Synthesis and toxicological evaluation of a chitosan‑L‑leucine conjugate for pulmonary drug delivery applications

Herein are reported the synthesis of a conjugate of chitosan with L-leucine, the preparation of nanoparticles from both chitosan and the conjugate for use in pulmonary drug delivery, and the in vitro evaluation of toxicity and inflammatory effects of both the polymers and their nanoparticles on the bronchial epithelial cell line, BEAS-2B. The nanoparticles, successfully prepared both from chitosan and the conjugate, had a diameter in the range of 10−30 nm. The polymers and their nanoparticles were tested for their effects on cell viability by MTT assay, on trans-epithelial permeability by using sodium fluorescein as a fluid phase marker, and on IL-8 secretion by ELISA. The conjugate nanoparticles had a low overall toxicity (IC50 = 2 mg/mL following 48 h exposure; no induction of IL-8 release at 0.5 mg/mL concentration), suggesting that they may be safe for pulmonary drug delivery applications.

Toxicological Research on Environmental Endocrine Disruptors

Most evidence in terms of endocrine dsiruptors (EDs) mainly originates from studies on reproductive organs. However, in veterbrates, the ability to attain reproductive and development success relays on the intact organization of a complex endocrine system. Disturbances in the regulation of the key hormones and receptors functioning along this system may cause detrimental effects on reproduction and development. Here we reviewed recent studies of EDs on endocrine system. EDs may act on key hormones and receptors along with the hypothalarnic-pituitary-gonald (HPG) axis and lead to reproductive failure. Thyroid disruption may be caused at different levels, for example, the synthesis, transport, binding and cellular uptake along with the hypothalamic-pituitary-thyroid (HPT) axis. Knowledge of model of action EDs is largely via receptors-mediated pathway and alternatively may affect on steroid hormone synthesis. Aquatic hypoxia can influence fish reproduction and thus it is also an endocrine disruptor. Molecular techniques, such as toxicomics, transgenic fish will be employed as powerful tools for environmental EDs risk assessment, as well as in elucidating mechanisms of model action.

High content analysis: A sensitive tool to detect and quantify the cytotoxic, synergistic and antagonistic effects of chemical contaminants in foods

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are important mycotoxins in terms of
human exposure via food, their toxicity and regulatory limits that exist worldwide. Mixtures of toxins can frequently be present in foods, however due to the complications of determining their combined toxicity,
legal limits of exposure are determined for single compounds, based on long standing toxicological
techniques. High content analysis (HCA) may be a useful tool to determine total toxicity of complex
mixtures of mycotoxins. Endpoints including cell number (CN), nuclear intensity (NI), nuclear area (NA),
plasma membrane permeability (PMP), mitochondrial membrane potential (MMP) and mitochondrial
mass (MM) were compared to the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium
bromide (MTT) and neutral red (NR) endpoints in MDBK cells. Individual concentrations of each
mycotoxin (OTA 3mg/ml, FB1 8mg/ml and AFB11.28mg/ml) revealed no cytotoxicity with MTTor NR but
HCA showed significant cytotoxic effects up to 41.6% (p0.001) and 10.1% (p0.05) for OTA and AFB1,
respectively. The tertiary mixture (OTA 3mg/ml, FB1 8mg/ml and AFB1 1.28mg/ml) detected up to 37.3%
and 49.8% more cytotoxicity using HCA over MTT and NR, respectively. Whilst binary combinations of
OTA (3mg/ml) and FB1 (8mg/ml) revealed synergistic interactions using HCA (MMP, MM, NI endpoints)
not detected using MTT or NR. HCA is a highly novel and sensitive tool that could substantially help
determine future regulatory limits, for single and combined toxins present in food, ensuring legislation is based on true risks to human health exposure.

