Galactomyces fermentation filtrate prevents T helper 2-mediated reduction of filaggrin in an aryl hydrocarbon receptor-dependent manner.

BACKGROUND: The aryl hydrocarbon receptor (AhR) recognizes diverse small molecules such as dioxins, tryptophan photoproducts and phytochemicals. It also plays crucial roles in epidermal homeostasis by upregulating epidermal barrier proteins. In preliminary screening, we found that Galactomyces fermentation filtrate (GFF), a cosmetic compound, was capable of activating AhR. AIM: To examine whether GFF upregulates the expression of the filaggrin and loricrin genes, FLG and LOR, in an AhR-dependent manner. METHODS: The activation (cytoplasmic to nuclear translocation) of AhR was confirmed by immunofluorescence study and by upregulation of an AhR-specific marker, cytochrome P450-1A1 (CYP1A1). Gene expression levels were compared by quantitative reverse transcription PCR with or without GFF, interleukin (IL)-4 or IL-13 in normal human keratinocytes. AhR or control knockdown was carried out by transfection with AhR or control small interfering RNA. The protein expression of FLG and LOR was examined by immunohistochemistry using a three-dimensional epidermal equivalent treated with or without GFF or T helper (Th)2 cytokines. RESULTS: GFF induced the nuclear translocation of AhR with significant and dose-dependent upregulation of CYP1A1, FLG and LOR gene expression. The enhancing effects of GFF were abolished in AhR-knockdown keratinocytes. Th2 cytokines decreased expression of genes for FLG and LOR, and this expression was completely restored in the presence of GFF. The downregulated expression of the FLG gene with its restoration by GFF was also evident in the epidermal equivalent. GFF also upregulated the gene expression of genes encoding occludin, claudin-1 and 4, and kallikrein 5 and 7. CONCLUSIONS: Use of GFF is feasible to prevent the Th2-mediated reduction of FLG in an AhR-dependent fashion.

Metabolic disposition of the new fluoroquinolone antibacterial agent DW116 in rats.

The metabolic disposition of the new fluoroquinolone antibacterial agent DW116 has been studied in Sprague-Dawley rats. The compound was absorbed well and demonstrated excellent oral bioavailability. The plasma kinetic profiles were characterized by monoexponential elimination with an elimination half life of 3-4 hr. The apparent mean total clearance (CL(T)) and the volume of distribution (V(SS)) ranged from 221 +/- 55 to 274 +/- 27 ml/hr/kg and 1.0+/-0.1 to 1.5+/-0.2 l/kg, respectively, and were independent of dose between 4 and 20 mg/kg levels. The renal (CL(R)) clearance was 64.5 ml/hr/kg and the biliary (CL(B)) clearance was 33.8 ml/hr/kg. The combined value accounted for approximately one-half of the total clearance, indicating that the remaining one-half of the administered dose was eliminated via hepatic clearance. The major metabolite excreted in the bile was identified as the glucuromide ester of parent drug using base-hydrolysis of the conjugate metabolite followed by co-HPLC with standard compound, 19F-NMR and LC-MS methods. The mean urinary recoveries of free and total (free plus glucuronide ester) DW116 were 28.6 +/- 2.7% and 36.4 +/- 1.8% of the administered dose and the corresponding biliary recoveries were 14.4 +/- 5.5% and 37.0 +/- 7.6%, respectively. The mass balance study after a single (100mg/kg) oral administration of 14C-DW116 indicated complete recovery of radioactivity over a 7-day period, accounting for approximately 60-70% in feces and 30-40% in urine. 14C-DW116 extensively distributed during a prolonged process into all tissues with a rather slower penetration into the brain, lung, and muscle. The compound also readily crossed the placenta and was transferred to the fetus.

Synthesis and biological activity of ketomethylene-containing nonapeptide analogues of snake venom angiotensin converting enzyme inhibitors.

