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Melatonin and colon carcinogenesis
Exp Toxic Pathol 1999; 51: 47-52 Gustav Fischer Verlag
ILaboratory of Experimental Tumors, N. N. Petrov Research Institute of Oncology, St. Petersburg, Russia 2Laboratory of Pathomorphology, Medical Radiological Research Center, Obninsk, Russia 3Section of Clinical Pharmacology, UniversiUits-Frauenklinik, Ttibingen, Germany
Melatonin and colon carcinogenesis II. Intestinal melatonin-containing cells and serum melatonin level in rats with 1,2-dimethylhydrazine-induced colon tumors v.
N. ANISIMOyl, I. M. KYETNOy 2 , N. K. CHUMAKOYA2 , T. V. KYETNAYA 2 , A. o. MOLOTKOy 2 , N. A. POGUDINA2 , I. G. POPOVICH!, V. V. POPUCHIEy2, M. A. ZABEZHINSKI 1, H. BARTSCH3, and C. BARTSCH3 With 2 figures and 3 tables Received: September 8, 1997; Accepted: November 3, 1997 Address for correspondence: Prof. V. N. ANISIMOV, M. D., D. Sc., Lab. Exp. Tumors, N. N. Petrov Research Institute of Oncology, Pesochny-2, St. Petersburg 189646, Russia; Tel. : 812/437-8607, Fax: 812/437-8947, E-mail: [email protected] Key words: Melatonin; Colon carcinogesis; Carcinogenesis, colon; Intestinum, melatonin-containing cells; 1,2-Dimethylhydrazine-induced colon tumors.
Summary Two-month-old outbred female LIO rats were injected weekly with a single dose of 1,2-dimethylhydrazine (DMH; 21 mglkg of body weight) administered s.c. for 15 consecutive weeks. From the day of the 1st injection of the carcinogen the part of rats were given five days a week during the night time (from 18.00 h to 08.00 h) melatonin dissolved in tap water, 20 mg/l. The experiment was terminated in 6 months after the first injection of the carcinogen. The concentration of melatonin in the serum was estimated by radioimmunoassay in rats exposed to DMH alone or in intact control rats in the morning (between 10.00 and 11.00 hours) and night (between 24.00 and 01.00 hours) time. Number of melatonin-containing cells (M-cells) and their optical density were estimated by immunohistology in normal mucosa of glandular stomach, duodenum, ileum and descending colon of tumor-bearing animals from groups exposed to DMH or DMH + melatonin. It was shown that serum melatonin levels in rats with colon tumors was increased as compared with controls. However there was no diurnal rhythm of serum melatonin of colon tumor-bearing animals as compared to intact controls. The number ofM-cells was decreased in all tissues studied in rats with DMH-induced colon tumors in comparison to corresponding controls: by 2.0 times in stomach, by 1.8 time in duodenum, by 1.3 times in ileum, and by 1.8 times in colon. In ileum and colon of rats treated with DMH+melatonin the number of M-cells was similar to control level whereas in stomach and duodenum this number was significantly higher than that in rats treated with DMH alone, but less than in corresponding controls.
Relative content of melatonin in enterochromaffin cells of all parts of gastrointestinal tract evaluated as optical density of the cells and was decreased in rats exposed with DMH alone in comparison to the controls and was normalized and similar to the norm level in rats treated with DMH + melatonin. Thus, exogenous melatonin prevent a decrease in numbers of melatonin-containing cells as was observed in gastrointestinal tract (OIT) of rats exposed to DMH. This preventive action of melatonin correlated well with its anticarcinogenic effect.
Introduction In our previous experiments we have demonstrated the inhibitory effect of melatonin on intestinal carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in rats (1). This effect was manifested by the decrease of the incidence and multiplicity of bowel tumors, mainly in colon, by a decreasing rate of invasion rate and dimensions of colon carcinomas and by an increase of its differentiation. The mechanism of this effect is unknown. It was shown that bowels is one of main sources of melatonin in the body (2, 3) and specific binding sites for melatonin in the mouse colon have been demonstrated (4). Pinealectomy was followed by the increase of the crypt cell proliferation in rat bowel (colon including) which persisted at least 6 months after the operation (5). Also melatonin exert an inhibitory effect on cell proliferation in the rodent colon Exp Toxic Patho151 (1999) 1
47
(6). There is some evidence of impaired circadian rhythm of melatonin in colon cancer patients (7) Clinical data have shown that melatonin may be effective as a secondary line of therapy in cancers of digestive tract, particularly metastatic colorectal cancer (8). Thus, we decided to study a content of melatonin in serum of rats with DMHinduced intestinal tumors and evaluate the effect of exogenous melatonin on a number of melatonin-containing cells in gastrointestinal tract (GIT) as well on the carcinogenesis induced by the carcinogen in bowels.
