Hormonal and genotoxic estrogen-androgen carcinogenesis in the NBL rat prostate: A role for aromatase.

BACKGROUND: Androgens are generally thought to cause prostate cancer, but the data from animal studies suggest that they must be aromatized to estrogen and act in concert with genotoxic estrogen metabolites. The objective of this study was to determine whether treatment with testosterone (T) combined with a nonestrogenic estrogen metabolite and a nongenotoxic estrogenic compound would all be necessary and sufficient for the induction of a high incidence of prostate cancer in the susceptible NBL rat strain. METHODS: NBL rats were treated with low-dose testosterone via slow-release Silastic implants and with the marginally estrogenic genotoxic catechol estrogen 4-hydroxyestradiol (4OH-E2) and the nongenotoxic estrogen 2-fluoroestradiol (2F-E2) and in one experiment the aromatase inhibitor letrozole via custom-made slow-release pellets. Animals were euthanized 52 weeks after implantation and their pituitaries and prostate complexes weighed and fixed in formalin. Hematoxylin and eosin (H&E)-stained step sections were prepared and examined microscopically for proliferative lesions. RESULTS: Animals treated with 2F-E2, with or without the other compounds, had enlarged pituitaries demonstrating its estrogenicity. Animals treated with T, with or without the other compounds, had enlarged prostates consistent with its androgenicity. Rats treated with T plus 2F-E2 and 4OH-E2 developed a high incidence of prostatic cancer (89%), while, surprisingly, rats treated with T plus only 2F-E2 also had a high incidence of prostate cancer (95%) contradicting our initial hypothesis. To test whether the formation of E2 from T by aromatase could lead to estrogen genotoxicity and prostate carcinogenesis we then rats treated with T and 2F-E2 also with letrozole and found that it reduced prostate cancer incidence by about 50%. CONCLUSIONS: These findings indicate that long-term treatment with a nongenotoxic estrogen (2F-E2) and T as well as uninhibited prostatic aromatase activity generating genotoxic E2 are all required for induction of a high incidence of prostatic adenocarcinomas in NBL rats. These and previous data indicate that androgen receptor-mediated action, estrogen receptor mediation, and estrogen genotoxicity are all required and sufficient for hormonal carcinogenesis in the NBL rat prostate. Interference with the estrogen genotoxicity is a potential approach to prostate cancer chemoprevention.

Sodium arsenite does not affect prostate carcinogenesis in a chemically-hormonally-induced rat model.

There is epidemiologic evidence to suggest that arsenic exposure is associated with risk of prostate cancer incidence and mortality. There are no studies indicating that arsenic can induce prostate cancer in animals. We evaluated whether drinking water exposure to sodium arsenite would affect prostate carcinogenesis in a rat model that depends on long-term low dose treatment with testosterone. WU rats received a sequential treatment with the antiandrogen flutamide followed by a single androgen administration to stimulate prostatic cell proliferation during which a single injection of N-methyl-N-nitrosourea was given. Two weeks later the animals received subcutaneous slow release testosterone-containing silastic tubing implants and provided with drinking water containing 5 mg/L sodium arsenite or with control drinking water for the duration of the 64 week-long experiment. Arsenite provided in drinking water did not modify the induction of prostate cancer in this rat model compared to control rats or survival. It also did not affect the growth of the animals or their drinking water intake. This animal study with drinking water exposure to 5 mg/L sodium arsenite does not support the notion that arsenic enhances prostate carcinogenesis.

The MNU Plus Testosterone Rat Model of Prostate Carcinogenesis.

Animal models of prostate cancer are essential to identify chemopreventive treatments against this major male malignancy. The N-methyl-N-nitrosourea (MNU) plus testosterone rat model of prostate carcinogenesis is a reliable animal model that recapitulates human prostate cancer in many respects and has been used extensively in chemoprevention studies with good predictive value for the results of human clinical trials. The objective of this article is to describe the induction protocol of this model, demonstrate its robustness and reproducibility over time and across rat strains, provide diagnostic criteria for the identification of prostate lesions, and present the current tumor induction protocol so that others can use this model in a reliable manner. The majority of accessory sex gland tumors in this model are adenocarcinomas originating in the anterior and dorsolateral prostate that metastasize to lungs and abdominal structures. The rat strain used is of critical importance, with the commercially available Wistar WU and Fischer F344 strains yielding the highest tumor incidences. Low dose, long-term testosterone treatment is essential for a high tumor incidence, but in advanced stage, large adenocarcinomas do not appear to be androgen dependent. This rat model is a robust and reproducible prostate cancer animal model of human prostate cancer.

