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Biomedical subjects

C F Wilkinson

Publications and source records attributed to C F Wilkinson.

At least 19 recordsLinked to original sources
PubMed1990

Selective induction of cytochrome P450 isozymes in rat liver by 4-n-alkyl-methylenedioxybenzenes.

To examine the structural requirements of cytochrome P450 induction by 4-n-alkyl-substituted methylenedioxybenzenes (MDBs), rats were treated in vivo with a series of MDBs that differed in alkyl carbon side-chain length (0, 1, 2, 3, 4, 5, 6, or 8). Expression patterns of specific P450 isozymes were evaluated with Western and Northern blotting, enzymatic assays, and solution hybridization assays. As determined by carbon monoxide difference spectroscopy, maximal hepatic induction of total P450 content occurred when rats were treated with MDB derivatives with alkyl chain lengths of five or six carbons. However, maximum induction of the specific P450s--P450IA1, P450IIB1, and P450IIB2--occurred with n-hexyl-MDB. In contrast to effects observed with phenobarbital, treatment with MDBs resulted in higher levels of P450IIB2 than of P450IIB1 in rat hepatic microsomes. Western blot quantitation of MDB-induced hepatic P450IIB1 and P450IIB2 apoenzymes did not correlate to measured levels of the corresponding P450 mRNAs. In fact, P450IIB1 and P450IIB2 apoenzyme levels were consistently lower than expected based on Northern blot and solution hybridization measures of the respective mRNAs. These data suggest that the n-alkyl-MDBs effect increases in levels of hepatic P450 in a complex manner, producing accumulation of P450 mRNAs concomitant with alterations in processes regulating steady-state levels of P450 apoenzyme.

Alkylation↗
PubMed1989

Thyroid follicular cell carcinogenesis.

Ample information in experimental animals indicates a relationship between inhibition of thyroid-pituitary homeostasis and the developmental thyroid follicular cell neoplasms. This is generally the case when there are long-term reductions in circulating thyroid hormones which have triggered increases in circulating thyroid stimulating hormone. Such hormonal derangements leading to neoplasms have been produced by different regimens, including dietary iodide deficiency, subtotal thyroidectomy, and administration of natural and xenobiotic chemical substances. The carcinogenic process proceeds through a number of stages, including follicular cell hypertrophy, hyperplasia, and benign and sometimes malignant neoplasms. Given the interrelationship between the thyroid and pituitary glands, conditions that result in stimulation of the thyroid can also result in stimulation of the pituitary, with the development of hyperplastic and neoplastic changes. The progression of events leading to thyroid (and pituitary) neoplasms can be reversed under certain circumstances be reestablishing thyroid-pituitary homeostasis. Most chemicals that have induced follicular cell tumors seem to operate through inhibition of the synthesis of thyroid hormone or an increase in their degradation and removal. For some of these compounds, it appears that genotoxic reactions may not be playing a dominant role in the carcinogenic process. A seemingly small group of thyroid carcinogens seems to lack influence on thyroid-pituitary status and may in part be operating via their genotoxic potential. In contrast with the well-established relationship between thyroid-pituitary derangement and follicular cell neoplasms in animals, the state of information in humans is much less certain. At this time, ionizing radiation is the only acknowledged human thyroid carcinogen, a finding well established in experimental systems as well. Although humans respond to goitrogenic stimuli as do animals, with the development of cellular hypertrophy, hyperplasia, and under certain circumstances nodular lesions, disagreement exists as to whether malignant transformation occurs in any predictable manner. It would seem that if humans develop thyroid tumors following long-term derangement in thyroid-pituitary status, they may be less sensitive than the commonly used animal models.

Adenocarcinoma↗
PubMed1987

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole.

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from beta-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation. 2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex. 3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c. 4. Both ethoxyresorufin and benzo[a]pyrene inhibited the development of the type I optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[a]pyrene was noncompetitive in nature. 5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type I optical difference spectrum non-competitively. 6. The results indicate that ethoxyresorufin and benzo[a]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.

Animals↗
PubMed1986

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: ontogeny in chick embryo liver.

Aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) is induced in chick liver very early during embryonic development if embryos are treated with 3-methylcholanthrene-type compounds such as 3,4,3'4'-tetrachlorobiphenyl. In mammals, AHH induction is known to be mediated by the Ah receptor. Liver from embryonic and newly hatched chicks was found to contain a cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has properties that are very similar to properties of the Ah receptor previously characterized in mammalian tissues. In chick embryo liver, cytosolic binding sites for TCDD were of high affinity (Kd for [3-H]-TCDD = 0.2 nM) and were specific for 3-methylcholanthrene-type inducers. The specific binding component sedimented at about 9S on sucrose density gradients prepared at low ionic strength. A high level of Ah receptor was detected in chick embryo liver by the fifth day of incubation (5 DI); this is at least 24 hours prior to the onset of AHH inducibility. The Ah receptor concentration increased from 5 DI to 8 DI, the period when chick liver is undergoing early morphological differentiation. After 8 DI, Ah receptor levels dropped substantially and remained low into the posthatching period. In contrast, AHH inducibility was high by 7 DI and remained high throughout embryonic development and into the posthatching period. The discrepancy between Ah receptor levels and the degree of AHH inducibility suggests that only a small fraction of the Ah receptor population is required for maximal AHH induction.

