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Current process-based approaches to regulation are no longer fit for purpose

The phenomenon of encountering instrument meteorological conditions (IMC) on a visual flight rules (VFR) flight has been the focus of several previous studies. Most of these past studies, though, have involved examining various databases quantitatively or via a mixed-methods approach in search of possible causal factors such as pilot characteristics, weather conditions, aircraft type, and time of day. Missing from the literature are qualitative studies that tell the story of pilots who actually experienced such flights. To help fill this gap in the aviation literature, the purpose of the current study was to describe the first-hand experiences of GA pilots who inadvertently flew VFR-into-IMC. Participants consisted of 11 male pilots who previously had flown from VFR-into-IMC inadvertently at some point during their aviation career. The study used a phenomenological approach to describe participants’ shared experiences and then applied grounded theory to develop a set of conjectures derived inductively from participants’ responses. Using Spradley’s (1979) domain analysis to categorize common themes and patterns, the major domains of Weather Considerations and Expectations, Thoughts and Actions, and Postflight Experiences emerged. Major findings from the first domain revealed that as part of their preflight actions prior to departure, participants received a weather briefing, gave little consideration to overall weather conditions, neither expected nor anticipated IMC, and used a variety of communication resources to keep current with weather related issues. Major findings from the second domain revealed that participants recognized changes in the weather en route, used various communication resources to assess their current condition, reacted to IMC by trying to avoid and escape it, expressed feelings of trepidation about what they should do, were surprised over how the weather was not what they expected, and reverted to their training to get out of IMC. Major findings from the third domain revealed that participants’ postflight actions ranged from doing nothing to submitting a report to NASA’s ASRS, and that lessons learned included acquiring a heightened sense of situational awareness, a need to do a better job in alternative planning, and a greater appreciation for the weather. A comparison of these findings to past studies and theory are discussed, and implications and recommendations for practice and research are provided.

Introduction Adults with limb amputation and other physical disabilities are less likely to participate in physical activity than adults in the general population and have elevated risk of heart disease and stroke. Swimming is a physical activity often recommended for persons with limb amputation. However, a standard economical swim prosthesis that facilitates easy transition from land to water does not exist. Objective The objectives were (1) to measure ease of first-time use and likability of a novel U.S. Food and Drug Administration (FDA)-cleared 510(k) three-dimensional (3D) printed device, the "FIN," in a recreational pool; and (2) to determine differences in time to complete basic swim tasks using the novel 3D printed amphibious lower limb prosthesis or a standard Swim Ankle prosthesis. Our hypotheses were the following: (1) that the novel 3D printed amphibious lower limb prosthesis would be easy and likeable upon first use; and (2) that basic swim tasks would take comparable time to complete with either device. Setting Academic medical center and community pool in New York. Participants Participants were (N = 10) English-speaking adults with a transtibial amputation who self-identified to swim comfortably in a recreational setting. Interventions Participants completed tasks typical of recreational swimming while wearing the novel 3D printed amphibious lower limb prosthesis or a Swim Ankle. Main outcome measurements Participants performed a series of recreational swim tasks at self-selected speeds: entering/exiting pool, walking, swimming, and treading water, and completed a survey to assess the primary outcomes: likability, ease of use, and adverse events (feasibility). Results Participants found the novel 3D printed amphibious lower limb prosthesis more likable compared to the Swim Ankle and easy to use. Time to exit the pool was significantly reduced with the novel 3D printed amphibious lower limb prosthesis, while time to complete a 25-m lap was comparable. Participants did not show significant changes in vital signs when using either prosthesis. Conclusions The novel 3D printed amphibious lower limb prosthesis was likable and easy to use upon first use. This study supports conducting a larger clinical trial to determine if the data are broadly reproducible.

PURPOSE Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing. METHODS Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 μL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays. RESULTS Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment. CONCLUSIONS Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.

Recent studies have uncovered cross talks between nuclear factor-κB (NF-κB)-regulated inflammatory pathways and nuclear receptor-regulated pathways. These interactions are mediated at least in part by interactions between various NF-κB family proteins and a group of ligand-dependent transcription factors with xenobiotic sensor functions including pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR). These interactions are particularly important in tissues/organs that come in contact with toxicants, such as the gastrointestinal (GI) tract. Research on these interactions is important for understanding diseases such as liver and colon carcinogenesis, where inflammation is a significant risk factor. In addition to regulation of detoxification pathways, novel functions of PXR have recently been discovered, which include regulation of cell proliferation, apoptosis, cancer cell growth and interaction with commensal microbiota. Collectively, these novel findings suggest a multitude of PXR functions in reducing mutagenic exposure through metabolic detoxification as well as in regulation of cell proliferation and cancer growth. PXR, AhR, and NF-κB are transcriptional factors that can be regulated by various natural and synthetic ligands, and research on their interactions may provide a basis for identification of therapeutic and preventive applications and development of novel compounds that target these proteins for treatment and prevention of human diseases.

Background: There has been increasing interest in the concept that exposures to environmental chemicals may be contributing factors to the epidemics of diabetes and obesity. On 11–13 January 2011, the National Institute of Environmental Health Sciences (NIEHS) Division of the National Toxicology Program (NTP) organized a workshop to evaluate the current state of the science on these topics of increasing public health concern. Objective: The main objective of the workshop was to develop recommendations for a research agenda after completing a critical analysis of the literature for humans and experimental animals exposed to certain environmental chemicals. The environmental exposures considered at the workshop were arsenic, persistent organic pollutants, maternal smoking/nicotine, organotins, phthalates, bisphenol A, and pesticides. High-throughput screening data from Toxicology in the 21st Century (Tox21) were also considered as a way to evaluate potential cellular pathways and generate -hypotheses for testing which and how certain chemicals might perturb biological processes related to diabetes and obesity. Conclusions: Overall, the review of the existing literature identified linkages between several of the environmental exposures and type 2 diabetes. There was also support for the “developmental obesogen” hypothesis, which suggests that chemical exposures may increase the risk of obesity by altering the differentiation of adipocytes or the development of neural circuits that regulate feeding behavior. The effects may be most apparent when the developmental exposure is combined with consumption of a high-calorie, high-carbohydrate, or high-fat diet later in life. Research on environmental chemical exposures and type 1 diabetes was very limited. This lack of research was considered a critical data gap. In this workshop review, we outline the major themes that emerged from the workshop and discuss activities that NIEHS/NTP is undertaking to address research recommendations. This review also serves as an introduction to an upcoming series of articles that review the literature regarding specific exposures and outcomes in more detail.

