Post-zygotic genomic changes in glutamate and dopamine pathway genes may explain discordance of monozygotic twins for schizophrenia.

BACKGROUND: Monozygotic twins are valuable in assessing the genetic vs environmental contribution to diseases. In the era of complete genome sequences, they allow identification of mutational mechanisms and specific genes and pathways that offer predisposition to the development of complex diseases including schizophrenia. METHODS: We sequenced the complete genomes of two pairs of monozygotic twins discordant for schizophrenia (MZD), including one representing a family tetrad. The family specific complete sequences have allowed identification of post zygotic mutations between MZD genomes. It allows identification of affected genes including relevant network and pathways that may account for the diseased state in pair specific patient. RESULTS: We found multiple twin specific sequence differences between co-twins that included small nucleotides [single nucleotide variants (SNV), small indels and block substitutions], copy number variations (CNVs) and structural variations. The genes affected by these changes belonged to a number of canonical pathways, the most prominent ones are implicated in schizophrenia and related disorders. Although these changes were found in both twins, they were more frequent in the affected twin in both pairs. Two specific pathway defects, glutamate receptor signaling and dopamine feedback in cAMP signaling pathways, were uniquely affected in the two patients representing two unrelated families. CONCLUSIONS: We have identified genome-wide post zygotic mutations in two MZD pairs affected with schizophrenia. It has allowed us to use the threshold model and propose the most likely cause of this disease in the two patients studied. The results support the proposition that each schizophrenia patient may be unique and heterogeneous somatic de novo events may contribute to schizophrenia threshold and discordance of the disease in monozygotic twins.

Integration of DNA sequence and DNA methylation changes in monozygotic twin pairs discordant for schizophrenia.

Schizophrenia is a complex mental disorder with high heritability (80%), extensive genetic heterogeneity, environmental contributions and only 50% concordance in discordant monozygotic (MZ) twins. Discordant MZ twins provide an exceptional opportunity to assess patient specific genome-wide genetic and epigenetic changes that may account for the disease phenotype. A combined analysis of genetic and epigenetic changes on the same twin pairs is expected to provide a more effective approach for two reasons. First, it is now possible to generate relatively reliable complete genome sequences as well as promoter methylation states on an individual level and second, the unaffected twin that originated from the same zygote provides a near perfect genetic match for contrast and comparison. This report deals with the combined analysis of DNA sequence data and methylation data on two pairs of discordant MZ twins that have been clinically followed for over 20 years. Results on Family 1 show that 58 genes differ in DNA sequence as well as promoter methylation in a schizophrenia-affected twin as compared to her healthy co-twin. The corresponding number for family 2 was 13. The two lists are over represented by neuronal genes and include a number of known schizophrenia candidate genes and drug targets. The results argue that changes in multiple genes via co-localized genetic and epigenetic alteration contribute to a liability threshold that is necessary for development of schizophrenia. This novel hypothesis, although logical, remains to be validated.

Opioid receptor mu 1 gene, fat intake and obesity in adolescence.

Dietary preference for fat may increase risk for obesity. It is a complex behavior regulated in part by the amygdala, a brain structure involved in reward processing and food behavior, and modulated by genetic factors. Here, we conducted a genome-wide association study (GWAS) to search for gene loci associated with dietary intake of fat, and we tested whether these loci are also associated with adiposity and amygdala volume. We studied 598 adolescents (12-18 years) recruited from the French-Canadian founder population and genotyped them with 530 011 single-nucleotide polymorphisms. Fat intake was assessed with a 24-hour food recall. Adiposity was examined with anthropometry and bioimpedance. Amygdala volume was measured by magnetic resonance imaging. GWAS identified a locus of fat intake in the µ-opioid receptor gene (OPRM1, rs2281617, P=5.2 × 10(-6)), which encodes a receptor expressed in the brain-reward system and shown previously to modulate fat preference in animals. The minor OPRM1 allele appeared to have a 'protective' effect: it was associated with lower fat intake (by 4%) and lower body-fat mass (by ∼2 kg, P=0.02). Consistent with the possible amygdala-mediated inhibition of fat preference, this allele was additionally associated with higher amygdala volume (by 69 mm(3), P=0.02) and, in the carriers of this allele, amygdala volume correlated inversely with fat intake (P=0.02). Finally, OPRM1 was associated with fat intake in an independent sample of 490 young adults. In summary, OPRM1 may modulate dietary intake of fat and hence risk for obesity, and this effect may be modulated by subtle variations in the amygdala volume.

