Small Extracellular Vesicles Promote Stiffness-mediated Metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiologic matrix stiffness affects the quantity and protein cargo of small extracellular vesicles (EV) produced by cancer cells, which in turn aid cancer cell dissemination. Primary patient breast tissue released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα2ß1, ITGα6ß4, ITGα6ß1, CD44) compared with EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix proteins including collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer-associated fibroblast phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment. SIGNIFICANCE: Here we show that the quantity, cargo, and function of breast cancer-derived EVs vary with mechanical properties of the extracellular microenvironment.

Small extracellular vesicles promote stiffness-mediated metastasis.

Tissue stiffness is a critical prognostic factor in breast cancer and is associated with metastatic progression. Here we show an alternative and complementary hypothesis of tumor progression whereby physiological matrix stiffness affects the quantity and protein cargo of small EVs produced by cancer cells, which in turn drive their metastasis. Primary patient breast tissue produces significantly more EVs from stiff tumor tissue than soft tumor adjacent tissue. EVs released by cancer cells on matrices that model human breast tumors (25 kPa; stiff EVs) feature increased adhesion molecule presentation (ITGα 2 ß 1 , ITGα 6 ß 4 , ITGα 6 ß 1 , CD44) compared to EVs from softer normal tissue (0.5 kPa; soft EVs), which facilitates their binding to extracellular matrix (ECM) protein collagen IV, and a 3-fold increase in homing ability to distant organs in mice. In a zebrafish xenograft model, stiff EVs aid cancer cell dissemination through enhanced chemotaxis. Moreover, normal, resident lung fibroblasts treated with stiff and soft EVs change their gene expression profiles to adopt a cancer associated fibroblast (CAF) phenotype. These findings show that EV quantity, cargo, and function depend heavily on the mechanical properties of the extracellular microenvironment.

A Feedback Loop between Hypoxia and Matrix Stress Relaxation Increases Oxygen-Axis Migration and Metastasis in Sarcoma.

Upregulation of collagen matrix crosslinking directly increases its ability to relieve stress under the constant strain imposed by solid tumor, a matrix property termed stress relaxation. However, it is unknown how rapid stress relaxation in response to increased strain impacts disease progression in a hypoxic environment. Previously, it has been demonstrated that hypoxia-induced expression of the crosslinker procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), in sarcomas has resulted in increased lung metastasis. Here, we show that short stress relaxation times led to increased cell migration along a hypoxic gradient in 3D collagen matrices, and rapid stress relaxation upregulated PLOD2 expression via TGFß-SMAD2 signaling, forming a feedback loop between hypoxia and the matrix. Inhibition of this pathway led to a decrease in migration along the hypoxic gradients. In vivo, sarcoma primed in a hypoxic matrix with short stress relaxation time enhanced collagen fiber size and tumor density and increased lung metastasis. High expression of PLOD2 correlated with decreased overall survival in patients with sarcoma. Using a patient-derived sarcoma cell line, we developed a predictive platform for future personalized studies and therapeutics. Overall, these data show that the interplay between hypoxia and matrix stress relaxation amplifies PLOD2, which in turn accelerates sarcoma cell motility and metastasis. SIGNIFICANCE: These findings demonstrate that mechanical (stress relaxation) and chemical (hypoxia) properties of the tumor microenvironment jointly accelerate sarcoma motility and metastasis via increased expression of collagen matrix crosslinker PLOD2.

Microfluidic platform for the real time measurement and observation of endothelial barrier function under shear stress.