The current study investigated the influence of ethanol and ethanol-containing mouthrinses on model chemical permeability in an in vitro oral buccal mucosal construct (EpiOral, ORL-200, MatTek). Innate ethanol transport and metabolism in the tissue construct was also studied. Caffeine flux in buccal tissue was measured after pre-treatment with < 26.9% ethanol or Listerine(®) products under conditions modeling a typical mouthwash rinsing. Specifically, a 30s exposure to alcohol products followed by a 10h non-treatment phase and then a second 30s exposure prior to addition of caffeine. At 10min specific intervals, media was collected from the basal part of the tissue insert for HPLC analysis of caffeine. The results demonstrated no increase in caffeine flux due to prior exposure to either ethanol or Listerine(®), and the flux and permeability constants were derived from the linear phase. No cytotoxicity or histopathological effects were observed in these tissues. We also studied the transepithelial transport and metabolism of ethanol in these tissues. Transport of ethanol was concentration-dependent with rate of diffusion proportional to the concentration gradient across the membrane. The potential metabolism of ethanol in the EpiOral construct was addressed by analyzing the remaining level of ethanol after incubation and de novo accumulation of acetaldehyde or acetic acid in culture media. Incubation for 30min incubation resulted in no change in ethanol level up to 2000mM, the highest concentration tested. No acetaldehyde or acetic acid was detected in culture media. In conclusion, ethanol and ethanol-containing mouthrinse treatment modeled after a typical daily mouthrinse pattern had no apparent effect on the permeability of the standard model chemical, caffeine. This exposure also had no effect on the viability of the tissue construct or histopathology, and uptake of ethanol was rapid into the tissue construct.

TBA, or 2,4,6-tribromoanisole, is a musty-smelling metabolite of 2,4,6-tribromophenol that is used as a flame retardant and an antifungal agent for wooden pallets and packaging materials. The compound can impart its peculiar, often offensive, odor on product packaging to the concern of consumers for the safety of the package contents. These studies were conducted to evaluate the safety of TBA to humans ingesting products tainted with TBA. In addition to the 28-day oral study, a bacterial reverse mutation study was conducted, and confirmed that TBA was not mutagenic. To evaluate oral safety, TBA was evaluated in single dose and 5-day and 4-week repeated dose oral toxicity studies in rats. The test article, administered in single gavage doses of 2000, 5000 and 7500 mg/kg body weight (bw), in 5 daily repeated doses of 1000, 2000 or 3000 mg/kg bw/day or in 28 daily oral gavage doses of 0 001, 0.01, 100, and 1000 mg/kg bw/day did not result in any deaths. Also, the single and repeat dose studies resulted in no significant differences between control and treated groups on body weight gain, food consumption, clinical observations, blood biochemical values, and hematology findings. Treatment-related adverse findings were only detected in male rats during repeated dose studies and were associated with high plasma concentrations of TBA. The test article-related finding of hyaline droplets in the cortical tubular epithelium of kidneys was associated with increases in α(2 μ)-globulin content in the kidneys as indicated by the intensity of immunohistochemical staining. These findings were correlated with an increased weight of kidneys in males administered 1000mg/kgbw/day for 28days. Chemical induction of hyaline droplets containing α(2μ)-globulin in the renal proximal tubule is a process unique to the male rat and is not relevant for human risk assessment. Findings of increased liver weight with minimal centrilobular to diffuse hepatocellular hypertrophy in males treated with TBA at 1000mg/kg bw/day for 28days were considered to be an adaptive metabolic response to xenobiotic administration. The increased volume of urine, noted in both males and females treated with 1000mg/kg bw/day was considered adaptive and necessary to excrete the high xenobiotic burden resulting from TBA administration. TBA appeared to be highly bioavailable since high concentrations of TBA were detected in plasma, at 1, 4 and 8h after administration of TBA at 100 and 1000mg/kg bw for 1 and 28days. Levels were dose-related but did not clarify the course of TBA elimination with time after administration. These studies indicate that TBA, administered orally to rats, produced male rat-specific, treatment-related toxicity at the highest orally administered dose in repeated dose (5-day at 3000mg/kg bw and 28-day at 1000mg/kg bw) studies. Therefore, the NOAEL for the 28-day oral study was determined to be 1000mg/kg bw/day for the rat.

The goals of this study were (1) to compare the injury at the basement membrane zone (BMZ) of rabbit corneal organ cultures exposed to half mustard (2 chloroethyl ethyl sulfide, CEES) and nitrogen mustard with that of in vivo rabbit eyes exposed to sulfur mustard (SM); (2) to test the efficacy of 4 tetracycline derivatives in attenuating vesicant-induced BMZ disruption in the 24-h period postexposure; and (3) to use the most effective tetracycline derivative to compare the improvement of injury when the drug is delivered as drops or hydrogels to eyes exposed in vivo to SM.Histological analysis of hematoxylin and eosin–stained sections was performed; the ultrastructure of the corneal BMZ was evaluated by transmission electron microscopy; matrix metalloproteinase-9 was assessed by immunofluorescence; doxycycline as drops or a hydrogel was applied daily for 28 days to eyes exposed in vivo to SM. Corneal edema was assessed by pachymetry and the extent of neovascularization was graded by length of longest vessel in each quadrant.Injury to the BMZ was highly similar with all vesicants, but varied in degree of severity. The effectiveness of the 4 drugs in retaining BMZ integrity did not correlate with their ability to attenuate matrix metalloproteinase-9 expression at the epithelial–stromal border. Doxycycline was most effective on organ cultures; therefore, it was applied as drops or a hydrogel to rabbit corneas exposed in vivo to SM. Eyes were examined at 1, 3, 7, and 28 days after exposure. At 7 and 28 days after SM exposure, eyes treated with doxycycline were greatly improved over those that received no therapy. Corneal thickness decreased somewhat faster using doxycycline drops, whereas the hydrogel formulation decreased the incidence of neovascularization.Corneal cultures exposed to 2-chloroethyl ethyl sulfide and nitrogen mustard were effective models to simulate in vivo SM exposures. Doxycycline as drops and hydrogels ameliorated vesicant injury. With in vivo exposed animals, the drops reduced edema faster than the hydrogels, but use of the hydrogels significantly reduced neovascularization. The data provide proof of principle that a hydrogel formulation of doxycycline as a daily therapy for ocular vesicant injury should be further investigated.

