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As part of a study conducted on the fate of xenobiotics in the environment, a selection of 100 strains of micromycetes (Ascomycetes, Basidiomycetes and Yeasts) have been cultivated in liquid synthetic medium with 3 phenylurea herbicides: chlortoluron and isoproturon (100mg L-1) and diuron (20mg L-1). While 17 strains depleted isoproturon over 50% only 4 depleted diuron and 2 chlortoluron at the same level. The best results were obtained with Bjerkandera adusta and Oxysporus sp which were the most efficient towards the 3 substrates. After 2 weeks Bjerkandera adusta depleted chlortoluron 98%, diuron 92% and isoproturon 88%.

Three different isolation routes (flask enrichment/flask degradation assay, flask enrichment/microplate degradation assay, MPN assay/microplate degradation assay) were used to obtain pure cultures of bacteria which degraded isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) as sole carbon and nitrogen source in a mineral salts medium from a field soil treated with isoproturon in the laboratory. All three isolation routes were successful, but the microplate assay of degradation was more successful than the flask assay. Characterization of 36 isolates indicated that they formed 16 distinct phenotypes (10 Gram-positive phenotypes, six Gram-negative phenotypes) which are likely to represent distinct species. Low concentrations of the degradation product 3-(4- isopropylphenyl)-1-methylurea (IPPMU) were occasionally found in the culture solutions. When provided as the sole source of carbon and nitrogen, the monomethyl degradation product was itself rapidly degraded by several of the isolates. Some isolates were also able to use the demethylated degradation product 3-(4-isopropylphenyl)-urea (IPPU) as sole source of carbon and nitrogen, although there was occasionally an extended lag-phase before rapid degradation commenced. One isolate was particularly active and degraded isoproturon, the monomethyl and demethylated degradation products of isoproturon, and demethylated the related phenylureas diuron and linuron.

New methods for docking, template fitting and building pseudo-receptors are described. Full conformational searches are carried out for flexible cyclic and acyclic molecules. QXP (quick explore) search algorithms are derived from the method of Monte Carlo perturbation with energy minimization in Cartesian space. An additional fast search step is introduced between the initial perturbation and energy minimization. The fast search produces approximate low-energy structures, which are likely to minimize to a low energy. For template fitting, QXP uses a superposition force field which automatically assigns short-range attractive forces to similar atoms in different molecules. The docking algorithms were evaluated using X-ray data for 12 protein-ligand complexes. The ligands had up to 24 rotatable bonds and ranged from highly polar to mostly nonpolar. Docking searches of the randomly disordered ligands gave rms differences between the lowest energy docked structure and the energy-minimized X-ray structure, of less than 0.76 A for 10 of the ligands. For all the ligands, the rms difference between the energy-minimized X-ray structure and the closest docked structure was less than 0.4 A, when parts of one of the molecules which are in the solvent were excluded from the rms calculation. Template fitting was tested using four ACE inhibitors. Three ACE templates have been previously published. A single run using QXP generated a series of templates which contained examples of each of the three. A pseudo-receptor, complementary to an ACE template, was built out of small molecules, such as pyrrole, cyclopentanone and propane. When individually energy minimized in the pseudo-receptor, each of the four ACE inhibitors moved with an rms of less than 0.25 A. After random perturbation, the inhibitors were docked into the pseudo-receptor. Each lowest energy docked structure matched the energy-minimized geometry with an rms of less than 0.08 A. Thus, the pseudo-receptor shows steric and chemical complementarity to all four molecules. The QXP program is reliable, easy to use and sufficiently rapid for routine application in structure-based drug design.

