Developmental biology meets toxicology: contributing reproductive mechanisms to build adverse outcome pathways.

An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function.

[COPD due to Occupational Noxae]. / COPD durch berufliche Noxen.

The chronic obstructive pulmonary disease (COPD) is characterised by mainly non-reversible bronchial obstruction with airflow limitation. Typically, it exhibits a progressive course. It is one of the leading causes of morbidity and mortality worldwide. In addition to dominating causative smoking and environmental exposures (especially biomass smoke from cooking with open fire stoves), about 15 % are due to occupational exposure. Relatively rare cases (ca. 6 %) do not show an external noxious influence. Occupational causes are frequently not recognised because a detailed occupational history has not been taken. This is especially evident by the discrepancy in the identified COPD prevalences and incidences shown in many studies on the one hand and relatively low numbers in the official statistics on reports, acknowledgements and compensations of the disorder on the other hand. Whether occupational exposures to inhalative noxae are - in addition to non-occupational factors (e. g. smoking) - causative according to legal definitions is frequently a challenging question. Respective decisions of social courts in litigations are presented.

Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2ß1-mediated collagen binding in thrombus formation.

Rare missense mutations in the von Willebrand factor (VWF) A3 domain that disrupt collagen binding have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been limited. Here, we have developed mouse models of collagen-binding mutants and analyzed the function of the A3 domain using comprehensive in vitro and in vivo approaches. Five loss-of-function (p.S1731T, p.W1745C, p.S1783A, p.H1786D, A3 deletion) and 1 gain-of-function (p.L1757A) variants were generated in the mouse VWF complementary DNA. The results of these various assays were consistent, although the magnitude of the effects were different: the gain-of-function (p.L1757A) variant showed consistent enhanced collagen binding whereas the loss-of-function mutants showed variable degrees of functional deficit. We further analyzed the impact of direct platelet-collagen binding by blocking glycoprotein VI (GPVI) and integrin α2ß1 in our ferric chloride murine thrombosis model. The inhibition of GPVI demonstrated a comparable functional defect in thrombosis formation to the VWF(-/-) mice whereas α2ß1 inhibition demonstrated a milder bleeding phenotype. Furthermore, a delayed and markedly reduced thrombogenic response was still evident in VWF(-/-), GPVI, and α2ß1 blocked animals, suggesting that alternative primary hemostatic mechanisms can partially rescue the bleeding phenotype associated with these defects.

Desmolaris, a novel factor XIa anticoagulant from the salivary gland of the vampire bat (Desmodus rotundus) inhibits inflammation and thrombosis in vivo.

The identity of vampire bat saliva anticoagulant remained elusive for almost a century. Sequencing the salivary gland genes from the vampire bat Desmodus rotundus identified Desmolaris as a novel 21.5-kDa naturally deleted (Kunitz 1-domainless) form of tissue factor pathway inhibitor. Recombinant Desmolaris was expressed in HEK293 cells and characterized as a slow, tight, and noncompetitive inhibitor of factor (F) XIa by a mechanism modulated by heparin. Desmolaris also inhibits FXa with lower affinity, independently of protein S. In addition, Desmolaris binds kallikrein and reduces bradykinin generation in plasma activated with kaolin. Truncated and mutated forms of Desmolaris determined that Arg32 in the Kunitz-1 domain is critical for protease inhibition. Moreover, Kunitz-2 and the carboxyl-terminus domains mediate interaction of Desmolaris with heparin and are required for optimal inhibition of FXIa and FXa. Notably, Desmolaris (100 µg/kg) inhibited FeCl3-induced carotid artery thrombus without impairing hemostasis. These results imply that FXIa is the primary in vivo target for Desmolaris at antithrombotic concentrations. Desmolaris also reduces the polyphosphate-induced increase in vascular permeability and collagen- and epinephrine-mediated thromboembolism in mice. Desmolaris emerges as a novel anticoagulant targeting FXIa under conditions in which the coagulation activation, particularly the contact pathway, plays a major pathological role.

Lifelong occupational exposures and hearing loss among elderly Latino Americans aged 65-75 years.