A recent study in rats investigated the retail sweetener product, Granulated SPLENDA No Calorie Sweetener (Splenda) (Abou-Donia et al., 2008. Splenda alters gut microflora and increases intestinal P-glycoprotein and cytochrome P-450 in male rats. J. Toxicol. Environ. Health A, 71, 1415-1429), which is composed of (by dry weight) maltodextrin ( approximately 99%) and sucralose ( approximately 1%). The investigators reported that Splenda increased body weight, decreased beneficial intestinal bacteria, and increased the expression of certain cytochrome P450 (CYP450) enzymes and the transporter protein, P-glycoprotein (P-gp), the latter of which was considered evidence that Splenda or sucralose might interfere with the absorption of nutrients and drugs. The investigators indicated that the reported changes were attributable to the sucralose present in the product tested. An Expert Panel conducted a rigorous evaluation of this study. In arriving at its conclusions, the Expert Panel considered the design and conduct of the study, its outcomes and the outcomes reported in other data available publicly. The Expert Panel found that the study was deficient in several critical areas and that its results cannot be interpreted as evidence that either Splenda, or sucralose, produced adverse effects in male rats, including effects on gastrointestinal microflora, body weight, CYP450 and P-gp activity, and nutrient and drug absorption. The study conclusions are not consistent with published literature and not supported by the data presented.

This study examines the relationship between scheduling (3-, 2-, and 1-day-per-week classes) and achievement in college algebra. The study is grounded in spacing effect theory, which examines how variations in the frequency and timing of instruction affect student learning, and involves 116 Florida community college students. Regression analyses controlling for student and teacher attributes show that the 1-day-per-week group consistently scores the lowest on unit tests and final examinations. The findings suggest that although many students may prefer intensive courses or compressed schedules that minimize the time they spend on campus, these scheduling options may not be optimal for learning, at least not in mathematics.

We studied the effect of administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by i.p. injection once every 2 weeks in combination with a high-fat (HF) diet for 8 or 16 weeks on the body and organ weight changes as well as on the hepatic enzyme activity for estrogen metabolism in C3H/HeN female mice. Administration of TCDD at 100 microg/kg b.w. once every 2 weeks for 8 weeks increased the body weight by 46% in the HF diet-fed animals, but not in the regular diet-fed animals. This is the first observation suggesting that TCDD at a high dose (100 microg/kg b.w.), but not at lower doses (1 or 10 microg/kg b.w.), may have a strong obesity-inducing effect in C3H/HeN mice fed an HF diet. While TCDD increased liver weight and decreased thymus weight in animals, these effects were enhanced by feeding animals an HF diet. Metabolism studies showed that TCDD administration for 8 or 16 weeks increased the liver microsomal activity for the 2- and 4-hydroxylation of 17 beta-estradiol in animals fed a control diet, but surprisingly not in animals fed an HF diet. Treatment with TCDD dose-dependently increased the hepatic activity for the O-methylation of catechol estrogens in both control and HF diet-fed animals, and it also decreased the levels of liver microsomal sulfatase activity for hydrolysis of estrone-3-sulfate. TCDD did not significantly affect the hepatic enzyme activity for the glucuronidation or esterification of endogenous estrogens. It is suggested that enhanced metabolic inactivation of endogenous estrogens by hepatic estrogen-metabolizing enzymes in TCDD-treated, control diet-fed animals contributes importantly to the reduced incidence of estrogen-associated tumors in animals treated with TCDD.

2-Methoxyestradiol (2ME) is an estradiol metabolite with anti-tumor and anti-angiogenic properties. We studied the effect of 2ME on apoptosis of MCF-7 breast cancer cells and explored a combination therapy using 2ME and a polyamine analogue, bis(ethyl)norspermine (BE-3-3-3). Determination of viable cells on day 4 of treatment with 2ME/BE-3-3-3 combinations showed synergistic effects by Chou-Talalay analysis. APO-BRDU analysis showed that there was only 1.5+/-0.5% apoptosis at 200 nM 2ME and 3.7+/-1.7% in the presence of 2.5 microM BE-3-3-3. Combination of 200 nM 2ME and 2.5 microM BE-3-3-3 resulted in 52.2+/-2.6% apoptosis. Up to 90% of the cells underwent apoptosis in the presence of 1000 nM 2ME and 2.5 microM BE-3-3-3. Combination treatments resulted in total disruption of microtubules and depletion of putrescine, spermidine and spermine. In addition, phosphorylation of Akt and nuclear localization of cyclin D1 were altered by 2ME/BE-3-3-3 combination. Our results suggest an important strategy to induce apoptosis of breast cancer cells, with potential applications in therapy.

Purpose: To determine if a commonly used soy protein supplement exhibits biological activity in vivo and in vitro, we evaluated an over-the-counter soy protein powder supplement using blood from healthy male volunteers and in an estrogen receptor in vitro assay. Subjects and Methods: We recruited healthy male volunteers 18 years of age or older that were in good health. Treatment consisted of consuming two scoops (56 g) of pure soy protein powder (Puritan's Pride, Oakdale, NY) daily for 28 days. Serum testosterone and luteinizing hormone (LH) levels were collected on days −7, 0, 14, and 28 of therapy, and day 42. A reporter estrogen receptor (ER) assay was used to determine the effect on ER-β and ER-α in vitro. Results: Twelve subjects were enrolled with a mean age of 32.25 years (range 25 to 47). Serum testosterone decreased 19%(±22%) during the 4-week use of soy protein powder (P = 0.021) and increased within 2 weeks after we discontinued soy protein powder. Serum LH concentrations decreased during the 4-week use of soy protein powder then increased within 2 weeks after we stopped the soy protein powder, but the changes did not reach statistical significance (P = 0.20). Soy protein powder was found to induce agonist activity to ER-β using a reporter estrogen receptor assay in yeast. Conclusion: Soy protein powder decreases serum testosterone levels in healthy men and acts as an ER-β agonist; the significance of this biological effect with respect to cancer prevention needs further study. (Cancer Epidemiol Biomarkers Prev 2007;16(4):829–33)

Estrogen receptors (ERα and ERβ) are ligand-activated transcription factors. We examined the effects of estradiol (E2), 4-hydroxytamoxifen (HT), and the estrogen response element (ERE) on the helical content and thermal unfolding of ERβ. A circular dichroism (CD) spectrum of ERβ showed changes at 210 and 222 nm that were due to the presence of E2, which is indicative of partial unfolding. In contrast, HT did not alter the CD spectrum of ERβ. The addition of E2 + ERE caused an increase in the α-helical content and an increase in the temperature midpoint of folding transition (TM) from 39 ± 0.7 °C to 57.2 ± 1 °C. The addition of E2 + mutant ERE, or E2 + control oligonucleotide, increased the TMof ERβ to 45 ± 2 °C only. In the presence of HT, ERβ yielded similar TMvalues (55–58 °C) with ERE, mutant ERE, or control oligodeoxynucleotide. The binding affinity of ERβ for ERE increased 125.7-fold as a result of the presence of E2, but only 4-fold as a result of HT. These results demonstrate coupled effects of E2and ERE on ERβ stability and binding affinity. The increased thermal stability of HT–ERβ–ERE was associated with reduced specificity of ERβ–ERE recognition, illustrating profound differences in conformational states of ERβ induced by E2and HT.