Electric cell-substrate impedance sensing (ECIS) is a quickly advancing field to measure the barrier function of endothelial cells. Most ECIS systems that are commercially available use gold electrodes, which are opaque and do not allow for real-time imaging of cellular responses. In addition, most ECIS systems have a traditional tissue culture Petri-dish set up. This conventional set-up does not allow the introduction of physiologically relevant shear stress, which is crucial for the endothelial cell barrier function. Here, we created a new ECIS micro-bioreactor (MBR) that incorporates a clear electrode made of indium tin oxide in a microfluidic device. Using this device, we demonstrate the ability to monitor the barrier function along culture of cells under varying flow rates. We show that while two cell types align in the direction of flow in responses to high shear stress, they differ in the barrier function. Additionally, we observe a change in the barrier function in response to chemical perturbation. Following exposure to EDTA that disrupts cell-to-cell junctions, we could not observe distinct morphological changes but measured a loss of impedance that could be recovered with EDTA washout. High magnification imaging further demonstrates the loss and recovery of the barrier structure. Overall, we establish an ECIS MBR capable of real-time monitoring of the barrier function and cell morphology under shear stress and allowing high-resolution analysis of the barrier structure.

Sex differences in the mitochondrial bioenergetics of astrocytes but not microglia at a physiologically relevant brain oxygen tension.

Biological sex is thought to influence mitochondrial bioenergetic function. Previous respiration measurements examining brain mitochondrial sex differences were made at atmospheric oxygen using isolated brain mitochondria. Oxygen is 160 mm Hg (21%) in the atmosphere, while the oxygen tension in the brain generally ranges from ∼5 to 45 mm Hg (∼1-6% O2). This study tested the hypothesis that sex and/or brain physiological oxygen tension influence the mitochondrial bioenergetic properties of primary rat cortical astrocytes and microglia. Oxygen consumption was measured with a Seahorse XF24 cell respirometer in an oxygen-controlled environmental chamber. Strikingly, male astrocytes had a higher maximal respiration than female astrocytes when cultured and assayed at 3% O2. Three percent O2 yielded a low physiological dissolved O2 level of ∼1.2% (9.1 mm Hg) at the cell monolayer during culture and 1.2-3.0% O2 during assays. No differences in bioenergetic parameters were observed between male and female astrocytes at 21% O2 (dissolved O2 of ∼19.7%, 150 mm Hg during culture) or between either of these cell populations and female astrocytes at 3% O2. In contrast to astrocytes, microglia showed no sex differences in mitochondrial bioenergetic parameters at either oxygen level, regardless of whether they were non-stimulated or activated to a proinflammatory state. There were also no O2- or sex-dependent differences in proinflammatory TNF-α or IL-1ß cytokine secretion measured at 18 h activation. Overall, results reveal an intriguing sex variance in astrocytic maximal respiration that requires additional investigation. Findings also demonstrate that sex differences can be masked by conducting experiments at non-physiological O2.

Collagen Fiber Architecture Regulates Hypoxic Sarcoma Cell Migration.

Collagen is prevalent in the microenvironment of many cancer types and has been demonstrated to play an important role during disease progression. We previously showed the importance of hypoxic gradients in sarcoma cell migration. Here, we utilized an oxygen gradient collagen gel platform to determine the impact of collagen fiber density and hypoxic gradient on sarcoma cell migration. The oxygen gradient was created by regulating the oxygen diffusion coefficient along with the cellular oxygen consumption rate. Collagen fiber density in the hydrogels is modified by changing the preincubation period of the collagen gel solution at 4 °C, controlling fiber density independently of collagen concentration and oxygen tension. High fiber density gels have wider and longer fibers but a similar microscale pore size with a larger nanoscale pore size and quicker stress relaxation time, compared to the low fiber density gel. Both gels have the same Young's modulus. We analyzed responses of sarcoma cells encapsulated in the different hydrogels for 3 days. In the nonhypoxic low fiber density constructs, sarcoma cells exhibit a larger aspect ratio, and the matrix has less fiber alignment compared to the nonhypoxic high fiber density constructs. Interestingly, we found a minimal effect of fiber density on cell migration and the ability of the cells to degrade the matrix in nonhypoxic constructs. When compared with hypoxic constructs, we observed the opposite trend, where cells in low fiber density constructs exhibit a lower aspect ratio and the matrix has more aligned fibers compared to hypoxic high fiber density constructs. Sarcoma cells encapsulated in high fiber density hypoxic gels migrated faster and degraded the matrix more rapidly compared to the low fiber density hypoxic constructs. Overall, we show that hypoxic cell migration and matrix degradation are enhanced in high fiber density gels, while hypoxic matrix alignment is prominent in low fiber density gels. Our results suggest that the differences in cellular responses under hypoxic gradients are due to the hydrogel architecture including fiber density, size (length and width), and stress relaxation.