The effects of temperature on the bioaccumulation of the phenylurea herbicide isoproturon (IPU) and on the growth inhibition induced on two freshwater rooted macrophytes--Elodea densa and Ludwigia natans--were investigated using indoor microcosms. The experimental protocol was based on five temperatures (12, 16, 20, 24, and 28 degrees C) and two contamination levels of the water column (30 and 60 micrograms IPU.liter-1), plus a control condition. Variations in temperature had little effect on the decrease in [IPU] in the water column during the 21-day experiment. The IPU concentration in the two macrophyte species was not significantly modified by the large range of temperatures when results were expressed using the concentration criterion. IPU burdens in the E. densa cuttings, on the other hand, increased significantly when the temperature rose from 12 to 28 degrees C; small differences were observed between the two exposure conditions, in relation to the antagonistic effects of IPU and temperature on the growth of the cuttings. Bioconcentration factors in the plants (stems + leaves) were close to 10 and 13 after exposure to 30 and 60 micrograms IPU.liter-1, respectively.

Field and laboratory tests were conducted to investigate the disappearance and decrease in availability of the herbicide isoproturon in soil with time. Monitoring the fate of 14C-labeled isoproturon gave the persistence of the herbicide under different conditions. The calculated half-life (DT50) of the extractable parent product was close to 19 days for the two experiments, whereas half-lives of exhaustive extractable 14C-residues averaged 22 days under controlled conditions versus 48 days in the field. Moreover, if nonextractable 14C-residues were taken into account in estimating isoproturon fate, the DT50 of radioactive residues in soil (extractable parent and degradation products + nonextractable residues) reached 1 to 1.4 years under field and controlled conditions, respectively. The variability of disappearance rates for the different kinds of residues is discussed within a wider definition of persistence.

As a part of a study conducted on the fate of xenobiotics in the environment, a selection of 90 strains of micromycetes, mostly isolated from soil and belonging to various taxonomic groups, have been cultivated in liquid synthetic medium with chlortoluron (100 mg.L-1), diuron (20 mg.L-1), and isoproturon (100 mg.L-1) for 5 days. Evaluation of the chemicals in the culture media was made by HPLC. Our results show a wide variation not only with taxonomic groups but also with the species, and with the tested chemicals. On the whole, 4, 7, and 11% of the strains depleted respectively chlortoluron, diuron, and isoproturon, at 50% or over. Rhizoctonia solani was the only strain that depleted each of the 3 substituted phenylureas over 70%, nevertheless, the growth of this fungus was slightly inhibited by diuron. The very fast disappearance of the chemicals from the culture media was only due to biotic phenomena, as no adsorption occurred on the fungal biomass. So, depletion translated a real biodegradation of the tested substrates.

The structure of the HIV-1 protease in complex with a pseudo-C2 symmetric inhibitor, which contains a central difluoroketone motif, has been determined with X-ray diffraction data extending to 1.7 A resolution. The electron density map clearly indicates that the inhibitor is bound in a symmetric fashion as the hydrated, or gemdiol, form of the difluoroketone. Refinement of the complex reveals a unique, and almost symmetric, set of interactions between the geminal hydroxyl groups, the geminal fluorine atoms, and the active-site aspartate residues. Several hydrogen bonding patterns are consistent with that conformation. The lowest energy hydrogen disposition, as determined by semiempirical energy calculations, shows only one active site aspartate protonated. A comparison between the corresponding dihedral angles of the difluorodiol core and those of a hydrated peptide bond analog, calculated ab-initio, shows that the inhibitor core is a mimic of a hydrated peptide bond in a gauche conformation. The feasibility of an anti-gauche transition for a peptide bond after hydration is verified by extensive molecular dynamics simulations. The simulations suggest that rotation about the C-N scissile bond would readily occur after hydration and would be driven by the optimization of the interactions of peptide side-chains with the enzyme. These results, together with the characterization of a transition state leading to bond breakage via a concerted exchange of two protons, suggest a proteolysis mechanism whereby only one active site aspartate is initially protonated. The steps of this mechanism are: asymmetric binding of the substrate; hydration of the peptidic carbonyl by an active site water; proton translocation between the active site aspartate residues simultaneously with carbonyl hydration; optimization of the binding of the entire substrate facilitated by the flexible structure of the hydrated peptide bond, which, in turn, forces the hydrated peptide bond to assume a gauche conformation; simultaneous proton exchange whereby one hydroxyl donates a proton to the charged aspartate, and, at the same time, the nitrogen lone pair accepts a proton from the other aspartate; and, bond breakage and regeneration of the initial protonation state of the aspartate residues.

