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1.
Eur J Clin Nutr ; 70(3): 358-63, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26373962

ABSTRACT

BACKGROUND/OBJECTIVES: There are several biomarkers for measuring iron deficiency (ID) in pregnancy, but the prevalence of ID and its association with inflammation and adverse pregnancy outcomes is inconclusive. The aim of this work was to describe the prevalence and determinants of first trimester ID and associations with pregnancy and birth outcomes. SUBJECTS/METHODS: A record-linkage cohort study of archived serum samples of women attending first trimester screening and birth and hospital data to ascertain maternal characteristics and pregnancy outcomes. Sera were analysed for iron stores (ferritin; µg/l), lack of iron in the tissues (soluble transferrin receptor (sTfR); nmol/l) and inflammatory (C-reactive protein (CRP); mg/dl) biomarkers. Total body iron (TBI) was calculated from serum ferritin (SF) and sTfR concentrations. Multivariate logistic regression analysed risk factors and pregnancy outcomes associated with ID using the definitions: SF<12 µg/l, TfR ⩾ 21.0 nmol/l, and TBI<0 mg/kg. RESULTS: Of the 4420 women, the prevalence of ID based on ferritin, sTfR and TBI was 19.6, 15.3 and 15.7%, respectively. Risk factors of ID varied depending on which iron parameter was used and included maternal age <25 years, multiparity, socioeconomic disadvantage, high maternal body weight and inflammation. ID, defined by SF and TBI but not TfR, was associated with reduced risk of gestational diabetes mellitus (GDM). ID defined using TBI only was associated with increased risk of large-for-gestation-age (LGA) infants. CONCLUSIONS: Nearly one in five Australian women begin pregnancy with ID. Further investigation of excess maternal weight and inflammation in the relationships between ID and GDM and LGA infants is needed.


Subject(s)
Anemia, Iron-Deficiency/epidemiology , Ferritins/blood , Pregnancy Outcome , Receptors, Transferrin/blood , Adult , Anemia, Iron-Deficiency/blood , Anemia, Iron-Deficiency/complications , Australia/epidemiology , Biomarkers/blood , C-Reactive Protein/metabolism , Cohort Studies , Diabetes, Gestational/blood , Diabetes, Gestational/epidemiology , Female , Humans , Infant, Newborn , Iron/blood , Logistic Models , Multivariate Analysis , Pregnancy , Prevalence , Risk Factors , Socioeconomic Factors
2.
Diabet Med ; 33(9): 1211-21, 2016 09.
Article in English | MEDLINE | ID: mdl-26670627

ABSTRACT

AIM: High iron measured using dietary intake and biomarkers is associated with Type 2 diabetes. It is uncertain whether a similar association exists for gestational diabetes mellitus. The aim of this systematic review was to conduct a cohort study examining first trimester body iron stores and subsequent risk of gestational diabetes, and to include these findings in a systematic review of all studies examining the association between maternal iron status, iron intake (dietary and supplemental) and the risk of gestational diabetes. METHODS: Serum samples from women with first trimester screening were linked to birth and hospital records for data on maternal characteristics and gestational diabetes diagnosis. Blood was analysed for ferritin, soluble transferrin receptor and C-reactive protein. Associations between iron biomarkers and gestational diabetes were assessed using multivariate logistic regression. A systematic review and meta-analysis, registered with PROSPERO (CRD42014013663) included studies of all designs published in English from January 1995 to July 2015 that examined the association between iron and gestational diabetes and included an appropriate comparison group. RESULTS: Of 3776 women, 3.4% subsequently developed gestational diabetes. Adjusted analyses found increased odds of gestational diabetes for ferritin (OR 1.41; 95% CI 1.11, 1.78), but not for soluble transferrin receptor (OR 1.00; 95% CI 0.97, 1.03) per unit increase of the biomarker. Two trials of iron supplementation found no association with gestational diabetes. Increased risk of gestational diabetes was associated with higher levels of ferritin and serum iron and dietary haem iron intakes. CONCLUSIONS: Increased risk of gestational diabetes among women with high serum ferritin and iron levels and dietary haem iron intakes warrants further investigation.


