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R J Edwards

Publications and source records attributed to R J Edwards.

At least 73 records · Page 4Linked to original sources
PubMed1997

Specific inhibition of human CYP1A2 using a targeted antibody.

The structural similarity of related forms of P450 makes selective immunoinhibition of individual forms notoriously difficult to achieve. We report the use of a targeted antibody to overcome this problem. An antibody was raised against the synthetic peptide, Ser-Lys-Lys-Gly-Pro-Arg-Ala-Ser-Gly-Asn-Leu-Ile, corresponding to residues 291-302 of human CYP1A2. This sequence of human CYP1A2 is located in a similar position to a proinhibitory region previously identified in rat CYP1A1 and CYP1A2. The antibody bound strongly and specifically to CYP1A2 in human hepatic microsomal fraction. Binding was unaffected by denaturation of the protein. The specificity of the antibody was demonstrated by immunoblotting of human hepatic microsomal fraction where a single immunoreactive band was identified at Mr 54,000. The intensity of this band correlated strongly with high-affinity phenacetin O-deethylase activity of the microsomal fractions. In addition, the antibody bound to a single protein at Mr 54,000 in the microsomal fraction of lymphoblastoid cells expressing human CYP1A2, but not to any other recombinant P450 enzyme. CYP1A2-dependent activity (high-affinity phenacetin O-deethylase) of human hepatic microsomal fraction was inhibited >90% by whole antiserum or purified immunoglobulin. This decrease in activity represents complete inhibition of CYP1A2 activity, residual phenacetin O-deethylase activity being due to low-affinity enzymes. In contrast, the antibody, which does not bind to rat CYP1A2, had no effect on CYP1A2-dependent activity (high-affinity phenacetin O-deethylase) of rat hepatic microsomal fraction. The antiserum also had no effect on human hepatic microsomal debrisoquine 4-hydroxylase (CYP2D6) or coumarin 7-hydroxylase (CYP2A6) activities, indicating that inhibition was specific to human CYP1A2. These results demonstrate the importance of the region comprising residues 291-302 of human CYP1A2 in the catalytic activity of this enzyme.

Amino Acid Sequence↗
PubMed1997

Anti-HIV type 1 activity of sulfated derivatives of dextrin against primary viral isolates of HIV type 1 in lymphocytes and monocyte-derived macrophages.

The anti-HIV-1 activity of sulfated derivatives of dextrin was tested in activated peripheral blood mononuclear cells and in monocyte-derived macrophages using low-passage syncytium-inducing and non-syncytium-inducing primary viral isolates of HIV-1. All four compounds blocked infection in a dose-dependent manner. Dextrin 2-sulfate blocked infection with a 90% inhibitory concentration (IC90) of 69 microg ml(-1). The IC90 for dextrin 3-sulfate was 50 microg ml(-1) and for dextrin 6-sulfate was 14 microg ml(-1). Increasing the number of sulfate groups to three per glucan molecule (dextrin 2-, 3-, and 6-sulfate) did not reduce the IC90 further (13 microg ml(-1)) compared to dextrin 6-sulfate. There was no significant difference in the concentration required to block infection of activated peripheral blood mononuclear cells when compared with monocyte-derived macrophages, irrespective of whether low-passage syncytium-inducing or non-syncytium-inducing primary viral isolates of HIV-1 were used. Dextrin 2-sulfate and dextrin 6-sulfate also reduced the transmission of HIV-1 in experiments performed using peripheral blood mononuclear cells from HIV-1-positive patients by 6- to 251-fold in a limiting dilution tissue culture infectious dose assay. Sulfated dextrins were not toxic to either primary lymphocytes or macrophages at the concentrations tested. Having previously shown that the cell surface binding of sulfated dextrins is dependent on the position of the negatively charged sulfate groups, we now show that their anti-HIV-1 activity in primary lymphocytes and macrophages is also dependent on the same arrangement. A phase I/II clinical trial of dextrin 2-sulfate is now in progress.

Anti-HIV Agents↗
PubMed1997

Risk factors associated with the development of peripheral arterial disease in smokers: a case-control study.

