A Self-Assembled Cage with Endohedral Acid Groups both Catalyzes Substitution Reactions and Controls Their Molecularity.

A self-assembled Fe4 L6 cage complex internally decorated with acid functions is capable of accelerating the thioetherification of activated alcohols, ethers and amines by up to 1000-fold. No product inhibition is seen, and effective supramolecular catalysis can occur with as little as 5 % cage. The substrates are bound in the host with up to micromolar affinities, whereas the products show binding that is an order of magnitude weaker. Most importantly, the cage host alters the molecularity of the reaction: whereas the reaction catalyzed by simple acids is a unimolecular, SN 1-type substitution process, the rate of the host-mediated process is dependent on the concentration of nucleophile. The molecularity of the cage-catalyzed reaction is substrate-dependent, and can be up to bimolecular. In addition, the catalysis can be prevented by a large excess of nucleophile, where substrate inhibition dominates, and the use of tritylated anilines as substrates causes a negative feedback loop, whereby the liberated product destroys the catalyst and stops the reaction.

Health effects of subchronic inhalation exposure to gasoline engine exhaust.

Gasoline engine emissions are a ubiquitous source of exposure to complex mixtures of particulate matter (PM) and non-PM pollutants; yet their health hazards have received little study in comparison with those of diesel emissions. As a component of the National Environmental Respiratory Center (NERC) multipollutant research program, F344 and SHR rats and A/J, C57BL/6, and BALBc mice were exposed 6 h/day, 7 days/week for 1 week to 6 months to exhaust from 1996 General Motors 4.3-L engines burning national average fuel on a simulated urban operating cycle. Exposure groups included whole exhaust diluted 1:10, 1:15, or 1:90, filtered exhaust at the 1:10 dilution, or clean air controls. Evaluations included organ weight, histopathology, hematology, serum chemistry, bronchoalveolar lavage, cardiac electrophysiology, micronuclei in circulating cells, DNA methylation and oxidative injury, clearance of Pseudomonas aeruginosa from the lung, and development of respiratory allergic responses to ovalbumin. Among the 120 outcome variables, only 20 demonstrated significant exposure effects. Several statistically significant effects appeared isolated and were not supported by related variables. The most coherent and consistent effects were those related to increased red blood cells, interpreted as likely to have resulted from exposure to 13-107 ppm carbon monoxide. Other effects supported by multiple variables included mild lung irritation and depression of oxidant production by alveolar macrophages. The lowest exposure level caused no significant effects. Because only 6 of the 20 significant effects appeared to be substantially reversed by PM filtration, the majority of effects were apparently caused by non-PM components of exhaust.

Trigeminal uptake and clearance of inhaled manganese chloride in rats and mice.

Inhaled manganese (Mn) can enter the olfactory bulbs via the olfactory epithelium, and can then be further transported trans-synaptically to deeper brain structures. In addition to olfactory neurons, the nasal cavity is innervated by the maxillary division of the trigeminal nerve that projects to the spinal trigeminal nucleus. Direct uptake and transport of inhaled metal particles in the trigeminal system has not been investigated previously. We studied the uptake, deposition, and clearance of soluble Mn in the trigeminal system following nose-only inhalation of environmentally relevant concentrations. Rats and mice were exposed for 10-days (6 h/day, 5 days/week) to air or MnCl2 aerosols containing 2.3 +/- 1.3 mg/m3 Mn with mass median aerodynamic diameter (MMAD) of 3.1 +/- 1.4 microm for rats and 2.0 +/- 0.09 mg/m3 Mn MnCl2 with MMAD of 1.98 +/- 0.12 microm for mice. Mn concentrations in the trigeminal ganglia and spinal trigeminal nucleus were measured 2 h (0-day), 7-, 14-, or 30-days post-exposure using proton induced X-ray emission (PIXE). Manganese-exposed rats and mice showed statistically elevated levels of Mn in trigeminal ganglia 0-, 7- and 14-days after the 10-days exposure period when compared to control animals. The Mn concentration gradually decreased over time with a clearance rate (t1/2) of 7-8-days. Rats and mice were similar in both average accumulated Mn levels in trigeminal ganglia and in rates of clearance. We also found a small but significant elevation of Mn in the spinal trigeminal nucleus of mice 7-days post-exposure and in rats 0- and 7-days post-exposure. Our data demonstrate that the trigeminal nerve can serve as a pathway for entry of inhaled Mn to the brain in rodents following nose-only exposure and raise the question of whether entry of toxicants via this pathway may contribute to development of neurodegenerative diseases.

