Selective binding by the RNA binding domain of PKR revealed by affinity cleavage.

The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.

Site-specific modification and RNA crosslinking of the RNA-binding domain of PKR.

RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR is activated in vitro by binding RNA molecules with extensive duplex structure. To further define the nature of the RNA regulation of PKR, we have prepared and characterized site-specifically modified proteins consisting of the PKR 20 kDa RNA-binding domain (RBD). Here we show that the two cysteines found naturally in this domain can be altered by site-directed mutagenesis without loss of RNA binding affinity or the RNA-regulated kinase activity. Introduction of cysteine residues at other sites in the PKR RBD allows for site-specific modification with thiol-selective reagents. PKR RBD mutants reacted selectively with a maleimide to introduce a photoactivatable cross-linking aryl azide at three different positions in the protein. RNA crosslinking efficiency was found to be dependent on the amino acid modified, suggesting differences in access to the RNA from these positions in the protein. One of the amino acid modifications that led to crosslinking of the RNA is located at a residue known to be an autophosphorylation site, suggesting that autophosphorylation at this site could influence the RNA binding properties of PKR. The PKR RBD conjugates described here and other similar reagents prepared via these methods are applicable to future studies of PKR-RNA complexes using techniques such as photocrosslinking, fluorescence resonance energy transfer and affinity cleaving.

Mutagenicity of tetranitroazoxytoluenes: a preliminary screening in Salmonella typhimurium strains TA100 and TA100NR.

Tetranitroazoxytoluenes are polynitroaromatic compounds that can be produced during the microbial reduction of the explosive, 2,4,6-trinitrotoluene (TNT). The three major tetranitroazoxytoluenes were synthesized and tested in Salmonella typhimurium strains TA100 and TA100NR. All compounds were mutagenic in TA100 but not in TA100NR, indicating the need for nitroreductase activity to induce mutagenicity. The most active chemical was 4,4',6,6'-tetranitro-2,2'-azoxytoluene (2735 rev/mumol) followed by 2',4,6,6'-tetranitro-2',4-azoxytoluene (929 rev/mumol) and 2,2'-6,6'-tetranitro-4,4'-azoxytoluene (320 rev/mumol). These chemicals were more active than the aminodinitrotoluenes (298 rev/mumol for 2-amino-4,6-dinitrotoluene and 115 rev/mumol for 4-amino-2,6-dinitrotoluene) and only 4,4',6,6'-tetranitro-2,2'-azoxytoluene was more active than the parent compound, TNT (1022 rev/mumol).

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 18O2 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.

Structure-activity relationship for the intrinsic hepatotoxicity of dinitrotoluenes.

The relation of various structural parameters to hepatotoxic potential was investigated by using six dinitrotoluene (DNT) isomers and isolated rat hepatocyte suspensions as the biological test system. DNT-induced hepatotoxicity was found to correlate with an inhibition of protein synthesis and an increase in lactate dehydrogenase (LDH) release but not with lipid peroxidation. With each isomer, protein synthesis inhibition was the most sensitive indicator of cytotoxicity. Regardless of the indicator, ortho- and para-substituted isomers were more hepatotoxic at the same concentration than meta-substituted isomers. High-performance liquid chromatograms (HPLC) on samples at 4 h revealed significant quantities of reduced metabolites in the medium. However, increased lipid peroxidation (formation of thiobarbituric acid reactants or evolution of ethane) in the cells was not consistently demonstrated. log EC50 for protein synthesis inhibition and log EC20 for LDH release were linearly correlated with the C atomic charge on the ring carbons bearing the nitro substituents by using molecular orbital (MNDO calculations) theory. The relation was used to predict the hepatotoxic potentials of untested nitrotoluenes, and the predictions were verified to a first approximation by using three trinitrotoluene isomers.

Comparative mutagenicity of halogenated pyridines in the Salmonella typhimurium/mammalian microsome test.

The Salmonella/microsome assay with strains TA97, TA98, TA100 and TA102 was used to examine the potential mutagenicity and structure-activity of 16 mono- and di-halogenated pyridines. The chemical reactivity of the halopyridines suggests that nucleophilic displacement of halogens can occur with halogens at positions 2, 4 and 6 being displaced in addition-elimination reactions. 2-Chloropyridine gave a positive result with rat-liver metabolic activation, and 2-fluoropyridine gave equivocal results under these conditions. Mutagenic responses were also obtained with 2-chloromethyl pyridine and 3-chloromethyl pyridine, in both the presence and absence of rat-liver S9. These results suggest that the halogenated pyridines, especially with halogens at the 2-position, and singly on a methyl substituent, have mutagenic activity in the Salmonella assay.