Pharmacometabonomic characterization of xenobiotic and endogenous metabolic phenotypes that account for inter-individual variation in isoniazid-induced toxicological response

An NMR-based pharmacometabonomic approach was applied to investigate inter-animal variation in response to isoniazid (INH; 200 and 400 mg/kg) in male Sprague-Dawley rats, alongside complementary clinical chemistry and histopathological analysis. Marked inter-animal variability in central nervous system (CNS) toxicity was identified following administration of a high dose of INH, which enabled characterization of CNS responders and CNS non-responders. High-resolution post-dose urinary (1)H NMR spectra were modeled both by their xenobiotic and endogenous metabolic information sets, enabling simultaneous identification of the differential metabolic fate of INH and its associated endogenous metabolic consequences in CNS responders and CNS non-responders. A characteristic xenobiotic metabolic profile was observed for CNS responders, which revealed higher urinary levels of pyruvate isonicotinylhydrazone and β-glucosyl isonicotinylhydrazide and lower levels of acetylisoniazid compared to CNS non-responders. This suggested that the capacity for acetylation of INH was lower in CNS responders, leading to increased metabolism via conjugation with pyruvate and glucose. In addition, the endogenous metabolic profile of CNS responders revealed higher urinary levels of lactate and glucose, in comparison to CNS non-responders. Pharmacometabonomic analysis of the pre-dose (1)H NMR urinary spectra identified a metabolic signature that correlated with the development of INH-induced adverse CNS effects and may represent a means of predicting adverse events and acetylation capacity when challenged with high dose INH. Given the widespread use of INH for the treatment of tuberculosis, this pharmacometabonomic screening approach may have translational potential for patient stratification to minimize adverse events.

Aluminium and breast cancer: sources of exposure, tissue measurements and mechanisms of toxicological actions on breast biology

This review examines recent evidence linking exposure to aluminium with the aetiology of breast cancer. The human population is exposed to aluminium throughout daily life including through diet, application of antiperspirants, use of antacids and vaccination. Aluminium has now been measured in a range of human breast structures at higher levels than in blood serum and experimental evidence suggests that the tissue concentrations measured have the potential to adversely influence breast epithelial cells including generation of genomic instability, induction of anchorage-independent proliferation and interference in oestrogen action. The presence of aluminium in the human breast may also alter the breast microenvironment causing disruption to iron metabolism, oxidative damage to cellular components, inflammatory responses and alterations to the motility of cells. The main research need is now to investigate whether the concentrations of aluminium measured in the human breast can lead in vivo to any of the effects observed in cells in vitro and this would be aided by the identification of biomarkers specific for aluminium action.

Modelling grouped survival times in toxicological studies using generalized additive models

A method for combining a proportional-hazards survival time model with a bioassay model where the log-hazard function is modelled as a linear or smoothing spline function of log-concentration combined with a smoothing spline function of time is described. The combined model is fitted to mortality numbers, resulting from survival times that are grouped due to a common set of observation times, using Generalized Additive Models (GAMs). The GAM fits mortalities as conditional binomials using an approximation to the log of the integral of the hazard function and is implemented using freely-available, general software for fitting GAMs. Extensions of the GAM are described to allow random effects to be fitted and to allow for time-varying concentrations by replacing time with a calibrated cumulative exposure variable with calibration parameter estimated using profile likelihood. The models are demonstrated using data from a studies of a marine and a, previously published, freshwater taxa. The marine study involved two replicate bioassays of the effect of zinc exposure on survival of an Antarctic amphipod, Orchomenella pinguides. The other example modelled survival of the daphnid, Daphnia magna, exposed to potassium dichromate and was fitted by both the GAM and the process-based DEBtox model. The GAM fitted with a cubic regression spline in time gave a 61 % improvement in fit to the daphnid data compared to DEBtox due to a non-monotonic hazard function. A simulation study using each of these hazard functions as operating models demonstrated that the GAM is overall more accurate in recovering lethal concentration values across the range of forms of the underlying hazard function compared to DEBtox and standard multiple endpoint probit analyses.

