Prescription pattern of antipsychotic drugs for schizophrenic inpatients in Japan: research on East Asia Psychotropic Prescription Pattern-Antipsychotics study.
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Adolescent↗
Biomedical subjects
Jun Nakamura
Publications and source records attributed to Jun Nakamura.
Effects of maternal uninephrectomy on the development of fetal rat kidney: apoptosis and the expression of oncogenes.
The present study was designed to explore whether maternal uninephrectomy affects development of the collecting ducts in fetal kidney. Localization of DNA fragmented cells in the kidney of fetal rats from uninephrectomized mothers were examined by the terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end labeling (TUNEL) method. Immunohistochemistry was used to examine the localizations of bcl-2 gene products. The gene expressions for bcl-2, p53, and WT1 mRNAs were examined by using the semi-quantitative reverse transcript-polymerase chain reaction. TUNEL positive cells were more numerous in the medullary collecting ducts of the fetuses from uninephrectomized mothers than in those of the fetuses from sham-operated ones. The expressions of bcl-2, p53, and WT1 mRNAs were lower in the fetuses from uninephrectomized mothers than in the fetuses from sham-operated ones. Cells in the medullary collecting ducts showed positive reactions to anti-bcl-2 gene products antibody with the reactions being weaker in the fetuses from uninephrectomized mothers. These results showed that maternal uninephrectomy accelerated the development of fetal rat kidney and it was associated with the lowered the expression of bcl-2 in fetal rat kidney.
Animals↗
Changes in composition and content of mycolic acids in glutamate-overproducing Corynebacterium glutamicum.
Overproduction of glutamate by Corynebacterium glutamicum is induced by biotin limitation or by the supplementation of specific detergents, sublethal amounts of penicillin, or cerulenin. But, it remains unclear why these different treatments, which have different sites of primary action, produce similar effects. In this study, it was found that the cellular content of mycolic acids--characteristic constituents of Corynebacterineae that are synthesized from fatty acids and form a cell surface layer--decreased under all conditions that induced glutamate overproduction. Furthermore, short mycolic acids increased under conditions of biotin limitation and cerulenin supplementation. These results suggest that different treatments produce the same effect that causes defects in the mycolic acid layer. This is perhaps one of the key factors in overproduction of glutamate by C. glutamicum.
Cerulenin↗
No association between a functional NAD(P)H: quinone oxidoreductase gene polymorphism (Pro187Ser) and tardive dyskinesia.
Several lines of evidence have indicated that free radicals may play a role in the pathophysiology of tardive dyskinesia (TD) (reviewed in Andreassen and Jorgensen, 2000). NAD(P)H: quinone oxidoreductase (NQO1) is an important enzyme in the human body that counteracts the oxidative stress-induced neuronal injury caused by the toxic free radicals such as dopamine-semiquinones. Taking the possible genetic predisposition to TD into account (Yassa and Ananth, 1981), the NQO1 gene is a good candidate gene that may confer increased susceptibility to TD. Based on this hypothesis, Pae et al. (2004) reported a significant association between the Pro187Ser polymorphism in the NQO1 gene and TD. In the present study, we attempted to replicate the findings of Pae et al. (2004) with the same polymorphism in 222 Japanese patients with schizophrenia. No significant difference was detected between patients with and without TD in the allelic distribution (chi2 = 0.070, d.f. = 1, p = 0.795) and in the genotypic distribution (chi2 = 0.910, d.f. = 2, p = 0.657). In addition, there was no significant difference in terms of total Abnormal Involuntary Movement Scale scores among the three genotype groups (p = 0.49). Our results suggest that the NQO1 gene polymorphism does not confer an increased risk of TD.
Adult↗
Association study between functional polymorphisms in the cytochrome P450 1A2 and 2D6 genes and polydipsia in schizophrenia.
