Elaine C.M. Silva-Zacarin

Early cytotoxic and genotoxic effects of atrazine on Wistar rat liver: a morphological, immunohistochemical, biochemical, and molecular study.

Risk assessments suggest that intermediate and long-term exposure to triazine herbicides and its metabolites through water can cause severe damage to human health. The objective of this study was to investigate the possible effects of atrazine on Wistar rats submitted to subacute treatment. For this purpose, the activity of catalase and alanine aminotransferase was quantified, and the effect of the herbicide on cell membranes was examined based on the measurement of lipid peroxidation and consequent formation of malondialdehyde and on the mRNA expression of antioxidant enzymes (Mn-superoxide dismutase [SOD] and GSTM1) and connexins. In addition, we evaluated histopathological alterations in the liver, cellular expression of SOD and glutathione (GST), activation of heat shock proteins (HSPs) by immunohistochemistry, and the induction of apoptosis. The genotoxic potential of the herbicide was investigated by the micronucleus test in bone marrow smears. Adult male Wistar rats were treated with an aqueous solution of atrazine at a concentration of 400mg/kg/day, by gavage, for 14 consecutive days. Control groups were also included. The results showed an increase of catalase levels and maintenance of the expression of antioxidant enzymes (SOD and GST). In addition, lipid peroxidation, hepatic tissue degeneration, activation of HSP90, increased levels of connexin mRNA, and genotoxicity were observed. In conclusion, atrazine induced early hepatic oxidative stress that triggered defense mechanisms to maintain the morphophysiological integrity of the liver. Further studies are needed to better understand the effects of this herbicide on human health.

Autophagy and its physiological relevance in arthropods: Current knowledge and perspectives

Autophagic process is one of the best examples of a conserved mechanism of survival in eukaryotes. At the molecular level there are impressive similarities between unicellular and multicellular organisms, but there is increasing evidence that the same process may be used for different ends, i.e., survival or death, at least at cellular levels. Arthropods encompass a wide variety of invertebrates such as insects, crustaceans and spiders, and thus represent the taxon in which most of the investigations on autophagy in non-mammalian models are performed. The present review is focused on the genetic basis and the physiological meaning of the autophagic process on key models of arthropods. The involvement of autophagy in programmed cell death, especially during oogenesis and development, is also discussed.

Effects of sublethal dose of fipronil on neuron metabolic activity of Africanized honeybees.

Fipronil is a neurotoxic insecticide that inhibits the gamma-aminobutyric acid receptor and can affect gustative perception, olfactory learning, and motor activity of the honeybee Apis mellifera. This study determined the lethal dose (LD50) and the lethal concentration (LC50) for Africanized honeybee and evaluated the toxicity of a sublethal dose of fipronil on neuron metabolic activity by way of histochemical analysis using cytochrome oxidase detection in brains from worker bees of different ages. In addition, the present study investigated the recovery mechanism by discontinuing the oral exposure to fipronil. The results showed that mushroom bodies of aged Africanized honeybees are affected by fipronil, which causes changes in metabolism by increasing the respiratory activity of mitochondria. In antennal lobes, the sublethal dose of fipronil did not cause an increase in metabolic activity. The recovery experiments showed that discontinued exposure to a diet contaminated with fipronil did not lead to recovery of neural activity. Our results show that even at very low concentrations, fipronil is harmful to honeybees and can induce several types of injuries to honeybee physiology.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

In vitro effects of thiamethoxam on larvae of Africanized honey bee Apis mellifera (Hymenoptera: Apidae)