Effects of green tea on prostate carcinogenesis in rat models and a human prostate cancer xenograft model.

BACKGROUND: There is evidence to suggest that green tea soy may have protective effects against prostate cancer, but there are several epidemiologic and clinical studies that did not identify such an effect. We tested the notion of protective effects in a rat model of prostate carcinogenesis that has been predictive of the effects of selenium and vitamin E in randomized clinical trials and a human prostate cancer xenograft model in nude mice and rat prostate tumor cells transplanted in immune-competent syngeneic animals. METHODS: Prostate cancer was induced in rats with methylnitrosourea and testosterone and tumor incidence was determined. Subcutaneous tumor growth was measured resulting from injected cells isolated from rat prostate cancers grafted in syngeneic animals and from the prostate-specific antigen (PSA)-producing human prostate cancer PC346 xenografted in nude mice. Brewed decaffeinated green tea infusion or caffeinated green tea extract and the same 300 mg/ml concentration of caffeine were provided in drinking water of the rats and nude mice. RESULTS: Caffeinated green tea extract and caffeine provided in drinking water did not modify the induction of prostate cancer in the rat model compared with control rats. The same drinking water treatments also did not affect the growth and PSA production of PC346 human prostate cancer xenografts in nude mice and the growth of two transplantable rat prostate cancer tumor lines in Wistar Firth rats. Brewed green tea infusion as drinking water did also not affect tumor growth in these xeno- and allograft models. CONCLUSION: These animal studies with drinking water exposure to green tea and caffeine do not support the idea that green tea is protective against prostate cancer.

Longitudinal Impact of WTC Dust Inhalation on Rat Cardiac Tissue Transcriptomic Profiles.

First responders (FR) exposed to the World Trade Center (WTC) Ground Zero air over the first week after the 9/11 disaster have an increased heart disease incidence compared to unexposed FR and the general population. To test if WTC dusts were causative agents, rats were exposed to WTC dusts (under isoflurane [ISO] anesthesia) 2 h/day on 2 consecutive days; controls received air/ISO or air only. Hearts were collected 1, 30, 240, and 360 d post-exposure, left ventricle total RNA was extracted, and transcription profiles were obtained. The data showed that differentially expressed genes (DEG) for WTC vs. ISO rats did not reach any significance with a false discovery rate (FDR) < 0.05 at days 1, 30, and 240, indicating that the dusts did not impart effects beyond any from ISO. However, at day 360, 14 DEG with a low FDR were identified, reflecting potential long-term effects from WTC dust alone, and the majority of these DEG have been implicated as having an impact on heart functions. Furthermore, the functional gene set enrichment analysis (GSEA) data at day 360 showed that WTC dust could potentially impact the myocardial energy metabolism via PPAR signaling and heart valve development. This is the first study showing that WTC dust could significantly affect some genes that are associated with the heart/CV system, in the long term. Even > 20 years after the 9/11 disaster, this has potentially important implications for those FR exposed repeatedly at Ground Zero over the first week after the buildings collapsed.

Longitudinal impact on rat cardiac tissue transcriptomic profiles due to acute intratracheal inhalation exposures to isoflurane.