OBJECTIVE: The purpose of this study is to determine the relationship between occupational exposures and hearing among elderly Latino Americans. DESIGN: A descriptive, correlational design used for this secondary analysis with the data from the Sacramento Area Latino Study of Aging (SALSA). STUDY SAMPLE: A total of 547 older adults were included. RESULTS: A majority of participants (58%) reported occupational exposures to loud noise and/or ototoxic chemicals. About 65% and over 90% showed hearing loss at low and high frequencies, respectively. Participants with occupational exposure to loud noise and/or ototoxic chemicals were, significantly, two times more likely to have hearing loss at high frequencies compared to those without exposure (OR = 2.29; 95% CI: 1.17 = 4.51, p = .016), after controlling for other risk factors of hearing loss such as age, gender, household income, current smoking, and diabetes. However, lifelong occupational exposure was not significantly associated with hearing loss at low frequencies (OR = 1.43; 95% CI: 0.94 = 2.18, p = .094). CONCLUSION: Lifelong occupational exposure to loud noise and/or ototoxic chemicals was significantly associated with hearing loss among elderly Latino Americans. Healthy work life through protection from harmful auditory effects of occupational exposures to noise and chemicals will have a positive impact on better hearing in later life.

[Risk assessment for food preparation, cooking and service]. / Rischi da lavoro nel settore della ristorazione.

The restaurant and food preparation, cooking and distribution sector includes hotels, restaurants, catering, fast food, ecc. The restaurant and food preparation, cooking and distribution sector form a significant part of the Italian economy; they provide employment for a large number of people, both direct employees as well as part-time and contract staff. In this sector there are many hazards that can lead to a broad range of injuries and/or diseases to the workers. For the safety these hazards principally are slick floors, open flames, high temperature cooking surfaces, steam, knives and other cutting instruments and machineries. For the health: cleaning and disinfecting chemicals substances, cooking fumes and vapors, biological agents, heavy loads handling, thermal comfort, ecc. The paper presents an overview of the hazards in the sector and then make a focus on chemical risks identification and assessment to evaluate the workers' exposure (by skin adsorption and inhalation).

[Chemical, physical and biological risks in law enforcement]. / Rischi chimici, fisici e biologici nelle Forze dell'Ordine.

Chemical, physical and biological risks among public safety and security forces. Law enforcement personnel, involved in routine tasks and in emergency situations, are exposed to numerous and several occupational hazards (chemical, physical and biological) whith likely health and security consequences. These risks are particularly high when the organization and preparation are inadequate, there is a lacking or insufficient coordination, information, education and communication and safety and personal protective equipment are inadequate or insufficient. Despite the objective difficulties, caused by the actual special needs related to the service performed or the organizational peculiarities, the risk identification and assessment is essential for worker health and safety of personnel, as provided for by Legislative Decree no. 81/2008. Chemical risks include airborne pollutants due to vehicular traffic (carbon monoxide, ultrafine particles, benzene, polycyclic aromatic hydrocarbons, aldehydes, nitrogen and sulfur oxides, lead), toxic gases generated by combustion process following fires (aromatic hydrocarbons, PAHs, dioxins and furans, biphenyls, formaldehyde, metals and cyanides), substances emitted in case of chemical accidents (solvents, pesticides, toxic gases, caustics), drugs (methylamphetamine), riot control agents and self-defence spray, lead at firing ranges, and several materials and reagents used in forensic laboratory. The physical hazards are often caused by activities that induce biomechanical overload aid the onset of musculoskeletal disorders, the use of visual display terminals and work environments that may expose to heat stress and discomfort, high and low pressure, noise, vibrations, ionizing and non-ionizing radiation. The main biological risks are blood-borne diseases (viral hepatitis, AIDS), airborne diseases (eg, tuberculosis, meningitis, SARS, anthrax), MRSA, and vector-borne diseases. Many of these risk factors are unavoidable or are not predictable; so a proper risk assessment is very important, especially in case of emergencies, and also the necessary preventive measures, a careful analysis of alternative options for action and decision-making, implementation of security measures due to the provision of appropriate PPE and effective management of risk communication have great importance. Another important aspect is the education and training of staff, as in emergency situations should be able to take protective measures as quickly as possible.

A platelet target for venous thrombosis? P2Y1 deletion or antagonism protects mice from vena cava thrombosis.