Material and methods Animals: Two-month-old outbred female LIO rats from the Animal Department of Petrov Research Institute of Oncology, St. Petersburg, were used in the experiments. The characteristics of these rats have been described elsewhere (9). Rats were kept in polypropylene cages, seven in each under a 14/10 h light/dark regimen at 22 ± 2 DC. They received standard lab chow and tap water ad libitum. Experiment: Rats were randomly subdivided into 3 groups. Animals from group 1 were once a week exposed to 15 subcutaneous injections of DMH dihydrochloride (Sigma, U.S.A.) at a single dose of21 mg/kg of body weight (calculated as base). DMH was ex tempore dissolved in normal saline and neutralized with sodium bicarbonate (pH = 7.0). Rats from the group 2 were exposed to DMH as animals from group 1 and additionally from the day of the 1st injection of the carcinogen the part of rats were given melatonin (Sigma, U.S.A.) 5 days a week during the night time (from 18.00 h to 08.00 h) dissolved in tap water, 20 mg/I. This regimen and dosage provide the rhythmical pattern of serum melatonin concentration followed by an anticarcinogenic effect (1, 10, 17). Rats from group 3 were once a week exposed to 15 injections of saline and served as a controls. The experiment was terminated in 6 months after the first injection ofthe carcinogen. Ten rats exposed to DMH alone (group 1) and 10 controls rats (group 3) were killed by decapitation between 10.00 and 11.00 hours and another 20 rats from groups 1 and 3 were sacrificed between 24.00 and 01.00 hours at a dim red light. Blood was collected, centrifugated and samples of serum were stored at -20 DC. The concentration of melatonin in the serum was estimated by radioimmunoassay as described earlier (11). Pathological investigation: All animals killed or found dead before the end of the experiment were autopsied. Intestines were open longitudinally. Tumor number, position and dimensions were recorded on special charts as described elsewhere (12). All tumors and other tissues with macroscopically revealed lesions were fixed in 10 % neutral formaline and, after routine histological treatment, were embedded into paraffin. Five-to-seven ~ thick sections through the middle part of each tumor were stained with haematoxylin and eosin. The neoplasms were classified according to the IARC recommendations (12). Samples of tumor-free tissues from glandular stomach, duodenum, ileum and descending colon from rats exposed to DMH alone, DMH+melatonin and from controls (10 per group) were collected for immunohistochemical study. Immunohistochemistry assay for melatonin-containing cells: Only histologically normal tissues were analyzed immunohistochemically. Deparaffined 5 ~ slides were 48
Exp Toxic Pathol51 (1999) 1
stained with haematoxylin and eosin, or with Masson's argentaffin method for detection of enterochromaffin (EC) cells containing melatonin and serotonin. Immunohistochemical reaction for melatonin was performed according to (13). Deparaffined and dehydrated slides were treated with 0,5 % HzO z in methanol during 30 min. for blockade of endogenous peroxidase. Then slides were washed in 0.1 M phosphate buffer (PB, pH = 7.2) and incubated in 10 % normal rabbit serum during 20 min. for attenuation of non-specific binding of antibodies. Immunohistochemical reaction was performed with ultraspecific rabbit polyclonal antimelatonin antiserum (CIDtech Research Inc., Mississauga, Canada). Slides were incubated during 2 hours with primary antiserum (titer 1:100) at the room temperature with subsequent washing (3 times) with PB. Then slides were incubated with biotinylated goat antirabbit IgG (Dako, Glostrup, Denmark, titer 1: 200) 1 hour and 3 times were washed with PB. Then slides were treated during 30 min. with peroxidase-conjugated avidin (Dako, titer 1: 1000). Staining was revealed after treatment of slides with 0,025 % 3,3-diaminobenzidine tetrahydrochloride (DAB, Dako) and 0,01 % HzO z during 10 min. Additional staining was made with Mayer hematoxylin. Morphometric analysis of melatonin-containing cells (M-cells): Morphometric assay was automatically performed with system of computer analysis for morphological images MORPHOSTAR with using of applied software COLQUANT program (Imstar S.A., Paris, France). This system is a comprehensive modular system for multimodal image acquisition, display processing and analysis and it was successfully used for true colour representation and interpretation of various histological data from the results of immunohistochemical study of structure-functional organization of APUD cells in tumor growth (14, 15). All parameters were detected on 3 slides from each sample in 10 randomly selected microscopic fields (each optical microscopic field was 0.785 mmZ) at magnification x200. The following morphometric parameters were evaluated: 1) Mucosa square, ~ z; 2) Square ofM-cells (total square occupied by M-cells), Ilz; 3) Ratio ofM-cells square to mucosa square; 4) Mean number of M -cells per 1 optical field; 5) Optical density ofM-cells (units). Optical density was calculated as D = ach, where D - optical density; a - apportioned parameters of an absorbtion of a compound; c - concentration of a compound; h - slide thickness. This parameter, together with a number of Mcells gives an important information on tissue hypo- or hyperplasia as well as on changes in its functional activity (14, 15). All caluclations were performed automatically using above mentioned program. Statistical analysis: Morphometric results were treated statistically using program STATISTICA 4.3 (Tulsa, U.S.A.) Wylcoxon U-test and Student's t-test (16) were used as well.
Results Effect of melatonin on DMH-induced intestinal carcinogenesis The majority of colon tumors were localized in descending colon. Macroscopically colon tumors were exo-
Table 1. Intestinal tumor incidence and localization in rats exposed to 1,2-dimethylhydrazine (DMH) and melatonin (MLT).