Isoflurane (ISO) is a widely used inhalation anesthetic in experiments with rodents and humans during surgery. Though ISO has not been reported to impart long-lasting side effects, it is unknown if ISO can influence gene regulation in certain tissues, including the heart. Such changes could have important implications for use of this anesthetic in patients susceptible to heart failure/other cardiac abnormalities. To test if ISO could alter gene regulation/expression in heart tissues, and if such changes were reversible, prolonged, or late onset with time, SHR (spontaneously hypertensive) rats were exposed by intratracheal inhalation to a 97.5% air/2.5% ISO mixture on two consecutive days (2 hr/d). Control rats breathed filtered air only. On Days 1, 30, 240, and 360 post-exposure, rat hearts were collected and total RNA was extracted from the left ventricle for global gene expression analysis. The data revealed differentially-expressed genes (DEG) in response to ISO (compared to naïve control) at all post-exposure timepoints. The data showed acute ISO exposures led to DEG associated with wounding, local immune function, inflammation, and circadian rhythm regulation at Days 1 and 30; these effects dissipated by Day 240. There were other significantly-increased DEG induced by ISO at Day 360; these included changes in expression of genes associated with cell signaling, differentiation, and migration, extracellular matrix organization, cell-substrate adhesion, heart development, and blood pressure regulation. Examination of consistent DEG at Days 240 and 360 indicated late onset DEG reflecting potential long-lasting effects from ISO; these included DEG associated with oxidative phosphorylation, ribosome, angiogenesis, mitochondrial translation elongation, and focal adhesion. Together, the data show acute repeated ISO exposures could impart variable effects on gene expression/regulation in the heart. While some alterations self-resolved, others appeared to be long-lasting or late onset. Whether such changes occur in all rat models or in humans remains to be investigated.

Toxicity of particles emitted by fireworks.

BACKGROUND: Particle matter (PM) has been associated with increased morbidity and mortality rates across the world. This study was designed to test the hypotheses that pyrotechnic firework displays introduce significant amounts of toxic metals into the atmosphere and are hazardous to human health. Size-selective emissions from 10 different fireworks displays were collected during particle generation in a dynamic, stainless steel chamber and tested for toxicity in cells. A subset of 2 particle types were tested in vivo in mice. At doses that did not produce cytotoxicity in an LDH assay, in vitro reactive oxygen species (ROS) formation was measured in bronchial epithelial airway (BEAS-2B) and human pulmonary microvascular endothelial (HPMEC-ST1.6R) cell lines treated with size-fractionated particles from the emissions of fireworks. RESULTS: Significant increases in ROS, in both cell types, were dependent upon the type of firework but not particle size. The in vitro ROS activity was correlated with lung inflammation produced in groups of mice treated by oropharyngeal aspiration with 0, 50, or 100 µg fireworks PM10/mouse. Trace metal analyses of the PM10 samples showed significant differences in metal content among fireworks type. Interestingly, the PM10 sample for the fireworks type producing the greatest in vitro ROS response in BEAS-2B cells contained ~ 40,000 and ~ 12,000 ppm of lead and copper, respectively. This sample also produced the greatest inflammatory response (i.e., increased neutrophils in bronchoalveolar lavage fluid) in mice. CONCLUSIONS: These findings demonstrate that pyrotechnic display particles can produce adverse effects in mammalian cells and lungs, thus suggesting that further research is needed to expand our understanding of the contribution of metal content to the adverse health effects of fireworks particles. This information will lead to the manufacture of safer fireworks.

Impact on rats from acute intratracheal inhalation exposures to WTC dusts.

Background: Studies have revealed the increased incidence of health disorders in First Responders (FR) who were at Ground Zero over the initial 72 hr after the World Trade Center (WTC) collapses. Previous studies in rats exposed to WTC dusts using exposure scenarios that mimicked FR mouthbreathing showed exposure led to altered expression of genes whose products could be involved in lung ailments. Nevertheless, it was uncertain if repeated exposures (as occurred in earliest days post-disaster) might have given rise to long-term changes in the lungs/other organs, in white blood cell (WBC) profiles, and/or systemic expression of select (mostly immune-related) proteins.Methods: To examine this, rats were exposed on 2 consecutive days (2 hr/d, intratracheal inhalation) to WTC dusts and then examined over a 1-yr period thereafter. At select times post-exposure, organ (lung, heart, liver, kidney, spleen) weights, WBC profiles, and blood levels of a variety of proteins were evaluated.Results: The study showed that over the 1-yr period, there were nominal effects on organ weights (absolute, index) as a result of the dust exposures. There were significant changes (relative to in naïve rats) in WBC profiles, with exposed rats having increased monocyte-macrophage and decreased lymphocyte percentages. The study also found that dust exposure led to significant systemic increases in many proteins, including MCP-1, RANTES, MMP-9, RAGE, and Galectin-3.Conclusions: These results provide further support for our longstanding hypothesis that the WTC dusts could potentially have acted as direct inducers of many of the health effects that have been seen in the exposed FR.

