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Biomedical subjects

A Aström

Publications and source records attributed to A Aström.

At least 37 records · Page 2Linked to original sources
PubMed1992

Human skin levels of retinoic acid and cytochrome P-450-derived 4-hydroxyretinoic acid after topical application of retinoic acid in vivo compared to concentrations required to stimulate retinoic acid receptor-mediated transcription in vitro.

Metabolism of retinoic acid to a less active metabolite, 4-hydroxyretinoic acid, occurs via cytochrome P-450 isozyme(s). Effect of a pharmacological dose of retinoic acid on the level of retinoic acid in skin and on cytochrome P-450 activity was investigated. A cream containing 0.1% retinoic acid or cream alone was applied topically to adult human skin for four days under occlusion. Treated areas were removed by a keratome and a microsomal fraction was isolated from each biopsy. In vitro incubation of 3H-retinoic acid with microsomes from in vivo retinoic acid treated sites resulted in a 4.5-fold increase (P = 0.0001, n = 13) in its transformation to 4-hydroxyretinoic acid in comparison to in vitro incubations with microsomes from in vivo cream alone treated sites. This cytochrome P-450 mediated activity was oxygen- and NADPH-dependent and was inhibited 68% by 5 microM ketoconazole (P = 0.0035, n = 8) and 51% by carbon monoxide (P = 0.02, n = 6). Cotransfection of individual retinoic acid receptors (RARs) or retinoid X receptor-alpha (RXR-alpha) and a chloramphenicol acetyl transferase (CAT) reporter plasmid containing a retinoic acid responsive element into CV-1 cells was used to determine the ED50 values for stimulation of CAT activity by retinoic acid and its metabolites. Levels of all trans and 13-cis RA in RA-treated tissues were greater than the ED50 values determined for all three RARs with these compounds. Furthermore, the level of all trans RA was greater than the ED50 for RXR-alpha whereas the 4-OH RA level was greater than the ED50 for RAR-beta and RAR-gamma but less than for RAR-alpha and RXR-alpha. These data suggest that there are sufficient amounts of retinoic acid in treated skin to activate gene transcription over both RARs and RXR-alpha.

Administration, Topical↗
PubMed1991

A simple procedure for isolation of eukaryotic mRNA polyadenylation factors.

We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.

Cell Nucleus↗
PubMed1991

Molecular cloning of two human cellular retinoic acid-binding proteins (CRABP). Retinoic acid-induced expression of CRABP-II but not CRABP-I in adult human skin in vivo and in skin fibroblasts in vitro.

Cellular retinoic acid binding proteins (CRABP) are low molecular weight proteins whose precise function remains unknown. To investigate the role of CRABP in human skin we have cloned the human CRABP-II cDNA as well as the coding region of human CRABP-I. The predicted amino acid sequences of human CRABP-I and CRABP-II demonstrated a 99.3 and 93.5% identity to mouse CRABP-I and CRABP-II, respectively. CRABP-I transcripts were undetectable in adult human epidermis by RNA blot hybridization, while the CRABP-II cDNA probe detected an approximately 1.2-kilobase mRNA transcript. External application of 0.1% retinoic acid cream in vivo for 16 h resulted in a 16-fold induction of CRABP-II transcripts, while CRABP-I mRNA remained undetectable. Expression of CRABP-II, but not CRABP-I mRNA, was also markedly increased (greater than 15-fold) by retinoic acid treatment of fibroblasts cultured from human skin, whereas no significant induction of CRABP-II mRNA was observed in human lung fibroblasts. Human CRABP-II but not CRABP-I mRNA was significantly increased by agents which are known to induce keratinocyte differentiation in vitro. The marked inducibility of the CRABP-II gene is compatible with the idea that this isoform is important in retinoic acid-mediated regulation of human skin growth and differentiation.

Amino Acid Sequence↗
PubMed1991

In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease.

We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.

Base Sequence↗
PubMed1990

Retinoic acid and synthetic analogs differentially activate retinoic acid receptor dependent transcription.

