3-NOP: ADME studies in rats and ruminating animals.

3-NOP (3-nitrooxypropanol) reduces enteric methane formation in ruminants. A series of ADME studies in rats, lactating goats and beef cattle was performed. 3-NOP was entirely absorbed from the GIT of rats: approximately 75% of the administered 3-NOP was eliminated as carbon dioxide via exhalation and approximately 20% were excreted via urine. 3-NOP is oxidized to 3-nitrooxypropionic acid (NOPA) which is then hydrolyzed to 3-hydroxypropionic acid (HPA) and inorganic nitrate, the major rat plasma metabolites. NOPA is also a plasma metabolite in beef. The metabolism of 3-NOP is fast as indicated by the negligible amounts of 3-NOP found in rat plasma 2 h after dosing. HPA is a naturally occurring metabolite. It is either metabolized into carbon dioxide and acetyl-CoA or into propanoyl-CoA, the latter serves as substrate for gluconeogenesis. Gluconeogenesis is very prominent in lactating ruminants which use propanoyl-CoA as their main carbon source. Thus, the formation of lactose from 3-NOP by lactating goats is not unexpected. Lactose was the major metabolite of 3-NOP in the aqueous phase of milk. The incorporation of 3-NOP into endogenous metabolism makes it difficult to derive a marker residue, however, conservative risk assessment could be based on the measured radioactivity in tissues.

3-NOP: Mutagenicity and genotoxicity assessment.

3-NOP (3-nitroxy-propanol) is a new development compound which reduces methane emission from ruminating animals. For registration purposes with emphasis on EU and North America data requirements, mutagenic and genotoxic potential was assessed following OECD protocols and respective guidance documents. 3-NOP mutagenicity and genotoxicity testing raised no flags with regard to these endpoints. In silico assessment of 3-NOP and its major plasma metabolite NOPA (3-nitroxy-propionic acid) were predicted negative with regard to the bacterial reverse mutation (Ames) test. Ames test, mouse lymphoma assay, in vitro micronucleus test, and the oral in vivo micronucleus test using rat bone marrow were all negative. Exposure of the rat bone marrow was verified by the presence of 3-NOP and its metabolites NOPA and HPA (3-hydroxy-propionic acid) a naturally occurring substance in mammals) in plasma following oral dosing. It is therefore concluded that 3-NOP and its metabolites pose no mutagenic and genotoxic potential.

Coincubation of tissue slices, a new way to study metabolic cooperation between organs: hepatorenal cooperation in the biotransformation of CGP 47 969 A.

INTRODUCTION: There is limited information on in vitro/ex vivo tools to be used for studying interorgan metabolic cooperation. We report here the use of the tissue slice technique for this purpose. METHODS: Rat liver and kidney slices were used to study metabolic cooperation for the metabolism of CGP 47 969, a potential anti-inflammatory compound which in vivo is extensively conjugated with glutathione and subsequently degraded via the mercapturic acid pathway. RESULTS: Upon incubation with liver slices, CGP 47 969 was extensively conjugated with GSH whilst degradation of the GSH conjugate was moderate. Upon incubation with kidney slices, conjugation of CGP 47 969 with GSH was moderate but degradation of the GSH conjugate was complete. Upon coincubation of CGP 47 969 with liver and kidney slices, both conjugation with GSH and its subsequent degradation were almost complete. Thus, coincubation of liver and kidney slices permitted the efficient in vitro reproduction of the complete biotransformation of CGP 47 969 via its GSH conjugate to the ultimate mercapturic acid metabolite in a one step procedure. DISCUSSION: This novel slice coincubation culture could serve as an in vitro model for interorgan cooperation in multistep metabolic processing.

Subchronic toxicity study with ethylene-bis-(oxyethylene)-bis-(3-tert-butyl-4-hydroxy-5-methylhydro cin namate) in the cynomolgus monkey: lack of stimulation of the pituitary-thyroid-liver axis.