A role for platelets in the pathogenesis of venous thrombosis was suggested by clinical and preclinical studies. However, examination of the platelet receptor, P2Y1, in this area has been limited. The goal of the current study was to examine effects of P2Y1 deletion, or selective antagonism with MRS2500, in oxidative venous thrombosis in mice. The P2Y12 antagonist, clopidogrel, was included as a reference agent. Anesthetized C57BL/6 or genetically modified mice underwent 3.5 or 5 % FeCl(3)-induced vena cava thrombosis. Pharmacokinetic properties of MRS2500 were defined for dose selection. Platelet aggregation and renal or tail bleeding times (BT) were measured to put antithrombotic effects into perspective. P2Y1 deletion significantly reduced (p < 0.001) venous thrombus weight by 74 % in 3.5 % FeCl(3) injury compared to P2Y1(+/+) littermates. MRS2500 (2 mg/kg, i.v.) significantly decreased (p < 0.001) thrombus weight 64 % in C57BL/6 mice. In the more severe 5 % FeCl(3)-induced injury model, thrombus weight significantly (p < 0.001) decreased 68 % in P2Y1(-/-) mice versus P2Y1(+/+) mice, and MRS2500 (2 mg/kg) was also beneficial (54 % decrease, p < 0.01). Renal BT doubled in P2Y1(-/-) versus P2Y1(+/+) mice, and increased threefold with MRS2500 compared to vehicle. Tail BT was markedly prolonged in P2Y1(-/-) mice (7.9X) and in C57BL/6 mice given MRS2500. The current study demonstrates that P2Y1 deletion or antagonism significantly reduced venous thrombosis in mice, suggesting that P2Y1 receptors play a role in the pathogenesis of venous thrombosis, at least in this species. However as with many antithrombotic agents the benefit comes at the potential price of an increase in provoked bleeding times.

Protective effects of bilirubin against cyclophosphamide induced hemorrhagic cystitis in rats.

PURPOSE: The end product of the heme oxygenase pathway, bilirubin, is the most abundant endogenous antioxidant in mammals. We report the heme oxygenase-1 mediated production of bilirubin and its cytoprotective roles in cyclophosphamide induced hemorrhagic cystitis in rats. MATERIALS AND METHODS: Female Sprague-Dawley rats received intraperitoneal administration of cyclophosphamide. In the first experiment hemin (an inducer of heme oxygenase-1) with or without zinc protoporphyrin IX (an inhibitor of heme oxygenase activity) was given before cyclophosphamide injection. Endogenous bilirubin production was analyzed in bladder tissues immunohistochemically. In another experiment bilirubin solution was administered before the cyclophosphamide injection. Changes in bladder weight, microscopic feature and expression levels of inducible nitric oxide synthase, proinflammatory cytokines and heme oxygenase were evaluated using polymerase chain reaction and immunostaining. RESULTS: Bilirubin was generated in bladders with cyclophosphamide induced cystitis, especially in the urothelium and suburothelium. Hemin pretreatment provided increased production of endogenous bilirubin, which was decreased by zinc protoporphyrin IX. In an evaluation of the roles of bilirubin exogenous bilirubin administration ameliorated cyclophosphamide induced inflammatory changes and reduced the increase in bladder weight. The elevated expression of inducible nitric oxide synthase and interleukin-1beta in cyclophosphamide induced cystitis was significantly down-regulated by exogenously applied bilirubin. The expression of heme oxygenase-1 and 2 was not modified by bilirubin administration. CONCLUSIONS: Cyclophosphamide induced hemorrhagic cystitis is accompanied by endogenous bilirubin production through heme oxygenase-1 induction in the bladder. Bilirubin has cytoprotective roles in association with the down-regulation of inducible nitric oxide synthase expression. Our results suggest that bilirubin may have therapeutic potential against bladder inflammatory insults such as cyclophosphamide induced cystitis.

Expression of pancreatitis associated proteins in urothelium and urinary afferent neurons following cyclophosphamide induced cystitis.

PURPOSE: We examined the expression profile of the members of the pancreatitis associated proteins/regenerating gene family in the bladder and in the primary afferent neurons of dorsal root ganglia using an animal model of cystitis. MATERIALS AND METHODS: We examined the expression of pancreatitis-associated protein-I and pancreatitis-associated protein-III in the bladder and the dorsal root ganglia of female rats 4 hours, 48 hours or 10 days after cyclophosphamide (Sigma) injection using immunohistochemistry and reverse transcriptase-polymerase chain reaction. RESULTS: No pancreatitis-associated protein-III immunoreactivity was identified in control bladders but prominent expression was observed in the urothelium of animals with chronic cystitis. Cells expressing pancreatitis-associated protein-I were seen in the dorsal root ganglia but not in the bladder. In normal dorsal root ganglia pancreatitis-associated protein-I was expressed in a minor population of small diameter neurons (2.4%) that were also positive for isolectin-B4. However, by 10 days following the onset of cystitis the number of pancreatitis-associated protein-I positive neurons was increased (7.6%) and pancreatitis-associated protein-I immunoreactivity was further observed in a slightly larger group of neurons and tyrosine kinase A positive small neurons. CONCLUSIONS: The current results suggest that pancreatitis-associated protein-III is associated with bladder inflammation and they implicate pancreatitis-associated protein-I in the abnormal sensation in cystitis.

