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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)-1β 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 β(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.

Hydroquinone impairs several leucocyte cell functions, which alter the immune response. Although endothelial cell functions are important for the development of immune responses, hydroquinone actions on endothelial cell have not been shown. Therefore, the effect of hydroquinone exposure (10 or 100 μM for 2 hr) on primary culture of microvascular endothelial cells (PMECs) obtained from the cremaster muscle of Wistar rats incubated in the presence or absence of lipopolysaccharide (LPS, 2 μg/mL) was investigated. Hydroquinone treatment induced the membrane expression of cell adhesion molecules (CAMs) from the immunoglobulin superfamilies ICAM-1 (intercellular), VCAM-1(vascular) and PECAM-1 (platelet endothelial) and induced the secretion of cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). The effects were dependent on transcriptional modifications because enhanced CAM mRNA expression as well as both cytokines and nuclear factor κB (NF-κB) nuclear activation was found. These effects may be due to the direct action of hydroquinone rather than its quinone metabolites, because endothelial cells do not present myeloperoxidase enzyme and hydroquinone incubation did not induce the expression of cytochrome P450 2E1 (CYP2E1) or prostaglandin H synthase 1. In addition, the incubation of endothelial cells with benzoquinone (10 μM, 2 hr) impaired PECAM-1 expression and did not modify NF-κB nuclear activation. Taken together, the data herein presented reveal that hydroquinone evokes pro-inflammatory properties in endothelial cells that are triggered by the enhancement of NF-κB nuclear translocation-dependent gene transcription., (© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

Programmed cell death or apoptosis is one of the defense mechanisms used by insect cells in response to baculovirus infection. Baculoviruses harbour antiapoptotic genes to prevent apoptosis and to maintain the normal course of infection. In this work, we showed that, like other baculoviruses, Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) has a functional inhibitor of apoptosis gene (iap-3). The iap-3 gene was cloned, sequenced and its transcription confirmed by RT-PCR. The putative iap-3 gene of the baculovirus AgMNPV has 864 nucleotides and codes an ORF of 287 amino acids. We have found two BIR motifs (baculoviral iap repeats) at the amino-terminal region and a carboxi-terminal RING finger motif. The IAP-3 protein of AgMNPV is closely related to IAP-3 proteins of baculoviruses and lepidopteran IAPs, with most amino acid identity (75%) with the IAP-3 protein of CfDefNPV (Choristoneura fumiferana DEF nucleopolyhedrovirus). Transcriptional analysis of the AgMNPV iap-3 gene showed that iap-3-specific transcripts could be detected early and late in the infection. The iap-3 gene of AgMNPV was shown to encode a functional IAP since insect cells transfected with increasing amounts of a plasmid containing the iap-3 of AgMNPV showed increased resistance to apoptosis induced by a AgMNPV mutant virus.

The helicase gene from Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) was identified and localized in the 58.85-65.90 m.u. of the viral genomic map. This gene encodes a putative polypeptide of 1221 amino acids containing motifs homologous to those found in the helicase superfamily. Expression of the AgMNPV helicase was observed as early as 4h post-infection (p.i.) up until 10 h p.i. A unique early transcription initiation site was observed upstream a putative TATA box. Phylogenetic analysis of the helicase genes of 23 baculoviruses indicated that the AgMNPV helicase is closely related to the helicase genes from Epiphyas postvitanna multicapsid nucleopolyhedrovirus and Choristoneura fumiferana defective nucleopolyhedrovirus.

The Anticarsia gemmatalis nucelopolyhedrovirus (AgMNPV) egt gene was cloned, sequenced and its expression characterized by RT-PCR and western blot analysis. Sequence analysis of the gene indicated the presence of an open reading frame (ORF) of 1482 nucleotides, which codes for a polypeptide of 494 amino acids. ATATA box and a conserved regulatory sequence (CATT) found in other baculovirus early genes were present in the promoter region of the egt gene. A poly-A consensus sequence was present in the 3' untranslated region (3'-UTR) of the gene. Homology comparisons showed that the EGT protein of AgMNPV is most closely related (95.9% amino acid sequence identity) to the EGT from the Choristoneura fumiferana DEF nucleopolyhedrovirus (CfDEF). Transcriptional analysis of the AgMNPV egt gene showed that egt-specific transcripts can be detected both early and late in infection. The EGT protein was detected, by western blot analysis, in the intra- (from 12 to 48 h post-infection) and extra-cellular (from 12 to 96 h post-infection) fractions of infected insect cells. The AgMNPV Bgl II-F fragment, which has homology to the AcMNPV ie-1 gene, was cloned and used to cotransfect SF21 cells with the cloned AgMNPV egt gene. EGT activity was observed, suggesting that AgMNPV ie-1 can transactivate egt expression.