Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics.

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity.

Identification of a yellow impurity in aged samples of aqueous butamben suspension: evidence for the oxidative degradation of poly(ethylene glycol).

Butamben (butyl p-aminobenzoate) has been formulated to provide long-acting treatment for chronic pain. The suspension, which contains poly(ethylene glycol) and polysorbate 80, was found to yellow under ambient conditions if not adequately protected from oxygen. The impurity responsible for the color was isolated and identified on the basis of nuclear magnetic resonance spectroscopy and mass spectrometry. The compound is an oxalamidine, which is formally the condensation product of oxalic acid with four equivalents of butamben, and may be formed by the reaction of butamben with an oxidation product of poly(ethylene glycol).

Solubilization and partial characterization of extensin fragments from cell walls of cotton suspension cultures. Evidence for a covalent cross-link between extensin and pectin.

Extensin, a major hydroxyproline (Hyp)-rich glycoprotein in walls of cultured cells of dicotyledonous plants, is very difficult to solubilize. To learn about the nature of the insolubilization, we have tested the ability of a variety of selective hydrolytic methods, and combinations of them, to liberate extensin or fragments of extensin from suspension-culture cell walls. After the complete deglycosylation of cotton (Gossypium hirsutum L.) walls, trypsinization solubilized 80% of the Hyp. The sequences of three abundant peptides were: (a) serine-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-serine-Hyp-Hyp-lysine, (b) serine-Hyp-Hyp-Hyp-Hyp-valine-lysine, and (c) serine-Hyp-Hyp-serine-alanine-Hyp-lysine. After a sequential treatment of walls with endopolygalacturonase, cellulase, -73 degrees C anhydrous hydrogen fluoride solvolysis, and ammonium bicarbonate extraction, only sugars indicative of rhamnogalacturonan I and protein remained insoluble. Trypsin treatment of this residue liberated 50% of the Hyp. A significant proportion of rhamnogalacturonan-associated sugars co-solubilized and co-purified along with the extensin fragments following the trypsinization. By sodium dodecyl sulfate gel electrophoresis and gel filtration, the glycopeptides fell into two classes. One class contained distinctly sized molecules with relative molecular weights in the range of 4,000 to 24,000. The other class did not enter the resolving gel and was hetero-disperse. After complete deglycosylation by a 0 degrees C anhydrous hydrogen fluoride treatment, the first class was little affected in its electrophoretic mobility, whereas the larger heterogeneous material mostly entered the separating gel. After further trypsinization of the deglycosylated peptides and analysis by capillary zone electrophoresis, the peptides in both size classes were shown to contain the sequences described above. From our observations we suggest that cotton extensin becomes insolubilized into cell walls in part by pectin-protein cross-links in addition to the protein-protein (or protein-phenolic-protein) cross-links that have been repeatedly suggested.

Arthropod venom citrate inhibits phospholipase A2.

Citrate has been identified as a major component of honey bee (Apis mellifera) venom by gas liquid chromatography-mass spectrometry. A citrate concentration of 9% was found for dried bee venom by a coupled enzyme assay, aconitase-isocitric dehydrogenase. A liquid honey bee venom would contain 140 mM citrate concentration (if the solids content were 30%). Bee venom phospholipase was inhibited at a 43% level with a citrate concentration of 20 mM and calcium ion at 3 mM with the enzyme assay. Citrate was also found in the venoms of bumble bee, Bombus fervidus, 7%; yellow jacket, Vespula maculifrons, 4%; scorpion, Centruroides sculpturatus, 8%; tarantula, Grammastola cala, 8% and brown recluse spider venom gland extract, Loxoceles reclusa, 1.5% based on dried venom solids. Citrate may serve as an endogenous inhibitor of divalent metal ion-dependent enzymes in arthropod venoms as described by Francis et al. (1992, Toxicon 30, 1239-1246). Many arthropod venoms contain calcium-dependent phospholipases. A direct effect of citrate as a venom component may be possible. The presence of citrate in venoms must be considered in research on receptors, ion channels and divalent ion-dependent toxins.

"SpectraGraph" and "SpectraSort": mass spectral display and interpretation software for the Macintosh.

Two computer programs entitled "SpectraGraph" and "SpectraSort" have been written for te Apple Macintosh. SpectraGraph allows graphical display, manipulation, storage, and printing of an input mass spectrum list that has been imported from a mass spectrometer or entered manually. SpectraGraph gives the user the ability to display, normalize, and multiply different mass ranges, annotate peaks, and perform various other operations on the spectral display. Also the mass spectrum and other graphics may be copied to and from other Macintosh application documents. SpectraSort has been developed to aid in the interpretation of mass spectra, particularly those of biopolymers, by calculating the mass differences between peaks in a mass spectrum. The user then has the option of matching the mass differences with masses of fragments or residues stored in several user-definable look-up tables.

Direct electron spin resonance detection of free radical intermediates during the peroxidase catalyzed oxidation of phenacetin metabolites.

