Cataract surgery: new techniques, better results.
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Aged↗
Biomedical subjects
S Murray
Publications and source records attributed to S Murray.
Enzymatic studies of the activation of heterocyclic food mutagens in man.
2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are amongst the most abundant of the heterocyclic aromatic amines formed during the cooking of beef. Both compounds are genotoxic and carcinogenic in rodents. These effects are manifested only after activation of the amines by P450. Human liver is very active at converting these amines to mutagenic products. Studies in vitro have established that, for both amines, mutagenicity with human liver microsomes is entirely via the N-hydroxylamine, essentially the only oxidation product of either amine. Both N-hydroxylation and mutagenicity of the amines can be almost completely inhibited by furafylline, a potent and highly selective inhibitor of CYP1A2 in man. These data, together with the work of others, show that the N-hydroxylation and hence the mutagenicity of both MeIQx and PhIP in man is catalyzed almost exclusively by CYP1A2. Liver from cynomolgus monkeys, unlike that from humans and marmosets, is very poor at activating MeIQx or PhIP to mutagenic products. Studies with anti-peptide antibodies of defined specificity revealed that this is due to the absence of CYP1A2, suggesting that this species should not be used to assess the possible risk posed to man by these amines. The marmoset would be a better species for this purpose.
Animals↗
Systemic exposure to dietary heterocyclic amines in man.
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-I-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed during the cooking of meat and account for a significant proportion of the mutagenic material present in cooked beef. The amines are not directly mutagenic but are converted to active intermediates by P450. Studies in vitro with human liver have shown that N-hydroxylation catalyzed by CYP1A2 is the major pathway of oxidation of MeQx and PhIP and is solely responsible for the generation of mutagenic species. In the studies reported in this paper it is demonstrated that both MelIQx and PhIP are well absorbed and extensively metabolized following ingestion of amine-containing beef by humans. Experiments with furafylline, a potent and selective inhibitor of human CYP1A2, reveal that more than 90% of MeIQx and 70% of PhIP are N-hydroxylated in vivo, probably presystemically in the liver.
Animals↗
CYP1A2-catalyzed conversion of dietary heterocyclic amines to their proximate carcinogens is their major route of metabolism in humans.
The contribution of CYP1A2 to the metabolism of the dietary heterocyclic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in vivo in humans, has been determined with furafylline, a highly selective inhibitor of this enzyme. The inhibitory potential of furafylline in vivo was first assessed by determining its effect on clearance of phenacetin to paracetamol by the model CYP1A2-dependent O-deethylation pathway. Furafylline inhibited this reaction by > 99% in all subjects, thus demonstrating its applicability to determining the contribution of CYP1A2 to a given reaction in vivo. A group of 6 healthy male volunteers received either placebo or 125 mg furafylline, in a double-blind balanced crossover design, 2 h prior to consuming a test meal of fried beef containing a known amount of amines. The excretion of PhIP and MeIQx in urine was determined during the subsequent 28 h, using gas chromatography-mass spectrometry. Following furafylline, the excretion of unchanged MeIQx increased 14.3-fold, while that of PhIP increased 4.1-fold (P < 0.01, paired t test). Elimination of both amines was first order and very rapid, with half-lives of < 5 h. The elimination rate constants did not change following furafylline, suggesting that total clearance is limited by hepatic blood flow. Because the elimination of the amines was first order, it was possible to calculate the contribution of CYP1A2 to the clearance of the amines. CYP1A2-catalyzed metabolism accounts for 91% of the elimination of ingested MeIQx and 70% of ingested PhIP, most likely via N-hydroxylation.
Adult↗
Genetically engineered antibodies for diagnostic pathology.
Antibody genes can be cloned, genetically manipulated, and expressed in both homologous and heterologous expression systems to produce viable antigen-binding proteins complete with natural effector functions. Manipulation of antibody genes permits the expression of fusion proteins or truncated proteins that retain antigen-binding activity. The new antibody technologies are becoming increasingly sophisticated, permitting the alteration of antigen-binding responses, the transfer of antigen specificity between antibodies, and the expression of minimal-size antigen-binding protein domains. These new molecules have been made mostly for studies on function or to provide molecules suited for in vivo diagnosis and therapy; very few have been specifically designed for, or used for, diagnostic histopathology. We describe here the adaptation of small antibody derivatives for use in immunohistochemistry. Molecules suitable for this purpose need only to possess specific antigen-binding ability and some means of detection of antigen-bound material. Detection could be by recognition of a genetically fused flag or tag epitope, by the fusion of an enzyme whose activity can be assayed, or by fusion with a protein that can interact with pre-existing histopathological reagents.