Two ketomethylene-containing nonapeptide analogues were synthesized to determine if ketomethylene analogues of the nonapeptide venom inhibitors of angiotensin converting enzyme (ACE) would have oral ACE inhibition activity. Both ketomethylene-containing nonapeptides 18 and 19 were potent inhibitors of rabbit lung ACE with I50s of 3.4 and 8.0 nM, respectively, compared to 340 nM for their parent nonapeptide and 450 nM for captopril. Peptide 18 was rapidly cleaved by trypsin, but 19 was reasonably stable to all enzyme degradation systems tested with maximum degradation of 50% by pepsin in 3 h. Both 18 and 19 when given iv to normotensive rats were between 3 and 10 times more potent than captopril in inhibiting an angiotensin I induced blood pressure increase. Peptide 19 showed no ACE inhibition activity in unanesthetized normotensive rats when administered orally at doses of 10 or 100 mg/kg. Experiments were conducted to determine whether 19 is adsorbed from the gastrointestinal track following oral administration. These experiments indicated that 19 is adsorbed. It is concluded that the lack of oral activity of 19 is probably due to its rapid excretion, probably into the bile.

Synthesis and biological activity of pentapeptide analogues of the potent angiotensin converting enzyme inhibitor 5(S)-benzamido-4-oxo-6-phenylhexanoyl-L-proline.

Two pentapeptide analogues of the ketomethylene-containing angiotensin converting enzyme (ACE) inhibitor 5(S)-benzamido-4-oxo-6-phenylhexanoyl-L-proline (1) were synthesized and evaluated as ACE inhibitors and antihypertensive agents. Compounds 14 and 15 were very potent ACE inhibitors with I50 values of 7.0 and 3.0 nM, respectively, compared to an I50 value of 70 nM for 1. Neither 14 nor 15 showed significant blood pressure lowering activity in renal hypertensive rats. Investigations conducted on a tritiated analogue of 14 showed that 70% of an oral dose of this compound is absorbed but is rapidly excreted from the blood with a half life of 24 min. Thin-layer chromatography of bile and urine contents in rats given tritiated 14 orally showed that it is excreted in greater than 90% unchanged form. This implies that a ketomethylene linkage can stabilize peptide amide linkages adjacent to it to peptidase degradation.

Absorption, metabolism, and excretion studies of carbon 14- and tritium-labeled derivatives of a ketomethylene containing tripeptide.

Tritium and Carbon 14 analogs of the angiotensin converting enzyme inhibitor ketoACE were synthesized and their oral absorption, metabolism and excretion in rats were investigated. KetoACE, a ketomethylene analog of the tripeptide Bz-Phe-Gly-Pro, was slowly absorbed at a 35% level upon oral administration. It is rapidly eliminated from the blood with a half-life of about 10 minutes. Its excretion is primarily via the bile duct and it is excreted as 80% unchanged drug. The only identified metabolite consisting of 5-10% of the excreted radioactivity was determined to be the reduced ketoACE in which the ketone group was reduced to a hydroxyl.

Metabolic and toxicologic study of an artificial sweetener, oxime V.

Metabolic disposition and subchronic oral toxicologic studies were conducted on a new synthetic sweetener, Oxime V. Based on radioactivity assay, the compound was readily absorbed and metabolized. Excretion was nearly quantitative 48 hours after dosing the rat, dog, and rhesus monkey. The major metabolites were formed by oxidation and reduction of the cyclohexadiene ring, oxidation of the aldoxime and dimethyl ether moieties followed by conjugation with glycine, thiomethylation of the ring, and O-glucuronidation of the aldoxime. A two-month feeding study was conducted with male adult rats. The average consumption of Oxime V was 396.5 mg/kg per day by rats fed a diet containing 0.6% of the test compound. No treatment related histopathologic lesion was observed in the liver, kidney, spleen, and testes. The liver weight relative to the body weight and serum bilirubin level were increased.

Metabolic disposition study of chlorinated hydrocarbons in rats and mice.

