Repair replication of DNA in the intact animal following treatment with dimethylnitrosamine and with methyl methanesulphonate, studied by fractionation of nuclei in a zonal centrifuge.

To relate repair of lesions in DNA with carcinogenesis, it is necessary to study repair in the intact animal under conditions which will and will not induce cancer. The methods used to study the replications stage of repair in isolated cells are not suitable for use in the intact animal. A method is presented which depends on the fact that during cell replication the nuclei enlarge, and may be separated from nuclei of non-replicating cells by rate zonal centrifugation on a sucrose gradient in a zonal rotor. Thus incorporation of [3H]thymidine as a result of de novo synthesis of DNA in replicating nuclei can be separated from incorporation due to repair synthesis in non-replicating nuclei. Treatment of animals with dimethylnitrosamine or with methyl methanesulphonate produced a "repair-type" profile, which contrasted with that given by liver nuclei from untreated animals, or from animals in which DNA synthesis had been reduced by treatment with cycloheximide, a compound which is not known to cause direct damage to DNA. Evidence is presented which suggests that the method is a rapid sensitive test for the occurrence of repair replication of some kind in the liver of the intact animal.

Effect of the esophageal carcinogen methylbenzylnitrosamine and of a putative potentiating factor, a trichothecene mycotoxin, on O6-methylguanine-dna methyl transferase in rat esophagus and liver.

Epidemiological evidence from China and South Africa has implicated Fusaria mycotoxins in the etiology of esophageal cancer, although treatment of animals with extracts of Fusaria cultures did not cause cancer of the esophagus. Fusaria are the major producers of trichothecenes, and animal experiments have shown that these mycotoxins can damage the esophagus but they have not been shown to cause esophageal cancer. A plausible concept is therefore that esophageal cancer is initiated by the potent environmental esophageal carcinogens, certain nitrosamines, but that the levels of exposure are too low to cause clinical cancer unless their effects are enhanced by additional risk factors. Among the most likely enhancing factors in the regions mentioned above are Fusaria mycotoxins. As trichothecenes are known to inhibit sulphydryl-dependent reactions and to inhibit protein synthesis, experiments were carried out to determine whether potentiation of cancer could be mediated via inhibition of the DNA repair protein O6-methylguanine-DNA methyl transferase (O6MG-MT). The effect of diacetoxyscirpenol (DS) on O6MG-MT was studied. Chronic or acute treatment with DS did not alter the level of O6MG-MT in esophagus, or affect the depletion which occurs after injection of methylbenzylnitrosamine, or alter the rate of reappearance of O6MG-MT. A high dose of DS induced O6MG-MT in liver. These results suggest that if trichothecenes are risk factors for esophageal cancer, the effect is unlikely to be mediated by inhibition of O6MG-MT. Induction of the repair protein in liver may be relevant in the animal toxicoses caused by consumption of trichothecenes, but is unlikely to be implicated in the etiology of liver cancer in man.

The effect of the trichothecene mycotoxin diacetoxyscirpenol on nitrosamine-induced esophageal cancer in the rat.

The fact that the only chemicals known to be potent carcinogens for the esophagus in animals are certain nitrosamines suggests that these environmental carcinogens could be a cause of human esophageal cancer. Epidemiological investigations support this concept. The level of exposure alone is not considered sufficient to account for the very high incidence of the disease in certain regions, but potentiating factors have been shown to have a dramatic effect on nitrosamine-induced esophageal cancer in animal experiments. A likely enhancing factor is consumption of food contaminated by molds, especially by Fusaria spp, a group known to produce trichothecene mycotoxins. The effect of simultaneous treatment with diacetoxyscirpenol (DS) on methyl-benzyl-nitrosamine (NMBzA)-induced esophageal cancer was studied. Feeding a diet containing DS at 10 ppm for 10 weeks caused thickening of the basal cell layer of the esophageal epithelium, but feeding DS (10 ppm) simultaneously with NMBzA (4, 8, 16 ppm) for 10 weeks, or feeding a lower dose of DS with NMBzA for a longer period, or administration of DS per os at intervals during NMBzA treatment, did not potentiate but possibly reduced esophageal tumors. Toxicity, revealed by reduced growth rate of DS-fed animals, may have inhibited carcinogenesis. In contrast to the rapid potentiating effect of zinc deficiency, DS does not appear to cause an early enhancement of esophageal cancer.

