectomy and the proportion of hepatocytes positive for proliferating cell nuclear antigen significantly decreased the number of aberrant crypt foci in the colon in a multiorgan carcinogenesis bioassay of rats. These results support SMC and cysteine as chemopreventive agents for hepatocarcinogenesis and colon carcinogenesis. Their intake may be of importance for cancer.
KEY WORDS: • chemoprevention • cacinogenesis • water-soluble organosulfur compounds • S-methylcysteine • cysteine • garlic
Environmental compounds are likely involved in the development of many human cancers. Their elimination would be expected to help with the prevention of cancer. However, this is not a practical proposition; therefore, it is important to discover naturally occurring or synthetic compounds that might suppress or prevent the process of carcinogenesis.
It is well-known that both oil-soluble and water-soluble
organosulfur compounds
(OSC)3
are contained in garlic and onions. Some of these, particularly
oil-soluble OSC, have been shown to be chemopreventive in the
initiation stage of carcinogenesis. For example, diallyl sulfide (DAS)
inhibits the development of colon carcinomas, esophageal carcinomas,
pulmonary adenomas and forestomach tumors in rodents when administered
before carcinogen exposure (Sparnins et al. 1988
,
Wargovich 1987
, Wargovich et al. 1998,
Wattenberg et al. 1989
). In addition, DAS has been found
to inhibit hepatocarcinogenesis when administered after the initiating
procedure (Wattenberg et al. 1989
). However, there is a
lack of data on the chemopreventive effect when administered during the
promotion stage of carcinogenesis. Administration of oil-soluble
OSC after initiation with diethylnitrosamine (DEN), either enhanced or
inhibited glutathione S-transferase placental form
(GST-P)-positive foci in rat liver (Takada et al. 1994a and 1994b
).
S-Methylcysteine (SMC), a water-soluble OSC, exists in
various plants, including Allium sativum (Suzuki et al. 1961
), Phaseolus vulgaris (Thompson et al. 1956
), and Cruciferae (Synge and Wood 1956
).
Although its biological characteristics have not received much
attention, Sumiyoshi and Wargovich (1990)
reported that
S-allylcysteine (SAC), an analog of SMC, can prevent
1,2-dimethylhydrazine (DMH)-induced colon cancer in mice.
In this study, the modifying potentials of five water-soluble OSC,
SMC and four analogs were examined in the rat liver medium-term
bioassay system of Ito et al. (1988)
and the multiorgan
carcinogenesis bioassay based on the two-stage carcinogenesis model
(Takahashi et al. 1992b
). The four analogs of SMC
examined in this study were cysteine, SAC, S-propylcysteine
(SPC), and S-ethylcysteine (SEC). In another experiment, the
modifying effects of SMC and cysteine given during the initiation stage
of rat hepatocarcinogenesis were investigated as previously described
(Tsuda et al. 1994
).
Cancer prevention in hepatocarcinogenesis
Ito et al. (1988 and 1989)
developed a
medium-term bioassay system to detect liver carcinogens and
promoters; it is of relatively short duration but results in sufficient
lesions to allow statistical comparisons. A series of experiments were
performed to optimize the different components of the model as well as
a liver medium-term bioassay for carcinogens (Ito test). A duration
of 8 wk was established. Importantly, this rat liver medium-term
bioassay can be applied to detect not only the carcinogenic potential
of chemicals, but also their postinitiation modifying effects. The rat
liver has the particular advantage of easy detection of preneoplastic
enzyme-altered foci, widely accepted as early indicators of cancer
(Bannasch 1986
, Farber and Cameron 1980
).
GST-P-positive foci in the rat liver correlate with hepatocellular
carcinomas; thus, they have been routinely employed as an end-point
marker in this assay system (Ito et al. 1988 and 1989
).
