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Chemoprevention of tongue carcinogenesis in rats
Chemoprevention of tongue carcinogenesis in rats Isao Inoue, MD, Yuzo Yamamoto, MD, Takashi Ito, MD, and Hiroaki Takahashi, MD, Osaka, Japan DEPARTMENT
OF OTOLARYNGOLOGY,
OSAKA
MEDICAL
COLLEGE
The inhibitory effects of vitamin A, selenium, butyiated hydroxytoiuene, and their combinations on tongue carcinogenesis induced by 4-nitroquinoline l-oxide were examined in Sprague-Dawley rats. After 4 months of administration of 4nitroquinoline l-oxide, a wide range of lesions from hyperplasia to early invasive carcinoma was seen. The lesions were fewer and the carcinomas were less advanced in the rats given preventive treatment. The chemopreventive effect of vitamin A plus butylated hydroxytoluene was most effective. After 7 months of 4-nitroquinoline l-oxide administration, the incidence, the macroscopic diameter, and the microscopic depth of invasion among carcinomas were smaller in the chemopreventive groups than in the nonchemopreventive group. We conclude that vitamin A, selenium, and butylated hydroxytoluene inhibit tongue carcinogenesis induced by 4-nitroquinoline l-oxide in rats. The inhibitory effect of combined chemopreventives is not always superior to that of each chemopreventive agent used alone. (ORAL SURC ORAL MED ORAL PATHOL 1993;76:608-15)
Cancer-prevention measures generally involve the reof causative agents. Because so many carcinogens are present mainly in food, it is very difficult to remove all of them. Therefore chemoprevention is now thought to be a very important way to protect against cancer. Chemoprevention of cancer is a method of cancer control in which the occurrence of this disease is prevented by the administration of one or several chemical compounds. Furthermore, combination chemoprevention with several compounds is often used because it reduces the toxicity of each compound and exerts a synergistic action. Wattenberg’ classified chemopreventive agents into three broad categories: (A) compounds that are effective against a complete carcinogen, (B) compounds that are effective against a tumor promoter, and (C) compounds that inhibit neoplasia when administered shortly before exposure to a carcinogenic compound. Modality A is further subdivided into three categories: (1) compounds that prevent the formation of carcinogens from precursor substances (A-I); (2) compounds that inhibit carcinogenesis by preventing carcinogenic compounds from reaching critical target sites in the tissues (A-II), these inhibitors are called “blocking agents” and exert a barrier function; (3) inhibitors that act after exposure to carcinogenic agents (A-III), these inhibitors are termed “suppressing agents.” Vitamin A (VA) is considered to have A-III, B and C activities, selenium (Se) has A-III activities, and phenols such as butylated hydroxytoluene (BHT) have A-II and B activities. moval
Copyright 3 1993 by Mosby-Year Book, 0030-4220/93/$1 .OO + .lO 7/14/48400
608
Inc
There are many reports on chemoprevention of carcinogenesis by these agents in several organs among animal models, but only a few have been tested on lingual carcinogenesis. Shklar et al2 reported that 9, 10 dimethyl-1,2 benzanthracene (DMBA)-induced tongue carcinoma in hamsters was inhibited by 13-cis-retinoic acid, and Goodwin et a1.3 noted that DMBA-induced tongue carcinomas in hamsters were inhibited by 13-cis-retinoic acid with and without Se. However, in these aforementioned experiments, tongue carcinomas were produced by topical application of DMBA three times weekly with a sable brush, and it is probable that carcinogenesis was influenced by the techniques of the clinicians. Our experimental method of inducing carcinogenesis by the oral administration of 0.00 1% 4-nitroquinoline 1-oxide (4NQO) in drinking water appears to be more physiologic and more similar to the formation of tongue cancer in humans than is the painting method, because cancers are produced by long-term ingestion of only small amounts of carcinogens. In the present study, the inhibitory effects of VA, Se, and BHT singly and in combination were examined in rats with tongue carcinomas induced by 4 or 7 months of 4NQ0 administration. MATERIAL Animals
AND METHODS
One hundred sixty Sprague-Dawley male rats (Shizuoka Experimental Animal Co., Shizuoka,Japan), each weighing about 250 gm at the start of the the experiment, were used. Three to five rats were housed together in a metal cage. The rats were divided into 16 groups of 10 each. MM3 (Funabashi Farm
Inoue et al.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
609
Volume 76, Number .5
Co. Ltd., Chiba, Japan) was provided as the basal diet. Tumor
Table
I. Treatment schedule modulators
inductilon
All of the rats received 0.001% 4NQ0 (Nakarai Chemical Co., Kyoto, Japan), added to their drinking water and taken ad libitum from a brown, lightshielded, polyvinyl bottle for 4 or 7 months. The consumption of 4NQO solution was checked three times a week, and the bottles were refilled with fresh 4NQ0 solution. Administration
of chemopreventive
L 1 2
9 10
3 4 5
11 12 13
6
14
I
15
8
16
None Sodium selenite in drinking water Vitamin A acetate in diet BHT in diet Sodium selenite in drinking water and vitamin A acetate in diet Sodium selenite in drinking water and BHT in diet Vitamin A acetate and BHT in diet Sodium selenite in drinking water, vitamin A acetate and BHT in diet