Acute and chronic effects of diacetoxyscirpenol on cell replication in rat esophagus and stomach.

The effect of the mycotoxin diacetoxyscirpenol (DS) on the upper alimentary tract was studied on account of the association between the consumption of food contaminated by Fusaria and esophageal cancer. Previously it had been shown that a single high dose of DS induced basal cell replication in esophagus and in squamous and glandular stomach. To assess the significance of this effect in relation to the levels of exposure likely to be encountered by man and agricultural animals, it was essential to examine the dose response relationship. Also, the long-term effect of repeated intubations of DS, and of chronic feeding of DS at 10 ppm in the diet, was studied. Intubation of progressively lower doses of DS produced a decreasing effect on replication in esophagus and stomach, but at 0.06 mg/kg replication in squamous and glandular stomach was still more than in the control animals. Intubation repeated weekly for 6-8 weeks produced no detectable change in esophagus or stomach in the surviving animals which were killed at 9 months. When DS was fed in the diet, there was marked hyperplasia in the squamous stomach of two of the four animals which survived for 9 months. These results suggest that DS per se is not carcinogenic for esophagus or for stomach, and that exposure to occasional high doses does not cause persisting abnormalities in replication. However, repeated exposure to high doses would cause repeated periods of hyperplasia, and chronic exposure in some animals could result in continuing hyperplasia. Any increase in replication is likely to promote cancer by increasing the vulnerability of the gastric and esophageal mucosa to carcinogens.

Stimulation of DNA replication in rat esophagus and stomach by the trichothecene mycotoxin diacetoxyscirpenol.

Consumption by man of cereals contaminated by high levels of Fusaria mycotoxins has caused alimentary toxic aleukia, while chronic consumption at lower levels of contamination has been implicated in esophageal cancer in China and South Africa. Dietary treatment of animals with extracts of Fusaria cultures or with the trichothecene diacetoxyscirpenol (DS) caused esophageal hyperplasia but not cancer. The explanation could be that esophageal cancer is initiated by other factors, possibly by nitrosamines, and that Fusaria mycotoxins act either as co-carcinogens or as promoters as a result of their ability to stimulate cell replication. The effect of DS on replication in esophagus was therefore studied. As squamous stomach has a very similar histological structure to esophagus, the effect of DS on stomach was studied also. A high dose of DS given by gavage was shown by the bromodeoxyuridine-antibody technique to increase DNA replication in the basal cells of the esophagus and of the squamous and glandular stomach. For stomach, this correlated with an increased incorporation of tritiated thymidine into DNA, and an increase in ornithine decarboxylase activity. These effects had returned to normal by 7 days. The increased replication was apparently not a result of cell damage and restorative hyperplasia. It is suggested that, as has been proposed recently for butylated hydroxyanisole, DS may enhance carcinogenesis when exposure is sufficient to stimulate cell replication. This contrasts with the non-threshold action of initiating carcinogens. For man, acute exposure to the critical dose of DS probably occurs only under exceptional circumstances, as during outbreaks of alimentary toxic aleukia. Prolonged exposure to lower dose levels is more likely to be relevant.

Effect of N-nitrosamines carcinogenic for oesophagus on O6-alkyl-guanine-DNA-methyl transferase in rat oesophagus and liver.