The underlying pathophysiology of polydipsia in schizophrenia is poorly understood. Several studies, however, have suggested that there might be a genetic predisposition to polydipsia. In the present study, using a case-control sample that is independent from the previous family sample, we examined a possible association between polydipsia and functional polymorphisms in the genes of cytochrome P450 (CYP) 1A2 and 2D6, primarily important enzymes to the pharmacokinetics of antipsychotic drugs. Japanese patients with schizophrenia (63 polydipsics and 78 nonpolydipsics) were genotyped for two functional polymorphisms, the 734C/A polymorphism in the CYP1A2 gene and the 2D6*10 allele of the CYP2D6 gene. Neither of the polymorphisms was found to be associated with polydipsia nor was any evidence found that the two polymorphisms have an additive effect on the liability to polydipsia. Our results suggest that the CYP1A2 and CYP2D6 polymorphisms are not likely to play a major role in the development of polydipsia in schizophrenia, although further studies testing other alleles of CYP1A2 and CYP2D6 using different ethnic populations are warranted.
Aged↗
[Risk factors for suicide: a questionnaire survey by psychiatrists in Fukuoka Prefecture].
In our previous study, we demonstrated a preliminary questionnaire survey to psychiatrists from university hospitals, psychiatric hospitals, psychiatric clinics, and departments of psychiatry in general hospitals in Fukuoka Prefecture. In that study, 324 psychiatric patients who committed suicide between January 1, 1998 and December 31, 2001 were ascertained. In the present study, we have recruited matched control patients from the same clinics/hospitals and further demonstrated a secondary questionnaire survey to the psychiatrists in order to reveal the various risk factors for suicide suggested by the literature. Associations between completed suicide and possible risk factors were examined in 192 completed suicide psychiatric patients and 356 non-suicidal psychiatric patients (controls). Significant association was found for history of admission to psychiatric hospitals, history of suicide attempts, history of substance abuse, cluster B personality disorders, and hopelessness in our sample. In male samples, history of suicide attempts, cluster B personality disorders, recent loss, and hopelessness were significantly associated with suicide. In females, history of admission to psychiatric hospitals, history of suicide attempts, and hopelessness were significant.
Female↗
Association study between a functional glutathione S-transferase (GSTP1) gene polymorphism (Ile105Val) and tardive dyskinesia.
A possible role for oxidative stress in the pathophysiology of tardive dyskinesia (TD) has previously been proposed (reviewed in Andreassen and Jorgensen [O.A. Andreassen, H.A. Jorgensen, Neurotoxicity associated with neuroleptic-induced oral dyskinesias in rats Implications for tardive dyskinesia? Prog. Neurobiol. 61 (2000) 525-541]). Long-term administration of antipsychotics alters dopaminergic turnover, which results in increased formation of reactive oxygen species (ROS). This is hypothesized to lead to TD through neuronal toxicity as a consequence of oxidative stress. In the present study, the relationship between TD and a functional polymorphism of the gene coding for human glutathione S-transferase P1 (GSTP1), an important antioxidant enzyme involved in the detoxification of ROS, was studied in 225 chronic treatment-refractory patients with schizophrenia. An isoleucine (Ile) to valine (Val) substitution at codon 105 (Ile105Val) in the GSTP1 gene was genotyped. No significant difference in total AIMS scores was found among patients in the three genotype groups (chi(2)=1.47, d.f.=2, p=0.48). Moreover, no significant differences in genotype (chi(2)=0.05, d.f.=2, p=0.98) or allele frequencies (chi(2)=0.00, d.f.=1, p=1.00) were observed between subjects with and without TD. Our results suggest that the GSTP1 gene polymorphism does not confer increased susceptibility to TD, although further studies are warranted before a conclusion can be drawn.
Adolescent↗
Pyrimido[1,2-a]-purin-10(3H)-one, M1G, is less prone to artifact than base oxidation.
Pyrimido[1,2-a]-purin-10(3H)-one (M1G) is a secondary DNA damage product arising from primary reactive oxygen species (ROS) damage to membrane lipids or deoxyribose. The present study investigated conditions that might lead to artifactual formation or loss of M1G during DNA isolation. The addition of antioxidants, DNA isolation at low temperature or non-phenol extraction methods had no statistically significant effect on the number of M1G adducts measured in either control or positive control tissue samples. The number of M1G adducts in nuclear DNA isolated from brain, liver, kidney, pancreas, lung and heart of control male rats were 0.8, 1.1, 1.1, 1.1, 1.8 and 4.2 M1G/10(8) nt, respectively. In rat liver tissue, the mitochondrial DNA contained a 2-fold greater number of M1G adducts compared with nuclear DNA. Overall, the results from this study demonstrated that measuring M1G is a reliable way to assess oxidative DNA damage because the number of M1G adducts is significantly affected by the amount of ROS production, but not by DNA isolation procedures. In addition, this study confirmed that the background number of M1G adducts reported in genomic DNA could have been overestimated by one to three orders of magnitude in previous reports.
