An evaluation of the sensitivity and specificity of energy expenditure measured by heart rate and the Goldberg cut-off for energy intake: basal metabolic rate for identifying mis-reporting of energy intake by adults and children: a retrospective analysis.

OBJECTIVE: To identify adults and children as under- (UR), acceptable (AR), or over-reporters (OR) of energy intake (EI) using energy expenditure measured by doubly labelled water (DLW) (EE(DLW)), and to use this as a reference to determine the sensitivity and specificity of (i) EE measured by heart rate (EE(HR)), and (ii) the Goldberg cut-off technique for classifying subjects into the same categories. DESIGN: Retrospective analysis of a dataset comprising concurrent measurements of EE(DLW), EE(HR), basal metabolic rate (BMR), and EI by weighed record (EI(WR)) on 14 adults and 36 children. EI by diet history (EI(DH)) was also measured in the children only. EI(WR):EE(DLW) provided the reference definition of subjects as UR, AR or OR. Three strategies for classifying mis-reporters based on EE(HR) and Goldberg cut-offs were then explored. Sensitivity and specificity were calculated respectively as the proportion of UR and non-UR correctly identified. RESULTS: Approximately 80% of all subjects were AR. For EI(WR) and EI(DH) respectively, the sensitivity of EE(HR) was 0.50 and 1.00, and specificity was 0.98 and 1.00. Although designating subjects as having low, medium or high activity levels (EE(HR):BMR(meas)) and calculating cut-offs based on appropriate WHO physical activity level PALs did not change sensitivity, specificity dropped to 0.98 (EI(WR)) and 0.97 (EI(DH)). Cut-offs based on a PAL of 1.55 reduced sensitivity to 0.33 (EI(WR)) and 0.00 (EI(DH)), but specificity remained unchanged. The sensitivity of all cut-offs based on physical activity level (PALs) for EI(WR) was 0.50 (adults) and 0.25 (children). CONCLUSIONS: If the precision of EE(HR) was improved, it may be useful for identifying mis-reporters of EI.

Effect of a dCTP:dTTP pool imbalance on DNA replication fidelity in Friend murine erythroleukemia cells.

Nucleotide pool imbalances have been reported to affect the fidelity of DNA replication and repair in prokaryotic and eukaryotic cells. We have reported previously that the mutagen-hypersensitive thymidine kinase (TK)-deficient Friend erythroleukemia (FEL) cells (subclones 707BUF and 707BUE), have a more than sixfold increase in the dCTP:dTTP pool ratio when compared to that of wild-type, TK-positive (TK(+)) clone 707 cells. In this study we present the results of an investigation of the effect of the dCTP:dTTP pool imbalance on the accuracy of DNA replication within 707BUF cells. We examined the spontaneous mutation spectra occurring at the adenine phosphoribosyltransferase (aprt) locus within clone 707 (TK(+)) and 707BUF (TK(-)) FEL cells. Mutations recovered at the aprt locus in FEL cells comprised: base substitutions (43:73), frameshifts (14:13.5), and deletions (43:13.5) [clone 707 (TK(+)):707BUF (TK(-)), respectively, expressed as percentages]. A comparison of the mutation spectra obtained for the two cell lines did not reveal any significant increase in misincorporation of dCTP, the nucleotide in excess, in 707BUF (TK(-)) cells, during DNA replication synthesis. These data suggest that the dCTP:dTTP pool imbalance does not alter the fidelity of DNA replication synthesis in 707BUF (TK(-)) FEL cells. Rather, the predominance of GC --> AT transitions (53%) in the 707BUF (TK(-)) spectrum may reflect a reduced efficiency of repair by uracil DNA glycosylase of uracil residues within these cells.

Dietary antioxidant supplementation and DNA damage in smokers and nonsmokers.

Deficiencies of antioxidant nutrients have been implicated in the etiology of lung and other cancers. However, most intervention trials with antioxidant nutrients have not shown beneficial effects, and some have indicated that beta-carotene may be deleterious. This randomized, double-blind, placebo-controlled study evaluated the effects of five short-term (4-wk) antioxidant nutrient supplement regimens [ascorbic acid (350 mg), RRR-alpha-tocopherol (250 mg), beta-carotene (60 mg), selenium (80 micrograms as sodium selenite), ascorbic acid (350 mg) + RRR-alpha-tocopherol (250 mg)] on plasma antioxidants and mononuclear leukocyte DNA damage in male smokers (n = 9) and nonsmokers (n = 12). Plasma concentrations of ascorbic acid and tocopherol were significantly increased by supplementation, but there was no significant change in plasma beta-carotene or blood glutathione peroxidase activity after supplementation with beta-carotene or selenium. DNA damage in mononuclear leukocytes, as assessed by comet assay, was not affected by any supplementation regimen. DNA damage, as assessed by 8-hydroxydeoxyguanosine in mononuclear leukocytes, was not influenced by ascorbic acid, alpha-tocopherol, or selenium supplementation in smokers or nonsmokers, but beta-carotene supplementation resulted in significant differences between smokers and nonsmokers in the level of oxidative DNA damage, with decreases in smokers and increases in smokers. This is a further indication of the differential effects of supplemental beta-carotene in smokers and nonsmokers.

