Biomedical subjects

T W Gant

Publications and source records attributed to T W Gant.

30 records · Page 2Linked to original sources

PubMed1994

Metabolic activation of 2-acetylaminofluorene is required for induction of multidrug resistance gene expression in rat liver cells.

P-Glycoprotein the multidrug resistance (mdr) efflux transporter is encoded by class 1 mdr genes (mdr1) in humans and rodent species. In rat liver and in rat hepatocytes in primary culture, expression of mdr1 genes can be induced with the carcinogenic aromatic amine 2-acetylaminofluorene (2-AAF). As a consequence, increased P-glycoprotein levels led to an accelerated efflux of vinblastine from the hepatocytes and to resistance towards vinblastine-mediated cytotoxicity. N-Hydroxylation, an obligatory initial step in the activation of 2-AAF into electrophilic DNA-binding metabolites is catalyzed predominantly by cytochrome P450 (CYP)1A2, an isozyme present in normal rat liver. In rat hepatocytes in primary culture, mdr1 induction with 2-AAF could be inhibited by addition of the CYP1A-inhibitor alpha-naphthoflavone, indicating the requirement for metabolic conversion of 2-AAF to act as an inducer of mdr1 gene expression. Both N-hydroxy-2-AAF and the mutagenic 2-AAF derivative N-acetoxy-2-AAF (AAAF) were more potent than 2-AAF as mdr1 inducers. mdr1 induction also decreased when deacetylation of AAAF, which strongly accelerates its conversion into a mutagen, was inhibited with paraoxon. Furthermore, rat liver epithelial cells stably transfected with mouse CYP1A2 showed inducibility of mdr1 gene expression with 2-AAF, whereas the parental cell line, which is devoid of CYP1A2 activity, did not. These findings indicate that electrophilic metabolites formed during 2-AAF or AAAF metabolism are responsible for mdr1 induction in rat hepatocytes. The increased mdr1 gene expression may reflect an adaptive cellular response to electrophiles which includes enhanced synthesis of P-glycoprotein aimed to protect the cell from further damage.

2-Acetylaminofluorene↗

PubMed1994

Contribution of CYP1A1 and CYP1A2 to the activation of heterocyclic amines in monkeys and human.

The activation of heterocyclic amines to mutagenic products by hepatic microsomal fractions from cynomolgus monkey, marmoset monkey and man was compared with the respective levels of cytochrome P450 enzymes CYP1A1 and CYP1A2. The rate of activation of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to mutagens by hepatic microsomal fraction from cynomolgus monkey was very low. This was associated with a lack of constitutive expression of CYP1A1 and CYP1A2. In contrast, human hepatic microsomal fraction readily activates these heterocyclic amines and this is associated with constitutive expression of CYP1A2. Treatment of cynomolgus monkey with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a very modest induction of CYP1A2, and a small increase in the activation of MeIQx and IQ. However, there was marked induction of CYP1A1 which was accompanied by > 10-fold increases in PhIP activation and 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and aryl hydrocarbon hydroxylase activities. Following treatment of cynomolgus monkey with 3-methylcholanthrene, induction of CYP1A1, but not CYP1A2, was evident. In untreated marmoset monkey the activations of MeIQx and PhIP, as well as phenacetin O-deethylase, EROD, MROD and aryl hydrocarbon hydroxylase activities, are similar to those in man, although the activations of IQ and coumarin 7-hydroxylase activity are lower than in man. The presence of constitutive CYP1A2, and the absence of CYP1A1, in the liver of this species correspond to the situation in man. Treatment of marmoset monkey with TCDD results in increased CYP1A2 levels (4-fold), accompanied by proportional increases in the activation of MeIQx and IQ and phenacetin O-deethylase, EROD and MROD activities. The activation of PhIP is increased disproportionately, by 8-fold, most likely due to the activity of CYP1A1 which is also induced by TCDD in this species. Overall, the hepatic metabolism of heterocyclic amines by CYP1A enzymes in the untreated marmoset monkey resembles that in human more closely than that in the cynomolgus monkey. Therefore, marmoset monkey may be a more suitable model than the cynomolgus monkey for carcinogenicity studies involving MeIQx and PhIP, but not IQ.