It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. Cytochrome P-450 3A4 cyp304 is responsible for the metabolism of over 50% of current prescription drugs, and cyp3a4 expression is transcriptionally regulated by pregnane X receptor (PXR), which is a ligand-dependent transcription factor. In this study, we report that NF-kappaB activation by lipopolysaccharide and tumor necrosis factor-alpha plays a pivotal role in the suppression of cyp3a4 through interactions of NF-kappaB with the PXR.retinoid X receptor (RXR) complex. Inhibition of NF-kappaB by NF-kappaB-specific suppressor SRIkappaBalpha reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-alpha. Furthermore, we showed that NF-kappaB p65 disrupted the association of the PXR.RXRalpha complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-kappaB p65 directly interacted with the DNA-binding domain of RXRalpha and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR.RXRalpha complex. This mechanism of suppression by NF-kappaB activation may be extended to other nuclear receptor-regulated systems where RXRalpha is a dimerization partner.

Estrogen receptors (ERs) are proteins that mediate the action of estradiol and a series of natural and synthetic chemicals that mimic the estradiol structure. Estrogenic action was initially attributed to a single type of ER, now known as ERalpha, but ERbeta was discovered in 1995. Tissue specific distribution and the intensity of expression of these proteins determine the first response of tissues to estrogenic compounds. Estrogens and ERs play a major role in the origin and progression of breast cancer, and antiestrogens that block ER function are useful for breast cancer prevention and treatment. Estrogen mimetics, however, do not fall into distinct categories of agonists and antagonists, since their action is regulated by tissue-specific expression of a number of auxiliary proteins called coactivators or corepressors. In addition, small molecules such as polyamines, fattyacids, and thioredoxin may modulate ER function. Estrogenic functions encompass multiple organ systems, including the reproductive, skeletal, cardiovascular, and nervous system. Estrogens are critical for bone remodeling and mineralization so that estrogen replacement therapy is proven to strengthen bone health in post-menopausal women. Ideally, selective blockade of ER function in breast epithelial cells should be accompanied by growth support on bone and cardiovascular systems. The details of estrogenic function in different organs are to be fully realized, in order to better utilize selective estrogen receptor modulators (SERMs) to fight not only breast cancer but also osteoporosis and cardiovascular diseases. Current research on SERMs points toward accomplishing this goal by exploiting ER as a versatile target against multiple diseases.

Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of Phase II detoxifying enzymes as well as anti-oxidative enzymes. In this study, we investigated the transactivation potential of different Nrf2 transactivation domain regions by using the Gal4-Nrf2 chimeras and Gal4-Luc reporter co-transfection assay system in HepG2 cells. The results indicated that chimera Gal4-Nrf2-(1–370), which contains the full transactivation domain showed very potent transactivation activity. The high transactivation activity of Gal4-Nrf2-(113–251) and the diminished transactivation activities of chimera Gal4-Nrf2-(1–126) and Gal4-Nrf2-(230–370) suggested that the Nrf2 N-terminal 113–251 amino acids region is critical in maintaining its transactivation activity. Overexpression of upstream MAPKs such as Raf, MEKK1, TAK1-ΔN, and ASK1 up-regulated the transactivation activities of Gal4-Nrf2-(1–370) and Gal4-Nrf2-(113–251) in a dose-dependent manner. Further investigation on the effects of the three MAPK pathways on Nrf2 transactivation domain activity demonstrated that both ERK and JNK signaling pathways stimulated the Gal4-Nrf2-(1–370) transactivation activity while the p38 pathway played a negative role. Site-directed mutagenesis studies on potential MAPK phosphorylation sites of Gal4-Nrf2-(113–251) showed no significant effect on its basal transactivation activity or the fold of induction by Raf. Interestingly, the nuclear transcription coactivator CREB-binding protein (CBP), which can bind to Nrf2 transactivation domain and can be activated by ERK cascade, showed synergistic stimulation with Raf on the transactivation activities of both the chimera Gal4-Nrf2-(1–370) and the full-length Nrf2. Taken together, this study clearly demonstrated that different segments of Nrf2 transactivation domain have different transactivation potential and different MAPKs have differential effects on Nrf2 transcriptional activity. It also suggested that the up-regulation of Nrf2 transactivation domain activity by upstream MAPKs such as Raf may not be mediated by direct phosphorylation of the Nrf2 transactivation domain, but rather by regulation of the transcriptional activity of coactivator CBP.

Human antiglomerular basement membrane autoantibody disease in XenoMouse II. Background Previous studies have identified regions within α3(IV) collagen in human antiglomerular basement membrane (anti-GBM) disease, however, information pertaining to the nature of the pathogenic human autoantibodies has been limited by a lack of a relevant disease model. Availability of engineered mice that produce antibodies (that is, XenoMouse II™ strains) provides an ideal opportunity to examine the human antibody response. Methods XenoMouse II™ mice that produce human IgG2 (γ2κ) in response to antigenic challenge were immunized with various forms of α3(IV)NC1 GBM collagen, including native bovine α3(IV) NCl collagen, E. coli expressed r α3(IV)NCl, and mammalian fetal kidney 293 cell expressed r α3(IV)NC1 preparations. The mice were evaluated for autoantibody (Ab) production and nephritis. Results All immunized XenoMouse II animals produced human anti-GBM Ab associated with proliferative glomerulonephritis, linear IgG deposits along the murine GBM and tubular basement membrane (TBM), C3 deposits (weaker). A fully human mAb (Ig γ 2 κ), produced from a mouse immunized with native bovine α3(IV)NCl collagen produced basement membrane deposits, nephritis and proteinuria on transfer to normal XenoMouse II. Furthermore, monoclonal antibodies (mAb) shared idiotypic properties with polyclonal autoantibodies derived from patients with anti-GBM disease, supporting a structural relationship among the antibodies. Conclusions The results further support the importance of α3(IV)NCl collagen in the pathogenesis of anti-GBM disease. Moreover, to our knowledge this is the first demonstration that experimentally induced, pathogenic human autoantibodies result in disease. This new model of anti-GBM disease, therefore, provides the means and unique reagents to both decipher the molecular basis of the human anti-GBM autoantibody response and the opportunity to test specific therapies aimed at modulation of either B cells producing human autoantibodies or the human pathogenic antibodies themselves, in vivo, prior to trial in patients with the spontaneous form of the disease.