O2-controllable hydrogels for studying cellular responses to hypoxic gradients in three dimensions in vitro and in vivo.

Oxygen (O2) acts as a potent upstream regulator of cell function. In both physiological and pathophysiological microenvironments, the O2 concentration is not uniformly distributed but instead follows a gradient that depends on distance from oxygen-carrying blood vessels. Such gradients have a particularly important role in development, tissue regeneration, and tumor growth. In this protocol, we describe how to use our previously reported gelatin-based O2-controllable hydrogels that can provide hypoxic microenvironments in vitro. The hydrogel polymeric network is formed via a laccase-mediated cross-linking reaction. In this reaction, laccase catalyzes diferulic acid (diFA) formation to form hydrogels with an O2-consuming reaction. Cells, such as cancer or endothelial cells, as well as tumor/tissue grafts, can be encapsulated in the hydrogels during hydrogel formation and then analyzed for cellular responses to 3D hypoxic gradients and to elucidate the underlying mechanisms governing these responses. Importantly, oxygen gradients can be precisely controlled in standard cell/tissue culture conditions and in vivo. This platform has been applied to study vascular morphogenesis in response to hypoxia and to understand how oxygen gradients mediate cancer cell behavior. Herein, we describe the means to validate the assay from polymer synthesis and characterization-which take 1-2 weeks and include verification of ferulic acid (FA) conjugation, rheological measurements, and O2 monitoring-to the study of cellular responses and use in rodent models. Time courses for biological experiments using this hydrogel are variable, and thus they may range from hours to weeks, depending on the application and user end goal.

Dual Cross-Linked Biofunctional and Self-Healing Networks to Generate User-Defined Modular Gradient Hydrogel Constructs.

Gradient hydrogels have been developed to mimic the spatiotemporal differences of multiple gradient cues in tissues. Current approaches used to generate such hydrogels are restricted to a single gradient shape and distribution. Here, a hydrogel is designed that includes two chemical cross-linking networks, biofunctional, and self-healing networks, enabling the customizable formation of modular gradient hydrogel construct with various gradient distributions and flexible shapes. The biofunctional networks are formed via Michael addition between the acrylates of oxidized acrylated hyaluronic acid (OAHA) and the dithiol of matrix metalloproteinase (MMP)-sensitive cross-linker and RGD peptides. The self-healing networks are formed via dynamic Schiff base reaction between N-carboxyethyl chitosan (CEC) and OAHA, which drives the modular gradient units to self-heal into an integral modular gradient hydrogel. The CEC-OAHA-MMP hydrogel exhibits excellent flowability at 37 °C under shear stress, enabling its injection to generate gradient distributions and shapes. Furthermore, encapsulated sarcoma cells respond to the gradient cues of RGD peptides and MMP-sensitive cross-linkers in the hydrogel. With these superior properties, the dual cross-linked CEC-OAHA-MMP hydrogel holds significant potential for generating customizable gradient hydrogel constructs, to study and guide cellular responses to their microenvironment such as in tumor mimicking, tissue engineering, and stem cell differentiation and morphogenesis.

Intratumoral oxygen gradients mediate sarcoma cell invasion.

Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoma mouse tumors (>300 mm(3)) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1-6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.

Endothelial progenitor cell recruitment in a microfluidic vascular model.