Animals↗
Management of chronic airflow obstruction: differences in practice between respiratory and general physicians.
An audit of inpatient care of diseases characterized by chronic airflow obstruction namely chronic bronchitis, emphysema and chronic obstructive airways disease (ICD Code Nos. 490-2 & 496) was performed and the practice of respiratory and general physicians compared. One hundred cases were sampled at random from 279 cases admitted to hospitals serving the West of Glasgow in 1988. Fifty cases were selected from those admitted under the care of respiratory physicians and 50 from those under general physicians; 89 were suitable for analysis. The main outcome measurements consisted of the use of routine respiratory investigations, comparison of the use of standard therapies during the admission and at discharge, length of stay, inpatient deaths, follow up and readmission rates. The groups were similar in age, smoking history, gender and there was no significant difference in admission arterial blood gas values. The pulse rate on admission was higher in the general group (102 beats per min) in comparison to the respiratory group (91 beats per min) (P < 0.004). A similar use of chest radiograph and arterial blood gas analysis was noted between the groups. Ninety-six per cent of respiratory patients had either spirometry or peak expiratory flow measured compared to 62% in the general group (P = 0.0001). No significant differences were noted in the use of antibiotics, bronchodilators, corticosteroids, oxygen or respiratory stimulants. The mean length of stay was similar. Two patients (4%) in the respiratory group compared with seven (18%) in the general group died during the admission (P = 0.01); there were no further early deaths at 1 month from discharge.(ABSTRACT TRUNCATED AT 250 WORDS)
Aged↗
Contribution of CYP1A1 and CYP1A2 to the activation of heterocyclic amines in monkeys and human.
The activation of heterocyclic amines to mutagenic products by hepatic microsomal fractions from cynomolgus monkey, marmoset monkey and man was compared with the respective levels of cytochrome P450 enzymes CYP1A1 and CYP1A2. The rate of activation of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to mutagens by hepatic microsomal fraction from cynomolgus monkey was very low. This was associated with a lack of constitutive expression of CYP1A1 and CYP1A2. In contrast, human hepatic microsomal fraction readily activates these heterocyclic amines and this is associated with constitutive expression of CYP1A2. Treatment of cynomolgus monkey with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a very modest induction of CYP1A2, and a small increase in the activation of MeIQx and IQ. However, there was marked induction of CYP1A1 which was accompanied by > 10-fold increases in PhIP activation and 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and aryl hydrocarbon hydroxylase activities. Following treatment of cynomolgus monkey with 3-methylcholanthrene, induction of CYP1A1, but not CYP1A2, was evident. In untreated marmoset monkey the activations of MeIQx and PhIP, as well as phenacetin O-deethylase, EROD, MROD and aryl hydrocarbon hydroxylase activities, are similar to those in man, although the activations of IQ and coumarin 7-hydroxylase activity are lower than in man. The presence of constitutive CYP1A2, and the absence of CYP1A1, in the liver of this species correspond to the situation in man. Treatment of marmoset monkey with TCDD results in increased CYP1A2 levels (4-fold), accompanied by proportional increases in the activation of MeIQx and IQ and phenacetin O-deethylase, EROD and MROD activities. The activation of PhIP is increased disproportionately, by 8-fold, most likely due to the activity of CYP1A1 which is also induced by TCDD in this species. Overall, the hepatic metabolism of heterocyclic amines by CYP1A enzymes in the untreated marmoset monkey resembles that in human more closely than that in the cynomolgus monkey. Therefore, marmoset monkey may be a more suitable model than the cynomolgus monkey for carcinogenicity studies involving MeIQx and PhIP, but not IQ.
Animals↗
Metabolism of the food derived mutagen and carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) by human liver microsomes.
