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Biomedical subjects

T C Orton

Publications and source records attributed to T C Orton.

At least 37 records · Page 2Linked to original sources
PubMed1986

Comparative pharmacokinetics and subacute toxicity of di(2-ethylhexyl) phthalate (DEHP) in rats and marmosets: extrapolation of effects in rodents to man.

Certain phthalate esters and hypolipidemic agents are known to induce morphological and biochemical changes in the liver of rodents, which have been associated with an increased incidence of hepatocellular tumors in these species. There is evidence that hypolipidemic agents do not induce these effects in either subhuman primates or man. The oral and intraperitoneal administration of di(2-ethylhexyl) phthalate (DEHP) to the marmoset monkey at doses up to 5 mmole DEHP/kg body weight/day for 14 days did not induce morphological or biochemical changes in the liver or testis comparable with those obtained in rats given the same amount of DEHP. In the marmoset, the excretion profile of [14C]-DEHP following oral, IP, and IV administration and the lower tissue levels of radioactivity demonstrated a considerably reduced absorption in this species compared to the rat. The urinary metabolite pattern in the marmoset was in many respects qualitatively similar to but quantitatively different from that in the rat; the marmoset excreted principally conjugated metabolites derived from omega- 1 oxidation. The pharmacokinetic differences between these two species indicate that the tissues of the marmoset are exposed to a level of DEHP metabolites equivalent to the complete absorption of a dose of Ca. 0.1 to 0.25 mmole DEHP/kg body weight/day without significant toxicological effects. These exposure levels are at least 100-fold greater than the worst estimates of incidental human exposure (ca. 0.0015 mmole/kg/day). They are comparable with the human therapeutic dose of many hypolipidemic drugs (ca. 0.15 mmole/kg/day), a dose at which it is claimed that there is an absence of morphological or biochemical changes to human or subhuman primate liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Practolol: aspects of its metabolism and ocular binding in the hamster.

The metabolism and ocular binding of practolol were investigated after oral administration of 14C-practolol to hamsters treated with three modifiers of mixed-function oxidase activity: piperonyl butoxide, cobalt chloride or phenobarbitone. The major urinary metabolites of practolol were 3-hydroxypractolol and polar metabolites which included glucuronide conjugates. A number of unidentified metabolites constituted a minor portion of urinary radioactivity. Each pretreatment modified both the urinary excretion pattern (0-24 h) of practolol and its metabolites and also the metabolite profile of eye extracts 24 h after an oral dose. None of the modifiers of mixed-function oxidase activity had any significant effect on the ocular binding (both extractable and non-extractable components) of practolol and its metabolites. The results indicated that the non-extractable component was neither practolol nor 3-hydroxypractolol.

Animals↗
PubMed1985

Practolol and its metabolites: tissue localization and retention in the hamster.

The disposition of [14C]practolol and its tissue association in the male Syrian hamster were investigated after p.o. administration. After an acute dose of 400 mg/kg, there was a marked persistence in the eye of total radioactivity (primarily practolol; approximate T 1/2 = 7 days) and nonextractable radioactivity which showed minimal decay over 7 days. Nonextractable radioactivity was also found in other tissues (liver, skin, small intestine and skeletal muscle examined). The level of total and nonextractable radioactivity was linearly related to dose (800 mg/kg highest dose examined). There was a marked accumulation of total and nonextractable radioactivity in the eye (7- and 14-fold, respectively) and skin (6- and 7-fold) after daily administration of 400 mg/kg for 22 days. The association of practolol metabolites with tissue macromolecules might explain the immunological changes in patients with the practolol-induced oculomucocutaneous syndrome. The adverse effects of practolol in humans have not yet been reproduced in any laboratory species and so the results must be interpreted with caution in relation to the toxicity of compound.

Animals↗
PubMed1984

Clobuzarit: species differences in the morphological and biochemical response of the liver following chronic administration.