Olfactory bulb uptake and determination of biotransfer factors in the California ground squirrel (Spermophilus beecheyi) exposed to manganese and cadmium in environmental habitats.

Manganese (Mn) and cadmium (Cd) profiles in olfactory bulbs of California ground squirrels (Spermophilus beecheyi) trapped at Lawrence Livermore National Laboratory's Site 300 facility in California were determined with proton induced X-ray emission (PIXE). As a reference, Mn profiles in olfactory bulbs from laboratory rats exposed via nose-only inhalation to 0.53 mg/m3 Mn in the form of MnCl2 were also determined with PIXE. Atomic absorption spectrophotometry was used to measure soil Mn and Cd contents from the trapping sites and Mn and Cd contents in ground squirrel liver and leg muscle tissues. The data from laboratory rats revealed that Mn uptake into the olfactory bulb occurs via inhalation exposure. Data from ground squirrels and knowledge of the collection sites indicate that although several routes of exposure may occur, fossorial rodent olfactory uptake affords a significant exposure route to Mn and Cd in soils. Measured biotransfer factors (ratio of leg muscle tissue metal content to soil metal content) for Cd in ground squirrels were 10(3)-fold greater than exposure modeling estimates based on oral Cd uptake data from livestock. The measurements for ground squirrel tissues show that when conducting ecological risk assessments for natural habitats considerable care should be taken in selecting transfer factors. Specifically, transfer factors derived from data pertaining to comparable exposure pathways and ecological setting should be used wherever possible.

The XRCC1 399 glutamine allele is a risk factor for adenocarcinoma of the lung.

Defects in the repair and maintenance of DNA increase risk for cancer. X-ray cross-complementing group 1 protein (XRCC1) is involved with the repair of DNA single-strand breaks. A nucleotide substitution of guanine to adenine leading to a non-conservative amino acid change was identified in the XRCC1 gene at codon 399 (Arg/Gln). This change is associated with higher levels of aflatoxin B1-adducts and glycophorin A somatic mutations. A case-control study was conducted to test the hypothesis that the 399Gln allele is positively associated with risk for adenocarcinoma of the lung. XRCC1 genotypes were assessed at codon 399 in 172 cases of lung adenocarcinoma and 143 cancer-free controls. Two ethnic populations were represented, non-Hispanic White and Hispanic. The distribution of XRCC1 genotypes differed between cases and controls. Among cases, 47.7% were Arg/Arg, 35.5% were Arg/Gln, and 16.9% were Gln/Gln. Among controls, XRCC1 allele frequencies were 45.5% for Arg/Arg, 44.8% for Arg/Gln, and 9.8% for Gln/Gln. Logistic regression analysis was used to assess the association between lung adenocarcinoma and the G/G genotype relative to the A/A or A/G genotypes. In non-Hispanic White participants, the lung cancer risk associated with the G/G genotype increased significantly after adjustment for age (OR=2.81; 95% CI, 1.2-7.9; P=0.03) and increased further after adjustment for smoking (OR=3.25; 95% CI, 1.2-10.7; P=0.03). Among all groups, a significant association was found between the G/G homozygote and lung cancer (OR=2.45; 95% CI, 1.1-5.8; P=0.03) after adjustment for age, ethnicity, and smoking. This study links a functional polymorphism in the critical repair gene XRCC1 to risk for adenocarcinoma of the lung.

Predicting lung cancer by detecting aberrant promoter methylation in sputum.

Despite the promise of using DNA markers for the early detection of cancer, none has proven universally applicable to the most common and lethal forms of human malignancy. Lung carcinoma, the leading cause of tumor-related death, is a key example of a cancer for which mortality could be greatly reduced through the development of sensitive molecular markers detectable at the earliest stages of disease. By increasing the sensitivity of a PCR approach to detect methylated DNA sequences, we now demonstrate that aberrant methylation of the p16 and/or O6-methyl-guanine-DNA methyltransferase promoters can be detected in DNA from sputum in 100% of patients with squamous cell lung carcinoma up to 3 years before clinical diagnosis. Moreover, the prevalence of these markers in sputum from cancer-free, high-risk subjects approximates lifetime risk for lung cancer. The use of aberrant gene methylation as a molecular marker system seems to offer a potentially powerful approach to population-based screening for the detection of lung cancer, and possibly the other common forms of human cancer.

Glutathione, albumin, cysteine, and cys-gly effects on toxicity and accumulation of mercuric chloride in LLC-PK1 cells.

