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Query: UNIPROT:P06889 (Mol)
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Alloxan at millimolar concentrations slightly inhibited the velocity of Ca2+ uptake by isolated rat liver mitochondria irrespective of the free Ca2+ concentration between 1 and 10 microM and was an effective concentration-dependent stimulator of mitochondrial Ca2+ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca2+ uptake but only at free Ca2+ concentrations above 5 microM. However, ninhydrin was a strong stimulator of mitochondrial Ca2+ efflux even at micromolar concentrations, 10-50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10-20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca2+ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca2+ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca2+ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca2+ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca2+ uptake. The direct effects of ninhydrin on mitochondrial Ca2+ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca2+ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca2+ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan.
Mol Cell Biochem 1992 Dec 16
PMID:Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport. 129 9

A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.
Mol Cell Biol 1992 May
PMID:Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription. 131 49

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.
Mol Ecol 1992 Aug
PMID:Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2,4-dichlorophenoxyacetate. 134 88

We describe a novel transcriptional suppressor element found in the control region of the gene that encodes rat microsomal epoxide hydrolase (mEH), an inducible xenobiotic metabolizing enzyme. This element consists of the juxtaposition of two distinct factor-binding regions. The first region is composed of a series of five tandemly repeated factor-binding sequences, and the second region is an unique AT-rich factor-binding sequence. Although each region binds its cognate factor(s) in vitro, a single region does not function as a suppressor independently of the other. Transcriptional suppression was observed only when the two regions were combined. Thus, we propose that this regulatory element is a bipartite suppressor, requiring two distinct factor-binding regions for its function. The element displayed position-independent but orientation-dependent suppressor activity. The level of suppressor activity was proportional to the number of repetitive sites in region 1. We speculate that this region could mediate the dose-response behavior of mEH gene expression induced by chemical carcinogens in vivo. A qualitative difference in the region 2 binding factor(s) was observed between normal liver cells and a hepatoma cell line or carcinogen-treated liver cells. The possible relationship between this observation and the deregulation of mEH gene expression during the course of hepatocarcinogenesis is discussed.
Mol Cell Biol 1992 Oct
PMID:A bipartite suppressor: conjunction of two distinct factor-binding sites is essential for down-regulation in rat epoxide hydrolase gene expression. 140 38

We isolated a mutant mammalian cell line lacking activity for the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). The mutant was isolated through its resistance to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (HmdUrd). The mutant incorporates HmdUrd into DNA to the same extent as the parent line but, lacking the repair enzyme, does not remove it. The phenotype of the mutant demonstrates that the toxicity of HmdUrd does not result from substitution of thymine in DNA by HmUra but rather from the removal via base excision of large numbers of HmUra residues in DNA. This finding elucidates a novel mechanism of toxicity for a xenobiotic nucleoside. Furthermore, the isolation of this line supports our hypothesis that the enzymatic repairability of HmUra derives not from its formation opposite adenine via the oxidation of thymine, but rather from its formation opposite guanine as a product of the oxidation and subsequent deamination of 5-methylcytosine.
Mol Cell Biol 1992 Dec
PMID:A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine. 144 84

Evidence is reported for the existence of a structurally and functionally related and probably evolutionarily conserved class of membrane-bound liver carbonyl reductases/hydroxysteroid dehydrogenases involved in steroid and xenobiotic carbonyl metabolism. Carbonyl reduction was investigated in liver microsomes of 8 vertebrate species, as well as in insect larvae total homogenate and in purified 3 alpha-hydroxysteroid dehydrogenase preparations of the procaryont Pseudomonas testosteroni, using the ketone compound 2-methyl-1,2 di-(3-pyridyl)-1-propanone (metyrapone) as substrate. The enzyme activities involved in the metyrapone metabolism were screened for their sensitivity to several steroids as inhibitors. In all fractions tested, steroids of the adrostane or pregnane class strongly inhibited xenobiotic carbonyl reduction, whereas only in the insect and procaryotic species could ecdysteroids inhibit this reaction. Immunoblot analysis with antibodies against the respective microsomal mouse liver metyrapone reductase revealed strong crossrections in all fractions tested, even in those of the insect and the procaryont. A similar crossreaction pattern was achieved when the same fractions were incubated with antibodies against 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The mutual immunoreactivity of the antibody species against proteins from vertebrate liver microsomes, insects and procaryonts suggests the existence of structural homologies within these carbonyl reducing enzymes. This is further confirmed by limited proteolysis of purified microsomal mouse liver carbonyl reductase and subsequent analysis of the peptide fragments with antibodies specifically purified by immunoreactivity against this respective crossreactive antigen. These immunoblot experiments revealed a 22 kDa peptide fragment which was commonly recognized by all antibodies and which might represent a conserved domain of the enzyme.
J Steroid Biochem Mol Biol 1992 Dec
PMID:Homologies between enzymes involved in steroid and xenobiotic carbonyl reduction in vertebrates, invertebrates and procaryonts. 147 59