Several O6-alkylGua adducts have been shown to be removed from DNA during its repair by transfer of the alkyl group to a cysteine residue in a specific AAP, with the formation of S-alkylcysteine. As the reaction is stoichiometric and irreversible, the AAP content of the cell can be reduced or depleted. In vivo depletion by a high dose of nitrosamine can be used to test for the formation of a repairable alkylation adduct at the O6-position of guanine. In addition, if the carcinogenic potency of a nitroso compound for a particular organ is related to the persistence of the adduct in DNA, potency would depend not on the level of alkylation attained after treatment, but on whether this was sufficient to deplete the AAP content of the organ concerned and so to slow down repair, i.e. depletion of AAP is a more relevant estimate of potency than is the initial extent of DNA alkylation. Dose-response studies on target and non-target organs showed that depletion of AAP correlated with organotropy for those nitrosamines known to methylate DNA, i.e. with NDMA for liver, and with NMBzA for oesophagus. With NDEA, the results supported the suggestion that other adducts in addition to O6-alkylGua may be involved. NMPhA, an oesophageal specific carcinogen, did not deplete AAP in oesophagus, and induced AAP in liver. This result adds to the evidence that NMPhA does not alkylate DNA.

Repair and replication of DNA in rat brain and liver during foetal and post-natal development, in relation to nitroso-alkyl-urea induced carcinogenesis.

The susceptibility of rat brain to induction of cancer by N-nitroso-N-ethyl-urea (NEU) increases dramatically from a very low level in the 12 day foetus to a maximum at the time of birth, and then decreases with age. Liver tumors are rarely induced by a single treatment with NEU at any age. If induction of cancer by nitroso-alkyl-ureas depends on replication of DNA containing the mispairing base O6-alkylguanine, susceptibility would reflect the balance between the protective effect of removal of the base by repair mechanisms and the potentiating effect of cell replication. The capacity of tissues to remove O6-alkyl-guanine from DNA depends on the amount of alkyl acceptor protein (AAP) present. To study the concept that carcinogenesis results from replication of alkylated DNA, the AAP contents and relative rates of DNA replication were studied in brain and liver of rats at various stages of development, from the 12 day foetus to the 48 week old rat. Replication in liver was approximately ten times higher than in intra-cranial contents at each stage of development studies. The AAP content was higher in the 12 day foetal brain than later, decreasing to low levels as susceptibility to NEU increased until the time of birth, and then remaining low in the adult. The peak sensitivity of brain therefore corresponds to the time at which AAP content is low and rate of DNA replication is high. With liver, AAP levels are low in the foetus, although higher than in brain, and increase after birth. The higher level of AAP in the foetal liver compared with that of brain is possibly sufficient to explain why cancer is not induced in liver in spite of the high rate of DNA replication. The results are consistent with the concept that replication of alkylated DNA is an essential event in initiation. There may be no quantitative relationship between replication and repair and susceptibility to cancer on comparison of different tissues, owing to the fact that, at the cellular level, cancer is a rare event. The amount of mispairing at replication necessary to bring it about may depend on the detailed organisation of the genome, and hence on cell type.

A clinical evaluation of the Cobas Fara clinical chemistry analyzer for some routine serum enzymes and glucose.

The authors evaluated the Cobas FARA centrifugal analyzer with respect to pipetting precision and accuracy, instrument temperature, spectrophotometric response, and analytic performance for the assay of five serum enzymes and glucose. Spectrophotometric response, temperature response, pipetting precision, and accuracy were satisfactory. However, sufficient time must be allowed for cuvet contents to reach a stable temperature before measurements are made. Total day-to-day imprecision (within plus between run) was less than 5% (coefficient of variation) for aspartate and alanine aminotransferases (AST; Enzyme Commission classification number [EC] EC 2.6.1.1; and ALT; EC 2.6.1.2); alkaline phosphatase (AP; EC 3.1.3.1); gamma-glutamyltransferase (GGT; EC 2.3.1.2); lactate dehydrogenase (LD; EC 1.1.1.17); creatine kinase (CK; EC 2.7.3.1); and glucose assays. Results compare well with those obtained with other current clinical chemistry analyzers; correlation coefficients were greater than 0.993. Sample-to-sample carryover was negligible, and method linearity was satisfactory for all tests.

The priority test request form: a method for improving communication between the physician and the Emergency Clinical Biochemistry Laboratory.

Two forms--a priority test request form and a telephone results form--provide improved communication between the physician and the emergency laboratory of the Clinical Biochemistry Department. The priority test request form contains a list of available tests and it allows the physician to tell the laboratory exactly when emergency test results are required. The telephone results form ensures that the physician will receive a report, by telephone, of a test result although it may not be an urgent test. This allows a greater control of work flow, both routine and emergency, through the laboratory.