This study examined the relationship between expected meteorological conditions as specified by TAF reports and actual ground conditions as specified by hourly METAR reports for Chicago-Midway (MDW) and Seattle-Tacoma (SEA) airports for the period September–December 2011. MDW and SEA were targeted because they had the highest and lowest percentage of delays, respectively, for 2011. The rationale was to determine if one of the contributing factors for the difference in percentage delays was because of the relationship between TAF and METAR reports. The primary hypothesis was that the relationship between the forecasts and actual ground conditions at MDW would be weaker than the corresponding relationship at SEA. TAF and METAR data were acquired from the respective TAF and METAR products pages at “Aviation Weather Charts Archive” (2012). Descriptive statistics revealed that MDW had less total departures than SEA (86,834 vs. 100,133) for all of 2011, but it also had nearly five times as many weather-related departure delays than SEA. Chi square analyses indicated that although the relationship between TAF and METAR at each airport was statistically significant, the corresponding Kappa agreement coefficients showed that this relationship was nearly twice as strong at MDW (.60) than at SEA (.35). Plausible explanations include that 70% of the weather conditions at MDW were VFR as opposed to only 56% at SEA, MDW had one-third the number of special METARS than SEA (374 vs. 917), and MDW had approximately one-fifth the number of LIFR conditions than SEA (70 vs. 337). The analysis also revealed that SEA had difficulty correctly forecasting IFR and LIFR conditions, especially under rapidly changing conditions. Based on the study’s findings, it appears that the relationship between TAF and METAR was not a contributing factor to departure delays at both MDW and SEA during the September– December 2011 period.

Herbal therapies are commonly used by patients with cancer, despite little understanding about biologically active chemical derivatives. We recently demonstrated that the herbal combination PC-SPES, which contains licorice root, had anti-prostate cancer activity attributable to estrogen(s) that produced a chemical castration. A recent study also demonstrated that licorice root alone decreased circulating testosterone in men. Other studies demonstrated antitumor activity of PC-SPES in vitro associated with decreased expression of the anti-apoptotic protein Bcl-2 and in patients independent of chemical castration, suggesting that other mechanisms of antitumor activity exist separate from chemical castration. In the present study, we assessed licorice root extract for effects on Bcl-2 to identify novel cytotoxic derivatives. Licorice root extract induced Bcl-2 phosphorylation as demonstrated by immunoblot and G2/M cell cycle arrest, similarly to clinically used antimicrotubule agents such as paclitaxel. Bioassay-directed fractionations resulted in a biologically active fraction for Bcl-2 phosphorylation. HPLC separation followed by mass spectrometry and NMR identified 6 compounds. Only one molecule was responsible for Bcl-2 phosphorylation; it was identified as 1-(2,4-dihydroxyphenyl)-3-hydroxy-3-(4'-hydroxyphenyl) 1-propanone (beta-hydroxy-DHP). The effect on Bcl-2 was structure specific, because alpha-hydroxy-DHP, 1-(2,4-dihydroxyphenyl)-2-hydroxy-3-(4'-hydroxyphenyl) 1-propanone, in contrast to beta-hydroxy-DHP, was not capable of Bcl-2 phosphorylation. Pure beta-hydroxy-DHP induced Bcl-2 phosphorylation in breast and prostate tumor cells, G2/M cell cycle arrest, apoptosis demonstrated by Annexin V and TUNEL assay, decreased cell viability demonstrated by a tetrazolium (MTT) assay, and altered microtubule structure. Therefore, these data demonstrate that licorice root contains beta-hydroxy-DHP, which induced Bcl-2 phosphorylation, apoptosis, and G2/M cell cycle arrest, in breast and prostate tumor cells, similarly to the action of more complex (MW >800) antimicrotubule agents used clinically.

Proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, interleukin-1beta, and lipopolysaccharides (LPS), suppress the gene expression of cytochrome P-450 1A1 (cyp1a1). The mechanism of the suppression is not well understood. In present study, we show that activation of nuclear factor-kappaB (NF-kappaB) is a critical event leading to the suppression of cyp1a1 gene expression, thus providing an underlying mechanism for the TNF-alpha- and LPS-induced cyp1a1 suppression. We demonstrated that: (i) inducible RelA expression down-regulated aryl hydrocarbon receptor (AhR) activated reporter gene; (ii) the suppressive effects of LPS and TNF-alpha on the AhR-activated reporter gene could be blocked by pyrrolidine dithiocarbamate, which is known to inhibit NF-kappaB action; and (iii) TNF-alpha and LPS-imposed repression could be reversed by the NF-kappaB super repressor (SRIkappaBalpha), thus demonstrating the specific involvement of NF-kappaB. Furthermore, nuclear receptor coactivators p300/CBP and steroid receptor coactivator-1 act individually as well as cooperatively to reverse the suppressive effects by NF-kappaB on the AhR-activated reporter gene, suggesting that these transcriptional coactivators serve as the common integrators for the two pathways, thereby mediating the cross-interactions between AhR and NF-kappaB. Finally, using the chromatin immunoprecipitation assay, we demonstrated that AhR ligand induces histone H4 acetylation at the cyp1a1 promoter region containing the TATA box, whereas TNF-alpha inhibits this acetylation, suggesting that AhR/NF-kappaB interaction converges at level of transcription involving chromatin remodeling.

In non-cancer risk assessment the goal traditionally has been to protect the majority of people by setting limits that account for interindividual variability in the human population. The Environmental Protection Agency (EPA) has assigned a default uncertainty factor (?UF) of 10 to account for interindividual variability in response to toxic agents in the general population. Previous studies have suggested that it is appropriate to equally divide this factor into sub-factors of 3.2 each for variability in human pharmacokinetics (PK) and pharmacodynamics (PD). As an extension of this model, one can envision using scientific data from the literature to modify the default sub-factors with compound-specific adjustment factors (AFs) and to create new and more scientifically based defaults. In this paper, data from published clinical trials on six pharmaceutical compounds were used to further illustrate how to calculate and interpret data-derived AFs. The clinical trial data were analyzed for content and the re...

We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells.

The generation time of HIV Type 1 (HIV-1)in vivohas previously been estimated using a mathematical model of viral dynamics and was found to be on the order of one to two days per generation. Here, we describe a new method based on coalescence theory that allows the estimate of generation times to be derived by using nucleotide sequence data and a reconstructed genealogy of sequences obtained over time. The method is applied to sequences obtained from a long-term nonprogressing individual at five sampling occasions. The estimate of viral generation time using the coalescent method is 1.2 days per generation and is close to that obtained by mathematical modeling (1.8 days per generation), thus strengthening confidence in estimates of a short viral generation time. Apart from the estimation of relevant parameters relating to viral dynamics, coalescent modeling also allows us to simulate the evolutionary behavior of samples of sequences obtained over time.

