Modulatory effect of troxerutin on biotransforming enzymes and preneoplasic lesions induced by 1,2-dimethylhydrazine in rat colon carcinogenesis

Colon cancer is the third most global oncologic problem faced by medical fraternity. Troxerutin, a flavonoid present in tea, coffee, cereal grains, and a variety of fruits and vegetables, exhibits various pharmacological and biological activities. This study was carried out to investigate the effect of troxerutin on xenobiotic metabolizing enzymes, colonic bacterial enzymes and the development of aberrant crypt foci (ACF) during 1,2-dimethylhydrazine (DMH) induced experimental rat colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control. Group 2 received troxerutin (50 mg/kg b.w., p.o. every day) for 16 weeks. Groups 3–6 received subcutaneous injections of DMH (20 mg/kg b.w.) once a week, for the first four weeks. In addition, groups 4–6 received different doses of troxerutin (12.5, 25, 50 mg/kg b.w., p.o. every day respectively) along with DMH injections. Our results reveal that DMH treated rats exhibited elevated activities of phase I enzymes such as cytochrome P450, cytochrome b5, cytochrome P4502E1, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase and reduced activities of phase II enzymes such as glutathione-S-transferase (GST), DT-diaphorase (DTD) and uridine diphospho glucuronyl transferase (UDPGT) in the liver and colonic mucosa of control and experimental rats. Furthermore, the activities of fecal and colonic mucosal bacterial enzymes, such as β-glucronidase, β-glucosidase, β-galactosidase and mucinase were found to be significantly higher in DMH alone treated rats than those of the control rats. On supplementation with troxerutin to DMH treated rats, the alterations in the activities of the biotransforming enzymes, bacterial enzymes and the pathological changes were significantly reversed, the effect being more pronounced when troxerutin was supplemented at the dose of 25 mg/kg b.w. Thus troxerutin could be considered as a good chemopreventive agent against the formation of preneoplastic lesions in a rat model of colon carcinogenesis.     

Ursolic acid and oleanolic acid suppress preneoplastic lesions induced by 1,2-dimethylhydrazine in rat colon

Ursolic acid (UA) and oleanolic acid (OA) are pentacyclic triterpenoid compounds found in plants used in the human diet and in medicinal herbs, in the form of aglycones or as the free acid. These compounds are known for their hepatoprotective, anti-inflammatory, antimicrobial, hypoglycemic, antimutagenic, antioxidant, and antifertility activities. In the present study, we evaluated the effects of UA and OA on the formation of 1,2-dimethyl-hydrazine (DMH)-induced aberrant crypt foci (ACF) in the colon of the male Wistar rat. The animals received subcutaneous (sc) injections of DMH (40 mg/kg body weight) twice a week for two weeks to induce ACF. UA, OA and a mixture of UA and OA were administered to the rats five times a week for four weeks by gavage at doses of 25 mg/kg body weight/day each, during and after DMH treatment. All animals were sacrificed in week 5 for the evaluation of ACF. The results showed a significant reduction in the frequency of ACF in the group treated with the triterpenoid compounds plus DMH when compared to those treated with DMH alone, suggesting that UA and OA suppress the formation of ACF and have a protective effect against colon carcinogenesis.     

Melatonin and colon carcinogenesis. III. Effect of melatonin on proliferative activity and apoptosis in colon mucosa and colon tumors induced by 1,2-dimethylhydrazine in rats.

Forty-eight two-month-old outbred female LIO rats were injected weekly with a single dose of 1,2-dimethylhydrazine (DMH; 21 mg/kg 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. 10 rats were treated similarly with solvents and served as control. The experiment was terminated 6 months after the first injection of the carcinogen. Colon tumors (mainly adenocarcinomas) developed in a hundred percent of rats exposed both to DMH or to DMH plus melatonin. However, descending colon carcinomas were observed in 65 % of rats exposed to DMH plus melatonin against 100% in those exposed to DMH alone (p < 0.01). The multiplicity of colon tumors was also reduced in rats under the influence of melatonin. This effect is correlated with the significant inhibitory effect of the pineal hormone on mitotic index and with stimulating effect of melatonin on the relative number of apoptotic cells (TUNEL-method) in colon tumors. Long-term treatment with melatonin was followed also by the decrease in the area of lymphoid infiltrates in the colon mucosa of tumor-bearing rats.     

Effect of age,castration, and pregnancy on carcinogenesis induced in CBA mice by 1,2-dimethylhydrazine

When injected subcutaneously in a dose of 8 mg/kg weekly into female CBA mice, 1,2-dimethylhydrazine (DMH) induced the development of tumors of the intestine, anal region, uterus, and liver. When DMH was injected into mice aged 12–13 months the appearance of sarcomas of the uterus was observed earlier (at 8 weeks) and the incidence of tumors of the anal region rose more rapidly than in mice aged 3 months. In mice receiving DMH against the background of repeated pregnancies, a statistically significant decrease in the frequency of sarcomas of the uterus was observed (10.3% compared with 48.3% in nonpregnant mice); pregnancy did not affect the frequency of tumors of other organs. Castration had no significant effect on the time of appearance or the frequency of tumors in all situations.Laboratory of Carcinogenic Substances, Department of Carcinogenic Agents, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 5, pp. 458–460, May, 1979.     