agents
Table I shows the experimental protocol. Animals were divided into 16 groups according to the chemopreventive regimen and the duration of administration: 4-month,s administration (groups 1 to 8) or 7-months administration (groups 9 to 16). The first variable was sodium selenite (Se) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in the drinking water. Animals in groups 1, 3, 4, 7, 9, 11, 12, and 15 were given 4NQ0 solution without Se;those in groups 2, 5, 6, 8, 10, 13, 14, and 16 were given water supplemented with 3 ppm Se. The secondvariable was BHT (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in the diet. Animals in groups 4, 6, 7, 8, 12, 14, 15, and 16 welrefed basal diet plus 5000 mg of BHT per kilogram of food. The third variable was VA acetate (Wako F’ure Chemical Industries, Ltd., Osaka, Japan) in the diet. Animals in groups 3,5,7,8, 11,13, 15, and 16 were fed basal diet plus 0.1 gm (100,000 IU) of VA per kilogram of diet. Animals in groups 1, 2, 9, and 10 were fed the basal diet without BHT or VA. Thus animals in groups 1 and 9 served as controls, wherieasthose in groups 2 and 10 were given Se alone, those in groups 3 and 11 were given VA alone, those in groups 4 and 12 were given BHT alone, those in groupis5 and 13 were given Se and VA, those in groups 6 and 14 were given Se and BHT, those in groups 7 and II5 were given VA and BHT, and those in groups 8 and 16 were given Se, VA, and BHT. Observations
The numbe:r of surviving rats 4 or 7 months after the beginning of the experiment was the “effective number.” At 4 months or 7 months, the rats were killed by ethelr inhalation and weighed. The tongue, palate, gingiva, buccal mucosa, pharynx, larynx, trachea, and esophagus were removed and observed grossly. For light microscopy, the tongues were fixed in 20% formalin solution, dehydrated with alcohol, and embedded in paraffin. Sections were cut in 3 pm slices and stained with hematoxylin and eosin.
Macroscopic and microscopic tumor progression
examination
of
At autopsy, the longest diameter of the carcinomatous lesions in the tongue was measured by observation of the gross appearance acrossthe surface of the specimen and was defined as the macroscopic diameter. Tongue tissues were cut vertically through the macroscopic diameter and formalin-fixed, paraffinembedded 3 pm sections that contained the cut surface were made and stained with hemotoxylin and eosin. In the hemotoxylin and eosin preparations, the vertical distance between the top and the bottom of the lesions was measured microscopically with a micrometer. The vertical distance was defined as the microscopic depth. The degree of tumor progression of squamous cell carcinomas was evaluated from the macroscopic diameter and the microscopic depth. Statistical
analysis
Cancer incidences were compared by the x2 test, Statistical comparisons of the macroscopic diameter and the microscopic depth of invasive carcinomas were performed with Student’s t test. RESULTS Histopathologic
findings
at 4 months
Table II shows the histopathologic characteristics and the incidence of lesions after 4 months of 4NQ0 administration. Normal tongue (Fig. l), hyperplasia (Fig. 2), dysplasia (Fig. 3), carcinoma in situ (Fig. 4), and invasive carcinoma (Fig. 5) were seen. In the
610
ORAL SURGERY ORAL MEDICINE ORAL PAlHOLOGY November 1493
Inoue et al.
Fig. 1. Histologic features of normal tongue of rat. (Hematoxylih-eosin stain; original magnification x200.)
Table II. Effects of vitamin A, selenium, BHT, and their combinations
on tongue carcinogenesis in rats
treated with 4NQ0 for 4 months Incidence % (Number of rats)
Group 1 control
Effective number
Mean jinal body weight
of rats
(9)
9
415.6
Intact
(:I
2 Se alone
10
405.6
20.0
3 VA alone
10
451.2
40.0
9
426.7
5Se+VA
10
413.5
6Se+BHT
9
407.2
(2) (4) 4 BHT alone
7VA+BHT
8
436.3
8Se+VA+BHT
9
394.2
A 20.0
Hyperplasia
iI 10.0 (1) 10.0 (1) 11.1 (1) 10.0
Dysplasia 55.6 (5) 20.0
(2) (2)
(2)
(3) 40.0 (4)
20.0
(21 11.1
44.4
(:I
(2)
(2)
nonchemopreventive group (group 1), dysplasia was seen in five rats and carcinoma in four rats. In contrast, in the chemopreventive groups, a wide range of lesions from hyperplasia to invasive carcinoma was seen, and some rats had no lesions. When the lesions were divided into five histologic groups according to grade of progression (normal, hyperplasia, dysplasia, carcinoma in situ, and invasive carcinoma), the lesions in the chemopreventive groups were inclined to be less advanced than those in the nonchemopreventive group, although the number of rats with lesions
33.3
(7) 40.0
77.7
(4) 22.2
A
Invasive carcinomas
10.0 (1) 11.1 (1) 10.0 (11
(1)
c:,
10.0 (1) 20.0
22.2
(4)
11.1 (1)
20.0
(2) 50.0
Carcinoma in situ
37.5
33.3
(3) 33.3
(3)
(3)
(:I
(1) c&
11.1 (1)
(41 12.5 (1) 22.2
(2)
in each chemopreventive group was nearly equal to that in the nonchemopreventive group. Tongue carcinogenesis was inhibited to the greatest degree in the VA plus BHT group. Histopathologic
findings
at 7 months
Table III shows the histopathologic characteristics and the incidence of lesions after 7 months of 4NQ0 administration. In the nonchemopreventive group (group 9), invasive cancers were produced in all rats. In the VA only group (group 11) and the BHT only
ORAL SURGERYXL MEDICINE ORAL PATHOLOGY Volume 76, Number S
Inoue et al.