The Ah receptor (AhR) mediates many of the toxic responses induced by polyhalogenated and polycyclic hydrocarbons (PAHs) which are ubiquitous environmental contaminants causing toxic responses in human and wildlife. NF-kappaB is a pleiotropic transcription factor controlling many physiological functions adversely affected by PAHs, including immune suppression, thymus involution, hyperkeratosis, and carcinogenesis. Here, we show physical interaction and mutual functional repression between AhR and NF-kappaB. This mutual repression may provide an underlying mechanism for many hitherto poorly understood PAH-induced toxic responses, and may also provide a mechanistic explanation for alteration of xenobiotic metabolism by cytokines and compounds that regulate NF-kappaB.

Herbal mixtures are popular alternatives to demonstrated therapies. PC-SPES, a commercially available combination of eight herbs, is used as a nonestrogenic treatment for cancer of the prostate. Since other herbal medicines have estrogenic effects in vitro, we tested the estrogenic activity of PC-SPES in yeast and mice and in men with prostate cancer.We measured the estrogenic activity of PC-SPES with transcriptional-activation assays in yeast and a biologic assay in mice. We assessed the clinical activity of PC-SPES in eight patients with hormone-sensitive prostate cancer by measuring serum prostate-specific antigen and testosterone concentrations during and after treatment.In complementary yeast assays, a 1:200 dilution of an ethanol extract of PC-SPES had estrogenic activity similar to that of 1 nM estradiol, and in ovariectomized CD-1 mice, the herbal mixture increased uterine weights substantially. In six of six men with prostate cancer, PC-SPES decreased serum testosterone concentrations (P0.05), and in eight of eight patients it decreased serum concentrations of prostate-specific antigen. All eight patients had breast tenderness and loss of libido, and one had venous thrombosis. High-performance liquid chromatography, gas chromatography, and mass spectrometry showed that PC-SPES contains estrogenic organic compounds that are distinct from diethylstilbestrol, estrone, and estradiol.PC-SPES has potent estrogenic activity. The use of this unregulated mixture of herbs may confound the results of standard or experimental therapies and may produce clinically significant adverse effects.

This article describes the personal transformations experienced by participants during a study of home Internet use by low-income families. The study was designed to collect data related to the barriers, benefits, and perceived worth of the Internet to low-income families. Specifically, it asked what families designated as informationally disadvantaged would actually do online given unrestricted home Internet access. This research project provided the prerequisite resources necessary for “ideal” home Internet use to six low-income urban families. The experiences of these participants between December 1994 and January 1996 provide research-based evidence affirming the National Telecommunications and Information Administration’s (1995) thesis that many of those “most disadvantaged in terms of absolute computer and modem penetration are the most enthusiastic users of online services that facilitate economic uplift and empowerment” (p. 3). The ethnographically informed results of this study indicate t...

The evaluation of health risk from chemical exposure is evolving in concept and practice. The ability to sensitively detect levels of chemicals in the environment has served as the traditional foundation for determining exposure levels and consequent health risks. More recently, however, other parameters have been constructed to probe the pathway between environmental levels of a chemical and the biological effects of subsequent exposure. Among these, two that are discussed in this paper are bioavailability and biomarker determinations. Chemicals in the environment often are associated with a medium such as airborne particulate, water, or soil. The interaction between the chemical and its medium is dependent on the physicochemical properties of the system. In some cases, such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in soil, the chemical becomes partially and irreversibly bound to the medium. Animalingestion studies of TCDD-contaminated soil suggest that some of the TCDD remains bound to the soil and does not cross the gastrointestinal barrier during digestion, and therefore only a fraction of the TCDD enters the blood and becomes bioavailable. The characterization of bioavailability provides for more accurate exposure assessment. Biomarker information potentially can validate exposure assessment information from bioavailability studies, elucidate specific biological effects from chemical exposure, and investigate genetic susceptibility issues that may increase the likelihood that an individual or population will experience increased health risks. Benzene-induced chromosome damage is discussed as an example of a significant biomarker that has demonstrated the potential for providing information useful for accurately prediction health risk.

Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.

The purpose of this study was to investigate the effect direct and unrestricted access to the Internet had on a group of high school teachers. Based on the naturalistic inquiry paradigm, this study explored the barriers these teachers encountered when using the Internet, how and when they elected to use the Internet, the factors that influenced their continued use of the Internet, and the transitions they experienced from using the Internet. Data collection was based on Patton's (1990) three approaches to interviewing; data analysis was based in part on Miles and Huberman's (1984) model of data reduction and display and on Spradley's (1979) task of domain analysis. Findings suggested: that teachers require ongoing Internet training, technical support, home Internet access, and time in which to learn and incorporate the Internet into their classes; that Internet use can increase teachers' self-esteem and improve their attitudes toward computers and education; and that use of the Internet by teachers encourages them to restructure their classes and schedules to accommodate Internet resources within their classrooms.

Polychlorinated biphenyl (PCB) congeners differentially reduce serum thyroxine (T(4)) in rats, but little is known about their ability to affect biliary excretion of T(4). Thus, male Sprague-Dawley rats were orally administered Aroclor-1254, Aroclor-1242 (32 mg/kg per day), PCB-95, PCB-99, PCB-118 (16 mg/kg per day), PCB-126 (40 μg/kg per day), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (3.9 μg/kg per day), or corn oil for 7 days. Twenty-four hours after the last dose, [(125)I]T(4) was administered intravenously, and blood, bile, and urine samples were collected for quantifying [(125)I]T(4) and in bile [(125)I]T(4) metabolites. Serum T(4) concentrations were reduced by all treatments, but dramatic reductions occurred in response to Aroclor-1254, PCB-99 [phenobarbital (PB)-type congener], and PCB-118 (mixed-type congener). None of the treatments increased urinary excretion of [(125)I]T(4). Aroclor-1254, PCB-118, TCDD, and PCB-126 (TCDD-type congener) increased biliary excretion of T(4)-glucuronide by 850, 756, 710, and 573%, respectively, corresponding to marked induction of hepatic UDP-glucuronosyltransferase (UGT) activity toward T(4). PCB-95 and PCB-99 did not induce UGT activity; therefore, the increased biliary excretion of T(4)-glucuronide was related to the affinity of congeners for the aryl hydrocarbon receptor. The disappearance of [(125)I]T(4) from serum was rapid (within 15-min) and was increased by Aroclor-1254, PCB-99 and PCB-118. Thus, reductions in serum T(4) in response to PCBs did not always correspond with UGT activity toward T(4) or with increased biliary excretion of T(4)-glucuronide. The rapid disappearance of [(125)I]T(4) from the serum of rats treated with PB-like PCBs suggests that increased tissue uptake of T(4) is an additional mechanism by which PCBs may reduce serum T(4).