Clobuzarit is structurally related to a number of hypolipidemic agents which produce characteristic changes in rodent liver morphology and biochemistry. Liver effects were determined in a number of rodents, the albino rat, C57BL/10J mouse, and Syrian hamster, and in other mammals, the beagle dog and marmoset monkey, following chronic (14-day) oral administration of clobuzarit at two dose levels. Expected (peak) serum levels of clobuzarit were achieved in all species except the marmoset in which levels were approximately half those predicted. Dose-dependent enlargement of rat and mouse liver cells (assessed qualitatively by light microscopy) was observed. This finding was accompanied by proliferation of peroxisomes and smooth endoplasmic reticulum (assessed by electron microscopy and enzymatically). In the rat and mouse, the fatty acid oxidation enzyme systems of peroxisomes and endoplasmic reticulum were markedly induced. The hamster showed no increase in liver cell size but there was a limited proliferation of peroxisomes (indicated by electron microscopy and enzymatically) and an increase in endoplasmic reticulum fatty acid oxidase. No proliferative effects were observed in the liver of the dog or marmoset. Lowering of plasma triglycerides and cholesterol was observed in the rat only. The differences in the morphological and biochemical responses to clobuzarit among the species used in this toxicity testing clearly illustrate the problems of predicting effects in man from animal data.

Animals↗
PubMed1984

Differentation of rat embryo cells in culture: response following acute maternal exposure to teratogens and non-teratogens.

An in vivo-in vitro test system with high sensitivity to teratogens has been developed and validated. A single acute intra-peritoneal injection of teratogens (18) and non-teratogens (13) was administered to pregnant rats on the 12th day after fertilisation, and uteri were removed after 16 h by laparotomy. 34-36 Embryos somites were selected, and mid-brain (CNS) and fore-limb buds (LB) were dissected free and dispersed as single-cell suspensions in Ham's F12 culture medium. The cells were cultured as micromass cell islands for 5 days, and discrete foci of neuronal cells differentiated in CNS cultures and chondrocytes in LB cultures. After 5 days, differentiation as determined by number of stainable foci of differentiated cells and 3H-GABA incorporation in CNS or 35SO4 incorporation in LB and growth (as determined by total protein) were measured. Both differentiation and growth of CNS and LB cultures were markedly reduced following exposure of the dam to teratogens, whereas no significant effect was observed with non-teratogens. One teratogen (amaranth) and one non-teratogen (nitrilotriacetic acid) were classified as false negative and positive, respectively; the sensitivity of the test (proportion of teratogens correct) was therefore 92% and the specificity (proportion of non-teratogens correct) was 94%. Inhibition of growth and differentiation in the rat embryo cell cultures following maternal exposure forms the basis of a short-term in vitro test for teratogens.

Animals↗
PubMed1984

An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells.

A short-term in vitro assay for teratogens has been evaluated and shown to have a high predictability (greater than 90%). Cultured cells derived from midbrain (CNS) and limb buds (LB) of 34 to 36 somite rat embryos were exposed to 46 compounds (27 teratogens, 19 nonteratogens) in a blind trial. Rat liver post mitochondrial supernatant fraction plus cofactors were included in the cultures to provide metabolizing enzymes. Differentiation of neurons from CNS cells and chondrocytes from LB cells was measured after 5 days of culture. Inhibition of differentiation (assessed by reduction of number of foci) was the indicator of potential teratogenicity. Variation between experiments was limited. In repeat experiments with two direct-acting teratogens, aldrin and diphenylhyantoin, interexperiment variability was low (coefficient of variation; range 7 to 24%). Of 27 teratogens 25 (93%) and only 2 of 19 nonteratogens (11%) inhibited differentiation (CNS or LB). Inhibition of differentiation in one cell type alone was less predictive (CNS: 85%, LB: 82%).

Animals↗
PubMed1983

Hepatic microsomal glucuronidation of clofibric acid in the adult and neonate albino rat.

Hepatic microsomal UDP glucuronyltransferase activity towards the acid substrate clofibric acid has been described in the adult and neonate albino rat. The enzyme was maximally activated, approximately 2-fold, in the presence of 0.1-0.4% (w/v) digitonin. Induction of the digitonin activated clofibric acid glucuronyltransferase was observed following phenobarbitone treatment in vivo (2.2-fold), and to a lesser extent, following beta-naphthoflavone treatment (1.3-fold). Clofibrate treatment in vivo (of which clofibric acid is the ester hydrolysis product) had no effect on clofibric acid glucuronidation in vitro. The activity of clofibric acid glucuronyltransferase in the liver of rat before and at birth was low (approx. 0.08 nmoles glucuronide formed/min/mg microsomal protein). The activity increased 5-fold during the first three post-natal days. After this time, the activity increased linearly reaching adult levels by four weeks after birth. The data indicated that clofibric acid glucuronyltransferase belongs to the neonatal cluster of enzymes and clofibric acid is a group 2 substrate. Clofibric acid, a common therapeutic agent, is a useful, acid substrate for the estimation of mammalian hepatic microsomal glucuronyltransferase activity.