611
Fig. 2. Histologic features of hyperplasia. Slight extension of rete pegsand thickening of epithelium are seen. (Hematoxylin-eosin stain; original magnification x200.)
group (group 12), significantly fewer invasive carcinomas developed than in the nonchemopreventive group (group 9). The inhibitory effect of the combined chemopreventives was not always superior to that of each chemopreventive agent alone. The tumors developed mainky on the dorsum of the tongue, especially around the torus linguae (Fig. 6). Most squamous cell carcinomas were well differentiated (Fig. 7); a few were moderately differentiated. No carcinoma was poorly differentiated. The macroscopic diameters of invasive carcinomas are listed in Table IV. They were significantly smaller in all chemopreventive groups (groups 10 to 16) than in the nonchemopreventive group (group 9). The microscopic depth of invasive carcinomas is shown in Table V. It tended to be less in all chemopreventive groups. In the. VA alone (group ll), Se plus BHT (group 14), and Se plus VA plus BHT (group 16) groups, it was significantly less than in the control group. DISCUSSION
The prevention of cancer is attracting much attention mainly through the institution of mass screening procedures. Success in this field is best exemplified by the dramatic decrease in the incidence of cervical carcinoma since the introduction of the Pap test. An alternative approach to cancer prevention is chemoprevention. It differs from cancer treatment in its goal, which is to lower the incidence of cancer by both the prevention of carcinogen exposure and the arrest
Fig. 3. Histologic features of dysplasia. Atypical cells are present with loss of polarity. (Hematoxylin-eosin stain; original magnification X200.)
612
Inoue et al.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY -November 1993
Fig. 4. Histologic features of carcinoma in situ. Epitheiium is occupied by malignant cells. (Hematoxylineosin stain; original magnification X200.)
Fig. 5. Histologic features of early invasive squamouscell carcinoma. Cancer nestsof various sizesdevelop from lower layers and rete pegs of epithelium.
stain; original magnification X200.)
(Hematoxylin-eosin
or cure of premalignant lesions. Various examinations are necessary to demonstrate that the approach is feasible: studies of the epidemiology of human cancer in relation to certain dietary factors, laboratory studies of the reduction of the incidence of experimental carcinogenesis by these agents, and finally preliminary clinical studies of these agents in highrisk persons or in those with preneoplasia.4 There have been many reports on the chemoprevention of carcinogenesis in several organs of animal models. McGarty5 described three criteria for choosing chemopreventive agents: (1) activity against a broad spectrum of carcinogens and tumor types in animal studies; (2) action via different, complementary mechanisms; and (3) nontoxicity of effective doses in animal studies. They found that butylated hydroxyanisole, Se, and retinoid fulfill these criteria very well. Recently, combined chemoprevention with several compounds has been studied widely, because it reduces the toxicity of each compound and takes advantage of their synergistic actions. Shklar et al2 reported that 9, 10 dimethyl- 1,2 benzanthracene (DMBA)-induced tongue carcinogenesis in hamsters was inhibited by 13-cis-retinoic acid. Goodwin et al3 confirmed their findings and reported inhibition by Se and by a combination of Se and 13-cis retinoic acid. However, they produced tongue carcinomas in hamsters by applying DMBA topically three times a week with a sable brush. In our study, we produced precancerous tongue lesions and carcinomas in rats by Ohne’s method,6 which more closely parallels the development of human tongue carcinomas. In addition
Inoue et al.
ORAL SURGER’YAL MEDICINE ORAL PATHOLOGY Volume 76, Number 5
613
Fig. 6. Gross features of tongue carcinoma in rat given 0.001% 4NQ0 solution for 28 w eeks.
Table 111.Effects of vitamin
A, selenium, treated with 4,NQO for 7 months
BHT, and their combinations on tongue carcinogenesis in rats Incidence % (Number of rats)
Group
Effective number of rats
Mean final body weight (&?I
Intact
Hyperplasia
Dysplasia
6
9 control
10
314.1
(:I
(i,
10 Se alone
8
418.7
(:I
12.5 (1)
11 VA alone
8
481.9
12 BHT alone
I
13 Se+VA 14 Se + BHT 15VA+BHT 16Se+VA-tBHT
c:, 12.5 (1) 28.6
I
Carcinoma in situ
(:I 12.5 (1) 25.0
403.6
12.5 (1) A
(:I 6,
(2)
(2)
4
371.0
(:I
A
(:I
50.0
10
378.0
(:I
1
391.9
10.0 (1) (i
30.0 (3) (i
10
397.4
(i
10.0 (1)
(2) 28.6
Invasive carcinoma
100 (10) 75.0
(6) *50.0 (4) *42.9 (3) 50.0
(2)
(2)
(:I
60.0
14.3 (1) 20.0
85.7
(6)
(2)
(6) 70.0 (7)
*Significantlydifferentfrom controlsat P < 0.05.
