The metabolism of n-octane in Fischer 344 rats

The urinary metabolites of n-octane in Fischer 344 rats given the hydrocarbon by gavage included 2-octanol, 3-octanol, 5-oxohexanoic acid, and 6-oxoheptanoic acid. The sex of the animals influenced the relative amounts of metabolites formed. Analyses were performed by gas-liquid chromatography (GC) and gas-liquid chromatography/mass spectrometry (GC/MS). This is the first reported finding of keto acids in hydrocarbon oxidative metabolism. No kidney damage was found as a result of n-octane dosing although the 2,2,4-trimethylpentane (iso-octane) isomer does cause kidney lesions in male rats.     

Lack of detectable metabolism for solubilized 2,3,4-trimethylpentane by rat kidney proximal tubules

Primary proximal tubule suspension cultures exposed to solubilized 2,3,4-trimethylpentane (2,3,4-TMP) resulted in a linear dose response, as determined by cellular lactate dehydrogenase leakage. The EC50 for 2,3,4-TMP was 16.3 mM. Metabolite analysis by gas chromatography/mass spectrometry of supernate and cell extracts from cultures exposed to 2,3,4-TMP (12.0 mM) failed to detect the presence of metabolites. Electron-microscopic examination of proximal tubules exposed to 2,3,4-TMP indicated ultrastructural changes that included increased mitochondrial swelling, increased vesiculation, decreased microvilli and pyknotic nuclei. This study indicates that kidney proximal tubules do not appear to metabolize 2,3,4-TMP.     

Studies on the metabolism of the antioxidant ethoxyquin, 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline in the rat

1. The metabolism of ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) in rat has been investigated. Urinary metabolites were identified by combined g.l.c.-mass spectrometry. 2. The major metabolic reaction was de-ethylation which gave rise to 6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline and an oxidation product, 2,2,4-trimethyl-6-quinolone. Other reactions were hydroxylation to four different hydroxylated metabolites and one dihydroxylated metabolite. A total of 95% of the dose (100 mg/kg) was accounted for.     

Urinary metabolites of 2,6-diisopropylnaphthalene in rats

Metabolism of 2,6-diisopropylnaphthalene (2,6-DIPN) in rats was found to proceed exclusively through oxidation of the isopropyl chain of the molecule. Five unconjugated metabolites were isolated from the urine and identified: 2,6-naphthalenedi(2-propan)-2-ol, 2-[6-(1-hydroxy-1-methyl)ethylnaphthalen-2-yl]-2-propionic acid, 2,6-naphthalenedi-2-propionic acid, 2-[6-(1-hydroxy-1-methyl)ethylnaphthalen-2-yl]-2-hydroxypropionic acid, and 2-[6-(1-hydroxy-1-methyl)ethylnaphthalen-2-yl]-1,2-propanediol, together with a small amount of the unchanged compound. The identification of these metabolites was made by means of mass, infrared, and nuclear magnetic resonance spectrometry. The presence of glucuronides of five metabolites described above was also suggested by thin-layer and gas-liquid chromatography of the extract that was obtained after hydrolysis by beta-glucuronidase. In addition, the quantitative determination of metabolites indicated that these five were the major urinary metabolites of 2,6-DIPN, and that the total urinary excretion of these metabolites in 24 hr after administration was about 23% of the dose.     

Urinary bivalent sulfur metabolites of phenanthrene excreted by the rat and guinea pig

After the administration of phenanthrene (50 mg/kg, ip) to young adult male rats and guinea pigs, a series of bivalent sulfur urinary metabolites were isolated and characterized by gas chromatography and gas chromatography-mass spectrometry. Seven methylthio metabolites were isolated from the neutral fraction of hydrolyzed rat urine, whereas only two were detected in guinea pig urine. The major methylthio metabolite excreted by each species was 9-hydroxy-10-methylthio-9, 10-dihydrophenanthrene. This was observed as a second-day metabolite in the rat, and its appearance was accompanied by 9-phenanthrol. In addition to the methylthio compounds, which were excreted predominantly as glucuronides, six acidic bivalent sulfur metabolites were isolated from hydrolyzed rat urine and identified by GC/MS; five were present in hydrolyzed guinea pig urine. The major acidic metabolite in hydrolyzed rat urine was the hydroxydihydromercapturic acid N-acetyl-S-(9-hydroxy-9, 10-dihydro-10-phenanthryl)-L-cysteine. The major acidic metabolite in guinea pig urine was the mercaptoacetic acid S-(9-hydroxy-9, 10-dihydro-10-phenanthryl)mercaptoacetic acid, but the hydroxydihydromercapturic acid was also present.     

