A single point mutation in ecdysone receptor leads to increased ligand specificity: implications for gene switch applications.

The ecdysone receptor (EcR), a member of the nuclear receptor superfamily, plays an important role in regulating development and reproduction in insects. The EcR binds to ecdysteroids and regulates transcription of genes that contain ecdysone response elements. The EcR has been used to develop inducible gene switches for efficient regulation of foreign genes in applications such as gene therapy, protein production, and functional genomics. An EcR [Choristoneura fumiferana EcR (CfEcR)] homology model was constructed, and 17 amino acid residues were identified as critical for 20-hydroxyecdysone binding. Mutation of these amino acids followed by analysis of these mutants in transactivation (in insect and mammalian cells and in vivo in mice) and ligand-binding assays identified one particular mutant (A110P) that failed to respond to steroids, but its response to the diacylhydrazine nonsteroidal ligands RG-102240 (GS(TM)E) and RG-102317 was unaffected. This steroid-insensitive EcR mutant has potential gene switch applications in insects and plants that have endogenous ecdysteroids. In addition, this mutant would be also useful for developing orthogonal EcR-ligand pairs for simultaneous regulation of multiple genes in the same cell.

Comparison of iron-catalyzed DNA and lipid oxidation.

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.

Mechanical vibration in neonatal transport: a randomized study of different mattresses.

OBJECTIVE: To test the hypothesis that a gel mattress is most effective in attenuating mechanical vibration in neonatal transport, we performed a randomized block study of four mattress combinations (none, foam, gel, gel on foam) using mannequins and an ambulance traveling on fixed routes (city, highway). STUDY DESIGN: Mechanical vibration was assessed by measuring vertical accelerations at two locations: the forehead of a 2000-gm mannequin and the transport incubator base. From time histories of these accelerations, root mean square (RMS) values and power spectral density functions were calculated. The effect of the mattress on the transmission of vibration was determined from ratios of the RMS values at the two locations. An RMS ratio of < 1.0 indicates attenuation, whereas a ratio of > 1.0 indicates accentuation of vibration. From the power spectral density functions, the natural frequency of the system was determined for each mattress combination in relation to the natural frequencies of the ambulance. To determine the effect of the weight of the mannequin on vibration, additional measurements were performed using a 300-gm mannequin. RESULTS: All the observed RMS ratios were > 1. The highest ratios were observed on the city route in the absence of the gel mattress. The gel mattress, used alone or with the foam mattress, in contrast to foam or no mattress, shifted the natural frequency of the system away from the natural frequencies of the ambulance, avoiding a large amplification of vibration. A decrease in the weight of the mannequin caused the gel mattress to be less effective in attenuating vibration. CONCLUSION: A gel mattress, used alone or with a foam mattress, results in the least accentuation of vibration, but vibration in ambulance transport is not attenuated by any of the mattress combinations. The hazard of vibration may be particularly relevant when transporting extremely low birth weight neonates. These findings indicate a need for study and design of more effective devices that can reduce the vibratory stress.

Glutathione content and turnover in rat nasal epithelia.

During inhalation exposure to airborne toxicants, the nasal epithelium may be subjected to local toxicity. Since glutathione (GSH) is often involved in xenobiotic metabolism, GSH status in these tissues has been examined. GSH content and apparent first-order rate constants for GSH turnover and synthesis were determined for respiratory epithelium covering the anterior ventral septum and naso and maxillo turbinates, olfactory epithelium covering the dorsal posterior septum, and olfactory epithelium of the dorsal meatus from male Fischer-344 rats. The three tissues had GSH concentrations that ranged from 2.9 to 4.2 mumol/g tissue as determined by the Ellman's assay and by HPLC equipped with an electrochemical detector. Animals were administered [35S]cysteine (Cys) by tail vein injection and rate constants for GSH turnover were estimated, after incorporation of Cys into tissue GSH pools, by the decrease in GSH-specific activity 1-102 hr after administration. Total [35S]GSH was analyzed by HPLC with a flowthrough radioactivity detector. The respiratory epithelium had an apparent biphasic rate of GSH turnover, with a rapid-phase half-life of 4.4 hr and a slow-phase half-life of 34 hr. The other epithelia had slower rates of GSH turnover, with half-lives greater than 30 hr. When half-lives of GSH turnover and GSH concentrations for nasal epithelia, and for 14 previously evaluated tissues, were compared it was found that tissues with high concentrations of GSH generally had a more rapid turnover of GSH than did tissues with low concentrations of GSH. However, apparent GSH turnover in the two regions of the olfactory epithelium poorly followed this trend.