Complementary biobank of rodent tissue samples to study the effect of World Trade Center exposure on cancer development.

World Trade Center (WTC) responders were exposed to mixture of dust, smoke, chemicals and carcinogens. New York University (NYU) and Mount Sinai have recreated WTC exposure in rodents to observe the resulting systemic and local biological responses. These experiments aid in the interpretation of epidemiological observations and are useful for understanding the carcinogenesis process in the exposed human WTC cohort. Here we describe the implementation of a tissue bank system for the rodents experimentally exposed to WTC dust. NYU samples were experimentally exposed to WTC dust via intratracheal inhalation that mimicked conditions in the immediate aftermath of the disaster. Tissue from Mount Sinai was derived from genetically modified mice exposed to WTC dust via nasal instillation. All processed tissues include annotations of the experimental design, WTC dust concentration/dose, exposure route and duration, genetic background of the rodent, and method of tissue isolation/storage. A biobank of tissue from rodents exposed to WTC dust has been compiled representing an important resource for the scientific community. The biobank remains available as a scientific resource for future research through established mechanisms for samples request and utilization. Studies using the WTC tissue bank would benefit from confirming their findings in corresponding tissues from organs of animals experimentally exposed to WTC dust. Studies on rodent tissues will advance the understanding of the biology of the tumors developed by WTC responders and ultimately impact the modalities of treatment, and the probability of success and survival of WTC cancer patients.

Prostate Cancer in World Trade Center Responders Demonstrates Evidence of an Inflammatory Cascade.

An excess incidence of prostate cancer has been identified among World Trade Center (WTC) responders. In this study, we hypothesized that WTC dust, which contained carcinogens and tumor-promoting agents, could facilitate prostate cancer development by inducing DNA damage, promoting cell proliferation, and causing chronic inflammation. We compared expression of immunologic and inflammatory genes using a NanoString assay on archived prostate tumors from WTC Health Program (WTCHP) patients and non-WTC patients with prostate cancer. Furthermore, to assess immediate and delayed responses of prostate tissue to acute WTC dust exposure via intratracheal inhalation, we performed RNA-seq on the prostate of normal rats that were exposed to moderate to high doses of WTC dust. WTC prostate cancer cases showed significant upregulation of genes involved in DNA damage and G2-M arrest. Cell-type enrichment analysis showed that Th17 cells, a subset of proinflammatory Th cells, were specifically upregulated in WTC patients. In rats exposed to WTC dust, we observed upregulation of gene transcripts of cell types involved in both adaptive immune response (dendritic cells and B cells) and inflammatory response (Th17 cells) in the prostate. Unexpectedly, genes in the cholesterol biosynthesis pathway were also significantly upregulated 30 days after acute dust exposure. Our results suggest that respiratory exposure to WTC dust can induce inflammatory and immune responses in prostate tissue. IMPLICATIONS: WTC-related prostate cancer displayed a distinct gene expression pattern that could be the result of exposure to specific carcinogens. Our data warrant further epidemiologic and cellular mechanistic studies to better understand the consequences of WTC dust exposure.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/8/1605/F1.large.jpg.

Role of Estrogen in Androgen-Induced Prostate Carcinogenesis in NBL Rats.

