Francisco Eduardo Martinez

Characterization of Chemically Induced Ovarian Carcinomas in an Ethanol-Preferring Rat Model: Influence of Long-Term Melatonin Treatment

Ovarian cancer is the fourth most common cause of cancer deaths among women, and chronic alcoholism may exert co-carcinogenic effects. Because melatonin (mel) has oncostatic properties, we aimed to investigate and characterize the chemical induction of ovarian tumors in a model of ethanol-preferring rats and to verify the influence of mel treatment on the overall features of these tumors. After rats were selected to receive ethanol (EtOH), they were surgically injected with 100 µg of 7,12-dimethyl-benz[a]anthracene (DMBA) plus sesame oil directly under the left ovarian bursa. At 260 days old, half of the animals received i.p. injections of 200 µg mel/100 g b.w. for 60 days. Four experimental groups were established: Group C, rats bearing ovarian carcinomas (OC); Group C+EtOH, rats voluntarily consuming 10% (v/v) EtOH and bearing OC; Group C+M, rats bearing OC and receiving mel; and Group C+EtOH+M, rats with OC consuming EtOH and receiving mel. Estrous cycle and nutritional parameters were evaluated, and anatomopathological analyses of the ovarian tumors were conducted. The incidence of ovarian tumors was higher in EtOH drinking animals 120 days post-DMBA administration, and mel efficiently reduced the prevalence of some aggressive tumors. Although mel promoted high EtOH consumption, it was effective in synchronizing the estrous cycle and reducing ovarian tumor mass by 20%. While rats in the C group displayed cysts containing serous fluid, C+EtOH rats showed solid tumor masses. After mel treatment, the ovaries of these rats presented as soft and mobile tissues. EtOH consumption increased the incidence of serous papillary carcinomas and sarcomas but not clear cell carcinomas. In contrast, mel reduced the incidence of sarcomas, endometrioid carcinomas and cystic teratomas. Combination of DMBA with EtOH intake potentiated the incidence of OC with malignant histologic subtypes. We concluded that mel reduces ovarian masses and the incidence of adenocarcinomas in ethanol-deprived rats.

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

BackgroundMelatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation.MethodsTwenty-four adult Wistar rats, 60 days old (+/- 250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [100 μg/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a.m.ResultsMelatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated.ConclusionsWe suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

Long-term melatonin treatment reduces ovarian mass and enhances tissue antioxidant defenses during ovulation in the rat

Melatonin regulates the reproductive cycle, energy metabolism and may also act as a potential antioxidant indoleamine. The present study was undertaken to investigate whether long-term melatonin treatment can induce reproductive alterations and if it can protect ovarian tissue against lipid peroxidation during ovulation. Twenty-four adult female Wistar rats, 60 days old (± 250-260 g), were randomly divided into two equal groups. The control group received 0.3 mL 0.9% NaCl + 0.04 mL 95% ethanol as vehicle, and the melatonin-treated group received vehicle + melatonin (100 µg·100 g body weight(-1)·day(-1)) both intraperitoneally daily for 60 days. All animals were killed by decapitation during the morning estrus at 4:00 am. Body weight gain and body mass index were reduced by melatonin after 10 days of treatment (P < 0.05). Also, a marked loss of appetite was observed with a fall in food intake, energy intake (melatonin 51.41 ± 1.28 vs control 57.35 ± 1.34 kcal/day) and glucose levels (melatonin 80.3 ± 4.49 vs control 103.5 ± 5.47 mg/dL) towards the end of treatment. Melatonin itself and changes in energy balance promoted reductions in ovarian mass (20.2%) and estrous cycle remained extensive (26.7%), arresting at diestrus. Regarding the oxidative profile, lipid hydroperoxide levels decreased after melatonin treatment (6.9%) and total antioxidant substances were enhanced within the ovaries (23.9%). Additionally, melatonin increased superoxide dismutase (21.3%), catalase (23.6%) and glutathione-reductase (14.8%) activities and the reducing power (10.2% GSH/GSSG ratio). We suggest that melatonin alters ovarian mass and estrous cyclicity and protects the ovaries by increasing superoxide dismutase, catalase and glutathione-reductase activities.

Melatonin Attenuates Her-2, p38 MAPK, p-AKT, and mTOR Levels in Ovarian Carcinoma of Ethanol-Preferring Rats.

Epidermal growth factor receptors 2 (Her-2) and 4 (Her-4) are closely associated with ovarian cancer (OC) progression and metastasis, and a more complete understanding of these signaling pathways allow the development of new therapeutic strategies. Melatonin (Mel) is recognized as having several anticancer properties and has been reported to modulate Her-2 system in aggressive tumors. Here, we investigated OC and the role of Mel therapy on the Her-2- and Her-4-signaling pathway related to downstream molecules in an ethanol-preferring rat model. To induce OC, the left ovary was injected directly with a single dose of 100 µg 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in 10 µL of sesame oil under the bursa. Right ovaries were used as sham-surgery controls. After developing OC, half of the animals received i.p. injections of Mel (200 µg/100 g b.w./day) for 60 days. While Mel therapy was unable to reduce Her-4 and phosphoinositide 3-kinase (PI3K) levels, it was able to suppress the OC-related increase in the levels of the Her-2, p38 mitogen-activated protein kinases (p38 MAPK), protein kinase B (phospho-AKT), and mammalian target of rapamycin (mTOR). In addition, Mel significantly attenuated the expression of Her-2, p38 MAPK, and p-AKT, which are involved in OC signaling during ethanol intake. Collectively, our results suggest that Mel attenuates the Her-2-signaling pathway in OC of ethanol-preferring rats, providing an effective contribution for further development of adjuvant therapies.