Subject(s)
C-Reactive Protein/metabolism , Diabetes, Gestational/epidemiology , Dietary Supplements , Ferritins/metabolism , Iron, Dietary/therapeutic use , Receptors, Transferrin/metabolism , Adult , Diabetes, Gestational/metabolism , Female , Humans , Logistic Models , Multivariate Analysis , New South Wales/epidemiology , Odds Ratio , Pregnancy , Prospective Studies , Risk Factors , Young Adult
3.
Toxicol Appl Pharmacol ; 266(3): 439-42, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23201461

ABSTRACT

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.


Subject(s)
Chemical Warfare Agents/toxicity , Glutamate-Cysteine Ligase/biosynthesis , Mustard Gas/analogs & derivatives , Mustard Gas/toxicity , Skin/drug effects , Thiocyanates/pharmacology , Animals , Enzyme Induction/drug effects , Female , Glutathione/biosynthesis , Glutathione Transferase/biosynthesis , Immunoblotting , Isothiocyanates , Mice , Mice, Inbred C57BL , Mutation , Skin/enzymology , Skin/metabolism , Sulfoxides
4.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 67(Pt 10): 1179-83, 2011 Oct 01.
Article in English | MEDLINE | ID: mdl-22102022

ABSTRACT

Respiratory syncytial virus (RSV) is a frequent cause of respiratory illness in infants, but there is currently no vaccine nor effective drug treatment against this virus. The RSV RNA genome is encapsidated and protected by a nucleocapsid protein; this RNA-nucleocapsid complex serves as a template for viral replication. Interest in the nucleocapsid protein has increased owing to its recent identification as the target site for novel anti-RSV compounds. The crystal structure of human respiratory syncytial virus nucleocapsid (HRSVN) was determined to 3.6 Å resolution from two crystal forms belonging to space groups P2(1)2(1)2(1) and P1, with one and four decameric rings per asymmetric unit, respectively. In contrast to a previous structure of HRSVN, the addition of phosphoprotein was not required to obtain diffraction-quality crystals. The HRSVN structures reported here, although similar to the recently published structure, present different molecular packing which may have some biological implications. The positions of the monomers are slightly shifted in the decamer, confirming the adaptability of the ring structure. The details of the inter-ring contacts in one crystal form revealed here suggest a basis for helical packing and that the stabilization of native HRSVN is via mainly ionic interactions.


Subject(s)
Nucleocapsid Proteins/chemistry , Respiratory Syncytial Virus, Human/chemistry , Crystallography, X-Ray , Models, Molecular , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Protein Structure, Secondary , RNA, Viral/chemistry
5.
Neurobiol Dis ; 42(1): 48-54, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21220022

ABSTRACT

Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.


Subject(s)
Calcium Channels/biosynthesis , Epilepsy/genetics , Neurons/metabolism , Receptors, AMPA/biosynthesis , Somatosensory Cortex/metabolism , Animals , Calcium Channels/genetics , Cell Membrane/genetics , Cell Membrane/pathology , Cell Membrane/physiology , Disease Models, Animal , Epilepsy/pathology , Epilepsy/physiopathology , Genetic Predisposition to Disease , Neurons/pathology , Neurons/physiology , Phenotype , Rats , Rats, Mutant Strains , Receptors, AMPA/genetics , Somatosensory Cortex/pathology , Somatosensory Cortex/physiopathology
6.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 64(Pt 11): 1019-23, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18997331

ABSTRACT

Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length selenomethionine-labelled HRSVN were obtained that diffracted to 3.6 and approximately 5 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 133.6, b = 149.9, c = 255.1 A, and space group P2(1), with unit-cell parameters a = 175.1, b = 162.6, c = 242.8 A, beta = 90.1 degrees , respectively. For unlabelled HRSVN, only crystals belonging to space group P2(1) were obtained that diffracted to 3.6 A. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced.