Androgens are thought to cause prostate cancer, but the underlying mechanisms are unclear. Data from animal studies suggest that for androgens to cause prostate cancer, they must be aromatized to estrogen and act in concert with estrogen metabolites. We tested the hypothesis that androgen-receptor and estrogen receptor-mediated effects of androgen and estrogen are necessary, as well as genotoxicity of estrogen metabolites. NBL rats were treated with androgenic and estrogenic compounds for 16-75 weeks through slow-release silastic implants or pellets. Testosterone alone induced cancer in the prostate of 37% of rats. 5α-Dihydrotestosterone, which cannot be converted to estradiol or testosterone, did not cause a significant prostate cancer incidence (4%). Addition of estradiol to 5α-dihydrotestosterone treatment did not markedly enhance prostate cancer incidence (14%), unlike adding estradiol to testosterone treatment which induced a 100% tumor incidence. Testosterone plus estradiol treatment induced a DNA adduct detectable by 32P-postlabeling, oxidative DNA damage (8-hydroxyguanosine), and lipid peroxidation at the site within the prostate where this treatment causes cancers, preceding later cancer formation. The non-estrogenic 4-hydroxy metabolite of estradiol, when combined with testosterone, induced prostatic dysplasia within 16 weeks and, after long-term treatment, a very low incidence of prostate cancer (21%). When an estrogen that cannot be hydroxylated (2-fluoroestradiol) was added to this combined treatment with testosterone and 4-hydroxyestradiol, dysplasia frequency after 16 weeks was doubled. These results strongly support the hypothesis, but additional definitive studies are needed which may identify new targets to interfere with these mechanisms that are clinically feasible in humans.

In vitro and in vivo toxicity of urban and rural particulate matter from California.

Particulate matter (PM) varies in chemical composition and mass concentration based on location, source, and particle size. This study sought to evaluate the in vitro and in vivo toxicity of coarse (PM10-2.5) and fine (PM25) PM samples collected at 5 diverse sites within California. Coarse and fine PM samples were collected simultaneously at 2 rural and 3 urban sites within California during the summer. A human pulmonary microvascular endothelial cell line (HPMEC-ST1.6R) was exposed to PM suspensions (50 µg/mL) and analyzed for reactive oxygen species (ROS) after 5 hours of treatment. In addition, FVB/N mice were exposed by oropharyngeal aspiration to 50 µg PM, and lavage fluid was collected 24 hrs post-exposure and analyzed for total protein and %PMNs. Correlations between trace metal concentrations, endotoxin, and biological endpoints were calculated, and the effect of particle size range, locale (urban vs. rural), and location was determined. Absolute principal factor analysis was used to identify pollution sources of PM from elemental tracers of those sources. Ambient PM elicited an ROS and pro-inflammatory-related response in the cell and mouse models, respectively. These responses were dependent on particle size, locale, and location. Trace elements associated with soil and traffic markers were most strongly linked to the adverse effects in vitro and in vivo. Particle size, location, source, and composition of PM collected at 5 locations in California affected the ROS response in human pulmonary endothelial cells and the inflammatory response in mice.

Impact of acute exposure to WTC dust on ciliated and goblet cells in lungs of rats.

Clinical studies and the World Trade Center (WTC) Health Registry have revealed increases in the incidence of chronic (non-cancer) lung disorders among first responders (FR) who were at Ground Zero during the initial 72 h after the collapse. Our previous analyses of rats exposed to building-derived WTC dusts using exposure scenarios/levels that mimicked FR mouth-breathing showed that a single WTC dust exposure led to changes in expression of genes whose products could be involved in the lung ailments, but few other significant pathologies. We concluded that rather than acting as direct inducers of many of the FR health effects, it was more likely inhaled WTC dusts instead may have impacted on toxicities induced by other rescue-related co-pollutants present in Ground Zero air. To allow for such effects to occur, we hypothesized that the alkaline WTC dusts induced damage to the normal ability of the lungs to clear inhaled particles. To validate this, rats were exposed on two consecutive days (2 h/d, by intratracheal inhalation) to WTC dust (collected 12-13 September 2001) and examined over a 1-yr period thereafter for changes in the presence of ciliated cells in the airways and hyperplastic goblet cells in the lungs. WTC dust levels in the lungs were assessed in parallel to verify that any changes in levels of these cells corresponded with decreases in host ability to clear the particles themselves. Image analyses of the rat lungs revealed a significant decrease in ciliated cells and increase in hyperplastic goblet cells due to the single series of WTC dust exposures. The study also showed there was only a nominal non-significant decrease (6-11%) in WTC dust burden over a 1-yr period after the final exposure. These results provide support for our current hypothesis that exposure to WTC dusts caused changes in airway morphology/cell composition; such changes could, in turn, have led to potential alterations in the clearance/toxicities of other pollutants inhaled at Ground Zero in the critical initial 72-h period.