While the liver consists of both parenchymal cells (PC) and nonparenchymal cells (NPC), virtually all studies on promutagen activation have been performed using PC. To evaluate the comparative roles of PC and NPC in promutagen activation, we cocultivated a cell line generally considered to have an insignificant level of xenobiotic metabolism, Chinese hamster ovary (CHO) cells, with either PC, NPC, or a combination of both. The mixed culture was treated with two promutagens: dimethylnitrosamine (DMN) and 3-methylcholanthrene (3-MC). The induction of 6-thioguanine resistant mutants was evaluated using the well-established CHO/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay. Activation of promutagens, as indicated by an increase in mutant frequency in CHO cells, was observed only when the PC were present with the CHO cells during the treatment period. No activation was observed with NPC. Coculturing of PC and NPC yielded essentially the same results as PC alone. P-450 mixed function monooxygenase activity measured by the 7-ethoxycoumarin-O-deethylase assay further substantiates that PC had a significantly higher xenobiotic metabolism activity than NPC. Our study therefore indicates that PC, not NPC, are the major cell population in the liver responsible for the activation of promutagens.
Environ Mol Mutagen 1992
PMID:Comparison of rat liver parenchymal and nonparenchymal cells in the activation of promutagens. 150 29

Aldehydes are highly reactive molecules that may have a variety of effects on biological systems. They can be generated from a virtually limitless number of endogenous and exogenous sources. Although some aldehyde-mediated effects such as vision are beneficial, many effects are deleterious, including cytotoxicity, mutagenicity, and carcinogenicity. A variety of enzymes have evolved to metabolize aldehydes to less reactive forms. Among the most effective pathways for aldehyde metabolism is their oxidation to carboxylic acids by aldehyde dehydrogenases (ALDHs). ALDHs are a family of NADP-dependent enzymes with common structural and functional features that catalyze the oxidation of a broad spectrum of aliphatic and aromatic aldehydes. Based on primary sequence analysis, three major classes of mammalian ALDHs--1, 2, and 3--have been identified. Classes 1 and 3 contain both constitutively expressed and inducible cytosolic forms. Class 2 consists of constitutive mitochondrial enzymes. Each class appears to oxidize a variety of substrates that may be derived either from endogenous sources such as amino acid, biogenic amine, or lipid metabolism or from exogenous sources, including aldehydes derived from xenobiotic metabolism. Changes in ALDH activity have been observed during experimental liver and urinary bladder carcinogenesis and in a number of human tumors, including some liver, colon, and mammary cancers. Changes in ALDH define at least one population of preneoplastic cells having a high probability of progressing to overt neoplasms. The most common change is the appearance of class 3 ALDH dehydrogenase activity in tumors arising in tissues that normally do not express this form. The changes in enzyme activity occur early in tumorigenesis and are the result of permanent changes in ALDH gene expression. This review discusses several aspects of ALDH expression during carcinogenesis. A brief introduction examines the variety of sources of aldehydes. This is followed by a discussion of the mammalian ALDHs. Because the ALDHs are a relatively understudied family of enzymes, this section presents what is currently known about the general structural and functional properties of the enzymes and the interrelationships of the various forms. The remainder of the review discusses various aspects of the ALDHs in relation to tumorigenesis. The expression of ALDH during experimental carcinogenesis and what is known about the molecular mechanisms underlying those changes are discussed. This is followed by an extended discussion of the potential roles for ALDH in tumorigenesis. The role of ALDH in the metabolism of cyclophosphamidelike chemotherapeutic agents is described. This work suggests that modulation of ALDH activity may an important determinant of the effectiveness of certain chemotherapeutic agents.(ABSTRACT TRUNCATED AT 400 WORDS)
Crit Rev Biochem Mol Biol 1992
PMID:Aldehyde dehydrogenases and their role in carcinogenesis. 152 60

