Maternal feed restriction during pregnancy in Wistar rats: Evaluation of offspring using classical and immunoteratology protocols.

Studies have revealed that impairment of the pregnant body weight reduces the fetal body weight and causes minor changes in skeletal development. The aim of the present study was to assess the effects of maternal feed restriction during pregnancy in offspring immune system development. Pregnant Wistar rats were distributed into 5 groups: 1 control in which dams received food ad libitum and 4 experimental groups in which dams were fed restricted amounts of rodent ration (16, 12, 9, or 6 g/rat/day) from the 6th to 17th gestation day. Teratogenicity was assessed using classical teratological evaluation and developmental immunotoxicology protocols. Maternal body weight gain, fetus weight, and placenta weight were reduced for feed-restricted females from the groups fed 12, 9, and 6 g/rat/day ( p < 0.05). No pup mortality was observed immediately after cesarean sections among the groups, and no visceral or skeletal malformations were detected. An immunoteratological study revealed an increase in the relative weight of the thymus and an increase in the phorbol myristate-acetate solution-induced hydrogen peroxide release by inflammatory cells in 21-day-old pups. Alterations in the delayed-type hypersensitivity response and the humoral immune response against sheep red blood cells were observed in pups from feed-restricted mothers. Feed restriction in Wistar rats during organogenesis did not promote structural malformations but resulted in offspring with lower birth weights and promoted significant changes in the immune responses of the rat pups.

Effects of long-term administration of Senna occidentalis seeds on the hematopoietic tissue of rats.

Senna occidentalis (S. occidentalis) is a toxic leguminous plant that contaminates crops and has been shown to be toxic to several animal species. All parts of the plant are toxic, but most of the plant's toxicity is due to its seeds. Despite its toxicity, S. occidentalis is widely used for therapeutic purposes in humans. The aim of the present work was to investigate, for the first time, the effects of the chronic administration of S. occidentalis seeds on hematopoietic organs, including the bone marrow and spleen. Fifty male Wistar rats were divided into five groups of 10 animals. Rats were treated with diets containing 0% (control), 0.5% (So0.5), 1% (So1), or 2% (So2) S. occidentalis seeds for a period of 90 days. Food and water were provided ad libitum, except to pair-fed (PF) group which received the same amount of ration to those of So2 group, however free of S. occidentalis seeds. It was verified that rats treated with 2% S. occidentalis seeds presented changes in hematological parameters. The blood evaluation also showed a significant decrease of the Myeloid/Erythroid (M/E) ratio. Chronic treatment with S. occidentalis promoted a reduction in the cellularity of both the bone marrow and spleen. Additionally, we observed changes in bone marrow smears, iron stores and spleen hemosiderin accumulation. Histological analyses of bone marrow revealed erythroid hyperplasia which was consistent with the increased reticulocyte count. These findings suggest that the long-term administration of S. occidentalis seeds can promote blood toxicity.

Toxicity study about a medicinal plant Casearia sylvestris: A contribution to the Brazilian Unified Health System (SUS).

ETHNOPHARMACOLOGICAL RELEVANCE: Casearia sylvestris S.w (Salicaceae) is catalogued by the Brazilian Unified Health System as a plant of interest for the Brazilian population with the purpose of treating inflammatory disorders, such as pain and gastrointestinal disorders based on the folk use and some literature about efficacy; however, no toxicological studies concerned the safety of extract fluid of this plant have been reported. AIM OF THE STUDY: The present study was carried out to evaluate the acute and subchronic toxicity of the hydroethanolic extract fluid (FE) obtained from leaves of C. sylvestris in Wistar rats. MATERIALS AND METHODS: In the acute toxicity test three female Wistar rats were treated with a single dose of FE (2000 mg/kg) administered by oral gavage and observed for 14 days in order to identify signs of toxicity or death. In subchronic toxicity study animals received, by daily gavage three doses 60, 120 and 240 mg/kg of the FE of the plant for 28 and 90 days. The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured weekly and at the end of treatment were analysed hematological, biochemical and histopathological parameters. Also was analysed the cellularity of bone marrow and spleen. Moreover, phytochemical analysis by HPLC-PDA-ESI(+)/MS and CG/MS/EI was carried out to qualify the constituents of the extract. RESULTS: The results of acute study indicated that the LD50 is higher than 2000 mg/kg and at 28 and 90 day oral toxicity showed that there were no toxic effects detected in any of the parameters evaluated: body weight and relative organ weight, general behavioral changes, haematological and biochemical parameters and histopathological examination. The analysis by HPLC-PDA-ESI(+)/MS and CG/MS/EI identified the flavonoids rutin, quercetin and luteolin and also chlorogenic on the extract. CONCLUSION: Based on this study the hydroethanolic fluid extract of C. sylvestris could be safe even when used over a long period for therapeutic uses proposed by the Brazilian Unified Health System.