Speciation plays a profound if not dominant role in both transport and toxicity of Hg(II). Hg(II) has a high affinity for sulfhydryl groups. The formation constant for Hg2+ and the anionic form of a sulfhydryl group R-S- is > or =10(10) higher than that for the carboxyl or amino groups. The kidneys are the target organ for Hg(II) toxicity and the primary site of Hg(II) accumulation. Sulfhydryl groups have been implicated in both transport and nephrotoxicity; however, the role endogenous thiol compounds play in these parameters is not clear. The roles that albumin, glutathione, and the glutathione-derived complexes cysteinylglycine and L-cysteine play in toxicity and accumulation of HgCl2 were studied in LLC-PK1 cells incubated with different Hg(II):thiol ratios. In cysteine-containing medium, almost all 1:2 Hg(II):thiol complexes protected against Hg(II) toxicity up to 120 microM Hg, increased membrane-bound Hg(II), and decreased intracellular Hg(II) accumulation. In cysteine-free medium, all 1:1 Hg(II):thiol complexes were as toxic as uncomplexed Hg(II), and almost all 1:2 Hg(II):thiol complexes protected at > or =20 microM Hg, except albumin, which protected at < or =20 microM Hg. In cysteine-free but cystine-containing medium, two 1:1 Hg(II):thiol complexes were toxic at > or =80 microM Hg and two provided complete protection. All 1:2 complexes provided protection at 80-160 microM Hg. This investigation used defined media to demonstrate that mercury cytotoxicity in LLC-PK1 cells was dependent on Hg(II) concentration, the ligand, and the presence of a cysteine source for the cells. These effects were only partially explained by intracellular Hg(II) levels.

Quantitative particle-induced X-ray emission imaging of rat olfactory epithelium applied to the permeability of rat epithelium to inhaled aluminum.

Neurotoxicity from chronic metal inhalation has been suggested as an underlying contributor to late-developing neurodegenerative diseases that have symptoms similar to Alzheimer's and Parkinson's syndromes. If inhaled metals contribute to pathogenesis of these diseases, identifying, localizing, and quantitating metal deposition(s) within specific target regions of the central nervous system will be critical to our understanding of the mechanisms. Standard analytical techniques used to date require exposure to extremely high concentrations of metals to meet analytical detection limits in small tissue areas. The relevance to lower-dose environmentally relevant exposures and potential protective barriers is therefore questionable. The feasibility of microbeam particle-induced X-ray emission is investigated as a method for rapidly scanning tissues to study the inhalation of metals, nasal permeability, and central nervous system deposition. The optimal beam spot and analysis time used to image the rat olfactory epithelium to facilitate the rapid detection of aluminum localizations were determined. Measurements of aluminum localizations in rat olfactory bulb and brain sections are also presented.

Toxicity and transport of three synthesized mercury-thiol-complexes in isolated rabbit renal proximal tubule suspensions.

Previous work has suggested that endogenous sulfhydryls, such as glutathione (GSH) and cysteine, are involved in the uptake and toxicity of HgCl2. To study this possibility, uptake and toxicity of synthesized Hg(SG)2, Hg(cysteinylglycine)2 [Hg(CYS-GLY)2] and Hg(CYS)2 were investigated in rabbit renal proximal tubule suspensions (RPT). The intracellular K+ was used as a toxicity indicator, and the mercury content in the tubules was measured by proton induced x-ray emission analysis. The toxicity rank order of the three synthesized mercury-thiol-complexes from the highest to the lowest was: Hg(CYS)2 > Hg(CYS-GLY)2 > Hg(SG)2. However, no significant difference among the mercury contents in the tubules exposed to these synthesized mercury-thiol-complexes was detected. Acivicin (0.25 mM), an inhibitor of gamma-glutamyltranspeptidase (GGT), decreased the toxicity of Hg(SG)2 in a manner that did not decrease the uptake of mercury in the tubules. This suggests that the toxicity of Hg(SG)2 requires processing to Hg(CYS-GLY)2 or Hg(CYS)2, while Hg(SG)2 may be taken up by the tubules via Na(+)-dependent GSH transporter since 10 mM acivicin, an inhibitor of this transporter dramatically decreased the uptake of Hg(SG)2. Organic anion transporter plays a minor role, if any, in the toxicity and uptake of Hg(SG)2 and Hg(CYS)2 since p-aminohippuric acid (PAH), an inhibitor of organic anion transporter, did not have significant effect on their uptake and toxicity. L-phenylalanine, an inhibitor of the neutral amino acid decreased the uptake of mercury, but to a lesser extent. This suggested that neutral amino acid transporter seemed to play a role, in part, in the toxicity and uptake of synthesized Hg(CYS)2. In summary, the data suggested that basolateral transport is important for the toxicity of the three synthesized mercury-thiol-complexes, and a variety of mechanisms are involved in the toxicity and uptake of these complexes in isolated rabbit RPT.