Crystal structure of Bacillus anthracis virulence regulator AtxA and effects of phosphorylated histidines on multimerization and activity.

The Bacillus anthracis virulence regulator AtxA controls transcription of the anthrax toxin genes and capsule biosynthetic operon. AtxA activity is elevated during growth in media containing glucose and CO(2)/bicarbonate, and there is a positive correlation between the CO(2)/bicarbonate signal, AtxA activity and homomultimerization. AtxA activity is also affected by phosphorylation at specific histidines. We show that AtxA crystallizes as a dimer. Distinct folds associated with predicted DNA-binding domains (HTH1 and HTH2) and phosphoenolpyruvate: carbohydrate phosphotransferase system-regulated domains (PRD1 and PRD2) are apparent. We tested AtxA variants containing single and double phosphomimetic (His→Asp) and phosphoablative (His→Ala) amino acid changes for activity in B. anthracis cultures and for protein-protein interactions in cell lysates. Reduced activity of AtxA H199A, lack of multimerization and activity of AtxAH379D variants, and predicted structural changes associated with phosphorylation support a model for control of AtxA function. We propose that (i) in the AtxA dimer, phosphorylation of H199 in PRD1 affects HTH2 positioning, influencing DNA-binding; and (ii) phosphorylation of H379 in PRD2 disrupts dimer formation. The AtxA structure is the first reported high-resolution full-length structure of a PRD-containing regulator, and can serve as a model for proteins of this family, especially those that link virulence to bacterial metabolism.

Molecular basis for the catalytic specificity of the CTX-M extended-spectrum ß-lactamases.

Extended-spectrum ß-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M ß-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL ß-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 ß-lactamase. Substitutions of Ser237 and Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. The conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.

Acute high-level exposure to WTC particles alters expression of genes associated with oxidative stress and immune function in the lung.

First responders (FR) present at Ground Zero in the first 72 h after the World Trade Center (WTC) collapsed have progressively exhibited significant respiratory injuries. The few toxicology studies performed to date evaluated effects from just fine (< 2.5 µm) WTC dusts; none examined health effects/toxicities from atmospheres bearing larger particle sizes, despite the fact the majority (> 96%) of dusts were > 10 µm and most FR likely entrained dusts by mouth breathing. Using a system that generated/delivered supercoarse (10-53 µm) WTC dusts to F344 rats (in a manner that mimicked FR exposures), this study sought to examine potential toxicities in the lungs. In this exploratory study, rats were exposed for 2 h to 100 mg WTC dust/m(3) (while under isoflurane [ISO] anesthesia) or an air/ISO mixture; this dose conservatively modeled likely exposures by mouth-breathing FR facing ≈750-1000 mg WTC dust/m(3). Lungs were harvested 2 h post-exposure and total RNA extracted for subsequent global gene expression analysis. Among the > 1000 genes affected by WTC dust (under ISO) or ISO alone, 166 were unique to the dust exposure. In many instances, genes maximally-induced by the WTC dust exposure (relative to in naïve rats) were unchanged/inhibited by ISO only; similarly, several genes maximally inhibited in WTC dust rats were largely induced/unchanged in rats that received ISO only. These outcomes reflect likely contrasting effects of ISO and the WTC dust on lung gene expression. Overall, the data show that lungs of rats exposed to WTC dust (under ISO) - after accounting for any impact from ISO alone - displayed increased expression of genes related to lung inflammation, oxidative stress, and cell cycle control, while several involved in anti-oxidant function were inhibited. These changes suggested acute inflammogenic effects and oxidative stress in the lungs of WTC dust-exposed rats. This study, thus, concludes that a single very high exposure to WTC dusts could potentially have adversely affected the respiratory system - in terms of early inflammatory and oxidative stress processes. As these changes were not compared with other types of dusts, the uniqueness of these WTC-mediated effects remains to be confirmed. It also still remains to be determined if these effects might have any relevance to chronic lung pathologies that became evident among FR who encountered the highest dust levels on September 11, 2001 and the 2 days thereafter. Ongoing studies using longer-range post-exposure analyses (up to 1-year or more) will help to determine if effects seen here on genes were acute, reversible, or persistent, and associated with corresponding histopathologic/biochemical changes in situ.