Selenium reverses Pteridium aquilinum-induced immunotoxic effects.

We have previously shown that bracken fern (Pteridium aquilinum) has immunomodulatory effects on mouse natural killer (NK) cells by reducing cytotoxicity. Alternatively, it has been demonstrated that selenium can enhance NK cell activity. Therefore, the aims of the present study were to evaluate if ptaquiloside, the main toxic component found in P. aquilinum, is responsible for the immunotoxic effects observed in mice, and if selenium supplementation could prevent or even reverse these effects. Male C57BL/6 mice were administered the P. aquilinum extract by daily gavage for 30 days, and histological analyses revealed a significant reduction in splenic white pulp area that was fully reversed by selenium treatment. In addition, mice administered ptaquiloside by daily gavage for 14 days demonstrated the same reduction of NK cell activity as the P. aquilinum extract, and this reduction was prevented by selenium co-administration. Lastly, non-adherent splenic cells treated in vitro with an RPMI extract of P. aquilinum also showed diminished NK cell activity that was not only prevented by selenium co-treatment but also fully reversed by selenium post-treatment. The results of this study clearly show that the immunosuppressive effects of P. aquilinum are induced by ptaquiloside and that selenium supplementation can prevent as well as reverse these effects.

Pharmacokinetics of tramadol and o-desmethyltramadol in goats after intravenous and oral administration.

The aim of this trial was to implement a method to obtain a tool for analyses of tramadol and the main metabolite, o-desmethyltramadol (M1), in goat's plasma, and to evaluate the pharmacokinetics of these substances following intravenous (i.v.) and oral (p.o.) administration in female goats. The pharmacokinetics of tramadol and M1 were examined following i.v. or p.o. tramadol administration to six female goats (2 mg/kg). Average retention time was 5.13 min for tramadol and 2.42 min for M1. The calculated parameters for half-life, volume of distribution and total body clearance were 0.94+/-0.34 h, 2.48+/-0.58 L/kg and 2.18+/-0.23 L/kg/h following 2 mg/kg tramadol HCl administered intravenously. The systemic availability was 36.9+/-9.1% and half-life 2.67+/-0.54 h following tramadol 2 mg/kg p.o. M1 had a half-life of 2.89+/-0.43 h following i.v. administration of tramadol. Following p.o., M1 was not detectable.

Effect of Senna occidentalis seeds on immunity in broiler chickens.

This study investigated possible immunotoxic effects of Senna occidentalis (So) seeds incorporated in broiler chicken rations at different concentrations (0.0%, 0.25%, 0.50% and 0.75%), for 28 or 42 days. We evaluated innate immune function (macrophage activities of spreading, phagocytosis, peroxide and nitric oxide production) and acquired immune function (humoral and cellular immune responses), as well as lymphoid organ weights and pathology. There was enhanced macrophage activity, as hydrogen peroxide production increased (P < 0.05) in cells of birds given 0.75%So, but there were no other pro-inflammatory effects. Birds receiving 0.75% of So in ration for 42 days gained less weight (P < 0.01), and showed a decrease in relative weight of the bursa of Fabricius (P < 0.05) and spleen (P < 0.01). In addition, morphological changes were also noted in these lymphoid organs, with depletion of lymphoid cells on the spleen and bursa of Fabricius, resulting in lower relative weight of both lymphoid organs. No impairment of humoral immune response against Newcastle disease and in cellular immune response after a phytohaemagglutinin challenge was found. It is probable that mitochondrial damage and related apoptosis may be responsible for the enhanced peroxide production and the reduced relative weight of the bursa of Fabricius and spleen.