Total IgE antibody production in BALB/c mice after dermal exposure to chemicals.

Chemicals that bind to protein may cause immunological responses that include respiratory hypersensitivity mediated by IgE antibodies. The BALB/c mouse model has been used to characterize chemicals that induce an IgE antibody response. This model may be a useful predictive tool for the evaluation and classification of chemicals that induce IgE antibody production in humans. Total serum IgE content was determined after dermal exposure to various concentrations of isophorone diisocyanate (IPDI), diphenylmethane-4,4'-diisocyanate (MDI), toluene diisocyanate (TDI), 2,4-dinitrochlorobenzene (DNCB), trimellitic anhydride (TMA), formaldehyde (FA), and glutaric dialdehyde (GA). Chemicals were generally administered in acetone:olive oil on Days 1 and 7. Mouse serum was collected 14 days after the initial administration and subsequently total IgE antibody content was evaluated by an enzyme-linked immunosorbent assay. Mice treated with TMA, IPDI, MDI, and TDI had statistically (p < 0.01) higher concentrations of serum IgE antibodies than control animals. Total serum IgE content was examined at various times after TMA or TDI administration. Mice treated with a total of 37.5 mg TMA or 3 mg TDI had elevated IgE antibodies for 8-41 days after initial administration. In other studies where various concentrations of TDI were administered 15 times over a 3-week period or 30 times over a 6-week period, the apparent TDI threshold for IgE antibody production significantly increased with an increase in the number of TDI applications.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of auricular lymph node cell proliferation in isothiazolone-treated mice.

Chemicals that bind to protein may cause immunological responses that include allergic contact hypersensitivity mediated by T cells. Various animal models have been used to predict chemical-mediated contact sensitization. One assay that has recently been developed utilizes the proliferation response induced in the draining auricular lymph node after exposure to contact sensitizers. This assay has been modified to a simpler method that results in the more rapid processing of samples with greater reproducibility. Isothiazolones are chemicals used as biocides in various applications which at greater than use concentrations may cause allergic contact hypersensitivity. The mouse auricular lymph node cell proliferation assay has been used to examine the potential of various isothiazolones to induce contact sensitization. The extent of lymph node cell proliferation was dependent on the concentration of isothiazolone, the vehicle used in the application, and the ability of isothiazolone to bind to protein. Kathon biocide contains two isothiazolones: 5-chloro-2-methyl-4-isothiazolin-3-one (CMI)2 and 2-methyl-4-isothiazolin-3-one (MI). In vivo studies have shown that protein binding correlates with auricular lymph node cell proliferation and an increase in auricular lymph node size. CMI, which binds to protein, induced an auricular lymph node cell proliferation response while MI, which poorly binds to protein, neither stimulated a proliferative response nor induced an increase in lymph node size at concentrations similar to CMI.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of DNA adducts and oxidative DNA damage in rats treated with 1,6-dinitropyrene.

In vitro metabolism studies have indicated that the tumorigenic environmental pollutant 1,6-dinitropyrene has the potential to bind covalently to DNA and to induce oxidative DNA damage. We have determined if 1,6-dinitropyrene treatment will cause both types of DNA damage in vivo. Female Sprague-Dawley rats were given a single intraperitoneal injection of 1,6-dinitropyrene, and covalent DNA adduct formation, as indicated by the presence of N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene, and oxidative DNA damage, as indicated by increases in 5-hydroxymethyl-2'-deoxyuridine and 8-hydroxy-2'-deoxyguanosine, were assessed at 3, 12, 24 and 48 h after dosing. 32P-postlabeling analyses of DNA isolated from liver, mammary gland, bladder and nucleated blood cells indicated the formation of N-(deoxy-guanosin-8-yl)-1-amino-6-nitropyrene, with the levels being highest in the bladder. 5-hydroxymethyl-2'-deoxyuridine was detected in DNA from each of these tissues, and the levels of this oxidized nucleoside were higher in the mammary glands and livers of 1,6-dinitropyrene-treated rats. 1,6-Dinitropyrene dosing did not affect the levels of 8-hydroxy-2'-deoxyguanosine in these two tissues. These results indicate that exposure to 1,6-dinitropyrene can result in increased levels of 5-hydroxymethyl-2'-deoxyuridine in addition to covalent DNA adduct formation.

Apparent rates of glutathione turnover in rat tissues.