The experimental approach to the effects of the phenylurea herbicide Isoproturon (IPU), on the photosynthesis activity of leaf cells of the freshwater macrophyte Elodea densa was based on the fast induction kinetics of the PSII chlorophyll a in vivo fluorescence. FI/FP ratios determined on the induction curves exhibited a noticeable effect at 5 micrograms IPU.liter-1. They appeared to be the most reliable parameter for the quantitative evaluation of the dissociation constant of the complex "IPU/D1 protein" (Kd = 1.2 x 10(-7) M) and of the concentration of free IPU either at the thylakoid level or in the surrounding medium of the leaf epithelial cells. Total IPU bioaccumulation at the whole plant level as a function of [IPU] in the medium indicated that the D1 protein represented the main binding site for IPU in this aquatic plant species.

For the purpose of biodegradation studies several microorganisms were isolated from soil and adapted under laboratory conditions in the presence of pesticides, with the following degradation results: isoproturon after 72 h-86%, chlortoluron after 72 h-93%, after 65 h-88% and fenitrothion after 72 h-66%. The cultures can be used for the biodegradation of pesticides in the lyophilized state with or without an organic carrier.

The present study describes the acute histopathological changes induced by methyl isocyanate (MIC) in the lungs of rats at 24 h after a single exposure to varied concentrations/doses of MIC by inhalation and subcutaneous (s.c.) routes and also delineates the effects due to the hydrolytic derivatives of MIC, viz., methylamine (MA) and N,N'-dimethyl urea (DMU). MIC, either inhaled or administered s.c., resulted in a wide range and extent of histopathological changes in the lungs, proportional to the exposure concentration/dose. The salient, effects of inhaled MIC are acute necrotizing bronchitis of the entire respiratory tract accompanied by varying degrees of confluent congestion, hyperemia and interstitial and intra-alveolar edema, while MIC administered s.c. led to prominent vascular endothelial damage, congestion and severe interstitial pneumonitis with apparently normal bronchial epithelium; and intra-alveolar edema only with the high dose. The only noteworthy lesion produced by MA and DMU (to some extent) was interstitial pneumonitis, suggesting their possible involvement in the subsequent inflammatory response of MIC. Except, for the endothelial changes, the overall spectrum of the histopathological lesions is quite comparable to those observed in the lungs of Bhopal victims during the acute phase.

This paper describes the long-term (subacute and chronic) histopathological effects in the lungs of rats subjected to a single exposure to methyl isocyanate (MIC) by both the inhalation and subcutaneous (s.c.) routes as well as the role of methylamine (MA) and N,N'-dimethyl-urea (DMU), the hydrolytic derivatives of MIC in eliciting the observed changes. At the subacute phase, the intraalveolar and interstitial edema were prominent only in the inhalation group as against the more pronounced inflammatory response in the s.c. route. With the progress of time the evolution of lesions appeared to be similar, culminating in the development of significant interstitial pneumonitis and fibrosis. MA, one of the hydrolytic derivatives of MIC, also caused interstitial pneumonitis progressing to fibrosis, albeit to a lesser extent than MIC, indicating its contribution to the long-term pulmonary damage. The diffuse interstitial pulmonary fibrosis observed at 10 weeks after a single exposure to MIC by either route is of greater significance in the context of the occurrence of pulmonary fibrosis in the late autopsies of Bhopal gas victims and also clinical sequelae in some of the survivors.