Subject(s)
Nucleocapsid Proteins/chemistry , Respiratory Syncytial Virus, Human/chemistry , Crystallization , Humans , Infant , Models, Molecular , Molecular Sequence Data , Protein Structure, Quaternary , Respiratory Syncytial Virus, Human/genetics , X-Ray Diffraction
7.
Neurobiol Dis ; 31(2): 261-5, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18556211

ABSTRACT

Stargazin is membrane bound protein involved in trafficking, synapse anchoring and biophysical modulation of AMPA receptors. A quantitative trait locus in chromosome 7 containing the stargazin gene has been identified as controlling the frequency and duration of absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Furthermore, mutations in this gene result in the Stargazer mouse that displays an absence epilepsy phenotype. GAERS stargazin mRNA expression is increased 1.8 fold in the somatosensory cortex and by 1.3 fold in the thalamus. The changes were present before and after the onset of absence seizures indicating that increases are not a secondary consequence of the seizures. Stargazin protein expression was also significantly increased in the somatosensory cortex after the onset of spontaneous seizures. The results are of significant importance beyond the GAERS model, as they are the first to show that an increase in stargazin expression may be pro-epileptic.


Subject(s)
Calcium Channels/metabolism , Cerebral Cortex/metabolism , Epilepsy, Absence/metabolism , Thalamus/metabolism , Up-Regulation/genetics , Animals , Calcium Channels/genetics , Cerebral Cortex/physiopathology , Disease Models, Animal , Epilepsy, Absence/genetics , Epilepsy, Absence/physiopathology , Genetic Predisposition to Disease/genetics , Mutation/genetics , Neural Pathways/metabolism , Neural Pathways/physiopathology , RNA, Messenger/metabolism , Rats , Rats, Mutant Strains , Somatosensory Cortex/metabolism , Somatosensory Cortex/physiopathology , Thalamus/physiopathology
8.
Neuropharmacology ; 46(6): 836-46, 2004 May.
Article in English | MEDLINE | ID: mdl-15033343

ABSTRACT

The mechanism underlying the development of tolerance to morphine is still incompletely understood. Morphine binds to opioid receptors, which in turn activates downstream second messenger cascades through heterotrimeric guanine nucleotide binding proteins (G proteins). In this paper, we show that G(z), a member of the inhibitory G protein family, plays an important role in mediating the analgesic and lethality effects of morphine after tolerance development. We blocked signaling through the G(z) second messenger cascade by genetic ablation of the alpha subunit of the G protein in mice. The Galpha(z) knockout mouse develops significantly increased tolerance to morphine, which depends on Galpha(z) gene dosage. Further experiments demonstrate that the enhanced morphine tolerance is not caused by pharmacokinetic and behavioural learning mechanisms. The results suggest that G(z) signaling pathways are involved in transducing the analgesic and lethality effects of morphine following chronic morphine treatment.


Subject(s)
Drug Tolerance/genetics , GTP-Binding Protein alpha Subunits , GTP-Binding Proteins/deficiency , GTP-Binding Proteins/genetics , Gene Deletion , Morphine/pharmacology , Protein Subunits/deficiency , Protein Subunits/genetics , Animals , Dose-Response Relationship, Drug , Female , GTP-Binding Proteins/physiology , Genotype , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pain Threshold/drug effects , Pain Threshold/physiology , Protein Subunits/physiology , Second Messenger Systems/drug effects , Second Messenger Systems/genetics
9.
J Med Chem ; 44(1): 78-93, 2001 Jan 04.
Article in English | MEDLINE | ID: mdl-11141091

ABSTRACT

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.