The oxidation of the phenacetin metabolites p-phenetidine and acetaminophen by peroxidases was investigated. Free radical intermediates from both metabolites were detected using fast-flow ESR spectroscopy. Oxidation of acetaminophen with either lactoperoxidase and hydrogen peroxide or horseradish peroxidase and hydrogen peroxide resulted in the formation of the N-acetyl-4-aminophenoxyl free radical. Totally resolved spectra were obtained and completely analyzed. The radical concentration was dependent on the square root of the enzyme concentration, indicating second-order decay of the radical, as is consistent with its dimerization or disproportionation. The horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of p-phenetidine (4-ethoxyaniline) at pH 7.5-8.5 resulted in the one-electron oxidation products, the 4-ethoxyaniline cation free radical. The ESR spectra were well resolved and could be unambiguously assigned. Again, the enzyme dependence of the radical concentration indicated a second-order decay. The ESR spectrum of the conjugate base of the 4-ethoxyaniline cation radical, the neutral 4-ethoxyphenazyl free radical, was obtained at pH 11-12 by the oxidation of p-phenetidine with potassium permanganate.

Free-radical metabolites of acetaminophen and a dimethylated derivative.

The oxidation of acetaminophen (4'-hydroxyacetanilide) to the corresponding N-acetyl-p-benzoquinone imines by plant and mammalian peroxidases is discussed. The acetaminophen free radical (N-acetyl-4-aminophenoxyl) has been reported as an intermediate. It is very reactive and forms melanin-like polymeric products. Application of a fast-flow system makes it possible to detect the transient species and clearly distinguish it from persistent paramagnetic melanin polymers. A model system, leading to more stable metabolites, can be obtained by introduction of methyl groups next to the oxygen, 3',5'-dimethylacetaminophen (3',5'-dimethyl-4'-hydroxyacetanilide). The ESR spectrum of the free radical formed could be completely analyzed and confirmed by deuterium substitution. The data are consistent with the assignment to a phenoxyl free radical (N-acetyl-2,6-dimethyl-4-amino-phenoxyl). Its formation is discussed in terms of substrate, hydrogen peroxide and enzyme concentration dependence. It is believed to be formed via a direct one-electron oxidation of 3',5'-dimethyl-4'-hydroxy-acetanilide. The radical does not form polymers or react with nucleophiles. Its redox behavior is discussed. The possible reaction of these phenoxyl free radicals with oxygen is thought to be negligible.

Formation of 4-aminophenoxyl free radical from the acetaminophen metabolite N-acetyl-p-benzoquinone imine.

N-Acetyl-p-benzoquinone imine, a hepatic metabolite of acetaminophen, and its analogue, N-acetyl-3,5-dimethyl-p-benzoquinone imine, were metabolized by rat liver microsomes and NADPH to their corresponding 4-aminophenoxyl free radicals. ESR spectra were recorded and unambiguously identified. As indicated by the purple color and confirmed by UV and mass spectroscopy, indophenols were formed as final products. The 4-aminophenoxyl free radical formation could be suppressed by the deacetylase inhibitors, sodium fluoride and paraoxon. Microsomal incubations of N-acetyl-2,6-dimethyl-p-benzoquinone imine and NADPH do not result in a detectable radical concentration; in addition, no indophenol was found. Substitution of NADPH-cytochrome P-450 reductase for rat liver microsomes eliminates the deacetylase activity and results in direct reduction of N-acetyl-3,5-dimethyl-p-benzoquinone imine to the N-acetyl-2,6-dimethyl-4-aminophenoxyl free radical. Neither the incubation of N-acetyl-p-benzoquinone imine nor that of N-acetyl-2,6-dimethyl-p-benzoquinone imine with NADPH-cytochrome P-450 reductase yielded a detectable concentration of the corresponding phenoxyl free radical. When starting material that had been exposed to the atmosphere was used, a previously reported free radical with a splitting constant of approximately 2 G was formed. This spectrum is identical with that of the 2,6-dimethyl-p-benzosemiquinone free radical, implying hydrolysis of the starting material. Neither the N-acetyl-4-aminophenoxyl nor the N-acetyl-2,6-dimethyl-4-aminophenoxyl radical reduces oxygen to form superoxide or react with oxygen in any other detectable way.

Mutagenicity and purative carcinogenicity tests of several polycyclic aromatic compounds associated with impurities of the insecticide methoxychlor.

Several polycyclic hydrocarbons, 3,6-dimethoxy-9,10-bis(p-methoxyphenyl)-phenathrene, tetrakis(p-methoxyphenylyethylene and 3,6,11,14-tetramethoxydibenzo(g,p)chrysene, which are associated as impurities in commerical samples of the insecticide methoxychlor, have been tested in the Ames mutagenicity test with strains of Salmonella thyphimurium, TA 1535, TA 1537, TA 1538, and TA 98. Activation by liver microsomes induced with either phenobarbitol or Aroclor was examined. The only active compound was 3,6,11,14-tetramethoxydibenzo(g,p)chrysene, mutagenic (0.39 revertants/nmol) tostrain TA 98.