Effects of substituting sedentary time with physical activity on body mass index in Japanese adults with Down syndrome: A cross-sectional study.

Objective: and Rationale: Obesity is a health challenge for adults with Down syndrome. Therefore, a physical activity promotion program is required to prevent or reduce obesity in adults with this condition. However, there is a lack of evidence of useful risk reduction initiatives. The objective of this study was to suggest a rationale for behaviors that should replace time of inactivity to reduce obesity in Japanese adults with Down syndrome. Methods: The participants were adults with Down syndrome, aged 18-48 years, living in Japan. The snowball sampling method was used. To detect an effect size of 0.20 for body mass index using an F-test, 80 participants were required, generating a statistical power of 0.8 and a risk level of 0.05. Survey items included sex, age, height, weight, body mass index, and physical activity (min/d). Physical activity was categorized by intensity and further divided into ambulatory and non-ambulatory activities. The body mass index categories were compared using analysis of covariance. An isotemporal substitution model was used to confirm the interdependence of behaviors. Results: Half of the participants were obese, with a body mass index of 25 kg/m2 or higher. The obese group had significantly fewer light physical activity, moderate-to-vigorous physical activity, and ambulatory moderate-to-vigorous physical activity times than the non-obese group. Replacing 10 min of sedentary behavior with ambulatory moderate-to-vigorous physical activity was significantly associated with a lower body mass index. Conclusions: This study suggests a rationale for behaviors that should replace time of inactivity to reduce obesity in adults with Down syndrome. Specifically, replacing 10 min of sedentary behavior with ambulatory moderate-to-vigorous physical activity time may contribute to obesity reduction.

The Amount of Light to Vigorous Physical Activity (Met's-Hours/Day) in Children with and without Down Syndrome Attending Elementary School in Japan.

Children with Down syndrome (DS) have physical characteristics such as hypotonus of the musculature. Therefore, their attainment rate of physical activity guidelines is low, and guidelines alone may not be sufficient in assessing the amount of physical activity in children with DS. Compared with normal children (NC) of the same grade, light physical activity (LPA) must be considered while assessing physical activity of children with DS, owing to muscle hypotonia. This study included 69 children with DS and 68 NC in grades 4−6 attending elementary school in Japan. The measurements for physical characteristics included age, height, weight, and body mass index. Physical activity was measured using a triaxial accelerometer, which indicated physical activity volume. Children with DS had less moderate-to-vigorous physical activity duration (DS: 53.1 min/day, NC: 65.0 min/day; p < 0.001) but significantly longer LPA duration (DS: 376.4 min/day, NC: 287.7 min/day; p < 0.001) than NC. Conversely, the amount of light to vigorous physical activity (Met's-hours/day) was greater in children with DS (DS: 16.0 Met's-hours/day, NC: 14.4 Met's-hours/day; p = 0.037). In children with DS with muscular hypotonia, vigorous physical activity is challenging, but LPA is feasible. Developing and validating educational programs that promote physical activity with intensity level depending on individual's physical characteristic are warranted.

Syntactic development and verbal short-term memory of children with autism spectrum disorders having intellectual disabilities and children with down syndrome.

Background and aims: Previous studies suggest that syntactic development in children with intellectual disabilities (ID) is positively correlated with verbal short-term memory (VSTM). This study investigated the characteristics of syntactic development and their relationships of VSTM in children with ID based on type. Methods: The participants were children with ID (N = 34), including 14 children with autism spectrum disorders (ASD), 20 with Down syndrome (DS), with chronological ages from 8 years 10 months to 18 years 4 months and nonverbal mental ages (MA) of over 4 years, and typically developing (TD) children (N = 21) with chronological ages from 5 years 0 months to 5 years 10 months. They were assessed using VSTM, syntactic comprehension, and expression tasks. Results: The results showed that both the ASD and DS groups performed significantly lower on the syntactic comprehension task and the syntactic expression task than the TD group with the same nonverbal MA in the complex aspect of grammatical structure. In the VSTM task, the ASD group showed significantly lower performance in sentence and story repetition tasks than the TD group of the same nonverbal MA. The DS group showed significantly lower performance in forward digit span, and word, nonword, sentence, and story repetition tasks than the TD group of the same nonverbal MA. Conclusions: These results suggest that children with ASD have difficulty in understanding and remembering linguistic information with complex semantic structures, and children with DS have a small capacity for VSTM, affecting their syntactic development.