Subject(s)
Guanylate Cyclase/metabolism , Indazoles/chemical synthesis , Nitric Oxide/metabolism , Pyrazoles/chemical synthesis , Animals , Enzyme Activation , Humans , In Vitro Techniques , Indazoles/chemistry , Indazoles/pharmacokinetics , Indazoles/pharmacology , Male , Platelet Aggregation Inhibitors/chemical synthesis , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/pharmacokinetics , Platelet Aggregation Inhibitors/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Rats , Rats, Sprague-Dawley , Solubility , Structure-Activity Relationship
10.
Circulation ; 102(7): 779-85, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10942747

ABSTRACT

BACKGROUND: Human herpesviruses have been implicated but not proven to be involved in the etiology of atherosclerosis. To determine whether there is a causal relationship, the effect of herpesvirus infection on the development of atherosclerosis was assessed in the apolipoprotein E-deficient (apoE-/-) mouse. METHODS AND RESULTS: In the present study, 3- to 4-week-old apoE-/- mice were infected with murine gamma-herpesvirus-68 (MHV-68). Atheroma formation was accelerated over a 24-week period in infected apoE-/- mice compared with control uninfected apoE-/- mice. Acceleration of atherosclerosis was reduced by antiviral drug administration. Histological analysis of the atheromatous plaques showed no difference between lesions of infected and control mice. Viral mRNA was present in the aortas of infected mice before lesion development on day 5 after infection. This suggests that the virus may initiate endothelial injury, which is believed to be an early event in the development of atherosclerosis. Therefore, the virus may play a direct role in atherosclerosis rather than be an "innocent bystander." CONCLUSIONS: These data demonstrate that a gamma-herpesvirus can accelerate atherosclerosis in the apoE-/- mouse. This study provides the first report of a murine model in which to study the causative role of herpesvirus infection in the development of atherosclerosis.


Subject(s)
Apolipoproteins E/deficiency , Arteriosclerosis/pathology , Arteriosclerosis/virology , Herpesviridae Infections/complications , Animals , Antibody Formation , Antiviral Agents/therapeutic use , Aorta/metabolism , Aorta/pathology , Apolipoproteins E/genetics , Cholesterol/blood , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Herpesviridae/genetics , Herpesviridae/isolation & purification , Herpesviridae Infections/drug therapy , Herpesviridae Infections/pathology , Herpesviridae Infections/virology , Mice , Mice, Knockout/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolism , Time Factors
11.
Diabetes Metab ; 24(3): 244-50, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9690058

ABSTRACT

We report a study of 10 candidate genes presumably involved in diabetes or insulin resistance or obesity among Pondicherian Tamil Indians, an isolated population with a high prevalence of diabetes. Forty-nine families with at least two affected patients in the sibship (567 individuals) were selected and tested by PCR-RFLP techniques for reported mutations in 10 diabetes or obesity candidate genes: glucagon receptor, insulin receptor substrate 1, insulin receptor, human beta 3 adrenergic receptor, fatty acid binding protein 2, mitochondrial tRNA(Leu(UUR)), sulphonylurea receptor, human uncoupling protein and the glycogen-associated regulatory subunit of protein phosphatase-1. Glucokinase gene was also screened for mutations. No mutations were found in glucokinase, glucagon receptor and mitochondrial genes in any of the 49 probands. Frequencies of polymorphisms at other loci were similar to those reported in Caucasian populations, except for 4 of the loci at which a higher frequency of variants was observed: human beta 3 adrenergic receptor, human uncoupling type 1 protein, fatty acid binding protein 2 and the glycogen-associated regulatory subunit of protein phosphatase-1. However, no evidence of association between any of these gene variants and non-insulin-dependent diabetes mellitus (NIDDM) or quantitative traits related to NIDDM (including body mass index, waist/hip ratio, insulinaemia, glycaemia, triglycerides and total cholesterol) was found in our sample. These results suggest that none of these gene variants commonly found in the Pondicherian Tamil population of South India is a major NIDDM predisposing locus, although it cannot be excluded that they may contribute to the polygenic background of the metabolic syndrome in Pondichery.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Gene Frequency , Genetic Testing , Polymorphism, Genetic , Adult , Aged , Aged, 80 and over , Analysis of Variance , Chromosome Mapping , Humans , India , Middle Aged , Mutation
12.
Toxicology ; 125(1): 1-11, 1998 Jan 16.
Article in English | MEDLINE | ID: mdl-9585095