Isolation and structure elucidation of the major photodegradation products of seratrodast

Photodegradation of seratrodast ([+/-]-7-(3,5,6-trimethyl-1,4-benzoquinone-2-yl)-7-phenyl-heptanoic acid, SD), a p-benzoquinone anti-asthmatic drug, has been investigated in different solutions. HPLC analysis showed that SD was degradated under UV irradiation at 254nm to afford three major products (SD1, SD2 and SD3) in methanol and in acetonitrile/H(2)O solutions, while only two of them (SD2 and SD3) could be detected in acetonitrile solution. Furthermore, SD1 was unstable even when the samples were protected from light, so the acetylated SD1 (Ace-SD1) together with SD2 and SD3 were isolated by semi-preparative reversed-phase HPLC. The structures were elucidated as 6-(2,5-diacetoxy-3,4,6-trimethyl-phenyl)-7-phenyl-hept-6-enoic acid (Ace-SD1), 7-phenyl-6-(2,4,5-trimethyl-3,6-dioxo-cyclohexa-1,4-dienyl)-hept-6-enoic acid (SD2) and 5-(5-hydroxy-4,6,7-trimethyl-2-phenyl-benzofuran-3-yl)-pentanoic acid (SD3) based on the spectral data of MS, UV spectrum, IR spectrum and NMR spectrum. Meanwhile, a possible route for the formation of these photoproducts was proposed, which might explain the generation procedure of SD1, SD2 and SD3 during UV irradiation.     

Accumulation and depuration of the synthetic antioxidant ethoxyquin in the muscle of Atlantic salmon (Salmo salar L.).

The biological fate of the fish feed additive, ethoxyquin (EQ) was examined in the muscle of Atlantic salmon during 12 weeks of feeding followed by a 2 weeks depuration period. Parent EQ (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline), quinone imine (2,6-dihydro-2,2,4-trimethyl-6-quinolone), de-ethylated EQ (6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline) and EQDM (EQ dimer or 1,8'-di(1,2-dihydro-6-ethoxy-2,2,4-trimethyl-quinoline) were found to be the ubiquitous metabolites of dietary EQ, with EQDM as a main metabolite. A rapid decrease in the level of EQ (2.4 days of half-life) was balanced by an increase in EQDM, giving an unchanged net sum following 2 weeks of depuration. The mandatory 14 days depuration period prior to slaughtering of farmed salmon in Norway was not sufficient for complete elimination of EQ-derived residuals. Post depuration, EQDM accounted for 99% of sum of the two compounds in all treatment groups; possible toxicological effects of EQDM are not known. The individual concentrations of EQ and EQDM and their sum are dependent on EQ level in the feed, consequently, their residual concentrations may be controlled. The theoretical amount of EQ and EQDM consumed in one meal of farmed salmon would be under the recommended ADI, provided that the fish were raised on feed with no more than 150 mg EQ/kg feed, which is the EU maximum limit for EQ in fish feed.     

Further structural analysis of urinary metabolites of suprofen in the rat

The urinary metabolites of 2-(4-(2-thienylcarbonyl)phenyl)propionic acid (suprofen, S) in rats were analyzed by radio-GC, GC/MS, or 1H NMR technique. Radio-GC analysis of trimethylsilylated materials after TLC separation of intact urine showed the presence of three radioactive peaks with the retention times corresponding to the authentic S, 2-(4-(2-thienylhydroxymethyl)phenyl)propionic acid, and 2-(4-carboxyphenyl)propionic acid. About 40% of the total radioactivity appearing in the 0-24-hr urine was accounted for by the three metabolites and their conjugates. The identification of these metabolites was confirmed by comparison of the MS spectra of urine, in which rats were administered an equimolar mixture of S and S[phenyl-d4], with those of synthetic standards. The labile metabolites of S, corresponding to about 32% of the total radioactivity appearing in the 0-24-hr urine, were isolated and purified by ether extraction from the fresh urine and GC/MS or HPLC. GC/MS of the methylated metabolite revealed the consistent presence of the ion peaks at m/z 304, 245, 217, and 141, indicative of a dimethylated product with monohydroxy group on the thiophene ring. Analysis of the 1H NMR spectrum demonstrated the metabolite to be 2-(4-(5-hydroxy-2-thienylcarbonyl)phenyl)propionic acid.     

