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People are exposed to pesticides through food, drinking water, and the environment. These compounds are associated with several disorders, such as inflammatory diseases, rheumatoid arthritis, cancer, and a condition related to metabolic syndrome. The immunotoxicants or immunotoxic compounds can cause a wide variety of effects on immune function, altering humoral immunity and cell-mediated immunity, resulting in adverse effects to the body. Here, immune system disorders are highlighted because they are closely linked to multiple organs, including the nervous, endocrine, reproductive, cardiovascular, and respiratory systems, leading to transient or permanent changes. Therefore, this study reviewed the mechanisms involved in the immunotoxicity of fungicides, herbicides, and insecticides in cells, animals, and humans in the past 11 years. According to the studies analyzed, the pesticides interfere with innate and adaptive immune functions, but the effects observed mainly on cellular and humoral immunity were highlighted. These compounds affected specific immune cells, causing apoptosis, changes in factor nuclear kappa B (NF-κB) expression, pro-inflammatory factors interleukin 6 (IL-6), interleukin 8 (IL-8), interferon-gamma (IFN-γ), chemokines (CXCL-c1c), and anti-inflammatory factor, such as interleukin 10 (IL-10). To verify the threats of these compounds, new evaluations with immunotoxicological biomarkers are necessary. HighlightsPesticides interfere with the innate and adaptive immune response.Cells, animals and human studies demonstrate the immunotoxicity of pesticides in the cellular and humoral immune response.Fungicides, herbicides, and insecticides alter the immune system by various mechanisms, such as pro-inflammatory and anti-inflammatory factors.

Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone which are used medically for several diseases. However, misuse is commonly observed by athletes to promote enhancement of strength and performance. AAS are frequently obtained through online black markets from clandestine drug manufacturing laboratories, without any quality standards, being potentially dangerous for users. The purpose of this work was the development and application of a fast and simple procedure for the quantitation of stanozolol by liquid chromatography coupled to quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) in tablets packs seized in Rio Grande do Sul state, Brazil. The samples of stanozolol were separated considering its dosage form. The internal standard (methyltestosterone) was added to the aliquots of the samples, dissolved in methanol and 5μL were injected into the analytical system. The newly developed method has been validated for lower limit of quantitation (LLOQ), linearity, accuracy, precision and selectivity. The LLOQ was 0.1 µg/mL. The developed method was successfully applied to 31 samples seized by the Secretaria da Receita Federal do Brasil (a Brazilian federal revenue service agency). According to the results, 90.3% of the suspected medicines (n=31) were adulterated, and 65% exhibited higher concentrations of stanozolol than those indicated in the label. This work successfully established a new method for quantification of stanozolol using LC-QTOF-MS. This method aims at contributing to the identification and quantification of this anabolic androgenic steroid frequently seized by federal inspection agencies.

Introduction The presence of anabolic-androgenic steroids (AAS) in illegal commercial products has been pointed as a global threat for public health. Due the correlation with adverse toxicological effects, there is a growing interest in the implementation of straightforward methods for the determination of AAS in seized products. This work exploited the development of a mass spectrometry approach to characterize the illegal oil formulations containing AAS. Methods The optimization of sample preparation was performed through a simplex-centroid design and the best condition was described as follow: an aliquot of 5 μL of sample were added with 995 μL of acetonitrile and water (75:25, v/v). The solution was vortexed and centrifuged. After that, 10 μL of supernatant were added with 35 μL of acetonitrile and water and internal standard (testosterone-d3, 1.25 ng). An aliquot of 5 μL was injected into the analytical system. Results The method developed was validated and successfully applied in 115 seized samples. Testosterone and its esters had the highest incidence, found in more than 50% of the samples. Besides that, drugs such as boldenone, methandienone, and trenbolone have also been found, where the low quality of the samples was evidenced by the wide variation in the concentration of the drugs, always quantified in sub-doses. Finally, at least one AAS was detected in each sample analyzed. The statistical results were grouped by principal components analysis, to better understand the profile of the seized samples. Conclusion This work successfully established a fast and simple method for determination of AAS and can be applied to verify the profile of seized samples.