Donepezil significantly improves abilities in daily lives of female Down syndrome patients with severe cognitive impairment: a 24-week randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Down syndrome (DS) patients share certain neuropathological features with Alzheimer disease patients. A randomized, double-blind, placebo-controlled study was performed to investigate the efficacy and safety of donepezil, an Alzheimer disease drug, for DS patients. METHOD: Twenty-one DS patients with severe cognitive impairment were assigned to take donepezil (3 mg daily) or a placebo for 24 weeks, and evaluated for activities in daily lives by concisely modified International Classification of Functioning, Disability and Health (ICF) scaling system. RESULTS: ICF scores significantly increased without any adverse effects in the donepezil group in comparison to those in the placebo control. Among the individual functions tested, there was a dramatic improvement in the global mental functions and in specific mental functions. CONCLUSIONS: Donepezil may effectively and safely improve overall functioning of DS patients with severe cognitive impairment.

Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide.

The main anticancer action of doxorubicin (DOX) is believed to be due to topoisomerase II inhibition and free radical generation. Our previous study has demonstrated that TAS-103, a topoisomerase inhibitor, induces apoptosis through DNA cleavage and subsequent H(2)O(2) generation mediated by NAD(P)H oxidase activation [H. Mizutani et al. J. Biol. Chem. 277 (2002) 30684-30689]. Therefore, to clarify whether DOX functions as an anticancer drug through the same mechanism or not, we investigated the mechanism of apoptosis induced by DOX in the human leukemia cell line HL-60 and the H(2)O(2)-resistant sub-clone, HP100. DOX-induced DNA ladder formation could be detected in HL-60 cells after a 7 h incubation, whereas it could not be detected under the same condition in HP100 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded the increase in Delta Psi m and caspase-3 activation. Poly(ADP-ribose) polymerase (PARP) and NAD(P)H oxidase inhibitors prevented DOX-induced DNA ladder formation in HL-60 cells. Moreover, DOX significantly induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, in HL-60 cells at 1 h, but not in HP100 cells. DOX-induced apoptosis was mainly initiated by oxidative DNA damage in comparison with the ability of other topoisomerase inhibitors (TAS-103, amrubicin and amrubicinol) to cause DNA cleavage and apoptosis. These results suggest that the critical apoptotic trigger of DOX is considered to be oxidative DNA damage by the DOX-induced direct H(2)O(2) generation, although DOX-induced apoptosis may involve topoisomerase II inhibition. This oxidative DNA damage causes indirect H(2)O(2) generation through PARP and NAD(P)H oxidase activation, leading to the Delta Psi m increase and subsequent caspase-3 activation in DOX-induced apoptosis.

Factors influencing the overestimation of plasma vancomycin concentrations measured by the Abbott TDx technique.

The Abbott TDx technique (TDx) has been reported to overestimate the plasma concentration of vancomycin (VCM) in patients with renal failure and also in those with normal renal function. The aim of this study was to investigate factors influencing the overestimation of plasma VCM concentrations measured by TDx compared with high-performance liquid chromatography (HPLC) as a reference technique. First, the precision and accuracy of TDx and HPLC were compared using 5 weighed-in concentrations of VCM. The coefficients of variation (CV) for both TDx and HPLC were less than 3% at weighed-in concentrations of 3.0, 7.5, 15.0, 30.0, and 60.0 microg/mL. The authors did not find overestimation of VCM concentrations by TDx in any weighed-in sample. Next, VCM concentrations measured by TDx were compared with those measured by HPLC in 253 plasma samples obtained from 83 patients receiving VCM. Regression analysis showed that VCM concentrations measured by TDx were closely correlated with those measured by HPLC, with a correlation coefficient of 0.963. Then, the authors divided plasma samples into 2 groups based on the time of sampling, ie, pre- and postdose. The mean TDx/HPLC ratio was significantly higher in the predose group (1.23 +/- 0.22) than in the postdose group (1.11 +/- 0.16) (P < 0.01). An Eksborg plot showed that the TDx/HPLC ratio decreased slightly with increased total bilirubin concentrations in the predose samples. These results indicated that plasma VCM concentrations measured by TDx were influenced by the time of sampling and the plasma bilirubin concentration. The overestimation of VCM concentrations in the predose group may have resulted from metabolites of VCM because the relative concentrations of the metabolites were likely to be greater in the predose group than in the postdose group. Thus, VCM postdose samples may provide useful information in cases in which a good clinical outcome has not been obtained using monitoring of the predose concentration alone.

