Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization

The N-nitrosodimethylamine demethylase (P450I-IE1) is induced severalfold in liver by giving rats ethanol, acetone, pyrazole, and other related small molecular weight compounds. This induction is not the result of an increase in IIE1 mRNA, but could be due to either an increase in translation rate or a decrease in protein degradation. To determine the mechanism of induction, we measured IIE1 synthesis and degradation rates in untreated and acetone-treated rats. This was accomplished by immunopurification of radiolabeled IIE1 protein using a specific monoclonal antibody subsequent to in vivo labeling of total cellular protein with either NaH14CO3 or [3H]leucine. We found that in rats fed acetone, the rate of IIE1 synthesis was not changed; however, IIE1 degradation was markedly altered. In untreated rats, IIE1 protein was degraded via a biphasic pathway consisting of both a rapid and slow component with approximate half-lives of 7 and 37 h, respectively. However, in acetone-treated rats, only a monophasic curve with a half-life of 37 h was observed. The abolition of the rapid degradation component of the IIE1 turnover cycle indicates that induction of IIE1 by acetone is primarily due to specific stabilization of IIE1 protein. Since acetone is also metabolized by IIE1, we believe that this may be a substrate-induced enzyme stabilization.     

Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.     

Characterization of the ethanol-inducible alc gene expression system in tomato

The efficacy of the ethanol-inducible alc transgene expression system, derived from the filamentous fungus Aspergillus nidulans, has been demonstrated in transgenic tomato. Two direct comparisons have been made. First, this study has utilized two transgenic lines carrying distinct reporter genes (chloramphenicol acetyltransferase and beta-glucuronidase) to distinguish aspects of induction determined by the nature of the gene/gene product rather than that of the plant. Second, comparisons have been made to data generated in other species in order to identify any species-specific effects. The induction profiles for different genes in different species have shown remarkable similarity indicating the broad applicability of this gene switch. While there are minor differences observed between species, these probably arise from diversity in their metabolism. A series of potential alternative inducers have also been tested, revealing that ethanol (through metabolism to acetaldehyde) is better than other alcohols and ketones included in this study. Expression driven by alc was demonstrated to vary spatially, the upper younger leaves having higher activity than the lower older leaves; this will be important for some applications, and for experimental design. The highest levels of activity from ethanol-inducible transgene expression were determined to be the equivalent of those from the constitutive Cauliflower Mosaic Virus 35S promoter. This suggests that the alc system could be an important tool for plant functional genomics.     

Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots

Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid-inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol-inducible promoter within C. roseus hairy roots is reported. This ethanol-inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6-fold increase in CAT reporter activity after 24 h of induction. The ethanol-inducible CAT activity increased 24-fold over a 72-h induction period with 0.5% ethanol.     

Studies on the mechanisms of induction of N-nitrosodimethylamine demethylase by fasting,acetone, and ethanol

Previous work has shown that induction of a high-affinity NADPH-dependent nitrosodimethylamine demethylase (NDMAd) in liver microsomes occurs in rats due to fasting, ethanol consumption, and streptozotocin-induced diabetes. Several lines of observations suggest that this is due to the induction of specific cytochrome P-450 isozymes. Induction of P-450 species by ethanol has also been observed by other investigators. Since each of the above altered metabolic states has in common elevated levels of ketone bodies, the possible role of acetone, a known inducer of NDMAd, in the induction of the demethylase activity was investigated. Levels of endogenous acetone in fasted rats correlated (r = 0.72) with a three- to fourfold increase in NDMAd activity. However, a dose-response experiment showed endogenous levels of acetone to be capable of causing at most 40% of the induction in fasted rats. This suggests that other ketone bodies or factors may have contributed to the induction. The induction of NDMAd by ethanol was enhanced by alcohol dehydrogenase inhibitors pyrazole and acetaldehyde oxime, suggesting that ethanol, rather than its metabolites, was responsible for the induction.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

Purification and characterization of ethanol-inducible human hepatic cytochrome P-450HLj