Aging↗
PubMed1983

Metabolic capability of rat hepatocytes isolated by perfusion and tissue slice techniques.

The metabolic capability of hepatocytes prepared by the perfusion method (P cells) and the tissue slice method (S cells) has been compared using standardised procedures. Yields of P cells were four times greater than for S cells. Trypan blue exclusion viability and oxygen utilization were similar although the viability of S cells deteriorated faster with time. P cells had a lower endogenous rate of glycogenolysis and showed better glucagon stimulation than S cells. Similarly, P cells performed gluconeogenesis at a higher rate. However, there was no significant differences in the metabolism of the xenobiotic ethoxycoumarin. It is concluded that while S cells are probably satisfactory for studies of drug metabolism their use for work involving surface receptor binding and energy demanding processes should be questioned.

Animals↗
PubMed1983

Metabolic activation and bacterial mutagenicity of aflatoxin B1 in two different test systems.

The metabolism of aflatoxin B1 (AFB1) by liver post-mitochondrial supernatant fraction from phenobarbitone treated male albino rats in liquid suspension and soft-agar plate incorporation bacterial mutation assay systems has been studied. The AFB1 concentration used was in the range of those added to bacterial mutagenicity tests with this mycotoxin. Three oxidative metabolites of AFB1 viz the Tris derivative of AFB1 8,9-diol (derived from the 8,9-epoxide), aflatoxin Q1 (AFQ1) and M1 (AFM1) were observed. The metabolite profile and time course of formation were qualitatively similar in both assay systems. The rate and overall formation of metabolites in the soft-agar system was approximately one half that in the liquid suspension system which was reflected in decreased AFB1 induced bacterial mutation in the former system. The AFB1 metabolite profile in these in vitro systems did not mirror completely the reported in vivo profile seen in the rat, as aflatoxin P1 (AFP1) and a glutathione conjugate were not detected. No evidence was found for a prolongation of metabolism in the soft-agar system.

Aflatoxin B1↗
PubMed1983

A TLC fluorescence derivatization assay for chlorpromazine and its non-conjugated hepatic microsomal metabolites.

Procedures for the assay of chlorpromazine (CPZ) and its metabolites were compared. An improved assay, involving organic extraction, thin-layer chromatography and fluorescence derivatization, was developed. The compounds were extracted from microsomal protein suspensions into 15% n-propanol in dichloromethane by successive extractions at pH 2 and pH 12. CPZ and its mono- and di-desmethyl, 7-hydroxy, N-oxide, sulphoxide and N-oxide-sulphoxide metabolites were separated using TLC on silica gel developed with methanol-acetone-ammonia (50:50:1 v/v/v). The compounds were extracted from the TLC plate with chloroform-methanol (2:1 v/v) and subjected to oxidation with hydrogen peroxide. The highly fluorescent oxidized derivatives were identified as sulphoxides by their fluorescence characteristics. Amounts of derivatized CPZ and metabolites were measured using derivatized promazine as the internal standard.

Journal Article↗
PubMed1983

Dynamics of xenobiotic metabolism by isolated rat hepatocytes using a multichannel perifusion system.

The multichannel perifusion system in recirculating and non-recirculating (single-pass) mode has been used to monitor the rate of oxidative metabolism of three model substrates--7-ethoxycoumarin, dichloronitroanisole and aldrin. With control hepatocytes, the rate of de-ethylation of 7-ethoxycoumarin derived from recirculating mode was essentially similar to the rate obtained with conventional flask-incubated cell suspensions. The formation of 7-hydroxycoumarin glucuronide and sulphate by hepatocytes exposed to 7-ethoxycoumarin demonstrated the retention of conjugative ability of cells in the perifusion system. The rate of demethylation of dichloronitroanisole to dichloronitrophenol was low whilst aldrin epoxidation to dieldrin was rapid using control hepatocytes in recirculating mode. The inductive effect of phenobarbitone on hepatic mixed-function oxidases was demonstrated by a marked increase in the rate of 7-ethoxycoumarin (nine-fold) and dichloronitroanisole (64-fold) dealkylation by hepatocytes from phenobarbitone-treated animals in recirculating mode. The rate of substrate oxidation by hepatocytes perifused in the recirculating and the single-pass mode were the same. With dichloronitroanisole as substrate and a single-pass mode, the rate of dichloronitrophenol formation declined rapidly on perifusion with substrate-free medium and rapidly re-attained steady state on re-introduction of the substrate; the presence of metyrapone effectively inhibited dichloronitroanisole metabolism. The perifusion system is recommended for the study of the dynamics of xenobiotic metabolism by isolated mammalian hepatocytes.