ABSTRACT

2,6-Dithiopurine (DTP) has been proposed as a possible chemopreventive agent because of its ability to react with electrophiles. Acrolein, an electrophilic metabolite of cyclophosphamide (CP) involved in the toxicities of this anticancer drug, can be scavenged by DTP. The present study examined the effect of DTP treatment on CP-mediated bladder and lung toxicity in male ICR mice. Mice fed a diet containing 4% DTP that were treated intraperitoneally (i.p.) with 350 mg/kg CP showed no significant bladder damage (measured as bladder blood content at 48 h) with respect to the group fed a control diet. DTP (50 and 100 mg/kg), given i.p. 0.5 and 7 h after the initial injection of CP, also prevented the bladder damage when compared with the group receiving CP alone. Surprisingly, although neither parenteral CP nor DTP alone caused any mortality at these doses, the combined treatment resulted in 67% mortality within 3 days. At 24 h after CP + DTP, blood urea nitrogen was elevated 6-fold and urine volumes decreased by 70%. Histopathological analyses revealed a diffuse myocardial degeneration and necrosis, severe granular degeneration in the liver, abundant cellularity and infiltrates in interalveolar spaces in the lung and swollen nephron epithelial cells with some necrosis. All mice survived treatment when the dose of CP was lowered to 250 and 25-75 mg/kg DTP was given i.p. 0.5 and 7 h after CP. These DTP regimens reduced the degree of CP-induced lung toxicity, measured by [3H]thymidine incorporation into lung DNA 7 days after CP, in a dose-dependent manner. DTP (75 mg/kg) also reduced CP-induced lung fibrosis estimated by lung hydroxyproline content 28 days after CP. Analyses of urine from mice given CP + DTP revealed large amounts of the metabolic product dithiouric acid, smaller amounts of the parent DTP and several smaller peaks. The major unique metabolite peak was collected and analyzed by mass spectrometry, but did not correspond to either acrolein-DTP or acrolein-dithiouric acid. Thus, either very small amounts of an acrolein adduct are generated, the adduct is broken down to an unidentified product, or the ability of DTP to prevent CP-induced lung and bladder damage is related to some other mechanism. The possibility that mercapturic acid metabolites of acrolein released the parent electrophile in the urine was not supported by the finding that probenecid did not prevent CP-induced bladder toxicity.


Subject(s)
Antineoplastic Agents, Alkylating/toxicity , Cyclophosphamide/toxicity , Cystitis/drug therapy , Lung Diseases/drug therapy , Purines/therapeutic use , Animals , Cystitis/chemically induced , Cystitis/pathology , Liver/drug effects , Liver/pathology , Lung/drug effects , Lung/pathology , Lung Diseases/chemically induced , Lung Diseases/pathology , Male , Mice , Mice, Inbred ICR , Probenecid/therapeutic use , Purines/urine , Urinary Bladder/drug effects , Urinary Bladder/pathology
13.
Mol Carcinog ; 20(2): 216-23, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9364211