Parameters
DMH
DMH+MLT
Number of rats Tumor localization: Duodenum: No. of tumor-bearing rats No. of tumors Jejunum and ileum: No. of tumor-bearing rats No. of tumors Colon (all parts): No. of tumor-bearing rats No. of tumors Ascending colon: No. of tumor-bearing rats No. of tumors De~cending colon: No. of tumor-bearing rats No. of tumors Rectum: No. of tumor-bearing rats No. of tumors
25
23
8 (32.0 %) 9
5 (21.7 %) 5
4 (16.0 %) 5
1 (4.3 %)* 1
25 (l00 %) 237
23 (100 %) 137
23 (92.0 %) 15 (65.2 %)** 17 56 25 (100 %) 164
23 (100 %) 101
11 (44.0 %) 11 (47.8 %) 17 19
The difference with rats exposed to DMH alone is significant. * p < 0.05; ** p< 0.01. Table 2. Serum melatonin level in rats with colon tumors induced by 1,2-dimethylhydrazine and in control rats.
Treatment Serum melatonin level [nM/lJ
P
10.00 hours
24.00 hours
10 hrs vs 24 hrs
Controls (saline)
115 ± 35.7 (n = 10)
307 ± 59.7 (n = 10)
< 0.005
DMH
361 ± 139.4 (n = 10)
606 ± 188.7 (n =8)
> 0.05
P (controls vsDMH) < 0.005
>0.05
phytic or endophytic, however some cases of ulcerativeinfiltrative forms were detected. Microscopically we found different types of malignant tumors of intestines with absolute prevalence of adenocarcinomas. All neoplasia in small intestines were adenocarcinomas. The data on intestinal tumor incidence and localization are presented in table 1. Administration of melatonin in drinking water failed influence the total incidence of colon tumors. However incidence of carcinomas in ascending colon was significantly reduced (p < 0.01). The multiplicity of total colon tumors per rat as well as the mean number of tumors in ascending and descending colon per rat were also decreased under the influence of melatonin (Group 2 vs Group 1, p < 0.01). In the same experiment melatonin
slightly decreased the depth of tumor invasion and increased number of highly differentiated colon carcinomas induced by DMH (for details see ref. 1). The percentage of small tumors in descending colon among rats from group 2 was higher than that in group 1 (1). Treatment with melatonin was followed also by the decrease in the multiplicity of DMH-induced tumors of duodenum (Group 2 vs Group 1, p < 0.05) and by the decrease in the incidence of jejunum and ileum tumors (Group 2 vs Group 1, p < 0.05) (table 1).
Serum melatonin level in rats with DMH-induced colon tumors The results of RIA of serum melatonin are given in the table 2. In general, melatonin level in rats with colon tumors (group 1) was increased as compared with controls (group 3). There is significant (by 2.7 times, p < 0.005) elevation of the night level of melatonin as compared to the morning level in control rats. In rats with DMH-induced colon tumors the morning level of melatonin was increased (p < 0.005) as compared with timematched controls. However there was no significant elevation of night level of melatonin in comparison to the morning in colon tumor-bearing animals.
Melatonin-containing cells in intestinal mucosa of rats with DMH-induced colon tumors Immunohistochemical study revealed that the number of melatonin-containing cells (M-cells) was different in various parts of gastrointestinal tract and was distributed as stomach> duodenum> colon> ileum. In all tissues of rats with DMH-induced colon tumors (group 1) the number of M-cells was decreased in comparison to corresponding controls (group 3): by 2.0 times in stomach, by 1.8 time in duodenum, by 1.3 times in ileum and by 1.8 times in colon (table 3; fig. 1). In ileum and colon ofrats treated with DMH + melatonin (group 2) the number of M -cells was similar to control level whereas in stomach and duodenum this number was significantly higher than that in rats treated with DMH alone, but less than in corresponding controls. A calculation of relative content of melatonin in enterochromaffin cells of all parts of gastrointestinal tract evaluated as optical density of the cells (14) reveals a tendency toward the decrease in rats exposed with DMH alone in comparison to the controls and was normalized and similar to the norm level in rats treated with DMH + melatonin (table 3). Some examples of immunohistochemical pattern of M-cells in gastrointestinal tract of control, DMH- and DMH + melatonin-treated rats are given on fig. 2 (a, b, c). Exp Toxic Pathol 51 (1999) 1
49
Table 3. Effect of 1,2-dimethylhydrazine (DMH) and melatonin (MLT) on morphometric parameters in gastrointestinal tract of rats. Organ
Treatment
Mucosa square analized flm2 (M±m)
Square ofM-cells flm 2 (M±m)
Ratio of M-cells square to mucosa square
Number of M-cells per optical field (M±m)
Optical density of M-cells, units (M±m)
Stomach
Control DMH DMH+MLT
10614 ± 741 11021 ± 567 11132 ± 646
122 ± 14 109 ± 12 119 ± 14
0.018 ± 0.002 0.010 ± 0.002 0.011 ± 0.002
35.1 ± 1.8 17.3 ± 1.3" 27.5 ± 2.2bc
0.145 ± 0.034 0.111 ± 0.021 0.139 ± 0.030
Duodenum
Control DMH DMH+MLT
11316 ± 732 10822 ± 1695 10046 ± 1384
120 ± 15 119 ± 27 112 ± 12
0.012 ± 0.001 0.013 ± 0.002 0.013 ± 0.003
28.2 ± 1.1 15.3 ± 1.5" 23 .5 ± 0.9 bd
0.139 ± 0.033 0.113 ± 0.026 0.172 ± 0.044
Ileum
Control DMH DMH+MLT
11644 ± 922 11435 ± 912 10564 ± 1011
92±9 87 ± 5 93 ±2
0.008 ± 0.001 0.008 ± 0.001 0.009 ± 0.002
16.3 ± 0.9 12.1 ± 0.7" 16.5 ± 1.1 e
0.116 ± 0.019 0.084 ± 0.016 0.119 ± 0.022
Colon
Control DMH DMH+ MLT
11076 ± 876 10978 ± 785 11679 ± 769
107 ± 12 86± 8 103 ± 10
0.010 ± 0.013 0.008 ± 0.011 0.009 ± 0.013
24.6 ± 1.7 13.9 ± 1.6' 22.7 ± 1.9c
0.139 ± 0.028 0.054 ± 0.03Sf 0.135 ± 0.026
M-cells: melatonin-containing cells. The difference with controls is significant: a: p < 0.001 ; b: p < 0.01; f: 0.1 < P < 0.05.