to the effect of VA and Se, we examined the effect of BHT, which is classified as a blocking agent.l It is known that blocking agents prevent carcinogenic compounds from reaching critical target sites in the tissues. Furthermore, we designed the present study to examine the inhibitory effects of the three agents in rats given 4NQO for 4 months and for 7 months and compared the findings in the two groups. In the 4-month group, the lesions in rats given chemopreventives tended to be less advanced than in those not given chemopreventives, but the differences were not statistically significant. In the BHT group, many rats had precarncerous lesions, but only one had
cancer. In the Se and VA groups, precancerous and cancerous lesions were seen in some rats, but in these groups some rats had no lesions. These results suggest that BHT acts on the process from the precancerous to the cancerous stage and that VA and Se act on the process from normal epithelium to the precancerous stage. Among all the chemopreventives, the inhibitory effect of VA combined with BHT was best, presumably because the two agents act on different processes and complement each other and because the amount of each was appropriate. Of the rats given 4NQ0 for 7 months, all those in the nonchemopreventive group developed invasive
614
Inoue et al.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY November
1993
Table IV. Macroscopic diameter of invasive carcinomas in rats given 4NQ0 for 7 months
Group
9 control 10 Se alone 11 VA alone 12 BHT alone 13 Se+VA 14 Se + BHT 15VA+BHT 16Se+VA+BHT
Number of invasive carcinomas 11 6 5 3 4 9 1 I
Diameter imm) (Mean + SD) 20.0 *11.8 +5.0 *10.7 *6.5 *9.8 713.9 *12.1
i f t * 2 ril i ?I
Range (mm)
3.6 4.1 2.1 3.1 6.1 6.6 6.8 5.2
15 5283245-
25 18 15 15 17 18 26 22
*Significantly different from controls at P < 0.01, ‘ISignificantly different from controls at P < 0.05.
Microscopic depth of invasive carcinomas in rats given 4NQ0 for 7 months
Table V.
Group 9 control 10 Se alone 11 VA alone 12 BHT alone 13 Se+VA 14Se+BHT 15VA+BHT 16Se+VA+BHT
Fig. 7. Histologicfeaturesofwell-differentiatedsquamous cell carcinoma. Cancer nestsof various sizes and shapesare seenin lamina propria mucosae.(Hematoxylin-eosin stain; original magnification X200.)
cancers, and the incidence was higher than in any of the chemopreventive groups. Moreover, the macroscopic diameters and microscopic depths of the carcinomas were greater than in any of the chemopreventive groups. Thus the chemopreventive agents used in the present study not only inhibit 4NQO-induced tongue carcinogenesis but also probably partially prevent the proliferation of malignant cells as assessed by extent of disease. In all the chemopreventive groups, the inhibition of carcinogenesis was evident. However, the inhibitory effects were not always in proportion to the number of chemopreventive agents used, and the inhibitory effects in the 4-month 4NQ0 groups did not coincide with those in the 7-month 4NQ0 groups. There are several problems related to the use of chemopreventive agents. They have side effects if used in excess or for a long period of time. The carcinogenicity of BHT has not yet been demonstrated, but Ito
Number of invasive ’ carcinomas 11 6 5 3 4 9 7 7
Depth (Me:? 4.40 3.17 *1.71 1.68 2.36 *2.21 2.76 **2.03
SD) 1 ? + f f i ii+
2.30 2.09 0.55 0.69 1.14 0.64 1.06 0.54
Tzy
1.71 1.84 1.00 0.73 1.37 1.16 1.14 1.37
-
9.57 j.77 2.45 2.37 4.18 2.94 4.41 2.79
“Significantly different from controls at P < 0.05. **Significantly different from controls at P < 0.01.
et aL7 reported that butylated hydroxyanisole-induced forestomach carcinomas in F344 male and female rats. Moreover, it has been reported that BHT has different effects on carcinogenesis according to the target organs and the timing of administration.8’ 9 With respect to the clinical application of chemopreventive agents, Wattenberg’ mentioned that chemopreventive agents with low toxicity should be chosen, but they may have little effect. Therefore he stated that retinoids should be selected for use in populations at increased risk for neoplasia. We conclude that when chemoprevention therapy of carcinogenesis is undertaken, it is important to carefully determine the kind of chemopreventive agents, their combination, amount, timing, and duration of administration on the basis of individual conditions. REFERENCES 1. Wattenberg LW. Chemoprevention of cancer. Cancer Res 1985;45:1-8. 2. Shklar G, Marefat P, Kornhauser A, Tricker DP, Wallace KD.
Inoue et al.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY Volume 76. Number 5
3.
4. 5.
6.
7.
Retinoid inhibition of lingual carcinogenesis. ORAL SURC ORALMED ORAL PATHOL 1980;49:325-32. Goodwin WJ, Bordash GD, Huijing F, Altman N. Inhibition of hamster tongue carcinogenesis by selenium and retinoic acid. Ann Otol Rhino1 Laryngol 1986;95:162-6. Bertram JS. The chemoprevention of human cancer: an overview. Prog Clin Biol Res 1990;354A:345-60. McCarty Ml:. A practical prescription for cancer preventionsynergistic use of chemopreventive agents. Med Hypotheses 1984;14:213-25. Ohne M, Satoh T, Yamada S, Takai H. Experimental tongue carcinoma of rats induced by administration of 4-nitroquinoline l-oxide (4NQO) in drinking water. ORAL SURC ORAL MED ORAL PATHOL 1985;59:600-7. Ito N, Fukushima !3, Hagiwara A, Shibata M, Ogiso T. Carcinogenicity of but$ated hydroxianisole in F344 rats. J Nat1 Cancer Inst 1983;70:343-52.