Early and late effects of wound healing on development of colon tumours in a model of colon carcinogenesis by 1,2-dimethylhydrazine in the rat

The early and late effects of wound healing on tumour development at a distant site were evaluated morphologically in an experimental model of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH). Random bred male Wistar rats were given subcutaneous injections of DMH (20 mg/kg) or saline, once a week, for eight weeks. One week after the last DMH injection the animals received a full thickness skin wound in their dorsal skin and the wound was left open to heal by second intention. Control and DMH treated rats, with or without skin wounds were sacrificed at the twelfth and twentieth weeks. The colons were removed and the incidence, distribution and morphology of any tumours were recorded. Tumours induced by DMH in the colonic mucosa increased in size during the experiment. At the twelfth week, just after healing of the skin wound was complete, the total number of tumours in the colonic mucosa and the number of tumours per rat was significantly higher in the skin-wounding DMH-treated group than in the unwounded group. No differences on tumour incidence and multiplicity were observed among the groups at the twentieth week. Histologically the number of poorly differentiated mucin-secreting carcinomas was increased in the skin-wounding DMH-treated group than in the unwounded group at the twelfth week. This effect was not observed at the twentieth week. The present results suggest that wound healing enhances tumour development at a distant site. This effect coincides with the period of repair and does not have a marked impact on later stages of tumour progression.     

Ultrastructure of renal-cell tumors induced in mice by 1,2-dimethylhydrazine

Fifteen primary renal adenomas induced with 1,2-dimethylhydrazine in CBA mice and 3 passages of adenomas transplanted subcutaneously were examined by electron microscopy. A well-developed brush border was found in one primary adenoma, this being in line with histochemical data on the presence of markers of the proximal canaliculi epithelium in this tumor. In other tumors a reduced brush border (5 cases) and apical canaliculi (14 cases) were revealed, which are typical of the proximal canaliculi epithelium. The ultrastructure of the types of adenoma cells is described. Atypical tumor cells with intracellular lumina were found in transplanted adenomas Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 9, pp. 286–290, September, 1995 Presented by Yu. N. Solov'ev, Member of the Russian Academy of Medical Sciences     

Intestinal carcinogenesis in rats using 1,2-dimethylhydrazine with or without degraded carrageenan.

The phenomena of proliferation, metaplasia, and neoplasia of the intestinal epithelium were studied in Sprague-Dawley rats, by using 1,2-dimethylhydrazine (DMH), which was injected once weekly, subcutaneously, at a dose of 30 mgm/kgm for up to 30 weeks. Degraded carrageenan (C16) was also given either in the diet (7.5 gm/kgm) or in drinking water (5% solution) alone or in combination with DMH for up to 30 weeks. Papillary adenomas, adenomatous polyps, and adenocarcinomas of various grades were observed as having been elicited by DMH. This compound, when given alone, causes the development of tumors that arise from the superficial protion of the intestinal mucosa; when given together with C16, DMH induces proliferation of the deep glandular areas. C16 alone induces transformation of the mucosal epithelium of the distal rectum into stratified squamous epithelium. The administration of C16 in the diet, in contrast to drinking water, elicits earlier changes in proliferation.     

Three new rat colon tumour cell lines from 1,2-dimethylhydrazine induced adenocarcinomas: morphology, karyotype and clonogenicity

Three different tumour cell lines (Per113, Per192, Per237) were established from colon adenocarcinomas induced by 1,2-dimethylhydrazine (DMH) inbred male Wistar/Wag rats. Each cell line had epithelial morphology and an abnormal karyotype. The three cell lines were clonogenic in semi-solid medium, but differed in growth factor response, culture characteristics and karyotype. Each cell line was stimulated differentially by epidermal growth factor (EGF) and bombesin in culture. Only Per192 would passage in vivo in athymic nude mice. Per 113 had modal chromosome number of 70 with multiple structural abnormalities; Per237 had 43 chromosomes with consistent trisomy of chromosome I; 20-25% of the cells in line Per192 were in the tetraploid range with multiple copies of chromosome I.     

Three new rat colon tumour cell lines from 1,2-dimethylhydrazine induced adenocarcinomas: morphology,karyotype and clonogenicity.

Three different tumour cell lines (Per113, Per192, Per237) were established from colon adenocarcinomas induced by 1,2-dimethylhydrazine (DMH) inbred male Wistar/Wag rats. Each cell line had epithelial morphology and an abnormal karyotype. The three cell lines were clonogenic in semi-solid medium, but differed in growth factor response, culture characteristics and karyotype. Each cell line was stimulated differentially by epidermal growth factor (EGF) and bombesin in culture. Only Per192 would passage in vivo in athymic nude mice. Per 113 had modal chromosome number of 70 with multiple structural abnormalities; Per237 had 43 chromosomes with consistent trisomy of chromosome I; 20-25% of the cells in line Per192 were in the tetraploid range with multiple copies of chromosome I.     