The test request form: a neglected route for communication between the physician and the clinical chemist?

The requirements for a clinical biochemistry test request form are reviewed. The interaction between the configuration of the main analysers and the number of individual tests, or profiles, that are ordered using different request form formats were monitored for three-month periods over a three-year period while the main analysers were being "reconfigured" or replaced. Although there was a significant increase in orders for individual tests (compared to profile requests) required on outpatients this did not occur with the inpatient ordering pattern. Instead, the numbers of discretionary tests dropped and more miniprofiles--for example, the electrolyte group, were ordered, although the total number of profiles (per patient day) did not increase because the "electrolyte-urea-creatinine" profile numbers markedly decreased during rhe period of the study. This shift in ordering patterns was assumed to be due to the faster turnaround of "priority" (emergency) test requests which could, due to improved instrumentation, be analysed as quickly as individual test requests. Glucose was dropped from the major profile and the numbers of discretionary glucose requests did not increase. It was concluded that, providing this single test can be performed efficiently, there is no need for glucose to be included in test profiles.

Unusual increases in serum lactate dehydrogenase isoenzyme-5 activity caused by severe congestive cardiac failure: two case reports.

Two cases of congestive cardiac failure had unusually high activities of serum lactate dehydrogenase and lactate dehydrogenase isoenzyme-5. Values for isoenzyme-5 exceeding 87% of the total serum activity, and increases of 228- to 380-fold of the upper reference limit, are very uncommon.

Clinical chemistry laboratory productivity: a comparison between a Canadian and a British teaching hospital.

the productivity of a clinical chemistry laboratory was measured in both a Canadian and a British tertiary care teaching hospital using 1977 data and the 1976 edition of the Canadian Schedule of Unit Values. Although productivity, measured as units produced per person or per paid hour, was lower in the British than in the Canadian teaching hospital-due to the British day-release system of staff-education-the output per actual worker hour was similar. We conclude, from this small study, that productivity in the laboratory services of the British National Health Service is not likely to be different from laboratory productivity elsewhere.

Patients with suspected myocardial infarction: their test request patterns for clinical biochemistry in a British and a Canadian cardiac care unit.

The total number of routine clinical biochemistry tests requested for patients admitted to a coronary care unit with a diagnosis of "query myocardial infarction" were recorded over four to eight months. There were 156 sequential admissions in a British teaching hospital and 163 in a Canadian counterpart; the incidence of confirmed myocardial infarction was 53% and 50%, respectively. The pattern of tests ordered was substantially similar in each unit, unlike the rate of testing. For example, total creatine kinase was requested five times less often per patient in the British hospital than in the Canadian unit in cases of confirmed myocardial infarction (2.17 and 10.17, respectively; p less than 0.0001): the difference was much less, but still significant, when there was no infarction (2.01 and 3.55; p less than 0.0001). This study suggests a significant international difference in the use of clinical biochemistry services between coronary care units. Physicians (clinical and laboratory) need to be more critical of their use of protocols, which may prove wasteful of limited health care resources.

Lability of human creatine kinase isoenzymes at 37 degrees C: a complication of electrophoretic separation.

The activity of the brain specific isoenzyme of creatine kinase is shown to fall off rapidly at 37 degrees C, particularly in the presence of albumin. Dithiothreitol cannot reverse this lability. The implications of this finding suggest that electrophoretic techniques which use incubation methods to detect the brain specific isoenzyme of creatine kinase may underestimate the true activity.

Liver-originating isoenzymes of alkaline phosphatase in the serum: a paraneoplastic manifestation of a malignant schwannoma of the sciatic nerve.

A case of malignant schwannoma of the sciatic nerve is described associated with hepatic dysfunction in the absence of hepatic metastases. An elevated serum alkaline phosphatase activity was present with an isoenzyme pattern indicating hepatic involvement. These abnormalities disappeared after extirpation of the tumour. The patient is well, with no evidence of metastases, over two years later. It is concluded that the abnormality of serum alkaline phosphatase was induced by the tumour, and that the liver can be involved in the paraneoplastic syndrome.