10
trol
.0\llH
nurn
II
m
In
Fig. 1. Number of melatonin-containing cells in gastrointestinal tracts of rats with colon tumors exposed to DMH and DMH + melatonin. * The difference between controls and experimental group is significant, p < 0.01; x The differences between rats exposed to DMH alone and DMH + melatonin is significant, p < 0.05.
Discussion Our data have shown that in rats with DMH-induced colon tumors the morning level of serum melatonin is increased in comparison to the controls. At the same time there are no clear-cut circadian rhythm of melatonin in colon tumor-bearing animals. These findings coincide with clinical observations, which demonstrated that serum level of this indole hormone is increased in colo50
Exp Toxic Pathol51 (1999) 1
rectal cancer patients and there were disturbances of circadian rhythm of melatonin excretion in such patients (2,7). At the same time, the decrease in the number ofMcells was observed in all part of gastrointestinal tract of animals with DMH-induced colon tumors and a relative decrease of melatonin content in these cells (table 3). It is possible to suggest that increased serum level of melatonin in DMH-treated rats (group 2) is a compensatory reaction of pineal gland on DMH-induced decrease in local level of melatonin in gastrointestinal tract. It has been shown that mammalian gastrointestinal tract contain much more melatonin than the pineal gland and enterochromaffine cells are the main source of melatonin in the organism (15). In spite of data demonstrating an active participation of melatonin in adaptive response and in pathophysiology, the normal function of extrapineal melatonin as well as the feedback mechanisms between pineal and GIT melatonin production are largely unknown. It is possible to suggest that decrease of number of Mcells and their functional activity in rat colon of rats exposed to DMH could play an important role in its carcinogenic effect. Treatment with melatonin prevent the decrease in the content ofM-cells in gastrointestinal tract of rat exposed to DMH (table 3; fig. 1) that correlated with anticarcinogenic effect of melatonin (1; table 1). The anticarcinogenic potential of melatonin has been demonstrated both in vivo and in vitro in relation to mammary carcinoma (17, 18). It has been suggested that melatonin regulates growth of mammary tumors through several mechanisms including direct modulation of mitotic activity, inhibition of serum estrogen and prolactin level,
point in its anticarcinogenic potential. It was shown also that melatonin inhibits the production of DNA-adducts of the carcinogen safrole in rodents (22). The production of highly reactive free radicals and decrease of activity of some enzymes of anti oxidative defence system has been observed during DMH-induced carcinogenesis in rats and mice (23-26). Antioxidative properties of melatonin (27, 28) may be also an important factor of it anticarcinogenic potential. In the same model we have shown that both in serum of rats exposed to DMH alone the level of diene conjugates and Schiff's bases was significantly increased as compared with controls. In colon tissue of rats with DMH-induced colon carcinomas the level of products of lipid and protein peroxidations as well as NO-synthase activity was significantly increased, and total antioxidative activity was decreased compared with controls, whereas treatment with melatonin resulted in the normalization of these parameters (29, 30). Recently it was demonstrated that melatonin inhibits mutagenic effect of DMH evaluated in two in vivo tests: chromosome aberration in bone marrow cells and anomal sperm heads (31). Thus, exogenous melatonin prevents a decrease in numbers of melatonin-containing cells as was observed in gastrointestinal tract (GIT) of rats exposed to DMH. This preventive action of melatonin correlated well with its anticarcinogenic effect. Some aspects of the problem need an additional experiments for elucidations. It is necessary to study a direct effect of DMH on melatonin production in rat colon as well as an effect of exogenous melatonin on the production of melatonin in colon. Also it is important to examine the effect of melatonin on activity of 0 6- alkylguanine transferase as well as a methylation of DNA bases in colon cells. We are intending to get anwer for these questions. Acknowledgements: This work was supported in part by grant 6/96 from The Russian Ministry of Science State Program "National Priorities in Medicine and Health". Authors are very thankful to Prof. G.M.Brown, Clarke Institute of Psychiatry, Toronto, Ontario, Canada for providing antiserum to melatonin. Fig. 2. Melatonin-containing cells in rat duodenum. Immunohistochemical staining, x 200. a: control (group 3); b: the decrease of cell number after DMH treatment (group 1); c: The increase (almost to control parameters) of cell number induced by exogenic melatonin in DMH-treated rats (group 2).
and the modulation of immune system (17, 18). The mechanism of the anti carcinogenic effect of melatonin on colon carcinogenesis is unknown. Melatonin inhibits cell proliferation in the rodent colon (6) and some positive response to melatonin treatment was observed in digestive tract of cancer patients (8). The presence of specific binding sites for melatonin in the mouse colon has been demonstrated as well (4). Melatonin also enhances cellto-cell junction contacts (19), and modify the activity of cytochromes b5 and P450 (20, 21) that may be a critical
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ILaboratory of Experimental Tumors, N. N. Petrov Research Institute of Oncology, St. Petersburg, Russia 2Laboratory of Pathomorphology, Medical Radiological Research Center, Obninsk, Russia 3Section of Clinical Pharmacology, UniversiUits-Frauenklinik, Ttibingen, Germany
Melatonin and colon carcinogenesis II. Intestinal melatonin-containing cells and serum melatonin level in rats with 1,2-dimethylhydrazine-induced colon tumors v.