Chlorinated hydrocarbons found in a bioassay to be carcinogenic to both B6C3F1 mice and Osborne-Mendel rats (1,2-dichloroethane), carcinogenic only to mice (1,1,2-trichloroethane, 1,1,2,2-tetrachloroethane, hexachloroethane, trichloroethylene, and tetrachloroethylene), and noncarcinogenic to either species (1,1-dichloroethane and 1,1,1-trichloroethane) were used to investigate the biochemical bases for tumorigenesis. Studies were conducted after chronic oral dosing of adult mice and rats with the MTD and 1/4 MTD of each compound. The extent to which the compounds were metabolized in 48 hr, hepatic protein binding, and urinary metabolite patterns were examined. Metabolism of the compounds (mmoles per kg body weight) was 1.7 to 10 times greater in mice than in rats. Hepatic protein binding (nanomole equivalents bound to 1 mg of liver protein) was 1.2 to 8.3 times higher in mice than in rats except for 1,2-dichloroethane and 1,1,1-trichloroethane. The noncarcinogens 1,1-dichloroethane and 1,1,1-trichloroethane exhibited 2 to 18 times more binding in mice than did the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethane. Urinary metabolite patterns of the compounds were similar in both species. The biochemical parameters measured provided no clue to differentiate the carcinogens from the noncarcinogens.

Pyrithione: plasma metabolite in man.

2-Methylsulfonylpyridine (2-MSP) has been identified as the terminal plasma pyrithione metabolite in rats, rabbits, dogs, and monkeys (W. B. Gibson, A. R. Jeffcoat, P. D. Rodriguez, T. S. Turan, P. F. Hughes, and M. E. Twine, Toxicol. Appl. Pharmacol. 62, 237-250 (1982); C. Mitoma, T. Steeger, J. Rogers, D. Thomas, and J. H. Wedig, Fundam. Appl. Toxicol. 3, 256-263 (1983]. This metabolite was detected in the systemic circulation of humans involved in the chemical manufacturing process. This confirms that the terminal pyrithione metabolite in plasma is identical among rat, rabbit, dog, monkey, and man.

Reproductive toxicological evaluation of Omadine MDS.

Reproductive toxicology studies with Omadine MDS were conducted using rats and rabbits, and plasma levels of the metabolite, 2-methylsulfonylpyridine (2-MSP), were assayed. In phase I (treated males, untreated females) of the fertility study, the no-effect level, for oral dosing, was 3.0 mg/kg; 7.5 mg/kg caused a decrease in male weight gain. In phase II (treated females, untreated males), the no-effect oral dose level was 1.0 mg/kg. At 3.0 mg/kg, decreases were seen in maternal body weight gain, the fertility index, and in total implantations and viable embryos at the 13-day uterine examination. Severe maternal toxicity including impaired motor function of limbs, decrease in weight gain, and mortality occurred at 7.5 mg/kg. In the perinatal/postnatal study, the no-effect level was 3.0 mg/kg. At 7.5 mg/kg, mortality was high, with a majority of animals dying during mid lactation. In the rat teratology study, the high dosage level, 30 mg/kg, dermally administered on Gestation Days 6 through 15 caused slight maternal toxicity, whereas 10 mg/kg produced none. These dosage levels corresponded to peak plasma levels on Gestation Day 10 of 470 and 1950 ng/ml 2-MSP at the 10- and 30-mg/kg dosage levels, respectively. In the rabbit, 5 mg/kg topically applied on Gestation Days 6 through 18 produced slight maternal toxicity while 1.5 mg/kg produced no maternal effects. The plasma levels of 2-MSP peaked on Days 12 and 15 of gestation. At 1.5 mg/kg these levels were 155 and 148 ng/ml; at 5.0 mg/kg, levels were 513 and 573 ng/ml. There was no teratogenic response seen in either species.

Metabolic disposition of pyrithiones.

The urinary pattern of pyrithione metabolites in urine of the rat, rabbit and rhesus monkey was similar to that of the swine after iv. administration of sodium pyrithione (Sodium Omadine) and the magnesium sulfate adduct of 2,2'-dithio-bis(pyridine-1-oxide), (Omadine MDS). The major metabolite accounting for 80% or more of the metabolites in urine was the S-glucuronide of 2-mercaptopyridine-N-oxide. After Omadine MDS administration, three transient metabolites and one persistent metabolite were observed in the plasma. The transient metabolites were tentatively identified as 2-methylthiopyridine-N-oxide, 2-methylsulfinylpyridine and 2-methylsulfinylpyridine-N-oxide. 2-Methylsulfonylpyridine was the only metabolite observed in the plasma 16 hr after Omadine administration. This metabolite could be detected 14 days after rats were treated repeatedly with a shampoo formulation containing Omadine MDS.