The subcutaneous administration of methyl isocyanate (MIC) to female rabbits, resulted in significant increases in haemoglobin concentration, erythrocyte volume fraction and leucocyte number in blood, as well as plasma total proteins, and urea. The present study was designed to investigate whether the hydrolytic products of MIC, methylamine (MA) and N,N'-dimethylurea (DMU) play any role in eliciting these changes. Both MA and DMU administered subcutaneously in an equimolar dose to that of 1.0 LD50 MIC, 2.2 mmol kg-1, had no influence on these parameters, although there was a marginal increase in the plasma urea level shortly after the administration of DMU. This study establishes that the observed haematological and biochemical changes induced by MIC intoxication in rabbits are mostly due to MIC.

N-(5-nitro-2-thiazolyl)-N'-carboxymethylurea (NTCU) has been identified as a urinary metabolite of the antischistosomal drug niridazole [1-(5-nitro-2-thiazolyl)-2-imidazolidinone]. When DBA/2J mice were treated with [14C]niridazole, a metabolite comprising 12-14% of the total radioactivity in 24-hr urine samples was resolved by HPLC. The compound was subsequently isolated from pooled urine of niridazole-treated patients. It was identified as NTCU by mass spectrometry, and the deduced structure was confirmed by chemical synthesis. NTCU is unique among known niridazole metabolites, because it lacks an intact imidazolidinone ring. Its structure allows for a ketoenol tautomerism in which the enolate is stabilized by conjugation with the nitrothiazole ring, as evidenced by a pH-dependent 80-nm red shift in the absorption spectrum. We hypothesized that NTCU arises via oxidation of an acyclic aldehyde tautomer of 5-hydroxyniridazole, one of two proximate oxidative niridazole metabolites. Indirect evidence for the aldehyde tautomer included the fact that 5-hydroxyniridazole displayed the same pH-dependent spectral shift as NTCU with a single isobestic point at 388 nm. The proposed precursor-product relationship was confirmed when we found that NTCU formation from 5-hydroxyniridazole was catalyzed by NAD(+)-dependent aldehyde dehydrogenase (EC 1.2.1.3). The activity copurified with benzaldehyde dehydrogenase activity from mouse liver cytosol. Furthermore, benzaldehyde was a competitive inhibitor of 5-hydroxyniridazole dehydrogenase activity. These results demonstrate that 5hydroxyniridazole is not an end product of niridazole metabolism. Because biotransformation of niridazole to its 4- and 5-hydroxy derivatives has been implicated in the drug's carcinogenicity and central nervous system toxicity, NTCU formation appears to represent a detoxication pathway in mammals.

Many microorganisms exhibit an adaptive response to mutagenic alkylation damage. In Escherichia coli the response is regulated by the inducible Ada protein. A sensitive immunoassay employing two anti-Ada monoclonal antibodies has been developed here to monitor low levels of induction of the Ada protein. This protein was detected in non-induced E. coli which contained an average of two molecules of Ada per cell. The occurrence of the adaptive response in bacteria signals the existence of an ecological niche in which cells are exposed to direct-acting methylating compounds, but the structure and identity of these agents are unknown. Using the immunoassay to search for possible candidates, a number of methylating agents and precursors of such agents have been investigated. Carbamyl phosphate and methylamine yield N-methylurea, which reacts subsequently with nitrite to generate the strong inducer N-methyl-N-nitrosourea. The antibiotic streptozotocin also is a potent inducer of the adaptive response. Moreover, the abundant environmental mutagen methyl chloride acts as an inducer.