Animal studies have shown that 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) undergoes both activation to a genotoxic metabolite and detoxication, catalysed by CYP enzymes. In this study, using direct chemical analysis, we have examined PhIP metabolism by the microsomal fraction of human liver for comparison to that occurring in animals. PhIP was incubated with human liver microsomes in the presence of an NADPH regenerating system and the reaction mixture then analyzed by HPLC. Only one metabolite, identified as N-hydroxy PhIP, was produced. The N-hydroxylation of PhIP by human liver microsomal fraction obeyed Michaelis-Menten kinetics, with a Km of 55 microM and a Vmax of 666 pmol/min/mg protein. Furafylline, a potent and specific inhibitor of CYP1A2 in man, inhibited this reaction by > 95%, with an IC50 of 0.6 microM. PhIP inhibited high affinity phenacetin O-deethylase activity of human liver microsomes, an activity catalysed specifically by CYP1A2, with an IC50 of about 80 microM. These data indicate that, in human liver microsomes, N-hydroxylation is the only route of oxidative metabolism of PhIP, yielding a genotoxic species, and that this reaction is catalysed almost exclusively by CYP1A2. Furthermore, the exclusive oxidative activation of PhIP by human liver is in direct contrast to PhIP metabolism in rodents and non-human primates where oxidative detoxication products predominate.
Carcinogens↗
Improving quality of care based on evaluation of the Western Ontario Provincial Lithotripsy Program by patients and their families.
A survey is an effective method for evaluating quality of care and generating cost-effective ideas for quality improvement. This article describes a survey of 138 patients and their families to evaluate the quality of care provided by a regional lithotripsy program. Although patients and their families generally gave high ratings to the care they received, areas for improvement were documented. The results were ultimately used to support change in nursing practice and care of patients and their families. The survey process described incorporates principles of research, quality assurance, and quality improvement.
Attitude to Health↗
Histopathology in erythroderma: review of a series of cases by multiple observers.
This study examines the utility of objective histopathological studies in the evaluation of adult patients with erythroderma. A series of 56 skin biopsies, from 40 erythrodermic patients, was reviewed sequentially by 4 Canadian dermatopathologists who were unaware of clinical details of the cases. The final diagnosis (gold standard), in each instance, had already been determined by others, based on clinicopathologic data and response to therapy. Direct comparison revealed that the mean accuracy of the histopathological diagnoses was 53% (range: 48-66%), a favorable result in view of the difficulty of the task at hand. Additional points of information which evolved from the study are as follows: (i) identification, by microscopy alone, of spongiotic dermatitis, cutaneous T-cell lymphoma and psoriasis, as underlying causes of erythroderma was more successful than that of drug eruptions and pityriasis rubra pilaris; (ii) the epidermotropism which characterizes cutaneous T-cell lymphoma may be mistaken for inflammatory interface changes seen in drug eruptions and vice versa, thus constituting a pitfall in diagnosis; (iii) finally, it appears that submission of multiple simultaneous biopsies, rather than a single specimen, from patients with erythroderma would be likely to enhance the accuracy of histopathological diagnosis.
Adult↗
Eruptive xanthomata with urate-like crystals.
The microscopic manifestations of eruptive xanthomata are variable and although they are most frequently characterized by aggregations of foamy macrophages in the dermis, they can, on occasion, simulate granuloma annulare. We have recently encountered a further unusual histopathological variant of eruptive xanthomata in 3 skin biopsies from 2 patients with hypertriglyceridemia. The lesions had a granuloma annulare-like pattern at low magnification, the exceptional feature being their content of polarizable, needle-shaped crystals. These bore a strong resemblance to monosodium urate monohydrate and displayed negative birefringence under polarized light. In the case of each patient, an initial pathological diagnosis of gout was rendered, and the true nature of the lesions came to light only after clinicopathologic correlation. While it is clear that the chemical composition of these crystals requires further study, recognition of this microscopic variant of eruptive xanthomata should help to reduce errors in diagnosis.
Adult↗
Impact of maternal infection on foetal growth and nutrition.
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Biology↗
An emergency nurses day celebration for children.
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Child↗
Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man.