Isolation and characterization of the urinary metabolites of arbaprostil in the male dog after intravenous administration

The profile of urinary metabolites of 3H-arbaprostil was characterized in the male dog after intravenous administration. The major metabolites were purified and their structures deduced by gas chromatography/mass spectrometry (GC/MS) studies after conversion to the methyl ester-methoxime-trimethylsilyl ether derivatives, aided by GC with simultaneous radioactivity monitoring. The identified metabolites accounted for 96% of the urinary excretion products. beta-Oxidation of the carboxy side-chain of arbaprostil to 15-methyl-2,3,4,5-tetranor PGE1, via the 15-methyl-2,3-dinor PGE2 intermediate, appeared to be the most significant metabolic pathway. In contrast to the rat, the following were observed in the dog: glucuronic acid conjugation of the 15-methyl-2,3,4,5-tetranor PGE, and PGA metabolites; detection of the 15-methyl-2,3-dinor PGE2 intermediate; absence of 19-hydroxyl-15-methyl-2,3,4,5-tetranor PGA, and PGB metabolites; oxidation at C-20; and excretion of some parent drug.     

Assessment of the genotoxic potential of unleaded gasoline and 2,2,4-trimethylpentane in human lymphoblasts in vitro

The potential of unleaded gasoline and 2,2,4-trimethylpentane to induce gene locus mutation and sister-chromatid exchange in human lymphoblasts in vitro was determined. Neither unleaded gasoline, at its maximum tolerated concentration in the medium, nor 2,2,4-trimethylpentane, at its limit of solubility, induced mutation at the thymidine kinase locus. Negative results were seen both in the presence and absence of a rat liver homogenate metabolizing system. Sister-chromatid exchange analyses of the cultures treated with unleaded gasoline and 2,2,4-trimethylpentane were also negative. Therefore the carcinogenicity and nephrotoxicity of unleaded gasoline and 2,2,4-trimethylpentane observed in vivo are not correlated with any marked genotoxicity in these in vitro tests.     

Synthesis and antiproliferative activity in vitro of novel 1,5-benzodiazepines. Part II

The reaction of 2,2,4-trimethyl-1H-2,3-dihydro-1,5-benzodiazepine (1) with cinnamoyl chloride leading to the formation of 1-cinnamoyl derivative 2 is described. Two novel benzodiazepines, 2,2,4-trimethyl-1H-2,3,4,5-tetrahydro-1,5-benzodiazepine (3) and 1-cinnamoyl-2,2,4-trimethyl-1H-2,3,4,5-tetrahydro-1,5-benzodiazepine (4), were synthesized by the reduction of 1 and 2 using NaBH4 in i-PrOH and two other derivatives 5 and 6 were obtained by reaction of 4 with equimolar and dimolar quantity of cinnamoyl chloride, respectively. The structures of 1-6 were confirmed by analytical and spectral data (IR, 1H NMR, and MS). 7-Carboxy-2,2,4-trimethyl-1H-2,3-dihydro-1,5-benzodiazepine (7) was synthesized and its crystals were subjected to X-ray analysis. Benzodiazepines 1-6 were evaluated for antiproliferative activity in vitro. Among the compounds tested, 4-6 exhibited cytotoxic activity against human cancer cell lines, namely SW707 (colon cancer), MCF-7 (breast cancer), A549 (lung cancer), and HCV29T (bladder cancer).     

Studies of the biliary excretion and metabolites of the antioxidant ethoxyquin, 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline in the rat

1. Biliary excretion and metabolites of ethoxyquin, and gastro-intestinal absorption of ethoxyquin were studied in rat. 2. An average of 28 and 36% of the dose of 14C following intragastric administration of [14C]ethoxyquin was recovered in the bile of bile-duct cannulated rats in 12 and 24 h, respectively. 3. By g.l.c.-mass spectrometry, 75 to 85% of the 14C excreted in the 12 h bile was identified as unchanged ethoxyquin, and the following metabolites were isolated and identified: 8-hydroxy-ethoxyquin, hydroxylated 8-hydroxy-ethoxyquin, 6-ethoxy-2,2,4-trimethyl-8-quinolone, hydroxylated 6-ethoxy-2,2,4-trimethyl-8-quinolone, 6-ethoxy-2,4-dimethylquinoline and 2,2,4-trimethyl-6-quinolone. 4. Three groups of rats were used in the biliary excretion experiments, and the effect of standardization of experimental conditions was demonstrated. Infusion of sodium taurocholate following bile-duct cannulation did not affect the biliary excretion kinetics of ethoxyquin. 5. Only about 3% of the radioactivity administered was absorbed from the gastrointestinal tract via the lymphatic pathway in thoracic-duct connulated rats within 24 h. It was concluded that ethoxyquin was absorbed primarily by the portal route.     