PURPOSE AND METHOD: A hospital based case-control study was designed to investigate what aspects of smoking and what co-factors of smoking are associated with the development of peripheral arterial disease (PAD). Cases were 291 smokers, newly referred with PAD, and controls were 828 age and sex matched smokers without PAD. RESULTS: Reported recent tobacco usage was similar in cases and controls but total tobacco exposure was associated with the risk of PAD-adjusted odds ratios (ORs) increasing with tertile of pack-years smoked to reach 1.63 (95% CI, 1.11-2.39; P = 0.011), for the highest tertile ( > 48 pack-years) compared with smokers in the lowest tertile (< 31 pack-years). Cases reported smoking significantly lower tar and nicotine yield cigarettes than controls, but tended to inhale more deeply, and had significantly higher plasma concentrations of cotinine. ORs for PAD were significantly and independently increased by systolic blood pressure > 160 mmHg (8.1 (5.2 13.0); P < 0.0001), history of hypertension (2.4 (1.5-3.2); P = 0.0003) and apolipoprotein B > 0.9 g/l(3.8 (2.3-7.6); P = 0.008). CONCLUSIONS: Increased total exposure to tobacco and the ability to smoke tobacco in a way which maximises nicotine yield are associated with increased risk of smokers developing PAD. There is no evidence that smoking low tar cigarettes reduces this risk, whereas both hypertension (particularly systolic) and high levels of apolipoprotein B, increase this risk.

Aged↗
PubMed1997

Reversal of phenobarbital-induced hyperplasia and hypertrophy in the livers of lpr mice.

Fas is a cell surface receptor that mediates apoptosis, and Fas mRNA has been demonstrated in hepatocytes. MRL/MP-lpr/lpr mice carry the mutated lymphoproliferation-associated gene, lpr, that codes for truncated Fas protein, resulting in reduced apoptotic potential in some circumstances. Phenobarbital treatment of experimental animals induces cytochrome P450 enzymes, and thus acts as a growth stimulus to the liver with both hyperplasia and hypertrophy; cessation results in reversion of liver to normal size with apoptosis playing a role. This study has determined the respective contributions of atrophy and apoptosis to this involution in Fas-defective and normal-FAs bearing animals. Between the first day and the fifth day after phenobarbital cessation, the weights of both Fas-defective (lpr/lpr) livers and control (lpr/+) livers reduced. Hepatocyte hypertrophy gradually reverted in both categories of mouse and this was the greater contribution to reduction in liver size. In lpr/lpr animals, there was a consistent level of apoptosis which remained relatively constant, while numbers of apoptotic cells in control livers increased over the period. This investigation has shown that in liver, a mechanism to execute apoptosis is operative even in Fas-defective mice, but it is not sensitive to signals activated by the removal of the growth stimulus. This is in contrast to mice which can mount a Fas-mediated response; thus a separate apoptotic pathway is indicated.

Animals↗
PubMed1997

Expression and localization of CYP3A4 and CYP3A5 in human lung.

Expression in the lung of procarcinogen-metabolizing P450 enzymes in the CYP3A subfamily may contribute to the initiation of pulmonary carcinogenesis by agents that require metabolic activation, such as tobacco-derived polycyclic aromatic hydrocarbons. Expression and localization of CYP3A4 and CYP3A5 proteins in human lung were determined by immunohistochemistry with three antibodies, one specific for members of the CYP3A subfamily and two antipeptide antibodies specific for CYP3A4 and CYP3A5, respectively. Positive immunostaining in one or several cell types of the lung was observed in all patients with anti-CYP3A4 and anti-CYP3A5 antibodies. With the anti-CYP3A4 antibody epithelial staining was observed in five cases and staining of alveolar macrophages in 12 of 27 cases. To determine which CYP3A genes are transcribed in lung tissue, analysis by reverse-transcriptase-polymerase chain reaction with gene-specific primers for CYP3A4, CYP3A5, and CYP3A7 was performed. CYP3A5 mRNA was detected in all eight samples studied, CYP3A4 mRNA in one sample, and CYP3A7 mRNA in none of the samples. CYP3A5 was localized by immunohistochemistry in the ciliated and mucous cells of the bronchial wall, bronchial glands, bronchiolar columnar and terminal cuboidal epithelium, type I and type II alveolar epithelium, vascular and capillary endothelium, and alveolar macrophages, whereas CYP3A4 was found in bronchial glands, bronchiolar columnar and terminal epithelium, type II alveolar epithelium, and alveolar macrophages. These data establish that CYP3A5 is the predominant CYP3A form in human lung, that CYP3A4 is expressed in about 20% of individuals, and considerable variation of pulmonary expression occurs in both CYPs between individuals.

