Cytotoxic effects of Euterpe oleraceae fruit oil (açaí) in rat liver and thyroid tissues

Abstract Euterpe oleracea Mart., Arecaceae, fruit (açaí) presents considerable potential for the development of new medicines due to its phytochemical composition and antioxidant activity. More recently, special attention has been given to the pharmacological potential of the fruit's oil. This study analysed the histological and histochemical effects of different dosages of açaí oil on rat's liver and thyroid cells, in order to evaluate its cytotoxic potential after administration for consecutive days. Male Wistar rats were treated with the açaí oil by gavage at doses of 30, 100 and 300 mg/kg, for 14 days, within a 24 h interval. Liver and thyroid fragments were collected for histology (hematoxylin and eosin) and histochemistry analysis (blue of Nilo (lipids), Baker (lipids), bromophenol blue (protein), PAS (polysaccharides)). The results showed that animals exposed to açaí oil presented alterations in the liver cells, where the integrity of the liver tissue was increasingly lost as the açaí oil doses increased. Nuclear pyknosis was observed in several hepatocytes, evidencing the occurrence of cell death. Alteration in the amount of lipids, polysaccharides, vacuoles in the cytoplasm, and proliferation of Kupffer cells were observed in histochemical analyzes. As for the thyroid of the treated rats, alterations were observed in the size of the follicular lumen and also in the connective tissue found between the follicles. Under the experimental conditions employed in the present study, the cytotoxicity observed in this work is worrying, specially considering the liver, when frequent or continuous damage could lead pathological disorders in this organ.

Effects of carvacrol on oocyte development in semi-engorged Rhipicephalus sanguineus sensu lato females ticks (Acari: Ixodidae).

Currently, the most commonly used method to control ticks is the use of synthetic acaricides. However, these compounds are potentially harmful to hosts and the environment, in addition to causing the selection of resistant individuals. Therefore, several studies have been conducted to find sustainable methods to control ticks, such as Rhipicephalus sanguineus sensu lato, the most important vectors of pathogens for dogs. There has been increasing research on natural products with acaricidal action, especially with regard to plant-derived compounds as carvacrol, an aromatic monoterpene with several biological properties, including repellent and acaricidal activities, besides exerting cytotoxic effects on the exposed ticks. The objective of the present study was to evaluate the action of different carvacrol concentrations on the ovaries morphophysiology in semi-engorged R. sanguineus s.l. females to determine its effect on oocyte development. The results showed the occurrence of significant morphological alterations in the shape of oocytes (from round-shaped to irregular) and in the germinal vesicles, in addition to extensive cytoplasmic vacuolation.These effects were observed after the application of carvacrol at a concentration of 20 µL/mL. The most significant alterations were observed at the highest concentration (100 µL/mL), at which the oocytes could not develop further than stage II (total absence of oocytes III, IV and V). These data showed that even though carvacrol was unable to kill all ticks at these concentrations, surviving females could have had an altered reproduction, which would hinder the generation of new individuals, resulting in a long-term control. Data regarding the inhibition of oocyte development are unprecedented and indicate the use of carvacrol as a natural product with the potential to control R. sanguineus s.l. ticks.

In vivo study of a homeopathic medicine against Rhipicephalus (Boophilus) microplus in dairy cow

ABSTRACT The tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) causes large economic losses to cattle breeders and its control is hampered by problems of resistance to the main commercial synthetic acaricides and the risk of residues in animal products. Alternative controls are important for the sustainability of cattle breeding in tropical regions, principally for organic milk production. This study evaluated the efficacy of a homeopathic preparation to control natural tick infestations. The evaluations were conducted with 24 taurine dairy cows during 12 months. The homeopathic medicine (30 CH + Sulfur 30 CH) was administered in a proportion of 1:100 (v/w) in a formulation with sugar, added to 30 kg of mineral salt. This preparation was supplied along with concentrated feed (2 kg/animal/day). The control group received the same feed mixture without the medicine. The groups were kept in separate paddocks. Data were collected on body weight, packed cell volume and number of engorged female ticks on each animal. Temperature, relative humidity and rainfall were recorded. Engorged females were collected from each group for in vitro comparison of reproductive parameters, and semi-engorged ticks for morpho-histological analysis. The mean results of body weight, packed cell volume and tick number were not statistically different between the control and treated groups: 616.5 kg and 618.6 kg; 27.9% and 27.3%; and 12.19 and 13.58 ticks, respectively. In the in vitro analyses, the average weights of the ticks (0.18 and 0.17 g) and eggs (0.07 and 0.06 g), larval hatching rate (70.1 and 76.6%) and reproductive efficiency index (54.3 and 60.6%) of the control and treated groups, respectively, also were not statistically different. Furthermore, no alteration in the ovaries was detected by microscopic analysis. Therefore, the homeopathic medicine was ineffective to control R. (B.) microplus, although the inclusion of agro-ecological practices might enhance its effect of homeopathy in controlling tick infestations.

Toxic action of Acmella oleracea extract on the male reproductive system of Amblyomma cajennense ticks.