We have developed an assay where the potency of retinoids in retinoic acid receptor (RAR) mediated transcriptional activation can be rapidly evaluated. In this assay hRAR-alpha, hRAR-beta and hRAR-gamma were expressed in CV-1 cells together with a reporter gene containing a retinoic acid responsive element (TRE3-tk-CAT). Concentrations required to obtain half-maximum induction (ED50) of CAT-activity were determined for several retinoids, e.g., all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), arotinoid acid (TTNPB) and m-carboxy-arotinoid acid (m-carboxy-TTNPB, an inactive arotinoid analog). The ED50 values for RA decreased in the order of RAR-alpha (24 nM) greater than RAR-beta (4.0 nM) greater than RAR-gamma (1.3 nM), while the ED50 values for TTNPB and 13-cis-RA decreased in the order of RAR-alpha (6.5 nM, 190 nM) greater than RAR-gamma (2.3 nM, 140 nM) greater than RAR-beta (0.6 nM, 43 nM), respectively. No significant inductions were obtained when cells were treated with m-carboxy-TTNPB, even at 10 microM concentrations. The fold induction of CAT-activity for all compounds tested decreased in the order of RAR-alpha greater than RAR-beta greater than RAR-gamma.

Animals↗
PubMed1990

Interferon down regulates the male-specific cytochrome P450IIIA2 in rat liver.

The aim of this study was to clarify the mechanism by which cytochrome P450 (P450)-mediated catalytic activity is decreased following interferon (IFN) administration. Microsomal steroid hydroxylation was assessed to test the hypothesis that IFN selectively decreases the activities of individual P450 isozymes in male rats. Thus, recombinant rat IFN gamma (r-rat IFN gamma) treatment produced 40% and 17% reductions in androst-4-ene-3,17-dione (androstenedione) 6 beta- and 16 beta-hydroxylation, respectively. Androstenedione 16 alpha- and 7 alpha-hydroxylation were unaltered following r-rat IFN gamma treatment. Similar changes in the androstenedione hydroxylation pathways were observed following administration of naturally derived rat IFN alpha/beta. Microsomal levels of P450IIIA2, the male-specific constitutive steroid 6 beta-hydroxylase, were lower after administration of r-rat IFN gamma (42% of control fractions). Furthermore, hepatic P450IIIA2 mRNA was found to be decreased to a similar extent by r-rat IFN gamma. These findings suggest that IFN selectively decreases the content of this isozyme by a mechanism involving altered mRNA regulation. Sex steroids were unlikely to have mediated the decrease in P450IIIA2 levels since serum estradiol and testosterone levels were unchanged by r-rat IFN gamma. In order to determine whether IFN alters the expression of P450IIIA1, a steroid-inducible member of the P450IIIA gene subfamily which is not expressed in untreated rat liver, adult female rats (which lack P450IIIA2) were coadministered pregnenolone 16 alpha-carbonitrile and r-rat IFN gamma. However, IFN failed to impair the induction of androstenedione 6 beta-hydroxylation produced by pregnenolone 16 alpha-carbonitrile. These findings suggest that although IFN decreases the expression of P450IIIA2, it may not down regulate the expression of other steroid-inducible P450IIIA proteins. In view of the existence of human P450IIIA orthologs which catalyze the metabolism of several important therapeutic agents, the findings of this study may help predict possible drug interactions in patients receiving IFN.

Animals↗
PubMed1987

Induction of liver microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic glutathione transferase in different rodent species by 2-acetylaminofluorene or 3-methylcholanthrene.

