Chemoprevention of N-Methyl-N-Nitrosourea Induced Mammary Carcinogenesis with Raloxifene and Melatonin: Metabolic Changes in Female Rats

Chamilová M., P. Kubatka, K. Kal ická, E. Adámeková, B. Bojková, I . Ahlers , E. Ahlersová: Chemoprevention of N-methyl-N-nitrosourea Induced Mammary Carcinogenesis with Raloxifene and Melatonin: Metabolic Changes in Female Rats. Acta Vet. Brno 2002, 71: 235–242. The aim of this work was to determine the selected parameters of carbohydrate and lipid metabolism in the mammary carcinogenesis induced with N-methyl-N-nitrosourea (NMU) in two doses, each by 50 mg/kg of body weight with a 7-day interval between them within the postnatal days 43 and 54 in female Sprague-Dawley rats. Chemoprevention started with the administration of melatonin (MEL, 4 μg/ml in water, from 15.00 h to 08.00 h) 12 days and raloxifene (RAL 5 mg/kg, 2 × weekly) 10 days before the application of NMU. Twenty-four weeks following the NMU administration the animals were killed, and the incidence, latency, frequency and volume of tumours were evaluated. The animals were divided into: tumour-bearing (TB) and non-tumourbearing (NTB) with the influence of RAL, MEL and their combination. While RAL and RAL plus MEL significantly decreased the incidence and frequency of tumours, the effect of isolated MEL was substantially lower. In the serum, an increase in the concentration of serum glucose in TB and also NTB animals was observed. In the liver of both the TB and NTB animals, the content of cholesterol (CH) and triacylglycerols (TG) decreased and the contents of phospholipids (PL) increased. RAL decreased the contents of CH and PL in the liver of NTB animals and increased the concentration of TG in both groups of animals. Administration of RAL to NTB animals decreased the concentrations of malondialdehyde (MDA) in the serum and thymus, in the bone marrow also in TB animals. MEL decreased the concentration of MDA in the bone marrow of TB animals. MEL increased the concentrations of serum glucose and glycogen content in the heart muscle of NTB animals. RAL plus MEL decreased the concentration of serum TG and PL and decreased the contents of CH and PL in the liver of TB as well as NTB animals. In the thymus and liver, combination of RAL+MEL decreased the MDA content compared with the RAL alone in NTB animals. The co-effect of two or more substances will be probably the optimal way in prevention of cancer. The co-effect of RAL and MEL shows to be a prospective way for influencing the mammary tumors. Breast cancer, female rats, raloxifene, melatonin, chemoprevention The hormonal therapy of the breast carcinoma is an inseparable part of the variety of therapeutic procedures. The substitution with estrogens has been considered for a long time as a dominant indication of therapy in postmenopausal women, and it has been recognized that approximately one third of women will have a benefit of this procedure. The use of estrogens protects these women against osteoporosis and decreases the cardiovascular risk, but on the other hand, increases the risk of breast and endometrium carcinoma (Col et al. 1997). With regard to the unfavourable effects of estrogens on the breast and endometrium tissue, it was necessary to develop substances which could have a favourable estrogenic effects on the bone tissue and cardiovascular system without increasing the risk of breast and endometrium carcinoma. Raloxifene (RAL) represents the second generation of the ACTA VET. BRNO 2002, 71: 235–242 Address for correspondence: Prof. MUDr. Ivan Ahlers, DrSc. Institute of Animal Physiology Faculty of Science, P. J. ·afárik University Moyzesova 11, 041 67 Ko‰ice, Slovak Republic Phone: +421 95 62 226 10 Fax: +421 55 62 221 24 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm substances called selective modulators of estrogen receptors (SERM). RAL is a non-steroid antiestrogen with the structure of benzothiophene originally developed for the treatment and prevention of osteoporosis in postmenopausal women. Preliminary results in women treated for osteoporosis indicate that RAL reduces the risk of the incidence of breast carcinoma without an unfavourable effect on the endometrium (Blum