Anxiolytic and sedative effects of extracts and essential oil from Citrus aurantium L.

Citrus aurantium L. is commonly used as an alternative treatment for insomnia, anxiety and epilepsy. Essential oil from peel (EOP) and hydroethanolic (70% w/v) extract (HE) from leaves were obtained. Hexanic (HF), dichloromethanic (DF) and final aqueous (AF) fractions were obtained from HE by successive partitions. Swiss male mice (35-45 g) were treated orally with 0.5 or 1.0 g/kg of these preparations 30 min before the experiments for the evaluation of the sedative/hypnotic activity (sleeping time induced by sodium pentobarbital-SPB: 40 mg/kg, i.p.), anxiolytic activity (elevated plus maze-EPM) and anticonvulsant activity (induced by pentylenetetrazole-PTZ: 85 mg/kg, se or by maximal electroshock-MES: 50 mA, 0.11s, corneal). The results showed that EOP (0.5 g/kg) increased the latency period of tonic seizures in both convulsing experimental models. This effect was not dose-dependent. Treatment with 1.0 g/kg increased the sleeping time induced by barbiturates and the time spent in the open arms of the EPM. Specific tests indicated that the preparation, in both doses used, did not promote deficits in general activity or motor coordination. HF and DF fractions (1.0 g/kg) did not interfere in the epileptic seizures, but were able to enhance the sleeping time induced by barbiturates. The results obtained with EOP in the anxiety model, and with EOP, HF and DF in the sedation model, are in accord with the ethnopharmacological use of Citrus aurantium L., which could be useful in primary medical care, after toxicological investigation.

Antiulcerogenic and analgesic effects of Maytenus aquifolium, Sorocea bomplandii and Zolernia ilicifolia

Maytenus aquifolium (Celastraceae), Sorocea bomplandii (Moraceae) and Zolernia ilicifolia (Fabaceae) are native plants from the Tropical Atlantic Forest (Mata Atlantica, Brazil) known a 'espinheira-santa'. These plants are traditionally used as analgesic and antiulcerogenic medicine, with the same traditional uses of the true 'espinheira-santa' (Maytenus ilicifolia, Celastraceae), an efficient antiulcerogenic agent. Pharmacological and toxicological studies with these plants have not been carried out. The purpose in this study was to evaluate the efficacy (analgesic and antiulcerogenic activities), safety (acute toxicity) and quality (phytochemical profile) of these three plants. The analgesic effect was analyzed by writhing and tail flick tests, while anti Ulcerogenic effect was performed through ulcer induction by ethanol and indomethacin/bethanecol assays. LD., and acute toxic effects, as well as phytochemical profiles of all plants also were carried. Surprisingly, the three plants showed analgesic and antiulcerogenic effects at dose of 1000 mg/kg, v.o. Maytenus aquifolium lowering all ulcerogenic parameters (ethanol test), but increased the ulcerogenic effects in the indoniethacin/bethanecol test. Sorocea bomplandii produced antiulcerogenic effects in both experimental models used, while Zolernia ilicifolia showed significant effects only in indomethacin/bethanecol-induced gastric lesions. Pre-treatment with Zolernia ilicifolia induced someone toxic effects, A phytochemical profile for each plant species was determined and its main chemical classes of compounds were described. (C) 2001 Elsevier B.V. Ireland Ltd. All rights reserved.