Aldrin↗
PubMed1982

Cytochrome P-450 induction by clofibrate. Purification and properties of a hepatic cytochrome P-450 relatively specific for the 12- and 11-hydroxylation of dodecanoic acid (lauric acid).

Hypolipidaemic drugs induce peroxisomal proliferation in the liver and many induce the formation of the hepatic endoplasmic reticulum in general and the formation of cytochrome P-450 in particular. We have induced the formation of rat liver microsomal cytochrome P-450 by the administration of the hypolipidaemic drug clofibrate, isolated the endoplasmic reticulum, solubilized the cytochrome P-450 from these membranes and subdivided the cytochrome P-450 into four fractions by the use of hydrophobic, anionic, cationic and adsorption chromatography. One of these fractions (cytochrome P-450 fraction 1) was highly purified to a specific content of 17nmol of cytochrome P-450/mg of protein and the protein was active in a reconstituted enzyme system towards the 12- and 11-hydroxylation of the fatty acid, dodecanoic (lauric) acid, with preferential activity towards the 12-hydroxy metabolite. This reconstituted activity was absolutely dependent on NADPH, NADPH-cytochrome P-450 reductase and cytochrome P-450, indicating the role of the mixed-function oxidase system in the metabolism of lauric acid. Another fraction of the haemoprotein (cytochrome P-450 fraction 2) preferentially formed 11-hydroxylauric acid, whereas a third fraction (cytochrome P-450 fraction 3) exhibited only trace laurate oxidase activity and was similar to the phenobarbitone form of the haemoprotein in that these last two cytochromes rapidly turned-over the drug benzphetamine. The molecular weights and spectral properties of these cytochrome P-450 fractions are reported, along with the phenobarbitone-induced form of the enzyme and the nature of the cytochrome(s) induced by clofibrate pretreatment are discussed in the terms of possible haemoprotein heterogeneity.

Animals↗
PubMed1982

A multichannel perifusion system for the continuous monitoring of glycogenolysis and gluconeogenesis in isolated rat hepatocytes.

A multichannel perifusion system for isolated rat hepatocytes entrapped in a Sephadex matrix is described and criteria for the choice of matrices are discussed. This system overcomes the usual problem of clogged filters and impaired flow rates encountered in suspension perifusion systems, and is assembled from standard widely available components. Gluconeogenic capability and mitochondrial respiratory control ratios were unaltered. Decreases in trypan blue viability index and respiration rate were small when compared with flask-incubated hepatocytes. The endogenous rate of glycogenolysis was slightly higher in perifused hepatocytes but hormone response, as judged by glucagon stimulation of glycogenolysis, was unimpaired. The potential of this system is indicated by experiments monitoring glycogenolysis and gluconeogenesis in recycling and non-recycling modes.

Animals↗
PubMed1981

Practolol metabolism. III. Irreversible binding of [14C]practolol metabolite(s) to mammalian liver microsomes.

The irreversible binding of [14C]practolol metabolites to mammalian liver microsomes was studied in vitro. Binding occurred by covalent linkage to amino acids of microsomal proteins. The binding reaction was dependent on oxygen, NADPH, reaction time, microsomal protein content and substrate concentration and was inhibited by carbon monoxide, glutathione and albumin. Albumin inhibition resulted in the binding of reactive metabolite(s) to this serum protein. Hamster (Syrian) liver microsomes had the highest rate of binding and 3-hydroxypractolol formation and marmoset the lowest. The reduced binding of [acetyl-14C]practolol compared to [phenyl-14C]practolol to hamster liver microsomes, but not rat, indicated that metabolites with and without the acetyl grouping were bound in this species. The binding of [14C]desacetylpractolol to hamster liver microsomes was greater than the binding of practolol. Sodium fluoride caused both an increase and a decrease of practolol binding to microsomes from pooled hamster livers, whereas there was a consistent increases in binding to microsomes from individual hamster livers. The collective data indicate that electrophilic metabolites of practolol, possibly N-hydroxylation of the acetyl-amino grouping are formed by the microsomal cytochrome P-450-dependent drug metabolizing enzyme system. These metabolites can react irreversibly with tissue and serum proteins which could be sufficiently altered to be recognized as antigens.

Animals↗