Animals↗
Effect of prolonged exposure to milnacipran on norepinephrine transporter in cultured bovine adrenal medullary cells.
The antidepressants milnacipran and paroxetine are used clinically worldwide. In the present study, we report here the effects of treatment with milnacipran and paroxetine on the functional activity, binding sites, and mRNA of the norepinephrine (NE) transporter (NET) in cultured bovine adrenal medullary cells. In acute treatment with antidepressants for 20 min, both milnacipran and paroxetine competitively inhibited NET function in cultured adrenal medullary cells. Prolonged treatment of adrenal medullary cells with milnacipran produced time (48-96h)- and concentration (35-355 nM)-dependent increases in [3H]NE uptake and [3H]DMI binding without any increase in NET mRNA. At a high concentration (800 nM, 72 h), paroxetine suppressed [3H]NE uptake. To examine whether milnacipran-induced [3H]NE uptake is mediated by newly synthesized mRNAs or proteins, we used actinomycin D, an inhibitor of DNA-dependent RNA polymerase, and cycloheximide, an inhibitor of ribosomal protein synthesis. Cycloheximide (1 micorM, 72 h) abolished the effect of milnacipran on [3H]NE uptake, while the stimulatory effect of milnacipran was observed in actinomycin D-treated cells. The present findings suggest that prolonged exposure to milnacipran up-regulates the NET function, probably through a post-transcriptional process of NET or other proteins.
Adrenal Medulla↗
Effects of ethylene oxide and ethylene inhalation on DNA adducts, apurinic/apyrimidinic sites and expression of base excision DNA repair genes in rat brain, spleen, and liver.
Ethylene oxide (EO) is an important industrial chemical that is classified as a known human carcinogen (IARC, Group 1). It is also a metabolite of ethylene (ET), a compound that is ubiquitous in the environment and is the most used petrochemical. ET has not produced evidence of cancer in laboratory animals and is "not classifiable as to its carcinogenicity to humans" (IARC, Group 3). The mechanism of carcinogenicity of EO is not well characterized, but is thought to involve the formation of DNA adducts. EO is mutagenic in a variety of in vitro and in vivo systems, whereas ET is not. Apurinic/apyrimidinic sites (AP) that result from chemical or glycosylase-mediated depurination of EO-induced DNA adducts could be an additional mechanism leading to mutations and chromosomal aberrations. This study tested the hypothesis that EO exposure results in the accumulation of AP sites and induces changes in expression of genes for base excision DNA repair (BER). Male Fisher 344 rats were exposed to EO (100 ppm) or ET (40 or 3000 ppm) by inhalation for 1, 3 or 20 days (6h/day, 5 days a week). Animals were sacrificed 2h after exposure for 1, 3 or 20 days as well as 6, 24 and 72 h after a single-day exposure. Experiments were performed with tissues from brain and spleen, target sites for EO-induced carcinogenesis, and liver, a non-target organ. Exposure to EO resulted in time-dependent increases in N7-(2-hydroxyethyl)guanine (7-HEG) in brain, spleen, and liver and N7-(2-hydroxyethyl)valine (7-HEVal) in globin. Ethylene exposure also induced 7-HEG and 7-HEVal, but the numbers of adducts were much lower. No increase in the number of aldehydic DNA lesions, an indicator of AP sites, was detected in any of the tissues between controls and EO-, or ET-exposed animals, regardless of the duration or strength of exposure. EO exposure led to a 3-7-fold decrease in expression of 3-methyladenine-DNA glycosylase (Mpg) in brain and spleen in rats exposed to EO for 1 day. Expression of 8-oxoguanine DNA glycosylase, Mpg, AP endonuclease (Ape), polymerase beta (Pol beta) and alkylguanine methyltransferase were increased by 20-100% in livers of rats exposed to EO for 20 days. The only effects of ET on BER gene expression were observed in brain, where Ape and Pol beta expression were increased by less than 20% after 20 days of exposure to 3000 ppm. These data suggest that DNA damage induced by exposure to EO is repaired without accumulation of AP sites and is associated with biologically insignificant changes in BER gene expression in target organs. We conclude that accumulation of AP sites is not a likely primary mechanism for mutagenicity and carcinogenicity of EO.