Effect of chronic intermittent exposure to AM radiofrequency field on responses to various types of noxious stimuli in growing rats.

There are several reports of altered pain sensation after exposure (from a few minutes to hours in single or repeated doses for 2-3 weeks) to electromagnetic fields (EMF) in adults. The commonly utilized noxious stimulus is radiant heat. The nociceptive responses are known to be influenced by characteristics of stimulus, organism, and environment. We studied the pattern of nociceptive responses to various noxious stimuli in growing rats exposed to radiofrequency field (73.5 MHz amplitude modulated, 16 Hz power density 1.33 mw/cm(2), SAR = 0.4 w/kg) for 45 d (2 h/d). Threshold current for stimulation of nociceptive afferents to mediate motor response of tail (TF), vocalization during stimulus (VD), and vocalization after discharge (VA); the withdrawal latency of tail (TFL) and hind paw (HPL) to thermal noxious stimulus and tonic pain responses were recorded in every rat. The TFL was not affected, HPL was decreased (p < 0.01), and the thresholds of TF and VD were not affected, while, that of VA was significantly decreased. The tonic pain rating was decreased (p < 0.01). A decrease in the threshold of VA (p < 0.01) is indicative of an increase in the emotional component of the response to the phasic pain, whereas a decrease in the pain rating indicates analgesia in response to the tonic pain. The results of our study suggest that chronic (45 d), intermittent (2 h/d) amplitude modulated RF field exposure to the peripubertal rat increases the emotional component of phasic pain over a basal eaualgesic state, while late response to tonic pain is decreased. The data suggest that amplitude modulated RF field differentially affects the mechanisms involved in the processing of various noxious stimuli.

Natural organic compounds as alternative to methyl bromide for nematodes control.

Thirty-three organic acids and furfural metabolites were examined for their nematicidal activity against plant-parasitic, free-living and predacious nematodes. Propionic acid, 2-methylhexanoic acid, lactic acid, maleic acid, and furic acid were the most effective nematicides among normal chain organic acids, branched organic acids, hydroxy/keto-acids, dicarboxylic acids and furfural metabolites, respectively. Seven of the tested compounds were found to have more than 90% mortality thus designating them as highly active nematicides. Of the highly active tested compounds, an average octanol/water log P of 0.97 was observed with a range from 0.28 to 2.64, and a Henry's Law constant averaging 2.6 x 10(- 7) atm.m3/mole. Tested chemicals with minor or low nematicidal activity showed an average log P of 1.76 with a range from 0.15 to 3.42 and a Henry's Law constant averaging 16.6 x 10(- 7) atm.m3/mole.

Nociception.

Nociception, the sensory mechanism that allows animals to sense and avoid potentially tissue-damaging stimuli, is critical for survival. This process relies on nociceptors, which are specialized neurons that detect and respond to potentially damaging forms of energy - heat, mechanical and chemical - in the environment. Nociceptors accomplish this task through the expression of molecules that function to detect and signal the presence of potential harm. Downstream of the nociceptive sensory input, the neural signals trigger protective (nocifensive) behaviors, and the sensory stimuli that reach the brain may be perceived as painful.

Odorant identification and quality perception following methyl bromide-induced lesions of the olfactory epithelium.

Using a 5-odorant identification confusion matrix task, the authors assessed the consequence of olfactory epithelial damage on odorant quality perception in the rat. After establishing prelesion identification performance, each rat's epithelium was subjected to 330 ppm methyl bromide gas for 6 hr. Comparison of prelesion and 3-day postlesion performance demonstrated a significant decrease in identification as a consequence of 95%-98% epithelial destruction. Further, there was a differential effect of lesion on the ability of different animals to identify the different individual odorants. Evaluation of the anatomical state of the epithelium relative to performance on the identification task demonstrated a significant relationship between the extent and location of anatomical sparing and changes in individual odorant identifications. Assessment of pre- and postlesion quality perception for the individual rats demonstrated a highly significant shift in quality perception that was independent of any decrease in performance. These results provide strong support for the proposition that the regional variations in mucosal sensitivities within and across olfactory receptor gene expression zones are fundamentally important for the encoding of odorant quality.