Analyses of genetic diversity in five Canadian dairy breeds using pedigree data.

The issue of loss of animal genetic diversity, worldwide in general and in Canada in particular, has become noteworthy. The objective of this study was to analyze the trend in within-breed genetic diversity and identify the major causes of loss of genetic diversity in five Canadian dairy breeds. Pedigrees were analyzed using the software EVA (evolutionary algorithm) and CFC (contribution, inbreeding, coancestry), and a FORTRAN package for pedigree analysis suited for large populations (PEDIG). The average rate of inbreeding in the last generation analyzed (2003 to 2007) was 0.93, 1.07, 1.26, 1.09 and 0.80% for Ayrshire, Brown Swiss, Canadienne, Guernsey and Milking Shorthorn, respectively, and the corresponding estimated effective population sizes were 54, 47, 40, 46 and 66, respectively. Based on coancestry coefficients, the estimated effective population sizes in the last generation were 62, 76, 43, 61 and 76, respectively. The estimated percentage of genetic diversity lost within each breed over the last four decades was 6, 7, 11, 8 and 5%, respectively. The relative proportion of genetic diversity lost due to random genetic drift in the five breeds ranged between 59.3% and 89.7%. The results indicate that each breed has lost genetic diversity over time and that the loss is gaining momentum due to increasing rates of inbreeding and reduced effective population sizes. Therefore, strategies to decrease rate of inbreeding and increase the effective population size are advised.

Genetic diversity of Guernsey population using pedigree data and gene-dropping simulations.

The objectives of this study were to analyze the trend of within-breed genetic diversity and identify major causes leading to loss of genetic diversity in Guernsey breed in three countries. Pedigree files of Canadian (GCN), South African (GSA) and American (GUS) Guernsey populations containing 130 927, 18 593 and 1 851 624 records, respectively, were analyzed. Several parameters derived from the in-depth pedigree analyses were used to measure trends and current levels of genetic diversity. Pedigree completeness index of GCN, GSA and GUS populations, in the most recent year (2007), was 97%, 74% and 79%, respectively, considering four generations back in the analysis. The rate of inbreeding in each population was 0.19%, 0.16% and 0.17% between 2002 and 2007, respectively. For the same period, the estimated effective population size for GCN, GSA and GUS was 46, 57 and 46, respectively. The estimated percentage of genetic diversity lost within each population over the last four decades was 8%, 3% and 5%, respectively. The relative proportion of genetic diversity lost due to random genetic drift in the three populations was 93%, 91% and 86%, respectively. In conclusion, the results suggested that GCN and GUS have lost more genetic diversity than GSA over the past four decades, and this loss is gaining momentum due to increasing rates of inbreeding. Therefore, strategies such as optimum contribution selection and migration of genetic material are advised to increase effective population size, particularly in GCN and GUS.

Expression of apoptosis regulatory genes and incidence of apoptosis in different morphological quality groups of in vitro-produced bovine pre-implantation embryos.

Apoptosis occurs during early development in both in vivo- and in vitro-produced embryos, and is considered as one of the causes of embryonic loss. The objectives of this study were, therefore, investigating stage-specific expression profiles of apoptosis regulatory genes in three quality groups of in vitro-produced bovine pre-implantation embryos; and analysing the relationship between cell number and DNA fragmentation with expressions of those genes. The relative abundance of mRNA of 9 pro- (Bax, caspase-9, Bcl-xs, P53, Caspase-3 and Fas) and anti- (Bcl-w and Mcl-1) apoptotic genes was analysed. Differential cell staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling were performed to analyse the variation in cell numbers and detect apoptotic nuclei respectively. Expression of Bax and Caspase-3 genes was significantly (p < 0.05) higher in poor quality pre-implantation embryos as compared with that of morphologically good quality embryos of the same developmental stages. Moreover, Mcl-1 expression was significantly higher in good quality immature oocytes than that in the poor quality group. Moreover, higher DNA fragmentation was evidenced in morphologically poor quality blastocysts. In conclusion, our study demonstrates that Bax, caspase-3 and Mcl-1 can be used as potential markers of embryo quality to evaluate in vitro-produced bovine embryos. Further studies are required to investigate specific molecular signatures that can be used in evaluating in vivo-derived embryos.