Administration, Inhalation↗
Oxidative DNA damage from potassium bromate exposure in Long-Evans rats is not enhanced by a mixture of drinking water disinfection by-products.
Public drinking water treated with chemical disinfectants contains a complex mixture of disinfection by-products (DBPs) for which the relative toxicity of the mixtures needs to be characterized to accurately assess risk. Potassium bromate (KBrO(3)) is a by-product from ozonation of high-bromide surface water for production of drinking water and is a rodent carcinogen that produces thyroid, mesothelial, and renal tumors. The proposed mechanism of KBrO(3) renal carcinogenesis involves the formation of 8-oxoguanine (8-oxoG), a promutagenic base lesion in DNA typically removed through base excision repair (BER). In this study, male Long-Evans rats were exposed via drinking water to carcinogenic concentrations of KBrO(3) (0.4 g/L), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (0.07 g/L), chloroform (1.8 g/L), bromodichloromethane (0.7 g/L), or a mixture of all these chemicals at the same concentrations for 3 weeks. Half of one kidney was processed for microscopic examination, and the remaining kidney was frozen for isolation of genomic DNA. Levels of 8-oxoG were measured using HPLC with electrochemical detection in DNA samples incubated with formamidopyrimidine-DNA glycosylase. Aldehydic lesions (e.g. abasic sites) in DNA samples were quantitated using an aldehyde-reactive probe slot-blot assay. Treatment with KBrO(3) produced a measurable increase of 8-oxoG in the kidney, and this effect was greater than that produced by treatment with the DBP mixture. No other single chemical treatment caused measurable increases of 8-oxoG. The mixture effect on the amount of 8-oxoG observed in this study suggests an interaction between chemicals that reduced the generation of oxidative DNA damage. No increases in abasic sites were observed with treatment, but a decrease was apparent in the rats treated with the DBP mixture. These data are consistent with previous studies where chronic exposure to this chemical mixture in drinking water resulted in a less than additive carcinogenic response in Tsc2 mutant Long-Evans rats.
Animals↗
The Arg280His polymorphism in X-ray repair cross-complementing gene 1 impairs DNA repair ability.
The contribution of three single nucleotide polymorphisms (SNPs) that substitute amino acids in the X-ray repair cross-complementing gene 1 (XRCC1) protein, Arg194Trp (R194W), Arg280His (R280H), and Arg399Gln (R399Q), to the risk of various types of cancers has been extensively investigated by epidemiological researches. To investigate whether two of these polymorphisms directly influence their repair ability, we established Chinese hamster ovary (CHO) EM9 cell lines transfected with XRCC1(WT), XRCC1(R194W), or XRCC1(R280H) genes and analyzed the DNA repair ability of these cells. The EM9 cells that lack functional XRCC1 proteins exhibit severe sensitivity to methyl methanesulfonate (MMS). Introduction of the human XRCC1(WT) and XRCC1(R194W) gene to EM9 cells restored the MMS sensitivity to the same level as the AA8 cells, a parental cell line. However, introduction of the XRCC1(R280H) gene partially restored the MMS sensitivity, resulting in a 1.7- to 1.9-fold higher sensitivity to MMS compared with XRCC1(WT) and XRCC1(R194W) cells at the LD(50) value. The alkaline comet assay determined diminished base excision repair/single strand break repair (BER/SSBR) efficiency in XRCC1(R280H) cells as observed in EM9 cells. In addition, the amount of intracellular NAD(P)H decreased in XRCC1(R280H) cells after MMS treatment. Indirect immunofluorescence staining of the XRCC1 protein showed an intense increase in the signals and clear foci of XRCC1 in the nuclei of the XRCC1(WT) cells, but a faint increase in the XRCC1(R280H) cells, after MMS exposure. These results suggest that the XRCC1(R280H) variant protein is defective in its efficient localization to a damaged site in the chromosome, thereby reducing the cellular BER/SSBR efficiency.