Swine were treated with cimetidine in order to quantify the reduction of nitrate to nitrite and the gastric formation of N-nitrosotrimethylurea (NTMU) under conditions similar to those in the achlorhydric human stomach. Gastric-fistulated swine were instilled with 6.0 mmol of nitrate in 50 ml water, after which gastric nitrate, nitrite and pH were monitored. Trimethylurea, 250 mumol in 50 ml water, was instilled via the fistula 10 min following the peak gastric nitrite concentration. Similar experiments were conducted with pentagastrin-stimulated animals, in order to quantitate the effect of gastric pH and microflora on the presence of nitrate, nitrite and NTMU formation. The stomachs of cimetidine-treated pigs (elevated pH) were colonized by nitrate reductase organisms to levels of 10(4)-10(7)/ml gastric fluid. Gastric nitrite concentration in cimetidine-treated animals reached a maximum of 370-2085 microM, 60 min following the nitrate dose. Trimethylurea was only marginally nitrosated (less than 0.1 mumol/l gastric fluid) in cimetidine- or pentagastrin-stimulated animals. The low yield of NTMU at elevated pH, in the presence of substantial nitrite, suggests that the nitrate-reducing bacteria present in the porcine stomach did not catalyze trimethylurea nitrosation in vivo.

Several plant phenolics, one instant coffee, one instant decaffeinated coffee, one roasted coffee, one Japanese tea, one black Indian tea, and one Chinese tea were examined for their inhibitory properties on mutagenicity resulting from the nitrosation of methylurea. Mutagenicity was estimated as the number of his+ revertants per survivor of Salmonella typhimurium TA1535 which was exposed in suspension to the nitrosation mixtures and the modulating agents for 20 min. Tannic acid, gallic acid and chlorogenic acid suppressed the mutagenicity of the model nitrosation system at concentrations similar to or even lower than ascorbic acid. The three tested coffees and three tested teas exerted an inhibitory effect on the mutagenicity of the test system at doses at which they are consumed.

14C-N-(2-Diisopropylaminoethyl)-N-(4,6-dimethyl-2-dimethyl- 2-pyridyl)-N',N'-dimethylurea (I) was administered orally to dogs and rats and to rhesus, African green, and squirrel monkeys. Radioactivity was excreted mainly in the urine (60-76%) of all species except the rat (25%). The plasma t1/2 of I in the dog was 0.8 hr. Five urinary metabolites were identified by TLC, GLC, GS/MS, and NMR spectrometry: N,N-dimethyl-N'-(2-diisopropylaminoethyl)-N'-(4-hydroxy-methyl-6-methyl-2-pyridyl)urea (II), N,N-dimethyl-N'-(2-diisopropylaminoethyl-N'-(4-methyl-6-hydroxymethyl-2-pyridyl )urea (III), N,N-dimethyl-N'-(2-isopropylaminoethyl)-N'-(4,6-dimethyl-2-pyridyl)urea (IV), N-methyl-N'-(2-diisopropylaminoethyl)-N'-(4,6-dimethyl-2-pyridyl)urea (V), and N-(2-diisopropylaminoethyl)-N-(4,6-dimethyl-2-pyridyl)urea (VI). II was a major urinary metabolite in dog and II and IV were major metabolites in the squirrel monkey. II was present in relatively high concentrations in testes and stomach tissues of dogs, but not rats, treated with I, a finding that may be related to species differences in toxicity observed with I in these species.

Nitrosomethylurea (NMU) formation was measured radioactively in the stomach contents of rats fed 3H-methylurea and sodium nitrite, mostly in semi-synthetic diets. When methylurea and sodium nitrite were added to various diets, the NMU concentration, averaged over 1-4 hours after the food was presented, was 4.6 micrograms/kg stomach contents for low-protein, 2.7 for control semi-synthetic, 2.4 for high-fat, 1.26 for bran, 1.24 for high-protein and 0.54 for the commercial diet. With the low-protein diet, the amount of NMU after 1 hour was 36.6 micrograms, corresponding to 5.3% conversion of methylurea. The decrease in yield as protein content increased was attributed to buffering action and competition for nitrite by the protein, as well as to the effects of the latter on consistency. It may also be correlated with the observation that human gastric cancer is associated with high-starch low-protein diets. Nitrite in diet was more effective in producing NMU than the same nitrite concentration in drinking water, except with sodium nitrite concentrations less than or equal to 0.5 g/kg vehicle, where the position was reversed. Sodium ascorbate added to a semi-synthetic diet at a level of 2.9 g/kg inhibited NMU production by 50%.