Control activities vary 12-fold for microsomal epoxide hydrolase, two-fold for UDP-glucuronyl transferase and five-fold for cytosolic glutathione (GSH) transferase among the different rodents (rat, hamster, guinea-pig, mouse) examined. For all three enzymes the activities in rat liver are towards the lower values. In these rodents, except for a 100% increase in microsomal epoxide hydrolase in guinea-pig liver, 2-acetylaminofluorene induces the three phase 2 enzymes only in rat. Treatment with 3-methylcholanthrene also produces the largest effects on these three enzyme activities in rat liver; exceptions are its failure to induce microsomal epoxide hydrolase in female rat and the large induction of cytosolic GSH transferase in hamster liver. Quantitatively, hepatic microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic GSH transferase activities, and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene, in male Sprague-Dawley rats are not representative for other rodent species or even, in all cases, for female rat.

2-Acetylaminofluorene↗
PubMed1986

Induction of cytochrome P-450 and related drug-metabolizing activities in the livers of different rodent species by 2-acetylaminofluorene or by 3-methylcholanthrene.

In general, large differences in the control levels of different cytochrome P-450-catalyzed activities (aminopyrine N-demethylase, benzo(a)pyrene monooxygenase, ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase and total 2-acetylaminofluorene metabolism and metabolite pattern) and in the inducibility of these activities in different rodent species (rat, hamster, guinea pig and mouse) and sexes were observed. For all the activities measured the lowest levels were observed in untreated rats. With a few minor exceptions, the only species tested in which cytochrome P-450-catalyzed activities were induced by treatment with 2-acetylaminofluorene was the rat. A larger number of the species tested were susceptible to induction by 3-methylcholanthrene. However, this xenobiotic proved also to induce most potently in the rat. There are relatively large differences between the male and female rat both in terms of control cytochrome P-450-catalyzed activities and in the inducibility of these activities by 2-acetylaminofluorene and 3-methylcholanthrene. In general, both of these xenobiotics proved to be more potent inducers in the female than in the male. Thus, it is quite clear that in quantitative terms the hepatic microsomal cytochrome P-450-catalyzed activities and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene in the male Sprague-Dawley rat are not representative for other rodent species or even for the female of the same species.

2-Acetylaminofluorene↗
PubMed1986

Subcellular and organ distribution of cholesterol epoxide hydrolase in the rat.

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.

Animals↗
PubMed1986

Induction of different isozymes of cytochrome P-450 and of microsomal epoxide hydrolase in rat liver by 2-acetylaminofluorene and structurally related compounds.

The amounts of five different forms of cytochrome P-450 and of microsomal epoxide hydrolase were determined immunochemically in rat liver microsomes before and after treatment of the animals with 2-acetylaminofluorene and 15 structurally related compounds. The amount of cytochrome P-450c was found to be increased about 60-fold after treatment with 2-aminofluorene and 3-aminofluorene. Administration of 1-aminofluorene, 4-aminofluorene, 2-acetylaminofluorene and nitrofluorene increased this isozyme about 15-19 times. 2-Aminofluorene was found to inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to a cytoplasmic receptor 50% at a concentration of 3.12 microM, while no such inhibition could be detected with 2-acetylaminofluorene. Induction of ethoxyresorufin O-deethylase activity was found to be highly correlated (+0.95) with the induction of cytochrome P-450c. Also correlated with the induction of this form was the amount of cytochrome P-450d (+0.84), which could be maximally increased about fourfold. Cytochromes P-450b + e were induced by 2-acetylaminofluorene, 4-acetylaminofluorene and fluorene (about tenfold), while 4-aminofluorene and 4-acetylaminofluorene were found to elevate cytochrome P-450PB/PCN-E about threefold. Microsomal epoxide hydrolase was induced by many of the compounds tested, with 2,7-diaminofluorene, 2,7-diacetylaminofluorene, 2-acetylaminofluorene and 2-(N-hydroxy)acetylaminofluorene being the most potent. No correlation of the induction of this enzyme with the induction of any isozyme of cytochrome P-450 was observed.

2-Acetylaminofluorene↗
PubMed1986

Pituitary regulation of cytochrome P-450-mediated metabolism of steroids and xenobiotics in rat liver microsomes.