BOUND
VOLUMES
AVAILABLE
615
8. McCormick DL, Major N, Moon RC. Inhibition of 7,12-dimethyl-benz(a)anthracene-induced rat mammary carcinogenesis by concomitant or postcarcinogen anti-oxidant exposure. Cancer Res 1984;44:2858-63. 9. lmaida K, Fukushima S, Shirai T, Ohtani M, Nakanishi K, Ito N. Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and inhibition of y-glutamyl transpeptidase-positive foci development in the liver of rats. Carcinogenesis 1983;4:895-9. Reprint requests: Isao Inoue, MD Department of Otolaryngology Osaka Medical College 2-7 Daigaku-cho, Takatsuki-City Osaka 569, Japan
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OF OTOLARYNGOLOGY,
OSAKA
MEDICAL
COLLEGE
The inhibitory effects of vitamin A, selenium, butyiated hydroxytoiuene, and their combinations on tongue carcinogenesis induced by 4-nitroquinoline l-oxide were examined in Sprague-Dawley rats. After 4 months of administration of 4nitroquinoline l-oxide, a wide range of lesions from hyperplasia to early invasive carcinoma was seen. The lesions were fewer and the carcinomas were less advanced in the rats given preventive treatment. The chemopreventive effect of vitamin A plus butylated hydroxytoluene was most effective. After 7 months of 4-nitroquinoline l-oxide administration, the incidence, the macroscopic diameter, and the microscopic depth of invasion among carcinomas were smaller in the chemopreventive groups than in the nonchemopreventive group. We conclude that vitamin A, selenium, and butylated hydroxytoluene inhibit tongue carcinogenesis induced by 4-nitroquinoline l-oxide in rats. The inhibitory effect of combined chemopreventives is not always superior to that of each chemopreventive agent used alone. (ORAL SURC ORAL MED ORAL PATHOL 1993;76:608-15)
Cancer-prevention measures generally involve the reof causative agents. Because so many carcinogens are present mainly in food, it is very difficult to remove all of them. Therefore chemoprevention is now thought to be a very important way to protect against cancer. Chemoprevention of cancer is a method of cancer control in which the occurrence of this disease is prevented by the administration of one or several chemical compounds. Furthermore, combination chemoprevention with several compounds is often used because it reduces the toxicity of each compound and exerts a synergistic action. Wattenberg’ classified chemopreventive agents into three broad categories: (A) compounds that are effective against a complete carcinogen, (B) compounds that are effective against a tumor promoter, and (C) compounds that inhibit neoplasia when administered shortly before exposure to a carcinogenic compound. Modality A is further subdivided into three categories: (1) compounds that prevent the formation of carcinogens from precursor substances (A-I); (2) compounds that inhibit carcinogenesis by preventing carcinogenic compounds from reaching critical target sites in the tissues (A-II), these inhibitors are called “blocking agents” and exert a barrier function; (3) inhibitors that act after exposure to carcinogenic agents (A-III), these inhibitors are termed “suppressing agents.” Vitamin A (VA) is considered to have A-III, B and C activities, selenium (Se) has A-III activities, and phenols such as butylated hydroxytoluene (BHT) have A-II and B activities. moval
Copyright 3 1993 by Mosby-Year Book, 0030-4220/93/$1 .OO + .lO 7/14/48400
608
Inc
There are many reports on chemoprevention of carcinogenesis by these agents in several organs among animal models, but only a few have been tested on lingual carcinogenesis. Shklar et al2 reported that 9, 10 dimethyl-1,2 benzanthracene (DMBA)-induced tongue carcinoma in hamsters was inhibited by 13-cis-retinoic acid, and Goodwin et a1.3 noted that DMBA-induced tongue carcinomas in hamsters were inhibited by 13-cis-retinoic acid with and without Se. However, in these aforementioned experiments, tongue carcinomas were produced by topical application of DMBA three times weekly with a sable brush, and it is probable that carcinogenesis was influenced by the techniques of the clinicians. Our experimental method of inducing carcinogenesis by the oral administration of 0.00 1% 4-nitroquinoline 1-oxide (4NQO) in drinking water appears to be more physiologic and more similar to the formation of tongue cancer in humans than is the painting method, because cancers are produced by long-term ingestion of only small amounts of carcinogens. In the present study, the inhibitory effects of VA, Se, and BHT singly and in combination were examined in rats with tongue carcinomas induced by 4 or 7 months of 4NQ0 administration. MATERIAL Animals
AND METHODS
One hundred sixty Sprague-Dawley male rats (Shizuoka Experimental Animal Co., Shizuoka,Japan), each weighing about 250 gm at the start of the the experiment, were used. Three to five rats were housed together in a metal cage. The rats were divided into 16 groups of 10 each. MM3 (Funabashi Farm
Inoue et al.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
609
Volume 76, Number .5
Co. Ltd., Chiba, Japan) was provided as the basal diet. Tumor
Table
I. Treatment schedule modulators
inductilon
All of the rats received 0.001% 4NQ0 (Nakarai Chemical Co., Kyoto, Japan), added to their drinking water and taken ad libitum from a brown, lightshielded, polyvinyl bottle for 4 or 7 months. The consumption of 4NQO solution was checked three times a week, and the bottles were refilled with fresh 4NQ0 solution. Administration
of chemopreventive
L 1 2
9 10
3 4 5
11 12 13
6
14
I
15
8
16