Evaluation of the antineoplastic activity of mitoxantrone-L-carnitine combination therapy on an experimental solid form of ehrlich tumour in mice.

We have commenced a series of experiments to evaluate the effect of carnitine derivatives on the antineoplastic activity of mitoxantrone (MX) on various animal cancers. This report describes the therapeutic effect of MX in combination with l-carnitine (LCAR) on the growth of a solid form of Ehrlich tumour inoculated into mice. LCAR was administered subcutaneously at doses of either 200 or 100mgkg(-1) on day 6 and 13 after tumour inoculation, 1h prior to the treatment with MX. Mitoxantrone was administered intravenously at doses of 3 or 6mgkg(-1). We found that LCAR had no potentiating effect on the efficacy of MX, in terms of either slowing tumour growth or increasing the survival of mice. Nevertheless, therapeutic effects can be assumed at higher doses of both drugs based on values calculated from an index of relative hazards.

[Plasma levels of troponin T in rabbits after administration of new antineoplastic agents (Oracin and Dimefluron) and daunorubicin]. / Plazmatické hladiny troponinu T u králíku po podání nových antineoplastických látek (Oracinu a Dimefluronu) s daunorubicinem.

Recently, cardiac troponin T (cTnT) has been shown to be a sensitive marker of anthracycline-induced cardiomyopathy. In our study, the cardiotoxicity of repeated i.v. administration (once a week, 10 administrations) of daunorubicin combined with new antineoplastic drugs (with mild side-effects) were followed in two groups of rabbits: 1) Dimefluron (3,9-dimethoxybenfluron hydrochloride-12 mg/kg) + daunorubicin (3 mg/kg), 2) Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H- indeno[1,2c]isoquinoline hydrochloride--10 mg/kg) + daunorubicin (3 mg/kg) and compared with the control group (saline--1 ml/kg) and the group with experimentally induced cardiomyopathy (daunorubicin--3 mg/kg). The concentration of cTnT in heparinized plasma samples was measured using commercial kit (Roche). In the control group, plasma levels of cTnT were always within the physiological range (i.e. lower than 0.1 microgram/l) during the experiment. During the development of daunorubicin-induced cardiomyopathy, after the eighth administration of drug, cTnT was significantly higher (0.31 +/- 0.11 microgram/l) in animals with premature deaths compared with the rest of the group (0.04 +/- 0.03 microgram/l). The animals with pathological values of cTnT were at higher risk of premature deaths (P = 0.0006). The combination of daunorubicin either with Oracin or with Dimefluron caused neither significant changes of cTnT levels nor significant deterioration of other followed-up parameters (especially, functional and toxicological parameters). Similarly to the daunorubicin group, the animals with pathological levels of cTnT after the eighth administration of antineoplastic drugs were at higher risk of premature death (P = 0.025). Our results show that the plasma concentration of cardiac troponin T could be a suitable predictive marker of cardiotoxicity of antineoplastic drugs.

[Effects of new antineoplastic agents (dimethoxybenflurone and Oracin) on the cardiovascular system and other parameters in the rabbit in vivo]. / Ucinky nových antineoplastických látek (dimethoxybenfluronu a Oracinu) na kardiovaskulární systém a dalsí parametry u králíku in vivo.