Effects of long-term cyanide ingestion by pigs.

Animal performance and health status are adversely affected by long-term cyanide ingestion; however, the effects of cyanide ingestion by pigs have not been fully determined. The aim of the present study was to determine the effects of prolonged exposure to different doses of potassium cyanide (KCN) in growing-finishing swine. Twenty-four pigs, 45 days of age, were divided into four equal groups and treated with different doses of KCN: 0, 2.0, 4.0 or 6.0 mg per kg body weight per day for 70 consecutive days. The results showed a significant alteration in thiocyanate, creatinine and urea levels and in alanine aminotransferase activity of swine dosed with 4.0 and 6.0 mg/kg/KCN. Thyroid weight was significantly increased in those pigs from 4.0 mg/kg KCN group, but no change in cholesterol, triiodothyronine or thyroline levels were observed. Body and carcase weights, body weight gain, and bacon thickness were not affected by KCN treatment. The histopathological study revealed increased numbers of vacuoles in the colloid of thyroid follicles, degeneration of cerebellar white matter and Purkinje cells, degeneration of renal tubular epithelial cells, caryolysis and pyknosis in hepatocytes, and disturbance of the normal lobular architecture of the liver in all treated pigs. Thus, long-term administration of KCN to swine affects several tissues and could adversely affect animal production.

Clinical and pathological effects of short-term cyanide repeated dosing to goats.

The purpose of this work is to determine and describe the effects of subacute cyanide toxicity to goats. Eight female goats were divided into two groups. The first group of five animals was treated with 8.0 mg KCN kg(-1) body weight day(-1) for seven consecutive days. The second group of three animals was treated with water as controls. Complete physical examination, including observation for behavior changes, was conducted before and after dosing. One treated animal was euthanized immediately after dosing. Later, two of the remaining treated animals and a control goat were euthanized after a 30-day recovery period. Euthanized animals were necropsied and tissues were collected and prepared for histologic studies. Clinical signs in treated goats were transient and included depression and lethargy, mild hyperpnea and hyperthermia, arrhythmias, abundant salivation, vocalizations, expiratory dyspnea, jerky movements and head pressing. Two goats developed convulsions after day 3 of treatment. One animal developed more permanent behavioral changes as she became less dominant and aggressive. Histologic changes included mild hepatocellular vacuolation and degeneration, mild vacuolation and swelling of the proximal convoluted tubules of the kidneys and spongiosis of the white matter (status spongiosis) of the cerebral white tracts, internal capsule, cerebellar peduncles, spinal cord and peripheral nerves. In summary, sub-lethal cyanide intoxication in goats resulted in behavioral changes, and during the treatment period animals showed delayed signs of toxicity. Significant histologic lesions in goats were observed and need to be characterized further.

Toxic effects of prenatal Ipomoea carnea administration to rats.

Chronic exposure of livestock to Ipomoea carnea, a toxic plant, promotes toxicosis characterized by lysosomal vacuolization of different organs, and is clinically manifested by CNS signs, abnormal endocrine and gastrointestinal functions, alteration of the immune system, and abnormal embryogenesis. The present study evaluated the effects of different doses of the plant extract on pregnant rats and their offspring after oral administration to the dams from day 6 to day 20 of gestation. Histopathology of thyroid, pancreas, liver and kidneys of dams on gestational day 21 showed characteristic vacuolization promoted by I. carnea toxicosis in these organs; the same was observed in the organs of 7-d-old pups. On the other hand, no alteration was found in these same organs of dams the 7th d after parturition. Although the lesions were reversed in the dams, the same did not occur in their pups. I. carnea administration also promoted decreased body weight, thymus atrophy and spleen enlargement in pups. The toxic principle of I. carnea (swainsonine) seems to pass through the placenta.

Effects of Ipomoea carnea aqueous fraction intake by dams during pregnancy on the physical and neurobehavioral development of rat offspring.