Structural plasticity of the coiled-coil domain of rotavirus NSP4.

UNLABELLED: Rotavirus (RV) nonstructural protein 4 (NSP4) is a virulence factor that disrupts cellular Ca(2+) homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian strain SA11 NSP4 forms a tetramer that binds Ca(2+) at its core, the CCD of human strain ST3 forms a pentamer lacking the bound Ca(2+) despite the residues (E120 and Q123) that coordinate Ca(2+) binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH, suggesting that preference for a particular oligomeric state is pH dependent and that pH could influence Ca(2+) binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca(2+) or pH. Biochemical, biophysical, and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high-affinity binding to Ca(2+) at neutral pH and forms a tetramer, it does not bind Ca(2+) at low pH and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca(2+) binding is necessary for the tetramer formation, as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca(2+) may form a basis for its pleiotropic functions during RV replication. IMPORTANCE: The nonstructural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis, and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states, a Ca(2+)-bound tetrameric state and a Ca(2+)-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical, and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca(2+)-bound tetramer to a Ca(2+)-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4.

A novel system to generate WTC dust particles for inhalation exposures.

First responders (FRs) present at Ground Zero within the critical first 72 h after the World Trade Center (WTC) collapse have progressively exhibited significant respiratory injury. The majority (>96%) of WTC dusts were >10 µm and no studies have examined potential health effects of this size fraction. This study sought to develop a system to generate and deliver supercoarse (10-53 µm) WTC particles to a rat model in a manner that mimicked FR exposure scenarios. A modified Fishing Line generator was integrated onto an intratracheal inhalation (ITIH) system that allowed for a bypassing of the nasal passages so as to mimic FR exposures. Dust concentrations were measured gravimetrically; particle size distribution was measured via elutriation. Results indicate that the system could produce dusts with 23 µm mass median aerodynamic diameter (MMAD) at levels up to ≥1200 mg/m(3). To validate system utility, F344 rats were exposed for 2 h to ≈100 mg WTC dust/m(3). Exposed rats had significantly increased lung weight and levels of select tracer metals 1 h after exposure. Using this system, it is now possible to conduct relevant inhalation exposures to determine adverse WTC dusts impacts on the respiratory system. Furthermore, this novel integrated Fishing Line-ITIH system could potentially be used in the analyses of a wide spectrum of other dusts/pollutants of sizes previously untested or delivered to the lungs in ways that did not reflect realistic exposure scenarios.

The effect of particle size, location and season on the toxicity of urban and rural particulate matter.

Particulate matter (PM) varies in chemical composition and mass concentration based on a number of factors including location, season, source and particle size. The aim of this study was to evaluate the in vitro and in vivo toxicity of coarse and fine PM simultaneously collected at three rural and two urban sites within the metropolitan New York City (NYC) region during two seasons, and to assess how particle size and elemental composition affect toxicity. Human pulmonary microvascular endothelial (HPMEC-ST1.6R) and bronchial epithelial (BEAS-2B) cell lines were exposed to PM (50 µg/mL) and analyzed for reactive oxygen species (ROS). Mice (FVB/N) were exposed by oropharyngeal aspiration to 50 µg PM, and lavage fluid was analyzed for total protein and PMN influx. The ROS response was greater in the HPMEC-ST1.6R cell line compared to BEAS-2B cells, but the responses were significantly correlated (p < 0.01). The ROS response was affected by location, locale and the location:size interaction in both cell lines, and an additional association for size was observed from HPMEC-ST1.6R cells. Urban fine PM generated the highest ROS response. In the mouse model, inflammation was associated with particle size and by a season:size interaction, with coarse PM producing greater PMN inflammation. This study showed that the aerodynamic size, locale (i.e. urban versus rural), and site of PM samples affected the ROS response in pulmonary endothelial and epithelial cells and the inflammatory response in mice. Importantly, these responses were dependent upon the chemical composition of the PM samples.

Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-ß-lactamase active site.

Metallo-ß-lactamases catalyze the hydrolysis of a broad range of ß-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ß-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ß-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ß-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 µM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.