Evaluation of manual and image analysis quantification of DNA damage in the alkaline comet assay.

The alkaline comet assay or single cell microgel electrophoresis assay is a sensitive method of detecting DNA strand breaks and alkali labile sites in individual cells. The results of this assay can be analysed by different methods. In this study we compared analyses of the same slides by a manual method and by image analysis, post-treatment of clone 707 Friend erythroleukaemia cells with H2O2. The parameters which were found to be particularly useful were comet area and comet length (measured manually) and percentage tail DNA, tail moment, tail length and tail length/head radius (L/H), measured using image analysis. The manual method for comet analysis presented in this paper would appear to provide good and reliable comet data. However, the image analysis comet system described offers an alternative analysis method which avoids the need for photomicrographs and tedious manual analysis. The image analysis parameters: % tail DNA, tail moment, tail length and L/H give good consistent results and for large-scale analysis it will, therefore, conceivably be the method of choice.

Activities of potential tumour marker enzymes during induced differentiation in HL-60 and U-937 cells.

HL-60 and U-937 cells were used as models to assess the involvement of the enzymes of thymidine metabolism in differentiation. Both cell types showed decreased thymidine kinase and thymidylate synthase but increased thymidine phosphorylase activities in response to the induction of differentiation by dimethylsulfoxide and 12-O-tetradecanoylphorbol 13-acetate. This was accompanied by a greater than three-fold increase in the stimulation of superoxide production in both cell lines. Thymidylate synthase and thymidine kinase activities were noted as potential markers of leukaemic cell proliferation while thymidine phosphorylase and superoxide production correlated well with differentiated phenotypes. Prolonged treatment of U-937 by 12-O-tetradecanoylphorbol 13-acetate resulted in a marked de-differentiation, indicating overstimulation of one or more of the isoforms of protein kinase C.

Mutagen sensitivity in thymidine kinase- and methyltransferase-deficient human lymphoblastoid cells.

In this study, the effect of thymidine kinase deficiency on the responses of the human lymphoblastoid cell line Raji to methyl methanesulphonate and mitomycin C was investigated. Mutagen sensitivity was measured in terms of cell survival and mutation to hypoxanthine-guanine phosphoribosyltransferase deficiency. Thymidine kinase-deficient Raji cells showed decreased survival and increased mutant frequency relative to wild-type cells following treatments with each of the mutagens used. It is suggested that this may be due to an imbalance in the supply of deoxyribonucleoside triphosphates to the excision repair process. The role of O6-methylguanine-DNA methyltransferase in the repair of DNA damage caused by these mutagens is also discussed.

Molecular mechanisms of mutagen hypersensitivity in adenine phosphoribosyl transferase-deficient Friend mouse erythroleukaemia cells.

Deficiency of the enzyme adenine phosphoribosyltransferase (APRT) has been associated with hypersensitivity to the mutagenic effects of ethyl methanesulphonate (EMS) and 254 nm ultraviolet (UV) radiation in clone 707 of Friend mouse erythroleukaemia (FEL) cells. The molecular nature of spontaneous EMS- and UV-induced mutations in the coding region of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was determined for wild-type FEL cells and two APRT-deficient mutant sub-clones which have significantly reduced ATP pool levels, and are mutagen-hypersensitive. Mis-sense base substitutions were the predominant type of spontaneous mutation. However, exon deletions, possibly involving aberrant splicing of HPRT mRNA, and a non-sense mutation were also observed. EMS-induced mutations in wild-type and APRT-deficient mutant sub-clones were GC-->AT transitions, which is consistent with O6-ethylguanine being the primary pre-mutagenic lesion. All UV-induced mutations in both cell types were targeted to dipyrimidine sites where the two most common classes of photoproducts (cyclobutane pyrimidine dimers and [6-4] photoproducts) are formed. The similarity in the mutations observed in both cell types indicates that the mutagen hypersensitivity of APRT-deficient cells may be the result of decreased efficiency in the excision repair processes due to reduced levels of ATP.

Serum thymidine kinase in colorectal neoplasia.