None Sodium selenite in drinking water Vitamin A acetate in diet BHT in diet Sodium selenite in drinking water and vitamin A acetate in diet Sodium selenite in drinking water and BHT in diet Vitamin A acetate and BHT in diet Sodium selenite in drinking water, vitamin A acetate and BHT in diet
agents
Table I shows the experimental protocol. Animals were divided into 16 groups according to the chemopreventive regimen and the duration of administration: 4-month,s administration (groups 1 to 8) or 7-months administration (groups 9 to 16). The first variable was sodium selenite (Se) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in the drinking water. Animals in groups 1, 3, 4, 7, 9, 11, 12, and 15 were given 4NQ0 solution without Se;those in groups 2, 5, 6, 8, 10, 13, 14, and 16 were given water supplemented with 3 ppm Se. The secondvariable was BHT (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in the diet. Animals in groups 4, 6, 7, 8, 12, 14, 15, and 16 welrefed basal diet plus 5000 mg of BHT per kilogram of food. The third variable was VA acetate (Wako F’ure Chemical Industries, Ltd., Osaka, Japan) in the diet. Animals in groups 3,5,7,8, 11,13, 15, and 16 were fed basal diet plus 0.1 gm (100,000 IU) of VA per kilogram of diet. Animals in groups 1, 2, 9, and 10 were fed the basal diet without BHT or VA. Thus animals in groups 1 and 9 served as controls, wherieasthose in groups 2 and 10 were given Se alone, those in groups 3 and 11 were given VA alone, those in groups 4 and 12 were given BHT alone, those in groupis5 and 13 were given Se and VA, those in groups 6 and 14 were given Se and BHT, those in groups 7 and II5 were given VA and BHT, and those in groups 8 and 16 were given Se, VA, and BHT. Observations
The numbe:r of surviving rats 4 or 7 months after the beginning of the experiment was the “effective number.” At 4 months or 7 months, the rats were killed by ethelr inhalation and weighed. The tongue, palate, gingiva, buccal mucosa, pharynx, larynx, trachea, and esophagus were removed and observed grossly. For light microscopy, the tongues were fixed in 20% formalin solution, dehydrated with alcohol, and embedded in paraffin. Sections were cut in 3 pm slices and stained with hematoxylin and eosin.
Macroscopic and microscopic tumor progression
examination
of
At autopsy, the longest diameter of the carcinomatous lesions in the tongue was measured by observation of the gross appearance acrossthe surface of the specimen and was defined as the macroscopic diameter. Tongue tissues were cut vertically through the macroscopic diameter and formalin-fixed, paraffinembedded 3 pm sections that contained the cut surface were made and stained with hemotoxylin and eosin. In the hemotoxylin and eosin preparations, the vertical distance between the top and the bottom of the lesions was measured microscopically with a micrometer. The vertical distance was defined as the microscopic depth. The degree of tumor progression of squamous cell carcinomas was evaluated from the macroscopic diameter and the microscopic depth. Statistical
analysis
Cancer incidences were compared by the x2 test, Statistical comparisons of the macroscopic diameter and the microscopic depth of invasive carcinomas were performed with Student’s t test. RESULTS Histopathologic
findings
at 4 months
Table II shows the histopathologic characteristics and the incidence of lesions after 4 months of 4NQ0 administration. Normal tongue (Fig. l), hyperplasia (Fig. 2), dysplasia (Fig. 3), carcinoma in situ (Fig. 4), and invasive carcinoma (Fig. 5) were seen. In the
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Inoue et al.
Fig. 1. Histologic features of normal tongue of rat. (Hematoxylih-eosin stain; original magnification x200.)
Table II. Effects of vitamin A, selenium, BHT, and their combinations
on tongue carcinogenesis in rats
treated with 4NQ0 for 4 months Incidence % (Number of rats)
Group 1 control
Effective number
Mean jinal body weight
of rats
(9)
9
415.6
Intact
(:I
2 Se alone
10
405.6
20.0
3 VA alone
10
451.2
40.0
9
426.7
5Se+VA
10
413.5
6Se+BHT
9
407.2
(2) (4) 4 BHT alone
7VA+BHT
8
436.3
8Se+VA+BHT
9
394.2
A 20.0
Hyperplasia
iI 10.0 (1) 10.0 (1) 11.1 (1) 10.0
Dysplasia 55.6 (5) 20.0
(2) (2)
(2)
(3) 40.0 (4)
20.0
(21 11.1
44.4
(:I
(2)
(2)
nonchemopreventive group (group 1), dysplasia was seen in five rats and carcinoma in four rats. In contrast, in the chemopreventive groups, a wide range of lesions from hyperplasia to invasive carcinoma was seen, and some rats had no lesions. When the lesions were divided into five histologic groups according to grade of progression (normal, hyperplasia, dysplasia, carcinoma in situ, and invasive carcinoma), the lesions in the chemopreventive groups were inclined to be less advanced than those in the nonchemopreventive group, although the number of rats with lesions
33.3
(7) 40.0
77.7
(4) 22.2
A
Invasive carcinomas
10.0 (1) 11.1 (1) 10.0 (11
(1)
c:,
10.0 (1) 20.0
22.2
(4)
11.1 (1)
20.0
(2) 50.0
Carcinoma in situ
37.5
33.3
(3) 33.3
(3)
(3)
(:I
(1) c&
11.1 (1)
(41 12.5 (1) 22.2
(2)
in each chemopreventive group was nearly equal to that in the nonchemopreventive group. Tongue carcinogenesis was inhibited to the greatest degree in the VA plus BHT group. Histopathologic
findings
at 7 months
Table III shows the histopathologic characteristics and the incidence of lesions after 7 months of 4NQ0 administration. In the nonchemopreventive group (group 9), invasive cancers were produced in all rats. In the VA only group (group 11) and the BHT only
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Fig. 2. Histologic features of hyperplasia. Slight extension of rete pegsand thickening of epithelium are seen. (Hematoxylin-eosin stain; original magnification x200.)