Anthracycline derivatives belong among the most effective antineoplastic drugs but their therapeutic use is limited by their side effects--a dose-related cardiotoxicity. The influence of repeated i.v. administration (once weekly, max. 10 weeks) of new antineoplastic agents--dimethoxybenfluron (DMB) (3,9-dimethoxybenfluron hydrochloride, C23H24O4NCl, M.w. 413.9, Institute of Experimental Biopharmaceutics, Czech Academy of Sciences, Hradec Králové, Czech Republic; 12 or 24 mg base/kg) and Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H- indeno[1,2c]isoquinoline hydrochloride), C20H19N2O3Cl, M.w. 370.84, Research Institute for Pharmacy and Biochemistry, Prague, Czech Republic; 5 or 10 mg/kg) on cardiovascular, biochemical, haematological and histological parameters were studied in rabbits in vivo. Data obtained in these groups were compared with the group with experimentally induced cardiomyopathy (daunorubicin 50 mg/m2 i.v.) and with the control group (saline 1 ml/kg). Only mild and mostly no significant changes of the cardiovascular parameters (DMB 12 group: PEP:LVET ratio--0.408-0.502, LV dP/dtmax.--1337.0 kPa/s; DMB 24 group: PEP:LVET ratio--0.407-0.433, LV dP/dtmax.--1438.2 kPa/s), biochemical parameters (decrease in natrium, ALP and increase in glucose, GPX and GSH levels) and haematological parameters (increase in erythrocytes and decrease in leukocytes after the larger dose of the drug) were found in the dimethoxybenfluron groups. Repeated administration of the lower dose of Oracin induced only mild and mostly no significant changes of parameters (PEP:LVET ratio--0.393-0.475, LV dP/dtmax.--1092.4 kPa/s) in comparison with the control group. Though significant in some intervals, only a mild oscillation of the PEP:LVET ratio (0.368-0.446), decrease in LV dP/dtmax. (991.2 kPa/s) and--in comparison with control group--significantly higher blood pressure and lower heart rate were found after the higher dose of Oracin. In the most of haematological and biochemical parameters (with the exception of chlorides, protein and albumin levels) no significant changes were present. Histological examination of the heart revealed normal structure of the myocardium including minute changes of myocardium following administration of antineoplastic agents in all groups. Administration of new antineoplastic agents induced mostly mild changes of the followed-up parameters (PEP:LVET ratio, LV dP/dtmax., heart rate, levels of cardiac troponin T, survival of animals, haematological and biochemical parameters); the values of parameters were mostly significantly different from those in rabbits with daunorubicin-induced cardiomyopathy. On the basis of our results it is possible to conclude that the administration of dimethoxybenflurone and Oracin did not induce signs of cardiotoxicity in rabbits in vivo. This observation is considered to be important from the viewpoint of possible further clinical use of these new antineoplastic agents.

Effect of acetyl-L-carnitine on leukemia L1210 resistant to mitoxantrone.

Supportive care in tumour chemotherapy is a subject of intensive research. The complications of cytostatic therapy are a cause of extensive research of their pharmacological interactions and side effects. The immunologic and biochemical changes accompanying tumours are the factor that is most responsible for the worsening of the physiology of the host. Regimens containing carnitine and it's acetyl-derivative are used in many cases, among others even for preventing hepatotoxicity. Our hypothesis was to verify the supporting metabolic effects of acetyl-L-carnitine hydrochloride (ALC) in combined therapy with mitoxantrone (MX) and hepatotoxic cytostatic drugs including alkylating agents. This present report describes the effect of ALC in combination with MX on DBA/2 male mice bearing a transplantable L1210 leukemia resistant to MX. The criterion for evaluation of effect was the length of survival time of experimental animals. The proportional-hazards model quadratic in the drug dose (7) was used for survival time evaluation and optimal dose calculation. The hazard functions and the index of relative hazard were determined using Weibull distribution after logarithmic transformation of the entered data in each particular group. The dose-response curve was represented by a second-degree polynomial without absolute term. The combination therapy revealed that the optimal dose of ALC was 186 mg/kg s.c. This relation is shown in Fig.1. A significant effect of ALC (s.c.) in combined therapy with MX (6 mg/kg i.v.) given to animals bearing an experimental form of leukemia L1210/MX resistant to MX was proven at a level of probability p < or = 0.001. The effect of ALC in monotherapy was not demonstrable.

Cardiac troponin T as a marker of myocardial damage caused by antineoplastic drugs in rabbits.