In a previous paper we reported on the influence of sex and pituitary hormones on the selection of diethylnitrosamine-initiated, enzyme-altered cells by 0.02% (w/w) 2-acetylaminofluorene (2-AAF) and partial hepatectomy in the resistant hepatocyte model (RH-model). The islands of enzyme-altered cells in this model grew faster in male than in female rat liver and the growth rate was markedly decreased in male rats bearing ectopic pituitary grafts during the 2-AAF selection period. Male rats are also generally more susceptible to 2-AAF carcinogenesis than female rats. In order to investigate whether the sex differentiated response to 2-AAF selection and 2-AAF carcinogenesis might be due to pituitary control of xenobiotic metabolism, as previously shown for rat liver metabolism of steroid hormones, we have studied the influence of age, sex and pituitary hormones on the cytochrome P-450-mediated hydroxylations of 2-AAF and benzo[a]pyrene (B[a]P), O-deethylation of 7-ethoxyresorufin and the metabolism of 4-androstene-3,17-dione (androstenedione) in rat liver microsomes. Microsomes from prepubertal rats had a generally higher capacity to metabolize the xenobiotic compounds whereas the capacity for androstenedione hydroxylation was low. In adult rats pronounced sex differences and a marked influence of pituitary hormones were observed in the microsomal formation of several 2-AAF metabolites as well as in B[a]P and androstenedione metabolism. The results clearly show that at least the oxidative pathways of 2-AAF and B[a]P metabolism are controlled by pituitary hormones in a similar way to the rat liver metabolism of steroids. These data do not, however, provide any explanation for the previously mentioned sex differences in the RH-model or in 2-AAF carcinogenesis. We therefore suggest that the pituitary regulation of other pathways of 2-AAF metabolism must be considered in order to clarify the biochemical background behind sexually differentiated 2-AAF carcinogenesis in rat liver.

2-Acetylaminofluorene↗
PubMed1986

Effects of neonatal and adult castration on the in vitro metabolism of steroids and xenobiotics in rat liver.

Previous studies in our laboratory have shown that the sex-differentiated metabolism of 4-androstene-3,17-dione and of several other steroid hormones in adult rat liver is "feminized" following neonatal castration of male rats, due to an influence via the hypothalamo-pituitary-liver axis. The metabolism of many xenobiotics is also sex differentiated, and an important question is whether endocrine ablations might alter hepatic carcinogen metabolism in a way explaining, for example, the decreased tendency of castrated male rats [Y.C. Toh, In: Shanmagarathnam et al. (eds.), Liver Cancer, Cancer Problems in Asian Countries, Proceedings of the Second Asian Cancer Conference, pp. 167-171. Singapore: Singapore Cancer Society, 1976] to form liver tumors following 2-acetylaminofluorene treatment. The results presented in this paper clearly show that neonatal castration of male rats, much more efficiently than adult castration, feminizes the cytochrome P-450-dependent, sex-differentiated, liver microsomal formation of 7-hydroxy-2-acetylaminofluorene, 9-hydroxy-2-acetylaminofluorene, 5-hydroxy-2-acetylaminofluorene, 1-hydroxy-2-acetylaminofluorene, and N-hydroxy-2-acetylaminofluorene from 2-acetylaminofluorene as well as the total microsomal formation of benzo(a)pyrene metabolites (male greater than female). O-Deethylation of 7-ethoxyresorufin was neither sex differentiated nor affected by castration. The capacity for in vitro sulfation of N-hydroxy-2-acetylaminofluorene in the postmicrosomal supernatant, markedly sex differentiated in the rat (male greater than female), was completely feminized by neonatal but not by adult castration. The results suggest that the influence of endocrine ablations on chemical carcinogenesis in rat liver might be mediated via the hypothalamo-pituitary regulation of certain pathways of hepatic xenobiotic metabolism.

2-Acetylaminofluorene↗
PubMed1985

Identification of the forms of cytochrome P-450 induced in rat liver by 2-acetylaminofluorene using immunoblotting and partial purification.