group (group 12), significantly fewer invasive carcinomas developed than in the nonchemopreventive group (group 9). The inhibitory effect of the combined chemopreventives was not always superior to that of each chemopreventive agent alone. The tumors developed mainky on the dorsum of the tongue, especially around the torus linguae (Fig. 6). Most squamous cell carcinomas were well differentiated (Fig. 7); a few were moderately differentiated. No carcinoma was poorly differentiated. The macroscopic diameters of invasive carcinomas are listed in Table IV. They were significantly smaller in all chemopreventive groups (groups 10 to 16) than in the nonchemopreventive group (group 9). The microscopic depth of invasive carcinomas is shown in Table V. It tended to be less in all chemopreventive groups. In the. VA alone (group ll), Se plus BHT (group 14), and Se plus VA plus BHT (group 16) groups, it was significantly less than in the control group. DISCUSSION
The prevention of cancer is attracting much attention mainly through the institution of mass screening procedures. Success in this field is best exemplified by the dramatic decrease in the incidence of cervical carcinoma since the introduction of the Pap test. An alternative approach to cancer prevention is chemoprevention. It differs from cancer treatment in its goal, which is to lower the incidence of cancer by both the prevention of carcinogen exposure and the arrest
Fig. 3. Histologic features of dysplasia. Atypical cells are present with loss of polarity. (Hematoxylin-eosin stain; original magnification X200.)
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ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY -November 1993
Fig. 4. Histologic features of carcinoma in situ. Epitheiium is occupied by malignant cells. (Hematoxylineosin stain; original magnification X200.)
Fig. 5. Histologic features of early invasive squamouscell carcinoma. Cancer nestsof various sizesdevelop from lower layers and rete pegs of epithelium.
stain; original magnification X200.)
(Hematoxylin-eosin
or cure of premalignant lesions. Various examinations are necessary to demonstrate that the approach is feasible: studies of the epidemiology of human cancer in relation to certain dietary factors, laboratory studies of the reduction of the incidence of experimental carcinogenesis by these agents, and finally preliminary clinical studies of these agents in highrisk persons or in those with preneoplasia.4 There have been many reports on the chemoprevention of carcinogenesis in several organs of animal models. McGarty5 described three criteria for choosing chemopreventive agents: (1) activity against a broad spectrum of carcinogens and tumor types in animal studies; (2) action via different, complementary mechanisms; and (3) nontoxicity of effective doses in animal studies. They found that butylated hydroxyanisole, Se, and retinoid fulfill these criteria very well. Recently, combined chemoprevention with several compounds has been studied widely, because it reduces the toxicity of each compound and takes advantage of their synergistic actions. Shklar et al2 reported that 9, 10 dimethyl- 1,2 benzanthracene (DMBA)-induced tongue carcinogenesis in hamsters was inhibited by 13-cis-retinoic acid. Goodwin et al3 confirmed their findings and reported inhibition by Se and by a combination of Se and 13-cis retinoic acid. However, they produced tongue carcinomas in hamsters by applying DMBA topically three times a week with a sable brush. In our study, we produced precancerous tongue lesions and carcinomas in rats by Ohne’s method,6 which more closely parallels the development of human tongue carcinomas. In addition
Inoue et al.
ORAL SURGER’YAL MEDICINE ORAL PATHOLOGY Volume 76, Number 5
613
Fig. 6. Gross features of tongue carcinoma in rat given 0.001% 4NQ0 solution for 28 w eeks.
Table 111.Effects of vitamin
A, selenium, treated with 4,NQO for 7 months
BHT, and their combinations on tongue carcinogenesis in rats Incidence % (Number of rats)
Group
Effective number of rats
Mean final body weight (&?I
Intact
Hyperplasia
Dysplasia
6
9 control
10
314.1
(:I
(i,
10 Se alone
8
418.7
(:I
12.5 (1)
11 VA alone
8
481.9
12 BHT alone
I
13 Se+VA 14 Se + BHT 15VA+BHT 16Se+VA-tBHT
c:, 12.5 (1) 28.6
I
Carcinoma in situ
(:I 12.5 (1) 25.0
403.6
12.5 (1) A
(:I 6,
(2)
(2)
4
371.0
(:I
A
(:I
50.0
10
378.0
(:I
1
391.9
10.0 (1) (i
30.0 (3) (i
10
397.4
(i
10.0 (1)
(2) 28.6
Invasive carcinoma
100 (10) 75.0
(6) *50.0 (4) *42.9 (3) 50.0
(2)
(2)
(:I
60.0
14.3 (1) 20.0
85.7
(6)
(2)
(6) 70.0 (7)
*Significantlydifferentfrom controlsat P < 0.05.