Treatment with Met-RANTES decreases bacterial translocation in experimental colitis.

BACKGROUND: During colitis, epithelial function is impaired, leading to increased bacterial translocation. Recent studies have shown the important role of proinflammatory cytokines and chemokines, including RANTES (regulated on activation, normal T-cell expressed and secreted), in inflammatory bowel diseases (IBDs). In this study, we evaluated the role of Met-RANTES, an antagonist of the RANTES receptor, on the impairment of bacterial translocation in a rat model of colitis. METHODS: Rats were randomly assigned to 3 groups. Group 1 = control, group 2 = experimental colitis, and group 3 = colitis plus Met-RANTES treatment. On day 7 after colitis was induced, plasma tumor necrosis factor-alpha colon tissue myeloperoxidase and portal blood endotoxin levels were measured. Lymph node, liver, and spleen culture quantified bacterial translocation. RESULTS: Met-RANTES treatment resulted in significant decreases in colonic damage as well as bacterial translocation in experimental colitis. CONCLUSIONS: These results suggest that chemokine receptor antagonists may potentially be useful in the treatment of IBDs.

Intestinal injury in the rat model of 17α-ethynylestradiol-induced intrahepatic cholestasis.

OBJECTIVE: Although the intimate relationship between liver and gut has been previously reported under physiological and pathological conditions, intestinal involvement in the process of intrahepatic cholestasis of pregnancy remains unclear. The aim of this study was to investigate intestinal changes in 17α-ethynylestradiol (EE)-induced cholestatic rat model. METHODS: Liver injury was assessed by HE stain and serum biochemical parameters were measured. Intestinal transit was determined using ink marks. Neuronal protein expressions in the intestine were analyzed by Western blot. RESULTS: EE treatment induced liver damage, including severe bile duct hyperplasia, portal edema, portal infiltration, a loss of hepatic structure in periportal areas and increased serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total bilirubin. Large areas of inflammatory cell infiltration and increased myeloperoxidase activity were observed in the intestine of EE-induced cholestatic rats. The EE-treated group showed increased intestinal transit and malondialdehyde levels, while the glutathione content and superoxide dismutase activity were notably decreased, together with decreased protein gene product 9.5 and neuronal nitric oxide synthase expression in the ileum and colon. Furthermore, choline acetyltransferase expression was significantly decreased in the ileum, whereas no change was observed in the colon of EE-treated rats. CONCLUSION: EE-induced liver damage is associated with oxidative stress, inflammation and neural loss in the intestine, which may lead to altered intestinal motility.

[Evaluation of the effects of occupational noxae on the cardiovascular system]. / Valutazione degli effetti di noxae lavorative sull'apparato cardiovascolare.

BACKGROUND: Working conditions and the environment may contribute to the multi-factorial aetiology of cardiovascular disease. OBJECTIVES: To provide a critical assessment of epidemiological and clinical methods and tools for evaluating the effects of occupational pathogenic noxae on the cardiovascular system. METHODS: A review was made of epidemiological and clinical studies published in the main scientific journals of occupational medicine and cardiology, in the period 1980-2003. Data sources were electronic medical data bases and conference proceedings. RESULTS AND CONCLUSION: Collecting case histories by means of free or questionnaire-structured interviews, observing specific physical signs, detecting changes in blood chemistry parameters and identifying morphological or functional abnormalities in the heart and vessels are all useful approaches. Some blood chemistry parameters that may be modified by occupational exposure or by particular conditions arising from work organization are cholesterol, triglycerides, apolipoproteins A and B, platelets, fibrinogen, factor VIIc, fibrinolysis products, plasminogen tissue activating factor, complement and glycated hemoglobin. They can all be measured easily and quickly and provide an estimate of the risk of cardiovascular disease. As high blood pressure is closely correlated to heart disease, blood pressure levels can be monitored in a working population using a standard mercury sphygmomanometer. Electronic measurement before and after a work shift and 24 hour Holter monitoring help reduce the "white coat effect" and provide further useful information. Occupational risk factors such as toxins (metals, solvents, pesticides), electromagnetic fields, extreme temperatures, noise, radiation and psychophysical stress can affect the cardiac neuro-autonomic balance of the exposed workers and cause cardiovascular abnormalities. These can be detected by long-term ECG monitoring, and are revealed as reduced heart rate variability and prolonged QT interval. Recently non-invasive systems have been adopted to detect cardiovascular lesions that are usually due to atherosclerosis. In occupational and environmental studies ultrasound measurement of intima-medial carotid thickness and brachial artery reactivity have been used to determine the effects of exposure to carbon disulfide and passive smoking. Occupational Medicine has yet to include the use of the very expensive electron-beam computed tomography for a rapid and non-invasive study of coronary artery disease.