Anthracycline derivatives are among the most effective antineoplastic drugs but their therapeutic use is limited by their adverse effects. The cardiac side-effects of antineoplastic drugs were investigated in rabbits in vivo from the viewpoint of release of cardiac troponin T (cTnT) measured by Elecsys Troponin T STAT immunoassay (Boehringer Mannheim, Germany). No increase in cTnT was found following administration of a single dose of daunorubicin (3 mg/kg i.v., n = 4). During development of daunorubicin-induced cardiomyopathy (daunorubicin 3 mg/kg i.v., once a week; maximum nine administrations, n = 7), the levels of cTnT were within the physiological range (i.e. cTnT <0.1 microg/l) at the beginning of the experiment and before and after the 5th administration, but the pathological values of cTnT after the 8th administration in 43% animals (0.22+/-0.08 microg/l) correlated with their premature death. In the control group, the levels of cTnT were always lower than 0.1 microg/l during the experiment. Following administration of a new antineoplastic drug - Oracin [6-[2-(2-hydroxyethyl) aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno [1,2-c]-isoquinoline hydrochloride, 10 mg/kg i.v., once weekly, ten administrations, n = 7], there was no increase in cTnT levels. These findings correlated with the PEP: LVET index, histological examination and no animal succumbing to premature death. It is possible to conclude that cTnT is a useful marker for the prediction of experimentally induced anthracycline cardiomyopathy and for the evaluation of cardiotoxic (and, possibly, cardioprotective) effects of new drugs in rabbits.

Lack of cardiotoxicity of a new antineoplastic agent, a synthetic derivative of indenoisochinoline: comparison with daunorubicin in rabbits.

The effect of repeated i.v. administration (once weekly, 12 administrations) of a new antineoplastic agent, Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5, 11-dioxo-5,6-dihydro-11 H-indeno [1,2-c]-isochinoline hydrochloride, 10 mg/kg) and daunorubicin (3 mg/kg) were investigated in rabbits in vivo. The criterion of occurrence of cardiotoxicity was compared with a control group of animals. Noninvasive polygraphic records were used to evaluate the function of the heart. The morphological changes of the heart were evaluated after the death of animals. There were no significant changes found in the ratio of the pre-ejection period/left ventricular ejection time (PEP: LVET ratio) after administration of Oracin (values between 0.3080 and 0.3310) or in the control group (values between 0.3425 and 0.3885). The administration of daunorubicin induced a significant, progressive increase in the PEP: LVET ratio (0.3775-0.9473), which was significantly different both from the Oracin-treated and the control group of animals. Histological examination of the hearts from the control group revealed normal structure of the myocardium including minute changes (dispersed cardiomyocytes with intensively eosinophilic cytoplasm, and several single cells with degenerated myofibrils) similar to the normal changes in muscle tissue. A very similar scenario was found in the Oracin group with the exception of one case where a slightly higher number of degenerated and necrotic cells was occurring. Administration of daunorubicin resulted in severe dispersed damage of the myocardium (myocytolysis with subsequent interstitial fibrosis), the changes being markedly different from those of the Oracin treatment and the control group. On the basis of our results it is possible to conclude that the administration of Oracin (10 mg/kg i.v.) did not induce signs of cardiotoxicity in rabbits in vivo.

Cloturin: effect on energy-producing processes in Ehrlich ascites and P388 murine leukaemia cells.

The main purpose of the present investigation was to study the effect of cloturin on aerobic glycolysis, endogenous and exogenous respiration and the level of ATP in both Ehrlich ascites carcinoma (EAC) and P388 murine leukaemia cells incubated in vitro. Also its effect on the level of total (T-SH) and non-protein (NP-SH) thiol groups was investigated. A significant inhibition of aerobic glycolysis was found only in P388 cells after 60 min of cloturin action. Cloturin inhibited both endogenous and exogenous respiration of EAC with succinate as substrate. Cloturin decreased the level of ATP after 2 h incubation in both types of tumour cell. The level of NP-SH was decreased more than that of T-SH in both types of cell.

Effects of benfluron and its two metabolites on respiratory processes in P388 murine leukemia and Ehrlich ascites cells.

This paper presents data on the effects of benfluron and its two metabolites DBF and NOBF on both endogenous and exogenous, respiration in the presence of succinate as substrate, of both P388 murine leukemia and Ehrlich ascites carcinoma cells. The most efficient inhibitors of endogenous and exogenous respiration were benfluron and DBF. NOBF did not interfere with respiratory processes in Ehrlich cells, even at quite high concentration. BF and DBF exert an almost identical inhibitory effect on exogenous respiration, the least effective being NOBF (Ehrlich cells). The decrease in the respiratory rates in cancer cells might be due to the effects of benfluron and its metabolites on the cell membrane. P388 murine leukemia cells are less "sensitive" than Ehrlich ascites cells.