An ESI-MS/MS method through direct infusion was validated for quantitative analysis of meropenem powder for injection. The validation parameters were established in a rapid analysis of 30 s. Drug stability was studied through the submission to stress testing, resulting on four degradation products. Under hydrolytic conditions, in acid, neutral and alkaline media, the major degradation product was formed through the cleavage of the β-lactam ring. Oxidation of the drug using H2O2 (3%) showed the formation of two degradation products from a decarboxylation reaction and N-oxide formation. Under high humidity conditions, there was detected a dimer product. The stability of meropenem after reconstitution was studied in conditions that simulate its clinical use. In samples reconstituted and diluted in infusion fluids, an extensive degradation was observed. At room temperature meropenem maintained its content > 90% for up to 4 h when prepared in 5% glucose and for up to 12 h when prepared in 0.9% NaCl. Through ESI-MS/MS analyzes it was observed a degradation product formed by β-lactam ring cleavage, detected in all conditions studied. It was also identified a degradation product formed only in 5% glucose, generated by the hydrolysis of β-lactam followed by the attachment of a glucose molecule to the nitrogen of the pyrrolidine ring. In general, all the results obtained in the stability studies contribute to the knowledge about this antibiotic and future candidates of this class.

Carbapenems show recognized instability in aqueous solutions; therefore some care must be taken in their handling and preparation and their use in the hospital environment. The stability and degradation products of imipenem were investigated from conditions that simulate its clinical use. For this, a simple stability-indicating method by HPLC-DAD was validated with a focus on the quantitation of drug concentration remaining from infusion solutions (sodium chloride 0.9% and glucose 5%). The degradation products formed were identified by high-resolution mass spectrometry (ESI-Q-TOF-MS/MS), with detection of the [M + H]+ ions at m/z 318 (DP-1), m/z 599 (DP-2) and m/z 658 (DP-3). The most probable elemental compositions were obtained with a high degree of confidence, where the error between the masses observed and calculated was 1.25 ppm for DP-1, -0.33 ppm for DP-2 and 1.82 ppm for DP-3. The DP-1 degradation product resulted from cleavage of the β-lactam ring; DP-2 corresponded to the drug dimer; and DP-3 was generated from the interaction between imipenem and cilastatin. The proposed method provides a safe and reliable alternative for the quantitation of imipenem, and the stability data obtained by ESI-Q-TOF help in understanding the drug behavior under the conditions of clinical use.