Animals↗
An open study of risperidone liquid in the acute phase of schizophrenia.
An open-label study was performed to investigate the clinical efficacy and mechanisms of risperidone liquid in ameliorating positive symptoms in the acute phase of schizophrenia. Eighty-eight patients (M/F: 50/38; age: 18-74 years;, mean +/- SD =32 +/- 16 years) meeting DSM-IV criteria for schizophrenia and treated with risperidone liquid (14 patients also used lorazepam) were evaluated with regard to their clinical improvement and extrapyramidal side effects using the positive and negative syndrome scale (PANSS) and the Simpson and Angus scale (SAS), while plasma concentrations of HVA and MHPG were analysed by HPLC-ECD before and 4 weeks after risperidone liquid administration. Patients showing a 50% or greater improvement in PANSS scores were defined as responders. An improvement in the PANSS scores related to excitement, hostility and poor impulse control was seen within 7 days after administration of risperidone liquid, and an improvement with regard to hallucinatory behaviour and uncooperativeness was seen within 14 days after its administration. Finally, 68% of patients were classified as responders 4 weeks after risperidone liquid administration. The scores of SAS were not changed after risperidone liquid administration. Pretreatment plasma homovanillic acid (HVA) levels in the responders (8.1 +/- 2.9 ng/ml) were higher than those in nonresponders (5.9 +/- 1.9 ng/ml). In addition, a negative correlation was seen between the changes in plasma HVA levels and the percentage of improvement in PANSS scores. On the other hand, there were no differences between pretreatment plasma 3-methoxy-4-hydroxyphenylglycol (MHPG) levels and those of nonresponders. These results suggest that risperidone liquid is effective and well tolerated for the treatment of acute phase schizophrenic patients, and that efficacy is related to its affects on dopaminergic activity, not noradrenergic activity.
Acute Disease↗
Effects of zotepine and olanzapine on noradrenaline transporter in cultured bovine adrenal medullary cells.
BACKGROUND: Previously, it was demonstrated that the inhibitory effects of atypical antipsychotic drugs such as clozapine and risperidone on noradrenaline transporter (NAT) might in part be associated with their clinical profile. The present study examined the effects of zotepine on NAT in the cells and compared them with those of olanzapine. MATERIALS AND METHODS: Adrenal medullary cells were isolated by a method of collagenase digestion of slices of fresh bovine adrenal medulla and the cells were plated at a density of 4 x 10(6) cells. Cells were incubated with [3H]noradrenaline (NA) in the presence or absence of zotepine or olanzapine. The amount of radioactivity taken into the cells was counted by a liquid scintillation counter. Plasma membranes of bovine adrenal medulla were prepared, and the binding of [3H]desipramine (DMI) was determined by incubating the membrane suspension in binding buffer together with zotepine or olanzapine. Specific binding of [3H] DMI was defined as that binding which was inhibited by nisoxetine. RESULTS: Both zotepine (10-1000 ng/ml) and olanzapine (10-1000 ng/ml) decreased [3H]NA uptake in a concentration-dependent manner. The IC50 values of zotepine and olanzapine on [3H]NA uptake were 10 +/- 4 and 14 +/- 8 ng/ml, respectively. Eadie-Hofstee analysis of [3H]NA uptake showed that treatment with zotepine and olanzapine decreased the V(max) of uptake without changing the K(m). Both zotepine (10-1000 ng/ml) and olanzapine (30-1000 ng/ml) inhibited [3H]DMI binding in a concentration-dependent manner. The IC50 values of zotepine and olanzapine on [3H]DMI binding were 50 +/- 18, and 120 +/- 38 ng/ml, respectively. Scatchard plot analysis of [3H]DMI binding showed that zotepine and olanzapine decreased the B(max) of binding without altering the K(d). CONCLUSIONS: The inhibitory effects of zotepine and olanzapine might be responsible in part for their clinical profile.
