Coumestrol and its metabolite in mares' plasma after ingestion of phytoestrogen-rich plants: potent endocrine disruptors inducing infertility.

Phytoestrogens exist in plants that are present in forages fed to horses. They may compete with 17-ß estradiol and influence the estrous cycle. Therefore, the objective was to determine whether coumestrol from clover-mixed pastures is present in mare's plasma after their ingestion (experiment I), and when this phytoestrogen was present in mare's plasma after ingestion (experiment II). The effect of a long-term ingestion of phytoestrogens on estrous cycle disruption was assessed (experiment III; clinical case). Experiment I was carried out in nonpregnant anestrous and cyclic Lusitano mares (n = 14) kept on clover and grass-mixed pastures, and supplemented with concentrate and hay or cereal straw. Blood and feedstuff were obtained from November to March. In experiment II, stabled cyclic Lusitano mares (n = 6) were fed for 14 days with increasing amounts of alfalfa pellets (250 g to 1 kg/day). Sequential blood samples were obtained for 8 hours after feed intake on Day 0 (control) and on Days 13 and 14 (1 kg/day alfalfa pellets). Experiment III mares were fed with a mixture of alfalfa and clover haylage for 5 months (group 1; n = 4) or for 9 months (group 2; n = 12). Estrous cycle was determined on the basis of plasma estradiol (E2), progesterone (P4), and ultrasound (experiment III). Concentrations of phytoestrogen coumestrol and its metabolite methoxycoumestrol were determined by high-performance liquid chromatography coupled with mass spectrometry. Phytoestrogens decreased in pasture from November until March (P < 0.01) (experiment I), but were always detected in mares' plasma. In experiment II, plasma-conjugated forms of coumestrol and methoxycoumestrol were higher on Days 13 and 14 than in control (P < 0.05). The highest concentrations of conjugated form of coumestrol were at 1.5 and 4 hours (P < 0.001), whereas its free forms peaked at 1 and at 3.5 hours after ingestion (P < 0.05). Methoxycoumestrol-conjugated form concentration was the highest at 1.5 and 5 hours (P < 0.001), whereas its free form peaked at 1 hour (P < 0.05) and at 1.5 hours (P < 0.001). Long-term intake of coumestrol caused lack of ovulation, uterine edema, and uterine fluid accumulation (experiment III). Coumestrol and methoxycoumestrol in both forms were higher in group 2 (while still ingesting haylage) than in group 1, after haylage withdrawal (P < 0.001). These data show that in the mare, coumestrol and its metabolite increase in blood after ingestion of estrogenic plants and can influence reproduction in mares as potent endocrine disruptors.

Effects of methyl jasmonate on accumulation of flavonoids in seedlings of common buckwheat (Fagopyrum esculentum Moench).

The jasmonates, which include jasmonic acid and its methyl ester (MJ), play a central role in regulating the biosynthesis of many secondary metabolites, including flavonoids, and also are signaling molecules in environmental stresses. Synthesis of anthocyanins pigments is a final part of flavonoids pathway route. Accumulation of the pigments in young seedlings is stimulated by various environmental stresses, such as high-intensity light, wounding, pathogen attack, drought, sugar and nutrient deficiency. The anthocyanins take part in defense system against excess of light and UV-B light, and therefore it is probably main reason why young plant tissues accumulate enlarged levels of the pigments. The effects of exogenously applied MJ on level of anthocyanins, glycosides of apigenin, luteolin, quercetin and proanthocyanidins in seedlings of common buckwheat (Fagopyrum esculentum Moench) were studied. MJ decreased contents of all the found cyanidin glycosides and its aglycone in hypocotyls of buckwheat seedlings. However contents of particular anthocyanins in cotyledons of buckwheat seedlings treated with the plant hormone were not significantly different from the control. Applied doses of MJ did not affect levels of quercetin, apigenin and luteolin glycosides in the analyzed parts of buckwheat seedlings: cotyledons and hypocotyls. On the other hand, treatment of buckwheat seedlings with MJ clearly stimulated of proanthocyanidins biosynthesis in hypocotyls. We suggest that methyl jasmonate induces in hypocotyls of buckwheat seedlings the leucocyanidin reductase or anthocyanidin reductase, possible enzymes in proanthocyanidins synthesis, and/or inhibits anthocyanidin synthase, which transforms leucocyanidin into cyanidin. According to our knowledge this is the first report regarding the effect of methyl jasmonate on enhancing the accumulation of proanthocyanidins in cultivated plants.

Antileukemic action of (-)-epicatechin in the spleen of rats with acute myeloid leukemia.

The aim of this study was to investigate whether (-)-epicatechin (EC) can induce DNA damage and apoptosis of cancer cells in the spleen of rat with acute myeloid leukemia. Healthy and leukemic rats were given EC by gavage at a dose of 40 mg/kg b.w. for 22 consecutive days. Spleen cells were subjected for analysis of DNA damage and apoptosis. The amount of DNA damage was estimated by the comet assay, while apoptosis was examined by flow cytometry using Annexin V staining. Leukemic cells were identified in the spleen cells by indirect immunofluorescence using RM-124 antibody followed by flow cytometry analysis. The results show that EC did not affect DNA damage in the splenocytes of healthy rats, but significantly increased the extent of DNA strand breaks in the spleen cells of leukemic animals. EC administration to leukemic rats induced a significant increase in the level of Annexin V-positive leukemic cells, but the level of non-leukemic Annexin V-positive cells remained unchanged in comparison to control. The percentage of leukemic cells decreased significantly under EC influence comparing to the untreated group. The results of the study reveal that EC could be used as an effective supplement of standard therapy against acute myeloid leukemia.