to the effect of VA and Se, we examined the effect of BHT, which is classified as a blocking agent.l It is known that blocking agents prevent carcinogenic compounds from reaching critical target sites in the tissues. Furthermore, we designed the present study to examine the inhibitory effects of the three agents in rats given 4NQO for 4 months and for 7 months and compared the findings in the two groups. In the 4-month group, the lesions in rats given chemopreventives tended to be less advanced than in those not given chemopreventives, but the differences were not statistically significant. In the BHT group, many rats had precarncerous lesions, but only one had
cancer. In the Se and VA groups, precancerous and cancerous lesions were seen in some rats, but in these groups some rats had no lesions. These results suggest that BHT acts on the process from the precancerous to the cancerous stage and that VA and Se act on the process from normal epithelium to the precancerous stage. Among all the chemopreventives, the inhibitory effect of VA combined with BHT was best, presumably because the two agents act on different processes and complement each other and because the amount of each was appropriate. Of the rats given 4NQ0 for 7 months, all those in the nonchemopreventive group developed invasive
614
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ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY November
1993
Table IV. Macroscopic diameter of invasive carcinomas in rats given 4NQ0 for 7 months
Group
9 control 10 Se alone 11 VA alone 12 BHT alone 13 Se+VA 14 Se + BHT 15VA+BHT 16Se+VA+BHT
Number of invasive carcinomas 11 6 5 3 4 9 1 I
Diameter imm) (Mean + SD) 20.0 *11.8 +5.0 *10.7 *6.5 *9.8 713.9 *12.1
i f t * 2 ril i ?I
Range (mm)
3.6 4.1 2.1 3.1 6.1 6.6 6.8 5.2
15 5283245-
25 18 15 15 17 18 26 22
*Significantly different from controls at P < 0.01, ‘ISignificantly different from controls at P < 0.05.
Microscopic depth of invasive carcinomas in rats given 4NQ0 for 7 months
Table V.
Group 9 control 10 Se alone 11 VA alone 12 BHT alone 13 Se+VA 14Se+BHT 15VA+BHT 16Se+VA+BHT
Fig. 7. Histologicfeaturesofwell-differentiatedsquamous cell carcinoma. Cancer nestsof various sizes and shapesare seenin lamina propria mucosae.(Hematoxylin-eosin stain; original magnification X200.)
cancers, and the incidence was higher than in any of the chemopreventive groups. Moreover, the macroscopic diameters and microscopic depths of the carcinomas were greater than in any of the chemopreventive groups. Thus the chemopreventive agents used in the present study not only inhibit 4NQO-induced tongue carcinogenesis but also probably partially prevent the proliferation of malignant cells as assessed by extent of disease. In all the chemopreventive groups, the inhibition of carcinogenesis was evident. However, the inhibitory effects were not always in proportion to the number of chemopreventive agents used, and the inhibitory effects in the 4-month 4NQ0 groups did not coincide with those in the 7-month 4NQ0 groups. There are several problems related to the use of chemopreventive agents. They have side effects if used in excess or for a long period of time. The carcinogenicity of BHT has not yet been demonstrated, but Ito
Number of invasive ’ carcinomas 11 6 5 3 4 9 7 7
Depth (Me:? 4.40 3.17 *1.71 1.68 2.36 *2.21 2.76 **2.03
SD) 1 ? + f f i ii+
2.30 2.09 0.55 0.69 1.14 0.64 1.06 0.54
Tzy
1.71 1.84 1.00 0.73 1.37 1.16 1.14 1.37
-
9.57 j.77 2.45 2.37 4.18 2.94 4.41 2.79
“Significantly different from controls at P < 0.05. **Significantly different from controls at P < 0.01.
et aL7 reported that butylated hydroxyanisole-induced forestomach carcinomas in F344 male and female rats. Moreover, it has been reported that BHT has different effects on carcinogenesis according to the target organs and the timing of administration.8’ 9 With respect to the clinical application of chemopreventive agents, Wattenberg’ mentioned that chemopreventive agents with low toxicity should be chosen, but they may have little effect. Therefore he stated that retinoids should be selected for use in populations at increased risk for neoplasia. We conclude that when chemoprevention therapy of carcinogenesis is undertaken, it is important to carefully determine the kind of chemopreventive agents, their combination, amount, timing, and duration of administration on the basis of individual conditions. REFERENCES 1. Wattenberg LW. Chemoprevention of cancer. Cancer Res 1985;45:1-8. 2. Shklar G, Marefat P, Kornhauser A, Tricker DP, Wallace KD.
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3.
4. 5.
6.
7.
Retinoid inhibition of lingual carcinogenesis. ORAL SURC ORALMED ORAL PATHOL 1980;49:325-32. Goodwin WJ, Bordash GD, Huijing F, Altman N. Inhibition of hamster tongue carcinogenesis by selenium and retinoic acid. Ann Otol Rhino1 Laryngol 1986;95:162-6. Bertram JS. The chemoprevention of human cancer: an overview. Prog Clin Biol Res 1990;354A:345-60. McCarty Ml:. A practical prescription for cancer preventionsynergistic use of chemopreventive agents. Med Hypotheses 1984;14:213-25. Ohne M, Satoh T, Yamada S, Takai H. Experimental tongue carcinoma of rats induced by administration of 4-nitroquinoline l-oxide (4NQO) in drinking water. ORAL SURC ORAL MED ORAL PATHOL 1985;59:600-7. Ito N, Fukushima !3, Hagiwara A, Shibata M, Ogiso T. Carcinogenicity of but$ated hydroxianisole in F344 rats. J Nat1 Cancer Inst 1983;70:343-52.
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8. McCormick DL, Major N, Moon RC. Inhibition of 7,12-dimethyl-benz(a)anthracene-induced rat mammary carcinogenesis by concomitant or postcarcinogen anti-oxidant exposure. Cancer Res 1984;44:2858-63. 9. lmaida K, Fukushima S, Shirai T, Ohtani M, Nakanishi K, Ito N. Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and inhibition of y-glutamyl transpeptidase-positive foci development in the liver of rats. Carcinogenesis 1983;4:895-9. Reprint requests: Isao Inoue, MD Department of Otolaryngology Osaka Medical College 2-7 Daigaku-cho, Takatsuki-City Osaka 569, Japan
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