Bronchi↗
PubMed1996

Selective localisation of P450 enzymes and NADPH-P450 oxidoreductase in rat basal ganglia using anti-peptide antisera.

Environmental or endogenous toxins may cause nigral cell death in Parkinson's disease (PD) due to altered expression of P450 enzymes. In rat brain, immunohistochemistry using anti-peptide antisera showed NADPH-P450 oxidoreductase and CYP2B1/2 in various hypothalamic nuclei and CYP1A1 in the globus pallidus, but neither enzyme was expressed in substantia nigra. No specific immunoreactivity to CYP2D1 or CYP3A1 was found in any brain region examined. In contrast, CYP2E1 was expressed in substantia nigra and in striatal blood vessels. Since CYP2E1 is associated with free radical production, it may contribute to the oxidative stress believed to underlie nigral degeneration.

Alleles↗
PubMed1996

Inducibility of cytochromes P-450 by dioxin in liver and extrahepatic tissues of the marmoset monkey (Callithrix jacchus).

Cytochrome P-450 induction was investigated in the marmoset monkey, a non-human primate, using dioxins as inducing agents. Animals received a single subcutaneous dose of 1.6 nmol tetrachlorodibenzo-p-dioxin or tetrabromodibenzo-p-dioxin/kg body weight. Microsomal fractions were prepared from liver, lung and kidney, and homogenates were prepared from gut and adrenal glands. Anti-peptide antibodies which bind to CYP1A1, CYP1A2, CYP2B6 and CYP3A4 in human were used to identify related forms in the marmoset. The results indicate that CYP1A2 is constitutively expressed in liver, but not in lung, kidney, gut or adrenal gland and that CYP1A1 is not expressed in any of these tissues in untreated animals. Treatment with dioxin induced both CYP1A1 and CYP1A2 in liver, but only CYP1A1 in lung. No induction of CYP1A1 or CYP1A2 was found in kidney, small intestine or adrenal glands. Methoxy-, ethoxy-, pentoxy- and benzoyloxyresorufin O-dealkylases and high affinity phenacetin O-deethylase activities were induced in the liver, whereas ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities were not affected by dioxin treatment. High-affinity phenacetin O-deethylase and CYP1A2 apoprotein were detected only in liver, consistent with this activity being specifically catalysed by CYP1A2. Furafylline was found to be a competitive inhibitor of methoxyresorufin O-demethylase activity with a Ki of 10 microM. In the lung the induction of CYP1A1 was accompanied by 15- and 23-fold increases in ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase activities, respectively, suggesting that both activities are catalysed by CYP1A1. In contrast, there was no induction of aryl hydrocarbon hydroxylase activity in lung or liver showing that, unlike in many other species, marmoset CYP1A1 does not catalyse this reaction efficiently. The expression, distribution, induction and substrate specificities of marmoset monkey P-450 enzymes differ from the situation found in rodents and other species, demonstrating that caution has to be exercised when making cross-species extrapolations.

Animals↗
PubMed1996

Expression of xenobiotic-metabolizing cytochrome P450 forms in human full-term placenta.

