CHEMOPREVENTIVE EFFECTS OF ROOT BARK EXTRACT OF C. PORTORICENSIS (BENTH.) IN N-NITROSO-N-METHYLUREA AND BENZO(A)PYRENE-INDUCED MAMMARY GLAND AND REPRODUCTIVE TOXICITY IN WISTAR RATS

Abstract

Breast cancer (BC) remains a disease with high morbidity and mortality. In Nigeria, BC represents about 23% of all cancer cases. Currently available chemotherapeutic interventions are associated with significant adverse effects, hence the need for safer options. C. portoricensis (CP) has been applied to manage breast diseases in ethnomedicine. However, there is paucity of scientific basis to justify this claim. In this study, an investigation of the effects of CP on N-nitroso-N-methylurea (MNU) and Benzo(a)pyrene (BP)-induced mammary and reproductive organ toxicity in Wistar rats was conducted. A root bark sample of CP was taken from Ikire, Osun State and authenticated at Forest Hebarium Ibadan (FHI:111949). The methanol extract of CP (MCP) was obtained from the powdered root. The MCP was partitioned to obtain n-hexane, chloroform, ethylacetate and butanol fractions. The chloroform fraction of CP (CCP) was subjected to biochemical assay using MCF-7 cells. Sixy-four female Wistar rats (30-40g) were divided into eight groups (n=8): Vehincle, MNU, [MNU+MCP (100 mg/kg)], [MNU+MCP (200 mg/kg)], [MNU+MCP (300 mg/kg)], MCP (300 mg/kg), [MNU+Vincasar (0.5 mg/kg)] and Vincasar only. In another study, fifty-six female rats were grouped into seven (n=8): Vehincle, [MNU+BP], [MNU+BP+CCP (50 mg/kg)], [MNU+BP+CCP (100 mg/kg)], CCP (100 mg/kg), [MNU+BP+ Vincasar] and Vincasar only. The MNU (50 mg/kg) and BP (50 mg/kg) were administered intraperitoneally at age 7, 10 and 13 weeks. The MCP and CCP were administered orally thrice weekly. Rats were sacrificed; blood and tissues (mammary and uterine) were obtained for analyses. Lactate dehydrogenase (LDH), Superoxide Dismutase (SOD), Catalase, Glutathione-S-Transferase (GST), Glutathione Peroxidase (GPx), malondialdehyde, Nitric Oxide (NO) and Myeloperoxidase were determined by visspectrophotometry. Cyclooxygenase-2, inducible Nitric Oxide Synthase (iNOS), B-cell lymphoma-2 (Bcl-2), p53, Interleukins-1β and 6 (IL-1β, IL-6), Estrogen and Progesterone receptors (ER+, PR+) and Epidermal Growth Factor Receptor-2 (EGFR- 2) were determined by immunohistochemistry. Micro-section of tissues were subjected to Hematoxylin & Eosin and examined under the light microscope. Data were analysed using ANOVA at α0.05.ix The CCP decreased levels of IL-1β (34%), LDH (24%), MPO (74%), malondialdehyde (55%) and increased the activities of SOD (48%) and catalase (49%) in MCF-7 cells. The MNU decreased activities of mammary SOD (22%), uterine CAT (24%) and, GST (25%) relative to Vehincle. The MCP (100 mg/kg) when compared with MNU significantly elevated mammary SOD (18%) and uterine GST (20%). Treatment with CCP (100 mg/kg) when compared with MNU+BP significantly increased the mammary catalase (24.42±4.86 vs 16.1p6±2.90), GPx (171.48±13.97 vs 93.68±5.06), SOD (25.16±4.34 vs 16.09±2.90) and uterine catalase (29.52±4.83 vs 15.04±2.41) and SOD (48.56±4.70 vs 21.42±0.35), respectively. Additionally, CCP (100 mg/kg) significantly decreased mammary and uterine MPO (73%, 57%), NO (21%, 26%) and malondialdehyde (10%, 31%) when compared with [MNU+BP]-treated rats. The CCP (100 mg/kg) decreased ER+, PR+, EGFR-2, cyclooxygenase-2, iNOS, IL-6, IL-1β, Bcl- 2 and increased p53 expression in the mammary tissues of [MNU+BP+CCP]-treated rats. Histology of mammary tissues showed atypical epithelia, high nucleocytoplasm and ductal carcinoma in MNU+BP rats, which were reversed in rats given CCP (100 mg/kg). C. portoricensis demonstrated chemopreventive effects via induction of apoptosis and reduction of inflammatory cytokines.