Isolation and characterization of urinary metabolites of arbaprostil in the rat

The urinary metabolites of arbaprostil-3H in the male rat were profiled, isolated, and purified. Their structures were deduced by gas chromatography/mass spectrometry (GC/MS) studies after conversion to the methyl ester-methoxime-trimethylsilyl ether derivatives, aided by GC with simultaneous radioactivity monitoring. The identified metabolites accounted for over 91% of the urinary excretion products. beta-oxidation of the carboxy side-chain of arbaprostil to 15-methyl-tetranor PGE1 appeared to be the most significant metabolic pathway. Conversion to the dinor A and B derivatives and further beta-oxidation of these to the 15-methyl-tetranor A and B metabolites also appeared to occur. C-19-hydroxylated tetranor A and B derivatives of arbaprostil-3H were excreted in the urine. No conjugated urinary metabolites were evident.     

Identification of metabolites of 3,4-methylenedioxymethamphetamine in rats

Liquid chromatography with electrochemical detection (LC/ECD) and gas chromatography/mass spectrometry (GC/MS) were used to identify metabolites of N-methyl-3,4-methylenedioxyamphetamine (MDMA) in samples of rat plasma and urine. Several potential metabolites, based on what is known about the metabolism of the desmethyl analog (i.e., MDA), were synthesized as standards to aid in the identification of the MDMA metabolites. MDA and N-methyl-1-(4-hydroxy-3-methoxy-phenyl)-2-aminopropane (3b) were identified in urine by HPLC and confirmed by GC/MS. 1-(4-Hydroxy-3-methyoxyphenyl)2-aminopropane, (3a), N-methyl-1-(3-hydroxy-4-methoxyphenyl)-2-aminopropane (2b) and 1-(3,4-dihydroxyphenyl)-2-aminopropane (4a) were tentatively identified by LC/ECD but insufficient sample size precluded confirmation by mass spectrometry. MDA was also identified in brain and plasma extracts. Because MDA is a metabolite of MDMA in humans, and because it has been speculated that the neurotoxic effects of MDA and MDMA may be due to a metabolite, the results of the present study may ultimately aid our understanding of the neurotoxic mechanism of these drugs of abuse.     

Metabolism and nephrotoxicity of indan in male Fischer 344 rats

Indan, a component of fuels, solvents, and varnishes, is metabolized in male Fischer 344 rats to 1-indanol, 2-indanol, 5-indanol, 1-indanone, 2-indanone, 2-hydroxy-1-indanone, cis-1,2-indandiol, and trans-1,2-indandiol. The metabolites were identified using the techniques of gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The rats treated with indan demonstrated the classic lesions of hydrocarbon-induced nephropathy. The kidney damage produced was less than that found for tetralin and other branched-chain acyclic hydrocarbons.     

Disposition of tilidine in a fatal poisoning in man

A fatal intoxication due to the ingestion of tilidine, a narcotic analgesic, in conjunction with ethanol, is described. Tilidine and its two active metabolites, nortilidine and bisnortilidine, were identified and quantitated in the biological fluids and tissues by thin-layer chromatography (TLC), gas-liquid chromatography with sensitive nitrogen-phosphorus detection (GLC/NPD) and gas-liquid chromatography with mass spectrometric detection (GC/MS). The toxicological results are compared with previously reported 14C-tilidine tissue distributions in rats following oral administration and limited tissue data in a previously reported human fatality. In the present case, the death was attributed to the combined central nervous system-depressing effects of ethanol and tilidine.     

Renal and hepatic lesions induced by 2,2,4-trimethylpentane

The renal changes produced by 2,2,4-trimethylpentane, a constituent of unleaded gasoline, are studied in Wistar rats. The compound was given at a dose of 2 ml/kg daily by gavage dissolved in corn oil (2:1) to nine Wistar Albino rats. Nine control male rats received an equivalent dose of corn oil. Six animals were housed in metabowls to allow the collection of urine for enzyme and urinanalysis. The remaining three animals of each group were housed in a normal animal cage. After 2 days of treatment, all the test rats were noted to have lost weight and were killed on this or on the subsequent day. Macroscopic examination of the livers and kidneys revealed no visible lesions on the kidney, but two of the rats had white, slightly raised patches on the liver. Microscopic examination demonstrated centrilobular and confluent necrosis, hydropic degeneration and vacuolation of hepatocytes. Microscopic examination of the kidneys indicated eosinophilic hyaline droplet accumulation in the cells of the tubules, and tubular dilation. Analysis of plasma alkaline phosphatase and aspartate transaminase activity revealed increases consistent with liver damage. Analysis of urinary N-acetyl-beta-glucosaminidase and alkaline phosphatase activity showed increases consistent with renal toxicity. An apparent increase in the amount of cellular debris in the urine was also found, when the sediment was examined microscopically. From this study, it would appear that 2,2,4-trimethylpentane possess hepatotoxic as well as nephrotoxic properties.     