Adrenal Medulla↗
Association between three functional polymorphisms of the dopamine D2 receptor gene and polydipsia in schizophrenia.
The underlying pathophysiology of polydipsia in schizophrenia is poorly understood. However, several studies suggest there may be a genetic predisposition to polydipsia, including our previous study demonstrating familial concordance of polydipsia among first-degree relatives with schizophrenia. Antipsychotic medications may contribute to the development of polydipsia and studies show that dopamine D2 receptors are involved in drinking behaviour pathophysiology. Our hypothesis is that polymorphisms in the dopamine D2 receptor gene (DRD2) may confer susceptibility to polydipsia in schizophrenia. We tested for an association between polydipsia in schizophrenia and three functional polymorphisms of DRD2. The three polymorphisms, -141C Ins/Del, Ser311Cys, and TaqIA, were genotyped in patients with polydipsia (n = 64) and in those without polydipsia (n = 91). Of the three polymorphisms, TaqIA was significantly associated with polydipsia [genotype: chi2 = 6.59, df = 2, p = 0.037; allele: chi2 = 6.52, df = 1, p = 0.011, OR 1.81, 95% CI 1.15-2.86]. Haplotype analysis of the three markers found increased significance of the association (global, p = 0.00091). Although based on a relatively small portion of the sample, individual comparison of the common haplotypes showed that haplotype Ins-Cys-A1 was significantly less frequent in patients with polydipsia (p = 0.00082). The present data suggests polymorphisms in DRD2 may confer susceptibility to polydipsia in schizophrenia. To confirm our findings, further studies are warranted on larger samples using more extensive biological measures for diagnosing the polydipsia phenotype.
Adult↗
Induction of cytotoxicity, aldehydic DNA lesions, and poly(ADP-ribose) polymerase-1 activation by catechol derivatives of pentachlorophenol in calf thymus DNA and in human breast cancer cells.
The purpose of this study was to investigate the degree of chlorination of catechol (CAT) derivatives of pentachlorophenol (PCP) on the induction of cytotoxicity and DNA damaging effects in calf thymus DNA (ct-DNA) and in two human breast carcinoma cell lines. Results indicated that with the addition of the transition metal copper(II), increases in the amount of aldehydic DNA lesions (ADL) were detected in ct-DNA exposed to PCP-derived CATs over the corresponding control. The DNA lesions induced by various degrees of chlorination of PCP-derived CATs decrease in the rank order CAT congruent with 4-chlorocatechol (4-ClCAT) > 4,5-dichlorocatechol (4,5-Cl2CAT) > 3,4,5-trichlorocatechol (3,4,5-Cl3CAT) > tetrachlorocatechol (Cl4CAT). In contrast, Cl4CAT was the only congeneric form of PCP-derived catechols that induced a significant increase in the number of ADL in human MCF-7 cells, and this only occurred when glutathione was depleted. Pretreatment with copper(I) and iron(II) chelators significantly reduced the formation of ADL in cells exposed to Cl4CAT. The data also indicated that the ADL induced by Cl4CAT in MCF-7 cells contain approximately 70% putrescine excisable ADL. This evidence confirmed that the ADL induced by Cl4CAT in MCF-7 cells were derived from oxidative events. In addition, we demonstrated that the depletion of NAD(P)H in human T47D cells exposed to chlorinated CATs decreased in the rank order Cl4CAT >> 4-ClCAT congruent with CAT. The depletion of NAD(P)H induced by Cl4CAT in T47D cells was partially blocked by catalase, superoxide dismutase, dimethyl sulfoxide, and copper(I) and iron(II) specific chelators. Additionally, the depletion of NAD(P)H in T47D cells exposed to Cl4CAT (1-10 microM) was completely blocked by three types of poly(ADP-ribose) polymerase-1 inhibitors. This evidence suggests that Cl4CAT induces an imbalance in DNA repair and the subsequent accumulation of DNA strand breaks in human cultured cells. Overall, these findings indicate that dechlorination may decrease the potentials of chlorinated catechols to induce oxidative DNA lesions and cytotoxic effects in living cells.