The intragastric host-mediated assay (h.m.a.) was devised and carried out with a view to assessing the formation of direct mutagens in the gastrointestinal tract of mammals. The h.m.a. consists in the injection of nitrosable compounds, NaNO2 and cells of the yeast S. pombe, by gavage into the animals' stomachs and in the recovery of the target cells from the faeces for mutation-induction analysis. Methylurea was chosen as a model nitrosable compound, and the effects of nitrosation modulators such as ascorbic acid and thiocyanate were studied. Cimetidine, a drug nitrosable in vitro, was tested with the system. Positive results were obtained only at very large doses and in artificially produced low pH. The new host-mediated assay seems to be efficient in revealing the formation, in vivo, of direct, short-living mutagens.

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 degrees C, kcat has the values 5870, 85, 0.55, and 0.075 s-1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato-enzyme intermediate.

The formation of N-nitrosomethylurea (NMU) from methylurea (MU) and sodium nitrite in the guinea-pig stomach and the disappearance of NMU from the stomach were studied using a previously described method for NMU determination (Yamamoto et al. Fd Chem. Toxic. 1986, 24, 247). Guinea-pigs were used since they have only glandular stomachs and the pH of the gastric juice (1-2) is similar to that of humans. NMU was relatively stable in the isolated gastric contents of this species. When 2 mumol NMU was injected into the pylorus-ligated stomach of fasting guinea-pigs, about 50 and 37% of the NMU remained at 20 and 30 min, respectively. Some 19 and 42% remained 30 min after NMU was given orally by stomach tube to fasting and feeding guinea-pigs, respectively. NMU was detected in most blood samples irrespective of the administration procedure, but it disappeared rapidly from the blood after iv injection. Nitrite disappeared rapidly from the pylorus-ligated stomach, residual nitrite being less than 20% of the dose in 2.5 min. when 7.5 mumol MU and 15 mumol NaNO2 were co-injected into the ligated stomach, 3.1 mumol NMU was detected 10 min after the injection, followed by a gradual decrease. When MU and NaNO2 were given orally to the animals, 0.7-1.0 mumol NMU was detected in the stomach 10 min after the treatment. Thus NMU was shown to be formed readily in the stomach of the guinea-pig and to be absorbed from the stomach into the blood.

The N-nitrosocarbamates are potent mutagens and carcinogens, and have been synthesized under acid conditions that prevail in the human stomach. However, it has never been documented that nitrosocarbamates are actually formed in vivo in the stomach of any mammalian species. Using 14C-labeled carbaryl and carbofuran, attempts were made to isolate the nitroso derivatives from the stomach contents of rats and guinea pigs treated orally with the carbamate and sodium nitrite. Only trace quantities of nitrocarbamate were recovered from the rat stomach, whereas 0.5 to 2.0% of the carbamate doses were isolated as the nitroso derivative from the contents of the guinea pig stomach. The rather low apparent yields resulted in part from the instability of the nitrosocarbamates and from absorption of the carbamate and/or nitrosocarbamate from the stomach. Higher rates of synthesis were indicated by incubating the carbamates with sodium nitrite in the presence of the stomach contents at 37 degrees C for 15 min. About 30% nitrosation occurred with the guinea pig and about 0.5% with the rat. The difference was attributed to the pH of the gastric contents. For the rat, the pH ranged from 3 to 5; gastric contents of the guinea pig had a pH between 1 and 2. Since the pH of the human stomach is also in the pH 1-2 range, it is likely that nitrosation of carbamates in humans would be very similar to that in the guinea pig.