Human hepatic cytochrome P-450HLj was purified in a catalytically active state from microsomes obtained from an ethanol-intoxicated man. The electrophoretically homogeneous preparation of HLj was compared to rat P-450j and found to have a slightly different apparent molecular mass (54 vs 51.5 kDa) but highly similar immunochemical, spectral, and catalytic properties. Purified HLj exhibited high activity toward N-nitro-sodimethylamine (NDMA) and aniline metabolism, low but measurable activity toward benzphetamine and 7-ethoxycoumarin, and no detectable activity toward benzo[a]pyrene, testosterone, and progesterone. Antibody against rat P-450j reacted with HLj in immunoblot analyses and, when added directly to HLj before reconstitution with NADPH-cytochrome P-450 reductase and lipid, the antibody inhibited (96%) NDMA metabolism by HLj almost completely. However, if HLj was reconstituted with the other components before the addition of the anti-P-450j IgG, the ability of the antibody to inhibit the metabolism of NDMA was greatly diminished. This suggests that the interactions between reductase and HLj are similar to those previously observed between rat P-450j and reductase, and appear to prevent the complete access of anti-P-450j. The addition of cytochrome b5 to reconstitution systems containing HLj resulted in a small increase in the Vmax from NDMA demethylation accompanied by a decrease in Km,app (1.3 to 0.3 mM) as has been observed in reconstitution systems with rat P-450j. Therefore, in reconstituted systems, cytochrome b5 appears to play an important role in the biotransformations mediated by HLj and P-450j. In conclusion, this study demonstrates that HLj is functionally related to ethanol-inducible rat P-450j and rabbit LM3a.     

Centrilobular expression of ethanol-inducible cytochrome P-450 (IIE1) in rat liver

Western blot analysis of digitonin eluates as well as immunohistochemical analysis revealed a 30-fold higher concentration of cytochrome P-450IIE1 in the centrilobular than in the periportal regions of the rat liver. Ethanol treatment caused a selective centrilobular induction of P-450IIE1, whereas phenobarbital induced P-450IIB1/2 in both liver lobule regions. The heterogeneous distribution pattern of P-450IIE1 was also observed in cells isolated from either region and correlated to the relative content of P-450IIE1 mRNA in the two cell types. The regiospecific expression and induction of P-450IIE1 may explain why several hepatotoxins, known to be metabolized by this isozyme, primarily damage the centrilobular region in the liver.     

Characterization of cells in the developing human liver

Human hepatic progenitor cells (HPCs) have been shown to co-express the hematopoietic stem cell (HSC) markers, CD117 and CD34. These cells differentiate not only into hepatocytes and cholangiocytes but also into pancreatic ductal and acinar cells under certain conditions. The fetal liver (FL) is rich in precursor/stem cells; however, little is known about (i) the markers expressed by liver cells during fetal development and (ii) whether an equivalent to the adult liver stem-like progenitors exists in the FL. Here, (i) FL tissue obtained from human 5-18-week-old fetuses were evaluated by means of flow cytometry, immunocyto-, and histochemistry for the emergence of cells expressing and co-expressing known hematopoietic, hepatic, and pancreatic cell markers, and (ii) isolated putative HPCs were phenotypically and molecularly characterized. We report that (i) red blood and endothelial cell precursors were most abundant in early gestation. Cells expressing HSC and pancreatic markers were found in the first trimester, while cells expressing hepatic markers appeared in the second trimester. Very few committed cells were present in FLs obtained early in the first trimester. In addition, cells expressing pancreatic markers co-expressed the HSC marker CD117. (ii) Isolated CD117+/CD34+/CD90- cells in vitro expressed both the genes and proteins for the hepatic markers such as albumin, alpha feto protein (AFP), alpha1-antitrypsin, and cytokeratin 19 (CK19). Our study suggests that hepatoblast and ductal plate/bile duct development mainly occurs during the second trimester. FLs in gestation weeks 5-9 had the highest numbers of precursor cells and the least committed cells. Cells that differentiate into Alb+ or CK19+ can be isolated from early FLs and may be appropriate progenitors for establishing novel systems to investigate basic mechanisms for cell therapy.     

Identification of lanosterol 14 alpha-methyl demethylase in human tissues

Lanosterol 14 alpha-methyl demethylase was investigated in human tissues using a radio-HPLC assay to detect the 4,4-dimethyl-5 alpha-cholesta-8, 14-dien-3 beta-ol (diene) metabolite. The sequence of events leading to the demethylated product in human liver microsomes involves the conversion of the diol to the aldehyde followed by diene formation. Enzyme activity displayed a greater than 10 fold variation among the 9 liver samples studied. Kinetic parameters were determined and shown to differ between two separate liver samples. Addition of inhibitors of yeast lanosterol 14 alpha demethylase, ketoconazole and miconazole, resulted in extensive inhibition of formation of the demethylated metabolite. The enzyme, detected in microsomes isolated from human kidney and lymphocytes, also catalyzed the conversion of dihydrolanosterol to oxylanosterol intermediates and the diene. The presence of this enzyme in microsomes from various human tissues suggests that it may play a role in cellular regulation of cholesterol synthesis.     