Amplification of C1027-induced DNA cleavage and apoptosis by a quinacrine-netropsin hybrid molecule in tumor cell lines.

We examined the effect of a newly synthesized DNA-binding ligand, quinacrine-netropsin hybrid molecule (QN), on cytotoxicity, apoptosis, and DNA strand breaks induced by an enediyne antitumor antibiotic, C1027. QN significantly enhanced C1027-induced cellular DNA strand breaks, caspase-3 activation, and DNA ladder formation, characteristic of apoptosis, in human HL-60 cells. Flow cytometry revealed that C1027-induced intracellular H(2)O(2) generation was enhanced by QN, suggesting that QN enhances C1027-induced cytotoxic effect through H(2)O(2)-mediated apoptosis. QN also significantly enhanced C1027-induced apoptosis in BJAB cells, and the inhibition of apoptosis was observed in BJAB cells transfected with Bcl-2 gene. The experiment using (32)P-labeled DNA fragments showed that the addition of QN enhanced C1027-induced double-stranded DNA cleavage at the 5'-AGG-3'/3'-TCC-5' sequence (cutting sites are underlined). These results suggest that QN enhances C1027-induced antitumor effect via DNA cleavage and apoptosis. The present study shows a novel approach to the potentially effective anticancer therapy.

Comparison of cyclosporine concentrations in renal transplant recipients using ACMIA and mFPIA methods.

OBJECTIVE: This study investigated the performance and data comparability of a new, relatively specific immunoassay method, affinity column mediated immunoassay (ACMIA) run on the Dimension Xpand-HM, and the established less specific monoclonal fluorescence polarization immunoassay (mFPIA) method on the TDx analyzer (mFPIA/TDx) in determining cyclosporine (CsA) concentrations. METHODS: Accuracy and within and between-run precision were tested. Then we measured CsA concentrations of 216 samples obtained from 51 patients and divided the 113 samples from 21 patients with renal transplants into two groups based on sampling time. RESULTS: Accuracy relative to the four weighed-in concentrations ranged from 99% to 104% and from 106% to 117% for ACMIA and mFPIA/TDx, respectively. The mean within-run precision (CV%) for the ACMIA and mFPIA/TDx methods was 4.31% and 2.57%, respectively. The mean recovery for ACMIA and mFPIA/TDx in the between-run study was 104.50% and 111.12%, respectively. The mFPIA/ACMIA ratio (the ratio of the concentration measured by mFPIA/TDx to that measured by ACMIA) of C2-3 (concentrations measured 2-3 h after oral administration) was 118.85%, which was significantly smaller than that (139.12%) of C8-12 (those measured more than 8 h after the administration) at each mean concentration. CONCLUSIONS: These results indicate that ACMIA is more accurate than mFPIA/TDx, and the difference in the mFPIA/ACMIA ratio between C2-3 and C8-12 was due to the difference in the relative cross-reactivity with CsA metabolites. Although mFPIA/TDx had an apparent calibration error, both methods had a clinically acceptable within and between-run precision.

DNA intrastrand cross-link at the 5'-GA-3' sequence formed by busulfan and its role in the cytotoxic effect.

Busulfan (1,4-butanediol dimethanesulfonate) has been used widely for the treatment of patients with chronic myelogenous leukemia. Busulfan is bifunctional and thus may effectively induce DNA damage, which may play an important role in the cytotoxicity. In this study, we compared the cytotoxicity of bifunctional busulfan with that of monofunctional ethyl methanesulfonate (EMS) in human promyelocytic leukemia HL-60 cells. Busulfan showed a significant inhibitory effect on cell growth, whereas the cells grew in the presence of EMS. To clarify the mechanism of cytotoxicity of busulfan, we investigated DNA damage induced by busulfan using 32P-5'-end-labeled DNA fragments obtained from the human p16 tumor suppressor gene. Busulfan induced DNA damage dose-dependently, whereas EMS caused little DNA damage. DNA-sequencing experiments using piperidine and 3-methyladenine DNA glycosylase indicated that busulfan caused double-base lesions mainly at 5'-GA-3' and, to a lesser extent, at 5'-GG-3' sequences. Time of flight mass spectrometry confirmed that busulfan forms an intrastrand cross-link at the 5'-GA-3' sequence, in addition to mono-alkylation. The mechanism and the role of cross-linking at the 5'-GA-3' sequence are discussed in relation to the cytotoxicity induced by busulfan.