Animals↗
Analysis of M1G-dR in DNA by aldehyde reactive probe labeling and liquid chromatography tandem mass spectrometry.
A novel method for the measurement of pyrimido[1,2-a]purin-10(3H)one (M1G) has been developed. Previous methods for analysis of M1G have been confounded by the fact that this lesion exists in equilibrium between a ring-closed form and a ring-opened aldehyde form. Poor detection sensitivity of the aldehydic form can result from loss of the adduct during analysis by its reaction with amines or proteins. We utilized the aldehyde reactive probe (ARP) to produce a stable ARP-M1G-deoxyribose (ARP-M1G-dR) conjugate to minimize adduct loss. This conjugate has increased the hydrophobicity that enhances separation of ARP-M1G-dR from unmodified DNA nucleosides by using solid phase extraction. In addition, measuring ARP-M1G-dR by selective reaction monitoring (SRM) of the [ARP-M1G-dR + H]+ (635) --> [M1G + H]+ (188) transition increases the detection sensitivity by nearly an order of magnitude relative to the measurement of M1G-dR by SRM. For accurate measurement, analytical standard (AS) DNA and internal standard (IS) DNA were used. High purity 15N-labeled DNA was isolated from Escherichia coli that had been grown in minimum salt medium containing (15NH4)2SO4. The 15N-DNA and calf thymus DNA were treated with malondialdehyde to induce a high number of M1G adducts to prepare the IS and AS DNA, respectively. A consistent calibration curve was established from the analysis of 200 microg of blank DNA, 23 ng of IS DNA (400 fmol of 15N5-M1G-dR), and AS DNA containing 0-810 fmol of M1G-dR. With the use of this novel IS DNA and selective labeling, this assay is a useful tool for monitoring oxidative stress-induced DNA damage from small amounts of DNA without the need of a specific antibody or laborious procedures. By this assay, two M1G adducts/10(8) guanines can readily be detected. Furthermore, this approach should be applicable to the analysis of other aldehydic DNA adducts as well as the measurement of an array of DNA lesions.
Antimetabolites, Antineoplastic↗
Effects of naphthalene quinonoids on the induction of oxidative DNA damage and cytotoxicity in calf thymus DNA and in human cultured cells.
The purpose of this study was to examine the differences in the induction of DNA damage and cytotoxic effects by quinonoid derivatives of naphthalene in calf thymus DNA (ct-DNA) and in human T47D breast cancer cells. Results indicated that copper(II) and NADPH were essential for causing oxidant-mediated aldehydic DNA lesions (ADLs), including abasic sites and aldehydic base/sugar lesions, in ct-DNA exposed to 1,2-naphthalenediol (NCAT), 1,4-naphthalenediol (NHQ), 1,2-naphthoquinone (1,2-NQ), and 1,4-naphthoquinone (1,4-NQ). The ADLs induced by naphthalene quinonoids in ct-DNA decrease in the rank order NCAT congruent with 1,2-NQ > NHQ >> 1,4-NQ. Results from the analyses in cells indicated that after 1.5-5 h of exposure all naphthalene quinonoids induced a cytotoxic response in T47D cells at concentrations 10-100 microM or above, where NHQ and 1,4-NQ were approximately 5-10 times more efficient than NCAT and 1,2-NQ in the induction of cell death. In addition, NHQ, 1,2-NQ, and 1,4-NQ were not able to produce measurable levels of ADLs in cells at concentrations up to 1.25 mM, whereas NCAT (0.75-1.25 mM) induced a significant increase in the number of ADLs in T47D cells after 1.5 h of exposure when compared to control. The specific type of ADLs induced by NCAT is resistant to cellular excision repair pathway. Results from the measurements of reactive oxygen species (ROS) indicated that all naphthalene quinonoids induced increases in ROS formation in T47D cells. The induction of ROS formation in cells by naphthalene quinonoids decreases in the rank order 1,4-NQ congruent with 1,2-NQ > NHQ > NCAT. Overall, results from our investigation suggest that naphthalene quinonoids cause cell death at concentrations well below the concentrations at which they induce the formation of ADLs, perhaps by altering intracellular redox status.
Aldehydes↗