N. ANISIMOyl, I. M. KYETNOy 2 , N. K. CHUMAKOYA2 , T. V. KYETNAYA 2 , A. o. MOLOTKOy 2 , N. A. POGUDINA2 , I. G. POPOVICH!, V. V. POPUCHIEy2, M. A. ZABEZHINSKI 1, H. BARTSCH3, and C. BARTSCH3 With 2 figures and 3 tables Received: September 8, 1997; Accepted: November 3, 1997 Address for correspondence: Prof. V. N. ANISIMOV, M. D., D. Sc., Lab. Exp. Tumors, N. N. Petrov Research Institute of Oncology, Pesochny-2, St. Petersburg 189646, Russia; Tel. : 812/437-8607, Fax: 812/437-8947, E-mail: [email protected] Key words: Melatonin; Colon carcinogesis; Carcinogenesis, colon; Intestinum, melatonin-containing cells; 1,2-Dimethylhydrazine-induced colon tumors.
Summary Two-month-old outbred female LIO rats were injected weekly with a single dose of 1,2-dimethylhydrazine (DMH; 21 mglkg of body weight) administered s.c. for 15 consecutive weeks. From the day of the 1st injection of the carcinogen the part of rats were given five days a week during the night time (from 18.00 h to 08.00 h) melatonin dissolved in tap water, 20 mg/l. The experiment was terminated in 6 months after the first injection of the carcinogen. The concentration of melatonin in the serum was estimated by radioimmunoassay in rats exposed to DMH alone or in intact control rats in the morning (between 10.00 and 11.00 hours) and night (between 24.00 and 01.00 hours) time. Number of melatonin-containing cells (M-cells) and their optical density were estimated by immunohistology in normal mucosa of glandular stomach, duodenum, ileum and descending colon of tumor-bearing animals from groups exposed to DMH or DMH + melatonin. It was shown that serum melatonin levels in rats with colon tumors was increased as compared with controls. However there was no diurnal rhythm of serum melatonin of colon tumor-bearing animals as compared to intact controls. The number ofM-cells was decreased in all tissues studied in rats with DMH-induced colon tumors in comparison to corresponding controls: by 2.0 times in stomach, by 1.8 time in duodenum, by 1.3 times in ileum, and by 1.8 times in colon. In ileum and colon of rats treated with DMH+melatonin the number of M-cells was similar to control level whereas in stomach and duodenum this number was significantly higher than that in rats treated with DMH alone, but less than in corresponding controls.
Relative content of melatonin in enterochromaffin cells of all parts of gastrointestinal tract evaluated as optical density of the cells and was decreased in rats exposed with DMH alone in comparison to the controls and was normalized and similar to the norm level in rats treated with DMH + melatonin. Thus, exogenous melatonin prevent a decrease in numbers of melatonin-containing cells as was observed in gastrointestinal tract (OIT) of rats exposed to DMH. This preventive action of melatonin correlated well with its anticarcinogenic effect.
Introduction In our previous experiments we have demonstrated the inhibitory effect of melatonin on intestinal carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in rats (1). This effect was manifested by the decrease of the incidence and multiplicity of bowel tumors, mainly in colon, by a decreasing rate of invasion rate and dimensions of colon carcinomas and by an increase of its differentiation. The mechanism of this effect is unknown. It was shown that bowels is one of main sources of melatonin in the body (2, 3) and specific binding sites for melatonin in the mouse colon have been demonstrated (4). Pinealectomy was followed by the increase of the crypt cell proliferation in rat bowel (colon including) which persisted at least 6 months after the operation (5). Also melatonin exert an inhibitory effect on cell proliferation in the rodent colon Exp Toxic Patho151 (1999) 1
47
(6). There is some evidence of impaired circadian rhythm of melatonin in colon cancer patients (7) Clinical data have shown that melatonin may be effective as a secondary line of therapy in cancers of digestive tract, particularly metastatic colorectal cancer (8). Thus, we decided to study a content of melatonin in serum of rats with DMHinduced intestinal tumors and evaluate the effect of exogenous melatonin on a number of melatonin-containing cells in gastrointestinal tract (GIT) as well on the carcinogenesis induced by the carcinogen in bowels.