Estrogens are major risk factors for the development of breast cancer; they can be metabolized to catechols, which are further oxidized to DNA-reactive quinones and semiquinones (SQs). These metabolites are mutagenic and may contribute to the carcinogenic activity of estrogens. Redox cycling of the SQs and subsequent generation of reactive oxygen species (ROS) is also an important mechanism leading to DNA damage. The SQs of exogenous estrogens have been shown to redox cycle, however, redox cycling and the generation of ROS by endogenous estrogens has never been characterized. In the present studies, we determined whether the catechol metabolites of endogenous estrogens, including 2-hydroxyestradiol, 4-hydroxyestradiol, 4-hydroxyestrone and 2-hydroxyestriol, can redox cycle in breast epithelial cells. These catechol estrogens, but not estradiol, estrone, estriol or 2-methoxyestradiol, were found to redox cycle and generate hydrogen peroxide (H(2)O(2)) and hydroxyl radicals in lysates of three different breast epithelial cell lines: MCF-7, MDA-MB-231 and MCF-10A. The generation of ROS required reduced nicotinamide adenine dinucleotide phosphate as a reducing equivalent and was inhibited by diphenyleneiodonium, a flavoenzyme inhibitor, indicating that redox cycling is mediated by flavin-containing oxidoreductases. Using extracellular microsensors, catechol estrogen metabolites stimulated the release of H(2)O(2) by adherent cells, indicating that redox cycling occurs in viable intact cells. Taken together, these data demonstrate that catechol metabolites of endogenous estrogens undergo redox cycling in breast epithelial cells, resulting in ROS production. Depending on the localized concentrations of catechol estrogens and enzymes that mediate redox cycling, this may be an important mechanism contributing to the development of breast cancer.

There is increasing evidence that receptor-mediated events impact one or more stages responsible for tumor development in experimental animals and humans. Although many chemicals and endogenous hormones require receptor interactions as a necessary event in their carcinogenic activity, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its structural analogs are the most visible examples of receptor-mediated carcinogens. TCDD, or dioxin as it is frequently called, interacts with the Ah receptor (AhR), which functions in a manner analogous to receptors for steroids. TCDD produces a wide spectrum of biochemical and toxic responses in in vitro and in vivo systems, and the Ah receptor is generally considered necessary for most if not all of these responses. Risk assessments for dioxin made by the United States and other countries throughout the world have been based on its carcinogenecity in experimental animals. Recently, epidemiology studies have indicated that TCDD is a human carcinogen at high doses. Because TCDD appears to be acting like a potent and persistent hormone agonist, it appears reasonable to incorporate mechanistic information on receptor-mediated events in risk assessments for TCDD. This information may be obtained from steroid receptor action and from molecular data on the Ah receptor. In this paper, we evaluate the scientific foundation on which mechanistic models for estimating dioxin's risks should be based. These models need to recognize the mechanisms possible for the diversity of biological responses that are initiated by a single receptor interacting with a single ligand. The U.S. EPA is currently reevaluating dioxin's risks by examining the possibility of developing biologically based models.(ABSTRACT TRUNCATED AT 250 WORDS) Images p36-a Figure 1. p42-a p42-b p42-c

The study of concept mapping as a research topic evolved from work conducted at Cornell University under the auspices of Novak (Novak and Gowin, 1984). Most recently reported is a 12-year longitudinal study of science concept learning in which Novak and his colleagues developed concept maps as a tool to represent knowledge structures (Novak and Musonda, 1991). Predicated on Ausubel’s assimilation theory of cognitive learning, these maps depicted the hierarchy and relationships among concepts. Data gathered from clinical interviews given before and after instruction were transformed from their raw, propositional form to concept maps. These “before and after” maps, which represented specific pre- and postinstruction concept meanings held by students, were then analyzed for changes in students’ cognitive structure. In summarizing the results of this study, Novak and Musonda reported that experimental students showed “many more valid conceptions and many fewer invalid conceptions” (p. 148) when compared to a similar sample of students who received no formal instruction in basic science concepts. In the research done since Novak developed this tool, concept mapping has become a viable educational medium. For example, there is evidence that concept maps can help teachers become more effective (Beyerbach and Smith, 1990; Hoz et al., 1990), and can serve as a heuristic for curriculum development (Starr and Krajcik, 1990). Perhaps most importantly, concept maps have been reported to be a potent instructional tool for promoting what Ausubel has described as meaningful learning. Meaningful learning refers to anchoring new ideas or concepts with previously acquired knowledge in a nonarbitrary way (Novak, 1977). It is this latter role of concept maps that is the focus of this study.

The cornea is highly sensitive to ultraviolet B (UVB) light-induced oxidative stress, a process that results in the production of inflammatory mediators which have been implicated in tissue injury. In the present studies, we characterized the inflammatory response of human corneal epithelial cells to UVB (2.5-25mJ/cm(2)). UVB caused a dose-dependent increase in the generation of reactive oxygen species in the cells. This was associated with increases in mRNA expression of the antioxidants Cu,Zn superoxide dismutase (SOD), Mn-SOD, catalase and heme oxygenase-1 (HO-1), as well as the glutathione S-transferases (GST), GSTA1-2, GSTA3, GSTA4, GSTM1, and mGST2. UVB also upregulated expression of the proinflammatory cytokines, IFNγ, IL-1β, TGFβ and TNFα, and enzymes important in prostaglandin (PG) biosynthesis including cyclooxygenase-2 (COX-2) and the PG synthases mPGES-2, PGDS, PGFS and thromboxane synthase, and in leukotriene biosynthesis including 5-lipoxygenase (5-LOX), 15-LOX-2, and the epidermal and platelet forms of 12-LOX. UVB was found to activate JNK and p38 MAP kinases in corneal epithelial cells; ERK1/2 MAP kinase was found to be constitutively active, and its activity increased following UVB treatment. Inhibition of p38 blocked UVB-induced expression of TNFα, COX-2, PGDS and 15-LOX-2, while JNK inhibition suppressed TNFα and HO-1. These data indicate that UVB modulates corneal epithelial cell expression of antioxidants and proinflammatory mediators by distinct mechanisms. Alterations in expression of these mediators are likely to be important in regulating inflammation and protecting the cornea from UVB-induced oxidative stress.