A gravidade dos efeitos causados pela exposição ambiental e ocupacional ao benzeno determinou o controle de sua utilização. No entanto, mesmo nestas condições, toxicidade ao sistema imune e nervoso tem sido descrita. A toxicidade do benzeno é determinada pelos seus produtos de biotransformação, em que fenol (FE) e hidroquinona (HQ) têm papel relevante na imunotoxicidade. Neste contexto, o presente trabalho mostra que a exposição de ratos Wistar, machos, a doses de 5 ou 10 mg/kg de HQ (via i.p., uma vez ao dia, 13 doses consecutivas, com intervalos de 2 dias a cada 5 doses) provocou reduções acentuadas no influxo de leucócitos polimorfonucleares (PMN) e mononucleares (MN) para o pulmão 24 horas após inalação de Lipopolissacarídeo (LPS) de Salmonella abortus. Diferentemente, a migração de leucócitos em animais expostos ao FE não foi alterada. A exposição a ambos os agentes químicos simultaneamente (dose de 5 mg/kg cada) manteve a redução na migração de MN detectada em animais expostos à HQ e potencializou o efeito inibitório da HQ sobre a migração de leucócitos PMN. Os prejuízos nas migrações de leucócitos não foram decorrentes de modificações no número destas células na circulação. É importante ressaltar que os efeitos foram induzidos por doses dos agentes químicos que não causaram prejuízo à função hepática ou renal, determinados pela atividade das transaminases hepáticas e a concentração de creatinina no soro. Em conjunto, os dados obtidos mostram a exposição a baixas doses de HQ não provoca alterações nos parâmetros empregados como indicadores de toxicidade. No entanto, os efeitos tóxicos são manifestados resposta do organismo ao trauma.The high toxicity induced by occupational and environmental benzene exposure lead to its use restriction. However, at these conditions, neuronal and immune toxicity has been described. It is well known that benzene metabolites, such as hydroxyl compounds phenol (PHE) and hydroquinone (HQ), are responsible for immunotoxicity. In this context, it has shown herein that male Wistar rats exposed to HQ (doses of 5 or 10 mg/kg/day; 13 days with 2-day intervals every 5 doses) presented marked reduction in the number of mononuclear (MN) and polymorphonuclear (PMN) leukocytes in the bronchoalveolar fluid 24 hours after inhalation of Lipopolyssacaride of Salmonella abortus (LPS; 100 µg/mL). On the other hand, leukocyte migration into inflamed lungs was not altered in FE exposed rats, since values obtained were similar to those detected in control animals. Simultaneous exposure to HQ and PHE (5 mg/kg each compound) maintained the decreased number of MN cells observed in HQ exposed rats and potentiated the reduction of PMN cells induced by HQ exposure. The impaired leukocyte migration into inflamed lung did not reflect alterations on number of circulating cells. Moreover, it is important to emphasize that schedule of intoxication did not alter the functional ability of liver and kidney, as detected by normal activity of transaminases and creatinine concentration in the serum. Therefore, it is shown herein that in vivo exposures to lower doses of HQ do not alter end points used as biological indicators of toxicity, nevertheless toxic effects are evident after a host defense.

Phenol (PHE) and hydroquinone (HQ) are metabolites of benzene that affect leukocytes after solvent intoxication. Hence, we investigated the effects of PHE or HQ exposure on neutrophil mobilization during an inflammatory response. Male Wistar rats received intraperitoneal injections of PHE, HQ or vehicle only and assays were performed 24 h after the last dose. Quantifications of bone marrow or circulating leukocytes showed that only HQ exposure induced neutrophilia, probably due to the accelerated mobilization from the bone marrow compartment, since reduced numbers of segmented cells in the last phase of maturation were detected there. Intravital microscopy showed that circulating leukocytes of HQ-exposed rats increased their rolling behavior and adherence to the mesenteric postcapillary venule wall in vivo. The enhanced leukocyte-endothelium interaction was not dependent on microvascular reactivity or perivascular mast cell degranulation. Instead, it was the result of neutrophil activation, demonstrated by a decrease in L-selectin and an increase in beta2 integrin expression on neutrophil membranes. This pattern of neutrophil activation may have contributed to the higher number of neutrophils in the subcutaneous inflammatory response of HQ-exposed rats after oyster glycogen injection. Taken together, our results indicate that HQ exposure alters neutrophil mobilization, which results in an exacerbated response after an injury. Although PHE is endogenously metabolized to HQ, PHE exposure only induced an increment in rolling behavior, which was not sufficient to alter the inflammatory response.

Este estudo avaliou o perfil hepático, renal e a glicemia de ratos submetidos à ingesta de queijo prato contendo extrato de erva-mate e culturas mesófilas. Os ratos receberam 5g de queijo prato por animal/dia contendo extrato de erva-mate (0,1% e 0,2%) e também probióticos por 30 dias. O grupo controle recebeu dieta basal e água ad libitum. O perfil hepático foi avaliado por medida da atividade das transaminases (AST e ALT), enquanto que o perfil renal foi avaliado pela dosagem de ácido úrico. A análise cardiovascular foi composta de análise morfométrica da estrutura das artérias coronárias, diâmetro e espessura do miocárdio e seus vasos sanguíneos. O nível glicemico foi avaliado pela dosagem de glicose sérica. Houve um aumento significativo dos níveis de glicose e ácido úrico nos animais que receberam queijo com extrato de erva-mate. Nos demais parâmetros analisados não houve alteração significativa.