9-Hydroxybenfluron: cytostatic effects and inhibition of macromolecular biosynthesis in Ehrlich ascites and P388 murine leukemia cells.

Primary screening in vitro and study on the mode of action of 9-hydroxybenfluron (HBF) in both murine P388 leukemia and Ehrlich ascites carcinoma cells have been performed. Metabolite HBF is approximately twice as effective as a reference drug (benfluron). To elucidate the biochemical mode of action, the effect of HBF on the biosynthesis of macromolecules indicated by the incorporation rate of [14C]adenine (in DNA and RNA), [14C]thymidine (in DNA), [14C]uridine (in RNA) and [14C]valine (in protein) was studied in concentration and time dependence. HBF inhibited incorporation of all four precursors into the trichloroacetic acid-insoluble fraction of Ehrlich ascites cells. The fact that incorporation of these four precursors is inhibited suggests that the effect of HBF lies at an underlying level of energy generation or transfer rather than at specific reactions in the biosynthesis of DNA and proteins.

Primary screening and inhibition of macromolecular biosynthesis in Ehrlich ascites cells by benzo(C)fluorene derivatives.

The main objective of the present investigation was to screen a series of new benzo(c)fluorene compounds for in vitro activity. It can be stated that each of the 9 newly synthesized benzo(c)fluorene derivatives was about 10 times as active as tilorone. To elucidate the biochemical mode of action, the effects of 2 new compounds (13468 and 14200) on biosynthesis of macromolecules indicated by the incorporation rate of [14C]adenine (DNA, RNA), [14C]-thymidine (DNA), [14C]uridine (RNA) and [14C]valine (protein) were studied in concentration and time dependence. Both compounds inhibited the incorporation of the 4 precursors into the TCA-insoluble fraction of Ehrlich ascites carcinoma cells.

Effect of new purine analog 6-purinyl-N-[2-chloroethyl] thiocarbamate (Cloturin) on immune system of mice.

The effect of purine analog 6-purinyl-N-[2-chloroethyl] thiocarbamate (Cloturin) was studied in a battery of immunological tests in mice. Cloturin exhibited pronounced suppressive nonselective effect on immune system and its functional capacity. Cloturin decreased cellularity of spleen and bone marrow, elicited leukocytopenia in peripheral blood, suppressed phagocytosis, graft-versus-host reaction and specific antibody production. In comparison with azathioprine, Cloturin had more pronounced activity. The immunosuppressive effect of Cloturin on T-dependent specific antibody production suggests that Cloturin possesses both alkylating and antimetabolic properties. For its properties Cloturin could be useful not only as a cytostatic drug, but also as an immunosuppressive agent.

Substances with antineoplastic activity. XCVI. Some pharmacological properties and therapeutic synergism of 6-purinyl-N-(2-chloroethyl)thiocarbamate with cytosine arabinoside.

The cancerostatic effect of 6-purinyl-N-(2-chloroethyl)thiocarbamate (Cloturin VUFB, VUFB-15686) was studied in detail in mice and rats bearing transplantable tumors. The cytotoxic activities were measured by determining the incorporation rate of 5-iodo-2'-deoxy[6-3H]uridine and uniformly labeled [U-14C]amino acid mixture into trichloroacetic acid-insoluble fraction of Yoshida ascites reticulosarcoma cells during a short-term incubation with the drug. From the results obtained it follows that the new substance is therapeutically more effective in comparison with 6-mercaptopurine (NSC-755). A therapeutic synergism of Cloturin with cytosine arabinoside (NSC-63978) was observed.

Effects of benfluron metabolites on glycolysis and level of thiol groups in P388 and Ehrlich ascites cells.

The effects of two benfluron metabolites, namely 7-dihydrobenfluron (DBF) and N-oxide of benfluron (NOBF), on the aerobic glycolysis of Ehrlich ascites carcinoma (EAC) and P388 cells were studied. Also their effect on the level of total (T-SH) and nonprotein (NP-SH) thiol groups was investigated. A significant inhibition of aerobic glycolysis in the presence of NOBF was found only in P388 cells. Both metabolites decreased more significantly the level of NP-SH than that of T-SH.