The concept of the vanishing zero, which was first discussed 50 years ago in relation to pesticide residues in foods and food crops, focused on the unintended regulatory consequences created by ever-increasing sensitivity and selectivity of analytical methods, in conjunction with the ambiguous wording of legislation meant to protect public health. In the interim, the ability to detect xenobiotics in most substrates has increased from tens of parts per million to parts per trillion or less, challenging our ability to interpret the biological significance of exposures at the lowest detectable levels. As a result the focus of risk assessment, especially for potential carcinogens, has shifted from defining an acceptable level, to extrapolating from the best available analytical results. Analysis of gene expression profiles in exposed target cells using genomic technologies can identify biological pathways induced or repressed by the exposure as a function of dose and time. This treatise explores how toxicogenomic responses at low doses may inform risk assessment and risk management by defining thresholds for cellular responses linked to modes or mechanisms of toxicity at the molecular level.

The pulmonary bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the enrichment of polychlorinated dioxins (PCDDs) and furans (PCDFs) in fine particles were evaluated to assess the implications that these factors have on risk and exposure assessments. Respirable subfractions of PCDD-contaminated soil from a former 2,4,5-trichlorophenoxyacetic acid manufacturing site were isolated by chemical dispersion and gravity sedimentation. Analysis of the subfractions revealed that there was a size-dependent enrichment of PCDDs and PCDFs, with smaller particles more highly contaminated. TCDD was enriched up to 33-fold as compared to unfractionated soil. Soil and laboratory-recontaminated gallium oxide, which served as the positive control, were administered by intratracheal instillation to female Sprague-Dawley rats. Animals were terminated up to 28 days following treatment and pulmonary bioavailability of TCDD was assessed by hepatic enzyme induction and TCDD concentration. Enzyme induction was dependent on the duration of exposure with up to 56 and 918% increases in cytochrome P450 and aryl hydrocarbon hydroxylase (AHH) activity, respectively, following exposure to PCDD-contaminated soil. There was no significant difference in AHH induction between animals which received contaminated soil and those treated with the positive control. Hepatic concentration of TCDD in soil-exposed rats was 115, 101, and 179% of positive controls at 1, 7, and 28 days post-treatment, suggesting that the soil or cocontaminants influenced retention of TCDD in the liver. These data indicate that the relative pulmonary bioavailability of TCDD on respirable soil particles is 100% as compared to laboratory-recontaminated gallium oxide and that PCDDs and PCDFs are highly enriched on respirable particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Female Sprague-Dawley rats were intratracheally instilled with TCDD-contaminated soil (74.4 ppm TCDD) or recontaminated gallium oxide (GaO; 72.2 ppm TCDD; positive control) and sacrificed 1, 7, or 28 days later. Enzyme activity in hepatic microsomes was time-dependent with up to 56% and 918% increases in cytochrome P-450 and aryl hydrocarbon hydroxylase (AHH), respectively. There was no significant difference in AHH induction between animals which received soil or GaO. Hepatic concentration of TCDD in soil-exposed animals was 115%, 101%, and 186% of positive controls at 1, 7, and 28 days, respectively, suggesting that (a) pulmonary bioavailability of TCDD from respirable soil particles is 100% and (b) soil or cocontaminants influenced hepatic retention of the compound.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a polychlorinated aromatic hydrocarbon with teratogenic and carcinogenic properties. Previous studies in our and other laboratories have demonstrated that TCDD has antiestrogenic properties. In order to elucidate the mechanism of action of TCDD on estrogen sensitive tissues, we studied its effects on serum estradiol and estrogen receptor (ER) levels in liver and uteri of CD1 mice. Treatment with TCDD did not result in alterations of serum estradiol levels at any of the doses tested (1.0-30 micrograms/kg). In contrast, TCDD treatment induced a dose-dependent decrease in hepatic and uterine ER protein as determined by an enzyme immunoassay and equilibrium binding assays. A decrease in cytosolic and nuclear ER levels in uteri occurred as early as 24 hr after initial treatment with 30 micrograms/kg TCDD and recovery occurred by 14 days. Hepatic cytosolic and nuclear ER also decreased at a dose of 30 micrograms/kg TCDD at 24 hr after treatment, but recovery occurred only by 21 days. Studies in ovariectomized mice indicate that the regulation of hepatic ER by TCDD is independent of ovarian factors, but ovariectomy inhibited the downregulation of uterine ER by TCDD. Furthermore, determination of TCDD-induced cytochrome P-450 levels indicates that the downregulation of uterine ER is uncoupled from induction of hepatic cytochrome P-450. This study indicates that the antiestrogenic effects of low doses of TCDD are mediated through its ability to decrease hepatic and uterine ER and are not due to alterations in serum estradiol levels. Our results on ovariectomized mice indicate that TCDD-induced downregulation of ER is tissue specific and may involve different mechanisms at transcriptional or posttranscriptional levels.

of the hemolytic agent. Although the concentrations of LPS used for Figure 1B are high, the actual concentration of LPS at the surface of the bacterium is far higher than the solution concentrations used in this trial. The possibility that the hemolytic agent does bind to gram-negative bacteria deserves further investigation. The ability to destroy foreign cells that come in contact with the blood is an important defense strategy for a variety of animals. In mammals, the cytolysis of foreign cells is conducted by the complement system, a multi-component ensemble of plasma proteins whose membrane attack elements are activated by a proteolytic cascade that, itself, is initiated by a variety of stimuli indicative of parasitic invasion (7). The complement system is found only in the deurostomate animals (i.e., the echinoderms and the chordates) and is absent from protostomate animals (8, 9). In the latter, the relatively few cytolytic systems that have been characterized are less complex than the vertebrate complement system, with some the province of a single protein that both recognizes and binds to the foreign cell and mediates its cytolytic destruction (5, 10). Only a few cytolytic systems have been reported in the plasma of molluscs (11, 12), and we have not found any reports for gastropods. The systems reported from bivalves (11, 12) are Ca+2dependent, suggesting that they are based on different effector molecules than the system described here from Busycon. Supported by Grant No. MCB-97-26771 from the National Science Foundation.

Lead chromate pigments can be encapsulated within an amorphous silica coating. This reduces both the chemical teachability and the biological availability of the constituents. The chromate moiety is inactive in in vitro mutagenicity assays, even in the presence of a strong chelating solubilizer under conditions where the unencapsulated material gives positive results. Rodent feeding studies demonstrate much reduced absorption of both lead and chromium from the encapsulated lead chromate compared to the analogous unencapsulated material. Encapsulation also reduces the leachability of both lead and chromium in tests used to define the applicable environmental disposal methods for lead chromate pigments. These findings have important implications for health and environmental issues surrounding the use of lead chromate as a pigment material.

An exchange hydrogenation reaction on trioxsalen yields two isomeric dihydro analogues for which a combined HPLC-supercritical fluid chromatography method supplies purified materials. These reduced compounds and a related benzodipyranone are biologically active and inhibit the binding of epidermal growth factor on HeLa cells to an even greater extent than trioxsalen. This observation suggests a non-DNA target may play a role in the overall effects of psoralens on cells.