ABSTRACT

Previous studies indicated that DNA adducts formed by a carcinogenic diol epoxide, 7r,8t-dihydroxy-9t, 10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. It was suggested that adduct-induced bends in the DNA were responsible for this behavior. The cell cycle-regulated transcription factor E2F is also known to bend DNA upon binding. When partially purified E2F was tested in a gel mobility-shift assay, binding to a target DNA containing two consensus E2F-binding sites was enhanced by prior modification of the DNA with BPDE. Recombinant human E2F1, E2F4, and DP1 fusion proteins were affinity purified from bacteria expressing these genes. A combination of either E2F1 or E2F4 with their dimerization partner, DP1, gave preparations that exhibited binding to the E2F site-containing DNA fragment. In both cases, the proteins exhibited much higher apparent affinity for BPDE-modified DNA than for unmodified DNA. In addition, BPDE-modified DNA was a better competitor for the binding than unmodified DNA. Heterologous DNA that contained no consensus E2F binding motifs also competed well for E2F binding when modified with BPDE. In contrast, transcription factor that does not bend DNA appreciably (GAL4) did not show enhanced affinity for BPDE-modified DNA. These findings suggest that numerous transcription factors that bend DNA may bind with anomalously high affinity to sequences that contain carcinogen-DNA adducts.


Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/metabolism , Carrier Proteins , Cell Cycle Proteins , DNA Adducts/metabolism , DNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins , Transcription Factors/metabolism , Binding Sites , DNA Probes , Dimerization , E2F Transcription Factors , E2F1 Transcription Factor , E2F4 Transcription Factor , Electrophoresis , Fungal Proteins/metabolism , HeLa Cells , Humans , Retinoblastoma-Binding Protein 1 , Transcription Factor DP1
14.
Chem Res Toxicol ; 9(8): 1298-304, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8951232

ABSTRACT

Purinethiols are a class of potential cancer chemopreventive agents that exhibit nucleophilic scavenging activity against the carcinogenic electrophile benzo[a]pyrene diol epoxide (BPDE). Of the purinethiols tested previously, 2,6-dithiopurine (DTP), exhibited the highest scavenging activity for BPDE when tested either in vitro or in vivo. Sulfur-based nucleophiles are typically classified as "soft" nucleophiles, showing selectivity in nucleophilic substitution reactions for "soft", easily polarizable electrophiles. It was of interest to determine whether electrophilic toxicants other than BPDE react facilely with DTP, and whether 2,6-dithiouric acid (DUA), the major in vivo metabolite of DTP, also has scavenging activity. Four diverse toxicants tested in the present work, acrolein, melphalan, dimethyl sulfate, and cisplatin, all react facilely with DTP in vitro near neutral pH. These toxicants are expected to react as "soft" electrophiles. Furthermore, each of these compounds, as well as BPDE, reacts with DUA with rate constants comparable to the analogous rate constants for reaction with DTP. In contrast, several toxicants classified as "hard" electrophiles (ethyl methanesulfonate, methylnitrosourea, ethylnitrosourea, 1-methyl-3-nitro-1-nitrosoguanidine) show no appreciable reaction with DTP. These results suggest that both DTP and its major metabolite act as "soft" nucleophiles in nucleophilic substitution reactions and may be effective in scavenging a wide range of toxicants that react as "soft" electrophiles.


Subject(s)
Anticarcinogenic Agents/chemistry , Purines/chemistry , Toxins, Biological/chemistry , Uric Acid/analogs & derivatives , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/chemistry , Acrolein/chemistry , Chemical Phenomena , Chemistry, Physical , Cisplatin/chemistry , Kinetics , Melphalan/chemistry , Nitroso Compounds/chemistry , Uric Acid/chemistry
15.
Mol Carcinog ; 16(1): 44-52, 1996 May.
Article in English | MEDLINE | ID: mdl-8634093