Mouriri elliptica: Validation of gastroprotective, healing and anti-Helicobacter pylori effects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Maternal exposure to aqueous extract of Jacaranda decurrens: Effects on reproductive system in male rats

Context: Jacaranda decurrens subsp. symmetrifoliolata Farias & Proenca (Bignoniaceae) is a species widely used for their medicinal properties. At least to our known, no study has been conducted concerning its toxicological profile after gestational and lactational exposure.Objective: The present study was carried out to evaluate the effects of J. decurrens on development of the reproductive system in male rats.Materials and methods: Pregnant rats were treated daily (gavage) with 250 or 500 mg/kg/day of aqueous extract of J. decurrens or vehicle, from day 12 of pregnancy to day 21 of lactation.Results and Discussion: Both doses of J. decurrens significantly anticipated (p < 0.05) the age of testicular descent to the scrotum, a parameter indicative of puberty initiation. Furthermore, at puberty, there was a significant reduction (p < 0.05) in testicular and epididymis weights in the offspring exposed to the higher dose of extract, without effect on sperm production and the histology of reproductive organs. on the other hand, at adulthood, the reproductive parameters analyzed did not differ among groups.Conclusions: J. decurrens, in this experimental model, interfered with the initial development of the reproductive system, but without lasting effects on sperm production in adulthood.

Labaditin, a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Toxicological evaluation of long-term intravenous administration of amitraz in horses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of diethylpropion treatment and withdrawal on aorta reactivity, endothelial factors and rat behavior

Diethylpropion (DEP) is an amphetamine-like compound used as a coadjutant in the treatment of obesity and which presents toxicological importance as a drug of abuse. This drug causes important behavioral and cardiovascular complications; however, the vascular and behavioral alterations during DEP treatment and withdrawal, have not been determined. We evaluated the effects of DEP treatment and withdrawal on the rat aorta reactivity to noradrenaline, focusing on the endothelium, and the rat behavior during DEP treatment and withdrawal. DEP treatment caused a hyporreactivity to noradrenaline in aorta, reversible after 2 days of withdrawal and abolished by both the endothelium removal and the presence of L-NAME, but not by the presence of indomethacin. Furthermore, DEP treatment increased the general activity of rats. Contrarily, DEP withdrawal caused a decrease in the locomotor activity and an increase in grooming behavior, on the 2nd and 7th days after the interruption of the treatment, respectively. DEP treatment also caused an adaptive vascular response to noradrenaline that seems to be dependent on the increase in the endothelial nitric oxide system activity, but independent of prostaglandins synthesis. The data evidenced chronological differences in the adaptive responses of the vascular and central nervous systems induced by DEP treatment. Finally, a reversion of the adaptive response to DEP was observed in the vascular system during withdrawal, whereas a neuroadaptive process was still present in the central nervous system post-DEP. These findings advance on the understanding of the vascular and behavioral pathophysiological processes involved in the therapeutic and abusive uses of DEP. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Pharmacological and toxicological studies of Drimys angustifolia Miers. (Winteraceae)

Drimys angustifolia Miers. (Winteraceae) is a Brazilian medicinal plant used as analgesic, antiulcer and anti-inflammatory without studies to assure its efficacy and safety Leaf and stem bark extracts were evaluated to determine the antiulcer, analgesic, antiinflammatory and antioxidant activities. Preliminary toxic effects and qualitative phytochemical profile were also performed. The antiulcer activity was detected in both extracts. Administration of the leaf extract at 250 mg/kg inhibited total lesion area by 76.50% (p < 0.01 in ethanol/HCl method), while carbenoxolone at 250 mg/kg reduced lesions by 69.48%. Stem bark extract (250 mg/kg) inhibited lesion by 81.42%, while carbenoxolone by 74.10%. Similar effects were observed in the ethanol-induced ulcer method, but no activity was observed in piroxican model. The effects involve nitric oxide in gastric protection, since the L-NAME treatment reversed the protection given by the extracts. Antioxidant effects suggest an involvement against oxidative stress. In the pain (writhing, tail-flick and hot-plate tests) and inflammation (carrageenan-induced paw edema) models, the extracts did not present any effect. The phytochemical studies demonstrated that both extracts contain flavonoids, saponins, glycosilated triterpenoids, fixed acids, cyanogenic glycosides, quinones, tannins, xanthone and steroidal aglycones. Toxicological studies showed that the extracts are safe at the effective antiulcer doses. (c) 2006 Elsevier B.V.. All rights reserved.