The effects of daily prenatal exposure to 0.0, 0.7, 3.0 and 15.0 mg/kg of the aqueous extract (AQE) of Ipomoea carnea dried leaves on gestational days 5-21 were studied in rat pups and adult offspring. The physical and reflex developmental parameters, open-field, plus-maze, social interaction, forced swimming, catalepsy and stereotyped behaviors, as well as striatal, cortical and hypothalamic monoamine levels (at 140 days of age) were measured. Maternal and offspring body weights were unaffected by exposure to the different doses of the AQE. High postnatal mortality, smaller size at Day 1 of life, reversible hyperflexion of the carpal joints and delay in the opening of both ears and in negative geotaxis were observed in the offspring exposed to the higher dose of AQE. At 60 and 90 days of age, open-field locomotion frequency was quite different between 0.0 and animals treated with 0.7 and 3.0 mg/kg AQE. No changes were observed in the plus-maze, social interaction, forced swimming, catalepsy, stereotyped behavior and central nervous system monoamines concentrations. Dams treated with the higher AQE dose showed severe cytoplasmic vacuolation in liver, kidney, pancreas and thyroid tissues, in contrast to the mild vacuolation observed in the other experimental groups. No alterations were observed in the histopathological study of the offspring of all experimental groups at 140 days of age. During adulthood, behavior was not modified in offspring exposed to the higher dose of AQE as well as no changes occurred in central nervous system neurotransmitters. The present data show that the offspring development alterations were not severe enough to produce behavioral and central monoamine level changes.

The effects of low doses of Senna occidentalis seeds on broiler chickens.

The effects of 0.5%, 0.3% and 0.1% w/w concentrations of Senna occidentalis (So) seed mixed with commercial ration were studied in 18 groups of 32 broiler chicks each, from 1 day to 49 days of age. Three groups were fed one of the rations throughout their lives (TL). Three other groups were fed one of the rations from the 1st to the 28th day of life (starter phase, SP), and the final 3 groups were fed one of the rations from the 29th to 49th day (finisher phase, FP). Each experimental group was matched by a control group fed the same diet over the same period but without the inclusion of So. All the animals were killed at 49 days of age, and blood was collected from 10 birds in each group for biochemical studies (ALT, AST, GGT, LDH, UA). A complete necropsy was performed on 3 birds from each group. No significant differences in the biochemical parameters in the serum were found between the control and experimental chicks, but animals treated with 0.5% So in groups FP and TL, gained less weight and chicks that received 0.3% So or 0.5% So in the ration throughout life (TL) had a larger feed conversion ratio. Besides this, degenerative changes were found in the striated skeletal muscle in the chest, in the myocardium and in the liver in the animals that received the higher concentrations of So seeds.

Evaluation of immunomodulatory activity of Ipomoea carnea on peritoneal cells of rats.

In the present study, animals of the experimental groups were treated with an aqueous fraction (AF) of Ipomoea carnea diluted in drinking water in order to obtain daily doses of 3gdryleaves/kg/body weight (bw) and 15g/kg/bw for 14 and 21 days, or by gavage 15g/kg/bw administered for 14 days, respectively. Peritoneal macrophages were collected and submitted to the spreading, phagocytosis, and hydrogen peroxide release tests. AF administration in drinking water for 14 and 21 days promoted increased macrophage phagocytosis activity and hydrogen peroxide release. However, the administration of 15g/kg/bw of AF by gavage for 14 days resulted in no alteration in macrophage activity. These results suggest that low dosages of Ipomoea carnea induced enhanced phagocytosis activity and hydrogen peroxide production by macrophages.

Neuropathologic study of long term cyanide administration to goats.