Apparent first-order rate constants for glutathione (GSH) turnover were determined for 14 tissues in male Fischer 344 rats after intravenous injection of [35S]cysteine ([35S]Cys). Rate constants for glutathione turnover were estimated by nonlinear least-squares iterative minimization from the decrease in GSH specific activity 1-102 hr after administration of [35S]Cys. Tissue nonprotein sulfhydryl concentrations were determined by Ellman's assay and compared with GSH and Cys levels detected by high-performance liquid chromatography equipped with an electrochemical detector. Additionally, total radiolabeled [35S]GSH was determined by high-performance liquid chromatography equipped with a flow-through radioactivity detector. There were substantial differences in the apparent rates of GSH turnover between the various tissues examined. For example, both the liver and the kidney had rapid turnover rates with half-lives of 1-5 hr, while those for heart, skeletal muscle, and blood were much slower with half-lives of 68-118 hr. Gastrointestinal tract tissues were shown to have intermediate turnover rates of the following order: glandular stomach = caecum > duodenum = small intestine = large intestine > colon > forestomach. [35S]GSH had a half-life in lung and skin of approximately 63 and 50 hr, respectively.

Detection of substituted benzenes in water at the pg/ml level using solid-phase microextraction and gas chromatography-ion trap mass spectrometry.

Solid-phase microextraction (SPME) is combined with gas chromatography-ion trap mass spectrometry (GC-IT-MS) for the analysis of benzene, toluene, ethyl benzene and xylene isomers (BTEX) in water. SPME is a recent technique for extracting organics from an aqueous matrix into a stationary phase immobilized on a fused-silica fiber. The analytes are thermally desorbed directly in the injector of a gas chromatograph. The wide linear dynamic range (five orders of magnitude) and pg sensitivity of the ion trap mass spectrometer in its full scan mode is an ideal detector for identifying and quantifying the analytes extracted with an SPME device. The combined method SPME-GC-IT-MS, using fibers coated with a 100-microns polydimethylsiloxane coating, showed a limit of quantitation (LOQ) of 50 pg/ml benzene in water. This corresponds to 5 pg of benzene absorbed onto the fiber. The limit of detection (LOD) was 15 pg/ml benzene. For o-xylene spiked at 50 pg/ml in water 50 pg were absorbed by the fiber indicating an LOQ and LOD 10 times better than for benzene. The detection limits obtained exceed the requirements of both the United States Environmental Protection Agency method 524.2 and the Ontario Municipal/Industrial Strategy for Abatement program, which range from 30 to 80 pg/ml and 500 to 1100 pg/ml, respectively. The linearity of the method extended over five orders of magnitude. Relative standard deviation ranged from 2.7 to 5.2% for 15 ng/ml BTEX in water and from 5.5 to 7.5% for 50 pg/ml BTEX in water. SPME-GC-IT-MS was used to evaluate the contamination level in laboratory, potable and wastewater sources.

Rates of ethyl acrylate binding to glutathione and protein.

Ethyl acrylate is a monomer used extensively in polymer manufacturing. Although ethyl acrylate is toxic at high concentrations, it is metabolized and detoxified rapidly at low concentrations. In the current studies, in vitro experiments have demonstrated that [14C]ethyl acrylate reacts with both glutathione (GSH) and protein to give either [14C]3-(glutathion-S-yl)ethylpropionate or covalently bound protein adducts, respectively. The second-order rate constant for [14C]ethyl acrylate conjugation with GSH was determined by quantification of [14C]3-(glutathion-S-yl)ethylpropionate using an HPLC system equipped with a flow-through radioactive detector. The rate constant for conjugation was 32.8 M-1 min-1. Additionally, the apparent second-order rate constants were determined for [14C]ethyl acrylate binding to the protein fraction of 14 whole tissue homogenates. Estimation of total protein binding sites was performed by reacting tissue homogenates with high concentrations of [14C]ethyl acrylate, while rates of binding were determined by reacting tissue homogenates with 200 microM [14C]ethyl acrylate at 37 degrees C for various periods of time. Apparent second-order rate constants for ethyl acrylate binding to protein homogenates were similar to that observed for GSH reacting with ethyl acrylate. The role of GSH-transferase in catalyzing 3-(glutathion-S-yl)ethylpropionate formation also was evaluated with whole tissue homogenates. In most tissues, the GSH-transferases poorly catalyzed the conjugation reaction. However, a significant increase in 3-(glutathion-S-yl)ethylpropionate formation was observed with liver homogenate.