Steroid-induced diabetes mellitus and related risk factors in patients with neurologic diseases.

STUDY OBJECTIVE: To determine the frequency of steroid-induced diabetes mellitus (SDM) and the related risk factors in patients with neurologic diseases who receive high doses of steroids. DESIGN: Retrospective chart review. SETTING: Neurology ward of a university-affiliated hospital. PATIENTS: Twenty-five patients with neurologic diseases who received prednisolone 30-60 mg/day orally after breakfast for more than 2 weeks. MEASUREMENTS AND MAIN RESULTS: Plasma glucose concentrations were determined immediately before and 2 hours after each meal. Steroid-induced diabetes mellitus was diagnosed if the patient had either a fasting glucose concentration of 126 mg/dl or greater, or a random glucose concentration of 200 mg/dl or greater. The patients were divided into two groups on the basis of whether SDM had developed (13 patients) or not (12 patients). Ages, body mass indexes, cumulative total doses and daily doses of prednisolone, duration of therapy, and serum cholesterol and triglyceride concentrations were compared between the groups. Thirteen of the 25 patients were identified with SDM, and all of them had plasma glucose concentrations of 200 mg/dl or greater 2 hours after lunch. Mean age (59.1 +/- 10.2 yrs) and cholesterol concentration after prednisolone treatment (226.8 +/- 36.4 mg/dl) in the SDM group were significantly higher than those values in the non-SDM group (41.3 +/- 18.0 yrs and 188.1 +/- 27.2 mg/dl, respectively, p<0.01). CONCLUSIONS: A close relationship among postprandial hyperglycemia, advanced age, and hypercholesterolemia is a characteristic of SDM in patients with neurologic diseases. Therefore, monitoring the plasma glucose concentration 2 hours after lunch may be useful to detect SDM in these patients.

[Successful treatment by tacrolimus in two patients with Osserman's grade III intractable myasthenia gravis and elderly IIb].

Tacrolimus, a potent immunosuppressive agent, has recently become available for the treatment of myasthenia gravis (MG). However, few reports have evaluated the usefulness of tacrolimus in the elderly and in intractable MG classified as Osserman's grade IIb or higher. In this study, we examined the effects of tacrolimus in two patients with postthymectomy Osserman's grade III and IIb MG. The effects of tacrolimus were evaluated using the modified quantitative MG score (QMG score) and anti-acetylcholine receptor antibody (AChRAb) titer. Patient 1 was a 39-year-old woman with Osserman grade III MG. Her MG score improved after tacrolimus administration (3 mg/day), allowing gradual decreases in the doses of prednisolone and pyridostigmine because of improvement in MG symptoms. The AChRAb titer also decreased from 100.0 to 25.7 nmol/l during tacrolimus treatment. The concentrations of tacrolimus in whole blood ranged from 5.5 to 8.2 ng/ml during treatment. Patient 2 was a 68-year-old man diagnosed with Osserman grade IIb MG. After the initiation of tacrolimus treatment, dysphagia improved dramatically, and he was able to swallow food without a nasal feeding tube. The AChRAb titer decreased from 42.4 to 23.4 nmol/l during the treatment period. The concentration of tacrolimus in whole blood was 7.0 ng/ml 6 days after the initiation of treatment. These findings suggest that tacrolimus is effective in intractable and elderly MG patients.

Fever as a risk factor causing delayed elimination of methotrexate in pediatric patients receiving high doses of cancer chemotherapy.

PURPOSE: Delayed elimination of methotrexate (MTX) has been reported to be caused by a number of factors. In order to identify these causes, we retrospectively investigated the risk factors for delayed elimination in pediatric patients who received high doses of MTX. SUBJECTS AND METHODS: The study included 69 courses of therapy involving 22 patients who received more than 1000 mg/m(2) of MTX. Plasma MTX concentrations 48 h (C48) and 72 h (C72) after infusion were used as indices of MTX elimination. RESULTS: Neither C48 nor C72 was directly proportional to the dose of MTX infused. Both C48 and C72 were significantly higher in patients who developed fever of above 38 degrees C within a 10-day period (i.e., from 7 days before to 3 days after infusion) than in patients with no fever. Subgroup analysis revealed that C72 was significantly higher in patients who either developed fever and received antipyretics or developed fever but did not receive antipyretics than in patients who did not develop fever and did not receive antipyretics. Changes in the serum creatinine concentrations before and after MTX infusion revealed that renal function was significantly decreased in patients who developed fever compared to patients without fever. CONCLUSIONS: The present results suggest that the development of fever is one of the main risk factors for the delayed elimination of MTX.