During vessel injury, endothelial progenitors cells (EPCs) are recruited from bone marrow and directed to the hypoxic injury site. The hypoxic conditions in the damaged blood vessel promote TNF-α, which upregulates intercellular adhesion molecule-1 (ICAM-1). EPCs attach to endothelial cell lining using ICAM-1. Here we aimed to examine EPC attachment to ECs in an injured-blood vessel conditions. We first determined ICAM-1 expression in stimulated HUVECs. We stimulated HUVECs with 21% oxygen (atmospheric), atmospheric with TNF-α-supplemented media, 1% oxygen (hypoxia), and hypoxia with TNF-α-supplemented media and found the highest ECFC attachment on HUVECs stimulated with TNF-α and hypoxia, correlating with the highest ICAM-1 expression. We next designed, fabricated and tested a three-dimensional microbioreactor (3D MBR) system with precise control and monitoring of dissolve oxygen and media flow rate in the cellular environment. We utilized a step-wise seeding approach, producing monolayer of HUVECs on all four walls. When stimulated with both TNF-α and hypoxia, ECFC retention on HUVECs was significantly increased under low shear stress compared to static controls. Overall, the 3D MBR system mimics the pathological oxygen tension and shear stress in the damaged vasculature, providing a platform to model vascular-related disorders.

Patterning microscale extracellular matrices to study endothelial and cancer cell interactions in vitro.

The extracellular matrix (ECM) of the tumor niche provides support to residing and migrating cells and presents instructive cues that influence cellular behaviours. The ECM protein fibronectin (Fn) enables vascular network formation, while hyaluronic acid (HA) is known to facilitate breast tumor development. To recapitulate aspects of the tumor microenvironment, we developed systems of spatially defined Fn and HA for the co-culture of endothelial colony forming cells (ECFCs) and breast cancer cells (BCCs). A micropatterned system was developed using sequential microcontact printing of HA and Fn. This approach supported the preferential adhesion of ECFCs to Fn, but did not support the preferential adhesion of BCCs to HA. Thus, we developed a microstructured analog to spatially organize BCC-laden HA micromolded hydrogels adjacent to ECFCs in fibrin hydrogels. These novel, miniaturized systems allow the analysis of the spatial and temporal mechanisms regulating tumor angiogenesis, and can be applied to mimic other microenvironments of healthy and diseased tissues.

Post-hire asthma among insect-rearing workers.

OBJECTIVE: To evaluate the incidence of post-hire asthma (PHA) among insect-rearing workers, defined as asthma, the symptoms of which appeared after hire at the current workplace. METHODS: We surveyed the health of workers at three insect-rearing facilities and an associated office facility. We calculated the incidence and estimated hazard ratios for PHA. RESULTS: Post-hire asthma incidence in 157 insect-rearing workers was 16.2 per 1000 person-years compared with 9.2 per 1,000 person-years in 70 office workers. Workers with predominant exposure to Lepidoptera had an incidence of 26.9 per 1000 person-years and a hazard ratio of 5.5 (95% confidence interval: 1.6 to 23.9) adjusted for sex, race, and parental asthma. In contrast, the presence of specific immunoglobulin E to Lepidoptera antigens was not associated with PHA. CONCLUSION: Insect-rearing workers had a high incidence of PHA, primarily accounted for by workplace exposure to Lepidoptera.

History and results of the two inter-laboratory round robin endotoxin assay studies on cotton dust.

