Ethanol-Extracted Brazilian Propolis Exerts Protective Effects on Tumorigenesis in Wistar Hannover Rats

<div><p>The present study was conducted over a course of 104 weeks to estimate the carcinogenicity of ethanol-extracted Brazilian green propolis (EEP). Groups of 50 male and 50 female Wistar Hannover rats, 6-week-old at commencement were exposed to EEP at doses of 0, 0.5 or 2.5% in the diet. Survival rates of 0.5% and 2.5% EEP-treated male and female rats, respectively, were significantly higher than those of respective control groups. Overall histopathological evaluation of neoplasms in rat tissues after 2 years showed no significant increase of tumors or preneoplastic lesions in any organ of animals administered EEP. Significantly lower incidences of pituitary tumors in 0.5% EEP male and 2.5% EEP female groups, malignant lymphoma/leukemia in both 2.5% EEP-treated males and females and total thyroid tumors in 0.5% EEP male group were found. Administration of EEP caused significant decreases of lymphoid hyperplasia of the thymus and lymph nodes in 2.5% EEP-treated rats, tubular cell hyperplasia of kidneys in all EEP groups, and cortical hyperplasia of adrenals in EEP-treated females. In the blood, significant reduction of neutrophils in all EEP-treated males and band neutrophils in 2.5% EEP-treated females was found indicating lower levels of inflammation. Total cholesterol and triglicerides levels were significantly lower in the blood of 2.5% EEP-treated female rats. In conclusion, under the conditions of the 2-year feeding experiment, EEP was not carcinogenic, did not induce significant histopathological changes in any organ, and further exerted anti-inflammatory and antitumorigenic effects resulting in increase of survival of Wistar Hannover rats.</p></div

Complete blood counts, hematology and biochemistry data.

<p>Complete blood counts, hematology and biochemistry data.</p

Survival curves of male and female rats fed 0.5% and 2.5% EEP-containing diet for 2 years.

<p>Survival curves of male and female rats fed 0.5% and 2.5% EEP-containing diet for 2 years.</p

Incidence of neoplastic lesions observed in the other organs or tissues of Wistar Hannover rats administered EEP for 2 years.

<p>Incidence of neoplastic lesions observed in the other organs or tissues of Wistar Hannover rats administered EEP for 2 years.</p

Incidences of lymphoma/leukemia and neoplastic (benign), neoplasmic (malignant) and preneoplastic (proliferative) lesions in the liver, kidneys, thymus, pituitary gland, thyroid and adrenals of Wistar Hannover rats administered EEP for 2 years.

<p>Incidences of lymphoma/leukemia and neoplastic (benign), neoplasmic (malignant) and preneoplastic (proliferative) lesions in the liver, kidneys, thymus, pituitary gland, thyroid and adrenals of Wistar Hannover rats administered EEP for 2 years.</p

Differentially expressed genes in the livers of rats treated with DEN and administered Valerian at doses of 50, 500 and 5000 ppm, identified by Affimetrix microarray analysis.

<p>TR: transcription regulation; CP: cell proliferation; CC: cell cycle; BS: brain segmentation; LM: lipid metabolism; ST: signal transduction; M: metabolism; CM: cell migration, D: differentiation; CG: cell growth; A: apoptosis; BAB: bile acid biosynthesis; XM: xenobiotic metabolism; T: transport; IM: inactivation of MAPK; R: receptor; ANG: angiogenesis.</p><p>Differentially expressed genes in the livers of rats treated with DEN and administered Valerian at doses of 50, 500 and 5000 ppm, identified by Affimetrix microarray analysis.</p

Numbers and areas of GST-P⁺ foci in the livers of rats treated with DEN or vehicle and administered Valerian at doses of 50, 500 and 5000 ppm.

<p>Data are Mean ± SD (all survived rats were subjected to analysis). ^**P<0.01; ^***P<0.0001 versus the DEN control group.</p

Alteration of cell proliferation and apoptosis in GST-P⁺ foci and surrounding liver tissue, 8-OHdG formation and GABA(A)RA1/GST-P⁺ foci numbers in the livers of rats treated with DEN or vehicle (saline) and administered Valerian at doses of 50, 500 and 5000 ppm.

<p>Data are Mean ± SD; ^aP<0.05; ^bP<0.01; ^cP<0.001; ^dP<0.0001 vs DEN control group; ^e(n = 10) for DEN, DEN→Val, 50 ppm, DEN→Val, 500 ppm, DEN→Val, 5000 ppm, Vehicle→Val, 5000 ppm groups and (n = 9) for Vehicle control group. *P<0.05 and **P<0.01 vs vehicle control group; ND: not detected.</p><p>Alteration of cell proliferation and apoptosis in GST-P⁺ foci and surrounding liver tissue, 8-OHdG formation and GABA(A)RA1/GST-P⁺ foci numbers in the livers of rats treated with DEN or vehicle (saline) and administered Valerian at doses of 50, 500 and 5000 ppm.</p

Double immunohistochemistry for GST-P (red) and PCNA (brown/black) (A, B), GST-P (red) and apoptosis (TUNEL) (black) (C, D), and immunohistochemical assessment in serial sections of GABA(A)RA1 (blue) and PCNA (brown/black) (E, F) and GST-P (red) (G, H) in the livers of F344 rats treated with DEN (A, C, E, G) or Valerian at a dose of 5000 ppm after DEN initiation (B, D, F, H). E and G, F and H are serial sections, respectively.

<p>Three preparations of liver tissue for each rat were used in the analysis of GABA(A)RA1⁺/GST-P⁺ foci numbers. 5000 cells on the normal-appearing liver tissue and total cells in the GST-P⁺ foci areas were checked for PCNA and apoptotic indices. Note the decreased number of PCNA positive cells and induction of apoptosis in GST-P⁺ foci in 5000 ppm Valerian treated rats, with membranous overexpression of GABA(A)RA1 in GST-P⁺ foci after DEN initiation.</p

Body, relative liver and kidney weights and AST and ALT levels in the blood serum of rats treated with DEN or vehicle (saline) and administered Valerian at doses of 50, 500 and 5000 ppm.

<p>Data are Mean ± SD; ^aP<0.05; ^bP<0.01; ^cP<0.0001 vs DEN control group ^d(n = 10) for DEN, DEN→Val, 50 ppm, DEN→Val, 500 ppm, DEN→Val, 5000 ppm, Vehicle→Val,5000 ppm groups and (n = 9) for Vehicle control group.</p><p>Body, relative liver and kidney weights and AST and ALT levels in the blood serum of rats treated with DEN or vehicle (saline) and administered Valerian at doses of 50, 500 and 5000 ppm.</p