Animals↗
PubMed1985

Identification of the Ah receptor in selected mammalian species and induction of aryl hydrocarbon hydroxylase.

The Ah receptor protein, important in the mechanism of induction of aryl hydrocarbon hydroxylase activity, has been identified and partially characterized in hepatic cytosolic preparations from rat, BALB/c mouse, gerbil, hamster, rabbit, ferret and guinea-pig by means of sucrose density centrifugation analysis and hydroxyapatite binding assays. Using 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin (TCDD) as the ligand, total specific binding capacities ranged over 74-691 fmol [3H]TCDD/mg cytosolic protein and apparent dissociation constants ranged over 0.30-7.8 nM. There was no quantitative correlation between the concentration of cytosolic Ah receptors and the 3-methylcholanthrene-mediated induction of aryl hydrocarbon hydroxylase activity in the species studied. Competitive binding studies with a series of monohydroxylated benzo[a]pyrene derivatives suggested the importance of electronic character in their ability to bind to the Ah receptor and to compete with TCDD for specific binding sites on the receptor.

Animals↗
PubMed1985

Cytochrome P-450 and monooxygenase activity in hepatic microsomes from N-phenylimidazole-treated rats.

Repeated administration of N-phenylimidazole (PI) to rats (3 daily doses of 200 mumol/kg/day) enhanced hepatic microsomal cytochrome P-450 levels (approx. 130%) and aminopyrine N-demethylase (APDM) and aniline p-hydroxylase (APH) activities (approx. 140%); aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) activities were not enhanced over control values under similar conditions. Spectral studies with PI-induced microsomes indicated that although type II PI-binding characteristics were similar to those observed in controls, the 427 nm/455 nm absorbance ratio of the type III dihydrosafrole metabolite-cytochrome P-450 complex was lower than that in control microsomes. The results suggest that the inducing characteristics of PI bear some resemblance to those of phenobarbital (PB).

7-Alkoxycoumarin O-Dealkylase↗
PubMed1985

Comparative studies of aryl hydrocarbon hydroxylase and the Ah receptor in nonmammalian species.

In vivo treatment of chicks, quail and rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene (MC) caused a dose-dependent increase in hepatic microsomal aryl hydrocarbon hydroxylase activity. A much lower level of AHH induction was observed following similar treatment of trout with high concentrations of TCDD or MC. No induction was apparent in midgut tissues from southern armyworm larvae exposed to the same inducers. A low level of receptor exhibiting specific binding of [3H]TCDD was demonstrated in chick hepatic cytosol, but no evidence of receptor was obtained with the other species. Although the specific binding of the receptor in chick cytosol was only 6-8 fmoles TCDD bound/mg protein compared to 135 fmoles/mg in rat hepatic cytosol, the chick receptor exhibited properties similar to those of Ah receptors in mammals.

Animals↗
PubMed1985

Induction of rat-hepatic microsomal cytochrome P-450 and aryl hydrocarbon hydroxylase by 1,3-benzodioxole derivatives.

Several 1,3-benzodioxoles (BD) and related compounds were studied in relation to their ability to generate metabolite complexes with hepatic cytochrome P-450 following administration in vivo to rats. BD derivatives that formed stable metabolite complexes with cytochrome P-450 were considerably more effective inducers of cytochrome P-450 and aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (AHH) activity than derivatives that did not form stable complexes. Linear regression analysis showed that AHH activity was well correlated (r = 0.980) with total (i.e. complexed plus uncomplexed) cytochrome P-450 content and was not correlated with levels of uncomplexed cytochrome P-450. Aminopyrine N-demethylase (APDM) activity in hepatic microsomes from rats treated with 1,3-benzodioxoles was moderately correlated in a linear relationship with uncomplexed levels of cytochrome P-450 and not with total cytochrome P-450.

Aminopyrine N-Demethylase↗
PubMed1985

Selective inhibitory interactions of alkoxymethylenedioxybenzenes towards mono-oxygenase activity in rat-hepatic microsomes.