To evaluate the toxicological profile of the phenolic antioxidant ethylene-bis-(oxyethylene)-bis-(3-tert-butyl-4-hydroxy-5-methyl- hydrocinnamate) (EOC) in a non-human primate, male cynomolgus monkeys (Macaca fascicularis) were treated for 4 weeks by oral administration of 0, 200, or 1000 mg/kg body weight/day. Special attention was directed to parameters of the pituitary-thyroid-liver axis. Moderately increased liver weights and minimal to moderate hepatocellular hypertrophy were observed in treated animals. Otherwise, no treatment-related changes were detected in hematological, clinical chemistry, or urinalysis parameters or upon histopathological examination. Except for a slight induction of microsomal testosterone 16beta-hydroxylation, liver xenobiotic-metabolising enzyme activities and peroxisomal fatty acid beta-oxidation remained unchanged. Likewise, serum levels of thyroid stimulating hormone, thyroxine, 3,3',5-triiodothyronine and 3,3',5'-triiodothyronine as well as 5'-monodeiodinase type 1 mRNA levels in the liver, heart, cerebral cortex, and thyroid were found unchanged. The results demonstrate that, in the Cynomolgus monkey, EOC is only a very weak inducer of liver xenobiotic-metabolizing enzymes and has no effect on thyroid function. In contrast, upon feeding rats at dose levels up to 1000 ppm (equivalent to between 50 and 100 mg/kg body weight/day), EOC has been identified as a strong phenobarbital- and peroxisome proliferator-type inducer of hepatic xenobiotic-metabolizing enzymes, interfering with thyroid hormone homeostasis, causing thyroid follicular hypertrophy, and, upon chronic treatment, inducing thyroid gland follicular cell tumors (Thomas et al., 1995. In Toxicology of Industrial Compounds, pp. 319-339. Taylor and Francis). Thus, the results of this study with EOC in the cynomolgus monkey show that effects of xenobiotics on the pituitary-thyroid-liver axis as frequently observed in rodents can not necessarily be extrapolated to primates including man.

4,5-bis(4-fluoroanilino)phthalimide: A selective inhibitor of the epidermal growth factor receptor signal transduction pathway with potent in vivo antitumor activity.

Deregulated signal transduction via the epidermal growth factor (EGF) receptor family of tyrosine protein kinase growth factor receptors is associated with proliferative diseases such as cancer and psoriasis. In an attempt to selectively block signal transduction from the EGF receptor, we have synthesized a new class of dianilino-phthalimide tyrosine protein kinase inhibitors with selectivity for the EGF receptor tyrosine protein kinase. 4, 5-Dianilino-phthalimide (DAPH 1) was metabolized in vitro by mouse liver fractions and in vivo. The major metabolite has been identified as 4-(4-hydroxyanilino)-5-anilino-phthalimide. To specifically block this biotransformation (hydroxylation), we have synthesized 4,5-bis(4-fluoroanilino)phthalimide (DAPH 2), a potent and selective EGF receptor tyrosine protein kinase inhibitor. DAPH 2 inhibits the EGF receptor and protein kinase C beta2 enzymes with equal potency. In cells, DAPH 2 inhibits signal output from the EGF receptor, but not from other classes of receptor protein tyrosine kinases, such as the platelet-derived growth factor receptor, fibroblast growth factor receptor, insulin-like growth factor I receptor, and insulin receptor. Selective antitumor activity was demonstrated in vivo at well-tolerated doses in mice. This publication describes the biological profile of DAPH 2 and investigates its cellular and in vivo mechanism of action.

Ring cleavage and degradative pathway of cyanuric acid in bacteria.

The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.

Qualitative analysis of waste-water from ametryne production.

s-Triazines in waste-water from the synthesis of ametryne (2-(ethylamino)-4-[(l-methylethyl) amino]-6-(methylthio)-1,3,5-triazine) were tentatively identified co-chromatographically by HPLC and by UV-spectra. Alkylated s-triazines (e.g. hydroxyametryne, 4-(ethylamino)-6-(methylthio)-1,3,5-triazine-2(1H)-one, and N-ethylammelide) were isolated by preparative chromatography on a reversed phase support, and were identified by mass spectrometry. Putative cyanuric acid was desalted on activated charcoal and its identity confirmed as the silylated derivative by GLC.

Biosynthesis of proline in Pseudomonas aeruginosa. Properties of gamma-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase.

gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.