and Cannon 1998). Both clinical and experimental studies have revealed the ability of RAL to decrease the total and LDL cholesterol in the serum. The use of RAL in hormonal therapy in women is dependent on the results of the presently running clinical studies (MORE, STAR). The other efficient substance in the treatment of breast carcinoma is melatonin, the principal hormonal product of the pineal gland. The function of this indolamine is closely connected with the neuroendocrine, biorhythmical and immune functions (Guerrero and Reiter 1992). Numerous authors have confirmed the oncostatic effects of melatonin in the treatment of breast carcinoma (Lissoni et al. 1989) and other types of carcinoma (Blask 1993). The aim of our work was to find the effects of raloxifene, melatonin and their combination on the selected metabolic parameters in N-methyl-N-nitrosourea induced mammary carcinoma in female Sprague-Dawley rats and to compare their effects with the metabolic effects of “classical” antiestrogen tamoxifen alone or in combination with melatonin (Chamilová et al. 2001). Materials and Methods Female Sprague-Dawley rats (Anlab, Prague, Czech Republic) were used in the experiment. The animals were delivered at the age of 34-38 days, and during the whole experiment they were kept under standard conditions (23 ± 2 °C, relative air humidity 60-70 %, light regimen L:D 12:12 with the beginning of the light part of the day at 07.00 h). During the experiment the animals (4-5 in a cage) were fed the PM diet (Top-Dovo, Dobrá Voda, Slovak Republic) and drank tap water ad libitum. Chemocarcinogen N-methyl-N-nitrosourea (NMU; Sigma, Deisenhofen, Germany) was administered to animals between the postnatal days 43 and 54 in two doses intraperitoneally, each at 50mg/kg of body weight within 7 days. Chemoprevention with raloxifene (RAL) and melatonin (MEL) started 10 days and 12 days before the administration of NMU, respectively, and lasted until the end of the experiment. RAL (LY 139481-HCl, Eli Lilly and Co., Indianapolis, USA) was administered twice a week subcutaneously in the dorsal region at a dose of 5 mg/kg of body weight of animals as 0.25 ml solution of the mixture of PEG 400: water = 1:1. The animals drank MEL (Biosynth, Staad, Switzerland) in tap water at a concentration of 4 μg/ml daily from 15.00 h to 08.00 h next day. For the preparation of 1 l solution 4 mg of MEL was dissolved in 0.3 ml of ethanol and supplemented with tap water to the volume of 1 l. During the experiment, the animals were weighed and palpated once a week, and their food and water intake was measured. At the end of the experiment in week 24, the animals were killed by a quick decapitation and subsequently the incidence and growth of mammary tumours and selected parameters of the lipid and carbohydrate metabolism were analysed. In the serum of the mixed blood, following parameters were determined: the concentration of triacylglycerols (TG), cholesterol (CH), phospholipids (PL) malondialdehyde (MDA) (as a measure of lipid peroxidation) and glucose; in the liver, the contents of TG, PL, CH, MDA and glycogen; in the bone marrow the concentrations of TG, PL, MDA; in the thymus only the content of MDA; in the heart muscle the glycogen content. The concentration of phospholipids was determined from the lipid phosphorus by Bar t le t t ’ s method (1959), total cholesterol according to Zla tk is et al. (1953), glycogen by the method of Roe and Dai ley (1966), malondialdehyde in a reaction with thiobarbituric acid (Satch 1978), triacylglycerols and glucose by the commercial sets (Lachema). At the evaluation 9 groups were compared: 1. NMU – non-tumour-bearing animals (NTB, n = 4) 2. NMU – tumour-bearing animals (TB, n = 4) 3. NMU + RAL – non-tumour-bearing animals (NTB+RAL, n = 14) 4. NMU + RAL – tumour-bearing animals (TB+RAL, n = 3) 5. NMU + MEL – non-tumour-bearing animals (NTB+MEL, n = 6) 6. NMU + MEL – tumour-bearing animals (TB+MEL, n = 8) 7. NMU + RAL + MEL – non-tumour-bearing animals (NTB+RAL+MEL, n = 11) 8. NMU + RAL + MEL – tumour-bearing animals (TB+RAL+MEL, n = 4) 9. Intact animals (INT, n = 10) Results were statistically evaluated by the one-way analysis of variance for p < 0.05. 236