Comparative evaluation of digoxin concentrations determined by three assay systems: TDx, IMx and OPUS.

Digoxin concentrations measured by three automated immunoassay systems, i.e. OPUS, TDx and IMx assays, were compared in order to evaluate precision and accuracy performance, and data compatibility. Coefficients of variation for all methods in within-run and between-run precision were less than 10% at weighed-in concentrations of 0.545, 1.090 and 2.180 ng/ml. The accuracy relative to the three weighed-in concentrations ranged from 97% to 123% for all methods. One hundred and three plasma samples from 60 patients receiving digoxin were used to evaluate the data compatibility. Digoxin concentrations measured by the three immunoassay systems correlated well with one another. These results suggest that there are few problems when switching between digoxin assay methods, and that IMx and OPUS are more useful than TDx because they do not require sample pretreatment. The digoxin concentrations of the plasma samples from one patient receiving both digoxin and potassium canrenoate were investigated as a case report. The digoxin concentrations measured by TDx and IMx became higher than those measured by OPUS after starting the combination treatment. In another patient suffering from bilirubinaemia, the digoxin concentrations measured by TDx or IMx were higher than those measured by OPUS. These results suggest that OPUS has a higher specificity for measuring the plasma digoxin concentrations compared with TDx or IMx.

Relationship between development of nephrotoxicity and blood concentration of cyclosporine A in bone-marrow transplanted recipients who received the continuous intravenous infusion.

This study was undertaken to investigate the relationship between blood concentration of cyclosporine A (CsA), administered intravenously by a 24-h continuous infusion, and drug-induced nephrotoxicity or hepatotoxicity. It was investigated retrospectively in 8 patients who had received an allogeneic bone marrow transplant (BMT). The correlation between daily doses and blood concentration of CsA was not significant. Then, the data of blood concentration of CsA and renal or liver function test result were divided into 5-d periods from the date of transplantation, and the mean value for each period was calculated. The maximum values of blood urea nitrogen (BUN) and serum creatinine (SCr) were consistently observed only after the period when the 5-d mean CsA concentration reached the peak level: the maximum BUN and SCr values were witnessed at Periods 2 to 10 and at Periods 1 to 9, respectively. On the other hand, no consistent correlation was found between the 5-d mean CsA concentrations and liver function test result. We also investigated the relationship between renal function and the cumulative dose or AUC of CsA. The parameters of renal function tests reached peak levels at the cumulative dose of 4000 to 10000 mg and at the cumulative AUC of 280000 to 660000 ng/ml.h. These results suggest that: 1) a deterioration of renal function occurs usually after the peak blood concentration of CsA is attained, and 2) the monitoring of the blood concentration of CsA is useful in predicting renal dysfunction in post-BMT patients.

Oxidative DNA damage induced by photodegradation products of 3(')-azido-3(')-deoxythymidine.

3(')-Azido-3(')-deoxythymidine (AZT) is carcinogenic to experimental animals and can cause the formation of 8-oxo-7,8-dihydro-2(')-deoxyguanosine (8-oxodG) in humans and animals. To clarify the mechanism of carcinogenesis by AZT, we investigated DNA damage induced by its photodegradation products, using 32P-5(')-end-labeled DNA fragments obtained from human genes. Following exposure to UVB, AZT induced DNA damage in the presence of Cu(II). Catalase inhibited DNA damage, indicating the involvement of H(2)O(2). UVB-exposed AZT plus Cu(II) induced 8-oxodG formation in a dose-dependent manner. Mass spectrum of UVB-exposed AZT demonstrated the generation of a hydroxylamine derivative. The colorimetric determination suggested that AZT was converted into the hydroxylamine derivative depending on UVB doses. UVB-exposed AZT induced double base damage at the 5(')-ACG-3(') sequence, complementary to a hot spot of the p53 gene. The basic compound, hydroxylamine, showed similar site specificity. The hydroxylamine derivative produced by photodegradation and/or possible metabolism of AZT induces oxidative DNA damage, which may participate in carcinogenesis.

Distinct mechanisms of site-specific oxidative DNA damage by doxorubicin in the presence of copper(II) and NADPH-cytochrome P450 reductase.