The present study evaluated through morphohistological and histochemical techniques the effects of different concentrations of crude ethanolic extract of A. oleracea (EEAO) (Jambu) on the male reproductive system of Amblyomma cajennense sensu stricto (s.s.) ticks. The toxicity of this natural chemical was stablished, signalizing the promising potential of the compound as a strategy to control ectoparasites in the near future. For the experiment, 100 males fed on host rabbits with homogeneous weight (p>0.05) were used. The ticks were divided into five groups (10 animals each): Control 1-exposed to distilled water; Control 2-exposed to ethanol 50% and DMSO 1%; Treatment 1-3-exposed to the concentrations of 6.2, 12.5 and 25mg/mL of the EEAO, respectively, diluted in ethanol 50% and DMSO 1%, with exposure by immersion. After exposure, the males were dissected for the removal of the reproductive system and subjected to routine histological analysis with HE staining and histochemical techniques (PAS for the detection of neutral polysaccharides and Bromophenol blue to detect total proteins). The exposed individuals showed alterations in the glandular complex cells; however, the testes remained intact. The secretory cells of the multilobulated accessory glands presented intense cytoplasmic vacuolation. Additionally, the synthesis and secretion were reduced in the secretion granules, mainly concerning the polysaccharides, glyco- and lipoprotein elements, substances that will constitute the seminal fluid and enable the capacitation of spermatozoa in the female genital tract and also necessary for the formation of the spermatophore, which will encapsulate the mature spermatids. The alterations were dose-dependent, i.e., more intense and severe as the concentration of the product increased. .This experiment confirmed the cytotoxic potential of A. oleracea ethanolic extract in the concentrations of 6.2, 12.5 and 25mg/mL on the reproductive system of A. cajennense s.s. male ticks.

Potential action of extract of Acmella oleracea (L.) R.K. Jansen to control Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) ticks.

The use of synthetic acaricides is currently the main method to control ticks. However, the indiscriminate use of these chemicals can lead to the selection of resistant individuals and in the accumulation of chemical residues in the environment, contaminating the soil and water streams, consequently affecting the flora, fauna, and the human beings as well. In this sense, the objective of this study was to investigate the acaricidal effect of crude ethanolic extract of Acmella oleracea (L.) R.K. Jansen aerials parts at different concentrations on fed males and semi-engorged females of A. cajennense s.s. An in vitro bioassay (Adult Immersion Test) was carried out to determine the lethal concentration 50 (LC50) of ethanolic extract, calculated by Probit analysis. The results showed that the fed males were sensitive to all the concentrations of A. oleracea ethanolic extract, and mortality rate progressively increased (15-65%) in higher ethanolic extract concentrations. However, semi-engorged females were not sensitive to all the concentrations used here. In the highest concentration (100mg/mL), a mortality rate of 100% was observed after 72h of exposure, indicating that the acaricidal effect would probably be dose-dependent. The LC50 values obtained for the fed A. cajennense s.s males and semi-engorged females were 29.4534mg/mL (limits: 24.4467-41.3847mg/mL) and LC50=17.6335mg/mL (limits: 5.2506-23.5335mg/mL), respectively.

Morphological alterations of epidermis of rabbits infested by R. sanguineus ticks and exposed to Selamectin (active principle of Pfizer Revolution(®) acaricide): a confocal microscopy study.

The present study analyzed, by means of confocal laser scanning microscopy, the epidermis of rabbits infested by the brown dog tick Rhipicephalus sanguineus and exposed to concentrations of 50%, 80% and 100% of a selamectin-based commercial acaricide (Pfizer's Revolution(®)). The results demonstrated that rabbits exposed to concentrations of 80% and 100% of the Revolution acaricide, which contains 12% selamectin, showed thinning of epithelial tissue of the epidermis with associated cellular disorganization. Individuals exposed to a 50% concentration showed lower epidermal tissue disorganization when compared to those exposed to the higher doses of the acaricide (80% and 100%). Whereas selamectin, when used in higher concentrations (80% and 100% Revolution(®)) can alter the morphology of the epidermis, at lower concentrations (50%), even though still able to eliminate ectoparasites, it causes less toxicity damage to the host. Selamectin can be considered a dose-dependent toxic agent, since higher concentrations increase the morphological changes in the epidermis of the host rabbits.

The occurrence of autophagic cell death in the tegument of rabbits pre-infested with Rhipicephalus sanguineus and exposed to selamectin (active principle of acaricide Pfizer Revolution®).