A physiologically based pharmacokinetic and pharmacodynamic model to describe the oral dosing of rats with ethyl acrylate and its implications for risk assessment.

A physiologically based pharmacokinetic and pharmacodynamic model has been developed to describe the absorption, distribution, and metabolism of orally dosed ethyl acrylate. The model describes the metabolism of ethyl acrylate in 14 tissues based on in vitro metabolic studies conducted with tissue homogenates. The routes of metabolism included in the model are carboxylesterase-catalyzed ester hydrolysis, conjugation with glutathione, and binding to protein. To adequately describe the rate and extent of glutathione depletion following gavage dosing, the steady-state rate of glutathione synthesis in the organs of interest was included. In vivo validation of the model was conducted by comparing the predictions of the model to the results of a variety of gavage dosing experiments with ethyl acrylate, including (1) the time course of glutathione depletion in a variety of tissues up to 98 hr following dosing at three dose levels, (2) the rate and extent of radiolabeled carbon dioxide excretion, and (3) protein binding in the forestomach. The very rapid metabolism predicted by the model was consistent with the observation that ethyl acrylate was metabolized too rapidly in vivo to be detected by common analytical techniques for tissue metabolite analysis. The validation data indicated that the model provides a reasonable description of the pharmacokinetics and the pharmacodynamic response of specific rat tissues following gavage dosing of ethyl acrylate. A dose surrogate, or measure of delivered dose, for ethyl acrylate was calculated and correlated with the incidence and severity of contact site toxicity (edema, inflammation, ulceration, and hyperplasia). The model provides a quantitative tool for evaluating exposure scenarios for their potential to induce contact-site toxicity, and it provides a quantitative approach for understanding the lack of toxicity in tissues remote from the dosing site.

Arachidonic acid-dependent peroxidative activation of carcinogenic arylamines by extrahepatic human tissue microsomes.

Prostaglandin H synthase (PHS), an arachidonic acid-dependent peroxidase, has been implicated in the peroxidative activation of carcinogenic aromatic amines in extrahepatic carcinogen target tissues of experimental animals. We have examined the arachidonic acid-dependent activation of [3H]benzidine to DNA-bound products by microsomal preparations from 75 normal human tissues obtained during necessary surgical procedures. For several samples of urinary bladder epithelium, prostatic epithelium, colonic mucosa, and peripheral lung tissue, an arachidonic acid-dependent, microsomal-catalyzed activation of benzidine was observed; and the activity could be inhibited appreciably by indomethacin, a known inhibitor of PHS. Little or no arachidonic acid-dependent activity was detected in human placenta, breast, or liver microsomes or the majority of colon microsomes. Substrate specificity was also examined with purified ram PHS and with human bladder and with active colon preparations. Purified PHS catalyzed the activation of benzidine much greater than 2-naphthylamine, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole greater than 4-aminobiphenyl greater than 2-amino-3-methylimidazo[4,5-f]quinoline greater than 3-amino-1-methyl-5H-pyrido[4,3-b] indole. In comparison, human bladder and colon microsomes catalyzed the activation of benzidine greater than 4-aminobiphenyl, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 2-naphthylamine greater than 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. To confirm the occurrence of PHS antigen in human extrahepatic tissues, an avidin/biotin-amplified competitive enzyme-linked immunoabsorbent assay was developed with purified ram PHS and a commercially available monoclonal antibody known to cross-react with human platelet PHS. The avidin/biotin-amplified enzyme-linked immunosorbent assay, which detected ng quantities of ram PHS, clearly established the presence of the PHS protein in human bladder, prostate, and lung microsomes. In contrast, PHS antigen was not detected in the liver or placental microsomes. The interindividual and tissue-dependent variability of PHS and its role in aromatic amine carcinogenesis are discussed.

Epitope characterization of acetaminophen bound to protein and nonprotein sulfhydryl groups by an enzyme-linked immunosorbent assay.