BACKGROUND: In the US cotton industry, airborne cotton dust levels are regulated, and other countries are moving to specify safety limits for airborne endotoxins. There is concern about potential respiratory health hazards associated with agricultural and other organic dusts. In laboratories, ranking which samples have high and low levels of endotoxin is usually in good agreement between laboratories. When different laboratories assay identical samples, the levels differ. The objective of this research was to evaluate the intra- and inter-laboratory variability for 13 laboratories measuring endotoxin in cotton dust. METHOD: Two inter-laboratory round robin endotoxin assay studies were conducted using cotton dust. In the first round robin, each laboratory used their normal in-house assay method and then used a common extraction protocol. In the second round robin, a common extraction protocol and endotoxin assay kit was used. RESULTS: The intra-laboratory results had small variations but inter-laboratory results had very high variations. The inter-laboratory results using a common extraction protocol showed reduced differences. Using the same extraction protocol and endotoxin assay kit, the intra-laboratory variation was small and inter-laboratory variation was reduced but not enough for inter-laboratory agreement. Most of the laboratories were able to discern between the high and low endotoxin concentration dusts. CONCLUSIONS: Standardization has reduced the differences in results between laboratories and possibly further standardization may bring closer inter-laboratory agreement.

The development of species-specific immunodiagnostics for Stachybotrys chartarum: the role of cross-reactivity.

Mold contamination and exposure to fungi in indoor environments has been associated with various adverse health effects but little is known about the significance of individual fungal species in the initiation or exacerbation of such effects. Using Stachybotrys chartarum as a model fungus we sought to demonstrate that monoclonal antibodies (mAbs) can provide species-specific diagnostic reagents and also be used to investigate immunological cross-reactivity patterns among fungi. Mice were immunized with S. chartarum spore walls and monoclonal antibodies were screened against 60 fungal species and 24 different isolates of S. chartarum using an indirect ELISA. One species-specific mAb (IgG(1)) reacted only with spore preparations but not mycelium of S. chartarum or propagules of any other fungus. Five cross-reactive mAbs (IgM) documented extensive cross-reactivity among nine related Stachybotrys species and several non-related genera including several species of Cladosporium, Memnoniella, Myrothecium and Trichoderma. We also found that the ELISA reactivity for cross-reactive antigens and different isolates of S. chartarum differed considerably for normalized total amounts of mycelial antigen. We demonstrate that mAbs and immunoassays have the potential to detect S. chartarum species-specifically. The observed reactivity patterns with cross-reactive mAbs suggest that several fungi may share common antigens and that the majority of antigens are expressed by spores and mycelia. The observed cross-reactivity patterns need to be considered for accurate interpretations of environmental and serological analyses.

New-onset asthma associated with exposure to 3-amino-5-mercapto-1,2,4-triazole.

OBJECTIVE: The authors conducted an investigation of a cluster of eight new-onset asthma cases identified in a chemical plant through the Sentinel Event Notification Systems for Occupational Risks (SENSOR) program. METHODS: Workplace investigation involved interviews with the asthma cases, review of medical records, and medical and industrial hygiene surveys in the plant. RESULTS: Altogether, 11 work-related asthma cases were identified among the plant workers-approximately 10% of the workers exposed to the potential causative agents: 3-amino-5-mercapto-1,2,4-triazole (AMT) or N-(2,6-difluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide (DE-498; trade name Flumetsulam). Of these cases, six had physician-diagnosed occupational asthma (OA) based on work-related respiratory symptoms and nonspecific bronchial hyperresponsiveness (NSBH), and of these, three had work-related expiratory peak flow changes. CONCLUSIONS: The findings of this investigation, together with findings from concurrent animal studies, suggest that this outbreak of new-onset asthma was associated with exposure to AMT. CLINICAL SIGNIFICANCE: A cluster of eight new-onset asthma cases was identified in a chemical plant through the SENSOR program. Subsequent workplace investigation identified AMT, used in the production of a herbicide N-(2,6-difluorophenyl)-5-methyl- [1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide, as the most likely causal agent.

Clinical use of immunoassays in assessing exposure to fungi and potential health effects related to fungal exposure.