ABSTRACT

Previous studies indicated that DNA adducts formed by the carcinogenic diol epoxide 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. Whether adducts that form in the normal binding site, the GC box sequence, increase the affinity of Sp1 for the modified GC-box was not determined. Starting with a 23-nt sequence that contains two natural GC box sequences, site-specifically modified oligonucleotides were prepared with a single(+)-BPDE-deoxyguanosine adduct at one of three positions: the center of each GC-box or in between the two boxes. Four modified oligonucleotides were studied, two derived from cis addition of BPDE to the exocyclic amino group and two from trans addition. For three of these site-specifically modified oligonucleotides, there was a diminution in Sp1 affinity, whereas Sp1 binding to the fourth modified oligonucleotide was abolished. Furthermore, random modification of the oligonucleotide to a level of about 1 BPDE adduct per fragment slightly decreased the affinity for Sp1, and no evidence was found for a subpopulation of molecules with high affinity. These findings suggest that BPDE modification of the GC box does not lead to an increased affinity for Sp1. This is consistent with a model in which a BPDE-induced bend in the DNA mimics the conformation of the normal GC box:Sp1 complex, leading to high-affinity binding of Sp1 to non-Gc box sites.


Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/metabolism , DNA Adducts/metabolism , Promoter Regions, Genetic , Sp1 Transcription Factor/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/chemistry , Base Sequence , Binding Sites , DNA-Binding Proteins/metabolism , Deoxyguanosine/analogs & derivatives , HeLa Cells , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/metabolism , Protein Binding
16.
Drug Metab Dispos ; 23(8): 854-60, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7493553

ABSTRACT

2,6-Dithiopurine (DTP) has been proposed as a possible chemopreventive agent because of its facile reaction with the electrophilic ultimate carcinogen, benzo[a]pyrene diol epoxide, and other reactive electrophiles. Previous studies in mouse skin indicated almost complete inhibition of benzo[a]pyrene diol epoxide-induced tumorigenesis by DTP, suggesting the possible utility of this compound as a chemopreventive agent. However, little is known of the metabolism of DTP or of its possible long-term toxicity. Mice were fed diets containing up to 4% DTP in AIN-76A for a period of 7 weeks, and possible toxicity was monitored by weight gain and histopathological examination of all major tissues. No toxicity was observed at any dose of DTP. DTP was found to be a good substrate in vitro for two enzymes known to metabolize 6-mercapto-purine: xanthine oxidase and thiopurine methyltransferase. The in vitro metabolites were 2,6-dithiouric acid and an apparent monomethylated derivative, respectively. In vivo, the major urinary metabolite was 2,6-dithiouric acid, which attained levels as high as 34 mM in the urine of mice receiving the 4% DTP diet. DTP was also excreted unchanged in the feces and urine. DTP, 2,6-dithiouric acid, and an unidentified, relatively nonpolar metabolite were also detected in the serum of experimental animals. Although large interindividual variation in the serum DTP concentration was found, there was a dose-dependent increase in serum DTP as the dietary level of DTP was increased. These results suggest that neither toxicity nor metabolism will severely limit the utility of DTP as a chemopreventive agent.


Subject(s)
Anticarcinogenic Agents/metabolism , Anticarcinogenic Agents/toxicity , Purines/metabolism , Purines/toxicity , Algorithms , Animals , Biotransformation , Diet , Feces/chemistry , Female , Injections, Intraperitoneal , Methyltransferases/metabolism , Mice , Mice, Inbred SENCAR , Weight Gain/drug effects , Xanthine Oxidase/metabolism
17.
Carcinogenesis ; 16(5): 975-83, 1995 May.
Article in English | MEDLINE | ID: mdl-7767996