Este estudo teve como objetivo avaliar o efeito do iogurte contendo extrato de erva-mate (Ilex paraguariensis) e com/sem culturas probióticas sobre o perfil lipídico, glicêmico, hepático e renal de ratos alimentados com esses tipos de iogurtes. Ratos da linhagem Wistar (42) foram divididos em três grupos (n=14), e receberam iogurte sem extrato de erva-mate, iogurte com extrato de erva-mate 0,1% e iogurte com extrato de erva-mate 0,1% e culturas probióticas, durante 30 dias. Não foi observado no presente estudo efeito significativo do extrato de erva-mate sobre os níveis de colesterol total, colesterol HDL, triglicerídeos, uréia, ácido úrico, creatinina, glicose e na atividade das enzimas fosfatase alcalina, aspartato aminotransferase e alanina aminotransferase. O extrato de erva-mate, 0,1% no iogurte, não interfere no metabolismo de ratos alimentados por 30 dias. The objective of this study was to evaluate the effect of yoghurt containing yerba-mate extract (Ilex paraguariensis), with and without probiotic cultures, on the lipidic, glycemic, hepatic and kidney profiles of rats fed these types of yoghurt. Wistar rats (42) were divided into three groups (n=14) and for 30 days were fed yoghurt without yerba-mate extract, yoghurt with 0.1% yerba-mate extract and yoghurt with 0.1% yerba-mate extract and probiotic cultures. No significant effect of the yerba mate extract on the levels of total cholesterol, HDL cholesterol, triglycerides, urea, uric acid, creatinine, glucose and the activity of the enzymes alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase was observed in the present study. The addition of 0.1% yerba mate extract to the yoghurt did not interfere with the metabolism of the rats during 30 days.

The vascular effects of nitrolinoleate (LNO2), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO2 was capable to deliver free radical nitric oxide (·NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO2 for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO2 were observed in vivo by intravital microscopy assays. LNO2 decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO2 reduced mRNA and protein expression of β2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO2 on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO2 involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO2 inhibited adhesion molecules expression and promoted ·NO inactivation of the CD40-CD40L system, both important processes of the inflammatory response. © 2010 Elsevier Inc. All rights reserved., This study was supported by Fundação de Apoio à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico/Instituto do Milênio-Redoxoma.