Investigation of Mode of Action of Anti Bacterial Activity of Salacia Oblonga Extract Against Drug Resistant Pathogen

Abstract Salacia oblonga Wall belonging to family Celastraceae contains vital phytoconstituents and has been used since long for the treatment of diabetes, inflammation and burn wounds. S. oblonga ethyl acetate root extract was evaluated for antibacterial activity towards drug resistant pathogens Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. Further 260 nm absorbing material was estimated in the control and treated cells. Interestingly 260 nm absorbance material is higher in the Staphylococcus aureus. Further the effect of the plant extract on drug resistant pathogen S. aureus was examined by scanning electron microscopy (SEM). SEM results have shown that treated bacterial cells have changed in morphology, size and reduced in number. Based on these results it can be concluded that S. oblonga extract acts on membrane of the drug resistant pathogen S. aureus.

A role for benzo[a]pyrene and Slug in invasive properties of fibroblast-like synoviocytes in rheumatoid arthritis: a potential molecular link between smoking and radiographic progression.

OBJECTIVES: To investigate the effects of benzo[a]pyrene (B[a]P), a major toxic component of cigarette smoke, on the expression of Slug and to determine the effect of B[a]P/Slug on the invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). METHOD: The expression of Slug was measured by real-time PCR following the stimulation of FLS with different concentrations of B[a]P or EGF. The phosphorylation of the key enzymes in the signaling pathway was analyzed by western blots. Inhibitors of PI3K/Akt/mTOR pathway were used to confirm the critical pathway for Slug expression. An in vitro cell invasion assay was performed using RA FLS treated with Slug cDNA, Slug small interference RNA, or control. RESULTS: Slug expression increased significantly following treatment with B[a]P or EGF in a dose-dependent manner. The stimulation of FLS with B[a]P or EGF induced the phosphorylation of Akt kinase, but not in ERK, JNK and p38. The Slug mRNA expression induced by B[a]P and EGF decreased significantly following the treatment with PI3K/Akt/mTOR inhibitors. Slug overexpression using Slug cDNA upregulated the invasive function of FLS, and Slug depletion using siRNA showed the opposite effect compared with the control. In addition, the stimulation with B[a]P increased the invasive function of the control siRNA-treated FLS but not in the Slug siRNA-treated FLS. CONCLUSION: Our data showed that B[a]P regulates the invasive properties of RA FLS through Slug expression. This mechanism may provide a novel molecular link underlying the association between smoking and increased radiographic progression in RA.

Adductomics: characterizing exposures to reactive electrophiles.

To understand environmental causes of disease, unbiased methods are needed to characterize the human exposome, which represents all toxicants to which people are exposed from both exogenous and endogenous sources. Because they directly modify DNA and important proteins, reactive electrophiles are probably the most important constituents of the exposome. Exposures to reactive electrophiles can be characterized by measuring adducts from reactions between circulating electrophiles and blood nucleophiles. We define an 'adductome' as the totality of such adducts with a given nucleophilic target. Because of their greater abundance and residence times in human blood, adducts of hemoglobin (Hb) and human serum albumin (HSA) are preferable to those of DNA and glutathione for characterizing adductomes. In fact, the nucleophilic hotspot represented by the only free sulfhydryl group in HSA (HSA-Cys(34)) offers particular advantages for adductomic experiments. Although targeted adducts of HSA-Cys(34) have been monitored for decades, an unbiased method has only recently been reported for visualizing the HSA-Cys(34) 'subadductome'. The method relies upon a novel mass spectrometry application, termed fixed-step selected reaction monitoring (FS-SRM), to profile Cys(34) adducts in tryptic digests of HSA. Here, we selectively review the literature regarding the potential of adductomics to partially elucidate the human exposome, with particular attention to the HSA-Cys(34) subadductome.