Fate of water-insoluble and water-soluble dichlorobenzidine-based pigments in Fisher 344 rats.

The fate of two water-insoluble (WI) dichlorobenzidine-based pigments, chlorodiane blue (CDB) and pigment yellow 12 (PY12), and of their sulfonated water-soluble (WS) analogs was studied in male Fischer 344 rats. Water-soluble analogs of chlorodiane blue and pigment yellow 12 were synthesized in order to study the effect of water solubility on the absorption and metabolism of dichlorobenzidine-based pigments. [14C]WI-CBD, [14C]WI-PY12, and the water-soluble analogs [14C]WS-CDB and [14C]WS-PY12 were administered by gastric intubation or dermal application at doses of 1.24-2.65 mumol/kg. Neither [14C]WI-CDB nor [14C]WI-[Y12 could be detected in any tissue at time points up to 1 d. The entire dose was accounted for in the feces after oral administration, and at the application site after dermal administration. Water-insoluble CDB is a component of a photoconductor (Weaver, 1981). Approximately 4.1% of [14C]CDB was observed and located primarily in the urine and liver after oral administration, but no detectable amount was absorbed after dermal application. Metabolites of [14C]WS-CDB identified in the urine were 3,3'-dichlorobenzidine diacetate, 3.3'-dichlorobenzidine and its glucuronide conjugate, and 3,3'-dichlorobenzidine monacetate and its glucuronide conjugate. Only 0.02% of [14C]WS-PY12 was absorbed after oral administration. Thus, some degree of water solubility was prerequisite for even a small amount of absorption or metabolism in vivo.

Subchronic inhalation toxicity of 1,3,5-trichlorobenzene.

Male and female rats were exposed to 0, 10, 100 or 1000 mg/m3 of 1,3,5-trichlorobenzene vapors for 6 hours daily, 5 days a week, for up to 13 weeks. After 4 and 13 weeks of exposure, animals were sacrificed and examined for changes in blood, clinical chemistry, internal organs, and tissues resulting from the 1,3,5-trichlorobenzene treatment. No treatment-related effects on the blood and clinical chemistry were evident. The only effects that were considered treatment-related were a squamous metaplasia and hyperplasia in the respiratory epithelium in the nasal passages of high-dose rats and the increased incidence of dried red material on the faces of these rats during exposures compared with other groups.

Evaluation of hepatocytes isolated by a nonperfusion technique in a prescreen for cytotoxicity.

Twenty-three chemicals, differing widely in cytotoxic (hepatotoxic) potency in vivo, were examined to determine their ability to release glutamic-oxaloacetic transaminase (GOT) from hepatocytes isolated by a nonperfusion method from rat liver. The test chemicals were carbon tetrachloride, chloroform, 1,1,2- and 1,1,1-trichloroethane, six bromobenzene analogs, tri-n-butyl tin, chlorpromazine, tetracycline, halothane, phenobarbital, L-ethionine, acetaminophen, thioacetamide, allyl alcohol, ethanol, ascorbic acid, dimethyl sulfoxide, and acetone. In all but two cases--thioacetamide and allyl alcohol--there was a good correspondence between chemicals active in the assay as now performed and those that elevate serum transaminase and cause liver injury on short-term exposure in vivo. These results indicate that with further effort it may be possible to develop an effective, inexpensive, and rapid prescreen to identify drugs and environmental chemicals that are potentially cytotoxic to animals and humans.

Metabolic disposition of chlorambucil in rats.