The objective of this study was to simulate in vitro at least some of the conditions that prevail in man during ingestion of nitrate and nitrosable compounds. Human saliva has been chosen because most chemicals ingested through food will interact with saliva. The nitrosation of methylurea was used as a model because the nitrosation products can be readily detected by their mutagenic (his+ revertants of S. typhimurium) and clastogenic (chromosome aberrations in CHO cells) properties. The results show that human saliva inhibits the formation of mutagenic and clastogenic nitrosation products when present during nitrosation. A 50% inhibition of mutagenicity results from the addition of a saliva sample diluted at 5% of the original concentration. In the test system used a similar inhibitory effect was obtained by 2.5 mM ascorbic acid or 2.0 mM chlorogenic acid. The main inhibitory agents seem to reside in a deproteinized fraction which was filtered through an ultrafilter UM2 (greater than 1000 MW). At strong acid levels (below pH 2) the saliva loses its inhibitory effect on the nitrosation of methylurea. The contribution of saliva to the inhibition of endogenous nitrosation within the oral cavity or stomach is discussed.

Permeability coefficients (P) measured at various penetrant concentrations (C) by the perturbation method can be plotted to distinguish simple diffusion, simple pore kinetics and simple carrier kinetics as follows: for simple diffusion, 1/P = constant; for a simple pore, 1/P = 1/Po + 1/Po[1/Kin + 1/Ko]C; for a simple carrier, 1/P = 1/Po + 1/Po[1/Kin + 1/Ko]C + 1/Po[1/(K3K4)] C2 where Po is the maximal permeability at zero penetrant concentration and the K's are combinations of kinetic constants defining each of the transport steps. (Kin and Ko are the half-saturation constant for zero-trans efflux and influx, respectively; K3 is the half-saturation constant for equilibrium exchange, and K4 is related to the mobility of the free carrier). In human erythrocytes, permeability coefficients for diethylene glycol were constant suggesting simple diffusion. For glucose, a plot of 1/P versus concentration was nonlinear indicating carrier kinetics. Plots of 1/P versus penetrant concentrations gave straight lines with positive slopes for urea in human and bovine erythrocytes and for methylurea in human red cells, indicating these penetrants follow simple pore kinetics or simple carrier kinetics in which K4 is very large.

The use of isotopic substitution to retard the oxidative metabolism of the gastric antisecretory agent N,N-dimethyl-N'-[2-(diisopropylamino)ethyl]-N'-(4,6-dimethyl-2-pyridyl)urea (1) and improve its antisecretory potency was examined. The pyridine ring methyl hydrogens of 1 were replaced with either deuterium or fluorine. The hexadeuterated analogue (12) was found to be approximately 2.1 times more potent than the protio form (1) as an inhibitor of gastric acid secretion stimulated by gastrin tetrapeptide. The hexafluoro analogue (11) was 0.4 times as potent as 1. A useful pyridine ring synthesis was developed to prepare the metabolites of 1, 10a (4-hydroxymethyl) and 10b (6-hydroxymethyl), and the hexafluoro analogue 11. These syntheses involved the condensation of 1,3-diketones with an appropriately N-substituted amidinoacetate.

The mineralization of several N-compounds, such as MMU, DMU, and HMT, as well as their respective nitrificide action, have been investigated. It has been proved that the amount of available formaldehyde, as well as its rate of release, are responsible for the specific action of these compounds on the N-mineralization process. Factors affecting the rate of release, namely molecular structure, solubility, and temperature, are discussed as well. Importance of these N-compounds as slow-release N-fertilizers based on a new molecular-microbiological approach is described.

The interactions of human albumin with urea, methyl-, N,N'-dimethyl-, ethyl-, N,N'-diethyl-, propyl-, and butylurea were studied by means of calorimetry and circular dichroism. It has been found that the enthalpies of interaction of the alkylureas with human serum albumin are distinctly different from those of urea. Thus, the transfer of the protein from water to aqueous urea solutions is accompanied by release of heat, i.e., the overall reaction is exothermic, whereas the transfer of the same protein of solutions of alkylureas is characterized by consumption of heat, i.e., the overall reaction is endothermic. By examining the far ultraviolet circular dichroism in behavior reflects the presence of the hydrophobic moiety in the urea molecule.