Digitisers: their use in the entry of orders, urinalysis results, and isoenzyme interpretation in a clinical biochemistry computer system.

The processes of order entry, urinalysis result, and isoenzyme interpretation entry into a laboratory computer system is a time consuming activity. We have designed a series of forms for use with a digitising pad that allow us rapidly to enter orders, urinalysis results, and isoenzyme interpretative comments into our laboratory computer system. We have shown that digitiser entry is always significantly faster than manual entry. Although there are many devices available to facilitate computer entry, we believe that the digitiser technique is an attractive option because of its ease of use, speed, reliability, and low cost.

Mailbox: program designed to improve communication in clinical laboratories.

A MUMPS program called MAILBOX allows information to be more efficiently disseminated in a hospital department of clinical biochemistry. Readily accessible to all staff as the department is equipped with video display units in every laboratory and office, MAILBOX allows laboratory staff to send and read departmental messages; send, read, and save personal messages; sign in to and out of the department; and locate all other staff. Departmental messages containing information of general interest are automatically displayed on signing on to MAILBOX. Personal messages are directed to specific people, who are so informed when signing on; they can only be read by the sender and intended recipient(s).

Errors in measuring enzyme activity by reaction-rate methods.

1. The two types of assay used in clinical enzymology -- reaction-rate and two-point assays -- are usually performed with different types of analyzers. A third type, the multi-point analyzer, has been introduced recently. 2. Reaction-rate and two-point enzyme assays have sources of error inherent in their methodologies. There are also major errors due to the analytical system itself; these are considered systematically.

Biochemical markers of myocardial damage.

OBJECTIVE: To assess various biochemical markers of myocardial damage. METHODS AND RESULTS: Before routinely using any test as a biochemical marker of myocardial damage, the published evidence for its diagnostic utility must be critically assessed. Such assessment includes receiver operator curve (ROC) curve analyses, confidence interval estimates of claimed sensitivity and specificity values, and the effects of testing in serial and parallel modes. It is also necessary to establish the test's rule-in (high specificity) and rule-out (high sensitivity) decision thresholds that may vary with time after the onset of symptoms. The spectrum of ischemic heart disease includes acute (sudden death, non-Q- and Q-wave infarctions) and chronic (stable, unstable, and variant angina) conditions. Biochemical markers of myocardial damage are of most value in the diagnosis of acute ischemic heart disease, although increasingly some of these markers are being found to possess a prognostic value in chronic ischemic heart disease. The markers of enzymatic activity include aspartate aminotransferase, creatine kinase (together with isoenzymes and isoforms), and lactate dehydrogenase and isoenzymes. Creatine kinase isoenzyme-2 may also be measured immunologically, and this type of assay is in increasing use both because of its speed and because its blood levels rise earlier than the corresponding activities. The commercially available nonenzymatic markers are myoglobin and troponin T; troponin I is expected to become available in late 1995. While myoglobin is a nonspecific indicator of myocardial damage, its diagnostic value is due to its early appearance in blood. Troponin T is more cardiac specific, but the published data appears to suggest that the cardiac specificity of troponin I is superior. Troponin levels become abnormal at about the same time after the onset of symptoms as mass assays of creatine kinase isoenzyme-2; therefore, they are not useful as early markers of myocardial damage. CONCLUSION: The availability of these nonenzymatic markers of myocardial damage must force a reassessment of the continued use of the enzymatic markers. Are they necessary, and if so, which ones should be retained?

The organization of a 24-hour blood alcohols (volatiles) screening service in a hospital laboratory.

We describe the organization that evolved in the Clinical Biochemistry Department of a tertiary-care hospital for handling blood (serum) alcohol (volatiles) determinations. We use a microprocessor-controlled capillary gas chromatography system which will detect and quantitate methanol, ethanol, isopropanol and acetone. Minimal operator intervention is required, allowing operation of the system 24 hours each day, thus permitting timely detection of these volatiles. A Specimen Trace Card has been devised to document continuity of sample handling from the time of blood collection until completion of the analysis. This has proved of value when laboratory records are used for legal purposes.