OBJECTIVE: To review and summarize current evidence regarding the proper role of immunoassays in clinical assessments of exposure to fungi and health effects related to fungal exposure. DATA SOURCES: We reviewed relevant scientific investigations and previously published reviews concerning this topic. STUDY SELECTION: The authors' clinical, laboratory, and public health experiences were used to evaluate relevant data for scientific merit. RESULTS: Testing to determine the presence of IgE to specific fungi may be a useful component of a complete clinical evaluation in the diagnosis of illnesses that can be caused by immediate hypersensitivity such as allergic rhinitis and asthma. Detection of IgG to specific fungi has been used as a marker of exposure to agents that may cause illnesses such as hypersensitivity pneumonitis. However, the ubiquitous nature of many fungi and the lack of specificity of fungal antigens limit the usefulness of these types of tests in the evaluation of potential building-related illness and fungal exposure. Specific serologic tests (such as tests for cryptococcal antigen, coccidioidal antibody, and Histoplasma antigen) have been shown to be useful in the diagnosis of some fungal infections, but these are the exception not the rule. CONCLUSIONS: There is currently not enough scientific evidence to support the routine clinical use of immunoassays as a primary means of assessing environmental fungal exposure or health effects related to fungal exposure. Health care providers who care for persons expressing concerns about the relationship of symptoms to potential exposure to fungi are advised to use immunoassay results with care and only as an adjunct to a comprehensive approach to patient care.

Asthmalike biphasic airway responses in Brown Norway rats sensitized by dermal exposure to dry trimellitic anhydride powder.

BACKGROUND: Trimellitic anhydride (TMA) can induce specific IgE and occupational asthma. The significance of dermal exposure to TMA in immunologic sensitization and on subsequent airway responses is not clearly known. An animal model displaying both an early-phase airway response (EAR) and a late-phase airway response (LAR) after sensitization and subsequent inhalation challenge to a low-molecular-weight chemical has not been previously reported. OBJECTIVE: The present study investigated EAR and LAR after TMA inhalation challenge in Brown Norway rats sensitized by skin exposure to TMA dry powder. METHODS: Twenty milligrams of dry TMA powder was applied to the skin of each clipped rat's dorsum on days 0, 7, 14, and 21 and occluded overnight with surgical tape. Rats were challenged for 10 minutes with 0.2 to 40 mg/m(3) of TMA aerosol after day 35. Enhanced pause (an index of airway resistance) was recorded overnight in a whole-body plethysmography system. Specific IgE and pulmonary eosinophilia were also measured. RESULTS: Concentration-dependent responses to TMA were observed: provocation with 0.2 mg/m(3) produced no response; 1 mg/m(3) induced only EAR; and 5 mg/m(3) and 40 mg/m(3) induced both EAR and LAR. Specific IgE was positive; airway eosinophilic inflammation was observed. CONCLUSION: TMA powder applied to the skin can lead to both immunologic sensitization and subsequent dose-dependent biphasic airway responses after TMA aerosol challenge.

Effect of diesel exhaust particulate (DEP) on immune responses: contributions of particulate versus organic soluble components.

The effect of diesel exhaust particulate (DEP) exposure on innate, cellular and humoral pulmonary immunity was studied using high-dose, acute-exposure rat, mouse, and cell culture models. DEP consists of a complex mixture of petrochemical-derived organics adsorbed onto elemental carbon particles. DEP is a major component of particulate urban air pollution and a health concern in both urban and occupational environments. The alveolar macrophage is considered a key cellular component in pulmonary innate immunity. DEP and DEP organic extracts have been found to suppress alveolar macrophage function as demonstrated by reduced production of cytokines (interleukin-1 [IL-1], tumor necrosis factor- alpha [TNF- alpha]) and reactive oxygen species (ROS) in response to a variety of agents, including lipopolysaccharide (LPS), interferon- gamma (IFN- gamma), and bacteria. Fractionation of DEP organic extract suggests that this activity was predominately in polyaromatic-containing and more polar (resin) fractions. Organic-stripped DEP did not alter these innate pulmonary immune responses. DEP also depressed pulmonary clearance of Listeria monocytogenes and Bacillus Calmette-Guerin (BCG). The contribution of the organic component of DEP is less well defined with respect to acquired and humoral immunity. Indeed, both DEP and carbon black enhanced humoral immune responses (specific immunoglobulin [Ig] E and IgG) in an ovalbumin-sensitized rat model. It is concluded that both the particulate and adsorbed organics may contribute to DEP-mediated immune alterations.