Several investigations have revealed the toxic effects that neonicotinoids can have on Apis mellifera, while few studies have evaluated the impact of these insecticides can have on the larval stage of the honeybee. From the lethal concentration (LC50) of thiamethoxam for the larvae of the Africanized honeybee, we evaluated the sublethal effects of this insecticide on morphology of the brain. After determine the LC50 (14.34 ng/μL of diet) of thiamethoxam, larvae were exposed to a sublethal concentration of thiamethoxam equivalent to 1.43 ng/μL by acute and subchronic exposure. Morphological and immunocytochemistry analysis of the brains of the exposed bees, showed condensed cells and early cell death in the optic lobes. Additional dose-related effects were observed on larval development. Our results show that the sublethal concentrations of thiamethoxam tested are toxic to Africanized honeybees larvae and can modulate the development and consequently could affect the maintenance and survival of the colony. These results represent the first assessment of the effects of thiamethoxam in Africanized honeybee larvae and should contribute to studies on honey bee colony decline.

Toxicological and Histopathological Effects of Boric Acid on Atta sexdens rubropilosa (Hymenoptera: Formicidae) Workers

ABSTRACT The current study compared the toxicity of different concentrations of boric acid in adult workers of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae), with toxicological bioassays, and examining the dose-dependent and time-dependent histopathological changes, of the midgut, Malpighian tubules, and postpharyngeal glands. Our results revealed the importance of conducting toxicological bioassays combined with morphological analyses of the organs of ants chronically exposed to insecticides used in commercial ant baits. In vitro bioassays showed that boric acid significantly decreases the survivorship of workers regardless of concentration, whereas the morphological data suggested progressive dose-dependent and time-dependent changes in the organs examined, which were evident in the midgut. The midgut is the first organ to be affected, followed by the postpharyngeal gland and Malpighian tubules. This sequence is in agreement with the absorption pathway of this chemical compound in the midgut, its transference to the hemolymph, possibly reaching the postpharyngeal glands, and excretion by the Malpighian tubules. These progressive changes might be due to the cumulative and delayed effect of boric acid. Our findings provide important information for the understanding of the action of boric acid in ant baits in direct and indirect target organs.

Size changes in honey bee larvae oenocytes induced by exposure to Paraquat at very low concentrations.

The effects of the herbicide Paraquat were investigated in honey bee larvae with attention focused on oenocytes. Honey bee larvae were exposed to Paraquat at different concentrations in the food: 0, 0.001, 0.01, 0.1 and 1 µg/kg. In controls, between 24 h and 48 h, oenocytes grew from 630.1 to 1643.8 µm2 while nuclei changed in size from 124.9 to 245.6 µm2. At 24 h, Paraquat induced a slight decrease in the size of oenocytes and nuclei. N-acetylcysteine (NAC), an antioxidant substance, slightly lowered the effects of Paraquat. At 48 h, Paraquat elicited a strong concentration-dependent decrease in the size of oenocytes, even at the lowest concentration. NAC reversed the effect of Paraquat at a concentration of ≥0.01 µg/kg. This reversion suggested different modes of action of Paraquat, with an oxidant action prevalent at concentrations ≥0.01 µg/kg. This study is the first which reports an effect of a pesticide at the very low concentration of 1 ng/kg, a concentration below the detection limits of the most efficient analytic methods. It shows that chemicals, including pesticides, are likely to have a potential impact at such exposure levels. We also suggest that Paraquat could be used as a suitable tool for investigating the functions of oenocytes.

Effects of Nosema ceranae and thiametoxam in Apis mellifera: A comparative study in Africanized and Carniolan honey bees.