Renal cell tumors induced in CBA male mice by 1,2-dimethylhydrazine.

Epithelial kidney tumors induced in CBA male mice by 1,2-dimethylhydrazine (DMH) were studied histochemically and immunohistochemically. A total of 48 tumors studied histologically were diagnosed as clear-cell, acidophilic, or mixed adenomas located in the renal cortex. Gamma-glutamyl transpeptidase (GGT) was strongly positive in normal proximal convoluted tubules, slightly positive in the cells of Bowman's capsule, and negative in 47 of 48 tumors examined. Antibodies against the new antigen obtained from the mouse liver oval cells, antigen A6, were also used. This antigen is negative in normal kidney proximal tubules but always positive in distal tubules and collecting ducts. It was also positive in all 47 GGT-negative tumors studied here. One tumor was GGT-positive and antigen A6-negative. Based on our data, it was concluded that the majority of renal cell adenomas induced by DMH in mice probably originate from the distal tubules or collecting ducts and not from the proximal tubules.     

The inhibitory effect of rosmarinic acid on complement involves the C5 convertase.

Rosmarinic acid (RA), a naturally occurring extract from Melissa officinalis, inhibits several complement-dependent inflammatory processes and may have potential as a therapeutic agent for the control of complement activation in disease. Rosmarinic acid has been reported to have effects on both the classical pathway C3-convertase and on the cobra venom factor-induced, alternative pathway convertase. In order to define the mechanism of inhibition, the effect of RA on classical and alternative pathway lysis, C1q binding, the classical pathway convertase, the alternative pathway convertase, membrane attack pathway lysis and the generation of fragments of C3 and C5 during activation, was tested in vitro. The results showed that RA inhibited lysis by the classical pathway more than by the alternative pathway. This effect was dose-dependent with maximum inhibition of classical pathway lysis observed at 2.6 mmoles of RA. There was little effect on C1q binding or on the classical and alternative pathway convertases. However, there was highly significant inhibition of lysis of pre-formed EA43b cells by dilutions of human or rabbit serum in the presence of RA (1 mM); this was accompanied by inhibition of C5a generation. We conclude that the inhibitory effect of RA involves the C5 convertase. Such inhibition could be advantageous to the host in disorders where the terminal attack sequence plays a role in pathogenesis.     

The effects of the carcinogen, 1,2-dimethylhydrazine,on turnover 3H-thymidine labeled cells from mucosal glands of mouse colon

1,2-dimethylhydrazine (DMH), administered weekly to mice for 20 weeks, induces tumors in the distal segment of colon. Tumors are preceded by enlargement of the mucosal glands resulting from increases in the number of total cells and ³H-thymidine labeled cells/crypt. Cells located in the crypt base normally undergo 2–3 divisions as they migrate toward the lumen, and they become post-mitotic in the upper crypt. It is not known if cells in these eniarged crypts have rates of turnover similar to cells in normal crypts. Groups of w/s female mice were treated with DMH (20 mg/kg body wt) for 3,8, or 16 weeks; controls were given 0.001 M EDTA. After treatment, the animals were injected with ³H-thymidine and killed one hour or 1,2,4,7 or 17 days later. Autoradiographs were prepared from sections of distal colon. The total cells/crypt column in 30 crypts/animals were counted. Crypts were divided into 10 equal segments based on the crypt length and the labeled cells/segment were counted. The relative number of labeled cells and the distribution of these cells within crypts were similar in DMH-treated and control animals after one hour. However, as the cells migrated toward the lumen, the number of labeled cells doubled after 2 days and tripled after 4 days in DMH-treated animals but only doubled during the 4 days in controls. This difference was caused by retention of an increased number of dividing cells in the lower 4 segments of the crypts and suggests an increase in those cells that divide twice. In addition, increased numbers of labeled cells were retained in the upper 3 segments of DMH-treated animals after 4 days. These findings indicate that the crypt cells of DMH-treated animals are generally more immature than those of controls and this immaturity contributes to the enlargement of mucosal glands during carcinogenesis.     

Effect of rhythm of administration of 1,2-dimethylhydrazine on its carcinogenic activity

The rhythm of injection of 1,2-dimethylhydrazine (DMH), in the same total dose, affects the ultimate carcinogenic effect. The most widespread development of intestinal tumors is observed if DMH is given once a week in a dose of 21 mg/kg body weight. Daily injection in a dose of 3 mg/kg leads to less marked tumor development, limited to the large intestine. However, under these conditions pronounced degenerative and necrobiotic, and in a few cases neoplastic changes develop in the liver. The frequent administration of small doses of the carcinogen leads to a marked decrease in the cytochrome P₄₅₀ content in the microsomes of the liver; this evidently delays the metabolism of DMH, its conversion into its end product, and binding of the latter with macromolecules of the enterocytes.Professor N. N. Petrov Research Institute of Oncology, Ministry of Health of the USSR, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 3, pp. 337–340, March, 1978.