Thymidine kinase (TK), an enzyme known to be associated with DNA synthesis, has been measured in the serum of patients with asymptomatic colorectal adenomas (n = 40), asymptomatic colorectal carcinoma (n = 21) and patients known to have hepatic metastases form colorectal tumours (n = 33); enzyme levels have been compared with an age-matched group of asymptomatic people with no evidence of colorectal neoplasia at screening colonoscopy (n = 26). TK activity in patients with metastatic disease (median 4.23; range 2.03-14.12 pmol/ml/h) and in patients with adenomas (median 2.33; range 1.59-8.73 pmol/ml/h) was significantly higher than in the normal controls (median 2.04; range 1.29-5.40 pmol/ml/h). However TK activity in patients with asymptomatic cancer (median 1.85; range 1.00-4.50 pmol/ml/h) was not significantly different from the control group.

Bleomycin-induced DNA damage and repair in wild-type and thymidine kinase-deficient Friend mouse erythroleukaemia cells.

In this paper the DNA damage and repair induced by the radiomimetic agent bleomycin are compared in murine Friend erythroleukaemia wild-type 707 cells and a thymidine kinase-deficient sub-clone BUF. Comparisons are made using results obtained from the alkaline comet assay and unscheduled DNA synthesis experiments. Further analysis to determine the fidelity of bleomycin-induced repair as indicated by mutagenesis to hypoxanthine-phosphoribosyltransferase deficiency was also conducted. Similar sensitivities to bleomycin treatments were observed in the two cell types with the comet assay, while similar levels of dose-dependent excision repair following bleomycin treatments were also detected in unscheduled DNA synthesis experiments. Comet assay and unscheduled DNA synthesis experimental results are in agreement. Survival and induced hypoxanthine-phosphoribosyltransferase mutant frequencies were observed to be unaffected by a thymidine kinase-deficiency in Friend erythroleukaemia cells. The results of this investigation suggest no overall difference in the repair capacities or the repair fidelity of Friend 707 relative to BUF cells following bleomycin treatments.

An investigation of the cytotoxic and mutagenic potential of low intensity laser irradiation in Friend erythroleukaemia cells.

The purpose of this study was to investigate the cytotoxic and genotoxic potential of low intensity laser irradiation (660 nm, 12 mW, 5 kHz) on mammalian cells. Thymidine kinase (TK)-positive and TK-deficient Friend erythroleukaemia (FEL) cells, clone 707 and subclone 707BUF respectively, were used in this investigation. Following irradiation of exponentially growing cells in suspension at doses of 2 and 20 J/cm2 a number of sensitive bioassays were used to facilitate the detection of laser-induced mutations, DNA damage and cell killing. Mutations were assessed by the examination of chromosome spreads, the determination of micronucleus frequency and by the determination of the mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. DNA damage was quantified using a sensitive ELISA. The cytotoxic effect of laser irradiation was assessed using a cloning assay. The results of this investigation did not show any significant increase in mutation frequency, DNA damage or cell survival in the laser-irradiated cells, compared to sham-irradiated controls. The lack of any demonstrable cytotoxic and genotoxic effects of low intensity laser irradiation on mammalian cells in culture would support it as being a safe modality for clinical use.

Effects of thymidine kinase and methyltransferase deficiency on mutagenesis in a human lymphoblastoid cell line.

In this study the effect of thymidine kinase (TK) deficiency on mutagen sensitivity was examined in the human lymphoblastoid cell line Raji. Wild-type and TK-deficient Raji cells were treated with a range of concentrations of ethyl methanesulphonate (EMS) and a range of doses of ultraviolet (UV) light, then examined for mutagen sensitivity as measured by cell survival and mutation to HGPRT deficiency. Dose-dependent responses were observed and TK-deficient cells exhibited decreased survivals and increased mutant frequencies relative to wild-type cells. TK-deficient Raji cells are also deficient in O6-methylguanine-DNA-methyltransferase. This may partially account for their sensitivity to EMS but does not account for the results obtained with UV. It is therefore likely that an additional factor, such as alterations in supply of deoxyribonucleoside triphosphates, may affect the mutagen sensitivity of Raji cells.

An investigation of mutation as a function of age in humans.

An accumulation of mutations on their own or together with other age-related changes may contribute to aging and the development of age-related pathologies. The aim of this investigation was to assess the extent of DNA mutations as a function of age in humans. The mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl-transferase (hgprt) locus was assessed in lymphocytes isolated from male volunteers in each of three age groups (35-39, 50-54 and 65-69 years). Results show that the mean MF in the 65-69 years group was approximately twice that in the 35-39 and 50-54 years groups (4.1/10(6) cells, 1.9/10(6) cells and 1.79/10(6) cells, respectively) increasing by about 1.33% per year, after 54 years. In addition, there was an increased frequency of chromosomal aberrations in the 65-69 years group compared to the other two age groups. The results of this investigation show an increase in DNA mutations in cultured human lymphocytes with age. Factors which may influence the extent of DNA damage in human lymphocytes are discussed.