Ticks of Rhipicephalus sanguineus species have great medical and veterinary importance for being a vector of various diseases. In an attempt to minimize their action on the host, people have resorted to chemical control by using various acaricides, such as selamectin. Although previous studies have demonstrated its toxic action in domestic animals, no studies focused on the detection of cell death when exposed to selamectin. For this reason, the technique for detecting autophagic cell death was used in order to demonstrate the responses of rabbits' skin tissues pre-infested with R. sanguineus and exposed to different concentrations of selamectin. The obtained results when exposed to 100 and 80% concentrations of selamectin showed a strong mark of acid phosphatase on the cells of the connective tissue of the dermis and hair follicles, whereas the ones exposed to the 50% concentration had a weak mark on the cells of the connective tissue of the dermis and moderate staining in hair follicles. It became clear that, when used at high concentrations (100 and 80%), selamectin is capable to induce a large scale occurrence of the autophagic cell death process. On the other hand, the concentration of 50% causes minor morphophysiological changes in the skin of rabbit hosts when evaluated the cell death process. Therefore, the data confirms that selamectin is a powerful dose-dependent toxic agent causes increased activity of the enzyme acid phosphatase.

Azadirachta indica A. Juss (neem) induced morphological changes on oocytes of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) tick females.

Many studies have been conducted with plants whose extracts have the potential to be used for pest control. One of these plants is Azadirachta indica (neem), whose main active ingredient is azadirachtin, a compound shown to have acaricide and insecticide activity. Rhipicephalus sanguineus (brown dog tick) is currently considered to be an "urban pest," because of its high levels of infestation and its ability to attack humans. In the present study partially and fully engorged R. sanguineus females were exposed to aqueous extracts of neem at concentrations of 10% and 20%, and to a control treatment. The results showed that differently from what was observed in the control, the pedicel cells of females exposed to neem at both concentrations lost their original shape. In the latter cases, the cytoplasm of the cells became fully vacuolated, especially near the germinal vesicle (oocyte nuclei) and in the oocyte pole, which is in contact with the cells of the pedicel. Oocytes in early stages of development (I and II) of ticks treated with both concentrations had irregular germinal vesicle, including the presence of two nucleoli as well as fragments of these. Oocytes in stages IV and V of exposed individuals showed full granular cytoplasm with bigger yolk granules when compared to the early stages. Chorion of mature oocytes was also altered, showing folds and deformations along their entire extension. The observed changes in cells of the reproductive system of R. sanguineus, especially in the oocytes, indicated the potential of neem as a new alternative method to control these ectoparasites.

The dynamics of RNA participation in the vitellogenesis of Rhipicephalus sanguineus ticks Latreille 1806 (Acari:Ixodidae). I. Nucleoli or Cajal bodies?

To understand the morphological and histological aspects of internal systems of ticks has become important matter since these arthropods have an impact in the areas of the economy and public health. In this context, this study has provided morphological data on female germinative cells of Rhipicephalus sanguineus ticks, ectoparasites of dogs that maintain a close relationship with human on a daily basis. Oocytes of engorged females were analyzed, through the PAS reaction (detection of polysaccharides) counterstained by methyl green (detection of RNA) revealing information that allowed to infer for the first time the presence of Cajal bodies, in the germinal vesicles (nuclei) of developing oocytes, as well as showing how the RNA and the polysaccharides are involved in the dynamics of the vitellogenesis in this species.

Action of the chemical agent fipronil on the reproductive process of semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). Ultrastructural evaluation of ovary cells.

The ovary of the tick Rhipicephalus sanguineus consists of a wall of epithelial cells and a large number of oocytes in five different developmental stages (I-V), which are attached to the wall by a pedicel. The present study provides ultrastructural information on the effects (dose-response) of the acaricide fipronil (Frontline) on ovaries of semi-engorged females of R. sanguineus, as well as it demonstrates some possible defense mechanisms used by oocytes to protect themselves against this chemical agent. Individuals were divided into four groups. Group I was used as control while groups II, III and IV were treated with fipronil at the concentrations of 1, 5 and 10 ppm, respectively. Fipronil at the concentration of 10 ppm had the strongest effect on the development of oocytes. At this concentration, even oocytes that reached the final developmental stage exhibited damaged cell structures. Moreover, the observation in fipronil-treated R. sanguineus ticks of damaged cellular components such as plasmic membrane, mitochondria and protein granules (due to alteration in the protein synthesis), and cellular defense mechanisms such as increase in the amount of cytoplasmic microtubules and large amounts of digestive vacuoles and myelin figures, were only possible by means of ultrastructure.

Morphological characterization of the ovary and vitellogenesis dynamics in the tick Amblyomma cajennense (Acari: Ixodidae).

In this work we describe the internal morphology of the female reproductive system of the cayenne tick Amblyomma cajennense. This system is represented by a panoistic ovary, which lacks nurse cells in the germarium. This ovary consists of a single tube, in which a large number of oocytes develop asynchronously, thus accompanying the processes of yolk deposition in the oocytes. The oocytes were classified into stages that varied from I to V, according to: cytoplasm appearance, presence of the germ vesicle, presence of yolk granules, and presence of chorion. The study of vitellogenesis dynamics suggest that the yolk elements are deposited in the oocyte following a preferencial sequence, in which the lipids are the first to appear, followed by proteins an finally by the carbohydrates. In this way the yolk of A. cajennense ticks have these three elements that may be free in the cytoplasm or chemically bounded forming glycoprotein or lipoprotein complexes.