Short-term oral toxicity of 2,4,6-trinitrotoluene in mice, rats, and dogs.

The short-term oral toxicity of 2,4,6-trinitrotoluene (alpha-TNT) was determined in dogs, rats, and mice. Single-dose oral LD50s for alpha-TNT in corn oil were 1320 and 794 mg/kg in male and female rats, respectively, and 660 mg/kg in both male and female mice. For multiple-dose studies, dogs were dosed daily for up to 13 wk with alpha-TNT at 0, 0.2, 2.0, or 20 mg/kg by capsule; rats received 0, 0.002, 0.01, 0.05, or 0.25% and mice received 0, 0.001, 0.005, 0.025, or 0.125% alpha-TNT in their diets over the same period. All species receiving the highest doses exhibited anemia, with reduced erythrocytes, hemoglobin, and hematocrit. Alterations were observed in organ weights, including enlarged spleens (accompanied by hemosiderosis) and livers, and depressed body weight and/or body weight gain (temporary in dogs and mice). Alterations in clinical chemistry values included elevated cholesterol and depressed serum glutamicpyruvic transaminase activity in dogs and rats; no effect on serum glutamic-oxaloacetic transaminase activity was observed. Some effects, such as SGPT depression in rats, appeared after 13 wk, suggesting a cumulative toxicity. Reduced testes size was observed in rats at the highest dose regardless of length of exposure. Most of the toxic effects were reversible, but testicular atrophy was not in rats allowed a 4-wk recovery period after treatment. Signs of anemia were present at intermediate dose levels. "No observable effects" levels for alpha-TNT were: dogs, 0.20; rats, 1.42; and mice, 7.76 mg/kg . d.

Single-dose and repeated-exposure toxicity of a complex wastewater from munitions manufacturing plants.

The single-dose and repeated-exposure toxicity of a synthetic mixture of 30 nitrotoluene analogs, representative of a complex industrial wastewater termed condensate water, was evaluated in dogs, rats, and mice. The single-dose oral LD50s for the synthetic condensate water (CW) were 447 and 295 mg/kg in male and female rats, respectively. In the repeated-exposure studies, dogs were given 0, 0.05, 0.5, or 5 mg of CW per kilogram of body weight by capsule daily for 26 wk. Rats and mice received 0, 0.001, 0.01, or 0.1% of this mixture in their diet for 4 or 13 wk. Groups of each rodent species were set aside for 4 wk to assess recovery. The most notable findings were a compensatory anemia with reticulocytosis (severe in rats), Heinz body formation, and related blood cell abnormalities and hemosiderin in the spleen; pigmentation in the liver cells; atrophy and aspermia in the testes; hyperplasia and inflammation in the female reproductive organs; and neurotoxic signs at the high doses. Rats and mice also experienced food intake and body weight depression and exhibited some alterations in organ weights (spleen, testes, and liver). The findings were referable principally to the two major components 2,4,- and 2,6-dinitrotoluene, but the "no observable effects" levels were lower for the mixture.

Short-term oral toxicity of a 2,4,6-trinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine mixture in mice, rats, and dogs.

The oral toxicity of a mixture of 2,4,6-trinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine (1:0.62, w/w) compounds typically found in munitions plant effluents, was evaluated in mammalian species. Single-dose oral LD50s of the mixture were 574 and 594 mg/kg in male and female rats and 947 and 1130 mg/kg in male and female mice, respectively. Long dispersion periods during preparation or ultraviolet irradiation of the mixture lowered the LD50s. In repeated-exposure studies, dogs were given 0.50, 5.0 or 50 mg/kg X d by capsule for up to 90 d. Rats and mice were fed the mixture in the diet at 0.005, 0.05, or 0.5% for 90 d; mice were also fed at 0.25%. Mortality resulted at the highest dose level in each species. All three species showed depression of body weight or body weight gain, depressed food intake, moderate to severe anemia, and alterations in the spleen (hemosiderosis), liver (hepatomegaly), and testes (atrophy) at the highest dose levels. Cholesterol was elevated in rats and dogs after 90 d. Several species differences were also noted. Uric acid values were elevated in rats but not in dogs, serum glutamic-pyruvic transaminase (SGPT) activity was low in dogs but unchanged in rats, and rats developed hypoplasia of the uterus but dogs did not. Signs of anemia were present at the intermediate dose levels. The lowest dose level in all three species was designated at a "no observable effects" level, based on the absence of clearly treatment-related effects. In a 4-wk study, the irradiated mixture fed to rats at 0.003, 0.03, or 0.3% in the diet was less toxic than the unirradiated mixture.