Naturally occurring para-aminobenzoic acid (PABA) inhibited the formation of N-methyl-N-nitrosourea (MNU) from a nitrosation mixture of N-methylurea and nitrite at pH 3. The suppressive effect of PABA on the formation of MNU is higher than that of ascorbic acid. The presence of the MNU was assayed by its mutagenicity in a higher plant, Arabidopsis thaliana. In addition, PABA markedly reduced the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine in A.thaliana, but had no or only a low inhibitory effect on the mutagenicity of preformed MNU and on the promutagen N-nitrosodimethylamine.

The formation of nitrite from nitrate was studied in potatoes incubated at room temperature. After 24 hours of incubation at 25 degrees, 112 ppm and 373 ppm of nitrite formed in homogenized cooked potatoes containing 284 ppm and 584 ppm of nitrate, respectively. In homogenized fresh potatoes incubated at 21 degrees and containing 284 ppm of nitrate, 103 ppm of nitrate formed within 24 hours. During the period of nitrite formation, nitrate levels decreased sharply, indicating that reduction of nitrate to nitrite occurred. Incubation at 2 degrees completely prevented nitrite formation in all cases. The formation of methylnitrosourea from added methylurea and nitrite was observed in potato incubated under simulated gastric conditions (37 degrees, pH 1.5). An ascorbate-nitrite mole ratio of 4 gave a 93% inhibition of methylnitrosourea formation. Ascorbate reacted directly with nitrite in potato incubated under identical conditions, yielding a 43% decrease in nitrite concentration. Ascorbate did not react with methylurea or the product, methylnitrosourea.

The metabolism of methylamine has been investigated in the rabbit and rat, and evidence has been presented to show that the metabolism of the compound leads to the formation and excretion of methylurea, a metabolite suspected, but never conclusively shown, to exist by previous investigators. Urinary methylurea, after the administration of methyl-14C-amine, was shown to have 14C activity at both the carbonyl and methyl portions (groups) of the molecule. 14C activity in the carbonyl group of methylurea was derived from the same metabolic CO2 pool that is used in the formation of urinary urea and respiratory carbon dioxide. Administration of methyl-14C-amine led to the formation of respiratory 14CO2, as noted by various investigators, and it was shown that during a 24-hr period the ratio of specific activity (urea/respiratory CO2) approached 1, in agreement with previous data reported by Mackenzie and du Vigneaud [J. Biol. Chem. 172, 353-354 (1948)]. In order to quantitatively investigate some aspects of metabolism of methylurea, a procedure was developed for the convenient synthesis of N-methyl-14C-carbonyl-urea. In a series of experiments, the urinary urea of the animals injected with N-methyl-14C-carbonyl-urea and N-methyl-14C-urea was examined for radioactivity. Only limited amounts of 14C activity were found in the carbonyl group of urea following administration of N-methyl-14C-urea. However, after the administration of N-methyl-14C-carbonyl-urea, urinary urea contained a 200-fold excess of 14C activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat kidney contains an enzyme which hydrolyzes NG-monomethyl-L-arginine to give rise to the formation of ornithine and N-methylurea. Confirmation of formation of these reaction products is carried out colorimetrically as well as radiochemically employing NG-monomethyl-L-[ornithine-14C(U)]arginine. This pattern of reaction products suggests that the enzyme responsible is an arginase type. However, the kidney enzyme is quite distinct from the hepatic arginase; commercial bovine hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is completely inactive toward NG-monomethyl-L-arginine. Among various rat tissues examined, the hydrolytic activity is the highest in kidney, followed by the activity in liver and pancreas.