Melatonin and ethanol intake exert opposite effects on circulating estradiol and progesterone and differentially regulate sex steroid receptors in the ovaries, oviducts, and uteri of adult rats

Chronic ethanol intake is associated with sex hormone disturbances, and it is well known that melatonin plays a key role in regulating several reproductive processes. We report the effects of ethanol intake and melatonin treatment (at doses of 100 μg/100 g BW/day) on sex hormones and steroid receptors in the ovaries, oviducts and uteri of ethanol-preferring rats. After 150 days of treatment, animals were euthanized, and tissue samples were harvested to evaluate androgen, estrogen, progesterone and melatonin receptor subunits (AR, ER-α and ER-β, PRA, PRB and MT1R, respectively). Melatonin decreased estradiol (E2) and increased progesterone (P4) and 6-sulfatoxymelatonin (6-STM), while an ethanol-melatonin combination reduced both P4 and E2. Ovarian AR was not influenced by either treatment, and oviduct AR was reduced after ethanol-melatonin combination. Oviduct ER-α, ER-β and uterine ER-β were down-regulated by either ethanol or melatonin. Conversely, ovarian PRA and PRB were positively regulated by ethanol and ethanol-melatonin combination, whereas PRA was down-regulated in the uterus and oviduct after ethanol consumption. MT1R was increased in ovaries and uteri of melatonin-treated rats. Ethanol and melatonin exert opposite effects on E2 and P4, and they differentially regulate the expression of sex steroid receptors in female reproductive tissues.

Structural evaluation of the effects of chronic ethanol ingestion on the testis of Calomys callosus.

Chronic exposure to ethanol may results in pathophysiologic changes in cellular function. The present work was designed to investigate the morphology of testis submitted to experimental ethanol ingestion. Experimental animals were divided into two groups. The control group (n=23) received a solid diet and tap water and the alcoholic group (n=23) received the same solid diet and ethanol P.A. diluted 20% in water (v/v). After 120 days of treatment, all animals were anesthetized, weighed and sacrificed. Testosterone and luteinizing hormone levels in serum were lower in the alcoholic group than in the control group. Histological and ultrastructural alterations were observed in the testicular alcoholic germinative cells like enormous spaces, lipid droplets accumulation, digestive vacuoles, irregular diameter of the seminiferous tubules and interstitial dilated blood vessels. It was concluded that 20% ethanol provokes lesions on the testis germinative epithelium probably inducing gonadal dysfunction.

Ovarian structure and hormonal status of the UChA and UChB adult rats in response to ethanol.

OBJECTIVES In females, chronic alcoholism has a current and dangerous incidence to fertility. This work had the goal of elucidating the alterations on the ovary of UChA and UChB adult rats (ethanol 10% (v/v) voluntary drinkers). STUDY DESIGN After the treatment period, 42 female rats divided into three experimental groups (UChA, UChB and Wistar) suffered decapitation and their ovaries were removed and processed to further analysis on light and electron microscopy. The ovary was entirely sliced and stained by hematoxylin-eosin, toluidine blue, periodic acid Schiff (PAS) and Massons tricromic. Thereby, the enzymatic reaction to acid and alkaline phosphatase, estral cyclicity, reproductive hormonal status and frequency in oestrous-related ovarian structures were assigned. RESULTS The UChB rats showed an increase in body mass gain index and the ovaries relative weight was significantly lower comparing to the other groups. UCh rats presented the longest estral cycle durations and also persistent oestrous phasis, with uninterrupted cycles. Advanced follicular atresia was common in UCh animals, and degenerating intracellular fragments could be observed through acid phosphatase and electron microscopy techniques. CONCLUSIONS There were some estral cyclicity irregularities caused by chronic ethanol intake in the UCh groups which were consequently reflected as morphologic injury in the ovary structure.

Ultrastructural study of the coagulating gland of Wistar rats submitted to experimental chronic alcohol ingestion.

Morphological changes of coagulating gland of Wistar rats submitted to the experimental chronic alcoholism were noted. Ultrastructurally, it was observed reduction of the granular endoplasmic reticulum cisternae and nuclei with basal position and irregular shape. The epithelial cells presented pycnotic nuclei and the mitochondrial cristae disappeared, characteristic of cellular degeneration.

Repercussions of castration and vasectomy on the ductal system of the rat ventral prostate

Diseases, such as cancer and benign prostatic hyperplasia, are related to disruption of the mechanism regulating the balance between cell proliferation and apoptosis in prostatic cells. Since castration and vasectomy might alter that balance, this study evaluates the cell proliferation, apoptosis and height of the secretory epithelium of the ventral‐prostate ductal system post‐castration and vasectomy. Immunohistochemical (PCNA and Ki67), cytochemical (Fuelgen reaction) and morphometric investigation have been carried out. Cell proliferation indices decreased significantly in both regions of the ventral‐prostate ductal system after castration compared to the sham‐operated group. The apoptotic index increased significantly after 48 h, declining 7 days post‐castration. The cell proliferation indices did not differ after 48 h significantly; however, they increased 7 days post‐vasectomy in both regions. The apoptotic index did not differ significantly in either time post‐vasectomy. Castration caused an imbalance in favor of apoptosis, whereas vasectomy caused an imbalance in favor of cell proliferation.

Mast cells and ethanol consumption: interactions in the prostate, epididymis and testis of UChB rats.

Citation 
Mendes LO, Amorim JPA, Teixeira GR, Chuffa LGA, Fioruci BA, Pimentel TA, de Mello W Jr, Padovani CR, Pereira S, Martinez M, Pinheiro PFF, Oliani SM, Martinez FE. Mast cells and ethanol consumption: interactions in the prostate, epididymis and testis of UChB rats. Am J Reprod Immunol 2011; 66: 170–178