Mutagenicity in Salmonella typhimurium and structure-activity relationships of wastewater components emanating from the manufacture of trinitrotoluene.

The mutagenicity of 36 polynitroaromatic compounds was investigated with five strains of Salmonella typhimurium. Isomeric trinitrotoluenes (TNT), with the exception of 2,4,6-TNT and 2,3,4-TNT, exhibit mutagenicity independently of nitroreductase enzymes, but isomeric aminodinitrotoluenes (ADNT) and isomeric dinitrotoluenes (DNT) need nitroreductase to induce mutation. Within groups of isomeric TNTs, DNTs, and ADNTs, mutagenic response was enhanced by a para orientation of nitro groups. The mutagenic response of isomeric DNTs was found to correlate with the compound's ability to undergo charge-transfer complexation with reductive enzymes, whereas further complexation (such as a Janovsky complex) appears to be required for inducing mutation in dinitrobenzenes. These results indicate that polynitroaromatic compounds in TNT wastewaters possess a significant potential for biologic activity.

Application of high-pressure liquid chromatography and thermal energy analyzer to analysis of trinitroglycerin and its metabolites in blood.

A highly selective and sensitive analytical procedure for the determination of trinitroglycerin and four metabolites in whole blood was developed. Trinitroglycerin and its metabolites were extracted from whole blood with ethyl acetate and analyzed by high-pressure liquid chromatography using the thermal energy analyzer detector. Linearity of response was observed over the 1-1000-ng range. The applicability of this method to the analysis of whole blood from dogs orally dosed with trinitroglycerin is described.

N-formyliminodiacetic acid, a new compound from the reaction of nitrilotriacetic acid and chlorine.

It has been proposed that nitrilotriacetic acid be substituted for trisodium polyphosphates in detergents as a way to reduce the rate of eutrophication in the Great Lake Basin. The reaction of nitrilotriacetic acid with chlorine-containing solutions produces a hitherto unknown degradation production, N-formyliminodiacetic acid, in high yield. The toxicological and environmental implications of this reaction are unclear.

Cometabolism of DDT analogs by a Pseudomonas sp.

A Pseudomonas sp. capable of growth on several nonchlorinated and mono-p-chloro-substituted analogs of DDT as a sole carbon source degraded bis(p-chlorophenyl)methane and 1,1-bis(p-chlorophenyl)ethane only in the presence of diphenylethane. The products p-chlorophenylacetic acid and 2-(p-chlorophenyl)-propionic acid were not further metabolized by the bacterium. Other chlorinated analogs of DDT were found to be recalcitrant to cometabolic degradation with diphenylethane.

Some observations on biodegradation of pollutants in aquatic systems.

The biodegradability of pollutants introduced into aquatic environments is subject to many variabilities characteristic of microbial processes. Some observations have been reported on studies with p-cresol, methyl parathion, benzo[b]thiophene, dibenzothiophene, 9H-carbazole, quinoline, benzo[f]quinoline, benz[a]anthracene, benzo[a]pyrene, 7H-dibenzo[c,g]carbazole, Mirex, 2,4-dichlorophenoxyacetic acid, Lindane and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane analogs. Water reservoirs included a eutrophic stream, a eutrophic pond, an oligotrophic lake and effluents from waste-water treatment plants. The first ten of the above compounds were studied only in aerobic mixed-culture systems, and Mirex was studied with mixed cultures under both aerobic and anaerobic conditions. Examples were presented of the significance of cometabolism, diauxic processes, film fermentations and microbial interactions in biodegradation studies. Caution is advised in protocols regarding biodegradability of pollutants in aquatic environments.

Metabolism of DDT analogues by a Pseudomonas sp.

A Pseudomonas sp. rapidly metabolized several nonchlorinated analogues of DDT, with the exception of 2,2-diphenylethanol, as the sole carbon source. Several of the mono-p-chloro-substituted diphenyl analogues were also metabolized as the sole carbon source by the bacterium. The resulting chlorinated aromatic acid metabolites were not further metabolized. The isolate was unable to metabolize p,p'-dichlorodiphenyl analogues as the sole carbon source.

Degradation of lindane by Escherichia coli.

Lindane was degraded by Escherichia coli isolated from rat feces. About 10% of the added lindane was metabolized by the bacterium in Trypticase soy broth containing the pesticide. A single metabolite, 2,3,4,5,6-pentachloro-1-cyclohexene, was detected and identified by gas chromatography and mass spectrometry.