Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils.

Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 µM; 2 h) and subsequently incubated with LPS (5 µg/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or ß(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor κB (NF-κB) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure.

In vivo hydroquinone exposure impairs MCP-1 secretion and monocyte recruitment into the inflamed lung.

Alveolar macrophages (AMs) are important cells in the resolution of the inflammatory process and they come into direct contact with inhaled pollutants. Hydroquinone (HQ) is an environmental pollutant and a component of cigarette smoke that causes immunosuppressive effects. In the present work, we showed that mice exposed to low levels of aerosolized HQ (25 ppm; 1 h/day/5 days) presented impaired mononuclear cell migration to the lipopolysaccharide (LPS)-inflamed lung. This may have been due to reduced monocyte chemoattractant protein-1 (MCP-1) secretion into bronchoalveolar lavage fluid (BALF), and it was not related to alterations to mononuclear cell mobilization into the blood or adhesion molecules expression on mononuclear cell membranes. Corroborating the actions of HQ on MCP-1 secretion, reduced MCP-1 concentrations were also found in the supernatant of ex vivo AM and tracheal tissue collected from HQ-exposed mice. A direct action of HQ on MCP-1 secretion, resulting from impaired gene synthesis, was verified by in vitro incubation of naive AMs or tracheal tissue with HQ. The role of reduced levels of MCP-1 in the BALF on monocyte migration was analysed in the human monocytic lineage THP-1 in in vitro chemotaxis assays, which showed that the reduced concentrations of MCP-1 found in the BALF or cell supernatants from HQ-exposed mice impaired cell migration. Considering the fact that MCP-1 presents a broad spectrum of actions on pathophysiological conditions and that resident mononuclear cells are involved in lung tissue homeostasis and in immune host defence, the mechanism of HQ toxicity presented herein might be relevant to the genesis of infectious lung diseases in smokers and in inhabitants of polluted areas.

In vivo hydroquinone exposure causes tracheal hyperresponsiveness due to TNF secretion by epithelial cells.

Hydroquinone (HQ) is the main oxidative substance in cigarette smoke and a toxic product of benzene biotransformation. Although the respiratory tract is an inlet pathway of HQ exposure, its effect on airway muscle responsiveness has not been assessed. We thus investigated the effects of low dose in vivo HQ-exposure on tracheal responsiveness to a muscarinic receptor agonist. Male Swiss mice were exposed to aerosolised 5% ethanol/saline solution (HQ vehicle; control) or 0.04 ppm HQ (1h/day for 5 days) and tracheal rings were collected 1h after the last exposure. HQ exposure caused tracheal hyperresponsiveness to methacholine (MCh), which was abolished by mechanical removal of the epithelium. This hyperresponsiveness was not dependent on neutrophil infiltration, but on tumour necrosis factor (TNF) secretion by epithelial cells. This conclusion was based on the following data: (1) trachea from HQ-exposed mice presented a higher amount of TNF, which was abrogated following removal of the epithelium; (2) the trachea hyperresponsiveness and TNF levels were attenuated by in vivo chlorpromazine (CPZ) treatment, an inhibitor of TNF synthesis. The involvement of HQ-induced TNF secretion in trachea mast cell degranulation was also demonstrated by the partial reversion of tracheal hyperresponsiveness in sodium cromoglicate-treated animals, and the in vivo HQ-exposure-induced degranulation of trachea connective tissue and mucosal mast cells, which was reversed by CPZ treatment. Our data show that in vivo HQ exposure indirectly exacerbates the parasympathetic-induced contraction of airway smooth muscle cells, mediated by TNF secreted by tracheal epithelial cells, clearly showing the link between environmental HQ exposure and the reactivity of airways.

Anti-inflammatory activity of crude extract and fractions of Nectandra falcifolia leaves.

The present study evaluated the effect of the crude extract of the leaves of Nectandra falcifolia (NEES) Castiglioni and its fractions in different experimental models of inflammation (paw edema, pleurisy, and ear edema). Carrageenan-induced edema of the paw and pleurisy were evaluated in Wistar rats (180-220 g), which were treated with different doses of the total extract (250, 500 mg.kg-1). Edema of the ear, induced by croton oil, and determination of myeloperoxidase activity were evaluated in Swiss mice (25-35 g). In this experiment, the crude extract of Nectandra falcifolia (Nf) (1.25, 2.5, 5.0, 7.5 mg) and the hexane, chloroform, ethyl-acetate and hydromethanol fractions (5.0 mg) were applied topically, immediately after application of the oil. The crude extract of Nf (500 mg.kg-1) significantly reduced edema of the paw compared to the control group. Similarly, at doses of 250 and 500 mg.kg-1 it significantly reduced the volume of pleural inflammatory exudate compared to the control animals. However, it did not change the number of migrated cells. At doses of 2.5, 5.0 and 7.5 mg, the crude extract significantly inhibited edema of the ear and the influx of neutrophils. The fractions from Nectandra falcifolia (hexane, chloroform, ethyl acetate and hydromethanol) also inhibited edema of the ear. Taken together, the results demonstrated that the crude extract and its fractions administered to animals orally or topically showed an anti-inflammatory effect.