Urinary benzo[a]pyrene and its metabolites as molecular biomarkers of asphalt fume exposure characterized by microflow LC coupled to hybrid quadrupole time-of-flight mass spectrometry.

As a step to study the health effects of asphalt fume exposure, an analytical method was developed to characterize benzo[a]pyrene and its hydroxy metabolites in the urine of asphalt fume-exposed rats. This method is based on microflow liquid chromatography (LC) coupled to hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometry (Q-TOFMS). Twenty-four female Sprague-Dawley rats were used in the experiment, with 8 as controls and 16 exposed to asphalt fumes in a whole-body inhalation chamber for 10 days (4 h/day). Generated at 150 degrees C, the asphalt fume concentration in the animal exposure chamber ranged 76-117 mg/m(3). In the urine of the asphalt fume-exposed rats, benzo[a]pyrene and its metabolites of 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol(+/-), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(+/-) were identified, and their concentrations were determined at 2.19 +/- 0.49, 16.17 +/- 0.3, 6.28 +/- 0.36, and 29.35 +/- 0.26 ng/100 mL, respectively. The metabolite concentrations from the controlled group, however, were either under the detection limits or at a relatively very low level (0.19 +/- 0.41 ng/100 mL for benzo[a]pyrene-7,8,9,10-tetrahydrotetrol metabolite). The results clearly indicate that the benzo[a]pyrene and its hydroxy metabolites were significantly elevated (p < 0.001) in the urine of asphalt fume-exposed rats relative to controls. The study also demonstrated that the combination of microflow LC separation and collision-induced dissociation leading to a characteristic fragmentation pattern by hybrid Q-TOFMS offers a distinct advantage for the identifications and characterizations of the benzo[a]pyrene metabolites.

Identification and quantification of urinary benzo[a]pyrene and its metabolites from asphalt fume exposed mice by microflow LC coupled to hybrid quadrupole time-of-flight mass spectrometry.

Prolonged, extensive exposure to asphalt fume has been associated with several adverse health effects. Inhaled polycyclic aromatic hydrocarbons (PAHs) from asphalt fume exposure have been suspected of inducing such effects. In this study, a bioanalytical method was proposed and evaluated to identify and quantify benzo[a]pyrene and its hydroxy-metabolites. This method is based on coupling a microflow liquid chromatography (LC) to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometry (Q-TOFMS). In the experiment, thirty-two B6C3FI mice were exposed to asphalt fume in a whole body inhalation chamber for 10 days (4 h day(-1)) and twelve other mice were used as controls. The asphalt fume was generated at 180 degrees C and the concentrations in the animal exposure chamber ranged 175-182 mg m(-3). Benzo[a]pyrene and its metabolites of 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol(+/-), benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide(+/-), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(+/-) in the urine of asphalt fume exposed mice were identified and found at 3.18 ng 100 mL(-1), 31.36 ng 100 mL(-1), 11.56 ng 100 mL(-1), 54.92 ng 100 mL(-1), and 45.23 ng 100 mL(-1) respectively. The results revealed that the urinary benzo[a]pyrene and its hydroxy-metabolites from exposed mice were at significantly higher levels (p < 0.001) than those from the control groups. Compared with several other technologies such as HPLC-UV and HPLC-fluorescence, the new method is more sensitive and selective, and it can also provide additional useful information on the structures of the metabolites. Hence, this method can be used to perform the assessment and to study the mechanisms of the adverse health effects. The fragmentation patterns established in this study can also be used to identify and quantify PAH metabolites in other biological fluids.