Cyanogenic glycosides, which release cyanide, are present in several plant species of high importance for animal production, such as cassava and sorghum. Several human neurological diseases have been associated with chronic cyanide exposure. On the other hand, these effects in ruminants are almost unknown. Thus, the objective of the present study was to determine the long-term lesions of the central nervous system (CNS) caused by daily administration of potassium cyanide (KCN) to goats. Thirty-four male goats were divided into five groups, respectively treated orally with 0 (control), 0.3, 0.6, 1.2 or 3.0 mg KCN/kg/day for 5 months. At the end of the experiment, the whole CNS of each animal was collected for histopathology and immunohistochemistry for apoptotic markers (BAX, BCl2 and CPP32) and for glial fibrillary acid protein (GFAP; vimentin). The results showed the presence of spheroids in the pons, medulla oblongata, and ventral horn of the spinal cord, gliosis and spongiosis in medulla oblongata, gliosis in the pons, and damaged Purkinje cells in the cerebellum from goats that received the higher cyanide dose. In goats from the 1.2 mg KCN/kg group we observed congestion and hemorrhage in the cerebellum, and spheroids in the spinal cord. Gliosis was confirmed by GFAP protein expression. Immunohistochemistry for apoptotic markers and typical apoptotic morphology suggested apoptosis did not participate in the pathogenesis of the observed lesions. Thus, chronic cyanide exposure can promote neuropathological lesions also in goats, and this species can be a useful ruminant model to study the neurotoxic effects of long-term cyanide exposure.

Physiopathological effects of the administration of chronic cyanide to growing goats--a model for ingestion of cyanogenic plants.

Ingestion of cyanogenic plants, such as cassava and sorghum, has been associated with goitre and tropical pancreatic diabetes in both humans and animals. Thus, the objective of the present study was to determine the toxic effects on the thyroid and pancreas in growing goats of prolonged exposure to potassium cyanide (KCN). Thirty-four male goats were divided into five groups dosed with KCN at 0 (control). 0.3, 0.6, 1.2 or 3.0 mg/kg daily for 5 months. Blood samples were obtained in order to determine the glucose, cholesterol, thyroxine (T4), triiodothyronine (T3) and thiocyanate concentrations and for haematological studies; pancreas and thyroid gland were collected for histopathological study. The group receiving the highest dose of cyanide showed lower body weight gains and carcase weights and a decrease in plasma T3 concentrations compared to the control group. Reabsorption vacuoles in follicular colloid and normocytic normochromic anaemia were observed in the experimental animals. Inhibition of peripheral conversion of T4 to T3 is suggested. However, no diabetogenic effects were observed.

Lack of protective action of cysteine against the fetotoxic effect of monocrotaline.

Monocrotaline (MCT), a pyrrolizidine alkaloid present in Crotalaria species, has hepatotoxic, nephrotoxic, pneumotoxic and fetotoxic effects. However, the toxic effects of exposure to MCT in adult rats can be prevented by cysteine. Thus, the present study was conducted to evaluate the possible prevention by cysteine of the toxic effects of MCT on pregnant rats. Thirty-six pregnant rats were used. The females in the experimental groups were fed ration containing 0.02% MCT, 0.02% MCT + 1% cysteine, or 1% cysteine from day 6 to day 21 of pregnancy; the control group was fed only common ration for the same period of time. All rats were killed on day 21 of pregnancy and their blood was collected for determination of liver and kidney function. General toxicity to pregnant dams was assessed. Fetuses were removed by caesarian section and embryofetotoxic parameters were examined. Results showed impaired body weight gain in rats fed MCT, with or without cysteine supplementation. Plasma levels of AST, ALT, LDH, GGT, urea and creatinine were increased in MCT animals compared to controls. The pathology study revealed lesions only in dams from the MCT group. The weights of the placentas and fetuses of the MCT and MCT + cysteine groups were significantly lower than those of the control group. Thus, the present data suggests some protective action of 1% of cysteine in ration against the toxic effects of MCT on the dams but not on the litter.

Does prolonged cyanide exposure have a diabetogenic effect?

Cyanide exposure through cassava consumption has been associated with the development of malnutrition-related diabetes mellitus (MRDM). However, there are few experimental reproductions of this disease. In the present study 42 rats received 0, 9.0 or 12.0 mg KCN/kg bw/d for 15 d, 26 pigs were dosed with 0, 2.0, 4.0 or 6.0 mg KCN/kg for 74 d, and 34 goats received 0, 0.3, 0.6, 1.2 or 3.0 mg KCN/kg for 5 mo. At the end of each experimental period, plasma samples were obtained for glucose and thiocyanate measurement, and the pancreas was collected for histopathologic study. No significant differences in plasma glucose concentrations occurred between groups. The pancreas had no pathology. Chronic cyanide exposure did not promote diabetogenic effects in rats, swine or goats, suggesting that cyanide is not responsible for MRDM in humans.