Rat and human lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic activities towards several xenobiotic chemicals, including procarcinogens, in different microsomal preparations. Rat lung microsomal P-450s were more active than the human P-450s in catalyzing most of the monooxygenation reactions. Human lung microsomal P-450 was solubilized and purified. Human lung microsomes contain approximately 10 pmol of P-450/mg of protein, on the basis of Fe2+.CO versus Fe2+ difference spectra of the eluates obtained from an octylamino-agarose column. The partially purified P-450 preparations from two human lung microsomal samples showed high activities for the conversion of both (+)- and (-)-isomers of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to genotoxic products. After DEAE-cellulose column chromatography, a partially purified P-450 fraction containing polypeptides of Mr 52,000 and 58,000 was obtained from the early fraction of the octylamino-agarose column eluate, and an electrophoretically homogeneous protein having a molecular weight of approximately 52,000 was recovered from a latter fraction. The amino-terminal amino acid sequences of the two peptides in the earlier fraction were determined; neither polypeptide appears to resemble any known P-450 protein. The protein from the latter octylamino-agarose fraction was immunoreactive with anti-rat P-450 1A2 and anti-human P-450 1A2 but not with antibodies raised against other P-450 enzymes or autoimmune antibodies that specifically recognize human P-450 1A2. A tryptic peptide was isolated from the preparation, and the amino acid sequence matched that of human P-450 1A1 perfectly (residues 31-48) but not that of human P-450 1A2. All of nine human lung microsomal samples examined contained proteins that were immunoreactive with rabbit anti-rat P-450 1A2 and catalyzed the activation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene. The activities could be inhibited by rabbit anti-rat P-450 1A2 and, to a lesser extent, by anti-rat P-450 1A1. The addition of 7,8-benzoflavone caused inhibition or stimulation, depending upon the particular human lung microsomal preparation. Thus, this work clearly shows that human lung microsomes contain at least two major P-450 enzymes; human P-450 1A1 is present in lungs and can actually catalyze the activation of environmental procarcinogens, including polycyclic aromatic hydrocarbons.
Mol Pharmacol 1992 May
PMID:Characterization of human lung microsomal cytochrome P-450 1A1 and its role in the oxidation of chemical carcinogens. 158 20

The resting pH of 7.14 +/- 0.02 within rat cortical synaptosomes is elevated in vitro by the insecticide chlordecone, in a dose-dependent manner. Chlordecone also reduces the rate of oxygen radical formation within synaptosomes. Both of these changes can also be demonstrated following in vivo treatment of rats with chlordecone (75 mg/kg body wt). Although chlordecone increases the permeability of the plasma membrane, the increase in pH observed is unlikely to be caused by this, since in vivo administration of chlordecone does not appreciably alter membrane order as evaluated by both a lipophilic probe, and a probe with an ionic segment. Another xenobiotic agent, methyl mercuric chloride, and a free radical generating system, an ascorbic acid-ferrous sulfate mixture, did not modulate synaptosomal pH, although membrane permeability was increased. Other evidence of the ability of synaptosomes to maintain homeostasis was the failure of mitochondrial inhibitors to significantly reduce pH. The drop in synaptosomal pH effected by amiloride, an inhibitor of Na+/H+ exchange, and the transient rise in pH caused by ammonium chloride further suggested that synaptosomes may be a good model in the study of the regulation of intracellular pH. The elevation of cytosolic pH, and depression of oxygen radical formation by chlordecone, may result from both the attenuation of respiratory metabolism and an impaired capacity of the plasma membrane to maintain ionic gradients.
Mol Chem Neuropathol
PMID:Changes in synaptosomal pH and rates of oxygen radical formation induced by chlordecone. 171 Apr 60


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