Animals↗

PubMed1994

Requirement for metabolic activation of acetylaminofluorene to induce multidrug gene expression.

Previously we have demonstrated that several xenobiotics can induce multidrug (mdr) gene expression in cultures of primary isolated hepatocytes. One of the best of these xenobiotic inducers in rat hepatocytes is 2-acetylaminofluorene (2-AAF), which induces mdr expression by an enhancement of mdr gene transcription. In all species studied to date, AAF is extensively and variously metabolized. In this study we have sought to determine if AAF per se or a metabolite is responsible for mediating the increase in mdr gene transcription and expression. This study demonstrates that AAF per se is not active, but that the effect of AAF we have observed on mdr gene transcription and expression in the rat is due to the formation of a reactive metabolite(s). Our data indicate that this reactive metabolite is probably N-acetoxy-2-aminofluorene or the sulfate ester of N-hydroxy-AAF. The requirement for the formation of one of these metabolites may explain the differences in species response to AAF, in terms of mdr gene expression, that we have observed. We hypothesize that the mechanism by which mdr gene transcription is increased in response to AAF involves a covalent interaction between a reactive metabolite and an mdr gene regulatory protein. Our current work is concerned with the exploration of this hypothesis.

2-Acetylaminofluorene↗

PubMed1993

Expression of multidrug resistance genes in rat liver during regeneration and after carbon tetrachloride intoxication.

We analyzed expression of multidrug resistance (mdr) genes in rat liver during regeneration after partial hepatectomy or carbon tetrachloride-induced necrosis. In situ hybridization revealed that in the normal liver the cellular distribution of mdr transcripts and protein is restricted to hepatocytes and that a gradient, highest in zone 1 and lowest in zone 3, exists in the level of the mdr transcripts in the liver acinus. Increased levels of mdr1a and mdr1b transcripts were observed 3 hr after administration of carbon tetrachloride and remained increased for the next 5 days. In contrast, increased expression of mdr1a and mdr1b was first observed 24 hr after partial hepatectomy. Use of gene-specific probes to compare the time courses of mdr1b and mdr2 expression after carbon tetrachloride administration showed distinctly different patterns of expression; mdr1b reached a maximum level of expression at 12 hr, whereas increased mdr2 expression was first observed 48 hr after administration. Nuclear run-on analysis at 12 and 24 hr after carbon tetrachloride administration demonstrated 10-fold and eightfold increases in mdr transcription, respectively. However, 72 hr after carbon tetrachloride treatment the rate of mdr transcription was back to the control level. The cellular patterns of mdr expression after partial hepatectomy and carbon tetrachloride administration were similar; the increase was first observed in zone 1 and gradually extended into zone 3. These data strongly suggest that the physiological roles of mdr1b and mdr2 are different and that liver regeneration is an appropriate model for elucidating these differences.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates.

P-glycoprotein, an energy-dependent plasma membrane drug-efflux pump capable of reducing the intracellular concentration of a variety of hydrophobic xenobiotics, is encoded by mdr1, a member of the multidrug-resistant (mdr) gene family. The physiological function of this protein is unknown. Because of its location on the bile canalicular domain of the hepatocyte, we and others have hypothesized that P-glycoprotein may have a physiological role as a biliary transporter of xenobiotics and endobiotics and that its expression may therefore be altered in cholestasis. Both obstructive and alpha-naphthylisothiocyanate-induced cholestasis increased mdr1a and 1b gene expression in rat liver. Hepatic P-glycoprotein levels were also increased, and the protein remained localized at the biliary hepatocyte domain. Induction of mdr1a and mdr1b gene expression in rat liver was accomplished by means of increased transcription. alpha-Naphthylisothiocyanate-induced cholestasis in cynomolgus monkeys increased hepatic expression of both the mdr1 and 2 genes. To investigate the possible role of P-glycoprotein as a biliary efflux transporter, biliary excretion of vinblastine, a representative substrate of P-glycoprotein, was studied in rats. Increased hepatic mdr messenger RNA and P-glycoprotein levels, mediated by the xenobiotic inducer 2-acetylaminofluorene, resulted in a significant increase in biliary excretion of vinblastine, which was antagonized by the P-glycoprotein inhibitor verapamil. These findings suggest that P-glycoprotein functions as a biliary efflux pump for xenobiotics and, possibly, for unidentified physiological inducers that may mediate increased transcription of the mdr gene observed during cholestasis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Regulation of P-glycoprotein gene expression in hepatocyte cultures and liver cell lines by a trans-acting transcriptional repressor.