Experiment 1 investigated the modifying effects of each OSC on the
second stage of hepatocarcinogenesis using the Ito test (Takada et al. 1997
). The experimental design is shown in Figure 1
. The rats in groups 1–6 (n = 10 rats/group) were given
a single intraperitoneal injection of DEN (200 mg/kg body) dissolved in
saline to initiate hepatocarcinogenesis. After 2 wk, they received SAC
(groups 1 and 7), SPC (groups 2 and 8), SEC (groups 3 and 9), SMC
(groups 4 and 10), or cysteine (groups 5 and 11), each at a dose of 100
mg/kg body dissolved in saline (4 or 8 mL/kg), and administered by
gastric gavage 5 times/wk for 6 wk. Rats were subjected to two thirds
partial hepatectomy (PH) at wk 3 to maximize any interaction between
proliferation and the effects of the compounds tested. Control rats
(group 6) were given DEN and PH, followed by saline without
administration of any OSC compounds. Groups 7–11 (n = 5/group) received saline instead of DEN solution, but were subjected to
administration of test compounds and PH. Rats in each group were killed
for examination at the end of wk 8. The livers were examined
immunohistochemically for GST-P positive-focus formation. No
significant intergroup variation was found in the final body weight of
rats treated or untreated with OSC. Figure 2
summarizes the data for numbers and areas of GST-P positive foci
per unit area of liver section after DEN initiation in Experiment 1.
Values for numbers and areas in groups 1–3 treated with test chemicals
tended to be decreased compared with those in controls. Values for both
parameters in the groups that were given SMC (group 4) or cysteine
(group 5) were significantly decreased. In particular, group 4 values
were less than half of those in controls. Livers from groups not given
DEN did not have GST-P-positive foci and revealed normal histology.
|
|
. Rats (n = 42) were divided into five groups; groups
1–3 were given a single intraperitoneal injection of DEN (20 mg/kg
body) dissolved in saline, whereas groups 4 and 5 received saline
instead of the DEN solution. SMC (groups 1 and 4) or cysteine (groups 2
and 5) were administered at 100 mg/kg body dissolved in saline, once a
day from 5 d before DEN or saline injection to 1 d after.
Rats in group 3 were given saline intragastrically instead of OSC. All
rats were fed 0.01% 2-acetylaminofluorene (2-AAF) in the diet from wk
2 to 4 and subjected to PH at wk 3. Numbers and areas of GST-P
positive foci in Experiment 2 are shown in Table 1
. Values for both parameters in groups 1 and 2 were decreased compared
with those in group 3. GST-P-positive foci occurred sporadically in
groups 4 and 5, without DEN and with 2-AAF.
|
|
, O’Brien et al. 1975
), were measured in
SMC- and cysteine-treated liver tissues after PH. The expression of
proliferating cell nuclear antigen (PCNA) was examined
immunohistochemically in SMC- and cysteine-treated livers. Because
cell proliferation is generally low in the normal liver of adult rats,
PH was used for the next two experiments.
In Experiment 3, the relation between cell proliferation and inhibitory
effects of SMC and cysteine on liver ODC and SAT activities in liver
was examined. Rats (n = 5) were fed SMC or cysteine
(100 mg/kg body, intragastrically) at 0, 24 and 48 h before PH.
The control group (n = 5) received saline alone. All
rats were killed 4 h after PH, and ODC and SAT activities were
assessed. The results for ODC and SAT activities in rat liver tissues
after PH in Experiment 3 are summarized in Table 2
. ODC activities 4 h after PH were significantly decreased in SMC-
or cysteine-treated liver tissue compared with controls. Mean SAT
activities were also lower in SMC- and cysteine-treated rats, but
were not significantly different from controls.
|
). Moreover, even after 24 h, some reduction was still apparent.
|
. Maximum expression of c-fos, c-jun,
c-myc in the rat liver without SMC treatment was observed at
2, 8 and 8 h, respectively, after PH. Northern blot analysis
revealed that only the expression of c-jun in the rat liver
was obviously altered by SMC treatment. SMC was associated with a
down-regulation of c-jun during the first 8 h after
PH; however, there was an increase at the 12-h time point.
|
). The area of GST-P positive foci in the group treated
with SMC (0.32 ± 0.19
mm2/cm2) was significantly
less than in those not receiving SMC (0.60 ± 0.39
mm2/cm2). The number of
GST-P positive foci in SMC-treated rats was lower than in
controls, although not significantly different. There were no
alterations in the amount and concentration of
N-nitrosomorpholine in the stomach between the two groups.
These data indicated that SMC can suppress hepatocarcinogenesis caused
by nitrites and amines possibly by altering their metabolism.