An anti-peptide antibody was raised against the sequence Thr-Gly-Ala-Leu-Phe-Lys-His-Ser-Glu-Asn-Tyr-Lys which occurs at positions 283-294 in the rat cytochrome P450 enzyme CYP1A2. Compared with its binding to the peptide used for immunization, the antibody bound with only slightly reduced affinity to the truncated peptides Thr-Gly-Ala-Leu-Phe-Lys-His-Ser and Leu-Phe-Lys-His-Ser. However, binding to the peptide Ser-Glu-Asn-Tyr-Lys-Asp-Asn, which overlaps with the C-terminal region of the immunizing peptide, was very low. Thus, a major epitope for the anti-peptide antibody is Leu-Phe-Lys-His-Ser, which corresponds to a region of CYP1A2 that is conserved in many species. The antibody was tested by immunoblotting for its ability to bind to hepatic microsomal fractions from a number of species. Where possible animals were treated with compounds which induce CYP1A2 and the results compared with those with untreated animals. It was found that the antibody bound to rat, mouse, rabbit, hamster, guinea pig, pig, marmoset monkey and human CYP1A2. No evidence was found for binding to dog CYP1A2. The region corresponding to the major epitope at residues 286-290 of rat CYP1A2 was identical in mouse, hamster, rabbit and human CYP1A2. The sequence of marmoset and guinea pig CYP1A2 are not known but are predicted to be very similar to the sequence in the rat. The lack of binding of the antibody to dog CYP1A2 may be explained by two differences in this region compared with rat CYP1A2. Maximum inhibition of CYP1A2 activity by this antibody, as measured by high-affinity phenacetin O-deethylase activity, was 20%. This is in contrast to a previously described anti-peptide antibody directed to an adjacent region which caused 65% inhibition of this activity. Thus, the edge of an inhibitory region on the surface of cytochrome P450 has been identified. The ability of the antibody to bind to CYP1A2 from a number of animals should make this antibody of use for studying the levels of CYP1A2 apoprotein in many species.
Amino Acid Sequence↗
Quantification of the carcinogens 2-amino-3,8-dimethyl- and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in food using a combined assay based on gas chromatography-negative ion mass spectrometry.
A gas chromatographic-mass spectrometric assay has been developed for the measurement of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in food. Stable isotope-labelled analogues of MeIQx and PhIP are used as internal standards and the synthesis of deuterated PhIP is described. The mass spectrometer is operated in the electron-capture negative ion chemical ionisation mode and the amines are chromatographed as their di-3,5-bistrifluoromethylbenzyl derivatives. All three compounds can be measured in a single chromatographic run and detection limits of 0.05, 0.1 and 0.2 ng/g for MeIQx, DiMeIQx and PhIP, respectively, in food are obtained. Various home-cooked and commercially prepared foodstuffs were analysed with this assay and several were found to contain measurable amounts of one or more of the three amines. These results are presented and discussed.
Animals↗
An inhibitory monoclonal anti-protein antibody and an anti-peptide antibody share an epitope on rat cytochrome P-450 enzymes CYP1A1 and CYP1A2.
A monoclonal antibody, 12/2/3/2, which was raised against purified rat CYP1A1 recognises specifically rat and mouse CYP1A1 and CYP1A2, but not any cytochrome P-450 present in hepatic microsomal fractions from rabbit, guinea pig, hamster or human. By comparing the primary sequences of cytochromes P-450 to which 12/2/3/2 does and does not bind, 10 possible locations for its epitope were found. Of these, one was extremely hydrophilic and, hence, predicted to be the most antigenic in the native protein. An antibody was produced against the synthetic peptide corresponding to this region (Gly-Arg-Asp-Arg-Gln-Pro-Arg-Leu: residues 356-363 and 350-357 of rat CYP1A1 and CYP1A2, respectively). The antibody bound to rat, mouse and hamster CYP1A1 and to rat and mouse CYP1A2, but did not bind to any protein present in hepatic microsomal fractions from the rabbit, guinea pig or human. The binding of the anti-peptide antibody to CYP1A1 or CYP1A2 was partially antagonised by the monoclonal antibody. However, whereas the monoclonal antibody inhibited both CYP1A1- (aryl hydrocarbon hydroxylase) and CYP1A2-(high-affinity phenacetin O-deethylase) dependent monooxygenase activity, the anti-peptide antibody was without effect on these activities. Antigen denaturation by 8 M urea or 0.05% (w/v) SDS had no effect on binding of the anti-peptide antibody to cytochrome P-450, whilst binding of the monoclonal antibody was reduced by more than 1000-fold. The anti-peptide antibody partially antagonised the binding of 12/2/3/2 to urea-denatured but not native cytochrome P-450. These data suggest that whilst the complete binding site for the monoclonal antibody is discontinuous, sufficient of the epitope is linear, so that when the antigen is denatured the monoclonal antibody is still able to bind and this binding is antagonised by the anti-peptide antibody. However, inhibition of catalytic activity by the monoclonal antibody must require binding to discontinuous residues.