Previously we have demonstrated that expression of the multidrug resistance (mdr) genes in rat liver and primary rat hepatocyte cultures is induced by exposure to 2-acetylaminofluorene and 3-methylcholanthrene. The mdr expression induced by both of these compounds occurs primarily via increased gene transcription. To determine the nature of possible regulatory proteins involved in mdr gene regulation we inhibited protein synthesis using cycloheximide or emetine in primary rat hepatocyte cultures, mouse (HePa 1), human (Hep G2) and rat (H4-II-E) cell lines. Each cell type responded by strongly increasing its steady state mdr1 mRNA levels. In hepatocytes increased mdr expression was observed after greater than 50% inhibition of protein synthesis, and was first detected after 2h of protein synthesis inhibition with maximal induction occurring by 24h. Nuclear run-on analysis showed that the increased steady state mRNA level was due to increased gene transcription without alteration of the transcription start site. Combined these data indicate that one regulatory mechanism by which mdr gene expression is controlled is via a trans-acting transcriptional repressor.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Adenovirus-mediated in vivo gene transfer and expression in normal rat liver.

Replication deficient, recombinant adenovirus (Ad) vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in hepatocytes. In vitro, primary cultures of rat hepatocytes infected with a recombinant Ad containing a human alpha 1-antitrypsin cDNA (Ad-alpha 1AT) synthesized and secreted human alpha 1AT for 4 weeks. In rats, in vivo intraportal administration of a recombinant Ad containing the E. coli lacZ gene, was followed by expression of beta-galactosidase in hepatocytes 3 days after infection. Intraportal infusion of Ad-alpha 1AT produced detectable serum levels of human alpha 1AT for 4 weeks. Thus, targeted gene expression has been achieved in the liver, albeit at low levels, suggesting that adenovirus vectors may be a useful means for in vivo gene therapy in liver disorders.

Adenoviruses, Human↗

PubMed1991

Cloning and characterization of a member of the rat multidrug resistance (mdr) gene family.

The rat mdr gene family [genes encoding P-glycoprotein (Pgp)] was characterized and the complete sequence of a rat mdr cDNA was determined based on seven independent cDNA clones that correspond to the same gene. The longest of these clones contains a 4.3-kb insert which represents a full-length rat mdr cDNA. The longest open reading frame of this sequence is 3933 bp; the first ATG is at 103 bp, making the deduced protein 1277 amino acids long (141 kDa). This correlates well with previously identified Pgp. The sequence of this gene has a very high, greater than 90%, degree of identity to the mouse mdr1b gene (also known as the mdr1 gene) therefore, we designate it the rat mdr1b gene. Transcription of this gene begins at a single start point 151 nucleotides upstream from the start codon. We show here that the rat gene family is comprised of three members, which is consistent with previous data on other rodent species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Regulation of 2-acetylaminofluorene-and 3-methylcholanthrene--mediated induction of multidrug resistance and cytochrome P450IA gene family expression in primary hepatocyte cultures and rat liver.