Material and methods Animals: Two-month-old outbred female LIO rats from the Animal Department of Petrov Research Institute of Oncology, St. Petersburg, were used in the experiments. The characteristics of these rats have been described elsewhere (9). Rats were kept in polypropylene cages, seven in each under a 14/10 h light/dark regimen at 22 ± 2 DC. They received standard lab chow and tap water ad libitum. Experiment: Rats were randomly subdivided into 3 groups. Animals from group 1 were once a week exposed to 15 subcutaneous injections of DMH dihydrochloride (Sigma, U.S.A.) at a single dose of21 mg/kg of body weight (calculated as base). DMH was ex tempore dissolved in normal saline and neutralized with sodium bicarbonate (pH = 7.0). Rats from the group 2 were exposed to DMH as animals from group 1 and additionally from the day of the 1st injection of the carcinogen the part of rats were given melatonin (Sigma, U.S.A.) 5 days a week during the night time (from 18.00 h to 08.00 h) dissolved in tap water, 20 mg/I. This regimen and dosage provide the rhythmical pattern of serum melatonin concentration followed by an anticarcinogenic effect (1, 10, 17). Rats from group 3 were once a week exposed to 15 injections of saline and served as a controls. The experiment was terminated in 6 months after the first injection ofthe carcinogen. Ten rats exposed to DMH alone (group 1) and 10 controls rats (group 3) were killed by decapitation between 10.00 and 11.00 hours and another 20 rats from groups 1 and 3 were sacrificed between 24.00 and 01.00 hours at a dim red light. Blood was collected, centrifugated and samples of serum were stored at -20 DC. The concentration of melatonin in the serum was estimated by radioimmunoassay as described earlier (11). Pathological investigation: All animals killed or found dead before the end of the experiment were autopsied. Intestines were open longitudinally. Tumor number, position and dimensions were recorded on special charts as described elsewhere (12). All tumors and other tissues with macroscopically revealed lesions were fixed in 10 % neutral formaline and, after routine histological treatment, were embedded into paraffin. Five-to-seven ~ thick sections through the middle part of each tumor were stained with haematoxylin and eosin. The neoplasms were classified according to the IARC recommendations (12). Samples of tumor-free tissues from glandular stomach, duodenum, ileum and descending colon from rats exposed to DMH alone, DMH+melatonin and from controls (10 per group) were collected for immunohistochemical study. Immunohistochemistry assay for melatonin-containing cells: Only histologically normal tissues were analyzed immunohistochemically. Deparaffined 5 ~ slides were 48
Exp Toxic Pathol51 (1999) 1
stained with haematoxylin and eosin, or with Masson's argentaffin method for detection of enterochromaffin (EC) cells containing melatonin and serotonin. Immunohistochemical reaction for melatonin was performed according to (13). Deparaffined and dehydrated slides were treated with 0,5 % HzO z in methanol during 30 min. for blockade of endogenous peroxidase. Then slides were washed in 0.1 M phosphate buffer (PB, pH = 7.2) and incubated in 10 % normal rabbit serum during 20 min. for attenuation of non-specific binding of antibodies. Immunohistochemical reaction was performed with ultraspecific rabbit polyclonal antimelatonin antiserum (CIDtech Research Inc., Mississauga, Canada). Slides were incubated during 2 hours with primary antiserum (titer 1:100) at the room temperature with subsequent washing (3 times) with PB. Then slides were incubated with biotinylated goat antirabbit IgG (Dako, Glostrup, Denmark, titer 1: 200) 1 hour and 3 times were washed with PB. Then slides were treated during 30 min. with peroxidase-conjugated avidin (Dako, titer 1: 1000). Staining was revealed after treatment of slides with 0,025 % 3,3-diaminobenzidine tetrahydrochloride (DAB, Dako) and 0,01 % HzO z during 10 min. Additional staining was made with Mayer hematoxylin. Morphometric analysis of melatonin-containing cells (M-cells): Morphometric assay was automatically performed with system of computer analysis for morphological images MORPHOSTAR with using of applied software COLQUANT program (Imstar S.A., Paris, France). This system is a comprehensive modular system for multimodal image acquisition, display processing and analysis and it was successfully used for true colour representation and interpretation of various histological data from the results of immunohistochemical study of structure-functional organization of APUD cells in tumor growth (14, 15). All parameters were detected on 3 slides from each sample in 10 randomly selected microscopic fields (each optical microscopic field was 0.785 mmZ) at magnification x200. The following morphometric parameters were evaluated: 1) Mucosa square, ~ z; 2) Square ofM-cells (total square occupied by M-cells), Ilz; 3) Ratio ofM-cells square to mucosa square; 4) Mean number of M -cells per 1 optical field; 5) Optical density ofM-cells (units). Optical density was calculated as D = ach, where D - optical density; a - apportioned parameters of an absorbtion of a compound; c - concentration of a compound; h - slide thickness. This parameter, together with a number of Mcells gives an important information on tissue hypo- or hyperplasia as well as on changes in its functional activity (14, 15). All caluclations were performed automatically using above mentioned program. Statistical analysis: Morphometric results were treated statistically using program STATISTICA 4.3 (Tulsa, U.S.A.) Wylcoxon U-test and Student's t-test (16) were used as well.
Results Effect of melatonin on DMH-induced intestinal carcinogenesis The majority of colon tumors were localized in descending colon. Macroscopically colon tumors were exo-
Table 1. Intestinal tumor incidence and localization in rats exposed to 1,2-dimethylhydrazine (DMH) and melatonin (MLT).
Parameters
DMH
DMH+MLT
Number of rats Tumor localization: Duodenum: No. of tumor-bearing rats No. of tumors Jejunum and ileum: No. of tumor-bearing rats No. of tumors Colon (all parts): No. of tumor-bearing rats No. of tumors Ascending colon: No. of tumor-bearing rats No. of tumors De~cending colon: No. of tumor-bearing rats No. of tumors Rectum: No. of tumor-bearing rats No. of tumors
25
23
8 (32.0 %) 9
5 (21.7 %) 5
4 (16.0 %) 5
1 (4.3 %)* 1
25 (l00 %) 237
23 (100 %) 137
23 (92.0 %) 15 (65.2 %)** 17 56 25 (100 %) 164
23 (100 %) 101
11 (44.0 %) 11 (47.8 %) 17 19
The difference with rats exposed to DMH alone is significant. * p < 0.05; ** p< 0.01. Table 2. Serum melatonin level in rats with colon tumors induced by 1,2-dimethylhydrazine and in control rats.