The expression of individual xenobiotic-metabolizing cytochrome P450 (CYP) genes in human placenta was studied at the mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR). mRNAs of CYP1A1, CYP2E1, CYP2F1, CYP3A3/4, CYP3A5, and CYP4B1 were detected by RT-PCR, and CYP1A2, CYP2A6/7, CYP2B6/7, CYp2C8-19, CYP2D6, and CYp3A7 were not detected. Several enzyme activity assays and immunoblasts were used to further characterize expression of forms producing detectable mRNA transcripts. The catalytic activities of 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) were substantially increased in response to maternal cigarette smoking, and paralleled the amount of CYP1A1 mRNA and protein. Aromatase activities were slightly lower in placentas exposed to cigarette smoke compared with nonexposed placentas. These data show that several xenobiotic-metabolizing CYP genes are expressed in human placenta at a low level. The significant of such low-level expression is unknown, but it may have local physiological or toxic consequences.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1996

Enzymic and interindividual differences in the human metabolism of heterocyclic amines.

Heterocyclic amines (HAs) present in cooked meat (PhIP and MeIQx) are activated only by CYP1A2 in the liver of most species, including man. This enzyme exhibits marked interindividual differences in its expression, due to induction and possibly also genetically. The absence of CYP1A2 appears to protect from HA-(PhIP and MeIQx) induced cancer, as exemplified by results in the cynomolgus monkey. Differences in the potency of these HAs are not due to differences in the kinetics of their activation. The catalytic efficiency of CYP1A2 towards HAs and their oxidative fate varies amongst species, in both cases increasing the susceptibility of humans compared to that of the rat. Interindividual and inter-organ differences in the further metabolism of N-hydroxy-HAs appear to be important determinants of cancer susceptibility, as does the glutathione S-transferase catalysed detoxication of esters of N-hydroxy-PhIP. There is a need for an effective means of quantifying the in vivo activation of HAs in man to enable the possible risk posed by these compounds to be assessed effectively.

Animals↗
PubMed1996

A quantitative comparison of plaque types in Alzheimer's disease and senile dementia of the Lewy body type.

In a previous study we reported no difference in the overall beta-amyloid protein (beta AP) load between Alzheimer's disease (AD) and senile dementia of the Lewy body type (SDLT). However, it is possible that differences in the morphology of beta AP plaque types exist, analogous to the differences in cytoskeletal pathology found in these two disorders. We have carried out a quantitative image analysis of plaque subtypes in the temporal lobe of AD (n = 8), SDLT (n = 9) and control (n = 11) cases. Measurements of beta AP load and plaque density were consistently higher in AD and SDLT than in controls. When AD and SDLT cases were compared no differences were seen in either the density or relative proportions of classic and diffuse plaques. Based on these results we suggest that the variation in the clinical course of these diseases reflects differences in the cytoskeletal pathology, whereas the final stages of profound dementia common to both disorders is associated with the deposition of beta AP.

Aged↗
PubMed1996

Risk factors for abdominal aortic aneurysms in smokers.

OBJECTIVE: To investigate features of the smoking habit and other cardiovascular risk factors associated with the development of abdominal aortic aneurysm in smokers. DESIGN AND SETTING: Case control study of smokers recruited, between 1989 and 1992, in a London teaching hospital. SUBJECTS: Consecutive smokers referred with abdominal aortic aneurysm (cases n=44) and 244 age and sex-matched smokers attending Dermatology, Orthopaedic and Urology clinics (controls). MAIN RESULTS: Hypertension was more common amongst the patients with aneurysm (30%) than the controls (19%). Measured systolic and diastolic blood pressures were significantly higher in the aneurysm patients. The relative risk of having an abdominal aortic aneurysm increased 3.4-fold (95% CI 1.06-10.89) for those with a diastolic blood pressure of >90mmHg, p=0.035 and 4.1-fold (95% CI 1.27-13.16) for those with a mean arterial pressure > or = 100mmHg, p=0.009. Plasma lipids, fibrinogen and cotinine were similar amongst patients with aneurysm and controls. The cumulative smoking exposure was similar in the patients with aneurysm and controls, median 49 and 44 pack-years respectively. The relative risk of developing an aneurysm increased with the number of cigarettes currently smoked, p=0.008, and with increasing depth of inhalation, p=0.025, but was not associated with the tar or nicotine yield of current cigarettes. CONCLUSIONS: In smokers, the risk of developing an abdominal aortic aneurysm increases with increasing mean arterial and diastolic blood pressure and two aspects of current smoking habit: the number of cigarettes currently smoked and the depth of inhalation.