Study on dermal toxicity and urinary metabolites of the new insect repellent N,N-diethylphenylacetamide in rabbits

Dermal toxicity of the new multi-insect repellent N,N-diethylphenylacetamide (DEPA) was studied in female rabbits. LD50 of DEPA was estimated to be 3505 mg/kg b.w. On daily topical application for 21 d at a dose of 50 mg/kg b.w., dermal irritancy score and blood chemistry changes indicated that the compound is non-irritant and non-toxic. N-Ethylphenylacetamide and conjugated phenylacetic acid were identified as the urinary metabolites of DEPA by gas-liquid chromatography.     

Studies on the Biliary Excretion and Metabolites of the Antioxidant Ethoxyquin, 6-Ethoxy-2,2,4-Trimethyl-1,2-Dihydroquinoline in the Rat

1. Biliary excretion and metabolites of ethoxyquin, and gastro-intestinal absorption of ethoxyquin were studied in rat.

2. An average of 28 and 36% of the dose of¹⁴C following intragastric administration of [¹⁴C]ethoxyquin was recovered in the bile of bile-duct cannulated rats in 12 and 24?h, respectively.

3. By g.l.c.-mass spectrometry, 75 to 85% of the ¹⁴C excreted in the 12?h bile was identified as unchanged ethoxyquin, and the following metabolites were isolated and identified: 8-hydroxy-ethoxyquin, hydroxylated 8-hydroxy-ethoxyquin, 6-ethoxy-2,2,4-trimethyl-8-quinolone, hydroxylated 6-ethoxy-2,2,4-trimethyl-8-quinolone, 6-ethoxy-2,4-dimethylquinoline and 2,2,4-trimethyl-6-quinolone.

4. Three groups of rats were used in the biliary excretion experiments, and the effect of standardization of experimental conditions was demonstrated. Infusion of sodium tauro-cholate following bile-duct cannulation did not affect the biliary excretion kinetics of ethoxyquin.

5. Only about 3% of the radioactivity administered was absorbed from the gastrointestinal tract via the lymphatic pathway in thoracic-duct cannulated rats within 24?h. It was concluded that ethoxyquin was absorbed primarily by the portal route.     

Characteristics of chemical binding to alpha 2u-globulin in vitro--evaluating structure-activity relationships.

alpha 2u-Globulin (alpha 2u) has been shown to accumulate in the kidneys of male rats treated with 2,2,4-trimethylpentane (TMP). 2,4,4-Trimethyl-2-pentanol (TMP-2-OH), a metabolite of TMP, is found reversibly bound to alpha 2u isolated from the kidneys of these treated rats. The objectives of the following study were to characterize the ability of [3H]TMP-2-OH to bind to alpha 2u in vitro and to determine whether other compounds that cause this protein to accumulate have the same binding characteristics. Although compounds that have been shown to cause the accumulation of alpha 2u in male rat kidneys compete in vitro with [3H]TMP-2-OH for binding to alpha 2u, they do so to varying degrees. The binding affinity (Kd) of the [3H]TMP-2-OH-alpha 2u complex was calculated to be on the order of 10(-7) M. The inhibition constant values (Ki) determined for d-limonene, 1,4-dichlorobenzene, and 2,5-dichlorophenol were all in the range 10(-4) M, whereas the Ki values for isophorone, 2,4,4- or 2,2,4-trimethyl-1-pentanol, and d-limonene oxide were determined to be in the range 10(-6) and 10(-7) M, respectively. TMP and 2,4,4- and 2,2,4-trimethylpentanoic acid did not compete for binding. This suggests that other factors, besides binding, are involved in the accumulation of alpha 2u. In this study the ability of a chemical to bind to alpha 2u was used as a measure of biological activity to assess structure-activity relationships among the chemicals tested and known to cause the accumulation of alpha 2u. The results so far suggest that binding is dependent on both hydrophobic interactions and hydrogen bonding.