Cancer prevention in carcinogenesis of organs other than the liver
An alternative assay for detecting carcinogenicity and modifying
(promoting or inhibiting) activity is the multiorgan carcinogenesis
bioassay (Ito et al. 1991
, Otoshi et al. 1995
, Takahashi et al. 1992a and 1992b
). It has
the advantage of being a short-term assay of whole-body
carcinogenic or modifying potential. Using the best multiorgan
carcinogenesis assay among several systems investigated to date, we
assessed the influence of SMC and cysteine on the postinitiation stage
(Fig. 5
).
|
). However, no equivalent depression in the induction of
colon tumors was observed. The results of quantitative evaluation of
GST-P-positive foci in the liver revealed that the numbers and areas
were significantly lower in the group treated with SMC or cysteine than
in controls. There were no differences in other organs among SMC,
cysteine and control rats.
DISCUSSION
Our results indicate that OSC such as SMC and cysteine exert
chemopreventive effects against chemical carcinogenesis in rats. SMC
and cysteine at the level of 100 mg/kg body was toxic. SMC is found at
a concentration of 
40 µg/g of fresh garlic (personal
communication from Wakunaga Pharmaceutical Co., Ltd.). Examination of
low dose-response relationships for inhibitory effects of SMC and
cysteine are warranted to evaluate their usefulness as chemopreventive
agents at lower intakes. Moreover, it is very important to determine
whether SMC and cysteine promote carcinogenesis in other organs.
The fact that SMC and cysteine prevented elevation of ODC and SAT in the liver suggests a possible mechanism accounting for the prevention of hepatocarcinogenesis by such OSC. These data suggest that a down-regulation of polyamine metabolism may be involved.
Expression of early response genes may be very important for tumor promotion. Expression of c-jun mRNA was down-regulated for the first 8 h after the final intragastric administration of SMC, whereas levels of c-fos mRNA transcripts were not changed compared with controls. These data suggest that formation of Fos-Jun heterodimers, which are strongly linked to AP-1 sites, is decreased in rat liver treated with SMC and that a suppression in preneoplastic lesions in rat liver might be related.
In conclusion, our results indicate that SMC and cysteine exert chemopreventive effects on chemical carcinogenesis of rats. Additional studies are warranted to define the minimum quantities necessary to produce this effect and the mechanism responsible.
ACKNOWLEDGMENTS
We would like to express our gratitude to Shuzo Otani and Yoshihisa Yano at the Department of Biochemistry, Osaka City University Medical School for helping with this study and to Wakunaga Pharmaceutical, Osaka for providing the chemicals.
FOOTNOTES
1 Presented at the conference "Recent Advances on the Nutritional Benefits Accompanying the Use of Garlic as a Supplement" held November 15–17, 1998 in Newport Beach, CA. The conference was supported by educational grants from Pennsylvania State University, Wakunaga of America, Ltd. and the National Cancer Institute. The proceedings of this conference are published as a supplement to The Journal of Nutrition. Guest editors: John Milner, The Pennsylvania State University, University Park, PA and Richard Rivlin, Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer Center, New York, NY. 
3 Abbreviations used: 2-AAF, 2-acetylaminofluorene; BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; DAS, diallyl sulfide; DEN, diethylnitrosamine; DHPN, dihydroxy-di-propylnitrosamine; DMBDD, combination treatment with DEN, MNU, BBN, DMH and DHPN; DMH, 1,2-dimethylhydrazine; GST-P, glutathione S-transferase placental form; MNU, N-methyl-N-nitrosourea; ODC, ornithine decarboxylase; OSC, organosulfur compounds; PCNA, proliferating cell nuclear antigen; PH, partial hepatectomy; SAC, S-allylcysteine; SAT, spermidine/spermine N'-acetyltransferase; SEC, S-ethylcysteine; SMC, S-methylcysteine; SPC, S-propylcysteine.
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, DEN, 200 mg/kg body
(intraperitoneal); 
, saline, 5 mL/kg body (intraperitoneal); 
,
two thirds partial hepatectomy; S, killing; 
, test chemicals
(intragastric) 5 times/wk; 
, saline, 1 mL/kg body (intragastric).
, test chemicals (intragastric); 
,
saline, 1 mL/kg body (intragastric); 2-AAF, 2-acetylaminofluorene.
, no treatment).
, dihydroxy-di-propylnitrosamine
(DHPN), 0.1% in the drinking water;