Why do most primary bladder neoplasms first appear around the ureteric orifices?

The majority of primary bladder neoplasms are known to arise within the mucosa around the ureteric orifices and bladder base. This may be due to the mucosa in this area being more susceptible to carcinogens than other areas of the bladder. Deficiency in the nucleotide salvage pathway enzyme thymidine kinase (TK), and especially its TK1 isozyme, has been shown to predispose cell lines to increased mutagenesis. Total TK and TK1 activities were measured in mucosal samples taken adjacent to the ureteric orifices and dome in 32 normal bladders and both total TK and TK1 were shown to be significantly decreased in the mucosa adjacent to the ureteric orifices. This may explain why primary bladder neoplasms occur more commonly in this site.

Thymidine kinases: the enzymes and their clinical usefulness.

Thymidine kinases (TK) convert thymidine, or deoxythymidine (dT) to the respective monophosphate. TK occurs in many different procaryotic and eucaryotic species and different TK isoenzymes are found within the same eucaryotic cell. One isoenzyme (foetal, cytoplasmic, TK1) is associated with cell division while the other (adult, mitochondrial, TK2) is cell cycle independent. The relative isoenzyme activities in a tissue thus reflect the fraction of proliferating cells. The gene encoding TK1 has been cloned for many species and regulation of its expression is known to be complex. Increases in TK activity appear to correlate with the presence of human neoplasia and disease progression and regression have been reported to correlate with TK levels in many cancer types. TK estimations in human lymphoproliferative diseases have implicated this enzyme as an early marker of maldifferentiation. TK levels may also be increased in non-dividing mammalian cells infected with RNA or DNA viruses. Some virus encoded TK has been shown to differ biochemically, immunologically and in substrate specificity from the corresponding TK isoenzymes in target host cells thus facilitating the development of specific antiviral therapeutics. Further, TK1 in leukemic cells may differ biochemically from normal cellular TK1 such that tumor-specific TK may provide a target for tumor detection and therapy. TK quantitation has conventionally been performed in assays of enzyme activity using radiolabeled (3H or 125I) nucleoside substrates. The development of TK1-specific, non-radioisotope based immunoassays and the measurement of TK mRNA in tumour tissue using TK (DNA or RNA) probes may prove sufficiently valuable to be incorporated into the routine clinical management of human cancer.

Breast tumour thymidine kinase levels and disease recurrence.

Thymidine kinase (TK) exists in two forms, TK1 and TK2. TK levels and oestrogen receptor status (OR) were measured in tumours from 86 patients with operable breast cancer and the patients were monitored for recurrence over 24 months. During the monitored period, 13 patients showed recurrence. These patients also exhibited higher total TK levels per mg tumour (P < 0.01) and higher TK1 levels (P < 0.001) than those who did not show recurrence. TK1 levels relative to TK2 were significantly higher (P < 0.05) in OR-negative tumours (n = 29) than in OR-positive tumours (n = 57). OR-negative (n = 9) and OR-positive (n = 4) patients who recurred had significantly higher TK1 levels relative to TK2 than did those who not recur (n = 20 and n = 53, respectively). These preliminary results indicate that breast tumour TK levels may have value in determining prognosis.

Daily energy expenditure in free-living children: comparison of heart-rate monitoring with the doubly labeled water (2H2(18)O) method.

Total energy expenditure (TEE) was measured simultaneously in 36 free-living children aged 7, 9, 12, and 15 y over 10-15 d by the doubly labeled water (DLW) method and for 2-3 separate days by heart-rate (HR) monitoring. The 95% confidence limits of agreement (mean difference +/- 2SD) were -1.99 to +1.44 MJ/d. HR TEE discrepancies ranged from -16.7% to +18.8% with 23 values lying within +/- 10% of DLW TEE estimates. Boys and girls spent 462 +/- 108 and 318 +/- 120 min/d, respectively, in total physical activity (P less than 0.01). Time spent in moderate and vigorous physical activity (MVPA) was 68 +/- 37 min/d by younger children (7-9 y) and 34 +/- 24 min/d by older children (12-15 y) (P less than 0.001). Younger boys engaged in MVPA (91 +/- 33 min/d) and vigorous physical activity (VPA) (35 +/- 15 min/d) significantly longer than younger girls (MVPA, 39 +/- 16 min/d, P less than 0.001; VPA, 10 +/- 4 min/d, P less than 0.01) as did older boys (MVPA, 52 +/- 21 min/d; VPA, 30 +/- 18 min/d) compared with older girls (MVPA, 15 +/- 10 min/d; VPA, 8 +/- 5 min/d). HR monitoring provides a close estimation of the TEE of population groups and objective assessment of associated patterns of physical activity.