Amino Acid Sequence↗
Variation in 1,25-dihydroxyvitamin D3 regulation of proliferation and alkaline phosphatase activity in late-passage rat osteoblastic cell lines.
The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2,D3], 1,25-dihydroxy-16ene-23yne-vitamin D3[1,25(OH)2-16ene-23yne-D3] (a synthetic analog) and retinoic acid on proliferation, protein synthesis, and alkaline phosphatase activity and mRNA were compared in two late-passage (P > 70) clonal rat osteoblastic cell lines (G2 and C12) in order to characterize variations in the basal and hormonally-regulated phenotypes. All agents inhibited proliferation (measured as cell number after 3 days of treatment) in late-passage (P > 70) G2 and C12 cells without inhibiting the rate of protein synthesis ([3H]leucine incorporation into TCA-precipitable protein) during the last 18 h of incubation. Basal and hormone-treated alkaline phosphatase activities were lower in late-passage G2 and C12 cells than those previously reported for early-passage G2 and C12 cells. 1,25(OH)2D3 and 1,25(OH)2-16ene-23yne-D3, up-regulated alkaline phosphatase activity in late-passage C12 cells and down-regulated it in late-passage G2 cells. The direction of these regulatory changes in late-passage cells was opposite to that reported for early passage of these clones, and changes were related to the levels of tissue-unspecific alkaline phosphatase mRNA normalized for actin mRNA. Effects of 1,25(OH)2D3 or 1,25(OH)2-16ene-23yne-D3 and retinoic acid were not additive, suggesting a competitive mechanism of action. It appears that increased sensitivity to the antiproliferative effects of regulatory hormones and defects in proliferation and specialization of the osteoblast are observed with increasing passage number in vitro in two model osteoblastic cell lines (G2 and C12).
Alkaline Phosphatase↗
Molecular dosimetry of the food-borne carcinogen MeIQx using adducts of serum albumin.
Incubation of mouse serum albumin with the food borne carcinogen [2-14C]-Amino-3,8,-dimethylimidazo[4,5-f]quinoxaline (14C-MeIQx) in the presence of mouse hepatic microsomes and an NADPH-regenerating system in vitro resulted in the formation of adducts of MeIQx with albumin, which increased proportionately with time for at least 120 min (approximately 1 pmol equivalents/mg of protein/min). We have previously shown in male Swiss Webster mice in vivo that 14C-MeIQx bound covalently to serum proteins and that the formation of adducts was dose dependent. 14C-MeIQx (100 mg/kg, i.p.) was administered to male (MF1) mice which were killed 24 h later. Serum albumin was purified by affinity chromatography and covalent binding of 14C-MeIQx was assessed. Total covalent binding of MeIQx to albumin was 14.0 +/- 5.2 pmol per mg albumin, which was 5-fold greater than to haemoglobin. Following mild acid hydrolysis, 1.25 pmol MeIQx per mg albumin was liberated as free amine, as determined by gas chromatography negative ion mass spectrometry (GC-MS). This represents 9% of total MeIQx adducted to albumin in vivo (cf 1.3% adducted to haemoglobin). These results suggested that adducts of MeIQx with serum albumin should provide a significantly more sensitive dosimeter than those with haemoglobin. We therefore investigated this approach with serum protein samples from three volunteers. Human serum albumin and non-serum albumin protein fractions were separated by affinity chromatography, before being subjected to GC-MS analysis for hydrolysable adducts of MeIQx. The levels of MeIQx in control samples, and from the release of the putative sulphinamide adducts in hydrolysed samples were below the limits of detection of the GC-MS assay (29 +/- 2.6 amol MeIQx/mg albumin). Despite an increase of 2 orders of magnitude in sensitivity, compared with haemoglobin, it is unlikely that the sulphinamide adduct of MeIQx with human serum albumin can be used as a dosimeter for human aminoimidazoazaarene exposure.
Adult↗