Óxido Nítrico produzido durante a sepse intensifica a redução nos valores da razão de "train-of-four" (TOF) induzida por d-Tubocurarina e pancurônio / Produced Nitric oxide during sepse intensifies the reduction in the values of the reason of “train-of-four” (TOF) induced for d-Tubocurarine and pancuronium

Durante a sepse, grandes quantidades de óxido nítrico (NO) são sintetizadas pela enzima NO sintase tipo II. Em preparações neuromusculares, o NO produz ou acentua a fadiga de transmissão. 'Train-of-four'(TOF) são pulsos elétricos de 2 Hz aplicados ao nervo motor durante 2 segundos (4 contrações musculares) e o quociente entre as tensões musculares produzidas pela quarta (T4) e primeira (T1) contrações musculares (RTOF = T4/T1) é utilizado para avaliação do bloqueio da transmissão neuromuscular. A redução de RTOF é determinada por bloqueio dos receptores nicotínicos (Nn) facilitatórios e por estimulação dos receptores muscarínicos (M2) inibitórios do terminal nervoso motor. Estudos utilizando preparações neuromusculares de animais sépticos tratadas com bloqueadores neuromusculares em doses que reduzem os valores de RTOF sem promover alterações nas amplitudes das contrações musculares a 0,2 Hz não foram executados. Assim, utilizando preparações nervo ciático-músculo tibial anterior de ratos, verificamos que as menores doses de d-tubocurarina (d-TC), pancurônio (PANC) e galamina (GAL) capazes de produzir 30 por cento de redução em RTOF nos animais sham foram, respectivamente: 54,29 ± 2,86 mg/kg (n=7), 69,02 ± 6,13 mg/kg (n=5), 2,94 ± 0,33 mg/kg (n=4). A dose de d-TC foi menor em animais sépticos (34,53 ± 2,76 mg/kg, n=4), que em animais sham, porém os efeitos foram mais intensos e prolongados na sepse. d-TC (26,50 ± 0,68 mg/kg (n=5), promoveu 30 por cento de redução em RTOF nos animais sham submetidos à infusão de nitroprussiato de sódio (10 mg/kg/min). O tratamento com S-metilisotiouréia (SMT, 13 mg/kg) antagonizou a redução de RTOF em animais sépticos. As menores doses de PANC e GAL que reduziram RTOF em 30 por cento foram semelhantes em todos os grupos experimentais estudados e GAL promoveu efeitos similares em todas as condições experimentais. A redução de RTOF produzida por PANC foi menor que a induzida por d-TC e foi antagonizada pela administração de SMT....

Óxido Nítrico produzido durante a sepse intensifica a redução nos valores da razão de "train-of-four" (TOF) induzida por d-Tubocurarina e pancurônio / Produced Nitric oxide during sepse intensifies the reduction in the values of the reason of “train-of-four” (TOF) induced for d-Tubocurarine and pancuronium

Durante a sepse, grandes quantidades de óxido nítrico (NO) são sintetizadas pela enzima NO sintase tipo II. Em preparações neuromusculares, o NO produz ou acentua a fadiga de transmissão. 'Train-of-four'(TOF) são pulsos elétricos de 2 Hz aplicados ao nervo motor durante 2 segundos (4 contrações musculares) e o quociente entre as tensões musculares produzidas pela quarta (T4) e primeira (T1) contrações musculares (RTOF = T4/T1) é utilizado para avaliação do bloqueio da transmissão neuromuscular. A redução de RTOF é determinada por bloqueio dos receptores nicotínicos (Nn) facilitatórios e por estimulação dos receptores muscarínicos (M2) inibitórios do terminal nervoso motor. Estudos utilizando preparações neuromusculares de animais sépticos tratadas com bloqueadores neuromusculares em doses que reduzem os valores de RTOF sem promover alterações nas amplitudes das contrações musculares a 0,2 Hz não foram executados. Assim, utilizando preparações nervo ciático-músculo tibial anterior de ratos, verificamos que as menores doses de d-tubocurarina (d-TC), pancurônio (PANC) e galamina (GAL) capazes de produzir 30 por cento de redução em RTOF nos animais sham foram, respectivamente: 54,29 ± 2,86 mg/kg (n=7), 69,02 ± 6,13 mg/kg (n=5), 2,94 ± 0,33 mg/kg (n=4). A dose de d-TC foi menor em animais sépticos (34,53 ± 2,76 mg/kg, n=4), que em animais sham, porém os efeitos foram mais intensos e prolongados na sepse. d-TC (26,50 ± 0,68 mg/kg (n=5), promoveu 30 por cento de redução em RTOF nos animais sham submetidos à infusão de nitroprussiato de sódio (10 mg/kg/min). O tratamento com S-metilisotiouréia (SMT, 13 mg/kg) antagonizou a redução de RTOF em animais sépticos. As menores doses de PANC e GAL que reduziram RTOF em 30 por cento foram semelhantes em todos os grupos experimentais estudados e GAL promoveu efeitos similares em todas as condições experimentais. A redução de RTOF produzida por PANC foi menor que a induzida por d-TC e foi antagonizada pela administração de SMT....