Carbon tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450

Treatment of rats with ethanol or rabbits with either imidazole or pyrazole, agents known to induce the ethanol-inducible form of liver microsomal cytochrome P-450 (P-450 LMeb), caused, compared to controls, 3-25-fold enhanced rates of CCl4-dependent lipid peroxidation or chloroform production in isolated liver microsomes. No significant differences were seen when the rate of CCl4-dependent lipid peroxidation was expressed relative to the amount of P-450 LMeb in the various types of microsomal preparations. In reconstituted membranous systems, this type of P-450 was a 100-fold more effective catalyst of CCl4 metabolism than either of the cytochromes P-450 LM2 or P-450 LM4. It is proposed that the induction of this isozyme provides the explanation on a molecular level for the synergism seen of ethanol on CCl4-dependent hepatotoxicity.     

Characterization of trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes

A crude microsomal fraction isolated from red hake (Urophycis chuss) muscle demethylated trimethylamine-N-oxide (TMAO). Two cofactor systems were capable of stimulating activity; the system of NADH and FMN required anaerobic conditions while the other system, composed of iron and cysteine and/or ascorbate functioned in the presence or absence of oxygen. The components of each cofactor system functioned synergistically and kinetic parameters were established for each. Of several amine compounds common to fish muscle, TMAO was the only substrate demethylated by the microsomes. Activity was inhibited by iodoacetamide, potassium cyanide, and sodium azide under certain conditions, but not by carbon monoxide. An enzymic nature of the reaction was demonstrated by the properties of heat lability, sensitivity to protease treatment, the requirement of microsomes for TMAO demethylation and by the exhibition of typical hyperbolic kinetics with respect to substrate (TMAO). Moreover, TMAO demethylation by the microsomes was 3 to 4 orders of magnitude faster than the non-enzymic reaction and the reaction was specific for dimethylamine (DMA) as product. It appears the two cofactor systems may share a common catalytic unit in the process of TMAO demethylation.     

Characterization of arylsulfatase C isozymes from human liver and placenta

Arylsulfatase C and steroid sulfatase were thought to be identical enzymes. However, recent evidence showed that human arylsulfatase C consists of two isozymes, s and f. In this study, the biochemical properties of the s form partially purified from human placenta were compared with those of the f form from human liver. Only the placental s form has steroid sulfatase activity and hydrolyses estrone sulfate, dehydroepiandrosterone sulfate and cholesterol sulfate. The liver f form has barely detectable activity towards these sterol sulfates. With the artificial substrate, 4-methylumbelliferyl sulfate, both forms demonstrated a similar KM but the liver enzyme has a pH optimum of 6.9 while the placental form displayed two optima at 7.3 and 5.5. The molecular weight of the native enzyme determined with gel filtration was 183,000 for the s form and 200,000 for the f form and their pI's were also similar at 6.5. However, the T50, temperature at which half of the enzyme activity was lost, was 49.5 degrees C for the f form and 56.8 degrees C for the s form. Polyclonal antibodies raised against the placental form reacted specifically against the s and not the f form. They immuno-precipitated concomitantly greater than 80% of the total placental arylsulfatase C and steroid sulfatase activities while less than 20% of the liver enzyme was immuno-precipitable. In conclusion, the two isozymes s and f of arylsulfatase C in humans purified from placenta and liver, respectively, have similar KM, pI' and native molecular weight. However, they are distinct proteins with different substrate specificity, pH optima, heat-lability and antigenic properties. Only the s form is confirmed to be steroid sulfatase.     

Induction of the ethanol-inducible form of rabbit liver microsomal cytochrome P-450 by inhibitors of alcohol dehydrogenase

Cytochrome P-450 LMeb was purified from liver microsomes obtained from rabbits treated with either benzene or imidazole and was shown to have identical N-terminal amino acid sequence as that of cytochrome P-450 LM3a. The amino acid compositions of the proteins were indistinguishable. Quantitation of P-450 LMeb in various types of microsomes using radial immunodiffusion, revealed that pyrazole- or imidazole-treatment of the animals caused a 2-3-fold induction of the enzyme, accompanied by 2-3-fold increases of the rates of ethanol and aniline oxidation.     

Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.

Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.     

Characterization of human stellate cell activation-associated protein and its expression in human liver

This study cloned cDNA of human homologue (hSTAP) of rat stellate cell activation-associated protein (rSTAP). hSTAP gene is on chromosome 17q and is composed of four exons. Various types of cells including hepatic stellate cells expressed hSTAP mRNA. Recombinant hSTAP was a heme protein with the activity of peroxidase. hSTAP can be used as a marker of quiescent stellate cells in human liver.