Glutathione effects on toxicity and uptake of mercuric chloride and sodium arsenite in rabbit renal cortical slices.

The mechanism of renal uptake of nephrotoxic heavy metals such as HgCl2 and NaAsO2 is not clear. The metals are known to react with endogenous sulfhydryls such as glutathione (GSH), so metal-GSH conjugates may be delivered to the kidney. To study this possibility, renal cortical slices from male New Zealand white rabbits were incubated with 10(-4) M HgCl2 or 10(-3) M NaAsO2 +/- stoichiometric amounts (1-3x) of GSH; or synthetic metal-GSH conjugates [10(-4) M Hg(SG)2 or 10(-3) M As(SG)3]. Incubations were performed at 37 degrees C in DME-F12 buffer (95/5 O2/CO2) for 8 hr. Hg(SG)2 reduced slice K+/DNA content, as an indicator of viability, significantly less than HgCl2. As(SG)3 exhibited a 2-hr delay in K+/DNA content reduction compared to NaAsO2. This delay in toxicity was not correlated to changes in uptake. Arsenic and mercury accumulation, determined by proton-induced X-ray emission, were also identical between the metal salts and the metal-GSH conjugates. Exogenous GSH decreased HgCl2 cytotoxicity and was correlated to a decrease in Hg accumulation in the slice. Exogenous GSH had limited if any protective effects against cytotoxicity by NaAsO2 and a decrease in As accumulation was not observed. Complex metal-GSH interactions appear to exist and impact on the uptake and toxicity of these metals.

Fortified antibiotic ointment in bacterial keratitis.

We conducted experiments to determine the effectiveness of fortified antibiotic ointment in the treatment of Pseudomonas keratitis in rabbits. We evaluated gentamicin ointment (3, 10, 20, and 40 mg/g), gentamicin solution (3 and 10 mg/ml), and placebo, each given every 30 minutes. We also examined the effectiveness of fortified ointment given in extended treatment intervals. In short-term trials, commercial-strength gentamicin solution (3 mg/ml) was therapeutically superior (P less than .001) to commercial-strength gentamicin ointment (3 mg/g) in reducing corneal bacterial colony counts. No significant difference in antimicrobial effect was noted between fortified gentamicin ointment and fortified gentamicin solution at 30-minute treatment intervals. Fortified gentamicin ointment reduced colony counts even at extended treatment intervals of up to four hours in a severe keratitis model.

Conditioned saccharin aversions in rats as a result of cutaneous nicotine or intraperitoneal nicotine administered in divided doses.

Nicotine base was used in a conditioned taste aversion (CTA) paradigm to avert male Sprague-Dawley rats to saccharin solution (0.1%, w/v). Experiments investigated different dose routes of nicotine administration and duration of action as determinants in nicotine-induced CTA. In Experiment 1 nicotine was injected intraperitoneally (IP) at doses of 0.5, 1.0, or 3.0 mg/kg 30 min after drinking saccharin solution. Using a two-bottle choice test, no CTA was observed, although all nicotine animals showed obvious symptoms of malaise including seizures in the highest dose group. Experiment 2 showed dose-related CTA when nicotine (10.0, 30.0, or 50.0 mg/kg) was cutaneously applied 30 min following saccharin drinking. Experiment 2B showed that the aversions were due to associative rather than nonassociative factors such as sensitization or enhanced neophobia. In Experiment 3, the following group treatments were begun 30 min after saccharin drinking to distribute identical total nicotine doses over an extended period of time: One IP injection of 2.0 mg/kg nicotine (in a saline vehicle) and four injections of saline solution, three injections of 0.67 mg/kg nicotine and two injections of saline, five injections of 0.40 mg/kg nicotine, or five injections of saline. All injections were spaced 30 min apart. Compared with saline-injected controls, CTA occurred in the rats receiving either three or five injections of nicotine but the group receiving one injection did not differ from the control group. There was no difference in CTA between the groups receiving three or five injections.

Factors that influence the efficacy of topical gentamicin prophylaxis for experimental Pseudomonas keratitis.

We evaluated the efficacy of topical gentamicin prophylaxis for experimental Pseudomonas keratitis in rabbits by applying gentamicin in concentrations of 0.3 and 4 mg/100 ml, in both solution and ointment vehicles, one or four hours after a superficial stromal scratch was infected topically with Pseudomonas organisms. Under these conditions the most effective prophylaxis was that given early, in solution, and in high concentration.