A series of eight 4-n-alkoxymethylenedioxybenzene (AMDB) derivatives were evaluated for their inhibitory effects on several mono-oxygenase reactions and their capacity to form metabolite complexes with cytochrome P-450 in vitro in hepatic microsomes from phenobarbital (PB)-and Beta-naphthoflavone (Beta NF)-induced rats. Ethoxyresorufin O-deethylase in Beta NF-induced microsomes and aminopyrine N-demethylase in PB-induced microsomes were most susceptible to inhibition by the test compounds. In contrast, aldrin epoxidation and arylhydrocarbon hydroxylase in PB-and Beta NF-induced microsomes, respectively, were not inhibited by derivatives of AMDB. All AMDB derivatives elicited spectral complexes with cytochrome P-450, the characteristics of which were influenced by the microsomes employed and by the length of the AMDB alkoxy side-chain. Derivatives containing short-chain alkoxy substituents (C1 to C3) formed unstable metabolite complexes and generated substantial quantities of carbon monoxide (CO), those with intermediate length alkoxy groups (C4 to C6) generated little CO and rapidly formed intense spectral complexes (large delta A max), and those with the largest alkoxy groups (C7 and C8) formed no CO and elicited complexes of high stability. Quantitative structure-activity analyses showed that the biological data could be described by parabolic equations in II, the hydrophobic constant of the alkoxy substituent, and suggested the importance to AMDB interactions of a lipophilic-binding region at the active centre of the cytochrome P-450. The alkoxy chain length for optimal mono-oxygenase inhibition and complex formation with cytochrome P-450 appeared to be about five or six carbon atoms. The data suggest that the capacity of AMDB compounds to form stable inhibitory complexes with cytochrome P-450 may not always be associated with their ability to inhibit mono-oxygenase activity.

Aminopyrine N-Demethylase↗
PubMed1985

Spectral and inhibitory interactions of methylenedioxyphenyl and related compounds with purified isozymes of cytochrome P-450.

Spectral and inhibitory interactions of two methylenedioxyphenyl (MDP) compounds (dihydrosafrole (DHS) and 4,5-dichloro-1,2-methylenedioxybenzene (DCMB] and 4-n-butyl dioxolane (BD) were studied in vitro in reconstituted systems incorporating cytochromes P-450b and P-450c, purified respectively from hepatic microsomes of phenobarbital (PB)- and beta-naphthoflavone (beta NF)-treated rats. In NADPH-fortified reconstituted systems containing P-450b, DHS yielded a stable type III spectral complex with peaks at 428 and 458 nm; a complex with a single 456 nm peak was formed in systems containing cytochrome P-450c. DCMB formed unstable 456-458 nm spectral complexes with both isozymes, and BD generated an unstable complex with a single Soret peak near 428 nm with cytochrome P-450b; no spectral interaction occurred between BD and cytochrome P-450c. Carbon monoxide was formed in incubations of DCMB with both isozymes but was not observed with either DHS or BD. Marked selectivity was observed in the ability of the test compounds to inhibit selected mono-oxygenase reactions in the reconstituted systems. Thus, while DHS was an effective inhibitor of cytochrome P-450b-mediated ethoxycoumarin O-deethylase (ECD), it failed to inhibit aldrin epoxidase (AE) in the same system; DCMB and BD inhibited both of these reactions. In reconstituted systems incorporating cytochrome P-450c, DHS and DCMB, but not BD, were effective inhibitors of ethoxyresorufin O-deethylase (ERD) activity but none of the compounds showed any inhibitory activity towards aryl hydrocarbon (benzo[alpha]pyrene)hydrolase (AHH) activity. The results indicate that metabolite complex formation with cytochrome P-450 is not the sole criterion for inhibition of mono-oxygenase activity by MDP and related compounds, and that in some cases type I competitive interactions at the substrate binding sites may be the primary contributing factor.

Animals↗
PubMed1985

Congenital malformations and variations in reproductive performance in the ferret: effects of maternal age, color and parity.

Demographic data of ferrets from a commercial breeding colony were analyzed for the effects of maternal age, parity and strain on reproductive performance and the frequency of gross congenital abnormalities observed at parturition. Litter size (mean +/- SEM) was found to be greatest for young, primiparous females (10.3 +/- 0.2) and decreased with advancing maternal age and parity to a cohort mean of 8.1 +/- 0.1 for third parity females 16 months of age. Age, parity or strain had no effect on 24-hour neonatal mortality (7%) or mortality from birth to weaning (20%) and an examination of the causes for death suggested that these rates can be reduced. The malformation rate from two cohorts of females whelping at different times of the year was low (less than 1.0%) and not significantly different. A higher frequency of malformed offspring was detected in females of low previous parity (0-2) than in those with three or more. Based on data obtained in this survey, the ferret would seem a valuable alternative, nonrodent species for teratologic investigations using currently recommended protocols.

Animals↗
PubMed1979

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

Adenosine Monophosphate↗
PubMed1977

Insecticide solvents: interference with insecticidal action.

Several commercial solvent mixtures commonly used as insecticide carriers in spray formulations increase by more than threefold the microsomal N-demethylation of p-chloro N-methylaniline in midgut preparations of southern army-worm (Spodoptera eridania) larvae exposed orally to the test solvents. Under laboratory conditions, the same solvent mixtures exhibit a protective action against the in vivo toxicity of the insecticide carbaryl to the larvae. The data are discussed with respect to possible solvent-insecticide interactions occurring under field conditions and, more broadly, to potential toxicological hazards of these solvents to humans.

Enzyme Induction↗