Treatment Serum melatonin level [nM/lJ
P
10.00 hours
24.00 hours
10 hrs vs 24 hrs
Controls (saline)
115 ± 35.7 (n = 10)
307 ± 59.7 (n = 10)
< 0.005
DMH
361 ± 139.4 (n = 10)
606 ± 188.7 (n =8)
> 0.05
P (controls vsDMH) < 0.005
>0.05
phytic or endophytic, however some cases of ulcerativeinfiltrative forms were detected. Microscopically we found different types of malignant tumors of intestines with absolute prevalence of adenocarcinomas. All neoplasia in small intestines were adenocarcinomas. The data on intestinal tumor incidence and localization are presented in table 1. Administration of melatonin in drinking water failed influence the total incidence of colon tumors. However incidence of carcinomas in ascending colon was significantly reduced (p < 0.01). The multiplicity of total colon tumors per rat as well as the mean number of tumors in ascending and descending colon per rat were also decreased under the influence of melatonin (Group 2 vs Group 1, p < 0.01). In the same experiment melatonin
slightly decreased the depth of tumor invasion and increased number of highly differentiated colon carcinomas induced by DMH (for details see ref. 1). The percentage of small tumors in descending colon among rats from group 2 was higher than that in group 1 (1). Treatment with melatonin was followed also by the decrease in the multiplicity of DMH-induced tumors of duodenum (Group 2 vs Group 1, p < 0.05) and by the decrease in the incidence of jejunum and ileum tumors (Group 2 vs Group 1, p < 0.05) (table 1).
Serum melatonin level in rats with DMH-induced colon tumors The results of RIA of serum melatonin are given in the table 2. In general, melatonin level in rats with colon tumors (group 1) was increased as compared with controls (group 3). There is significant (by 2.7 times, p < 0.005) elevation of the night level of melatonin as compared to the morning level in control rats. In rats with DMH-induced colon tumors the morning level of melatonin was increased (p < 0.005) as compared with timematched controls. However there was no significant elevation of night level of melatonin in comparison to the morning in colon tumor-bearing animals.
Melatonin-containing cells in intestinal mucosa of rats with DMH-induced colon tumors Immunohistochemical study revealed that the number of melatonin-containing cells (M-cells) was different in various parts of gastrointestinal tract and was distributed as stomach> duodenum> colon> ileum. In all tissues of rats with DMH-induced colon tumors (group 1) the number of M-cells was decreased in comparison to corresponding controls (group 3): by 2.0 times in stomach, by 1.8 time in duodenum, by 1.3 times in ileum and by 1.8 times in colon (table 3; fig. 1). In ileum and colon ofrats treated with DMH + melatonin (group 2) the number of M -cells was similar to control level whereas in stomach and duodenum this number was significantly higher than that in rats treated with DMH alone, but less than in corresponding controls. A calculation of relative content of melatonin in enterochromaffin cells of all parts of gastrointestinal tract evaluated as optical density of the cells (14) reveals a tendency toward the decrease in rats exposed with DMH alone in comparison to the controls and was normalized and similar to the norm level in rats treated with DMH + melatonin (table 3). Some examples of immunohistochemical pattern of M-cells in gastrointestinal tract of control, DMH- and DMH + melatonin-treated rats are given on fig. 2 (a, b, c). Exp Toxic Pathol 51 (1999) 1
49
Table 3. Effect of 1,2-dimethylhydrazine (DMH) and melatonin (MLT) on morphometric parameters in gastrointestinal tract of rats. Organ
Treatment
Mucosa square analized flm2 (M±m)
Square ofM-cells flm 2 (M±m)
Ratio of M-cells square to mucosa square
Number of M-cells per optical field (M±m)
Optical density of M-cells, units (M±m)
Stomach
Control DMH DMH+MLT
10614 ± 741 11021 ± 567 11132 ± 646
122 ± 14 109 ± 12 119 ± 14
0.018 ± 0.002 0.010 ± 0.002 0.011 ± 0.002
35.1 ± 1.8 17.3 ± 1.3" 27.5 ± 2.2bc
0.145 ± 0.034 0.111 ± 0.021 0.139 ± 0.030
Duodenum
Control DMH DMH+MLT
11316 ± 732 10822 ± 1695 10046 ± 1384
120 ± 15 119 ± 27 112 ± 12
0.012 ± 0.001 0.013 ± 0.002 0.013 ± 0.003
28.2 ± 1.1 15.3 ± 1.5" 23 .5 ± 0.9 bd
0.139 ± 0.033 0.113 ± 0.026 0.172 ± 0.044
Ileum
Control DMH DMH+MLT
11644 ± 922 11435 ± 912 10564 ± 1011
92±9 87 ± 5 93 ±2
0.008 ± 0.001 0.008 ± 0.001 0.009 ± 0.002
16.3 ± 0.9 12.1 ± 0.7" 16.5 ± 1.1 e
0.116 ± 0.019 0.084 ± 0.016 0.119 ± 0.022
Colon
Control DMH DMH+ MLT
11076 ± 876 10978 ± 785 11679 ± 769
107 ± 12 86± 8 103 ± 10
0.010 ± 0.013 0.008 ± 0.011 0.009 ± 0.013
24.6 ± 1.7 13.9 ± 1.6' 22.7 ± 1.9c
0.139 ± 0.028 0.054 ± 0.03Sf 0.135 ± 0.026
M-cells: melatonin-containing cells. The difference with controls is significant: a: p < 0.001 ; b: p < 0.01; f: 0.1 < P < 0.05.