1. The metabolic dispositionof chlorambucil, 4-p-(di-2-chloroethyl)aminophenylbutyric acid, was studied in the rat. 2. After oral administration of [14C]chlorambucil to rats, plasma, liver, and kidney showed the highest concentration of 14C. After intravenous administration, plasma and kidney were heavily labelled. Although plasma contained as much as 10% of the administered dose in the first few hours after administration, the level decreased to 1% by 24 h. Elimination of radioactivity was mainly through the kidney. 3. Ten metabolites were isolated and characterized by mass spectrometry. Most metabolites had undergone oxidation on the butyric acid side-chain to form phenylacetic acid and benzoic acid derivatives. Spontaneous degradation products of [14C]chlorambucil were analysed and compared with the metabolites.

Metabolic disposition of cytembena in rats and dogs.

1. The metabolic disposition of cytembena (sodium cis-3-p-methoxybenzoyl-3-bromoacrylate)labelled at both carbonyl carbon atoms was studied in rats and dogs. 2. Rats excreted over 70% and dogs excreted over 50% of the radioactive dose in 24 h. Most of the radioactivity was found in urine. Kidney retained the highest level of radioactivity after 24 h. Renal cortex was responsible for the observed high retention. Both species exhibited similar tissue distribution and urinary metabolic patterns. 3. Cytembena was metabolized by demethylation catalysed by microsomes and by debromination and double bond saturation catalysed by the 100,000 g supernatant fraction in the presence of glutathione and NADPH. Tentative structures of the metabolites identified by mass spectrometry and the probable metabolic pathway of cytembena are presented,

Influence of fasting on the absorption and effects of delta9-tetrahydrocannabinol after oral administration in sesame oil.

Tissue levels of 3H were higher 2 hr after oral administration of 3H-delta9-THC (10 mg/kg in sesame oil) to male Fischer rats in the morning compared with treatment in the afternoon. A corresponding reduction in potency was seen for the impairing effect of delta9-THC on performance of a conditioned avoidance response (CAR). The hypothesis that these effects were related to the interval between feeding (which normally occurs during the night in the nocturnal rat) and drug administration was supported when they were mimicked in overnight fasted and ad lib fed rats. Food deprivation decreased the rate of gastrointestinal absorption of 14C-delta9-THC in sesame oil. Peak plasma levels of 14C occurred 2-4 hr after administration in fed rats compared with 8 hr in fasted rats. When tested 2 hr after oral administration, delta9-THC caused significantly greater impairment of CAR performance in fed than fasted rats, whereas the opposite was found after 8 hr. Extraction and subsequent thin layer chromatography of plasma and brain from fed and fasted rats sacrificed 2 or 8 hr after oral administration of 10 mg/kg 14C-delta9-THC showed that brain levels of 11-hydroxy-delta9-THC rather than delta9-THC were correlated with the behavioral effect.

Rauscher virus-induced reticulum-cell sarcomas: Their growth in vitro and erythropoietic differentiation.

A transplantable reticulum-cell sarcoma induced by Rauscher virus (RV) in (C57BL/6 X DBA/2)F1 (BDF1) mice was grown in tissue culture. Four separate cell lines were established, all of which grew predominantly in suspension. The doubling time of the cells from these cultures ranged from 17 to 32 h. Each culture continued to replicate RV, as indicated by the infectivity in newborn mice of all fluids tested up to the 75th passage. Since the morphological appearance of the cells in vitro was consistent with that of proerythroblasts, all cultures were tested for their ability to differentiate along the erythrocytic line under the influence of dimethylsulphoxide (DMSO). One of the cultures produced small quantities of haemoglobin independently of DMSO. Another was shown to produce haemoglobin, as well as to take up 59Fe and incorporate it into haem, only in the presence of DMSO. The 2 remaining cultures failed to produce haemoglobin, either spontaneously or in the presence of DMSO. Cells from each of the RV-induced cultures, when inoculated back into BDF1 mice, induced typical reticulum-cell sarcomas, without in vivo evidence of erythroid differentiation. In contrast, 2 morphologically identical but non-infectious cell lines derived from Friend virus-induced reticulum-cell sarcomas did not show erythroid differentiation in vivo or in vitro, either in the absence or presence of DMSO.