Multiple stressors, such as chemicals and pathogens, are likely to be detrimental for the health and lifespan of Apis mellifera, a bee species frequently exposed to both factors in the field and inside hives. The main objective of the present study was to evaluate comparatively the health of Carniolan and Africanized honey bees (AHB) co-exposed to thiamethoxam and Nosema ceranae (N. ceranae) spores. Newly-emerged worker honey bees were exposed solely with different sublethal doses of thiamethoxam (2% and 0.2% of LD50 for AHB), which could be consumed by bees under field conditions. Toxicity tests for the Carniolan bees were performed, and the LD50 of thiamethoxam for Carniolan honey bees was 7.86 ng bee(-1). Immunohistological analyses were also performed to detect cell death in the midgut of thiamethoxam and/or N. ceranae treated bees. Thiamethoxam exposure had no negative impact on Nosema development in experimental conditions, but it clearly inhibited cell death in the midgut of thiamethoxam and Nosema-exposed bees, as demonstrated by immunohistochemical data. Indeed, thiamethoxam exposure only had a minor synergistic toxic effect on midgut tissue when applied as a low dose simultaneously with N. ceranae to AHB and Carniolan honey bees, in comparison with the effect caused by both stressors separately. Our data provides insights into the effects of the neonicotenoid thiamethoxam on the AHB and Carniolan honey bee life span, as well as the effects of simultaneous application of thiamethoxam and N. ceranae spores to honey bees.

Toxicological and histopathological effects of hydramethylnon on Atta sexdens rubropilosa (Hymenoptera: Formicidae) workers

The leaf-cut ants are important agricultural pest, because they can cause intense defoliation in plants and destroy large areas cultivated. Although there are several works for the control of these insects by examining the toxicity of natural chemical compounds on various species of ants, few are focused on analyses of morphological changes caused in the affected organs. The aim of this study was to evaluate the effects of hydramethylnon on Atta sexdens rubropilosa workers through toxicological bioassays and morphological analysis of the post-pharyngeal glands, midgut, and Malpighian tubules of these ants. Hydramethylnon dissolved either in acetone (HA) or in a mixture of acetone and soy oil (HAO) was added to the artificial diet at a concentration of 200μg/mL. The workers fed daily with the diet containing hydramethylnon showed higher mortality than the controls, especially when HAO was used. Moreover, light and electron microscopy revealed morphological alterations in the midgut and Malpighian tubules of workers treated with HA, whereas alterations of the post-pharyngeal glands were observed in the HAO-treated group. These results indicated that the presence of soy oil provided an alternate route for the ingestion of the formicides active ingredient and corroborated previous studies that suggested a role for the post-pharyngeal glands in lipid metabolism. Our findings suggest that the oil may carry hydramethylnon to the gland lumen, resulting in lower quantity of the active ingredient in the intestinal lumen and Malpighian tubules that explains the lower degree of morphological alterations in these structures in the workers treated with HAO. These results may provide insight into the toxicological effects of hydramethylnon on leaf-cutting ants and the use of vegetable oil as an adjuvant in baits to control ants.

Negative impact of a cadmium concentration considered environmentally safe in Brazil on the cardiac performance of bullfrog tadpoles

A drastic amphibian decline has been observed worldwide, which can be attributed (among other factors) to exposure to pollutants. Considering that cadmium corresponds to the most rapidly increasing trace metal in the environment, the aim of this work was to evaluate whether the exposure (2 and 16 days) of bullfrog tadpoles to this trace metal, at the concentration currently considered environmentally safe (at 1ppb) in class 1 and 2 waters by the Brazilian Environmental Council, can affect the cardiac performance of these animals. The acute exposure (2 days) of tadpoles to cadmium resulted in a marked bradycardic response, which was correlated with an incomplete cardiac relaxation, without any compensation by improved cardiac twitch force (Fc) or contraction velocity (TPT), nor even by cardiac hypertrophy. Indeed, after 16 days of exposure, the cardiac function of tadpoles became even more depressed due to a marked decrease in Fc, a prolongation of TPT, and also incomplete relaxation (i.e. increases in the ventricle resting tension), without changes in ventricle relative mass. Altogether, the cardiodepressive effects of cadmium (especially after more prolonged exposure periods) impose negative alterations on a tadpole׳s development and also impede adequate homeostatic adjustments to respond appropriately to the exposure to cadmium with increase in energetic demand to counteract the deleterious effects of the xenobiotic. These disturbances can impair tadpoles׳ growth, development and reproduction. It is a fact that allows us to strongly suggest that cadmium concentrations, which are currently considered environmentally safe in Brazil, should be revised.