The anticancer mechanism of doxorubicin (DOX), an anthracycline antibiotic, is believed to involve DNA damage through topoisomerase II inhibition and free radical generation. The free radical generation may also participate in genotoxicity, as well as cardiotoxicity, in normal human cells. The present study showed that DOX generates 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), an indicator of oxidative DNA damage, in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. Since DOX has both p-quinone and p-hydroquinone residues, free radical generation can be initiated by either reduction or oxidation of DOX. To clarify whether the oxidized or reduced form is more important for DOX-induced H(2)O(2) generation, we investigated the site-specific DNA damage induced by DOX in the presence of Cu(II), in comparison with that in the presence of cytochrome P450 reductase, using (32)P-labeled DNA fragments. DOX caused DNA damage in the presence of Cu(II) or cytochrome P450 reductase. The degree of Cu(II)-mediated DNA damage, including 8-oxodG formation, was much greater than that of cytochrome P450 reductase-mediated DNA damage. DOX plus Cu(II) caused DNA damage specifically at guanine, thymine and cytosine residues, particularly at 5'-GG-3', 5'-GT-3' and 5'-TG-3' sequences. Scavenger experiments suggested the involvement of reactive species generated from H(2)O(2) and Cu(I). When cytochrome P450 reductase and NADPH were used instead of Cu(II), every nucleotide was uniformly damaged, suggesting the participation of.OH. We conclude that DOX may induce carcinostatic and genotoxic effects through oxidation of its p-hydroquinone moiety by metal ion rather than through p-quinone reduction by cytochrome P450 reductase.

Clinical efficacy of therapeutic drug monitoring in patients receiving vancomycin.

Efficacy of therapeutic drug monitoring (TDM) of vancomycin (VCM) was retrospectively investigated in 184 patients with methicillin-resistant Staphylococcus aureus (MRSA) infection. The incidence of nephrotoxicity was compared between the patients who received TDM practice (TDM group, n=73) and did not (non-TDM group, n=111). Creatinine clearance (CLcr) values decreased significantly after the VCM therapy in the non-TDM group (p<0.05). The patients with MRSA bacteremia or pneumonia were classified into two groups according to peak concentrations of VCM: above 25 microg/ml (Group A: n=29) and below (Group B: n=24). Mean duration of VCM therapy (14.1 d) in Group A was significantly shorter than that (27.0 d) in Group B. Mean cumulative total VCM doses (13.3 g) in Group A was significantly less than that (25.0 g) in Group B. These results indicate that monitoring peak concentration is essential to obtain better clinical effects for VCM therapy, and that the peak concentration above 25 microg/ml is more effective.

Mechanism of apoptosis induced by a new topoisomerase inhibitor through the generation of hydrogen peroxide.

TAS-103, a new anticancer drug, induces DNA cleavage by inhibiting the activities of topoisomerases I and II. We investigated the mechanism of TAS-103-induced apoptosis in human cell lines. Pulsed field gel electrophoresis revealed that in the leukemia cell line HL-60 and the H(2)O(2)-resistant subclone, HP100, TAS-103 induced DNA cleavage to form 1-2-Mb fragments at 1 h to a similar extent, indicating that the DNA cleavage was induced independently of H(2)O(2). TAS-103-induced DNA ladder formation in HP100 cells was delayed compared with that seen at 4 h in HL-60 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded increases in mitochondrial membrane potential (DeltaPsim) and caspase-3 activation. Inhibitors of poly(ADP-ribose) polymerase (PARP) prevented both TAS-103-induced H(2)O(2) generation and DNA ladder formation. The levels of NAD(+), a PARP substrate, were significantly decreased in HL-60 cells after a 3-h incubation with TAS-103. The decreases in NAD(+) levels preceded both increases in DeltaPsim and DNA ladder formation. Inhibitors of NAD(P)H oxidase prevented TAS-103-induced apoptosis, suggesting that NAD(P)H oxidase is the primary enzyme mediating H(2)O(2) formation. Expression of the antiapoptotic protein, Bcl-2, in BJAB cells drastically inhibited TAS-103-induced apoptosis, confirming that H(2)O(2) generation occurs upstream of mitochondrial permeability transition. Therefore, these findings indicate that DNA cleavage by TAS-103 induces PARP hyperactivation and subsequent NAD(+) depletion, followed by the activation of NAD(P)H oxidase. This enzyme mediates O(2)(-)-derived H(2)O(2) generation, followed by the increase in DeltaPsim and subsequent caspase-3 activation, leading to apoptosis.