ABSTRACT

Covalent binding of the carcinogen, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), to DNA causes changes in the conformation of the DNA around the site of the adduct. However, the influence of such carcinogen-DNA adducts on interactions of the DNA with specific proteins has received little attention. Binding of the transcription factor, Sp1, to GC-box sequences in the promoter of the hamster adenosine phosphoribosyl transferase gene is a useful model system. Electrophoretic mobility shift assays, competition experiments and DNase I footprinting demonstrated specific binding of affinity-purified, human Sp1 to two adjacent GC-boxes in the promoter fragment. Unexpectedly, modification of this DNA fragment to high levels (approximately 7% of the nucleotides) with BPDE caused a substantial (5- to 10-fold) increase in the apparent affinity of Sp1. A heterologous DNA fragment that contained no GC-boxes did not compete for the binding of Sp1 to the promoter, unless it was previously modified with BPDE. In addition, two DNA fragments that contained no GC-boxes exhibited Sp1-dependent mobility shifts only when modified by BPDE. DNase I footprinting of the BPDE-modified, Sp1-bound promoter fragment did not reveal specific sites of binding, suggesting that numerous BPDE-DNA adduct sites can interact with the protein. A model in which Sp1 binding to non-target sites is enhanced by a static bend or an induced flexibility at the site of an adduct is discussed.


Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/metabolism , DNA Adducts/metabolism , Sp1 Transcription Factor/metabolism , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/isolation & purification , Base Sequence , Binding Sites , Binding, Competitive , Consensus Sequence , DNA Adducts/isolation & purification , Deoxyribonuclease I , HeLa Cells , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/metabolism , Sp1 Transcription Factor/isolation & purification
19.
Genomics ; 21(1): 254-6, 1994 May 01.
Article in English | MEDLINE | ID: mdl-8088798

ABSTRACT

The gene encoding the human G-protein-coupled inwardly rectifying potassium channel designated GIRK1 (gene symbol, KCNJ3) was mapped to chromosome 2 by analyzing its segregation in a panel of human-hamster somatic cell hybrids. This assignment was confirmed by fluorescence in situ hybridization to metaphase chromosomes, and the gene was further localized to band 2q24.1. A highly informative simple tandem repeat DNA polymorphism of the form (CA)n was identified and used to localize KCNJ3 within the genetic map of the long arm of chromosome 2.


Subject(s)
Chromosomes, Human, Pair 2 , Polymorphism, Genetic , Potassium Channels/genetics , Receptors, Muscarinic/genetics , Repetitive Sequences, Nucleic Acid , Animals , Base Sequence , Chromosome Mapping , Cricetinae , GTP-Binding Proteins , Genes , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Lod Score , Male , Molecular Sequence Data
20.
Biochemistry ; 33(10): 2977-87, 1994 Mar 15.
Article in English | MEDLINE | ID: mdl-8130212

ABSTRACT

A metabolite of benzo[a]pyrene, 9-r,10-t-dihydroxy-7,8-c-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-III), that is not thought to be involved in carcinogenesis has nevertheless been shown to bind extensively to DNA in vitro. The adducts formed by this non-bay-region diol epoxide in Chinese hamster ovary cells are much less mutagenic than those formed by an isomeric diol epoxide that is carcinogenic. We have isolated and characterized three major adducts formed by in vitro reaction of BPDE-III with DNA. The major adduct, accounting for over half of the total is formed by reaction of BPDE-III with the N7 position of dGuo and is recovered after enzymatic digestion as an N7-Gua adduct. A second major adduct involves the N2 position of dGuo, while the third adduct is tentatively identified as a C8-substituted dGuo. Little or no reaction with deoxyadenosine residues is detected. The N7 adduct is unstable in DNA at 37 degrees C and is released as the modified base with a half-life of about 24 h. This adduct lability apparently leads to single-strand breaks and alkali-sensitive sites in the DNA and may account in part for some of the biological properties of BPDE-III adducts. This represents the first description of an N7-dGuo adduct that is formed in DNA as the major adduct by a diol epoxide derived from a carcinogenic polycyclic aromatic hydrocarbon.


Subject(s)
Benzopyrenes/metabolism , DNA Damage , DNA/chemistry , DNA/metabolism , Deoxyguanosine , Animals , Benzopyrenes/chemical synthesis , CHO Cells , Cricetinae , Magnetic Resonance Spectroscopy , Mass Spectrometry , Plasmids , Spectrometry, Mass, Fast Atom Bombardment
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