The amounts of 5 different forms of cytochrome P-450 in liver microsomes from rats treated with 2-acetylaminofluorene were determined and compared with the corresponding patterns in microsomes from control, 3-methylcholanthrene- and phenobarbital-treated animals. 2-Acetylaminofluorene was found to increase the amount of cytochromes P-450b + e 10-fold and of cytochrome P-450d 3-fold, while there was a 54% increase in the level of cytochrome P-450 PB/PCN-E. Cytochrome P-450c was increased from a level too low to detect (less than 0.001 pmol/mg protein) to 0.019 pmol/mg protein. These findings were also confirmed by partial purification of cytochromes P-450b + e and c after 2-acetylaminofluorene treatment.

2-Acetylaminofluorene↗
PubMed1985

Metabolism of 2-acetylaminofluorene by eight different forms of cytochrome P-450 isolated from rat liver.

Eight different forms of cytochrome P-450 have been isolated from the liver microsomes of male Sprague-Dawley rats and compared with previously characterized forms on the basis of chromatographic behavior, absorption maximum of the carbon monooxide complex, apparent molecular weight upon SDS-disc gel electrophoresis, and substrate specificity. With respect to 2-acetylaminofluorene metabolism in a reconstituted system involving these cytochromes P-450, form c was approximately 16 times as active as form d, which was in turn at least 30 times as active as any of the other forms. The patterns of metabolites obtained with all of the cytochromes P-450 were determined by h.p.l.c. and were all unique. Form d was as active as form c in producing N-hydroxy-2-acetylaminofluorene. This metabolite was observed with only one other form of the cytochrome, i.e., from PB/PCN-E, which gave rise to it at a very low rate.

2-Acetylaminofluorene↗
PubMed1984

Induction of cytosolic glutathione transferase and microsomal epoxide hydrolase activities in extrahepatic organs of the rat by phenobarbital, 3-methylcholanthrene and trans-stilbene oxide.

The effects of treating male Sprague-Dawley rats with phenobarbital, 3-methylcholanthrene or trans-stilbene oxide on cytosolic glutathione transferase and microsomal epoxide hydrolase activities in the liver, intestine, kidney, lung, testis, adrenal, spleen, heart and brain have been investigated. Studies on the time-courses of induction in liver demonstrate that these are complete after five days' treatment at the doses used. Phenobarbital induces both cytosolic glutathione transferase and microsomal epoxide hydrolase activities significantly only in liver and intestine. 3-Methylcholanthrene induces these activities in liver only. Trans-Stilbene oxide induces both of these activities in liver and kidney, and cytosolic glutathione transferase activity in adrenal as well.

Animals↗
PubMed1983

Characterization of drug-metabolizing systems in hyperplastic nodules from the livers of rats receiving 2-acetylaminofluorene in their diet.

A number of drug-metabolizing systems were measured in hyperplastic noduli from the livers of rats receiving 2-acetyl-aminofluorene in their diet and compared with corresponding activities in control liver. The level of microsomal cytochrome P-450 is reduced 54% in the nodular tissue, while 5 activities catalyzed by the cytochrome P-450 system (i.e., aminopyrine N-demethylase, benzo[a]pyrene monooxygenase, ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase, and 2-acetylaminofluorene N-hydroxylase) are all present at levels corresponding to 5-44% of the control levels. The pattern of 2-acetylaminofluorene metabolites formed by nodule microsomes also differs from the pattern observed with control microsomes. Microsomal epoxide hydrolase is increased 415%, cytosolic glutathione S-transferases 203-576%, microsomal UDP-glucuronyltransferase activity about 200%, and cytosolic DT-diaphorase 1210% in the nodules. The same changes are seen in nodules of different sizes and in individual nodules of the same size. Finally, of all of these changes only the full increase in epoxide hydrolase can be seen after 1-3 weeks of exposure to 2-acetylaminofluorene.

2-Acetylaminofluorene↗