Previous studies by this laboratory have indicated that expression of the multidrug resistance (mdr) gene can be increased in vivo by exposure to a variety of xenobiotics. Because of the nature of these compounds, it was proposed that mdr gene expression might, at least in part, be regulated by the arylhydrocarbon (Ah) receptor. In the present study, we used a primary hepatocyte culture model to examine the relationship between induction of cytochrome P450IA and mdr expression in vitro. Both 3-methylcholanthrene (MC) and 2-acetylaminofluorene (AAF) were efficient inducers of mdr expression in this model. Induction of mdr gene expression by both MC and AAF obeyed a log10 concentration/response relationship. In contrast, 2,3,7,8-tetrachlorodibenzo-P-dioxin did not induce mdr expression at concentrations that yielded maximum induction of cytochrome P450IA expression. These data suggest that mdr induction was not mediated via the Ah receptor. Nuclear run-off analysis indicated that both AAF and MC induced mdr expression by increasing transcription. Primer extension analysis indicated that mdr gene transcription was initiated at one major site 151 bp upstream of the ATG site in both the uninduced and induced state in vivo and in vitro. The sequence of the primer and the site of initiation of gene transcription indicate that the main gene induced was the mdr 1b gene.

2-Acetylaminofluorene↗

PubMed1991

Multidrug resistance gene family and chemical carcinogens.

The data discussed in this review indicate that the coordinated induction of both the mdr gene family and a subfamily of the cytochrome P-450 supergene family provide a unified response of the organism to prevent lethal accumulation of xenobiotics. Consequently, a distinct physiological role for the mdr multigene family now exists. Furthermore, recent evidence suggests the existence of multiple receptors with overlapping substrate specificity that are involved in the induction of both mdr and P-4501A gene families. The increased expression of mdr gene(s) in the early stages of liver carcinogenesis and presumably in other tissues is associated with the development of xenobiotic resistance that is observed in the preneoplastic cell populations. These observations may have important clinical implications and may provide an explanation for resistance to chemotherapy of tumors in organs such as liver and colon that are frequently exposed to both environmental and dietary xenobiotics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1988

Redox cycling and sulphydryl arylation; their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes.

Quinones are believed to be toxic by a mechanism involving redox cycling and oxidative stress. In this study, we have used 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ), which redox cycles to the same degree as menadione, but does not react with free thiol groups, to distinguish between the importance of redox cycling and arylation of free thiol groups in the causation of toxicity to isolated hepatocytes. Menadione was significantly more toxic to isolated hepatocytes than 2,3-diOMe-1,4-NQ. Both menadione and 2,3-diOMe-1,4-NQ caused an extensive GSH depletion accompanied by GSSG formation, preceding loss of viability. Both compounds stimulated a similar increase in oxygen uptake in isolated hepatocytes and NADPH oxidation in microsomes suggesting they both redox cycle to similar extents. Further evidence for the redox cycling in intact hepatocytes was the detection of the semiquinone anion radicals with electron spin resonance spectroscopy. In addition we have, using the spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide), demonstrated for the first time the formation of superoxide anion radicals by intact hepatocytes. These radicals result from oxidation of the semiquinone by oxygen and further prove that both these quinones redox cycle in intact hepatocytes. We conclude that while oxidative processes may cause toxicity, the arylation of intracellular thiols or nucleophiles also contributes significantly to the cytotoxicity of compounds such as menadione.

Animals↗

PubMed1985

Metabolism of 1-naphthol by tyrosinase.

1-Naphthol was metabolized by the polyphenol oxidase, tyrosinase, primarily to 1,2-naphthoquinone and to small amounts of 1,4-naphthoquinone as well as to covalently bound products. The inhibition of covalent binding by ethylenediamine, which reacts specifically with 1,2-naphthoquinone but not 1,4-naphthoquinone, suggested that most of the covalent binding was due to 1,2-naphthoquinone or a metabolite of similar structure. The activation by tyrosinase of 1-naphthol to covalently bound products suggested that it may alter the reaction kinetics of the enzyme. This was investigated by studying the effects of 1-naphthol on the tyrosinase-catalysed oxidation of 4-hydroxyanisole. Preincubation of tyrosinase with 1-naphthol increased the lag period of the oxidation of 4-hydroxyanisole, which may be due to a decrease in the amount of active enzyme, as well as to a reaction of 1-naphthol with 3,4-anisylquinone, an oxidation product of 4-hydroxyanisole. The metabolic activation of 1-naphthol by tyrosinase to covalently bound species suggests that 1-naphthol or a structurally related derivative may be of potential therapeutic application in the treatment of cells high in tyrosinase activity, such as certain melanomas.

Anisoles↗