Hidroquinona (HQ) é um dos metabólitos do benzeno responsáveis pelos efeitos tóxicos da exposição ao solvente, além de ser componente da dieta, medicamentos, cigarro e poluente do meio ambiente. Considerando a imunotoxicidade desta substância, o grupo de pesquisa do laboratório investiga o papel da exposição à HQ por período prolongado de tempo sobre respostas inflamatórias agudas (RIA). Neste contexto, o presente trabalho avaliou os efeitos desta exposição sobre os mecanismos vasculares e celulares da RIA de diferentes doses diárias de HQ (5, 10 ou 50 mg/kg) em ratos Wistar machos, via i.p., por 17 ou 22 dias, uma vez ao dia, com intervalos de 2 dias a cada 5 doses. Animais controles receberam o veículo (salina com 5% etanol). A resposta inflamatória aguda inata foi induzida pela administração de glicogênio de ostra (1% em PBS, 5 mL) na bolsa subcutânea dorsal ou pela instilação de lipopolissacarídeo de Salmonella abortus (LPS; 100 µL de solução 100 µg/mL); A resposta inflamatória aguda adquirida foi provocada pela inalação de ovalbumina (10 mL de solução de OA 1% PBS, 15 min) em animais previamente sensibilizados (10 µ/100mg AI(OH)3 no décimo dia de exposição). Os resultados obtidos mostraram que: 1) o aumento do número de leucócitos na bolsa dorsal de animais expostos a 50 mg/kg de HQ é dependente, pelo menos em parte, da maior interação de leucócitos circulantes à parede vascular da microcirculação, mas não é decorrente de alterações na reatividade microvascular; o aumento de expressão de moléculas de adesão (β2 integrina), que pode ser a responsável pelo aumento de interaçãoleucócito-endotélio e migração celular; 2) a redução da migração de leucócitos para o pulmão inflamado pelo LPS em animais expostos a HQ, nas menores doses, não foi decorrente de modificações na interação leucócito-endotélio, nem da expressão de moléculas de adesão nos leucócitos circulantes ou na célula endotelial do tecido pulmonar; 3) a redução da migração celular para o pulmão durante a RIA alérgica em animais expostos a 5, 10 ou 50 mglkg de HQ é dependente, pelo menos em parte, da menor concentração de anticorpos anafiláticos circulantes e conseqüentemente da desgranulação reduzida de mastócitos teciduais, visualizados no leito mesentérico após desafio in situ pela OA. A menor produção de anticorpos anafiláticos pode ser decorrente da ação da HQ em diferentes tipos celulares, uma vez que foi observada expressão reduzida de moléculas co-estimulatórias em linfócitos do baço (CD45R e CD6), menor atividade microbicida de macrófagos peritoniais frente a Candida albicans e menor secreção de interferon-γ por células do peritônio. Em conjunto, os resultados apresentados mostram os mecanismos da HQ sobre a resposta do organismo ao trauma de diferentes origens, interferindo com tipos celulares distintos envolvidos nas reações. Hydroquinone (HQ) is one of the metabolites of benzene responsible for the toxic effects of exposure to solvent, as well as being part of the diet, medicines, tobacco and polluting the environment. Considering the immunotoxicity of this substance, our laboratory has investigated the role of exposure to HQ by prolonged period of time on acute inflammatory responses (AIR). In this context, this study evaluated the effects of this exposure on the vascular and cellular mechanisms of AIR of different daily doses of HQ (5, 10 or 50 mg/kg) in male rats, via ip, for 17 or 22 days, a once a day, with intervals of 2 days every 5 doses. Control animais received the vehicle (saline with 5% ethanol). Innate AIR was induced by the administration of oysters glycogen (1% in PBS, 5 mL) into subcutaneous back pouch or by instillation of lipopolysaccharide of Salmonella aborius (LPS, 100 µL of solução100 µg/mL); Allergic AIR gained was caused by inhalation of ovalbumin (10 mL solution of 1% OA in PBS, 15 min) in previously sensitized animal (10 µ/100mg AI (OH)3 on the tenth day of exposure). Results showed that: 1) the increased number of white blood cells back into the pouch of animais exposed to 50 mg/kg of HQ is dependent, at least in part, of higher interaction of circulating leukocytes to the vascular wall of the microcirculation, but is not due to changes in microvascular reactivity; an increase of expression of molecules of accession (β2 integrin), which may be responsible for the enhanced interaction of leukocyteendothelial and cell migration 2) the reduced migration of leukocytes into the lung inflamed by LPS in animals exposed to 5 or 10 mg/kg was not due to changes in the interactions of leukocyte to endothelium, either the expression of adhesion molecules in circulating white blood cells or in cell endothelial lung tissue, 3) the reduced cell migration to the lungs during the AIR allergic to animais exposed to HQ, in lower doses, is dependent on lower concentration of· circulating anaphylactics antibodies which may be responsible for the mast cell desgranulation. The lower amount of anaphylatic antibodies in the circulation may be due to action of HQ in different cells, as reduced expression of co-stimulatory molecules by Iymphocytes (CD45R and CD6), lower microbicide activity of macrophages and reduced secretion of interferon-γ by peritoneal cells were detected. Together, the results show the toxicity of HQ on the body\'s response to trauma from different sources, interfering with different cell types involved in the reactions.