10
trol
.0\llH
nurn
II
m
In
Fig. 1. Number of melatonin-containing cells in gastrointestinal tracts of rats with colon tumors exposed to DMH and DMH + melatonin. * The difference between controls and experimental group is significant, p < 0.01; x The differences between rats exposed to DMH alone and DMH + melatonin is significant, p < 0.05.
Discussion Our data have shown that in rats with DMH-induced colon tumors the morning level of serum melatonin is increased in comparison to the controls. At the same time there are no clear-cut circadian rhythm of melatonin in colon tumor-bearing animals. These findings coincide with clinical observations, which demonstrated that serum level of this indole hormone is increased in colo50
Exp Toxic Pathol51 (1999) 1
rectal cancer patients and there were disturbances of circadian rhythm of melatonin excretion in such patients (2,7). At the same time, the decrease in the number ofMcells was observed in all part of gastrointestinal tract of animals with DMH-induced colon tumors and a relative decrease of melatonin content in these cells (table 3). It is possible to suggest that increased serum level of melatonin in DMH-treated rats (group 2) is a compensatory reaction of pineal gland on DMH-induced decrease in local level of melatonin in gastrointestinal tract. It has been shown that mammalian gastrointestinal tract contain much more melatonin than the pineal gland and enterochromaffine cells are the main source of melatonin in the organism (15). In spite of data demonstrating an active participation of melatonin in adaptive response and in pathophysiology, the normal function of extrapineal melatonin as well as the feedback mechanisms between pineal and GIT melatonin production are largely unknown. It is possible to suggest that decrease of number of Mcells and their functional activity in rat colon of rats exposed to DMH could play an important role in its carcinogenic effect. Treatment with melatonin prevent the decrease in the content ofM-cells in gastrointestinal tract of rat exposed to DMH (table 3; fig. 1) that correlated with anticarcinogenic effect of melatonin (1; table 1). The anticarcinogenic potential of melatonin has been demonstrated both in vivo and in vitro in relation to mammary carcinoma (17, 18). It has been suggested that melatonin regulates growth of mammary tumors through several mechanisms including direct modulation of mitotic activity, inhibition of serum estrogen and prolactin level,
point in its anticarcinogenic potential. It was shown also that melatonin inhibits the production of DNA-adducts of the carcinogen safrole in rodents (22). The production of highly reactive free radicals and decrease of activity of some enzymes of anti oxidative defence system has been observed during DMH-induced carcinogenesis in rats and mice (23-26). Antioxidative properties of melatonin (27, 28) may be also an important factor of it anticarcinogenic potential. In the same model we have shown that both in serum of rats exposed to DMH alone the level of diene conjugates and Schiff's bases was significantly increased as compared with controls. In colon tissue of rats with DMH-induced colon carcinomas the level of products of lipid and protein peroxidations as well as NO-synthase activity was significantly increased, and total antioxidative activity was decreased compared with controls, whereas treatment with melatonin resulted in the normalization of these parameters (29, 30). Recently it was demonstrated that melatonin inhibits mutagenic effect of DMH evaluated in two in vivo tests: chromosome aberration in bone marrow cells and anomal sperm heads (31). Thus, exogenous melatonin prevents a decrease in numbers of melatonin-containing cells as was observed in gastrointestinal tract (GIT) of rats exposed to DMH. This preventive action of melatonin correlated well with its anticarcinogenic effect. Some aspects of the problem need an additional experiments for elucidations. It is necessary to study a direct effect of DMH on melatonin production in rat colon as well as an effect of exogenous melatonin on the production of melatonin in colon. Also it is important to examine the effect of melatonin on activity of 0 6- alkylguanine transferase as well as a methylation of DNA bases in colon cells. We are intending to get anwer for these questions. Acknowledgements: This work was supported in part by grant 6/96 from The Russian Ministry of Science State Program "National Priorities in Medicine and Health". Authors are very thankful to Prof. G.M.Brown, Clarke Institute of Psychiatry, Toronto, Ontario, Canada for providing antiserum to melatonin. Fig. 2. Melatonin-containing cells in rat duodenum. Immunohistochemical staining, x 200. a: control (group 3); b: the decrease of cell number after DMH treatment (group 1); c: The increase (almost to control parameters) of cell number induced by exogenic melatonin in DMH-treated rats (group 2).
and the modulation of immune system (17, 18). The mechanism of the anti carcinogenic effect of melatonin on colon carcinogenesis is unknown. Melatonin inhibits cell proliferation in the rodent colon (6) and some positive response to melatonin treatment was observed in digestive tract of cancer patients (8). The presence of specific binding sites for melatonin in the mouse colon has been demonstrated as well (4). Melatonin also enhances cellto-cell junction contacts (19), and modify the activity of cytochromes b5 and P450 (20, 21) that may be a critical
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