The oxidation of acetaminophen to N-acetyl-p-benzoquinone imine and its subsequent binding to protein sulfhydryl groups may be important in the observed toxicity of acetaminophen. An avidin biotin-amplified competitive enzyme-linked immunosorbent assay has been developed which utilizes solid phase acetaminophen bound to metallothionein and antiserum obtained from rabbits that were immunized with 3-(N-acetyl-L-cystein-S-yl)acetaminophen covalently bound to keyhole-limpet hemocyanin. Over 25 synthetic analogs of acetaminophen conjugates and structurally similar compounds have been ranked relative to their ability to compete in the avidin biotin-amplified enzyme-linked immunosorbent assay. The most effective inhibitor was 3-(N-acetyl-L-cystein-S-yl)acetaminophen, which had an observed 50% inhibitory concentration of 120 fmol/well. Approximately 6200-fold higher concentrations of unbound acetaminophen and 5.2 x 10(6)-fold higher concentrations of N-acetyl-L-cysteine were required for comparable inhibition. It was demonstrated with acetaminophen analogs that the hydroxyl group and the N-acetyl moiety of acetaminophen were important in epitope recognition. A 5000-fold decrease in detection was observed when the analog did not contain the hydroxyl group or when the N-acetyl moiety was replaced with a hydroxyl substituent. Recognition by antibody was also dependent upon the stereochemistry of the analogs. The 50% inhibitory concentration for 3-(L-cystein-S-yl)acetaminophen was 2300 fmol/well, whereas a 25-fold higher concentration of 3-(D-cystein-S-yl)acetaminophen was required for 50% inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen adducts in serum and liver proteins of acetaminophen-treated mice.

Using a recently developed enzyme-linked immunosorbent assay specific for 3-(cystein-S-yl)acetaminophen adducts we have quantitated the formation of these specific adducts in liver and serum protein of B6C3F1 male mice dosed with acetaminophen. Administration of acetaminophen at doses of 50, 100, 200, 300, 400 and 500 mg/kg to mice resulted in evidence of hepatotoxicity (increase in serum levels of alanine aminotransferase and aspartate aminotransferase) at 4 hr in the 300, 400 and 500 mg/kg treatment groups only. The formation of 3-(cystein-S-yl)acetaminophen adducts in liver protein was not observed in the groups receiving 50, 100 and 200 mg/kg doses, but was observed in the groups receiving doses above 300 mg/kg of acetaminophen. Greater levels of adduct formation were observed at the higher doses. 3-(Cystein-S-yl)acetaminophen protein adducts were also observed in serum of mice receiving hepatotoxic doses of acetaminophen. After a 400 mg/kg dose of acetaminophen, 3-(cystein-S-yl)acetaminophen adducts in the liver protein reached peak levels 2 hr after dosing. By 12 hr the levels decreased to approximately 10% of the peak level. In contrast, 3-(cystein-S-yl)acetaminophen adducts in serum protein were delayed, reaching a sustained peak 6 to 12 hr after dosing. The dose-response correlation between the appearance of serum aminotransferases and 3-(cystein-S-yl)acetaminophen adducts in serum protein and the temporal correlation between the decrease in 3-(cystein-S-yl)acetaminophen adducts in liver protein and the appearance of adducts in serum protein are consistent with a hepatic origin of the adducts detected in serum protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct analysis of rat bile for acetaminophen and two of its conjugated metabolites via thermospray liquid chromatography/mass spectrometry.

Bile from rats treated with acetaminophen was analyzed by direct injection onto a thermospray liquid chromatography/mass spectrometry (LC/MS) system. Two conjugated metabolites of acetaminophen were separated by the high-pressure liquid chromatographic system and analyzed by mass spectrometry. The conjugates were identified as the glutathione-acetaminophen conjugate and the glucuronide-acetaminophen conjugate by comparison of the chromatographic retention times and the mass spectra to that of the synthetic standards. No evidence of acetaminophen metabolites was observed when bile samples were subjected to direct analysis by fast atom bombardment mass spectrometry.

Fast atom bombardment mass spectrometry and fast atom bombardment mass spectrometry/mass spectrometry of three glutathione conjugates of acetaminophen.

Three glutathione conjugates of acetaminophen were characterized by fast atom bombardment/mass spectrometry (FAB/MS) and fast atom bombardment/mass spectrometry/mass spectrometry (FAB/MS/MS). The conjugates, 3-(glutathion-S-yl)acetaminophen, 3-(glutathion-S-yl)diacetaminophen and 3-(diglutathion-S-yl)diacetaminophen showed intense [MH]+ ions at m/z 457, 606 and 911, respectively. Only 3-(glutathion-S-yl)acetaminophen showed any fragmentation by FAB/MS. Structurally characteristic fragmentation was observed with all three conjugates when the [MH]+ ions were collisionally activated. The loss of the glycine (GLY) and glutamic acid (GLU) moieties indicated the presence of at least one glutathione (GSH) group. Multiple losses, some of which could only occur via cleavages in both GSH moieties, were observed with the diglutathione conjugate.