Fetotoxicity and reproductive effects of monocrotaline in pregnant rats.

Four groups of 12 pregnant Wistar rats each were fed with rations containing 0, 0.01, 0.015 and 0.02% of monocrotaline (MCT) from day 6 to 21 of gestation. Liver weights of the dams from the three experimental groups were significantly lower than those from the control group. Serum levels of aspartate aminotransferase; alkaline phosphatase; lactate dehydrogenase; gamma glutamyltransferase, urea and creatinine were significantly higher in dams from MCT 0.02% group. The weights of the placenta, fetuses and fetal lungs of the 0.02% MCT group were significantly lower than those of the control group. A mild to moderate interstitial pneumonia and liver lesions were observed in dams ingesting 0.02% of MCT. These results showed the toxicity of MCT to the females that ingested 0.02% and their fetuses. Because there was no differences on the weight gains and food and water consumption of the dams it is suggested that this toxic effects in the fetuses was caused by the diffusion of MCT through the placenta. No significant differences were observed in the frequency of skeletal and visceral malformation or anomalies between the control and treated groups suggesting that MCT had no teratogenic effect.

Toxicity testing of Senna occidentalis seed in rabbits.

The effect was investigated of administering ground Senna occidentalis seeds to rabbits in different concentrations (1%, 2%, 3% and 4%) in the ration. The experiment lasted 30 days and the toxic effects of the plant were evaluated on the basis of weight gain, histopathological, biochemical and morphometric parameters, as well as histochemistry and electron microscopy. Animals that received the ration containing 4% ground S. occidentalis seeds gained less weight (p < 0.05) and died in the third week. Histopathology revealed that the heart and liver were the main organs affected, with myocardial necrosis and centrolobular degeneration. There was a reduction in cytochrome oxidase activity in the glycogenolytic fibres, together with muscle atrophy, confirmed by the morphometric studies. Electron microscopy of the liver cells revealed dilated mitochondria, with destruction of the internal cristae.

Evaluation in rabbits of the fetal effects of maternal ingestion of Solanum malacoxylon.

A study was performed to determine the possible toxic effects on the young of does that had been fed during the gestational period on a ration containing Solanum malacoxylon (Sm), a calcinogenic plant that contains a vitamin D3-glycoside conjugate. Experimental animals received a ration containing 0.03% or 0.04% of Sm leaves on days 6 to 30 of gestation. The levels of calcium, phosphorus and alkaline phosphatase in their sera, as well as their feed intake and body weight, were evaluated weekly. The does were euthanized 3 days after parturition and paraffin sections stained with haematoxylin and eosin were prepared from their heart, lungs, kidneys and aorta for histopathological examination. The young from does in the Sm 0.03% group were euthanized 3 days after birth and biochemical and histopathological determinations were performed, as described for the does. The does in both experimental groups showed decreased feed consumption and those in the Sm 0.04% group showed lower body weights throughout their gestation. Animals treated with Sm 0.04% presented a high incidence of abortion and stillbirth. There were biochemical and histopathological alterations in both experimental groups, which were more prominent in the does in the Sm 0.04% group. Litters from does treated with Sm 0.03% showed mineralization of soft tissue and an increase in phosphorus and calcium levels. These findings indicate that the vitamin D3-glycoside passes through the placental barrier to the fetus.

Effects of milk from goat fed Crotalaria spectabilis seeds on growing rats / Efeitos da administração de ração contendo leite de cabra alimentada com sementes de Crotalaria spectabilis a ratos em crescimento

Seeds of Crotalaria spectabilis, containing the pyrrolizidine alkaloid (PA) monocrotaline (MCT), were fed to a lactating dairy goat. Milk from this goat was fed to rats for 8 weeks to determine whether MCT or its toxic metabolites are transferred into the goat's milk. Rats from the experimental group showed significantly higher (p<0.05) serum levels of ALT, AST, GGT and LDH and less weight gains (p<0.05) than control rats. The most significant lesions in rats consuming the experimental ration were mild to moderate interstitial pneumonia and a vacuolar degeneration and occasionally necrosis of periportal hepatocytes. The results of this study indicate that the PA and/or its metabolites are eliminated in milk.(AU)