Aged↗
PubMed1996

Heterocyclic amines: evaluation of their role in diet associated human cancer.

1. Heterocyclic amines are formed in parts per billion levels when meat is cooked. 2. The heterocyclic amines MeIQx and PhIP are efficiently absorbed into the systemic circulation after ingestion of cooked food. 3. We have shown that MeIQx and PhIP, both in vitro and in vivo, are substrates for human hepatic CYP1A2, which exclusively and efficiently catalyses their conversion to genotoxic hydroxylamines. 4. MeIQx and PhIP are promutagens. MeIQx is a very powerful bacterial mutagen whereas PhIP is a more potent mammalian cell mutagen. Using a mammalian cell target gene, hprt, we have shown that PhIP induces a characteristic mutational 'fingerprint'. 5. MeIQx and PhIP are carcinogenic in bioassays. The PhIP mutational 'fingerprint' has been detected in the Apc gene of 5/8 colonic tumours induced by PhIP in rats.

Animals↗
PubMed1996

Dissecting the function of cytochrome P450.

1. The role that P450 plays in the metabolism of drugs and other chemicals is often pivotal in determining the duration and magnitude of their biological effects, so that it is important to identify the specific forms of the enzyme involved. 2. The similarity between different forms of P450 is such that polyclonal antibodies raised by conventional means, even against a homogeneous preparation of the enzyme will often react with several different P450s. 3. Whilst monoclonal antibodies are often more specific, their selectivity cannot be determined prior to their use. 4. The use of short sequences of amino acids (4-7 residues) predicted to occur in surface loop regions of the protein as immunogens, affords a means of targeting antibodies to specific forms of P450. 5. The terminal amino acid of a peptide, here the C-terminal residue, is very immunodominant in determining the specificity of the resultant antibody. Hence, antibodies against the C-terminal residues of P450 have a high affinity and, in most cases, will be specific. 6. Application of anti-peptide antibodies to the major forms of P450 in human liver revealed the anticipated interindividual variation. However, evidence was found that the polymorphism in CYP3A5 expression is quantitative rather than qualitative. 7. A putative pro-inhibitory region on the surface of mammalian P450, possibly involved in intramolecular electron transport, has been identified, using a combination of directed anti-peptide antibodies and sequence alignment based on secondary structure. 8. Antibodies directed against defined regions of P450 enzymes are proving invaluable in exploring the regulation, specificity and function of these key enzymes.

Amino Acid Sequence↗
PubMed1996

Liver damage in the rat induces hepatocyte stem cells from biliary epithelial cells.

BACKGROUND & AIMS: When rat hepatocyte regeneration after partial hepatectomy is blocked by 2-acetylaminofluorene, a proliferation of biliary epithelia sends out ductules into the parenchyma. The ability of these neoductules to act as a significant progenitor compartment for hepatocytes is in dispute. This study aims to resolve this question by varying the amount of 2-acetylaminofluorene administered. METHODS: Rats were fed 2-acetylaminofluorene fr 6 days before and up to 7 days after partial hepatectomy was performed at a dose of either 2.5 (low) or 5 (high) mg/kg(-1)/day(-1). The response was monitored by the immunohistochemical expression of intermediate filaments and cytochrome P450 enzymes. RESULTS: No regeneration by mature hepatocytes occurred with either dose, and new ductules expressed the biliary cytokeratins 7, 8, 18, and 19 and, in addition, vimentin. At the high dose, hepatocytic differentiation was infrequent, whereas apoptosis and intestinal differentiation were common. At the low dose, almost all ductules differentiated into hepatocytes within 14 days of hepatectomy. CONCLUSIONS: Biliary epithelium is an effective and substantiative hepatocyte progenitor compartment under appropriate conditions.

2-Acetylaminofluorene↗